JPH0640953A - Method for aminating saccharide - Google Patents

Abstract

PURPOSE:To obtain an aminated saccharide derivative useful for synthesizing a reduced end labeled saccharide and an artificial complex glucide by reducing a 2-aminopyridine derivative saccharide and decomposing the resultant compound under an alkali condition. CONSTITUTION:A compound of the formula (R-CH2 is group derived from a saccharide of the formula R-CHO containing reduced end made into an aldehyde; R<1> to R<4> are H or lower alkyl) is reduced by using a catalyst such as palladium black at normal temperature to 100 deg.C and then decomposed in the presence of hydrazine at room temperature to 100 deg.C to give an aminated saccharide derivative of the formula R-CH2-NH2. A reduced end labeled saccharide obtained by reacting a saccharide containing an amino group at the reduced end, namely an aminated saccharide derivative with a labeled compound capable of reacting an amino group is useful for studying a receptor of a saccharide of organism tissue and studying lectin saccharide. An artificial complex glucide synthesizes neoglycoprotein, neoglycolipid, etc., and is useful as a medicine, an immune source, a studying reagent, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sugar amination method for producing an aminated sugar derivative, and is particularly useful for combining a sugar having a specific structure with various reagents capable of reacting with an amino group. The present invention relates to a method for providing an aminated sugar derivative, and a method for producing the conjugate, for example, a sugar having a reducing end labeled, an artificial glycoconjugate.

[0002]

2. Description of the Related Art In recent years, in order to study the relationship between the structure of sugar chains and their functions, various methods with higher sensitivity and capable of quantitative assay with a small amount of sample have been proposed. For example, a method for microanalyzing sugar chains is to introduce tritium when converting the reducing end into sugar alcohol and utilize its radioactivity. This detection sensitivity is good at the pmol level, but it has various restrictions because it is a radioactive substance.

As a highly sensitive assay method without this limitation, 2-
A method using an organic compound such as aminopyridine as a fluorescent labeling agent is known (for example, Biochem. Biophys. Re
S. Commun., 85 , 257-263 (1978)). In this method, the reducing end of the sugar or sugar chain is reacted with the amino group of the 2-aminopyridine to form a Schiff base, which is then reduced and detected by fluorescence. Fractionation by HPLC and detection with a fluorescence detector are performed. It is disclosed that the sensitivity at the time of detection is several tens of fmol level.

However, the fluorescent labeling agent is excellent in that the reducing end of the sugar chain can be easily fluorescently labeled, but the fluorescence intensity of the obtained fluorescently labeled saccharide is weak, and for example, for histochemical analysis of saccharides. It wasn't enough to use. On the other hand, it contains many types of sugar chains separated from the inside of the body to synthesize artificial glycoconjugates that are expected to be used as immunogens for producing antibodies against sugar chains or as pharmaceuticals. Fractionation of sugar chains with a specific structure from the mixture, sugar chains with a specified structure and proteins, peptides,
There is a need for an effective method of binding an organic compound such as a lipid or a polymer resin. For this purpose, it is possible to introduce an amino group into the reducing end of the sugar chain and combine the amino group with the above organic compound. In the case of a single sugar chain, the reducing end of the sugar chain may be directly aminated, but it is difficult to separate the sugar chain having a specific structure from a mixture of many kinds of sugar chains obtained from nature. . Further, it is very time-consuming and extremely difficult to artificially synthesize a large amount of sugar chains having a complicated structure as naturally occurring.

Therefore, a method for efficiently fractionating a complex sugar chain having a specific structure existing in a living body and easily aminating a sugar compound having a single structure has been desired.

[0006]

SUMMARY OF THE INVENTION The first object of the present invention is to provide a method for amination of sugars which aminates sugars by a simple method to obtain aminated sugar derivatives. A second object of the present invention is to provide a method for easily producing an aminated sugar derivative having a single structure and a labeled form thereof from a mixture of sugars, or to react the aminated sugar derivative with another organic compound such as a protein. It is to provide a method for producing an artificial glycoconjugate.

[0007]

The present invention provides a general formula (a) represented by the following chemical formula 3.

[0008]

[Chemical 3]

(In the formula, R—CH ₂ — represents a group derived from a saccharide which can be represented by R—CHO when the reducing end is aldehydized, and R ¹ , R ² , R ³ and R ⁴ are the same as each other. A 2-aminopyridine derivatized saccharide represented by hydrogen, a lower alkyl group, which may be the same or different and each represents a group selected from hydrogen and lower alkyl groups.) Is subjected to a reduction reaction, and then subjected to a decomposition reaction under alkaline conditions. To obtain an aminated sugar derivative represented by the following general formula (b) R—CH ₂ —NH ₂ (b) (wherein R—CH ₂ — is as defined above). It provides an amination method.

Further, the present invention is characterized in that the compound represented by the general formula (b) obtained by the above method is reacted with a labeled compound capable of reacting with an amino group, and the reducing end is labeled. The present invention provides a method for producing a modified saccharide (hereinafter, also referred to as a reducing end labeled saccharide). Further, the present invention provides a method for producing an artificial glycoconjugate, which comprises reacting the compound represented by the general formula (b) obtained by the above method with a protein, peptide or lipid. It is a thing.

That is, the present invention provides a method for reducing and decomposing a 2-aminopyridine derivative saccharide represented by the general formula (a) to introduce an amino group into the reducing end of the saccharide. After the reduction reaction, the decomposition reaction is carried out under alkaline conditions, preferably in the presence of hydrazine. Here, the 2-aminopyridine derivatized saccharide used in the present invention may be a single species or a plurality of species. That is,
The R group (the same applies to the R—CH ₂ group) may or may not be single.

Therefore, in the present invention, even if a substantially single 2-aminopyridine derivatized saccharide is selected as a starting material to obtain a substantially single aminated saccharide derivative, many kinds of amino groups can be obtained. These may be singularized after obtaining the derivatized sugar derivative. In the former case, the starting saccharide is fluorescently labeled with a 2-aminopyridine compound to give a 2-aminopyridine derivatized saccharide having a sugar chain of a specific structure. After the fractionation, there is a method of converting the fluorescently labeled sugar chain into a sugar chain having an amino group at the reducing end, and in the latter case, the produced aminated sugar derivative mixture is preferably labeled. It is remarkable that a single aminated saccharide derivative or a reducing end-labeled saccharide and an aminated saccharide derivative by delabeling thereof can be isolated with high purity and sensitivity by separating from the product by chromatography. With the results.

The present invention will be specifically described below. [Synthesis of aminated sugar derivative (reducing terminal aminated sugar)] The 2-aminopyridine derivatized sugar represented by the general formula (a) is a known method [JP-A-1 (Sho-64) -10177; Agric. Bio
l. Chem., 54 (8), 2169-2170 (1990); Biochem. Biophys.
Res. Commun., 85 , 257-263 (1978); J. Biochem., 90 , 40
7-414 (1981); J. Biochem., 95 , 197-203 (1984); J. Bioc
hem., 112, No. 1, 122-126 (1992)].

That is, the general formula (d) represented by the following chemical formula 4

[0015]

[Chemical 4]

(In the formula, R ¹ , R ² , R ³ and R ^{4, which} may be the same or different, each represents a group selected from hydrogen and a lower alkyl group.) The reducing terminal of the amino group at the 2-position of the derivative is converted to an aldehyde to give a compound represented by the general formula (c); R-CHO (wherein R represents a sugar residue. However, R-CHO is a single sugar compound or plural kinds thereof). Of the saccharide compound (monosaccharides, oligosaccharides, polysaccharides, glycosaminoglycans, etc.) that can be represented by (1) to form a Schiff base, and then by reducing the aminoalkyl ( -CH ₂ NH-)
By forming a bond, a 2-aminopyridine derivatized saccharide represented by the general formula (a), which is a complex of the saccharide and the compound (d), can be synthesized.

In the present invention, the lower alkyl group means an alkyl group having about 1 to 6 carbon atoms, specifically, methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group and isomers thereof. Is mentioned. When a single compound (a) is used in the sugar amination method of the present invention, the 2-aminopyridine derivatized saccharide mixture is preferably subjected to high performance liquid chromatography (HPLC) or the like (eg, reverse phase HPLC). ) Separation and purification by
An example is a method of detecting a target complex by a fluorescence spectrum and using a single fraction.

The Schiff base forming reaction is carried out at room temperature to 100 ° C. in an inorganic acid such as hydrochloric acid or hydrofluoric acid or an acetic acid, an organic acid such as trifluoroacetic acid, an organic solvent such as pyridine or an aqueous solvent. Under a reaction condition of several minutes to several hours (preferably about 90 ° C., 1 to 3 hours, pH 3 to 6.4), using about 20 to 100 equivalents of the compound (d) with respect to the saccharide to react. Thus, a method of generating a Schiff base can be exemplified. For the reduction of the Schiff base, a reducing agent usually used for the reduction of the Schiff base can be used, and particularly volatile borane complex (for example, borane dimethylamine complex, borane triethylamine complex, borane pyridine complex, etc.), Sodium borohydride, sodium cyanoborohydride (NaBH ₃ CN)
Etc. are preferred. Especially in the case of sugars containing sialic acid,
It is preferable to react the borane dimethylamine complex with a reducing agent containing water and acetic acid because elimination of sialic acid can be prevented. The reduction reaction is from room temperature to 100 ° C, 1
It is completed in 10 hours to 10 hours (preferably about 80 to 90 ° C. for about 1 hour).

The saccharides may be saccharides cut out from complex carbohydrates such as glycoproteins and glycolipids. As a method for cleaving the sugar from the glycoconjugate, a method of decomposing the glycoconjugate in the presence of a base such as hydrazine (under alkaline conditions) and optionally N-acetylating (Biochem. Biophys.
Acta, 121, 417-420 (1966)), a method of decomposing trifluoroacetic acid, a method of digesting with an enzyme (endoglycosidase, glycopeptidase, endoglycoceramidase) and the like.

According to the present invention, the reaction for converting the pyridylamino group (which may have a substituent) of the 2-aminopyridine derivatized saccharide represented by the above general formula (a) into an amino group is first carried out. It can be carried out by subjecting the modified sugar to a reduction reaction and then a decomposition reaction under alkaline conditions. The above reduction reaction can be carried out in an acidic aqueous solution (for example, pH is adjusted to 3 to 4 using acetic acid or the like) using hydrogen gas in the presence of a hydrogenation catalyst (palladium black or the like), and the reaction can be performed at room temperature near several It is completed in time (preferably 3 to 4 hours).

After the above reaction, the catalyst is separated and removed by means such as filtration and centrifugation, concentrated to dryness, and the reaction product is not separated but continuously under alkaline conditions (for example, hydrazine (N ₂
In the presence of H ₄ )), a decomposition reaction is performed to synthesize the desired aminated sugar derivative represented by the above general formula (b). The reaction, the product of the reduction reaction, for example, in the absence of water,
Using anhydrous hydrazine, the temperature is from room temperature to about 100 ° C. (preferably 60 to 100 ° C., more preferably about 70 ° C.) for several seconds to one hour (preferably several minutes to 20 minutes, more preferably 2-3 minutes). It can be performed by heating or heating. Since hydrazine also acts as a reaction solvent, it may be used in an excess amount with respect to the raw material compound. When the decomposition reaction under alkaline conditions is carried out using hydrazine, hydrazine can be removed by subjecting the reaction solution to reduced pressure treatment after the reaction, which is preferable, but ammonia water, hydroxylamine, sodium hydroxide or the like is used instead of hydrazine. A decomposition reaction can also be performed.

The reaction scheme of the above method is shown in Chemical Formula 5 below for an example of a sugar chain in which the reducing terminal sugar residue is N-acetylglucosamine. The intermediates marked with * are putative and their structures have not been confirmed.

[0023]

[Chemical 5]

After completion of the reaction, the target compound can be separated and purified by chromatography such as HPLC, TLC (thin layer chromatography), gel chromatography and the like. [Synthesis of reducing end labeled sugar] The reducing end is synthesized by reacting a sugar having an amino group at the reducing end, that is, an aminated sugar derivative synthesized by the method of the present invention, with a labeling compound capable of reacting with the amino group. It is possible to synthesize a saccharide labeled with. Here, before separation and purification by the chromatography, the amino group of the aminated sugar derivative is labeled with a labeling compound, for example, a fluorescent labeling compound preferably exemplified below, and a plurality of labeled sugar derivatives mixed by the fluorescence. Can be fractionated to fractionate the reducing-end labeled sugar derivative having a single structure. If desired, this reducing end labeled sugar derivative can be delabeled to obtain an aminated sugar derivative.

The thus-synthesized labeled saccharide having the reducing end of a single specific sugar chain labeled is useful for the study of saccharide receptors in living tissues and the study of lectin sugar-binding specificity. is there. As the labeling compound, a compound having a functional group capable of binding to an amino group and having a fluorescent group, a chemiluminescent group, a color forming group, a radioactive isotope, etc. is used, and is not particularly limited. Specifically, for example, a biotinylation reagent (for example, active ester such as biotin-sulfo-N-hydroxysuccinimide ester), fluorescein isothiocyanate (FITC), phenylisothiocyanate, dansyl (DNS) reagent, dinitrophenyl ( DN
P) a chemical reagent and the like.

The aminated sugar derivative can be reacted with a labeled compound capable of reacting with an amino group by a known method. For example, biotin sulfo-N
-Reaction with hydroxysuccinimide ester is carried out at around room temperature under weakly basic (eg, saturated sodium hydrogen carbonate solution) or neutral conditions, and if necessary, acid (eg,
A strong acid cation exchange resin) is used for neutralization (J. Nucl. Med., 28 , 1294-1302 (1987)). Further, for example, the reaction with FITC may be carried out by heating under a basic (eg, in pyridine) or neutral condition to carry out the reaction (Am. J. Pathol., 34 , 1081 (1958)). [Synthesis of artificial glycoconjugate] A saccharide having an amino group at the reducing end synthesized by the method of the present invention and a protein, a peptide, a lipid, a polymer resin, or the like, directly or through a bifunctional crosslinking agent. Can be combined. Such conjugates are expected to have various uses such as pharmaceuticals, immunogens, research reagents, etc. as artificial glycoconjugates such as neoglycoprotein and neoglycolipid. Specifically, the following uses can be listed. (1) Use as a sugar chain probe for analyzing a molecular recognition phenomenon (specific structure recognition phenomenon) of a sugar chain by a physiologically active substance such as a lectin, a sugar-binding protein, an antibody, and a glycosyltransferase. (2) Use as an immunogen for producing an antibody against a sugar chain. (3) Use as a solid-phase artificial glycoconjugate for screening antibodies against sugar chains by ELISA or the like. (4) Use as a sugar chain probe for functional analysis of cell surface glycoprotein sugar chains in cell-cell interaction. (5) Use as a sugar chain-fixing affinity carrier.

The form in which the artificial glycoconjugate is used for each purpose is not particularly limited, but examples thereof include the following. -Immobilize (immobilize) on TLC plate, plastic plate, or various carriers.・ Use liposomes and lipid microspheres.

Regarding the use of the artificial composite material of the present invention, for example, Trends in Glycoscience and Glycotech
nology (TIGG) Vol.3, No.14, 435-437 (1991), "Shinsei Chemistry Laboratory 3" Carbohydrate I Glycoprotein, Artificial glycoconjugate (Pages 743-760) (published by Tokyo Kagaku Doujin Co., Ltd.) ) Can be referred to. As a cross-linking agent, a cross-linking agent that cross-links amino groups such as diisocyanate compounds, diisothiocyanate compounds, dihalogenated compounds, and glutaraldehyde; cross-linking that cross-links amino groups and thiol groups such as N- (m-maleimidobenzoyloxy) succinimide. Agent: A crosslinking agent that crosslinks amino groups and hydroxyl groups, such as dicarboxylic acid, can be used (“Seikagaku Chemistry Laboratory” 5, Immunobiochemistry Research Method, pages 83-87, 1986, issued by Tokyo Kagaku Dojin). .

Specifically, for example, compounds having a carboxyl group (proteins, peptides, lipids (for example, lysolecithin having a dicarboxylic acid crosslinking agent introduced in the hydroxyl group; phosphatidylethanolamine having a dicarboxylic acid crosslinking agent introduced in the amino group, etc. )) Carboxyl group is N-hydroxysuccinimide ester (for example, the compound described in Chemical formula 6), 1-hydroxybenzotriazole ester, p
-Active esters such as nitrophenyl ester ("Basics and experiments of peptide synthesis", 1985, published by Maruzen Co., Ltd.)
For example, it can be reacted with the saccharide having an amino group at the reducing end of the present invention. In addition, a compound having an amino group (for example, phospholipid such as phosphatidylethanolamine, or protein, peptide, etc.) is reacted with a dialdehyde (for example, glutaraldehyde), and then,
It is also possible to react with the saccharide having an amino group at the reducing end of the present invention.

[0030]

[Chemical 6]

[0031]

[Example 1: Synthesis of reducing end aminated monosaccharide]

Example 1-1 A known method using lactose and 2-aminopyridine [hereinafter sometimes abbreviated as "PA"] as raw materials (Biochem. Bioph.
ys. Res. Commun., 85 , 257-263 (1978)) 1-
4.5 μmol of pyridylamino-1-deoxylactitol [2-aminopyridine-bound lactose; sometimes referred to as “PA-lactose” below) was dissolved in 1 ml of water, and the pH was adjusted to 3 with acetic acid. A small amount of palladium black was added to this solution, and the reduction reaction was carried out in a hydrogen gas stream under normal pressure for 3 hours. The reaction solution was TLC [DC-Alufo
lien Kieselgel 60 (Merck); methanol / water /
It was developed using aqueous ammonia (6: 0.3: 0.1 v / v), and the target substance was colored by using sulfuric acid and heating] to analyze the progress of the reaction. A part (55 nmol) of the reaction product was evaporated to dryness under reduced pressure, and anhydrous hydrazine was added thereto to form a solution, which was heated in a sealed tube at 70 ° C. for 2 minutes. Then, excess hydrazine was distilled off under reduced pressure to obtain 1-amino-1-deoxylactitol (yield 9
5%). This is the other method (Anal. Biochem., 97 ,
It was identified by an amino acid analyzer as the same substance as 1-amino-1-deoxylactitol synthesized in 166-172 (1979)).

Example 1-2 Almost the same as Example 1-1 above, except that 1-pyridylamino-1-deoxy-N-acetylglucosaminitol was used as a starting material and decomposition with hydrazine was carried out under milder conditions. 1-amino-1-deoxy-N-acetylglucosaminitol (a compound of the formula (b1) in which Ra is H) was synthesized by the method.

[Example 2: Synthesis of reducing end aminated oligosaccharides] A known method (J. Biochem., 90 , 90 , using an oligomannose type sugar chain (M6B) of the following formula separated and purified from ovalbumin as a raw material. 407-414 (1981); J. Biochem., 9
5, 197-203 (1984)) 2-aminopyridine in the synthesis reducing end residue (terminal GlcNAc) in accordance with bound M6B
(M6B-PA) was used in an amount of 1.9 μmol, and the reducing end was aminated in the same manner as in Example 1 (M6B-M6B-PA).
N) (a compound in which Ra is a group consisting of a moiety excluding the terminal GlcNAc of M6B to be aminated in the formula (b1)) was synthesized. The reduction was performed by using palladium black as a catalyst and reacting in a hydrogen gas stream under normal pressure for 3 hours. In addition, decomposition with hydrazine
The reaction was carried out by using anhydrous hydrazine at 70 ° C. for 2 minutes in a sealed tube. After completion of the reaction, the same treatment as in Example 1-1 was carried out to purify the target compound by TLC (yield: about 5
0%).

Structure of M6B: Manα1-6 (Manα1-3) Manα1
-6 (Manα1-2Manα1-3) Manβ1-4GlcNAc β1-4GlcNAc mass spectrometry [M + Na] ⁺ : measured value m / z = 1420.3
(Calculated value 1420.5) [Example 3: Synthesis of fluorescein isothiocyanate-labeled oligosaccharide] M6B-N 20n synthesized in Example 2
The reaction liquid containing mol was concentrated to dryness in a test tube, and 10 μl of pyridine and 1.3 mg of fluorescein isothiocyanate (FITC) were added thereto. It heated at 70 degreeC for 2 minutes, and dried the reaction liquid under reduced pressure. Dissolve the concentrated residue in a small amount of water and use it for a TLC plate [DC-Alufolien Kieselgel6
0,4.5 × 7cm], methanol / ethanol /
It was developed with acetic acid (2: 1: 0.05 v / v). Then, the fluorescent band was eluted with water to obtain an aqueous solution of M6B (M6B-F) in which the amino group of the reducing terminal residue was labeled with FITC.

Example 4: Synthesis of biotin-labeled oligosaccharide The reaction solution containing 20 nmol of M6B-N synthesized in Example 2 was concentrated to dryness in a test tube, and 10 μl of saturated sodium hydrogen carbonate solution and biotin Sulfo-N-
2 mg of hydroxysuccinimide ester was added. This was reacted for 15 minutes at room temperature with occasional stirring. After the reaction, strong acid cation exchange resin Dowex (Do
Wex) 50 × 2 (manufactured by Dow Chemical Co., 100 to 200 mesh, H ⁺ type) was added to adjust the pH to 3. The resin was filtered off, washed with 5 volumes of water, and the filtrate and washings were combined and fractionated using TLC as in Example 3. The band of the desired compound was eluted with water to give An aqueous solution of M6B (M6B-B) in which the amino group was biotinylated was obtained.

[Example 5: Synthesis of artificial glycoconjugate] Example 5-1: Synthesis of complex of aminated sugar derivative and lysolecithin 2- (4-hydroxycarbonylbutyroyl) lysolecithin [1st position is palmitoyl group] Dissolved in DMF,
Cool to ° C and add 1 equivalent of N-hydroxysuccinimide and 1 equivalent of dicyclohexylcarbodiimide (DCC) to the lysolecithin. Adjust the pH to 6-7 with triethylamine and stir at room temperature for 15 hours. The insoluble matter is filtered through zeolite to obtain a DMF solution of active ester form.

The solvent of the active ester solution was distilled off,
1 equivalent of M synthesized in Example 2 with respect to the active ester form
An aqueous solution of 6B-N is added, and the mixture is reacted at 0 ° C. for 1 hour and further at room temperature overnight to obtain a bound product of M6B-N and the above lysolecithin derivative. Example 5-2 Synthesis of complex of aminated derivative and phosphatidylethanolamine 1 equivalent of succinic anhydride and 2 equivalents of triethylamine were added to dipalmitoylphosphatidylethanolamine dissolved in a chloroform solution, and the mixture was reacted at room temperature for 24 hours. Purify by silica gel column chromatography (eluent: chloroform-methanol). Then
In the same manner as in Example 5-2, N-hydroxysuccinimide and DCC were used to obtain an active ester form, and this was reacted with M6B-N synthesized in Example 2 to obtain a conjugate with phosphatidylethanolamine.

Reference Example: Detection of Lectin by Dot Blot Method Known method (Proc. Natl. Acad. Sci. USA,
76 , 4350-4354 (1979); Biochem. J., 257, 43-49 (1989).
), A concanavalin A (ConA; manufactured by Seikagaku Corp.) is used as a nitrocellulose membrane (manufactured by Bio-Rad).
Fixed on top (Spot A, 1 μg (10 pmol); Spot B, 0.5 μg (5 pmol); Spot C,
0.1 μg (1 pmol)), bovine serum albumin (B
Blocked with SA). This membrane was mixed with the M6B-F (0.05 mM) aqueous solution obtained in Example 3 or the M6B-B (0.1 mM) aqueous solution obtained in Example 4 in a 20 mM Tris-HCl buffer (pH 7.5; 1 mM chloride). Calcium, 1
Incubated for 30 minutes in mM magnesium chloride and 0.15 M sodium chloride).

After the reaction, UV was used when M6B-F was used.
The lamp was illuminated and the ConA spot was detected visually. As a result, it was possible to detect all ConA in the spots A to C although the spot C had a slightly low concentration. When M6B-B is used, a streptavidin-horseradish peroxidase conjugate (hereinafter abbreviated as “St.Av-HRP”; vector laboratory company) is further reacted to give 4-chloro-1-. Color is developed using a substrate containing naphthol and is detected macroscopically, or streptavidin-fluorescein conjugate (hereinafter referred to as "S
Abbreviated as "t.Av-FITC"; manufactured by Peace Co., Ltd.) and reacted with a UV lamp for macroscopic detection. As a result, St.Av-HR
When P is used, Con A can be detected in all of Spots A to C although the concentration of Spot C is slightly low.
When t.Av-FITC was used, spot B was near the detection limit and spot A was clearly detected. ConA was not detected in any of the controls incubated with water instead of M6B-B.

From the above results, 5 pmol or less of ConA was detectable by the dot blot method when the above-mentioned sugar-labeled product according to the present invention was used. On the other hand, using the M6B-PA, the dot blot method (UV lamp detection) was performed in the same manner.
When A was visually detected, 0.5 nmol was the detection limit.

[0041]

EFFECTS OF THE INVENTION A labeled 2-amino-derivatized sugar such as a pyridyl-aminated sugar chain (2-aminopyridine-derivatized oligosaccharide) is easily singly purified from a natural glycoconjugate using HPLC or the like. The sugar chain having an amino group at the reducing terminal obtained from such a pyridyl aminated sugar chain or the like by the method of the present invention can be easily reacted with a labeling compound, protein, peptide, etc. It is useful for histochemical studies on one structure, studies on lectins, etc., preparation of neoglycoprotein, neoglycolipid, etc.

Claims (7)

[Claims] 1. A general formula (a) represented by the following chemical formula 1 (Wherein, R-CH ₂ -, the reducing end is aldehyde represents a group derived from a saccharide which may be represented by R-CHO, R
¹ , R ² , R ³ and R ^{4, which} may be the same or different, each represents a group selected from hydrogen and a lower alkyl group. A 2-aminopyridine derivatized saccharide represented by the formula (1) is subjected to a reduction reaction, and then subjected to a decomposition reaction under alkaline conditions to give a compound represented by the following general formula (b) R—CH ₂ —NH ₂ (b) (wherein R-CH ₂ - is amination of the sugar, characterized in that to obtain the aminated sugar derivative represented by the same defined above).. 2. The method for amination of sugar according to claim 1, wherein the decomposition reaction under alkaline conditions is a decomposition reaction in the presence of hydrazine. 3. The general formula (a) represented by the chemical formula 1 of claim 1.
The compound of (1) has a reducing terminal aldehyde, and a saccharide that can be represented by the following general formula (c) R-CHO (c) (wherein R represents a saccharide residue) and a general formula shown by the following chemical formula 2. (D) [Chemical formula 2] (In the formula, R ¹ , R ² , R ³ and R ⁴ may be the same or different and each represents a group selected from hydrogen and a lower alkyl group.) And a 2-aminopyridine derivative The method for amination of sugar according to claim 1, which is a 2-aminopyridine derivatized saccharide obtained by reacting with a Schiff base to form a Schiff base, and then subjecting it to a reduction reaction. 4. The compound of the general formula (a) has a substantially single R group obtained by subjecting a mixture of plural kinds of 2-aminopyridine derivatized saccharides having different structures to fluorescence chromatography. The method for aminating sugar according to claim 1, which is a fractionated 2-aminopyridine derivatized saccharide. 5. A method for producing a saccharide labeled at a reducing end, which comprises reacting a compound represented by the general formula (b) according to claim 1 with a labeled compound capable of reacting with an amino group. . 6. The method according to claim 7, wherein the labeled compound is fluorescein isothiocyanate, phenyl isothiocyanate or biotin. 7. A method for producing an artificial glycoconjugate, which comprises reacting the compound represented by the general formula (b) according to claim 1 with a protein, a peptide or a lipid.