JP4982755B2 - Determination of absolute configurations of sugars and related aldehyde compounds by high-performance liquid chromatography. - Google Patents

Description

  The present invention relates to a method for determining the absolute configuration of sugars and related aldehyde compounds.

Many biological compounds have sugar residues such as glycosides, and determination of the absolute configuration of the sugar is important.
As a method for determining the absolute configuration of sugar, a method is known in which sugar is reacted with L-cysteine methyl ester, then trimethylsilylated, and measured by gas chromatography (see Non-Patent Document 1). This method requires gas chromatography, but maintenance and management of gas chromatography such as loading and unloading of gas cylinders is difficult.
As a method for determining the absolute configuration of sugar without using gas chromatography, the method described in Non-Patent Document 2 using liquid chromatography is also known, but the operation is complicated and skill is required. It is.
Therefore, development of a method for determining the absolute configuration of sugars by a simple operation is desired.
Chem. Pharm. Bull. , 35 (2), 501-506, 1987 CHEMISTRY LETTERS, 943-946, 1981

  An object of the present invention is to provide a method for determining the absolute configuration of sugars and related aldehyde compounds by a simple operation, and a kit for carrying out the method.

  As a result of intensive studies, the present inventors can determine the absolute configuration of sugars and related aldehyde compounds by a simple operation by subjecting a compound represented by the formula (I) described later to high performance liquid chromatography. The present inventors have found that the present invention can be accomplished and have completed the present invention. That is, the present invention is as follows.

[1] Formula (I):

[Where:
R1 represents an optionally substituted hydrocarbon group,
R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group;
Z represents an organic residue]
A compound represented by formula (II): wherein the compound represented by formula (hereinafter also referred to as “compound (I)”) is subjected to high performance liquid chromatography:

[Wherein Z is as defined above]
A method for determining an absolute configuration of an optically active aldehyde represented by formula (hereinafter also referred to as “optically active aldehyde (II)”).
[2] Formula (II):

[Wherein Z is as defined in claim 1]
An optically active aldehyde represented by the formula (III):

[Wherein R 1 has the same meaning as in claim 1]
And a salt thereof (hereinafter also referred to as “cysteine derivative (III)”), and then the formula (IV):

[Wherein R2 has the same meaning as in claim 1]
Formula (I) obtained by reacting with a compound represented by the formula (hereinafter also referred to as “compound (IV)”):

[Wherein each symbol has the same meaning as in claim 1]
The method according to [1], wherein the compound represented by the formula is subjected to high performance liquid chromatography.
[3] The L- or D-cysteine derivative has the formula (V):

[Wherein R 1 has the same meaning as in claim 1]
The method according to [2], which is produced using an L- or D-cystine derivative represented by the formula (I) or a salt thereof (hereinafter also referred to as “cystine derivative (V)”) and dithiothreitol.
[4] Z is the formula:

[Where:
R3 represents a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group. ,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The method as described in any one of [1]-[3] which is group represented by these.
[5] R1 represents an alkyl group;
The method according to any one of [1] to [4], wherein R2 represents an optionally substituted aryl group or an optionally substituted aralkyl group.
[6] Formula (I):

[Where:
R1 represents an optionally substituted hydrocarbon group,
R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group;
Z represents an organic residue]
A compound represented by
[7] Z is the formula:

[Where:
R3 represents a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group. ,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The compound of [ 6 ] description which is group represented by these.
[8] R1 represents an alkyl group,
The compound according to [ 6 ] or [ 7 ], wherein R 2 represents an optionally substituted aryl group or an optionally substituted aralkyl group.
[9] (a) Formula (III):

[Wherein R1 represents an optionally substituted hydrocarbon group]
An L- or D-cysteine derivative represented by the formula (IV):

[Wherein R 2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group]
A kit for analyzing an optically active aldehyde, comprising a compound represented by:
[10] The L- or D-cysteine derivative has the formula (V):

[Wherein R 1 has the same meaning as in claim 9]
The kit according to [9], which is produced using an L- or D-cystine derivative represented by the formula:
[11] (c) Formula (I) derived from an optically active aldehyde represented by the formula (II): Z—CHO having a known absolute configuration as a standard product :

[Where:
R1 represents an optionally substituted hydrocarbon group,
R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group;
Z represents an organic residue]
The kit according to [9] or [10], further comprising a compound represented by:
[12] Z is the formula:

[Where:
R3 represents a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group. ,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The kit according to [11], which is a group represented by:
[13] R1 represents an alkyl group;
The kit according to any one of [9] to [12], wherein R2 represents an aryl group which may be substituted or an aralkyl group which may be substituted.

  By the method of the present invention, the absolute configuration of the sugar and the related aldehyde compound can be determined by a simple operation.

  Further, according to the method of the present invention, the molar ratio of the mixture of sugar and related aldehyde compound can be estimated by a simple operation.

  The definitions of each group and each symbol used in the present specification are as follows.

Examples of the “halogen atom” include fluorine, chlorine, bromine and iodine.
Examples of the “alkyl group” include linear or branched C _1-6 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like. Groups.
Examples of the “alkenyl group” include linear or branched C ₂₋ such as vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 1-hexenyl and the like. ₆ alkenyl group is mentioned.
Examples of the “alkynyl group” include straight chain or branched C ₂ -2 such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 1-hexynyl and the like. _{A 6} alkynyl group is mentioned.
Examples of the “cycloalkyl group” include C _3-6 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
Examples of the “aryl group” include C _6-14 aryl groups such as phenyl, 1-naphthyl, 2-naphthyl, and anthryl.
Examples of the “aralkyl group” include C _7-14 aralkyl groups such as benzyl, 1-phenylethyl, 2-phenylethyl, naphthylmethyl and the like.
Examples of the “alkoxy group” include linear or branched C _1-6 alkoxy groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like. Is mentioned.

R1 represents an optionally substituted hydrocarbon group.
Examples of the “hydrocarbon group” of the “optionally substituted hydrocarbon group” represented by R 1 include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an aralkyl group, and the like.

Examples of the substituent that the “hydrocarbon group” of the “optionally substituted hydrocarbon group” represented by R 1 may have (1) a halogen atom,
(2) A hydroxy group etc. are mentioned. The number of substituents is 1-5, for example, Preferably it is 1-3, and when the number of substituents is two or more, each substituent may be the same or different.

R1 is preferably an _optionally substituted alkyl group, more preferably a C _1-6 alkyl group, and particularly preferably a methyl group or an ethyl group.

R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group.
As the substituent that the “aryl group” of the “optionally substituted aryl group” represented by R2 and the “aralkyl group” of the “optionally substituted aralkyl group” may have,
(1) a halogen atom,
(2) a hydroxy group,
(3) an alkyl group (preferably a methyl group, etc.),
(4) Alkoxy group (preferably methoxy group etc.)
Etc. The number of substituents is 1-5, for example, Preferably it is 1-3, and when the number of substituents is two or more, each substituent may be the same or different.

  The “fluorescent group” represented by R2 is not particularly limited as long as it emits fluorescence. For example, a 4- (N, N-dimethylaminosulfonyl) -2,1,3-benzoxadiazol-7-yl group And the like.

R2 is preferably an optionally substituted aryl group or an optionally substituted aralkyl group, and is substituted with one or more substituents selected from a C _1-6 alkyl group and a C _1-6 alkoxy group. An _{optionally substituted} C _6-14 aryl group and C _7-14 aralkyl group are more preferred. Of these, a phenyl group, a p-tolyl group, an o-tolyl group, a 3,4-dimethoxyphenyl group, a benzyl group, and a 2-phenylethyl group are particularly preferable.

  The “organic residue” represented by Z is not particularly limited as long as the aldehyde represented by the formula (II) is an optically active aldehyde. For example, the alkyl group, cycloalkyl, which may be substituted, respectively. Group, heterocyclic group and the like.

  Here, examples of the “heterocyclic group” include a 3 to 7-membered heterocyclic group containing 1 to 5 heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in addition to a carbon atom. Examples include pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, pyridyl, piperidinyl, piperazinyl, morpholinyl, quinuclidinyl and the like.

As the substituent that the “alkyl group” exemplified as the “organic residue” represented by Z may have, for example,
(1) an optionally substituted hydroxy group,
(2) a substituted amino group,
(3) carboxyl group,
(4) A halogen atom etc. are mentioned. The number of substituents is 1-5, for example, Preferably it is 1-3, and when the number of substituents is two or more, each substituent may be the same or different.
Examples of the “optionally substituted hydroxy group” include hydroxy groups that may be substituted with alkyl-carbonyl groups (eg, C _1-6 alkyl-carbonyl groups such as acetyl and propionyl) and the like. .
In addition, two of “optionally substituted hydroxy groups” are joined together,

Etc. may be formed.

Examples of the “substituted amino group” include an amino group mono- or di-substituted with an alkyl group, an alkyl-carbonyl group (eg, a C _1-6 alkyl-carbonyl group such as acetyl, propionyl, etc.) and the like. It is done.

  As the substituent that the “cycloalkyl group” or “heterocyclic group” exemplified as the “organic residue” represented by Z may have, the “optionally substituted aryl group” represented by R2 The same thing as the substituent which "aryl group" may have is mentioned. The number of substituents is 1-5, for example, Preferably it is 1-3, and when the number of substituents is two or more, each substituent may be the same or different.

  Among the “organic residues” represented by Z, an alkyl group which may be substituted is preferable, and among them, the formula:

[Where:
R3 represents a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group. ,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
Is more preferable.

  As the substituent of the “optionally substituted hydroxy group” represented by R3 or R5, the “alkyl group” of the “optionally substituted alkyl group” exemplified as the “organic residue” represented by Z What was illustrated as a substituent is mentioned.

  As the substituent of the “substituted amino group” represented by R3 or R5, the substituent of the “alkyl group” of the “optionally substituted alkyl group” exemplified as the “organic residue” represented by Z What was illustrated is mentioned.

  Examples of the “optionally substituted hydroxy group and the alkyl group substituted with a substituent selected from a substituted amino group” represented by R3 or R3 ′ include the above “optionally substituted hydroxy group”, Examples thereof include an alkyl group substituted with the above-mentioned “substituted amino group”. Specific examples include a hydroxymethyl group, an acetyloxymethyl group, a propionyloxymethyl group, and an acetylaminomethyl group.

  Further, substituted with a substituent selected from “optionally substituted hydroxy group” represented by R3 or R5 and “optionally substituted hydroxy group and substituted amino group represented by R3 or R3 ′” Any two of the “optionally substituted hydroxy groups” that are substituents of the alkyl group of

Etc. may be formed.

As the compound (I) of the present invention,
Z is

[Where:
R3 represents a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group. ,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The compound (I) which is group represented by these is preferable.

R1 is an alkyl group;
A compound (I) in which R 2 is an optionally substituted aryl group or an optionally substituted aralkyl group is also preferred.

Above all,
R1 is a C _1-6 alkyl group,
R2 is a C _6-14 aryl group which may be substituted with one or more substituents selected from a C _1-6 alkyl group and a C _1-6 alkoxy group, or a C _7-14 aralkyl group,
Z is the formula:

[Where:
R3 represents a hydrogen atom, a hydroxy group, an acetylamino group or a hydroxymethyl group,
R3 ′ is a hydrogen atom,
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom or a hydroxy group), or a carboxyl group,
n represents an integer of 1 to 4 (except that all of n R3 and R5 are hydrogen atoms)]
Compound (I) which is a group represented by the formula is particularly preferred.

The present invention provides a method for determining the absolute configuration of optically active aldehyde (II) by subjecting compound (I) to high performance liquid chromatography.
Specifically, the method for determining the absolute configuration of the present invention is:
(A) obtaining compound (I) from optically active aldehyde (II),
(B) A step of subjecting the obtained compound (I) to high performance liquid chromatography, and (c) a step of comparing the retention time of the compound (I) with the retention time of the standard product.

  Step (a) is a step of obtaining compound (I) from optically active aldehyde (II). This step is not particularly limited as long as compound (I) is obtained from optically active aldehyde (II). For example, optically active aldehyde (II) is reacted with cysteine derivative (III), and then compound (IV). The method of obtaining compound (I) by making it react with is mentioned.

  The reaction between the optically active aldehyde (II) and the cysteine derivative (III) is usually performed in a solvent. The solvent that can be used is not particularly limited as long as the reaction proceeds, and examples thereof include pyridine. The amount of cysteine derivative (III) to be used is generally 1 to 20 mol, preferably 1 to 5 mol, per 1 mol of optically active aldehyde (II). The reaction temperature is usually 20 ° C to 100 ° C, preferably 40 ° C to 70 ° C. The reaction time varies depending on the amount of reagent and solvent to be used and the reaction temperature, but is usually 0.1 hour to 2 hours, preferably 0.5 hour to 1 hour. The compound obtained by this reaction can be isolated by a method known per se, but may be reacted with compound (IV) as it is without isolation.

  Next, compound (I) is obtained by reacting the obtained compound with compound (IV) in a solvent. The solvent that can be used is not particularly limited as long as the reaction proceeds, and examples thereof include pyridine. The amount of compound (IV) to be used is generally 1 to 20 mol, preferably 1 to 5 mol, per 1 mol of optically active aldehyde (II). The reaction temperature is usually 20 ° C to 100 ° C, preferably 40 ° C to 70 ° C. The reaction time varies depending on the amount of reagent and solvent to be used and the reaction temperature, but is usually 0.1 hour to 2 hours, preferably 0.5 hour to 1 hour.

  The optically active aldehyde (II) is not particularly limited as long as it is an optically active substance. For example, those in which Z is an alkyl group, a cycloalkyl group, a heterocyclic group, or the like, each of which may be substituted, are available. Can be mentioned. Of these, the formula (II ′):

[Where:
R3 represents a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group. ,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
Or an aldose thereof or a derivative thereof (hereinafter also referred to as “aldose or a derivative thereof (II ′)”) can be preferably used.

  Some aldoses or derivatives thereof (II ′) have specific names, such as glucose, galactose, mannose, fucose, arabinose, xylose, rhamnose, glucuronic acid, allose, altrose, apiose, Chammelose etc. are mentioned.

  In addition, aldose or a derivative thereof (II ′) is not an aldehyde form represented by the formula (II ′) but may exist in a form in which the C-1 position forms a ring by binding a hemiacetal. Such a cyclic body is also included in aldose or its derivative (II ′).

  As the cysteine derivative (III), either L-form or D-form can be used. The optical purity of the cysteine derivative (III) is preferably 99% ee or higher. As the cysteine derivative (III), L-cysteine methyl ester and L-cysteine ethyl ester are preferable, and L-cysteine methyl ester is particularly preferable from the viewpoint of easy availability of raw materials and easy synthesis. . Examples of the salt of the cysteine derivative (III) include acid addition salts such as hydrochloride and sulfate.

  Compound (IV) may be selected according to the detector of the high performance liquid chromatography to be used. For example, when an ultraviolet-visible spectroscopic detector (hereinafter referred to as a UV detector) is used, an aryl group (preferably phenyl, p-tolyl, o-tolyl, 3,4-dimethoxy) in which R2 may be substituted. Compound (IV) which is a phenyl or the like or an aralkyl group (preferably benzyl, 2-phenylethyl or the like) may be selected. When a fluorescence detector is used, the compound (IV) where R2 is a fluorescent group is selected. Just choose.

  Cysteine derivative (III) can be synthesized by a method known per se. If there is a commercially available product, the commercially available product may be used as it is, but it must be produced using cystine derivative (V) and dithiothreitol. Is also possible.

  As the cystine derivative (V), either L-form or D-form can be used. The optical purity of the cystine derivative (V) is preferably 99% ee or more. As the cystine derivative (V), D-cystine dimethyl ester, D-cystine diethyl ester, L-cystine dimethyl ester, L-cystine diethyl are used from the viewpoint of easy availability of raw materials and easy synthesis. Esters are preferred, with D-cystine dimethyl ester and L-cystine dimethyl ester being particularly preferred. Examples of the salt of the cystine derivative (V) include acid addition salts such as hydrochloride and sulfate.

  The reaction between the cystine derivative (V) and dithiothreitol is not particularly limited as long as the corresponding cysteine derivative (III) is obtained, but is usually performed in a solvent. The solvent that can be used is not particularly limited as long as the reaction proceeds, and examples thereof include pyridine. The amount of cystine derivative (V) and dithiothreitol used is usually 1 mol to 10000 mol, preferably 1 mol to 1000 mol, more preferably 1 mol, respectively, per 1 mol of optically active aldehyde (II). -100 mol, most preferably 1 mol-10 mol each. The reaction temperature is usually 20 ° C to 100 ° C, preferably 40 ° C to 70 ° C. The reaction time varies depending on the amount of reagent and solvent to be used and the reaction temperature, but is usually 0.1 hour to 2 hours, preferably 0.5 hour to 1 hour. The cysteine derivative (III) obtained by this reaction can be isolated by a method known per se, but may be reacted as it is with optically active aldehyde (II) without isolation.

  The reaction between the cystine derivative (V) and dithiothreitol can also be carried out under the same conditions as the reaction between the optically active aldehyde (II) and the cysteine derivative (III). Therefore, both reactions can be carried out simultaneously by mixing optically active aldehyde (II), cystine derivative (V) and dithiothreitol and reacting them under the above conditions.

  Compound (IV) can be synthesized by a method known per se. If there is a commercially available product, the commercially available product may be used as it is.

  Step (b) is a step of subjecting the obtained compound (I) to high performance liquid chromatography. The compound (I) obtained in the step (a) does not need to be isolated, and can be measured by high performance liquid chromatography as it is.

In high performance liquid chromatography measurement, conditions such as sample charge amount, separation column type, separation column diameter and length, separation column temperature, mobile phase composition, mobile phase flow rate, etc. are selected as appropriate. The conditions most suitable for the separation of compound (I) may be selected. Such conditions are preferably such that the difference in retention time of each isomer of compound (I) is about 0.5 minutes or more (preferably 1 minute or more).
As the separation column, it is not necessary to use a column for optical isomer separation, and a commonly used column such as a column packed with octadecyl silica gel, phenyl silica gel, octyl silica gel or the like can be used.

Step (c) is a step of determining the absolute configuration of optically active aldehyde (II) by comparing the retention time of compound (I) measured in step (b) with the retention time of a standard product. is there.
Here, the “standard product” is a compound (I) derived from an optically active aldehyde (II) having a known absolute configuration.

  By this method, the absolute configuration of an optically active aldehyde, in particular, aldose or a derivative thereof can be easily determined.

  The optically active aldehyde (II) can be identified (including the absolute configuration) by comparing the retention time of the compound (I) with the retention time of the standard product.

  By the way, the retention time of the enantiomer of compound (I) is the same in high performance liquid chromatography. Therefore, by utilizing this, even when only a standard product derived from optically active aldehyde (II) having one absolute configuration is available, it is possible to know both retention times.

  For example, even when only an optically active aldehyde (II) having an absolute configuration D is available, among the standard products derived therefrom, D-cysteine methyl ester (D-cystine dimethyl ester and dithio) is used as cysteine derivative (III). An optical activity in which the absolute configuration is L-type, and a standard product (hereinafter also referred to as “D (II) -D (III) standard product”) derived using The retention time of compound (I) derived from aldehyde (II) using L-cysteine methyl ester [hereinafter also referred to as “L (II) -L (III) standard product]” is the same. Therefore, the retention times of D (II) -D (III) standards, which are derived as standards, are equal to the retention times of L (II) -L (III) standards and D (II)- By comparing the retention time of the L (III) standard product with the retention time of the compound (I), it becomes possible to identify the optically active aldehyde (II) (including the absolute configuration).

  Further, when the optically active aldehyde (II) is a mixture of optical isomers, by determining the peak area or height of each isomer of the compound (I) in the chromatogram based on the output result from the detector, It is also possible to measure optical purity.

  Further, when the optically active aldehyde (II) is a mixture of optical isomers, the molar area of the mixture is determined by determining the peak area ratio of each isomer of compound (I) in the chromatogram based on the output from the detector. It is possible to infer the ratio.

The present invention
(A) Cysteine derivative (III), and (b) Compound (IV)
A kit for determining the absolute configuration of an optically active aldehyde is provided.

  As the cysteine derivative (III) contained in the kit of the present invention, either L-form or D-form can be used. The optical purity of the cysteine derivative (III) is preferably 99% ee or higher. As the cysteine derivative (III), L-cysteine methyl ester and L-cysteine ethyl ester are preferable, and L-cysteine methyl ester is particularly preferable from the viewpoint of easy availability of raw materials and easy synthesis. .

  The cysteine derivative (III) contained in the kit of the present invention can be produced using the L-form or D-form cystine derivative (V) and dithiothreitol. The optical purity of the cystine derivative (V) is preferably 99% ee or more. As the cystine derivative (V), D-cystine dimethyl ester and D-cystine diethyl ester are preferable, and D-cystine dimethyl ester is particularly preferable from the viewpoint of easy availability of raw materials and easy synthesis. . Examples of the salt of the cystine derivative (V) include acid addition salts such as hydrochloride and sulfate.

Therefore, the present invention
(A) cystine derivative (V) or a salt thereof and dithiothreitol, and (b) compound (IV)
A kit for determining the absolute configuration of an optically active aldehyde is provided.

  The compound (IV) contained in the kit of the present invention may be selected in accordance with the high performance liquid chromatography detector to be used. For example, when a UV detector is used, an aryl group (preferably phenyl, p-tolyl, o-tolyl, 3,4-dimethoxyphenyl, etc.) or an aralkyl group (preferably, R2) may be substituted. Compound (IV) that is benzyl, 2-phenylethyl, etc.) may be selected. When a fluorescence detector is used, compound (IV) in which R2 is a fluorescent group may be selected.

The kit of the present invention may further contain a compound (I) derived from an optically active aldehyde (II) having a known absolute configuration as a standard product.
In addition, the kit of the present invention includes a solvent (for example, pyridine and the like) used for the reaction between the optically active aldehyde (II) and the cysteine derivative (III), the reaction with the compound (IV), a method for using the kit, and the like. The described items, reaction vessels, and the like may be further included.

  Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the scope of the present invention is not limited thereto.

Example 1
720 mg (4.0 × 10 ⁻³ mol) of D-glucose and 900 mg of L-cysteine methyl ester hydrochloride were dissolved in 5 ml of anhydrous pyridine in a 20 mL screw-capped test tube and reacted at 60 ° C. for 60 minutes in a water bath. After returning to room temperature, 900 μl of phenyl isothiocyanate was added, and the mixture was further reacted at 60 ° C. for 60 minutes. After distilling off pyridine with a rotary evaporator, an operation of adding a small amount of toluene and evaporating again was repeated twice. The residue was dissolved in a small amount of MeOH, and a silica gel column (inner diameter) was prepared using chloroform: methanol: water (90: 10: 1, 85: 15: 1, 80: 20: 2 gradient elution stepwise in steps of 200 mL each) as a solvent. Separation was performed at 3 cm × length 25 cm. The eluent was fractionated by 15 mL each with a fraction collector, and was subjected to thin layer chromatography (plate: silica gel 60 F ₂₅₄ , developing solvent: chloroform: methanol: water (80: 20: 2), detection: heating after spraying with 2% sulfuric acid). The portion containing only the product was analyzed, concentrated with a rotary evaporator, and dried with a vacuum drier. D-glucose derivative (A) 697.8 mg (1.6 × 10 ⁻³ mol, yield 40%) Was isolated.
L-glucose 300 mg (1.7 × 10 ⁻³ mol) and L-cysteine methyl ester hydrochloride 375 mg were dissolved in 3 ml of anhydrous pyridine, and the same operation as described above was performed to obtain 98.8 mg of L-glucose derivative (B). (2.3 × 10 ⁻⁴ mol, yield 14%) was obtained.

The L-glucose derivative (A) and the L-glucose derivative (B) both showed an [M + H] ⁺ ion peak at m / z 433 in FAB-MS.
Each derivative was subjected to ¹ H-NMR, ¹³ C-NMR, and HPLC measurements.

D-glucose derivative (A)

UV λmax 251 nm (HPLC photodiode array detector).
¹ H-NMR (400 MHz, pyridine-d ₅ ) δ: 11.36 (1H, s, H-7 ″), 7.59 (2H, br d, J = 7.5 Hz, H-2 ″, 6 "), 7.20 (2H, br t, J = 7.5 Hz, H-3", 5 "), 7.03 (1H, br t, J = 7.5 Hz, H-4"), 6.53 (1H, t, J = 7.5 Hz, H-2 ′), 6.33 (1H, d, J = 10.0 Hz, H−1), 5.03 (1H, dd, J = 3.0, 1.5 Hz, H-3), 4.82 (1H, dd, J = 10.0, 1.5 Hz, H-2), 4.63 (1H, dd, J = 8 0.0, 3.0 Hz, H-4), 4.58 (1H, ddd, J = 8.0, 5.3, 3.8 Hz, H-5), 4.52 (1H, dd, J = 11 0.0, 3.8 Hz, H-6a), 4.37 (1H, dd, J = 11.0, 5.3 Hz, H-6b), 3.53 (3H, s, OMe), 44 (1H, dd, J = 12.0, 7.5 Hz, H-3′a), 3.42 (1H, dd, J = 12.0, 7.5 Hz, H-3′b).
¹³ C-NMR (100 MHz, pyridine-d ₅ ) δ: 183.8 (C-8 ″), 171.6 (C-1 ′), 141.0 (C-1 ″), 128.6 (C− 3 ", 5"), 124.7 (C-4 "), 124.3 (C-2", 6 "), 78.6, 75.7, 73.2, 70.9, 69.9, 68.6, 64.9 (glucose-C, C-2 ′), 52.4 (OMe), 31.9 (C-3 ′).

L-glucose derivative (B)

UV λmax 244 nm (HPLC photodiode array detector).
¹ H-NMR (400 MHz, pyridine-d ₅ ) δ: 11.45 (1H, s, H-7 ″), 7.80 (2H, br d, J = 7.5 Hz, H-2 ″, 6 "), 7.22 (2H, br t, J = 7.5 Hz, H-3", 5 "), 7.07 (1H, br t, J = 7.5 Hz, H-4"), 6.66 (1H, t, J = 8.0 Hz, H-2 ′), 6.15 (1H, d, J = 9.2 Hz, H−1), 5.18 (1H, br s, H-3), 4.64 (2H, br s, H-4, 5), 4.60 (1H, br d, J = 9.2 Hz, H-2), 4.56 (1H, dd, J = 11.0, 2.7 Hz, H-6a), 4.38 (1H, dd, J = 11.0, 5.1 Hz, H-6b), 3.60 (5H, m, OM) , H-3a, 3b)
¹³ C-NMR (100 MHz, pyridine-d ₅ ) δ: 183.0 (C-8 ″), 171.7 (C-1 ′), 141.4 (C-1 ″), 128.5 (C− 3 ", 5"), 125.1 (C-2 ", 6"), 125.0 (C-4 "), 75.9, 75.9, 72.7, 70.7, 70.3, 68.5, 65.0 (glucose-C, C-2 ′), 52.6 (OMe), 31.4 (C-3 ′)

  Although the derivatives A and B are relatively stable in pyridine, they undergo a demethanol reaction in the process of separation and purification, and are changed to phenylthiohydantoin derivatives A ′ and B ′, respectively. It was particularly remarkable with L-glucose derivatives. Their retention times in HPLC were 8.87 min from D-glucose and 9.29 min from L-glucose, which was opposite to the elution order of A and B. These were also detected slightly in the pyridine reaction solution after the synthesis of the derivative.

D-glucose thiohydantoin derivative (A ') and L-glucose thiohydantoin derivative (B')

Spectral data of L-glucose thiohydantoin derivative (B ')

UV λmax 255 nm (HPLC photodiode array detector).
FAB-MS m / z 401 [M + H] ^+
1 H-NMR (300 MHz, pyridine-d ₅ ) δ: 7.30-7.40 (5H, m, phenyl-H), 6.70 (1H, d, J = 4.8 Hz, H-1) , 5.42 (1H, t, J = 8.3 Hz, H-3), 5.00 (H-2, H-4, overlapped with HOD signal), 4.67 (1H, m, H-5) ), 4.53 (1H, dd, J = 9.3, 7.8 Hz, H-2 ′), 4.51 (1H, dd, J = 10.3,
3.9 Hz, H-6a), 4.37 (1H, dd, J = 10.3, 5.4 Hz, H-6b), 3.53 (1H, dd, J = 9.3, 7. 8 Hz, H-3′a), 3.36 (1H, t, J = 9.3 Hz, H-3′b)
¹³ C-NMR (75 MHz, pyridine-d ₅ ) δ: 186.3 (C-8 ″), 172.2 (C-1 ′), 134.3 (C-1 ″), 129.2
128.9, 124.3 (C-2 ″, 3 ″, 4 ″, 5 ″, 6 ″), 78.1, 74.2, 73.4, 71.0, 67.5, 66.3 65.1 (glucose-C, C-2 ′), 32.2 (C-3 ′)

Example 2
Standard samples of glucose, galactose, mannose, fucose, fructose, arabinose, and xylose, D-form and L-form, L-rhamnose, D-glucuronic acid, 5 mg each were placed in a 10 mL screw cap test tube, and 5 mg L-cysteine methyl. After adding 1 mL of pyridine containing ester hydrochloride and heating at 60 ° C. for 1 hour, 0.5 mL of pyridine containing 5 mg of phenyl isothiocyanate was added to the reaction solution, and further heated at 60 ° C. for 1 hour. After returning the reaction solution to room temperature, 2 μL was analyzed by HPLC.

HPLC analysis condition column: Cosmosil 5C ₁₈ ARII (manufactured by Nacalai Tesque, 4.6 × 250 mm),
Column temperature: 35 ° C.
Mobile phase: A; 50 mM phosphoric acid aqueous solution containing 25% CH ₃ CN,
Flow rate: 0.8 ml / min,
Detection: Photodiode array detection (Max Absorbance)

  The results of HPLC analysis of the D-glucose derivative and L-glucose derivative are shown in FIG. Table 1 shows the retention time of each derivative including other sugars.

注１ ケトースであるフルクトースには適用できない。
注２ 標準品がないため測定できなかった。

Note 1 Not applicable to fructose, which is ketose.
Note 2 Measurement was not possible because there was no standard product.

  It was found that D-form and L-form were well separated in glucose, galactose, mannose, fucose, arabinose and xylose. In addition, no derivative was produced with fructose, which is ketose. As for rhamnose and glucuronic acid, no preparations of enantiomers are available on the market and comparison was not possible.

Example 3
In place of phenyl isothiocyanate, benzyl isothiocyanate, benzoyl isothiocyanate, 3,4-dimethoxyphenyl isothiocyanate, 2-phenylethyl isothiocyanate, p-tolyl isothiocyanate, o-tolyl isothiocyanate is used for derivatization reaction. D-glucose and L-glucose were identified. The reaction conditions and analysis conditions are the same as in Example 2.

  Table 2 shows the retention times of the respective derivatives obtained as a result of analyzing the D-glucose derivative and the L-glucose derivative by HPLC.

注１ Ｈ：チオヒダントイン誘導体（Ａ’，Ｂ’），Ｔ：テトラヒドロチアゾール誘導体
（Ａ，Ｂ）
注２ ２１１ｎｍを極大とする吸収の肩として観察された。

Note 1 H: Thiohydantoin derivative (A ′, B ′), T: Tetrahydrothiazole derivative (A, B)
Note 2 Observed as an absorption shoulder with a maximum at 211 nm.

  Of the isothiocyanates used in Example 3, benzoyl isothiocyanate did not produce the desired derivative. The formation of thiohydantoin derivatives was observed in all samples other than o-tolyl isothiocyanate, particularly with 3,4-dimethoxyphenyl and 2-phenylethyl derivatives. The reason why the thiohydantoin derivative is less likely to be formed with o-tolyl isothiocyanate was considered to be that the nitrogen atom in the tolyl group is less likely to attack the ester carbonyl of the nitrogen atom.

Example 4
Glucose, mannose, galactose, xylose, fucose, and arabinose D-form and L-form, L-rhamnose, D-glucuronic acid 1 mg each was put into a 5 mL screw cap test tube, 1 mg L-cysteine methyl ester hydrochloride After adding 1 mL of pyridine containing a salt and heating at 60 ° C. for 1 hour, 0.1 mL of pyridine containing 1 mg of o-tolyl isothiocyanate was added to the reaction solution, and the mixture was further heated at 60 ° C. for 1 hour. After returning the reaction solution to room temperature, 2 μL was analyzed by HPLC. The HPLC conditions are the same as in Example 2. Table 3 shows the retention time of each derivative.

Example 5
1 mg of each standard sample of glucose, galactose, and arabinose in D and L standards is put into a 5 mL screw cap test tube, and 1 mL of pyridine containing 1 mg of L-cysteine ethyl ester hydrochloride is added and heated at 60 ° C. for 1 hour. Then, 0.1 mL of pyridine containing 1 mg of o-tolyl isothiocyanate was added to the reaction solution, and the mixture was further heated at 60 ° C. for 1 hour. After returning the reaction solution to room temperature, 2 μL was analyzed by HPLC. The HPLC conditions are the same as in Example 2. Table 4 shows the retention time of each derivative.

Example 6: Application example Glycoside 4-allyl-1,2-dihydroxybenzene 2-O- (6′-O-α-arabinofuranosyl) -β-glucopyranoside 0.5 mg whose absolute configuration of sugar is unknown Was placed in a 0.3 mL screw cap vial, dissolved in 50 μL of 0.5 N hydrochloric acid, sealed, and hydrolyzed by heating at 90 ° C. for 2 hours. After cooling to room temperature, cation exchange resin IRA400 (treated with dilute aqueous sodium hydroxide solution and washed thoroughly with purified water) was added to neutralize the acid. The reaction solution was transferred to a 1 mL screw cap vial with 0.1 mL of purified water, blown with a nitrogen stream to distill off the water, and then dried in a vacuum desiccator for 3 hours. The residue was heated with 60 μL of pyridine containing 1 mg of L-cysteine methyl ester hydrochloride at 60 ° C. for 1 hour, 2 μL of phenyl isothiocyanate was added, and the mixture was further heated for 1 hour. After cooling the reaction solution to room temperature, 2 μL was analyzed by HPLC. The analysis conditions are the same as in Example 2. Since the observed retention times of the two peaks were identical to those of D-glucose and L-arabinose, respectively, it was revealed that glucose was D-form and arabinose was L-form. Since an extremely large peak was observed on HPLC with an unknown glycoside of only 0.5 mg, discrimination was sufficiently possible even with about 0.1 mg of glycoside.

Example 7: Application example Other four glycosides whose 2-dimensional structure was determined by various instrumental analyzes but whose absolute configuration of sugar was unknown, 2-allyl-4,5-methylenedioxyphenol 1 -O-β-glucopyranoside (1), 2-allyl-4,5-methylenedioxyphenol 1-O- (6′-O-α-xylopyranosyl) -β-glucopyranoside (2), 2-allyl-4, 5-methylenedioxyphenol 1-O- (6′-O-α-arabinopyranosyl) -β-glucopyranoside (3) and 1,2-dihydroxy-3-methoxy-5-allylbenzene 1-O Regarding -β-glucopyranoside (4), the absolute configuration of the sugar was determined using o-tolyl isothiocyanate. The hydrolysis, derivatization reaction, and analysis conditions are exactly the same as in Example 5, except that o-tolyl isothiocyanate is used instead of phenyl isothiocyanate.

  Table 5 shows the retention times of the derivatives obtained from each compound and the absolute configuration of the sugar.

Reference Example 1 mg of a standard sample of D-form and L-form of xylose was put into a 5 mL screw cap test tube, and 1 mL of pyridine containing 1 mg of D-cystine dimethyl ester hydrochloride and 2 mg of dithiothreitol was added. After heating for a period of time, 0.1 mL of pyridine containing 1 mg of o-tolyl isothiocyanate was added to the reaction solution, and the mixture was further heated at 60 ° C. for 1 hour. After returning the reaction solution to room temperature, 2 μL was analyzed by HPLC. The HPLC conditions are the same as in Example 2. As a result, the retention time of the derivative obtained from D-xylose was 19.1 minutes, which coincided with the retention time of the L-xylose derivative when L-cysteine methyl ester was used. On the other hand, the retention time of the derivative obtained from L-xylose was 20.5 minutes, which coincided with the retention time of the D-xylose derivative when L-cysteine methyl ester was used.
This is because the derivatives produced by the reaction of D-cysteine methyl ester produced during the reaction with D- and L-xylose are the derivatives produced by the reaction of L-cysteine methyl ester with L- and D-xylose, respectively. Although it becomes an enantiomer, it shows that these enantiomers cannot be distinguished by ordinary HPLC.
By utilizing this, D-cysteine methyl ester alone or in combination with D-cystine dimethyl ester and dithiothreitol, either D- or L-form can be used as a standard sugar. If there is an optically active aldehyde (II) in which one of the optical isomers is not available by comparing with the results obtained using L-cysteine methyl ester, the derivatives derived from both optically active isomers The retention time can be determined.

Example 8: Application example Although r-form is commercially available for rhamnose, D-form is difficult to obtain and synthesize. 2 mg of a standard sample of L-rhamnose, 4.6 mg of D-cystine dimethyl ester hydrochloride and 6.2 mg of dithiothreitol are placed in a 5 mL screw cap test tube, dissolved by adding 1.2 mL of pyridine, and heated at 60 ° C. for 1 hour. did. To the reaction solution was added 0.24 mL of pyridine containing 2.4 mg of o-tolyl isothiocyanate, and the mixture was further heated at 60 ° C. for 1 hour. After returning the reaction solution to room temperature, 2 μL was analyzed by HPLC. The HPLC conditions are the same as in Example 2. A peak was detected at 15.8 minutes by HPLC.
The derivative obtained here is an enantiomer of a derivative obtained from L-cysteine methyl ester and D-rhamnose, and the retention time is the retention time when D-rhamnose is derivatized with L-cysteine methyl ester. Matches. On the other hand, when the retention time of the L-rhamnose derivative was measured in the same manner as in Example 2 using L-cysteine methyl ester, it was 29.4 minutes. Therefore, L- and D-rhamnose can be discriminated by comparing with both retention times.

Example 9: Application example (quantitative property (1))
100 μl of a 0.05 mol / l pyridine solution of each of D-glucose, D-mannose, D-galactose, D-xylose, D-arabinose and D-fucose was placed in a 1 ml screw cap vial, 100 μl of L-cysteine methyl ester hydrochloride pyridine solution was added and heated at 60 ° C. for 1 hour. 200 μl of an o-tolyl isothiocyanate pyridine solution having a concentration of 10 mg / ml was added to the reaction solution, and the mixture was further heated at 60 ° C. for 1 hour. The reaction solution was analyzed by HPLC (analysis conditions are the same as in Example 2), and the peak areas at 251 nm were compared. Each was repeated three times and averaged. As a result, it was found that except for mannose, the peak areas were almost the same, and the molar ratio of the constituent sugars could be estimated from the peak area ratio. (FIG. 2) For mannose, the peak area was small due to the large amount of hydantoin derivatives produced.

Example 10: Application example (quantitative property (2))
A sugar solution was prepared by diluting a D-glucose 10 mg / ml pyridine solution with pyridine 2, 5, 10 and 50 times. 100 μl of each sugar solution was placed in a 1 ml screw cap vial, 200 μl of 10 mg / ml pyridine solution of L-cysteine methyl ester hydrochloride was added, and heated at 60 ° C. for 1 hour. 200 μl of an o-tolyl isothiocyanate pyridine solution having a concentration of 10 mg / ml was added to the reaction solution, and further heated at 60 ° C. for 1 hour. The reaction solution was analyzed by HPLC (analysis conditions are the same as in Example 2), and the peak areas at 251 nm were compared. As a result, the glucose concentration and the peak area showed a quantitative increase.

  According to the present invention, the absolute configuration of sugars and related aldehyde compounds can be determined by a simple operation.

Chromatogram of D-glucose derivative and L-glucose derivative. The graph which compared the peak area of the chromatogram which each sugar derivative shows. The graph which compared the glucose peak and the peak area of the chromatogram which a glucose derivative shows.

Claims (13)

Formula (I):

[Wherein R1 represents an optionally substituted hydrocarbon group;
R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group;
Z represents an organic residue]
The compound represented by formula (II) is subjected to high performance liquid chromatography:

[Wherein Z is as defined above]
A method for determining an absolute configuration of an optically active aldehyde represented by the formula: Formula (II):

[Wherein Z is as defined in claim 1]
An optically active aldehyde represented by the formula (III):

[Wherein R 1 has the same meaning as in claim 1]
And then reacting with an L- or D-cysteine derivative represented by the formula (IV):

[Wherein R2 has the same meaning as in claim 1]
Obtained by reacting with a compound represented by formula (I):

[Wherein each symbol has the same meaning as in claim 1]
The method according to claim 1, wherein the compound represented by the formula is subjected to high performance liquid chromatography. The L- or D-cysteine derivative has the formula (V):

[Wherein R 1 has the same meaning as in claim 1]
The method of Claim 2 produced | generated using the L- or D-cystine derivative represented by these, or its salt, and dithiothreitol. Z is the formula:

[Wherein R3 is substituted with a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or a substituent selected from an optionally substituted hydroxy group and a substituted amino group. Represents an alkyl group,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The method as described in any one of Claims 1-3 which is group represented by these. R1 represents an alkyl group,
The method as described in any one of Claims 1-4 in which R2 shows the aryl group which may be substituted, or the aralkyl group which may be substituted. Formula (I):

[Wherein R1 represents an optionally substituted hydrocarbon group;
R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group;
Z represents an organic residue]
A compound represented by Z is the formula:

[Wherein R3 is substituted with a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or a substituent selected from an optionally substituted hydroxy group and a substituted amino group. Represents an alkyl group,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The compound of Claim 6 which is group represented by these. R1 represents an alkyl group,
The compound according to claim 6 or 7 , wherein R2 represents an aryl group which may be substituted, or an aralkyl group which may be substituted. (A) Formula (III):

[Wherein R1 represents an optionally substituted hydrocarbon group]
An L- or D-cysteine derivative represented by the formula (IV):

[Wherein R 2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group]
A kit for analyzing an optically active aldehyde, comprising a compound represented by: The L- or D-cysteine derivative has the formula (V):

[Wherein R 1 has the same meaning as in claim 9]
The kit of Claim 9 produced | generated using the L- or D-cystine derivative represented by these, or its salt, and dithiothreitol. (C) Formula (I) derived from an optically active aldehyde represented by the formula (II): Z-CHO having a known absolute configuration as a standard product :

[Wherein R1 represents an optionally substituted hydrocarbon group;
R2 represents an optionally substituted aryl group, an optionally substituted aralkyl group or a fluorescent group;
Z represents an organic residue]
The kit of Claim 9 or 10 which further contains the compound represented by these. Z is the formula:

[Wherein R3 is substituted with a hydrogen atom, an optionally substituted hydroxy group, a substituted amino group, or a substituent selected from an optionally substituted hydroxy group and a substituted amino group. Represents an alkyl group,
R3 ′ represents a hydrogen atom or an alkyl group substituted with a substituent selected from an optionally substituted hydroxy group and a substituted amino group;
R4 represents a group represented by the formula: —CH ₂ R5 (R5 represents a hydrogen atom, an optionally substituted hydroxy group or a substituted amino group), or a carboxyl group;
n represents an integer of 1 to 4 (provided that all of n R3, R3 ′ and R5 are hydrogen atoms)]
The kit of Claim 11 which is group represented by these. R1 represents an alkyl group,
The kit according to any one of claims 9 to 12, wherein R2 represents an optionally substituted aryl group or an optionally substituted aralkyl group.

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