JPH07252288A - Method for fluorescent labeling of saccharide and production of artificial complex glycide - Google Patents

Abstract

PURPOSE:To provide a method for introducing a 2-aminopyridine derivative in a saccharide under a mild condition, to easily produce a reaction agent capable of being used for synthesizing an artificial complex glycide and to produce the artificial complex glycide using the reaction agent. CONSTITUTION:(1) A method for fluorescent labeling specified by bonding a 2-aminopyridine derivative having an aminoalkyl group, which has an amino protecting group, at 6-position to a saccharide, at least a terminal of which consists of a reduced saccharose, by reducing amination reaction and, then, eliminating the protecting group. (2) A method for producing an artificial complex glycide by obtaining a bonded compound of 2-aminopyridine derivative having an aminoalkyl group at 6-position to a saccharide by eliminating the protecting group in the process (1), then, directly reacting to bond the amino group in the 6-position amino alkyl group of the bonded compound with an organic compound having a functional group capable of bonding the amino group or reacting with the organic compound having the functional group capable of bonding the amino group through a spacer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent labeling method for sugar and a method for producing an artificial glycoconjugate, and more particularly to the method using a 2-aminopyridine derivative.

[0002]

2. Description of the Related Art In recent years, in order to study the relationship between the structure of glycoconjugates such as glycoproteins and their functions, various methods have been proposed which are more sensitive and capable of quantitative assay with a small amount of sample. 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 of using an organic compound such as aminopyridine as a fluorescent labeling agent is known (for example, S. Hase et al., Journal of Biochemistry, Vol. 95, 197-).
203 (1984)). This is a method in which the 2-amino group is reacted with the reducing end of a sugar or a sugar chain to form a Schiff base, which is then reduced and detected by fluorescence, and the sensitivity is at the pmol level as described above. Has been done.

However, although the fluorescent labeling agent is effective for structural analysis of sugar chains, since it is monofunctional, it can be used for other biological substances such as proteins, peptides, amino acids, lipids,
It was not possible to form a complex of nucleic acid, synthetic polymer and the like with sugar, and therefore it could not be said to be sufficient for comprehensive analysis of them. Therefore, the present applicant has filed Japanese Patent Application Laid-Open No.
-255253 discloses a 2-aminopyridine derivative in which a functional group capable of reacting with the biological substance is introduced at the 6-position, and it is shown that various artificial glycoconjugates can be synthesized.

However, a method of synthesizing an artificial glycoconjugate via an amino group produced by reduction of a cyano group after separating and purifying a labeled sugar chain using a 2-aminopyridine derivative into which a cyano group is introduced However, in the reduction of the cyano group, there has been a problem that Pd in concentrated aqueous ammonia has to be subjected to pressure catalytic reduction for a long time.

[0006]

The first object of the present invention is to provide a fluorescent labeling method for introducing a 2-aminopyridine derivative into a sugar under mild conditions and a reaction that can be used for the synthesis of artificial glycoconjugate. It is to provide a method for easily producing a conjugate of a 2-aminopyridine derivative as an agent and a sugar, and secondly to provide a method for producing an artificial glycoconjugate using the reaction agent.

[0007]

According to the present invention, a 2-aminopyridine derivative having an aminoalkyl group having an amino-protecting group at the 6-position and a sugar compound having at least a reducing sugar at the end are bound by a reductive amination reaction. , And then a method for fluorescently labeling a sugar, which comprises removing an amino-protecting group, wherein the amino-protecting group is a urethane-type amino-protecting group or a trihaloacetyl group, the fluorescent-labeling method for a sugar as described above, The amino-protecting group is a t-butoxycarbonyl group, a benzyloxycarbonyl group or a trifluoroacetyl group. The above-mentioned method for fluorescently labeling sugars, wherein the elimination reaction of the amino-protecting group is carried out under acidic conditions or basic conditions. The treatment according to the conditions, or the fluorescent labeling method of sugar according to any one of the above 1 to 3 performed by a reduction reaction, the treatment under acidic conditions, The above-mentioned method for labeling a sugar with fluorinated acetic acid, the treatment under basic conditions is a method for labeling a sugar with an aqueous solution of piperidine described above, and the reduction reaction is carried out by a catalytic reduction. A method for fluorescently labeling a sugar, wherein a 2-aminopyridine derivative having an aminoalkyl group having an amino-protecting group at the 6-position and a sugar compound having at least a reducing sugar at the end are linked by a reductive amination reaction, and then an amino-protecting group Removal is performed to obtain a conjugate of a 2-aminopyridine derivative having an aminoalkyl group at the 6-position and a sugar compound, and then the amino group of the 6-position aminoalkyl group of pyridine of the conjugate is bound to the amino group. Or a direct reaction with an organic compound having a functional group capable of binding,
Alternatively, a method for producing an artificial glycoconjugate characterized by reacting with an organic compound through a spacer having a functional group capable of binding to an amino group and binding the same, the organic compound is a sugar, a protein, a peptide, an amino acid,
Lipids, nucleic acids, nucleotides, nucleosides, biotin,
Alternatively, the method for producing an artificial glycoconjugate as described above, which is a synthetic polymer compound, wherein the organic compound or the spacer is a compound having a carboxyl group, and the amino group of the aminoalkyl group at the 6-position of pyridine is activated by activating the carboxyl group. The method for producing an artificial glycoconjugate as described above or described, which comprises reacting with a group.

The fluorescent labeling method for sugars of the present invention comprises a 2-aminopyridine derivative having an aminoalkyl group having an amino protecting group at the 6-position (hereinafter also referred to as a protecting group-containing 2-aminopyridine derivative). The terminal is bound by a reductive amination reaction with a sugar compound consisting of at least a reducing sugar,
Then the amino protecting group is removed. The present invention is
As a result of this elimination, a conjugate of a 2-aminopyridine derivative having an aminoalkyl group at the 6-position and a sugar compound can be obtained, and this conjugate can bind any other organic compound to the amino group at the 6-position. The method of the present invention also provides a method for producing a fluorescent labeling reagent because it has a function as a fluorescent labeling reagent for binding to obtain an artificial glycoconjugate.

Examples of the protective group-containing 2-aminopyridine derivative used in the present invention include compounds represented by the following general formula (1) (hereinafter, also referred to as compound 1).

[0010]

[Chemical 1]

In the formula, R represents a protecting group which is an organic group,
n is an integer of 2 to 20, preferably 2 to 8. Examples of the protecting group include urethane type amino protecting group and trihaloacetyl group, and examples of the urethane type amino protecting group include t-butoxycarbonyl group (hereinafter referred to as Boc) or benzyloxycarbonyl group (hereinafter referred to as Z). A trifluoroacetyl group (hereinafter referred to as Tfa) is particularly preferable as the trihaloacetyl group,
It is not limited to this.

Further, in the formula, hydrogen at the 3 to 5 positions is an arbitrary organic group, preferably a hydrocarbon group, and particularly preferably a carbon number of 1.
It may be substituted by a lower alkyl group of -4. In the present invention, the reductive amination reaction of Compound 1 and a sugar compound having at least a reducing sugar at the terminal condenses the amino group at the 2-position of Compound 1 with the sugar compound to form a Schiff base,
It is to form -CH ₂ NH- bond by reducing it then. The resulting reaction product (bound product) of Compound 1 and a sugar compound is also referred to as a sugar residue-containing compound 1.

The sugar residue-containing compound 1 thus obtained is subjected to a reaction for eliminating the amino-protecting group at the 6-position. The elimination reaction is not particularly limited, but the following method is preferable. A method of treating a sugar residue-containing compound 1 under acidic conditions using trifluoroacetic acid, anhydrous hydrogen chloride, formic acid, or the like. In particular,
It is suitable when the protecting group is Boc. A method for catalytically reducing the sugar residue-containing compound 1 in the presence of a catalyst such as Pd (palladium) with hydrogen under atmospheric pressure or pressure.
Alternatively, a method of catalytic hydrogen transfer reduction using an organic hydrogen donor in the presence of Pd. It is particularly suitable when the protecting group is Z. Examples thereof include a method of treating the sugar residue-containing compound 1 under basic conditions with an aqueous solution of a base, for example, piperidine, ammonia, sodium hydroxide and the like. In particular, the protecting group is Tf
It is suitable for a.

In the case of removing the protecting group using trifluoroacetic acid, the solution of the sugar residue-containing compound 1 in trifluoroacetic acid is kept at room temperature, specifically 10 to 25 ° C.
Stirring for about 30 minutes and then removing trifluoroacetic acid while blowing nitrogen gas. The conditions for removing the protecting group using Pd catalyst in
Pd is added to the aqueous solution of the sugar residue-containing compound 1, and the mixture is stirred at room temperature for 30 to 90 minutes under a hydrogen atmosphere at 1 to 10 atm.

In the case of removing the protecting group with piperidine, the 0.2 to 2 M piperidine aqueous solution of the sugar residue-containing compound 1 is stirred at 0 to 10 ° C. for 0.5 to 2 hours. To be The sugar residue-containing 2-aminopyridine derivative (hereinafter, also referred to as “sugar residue-containing compound 1r”) in which the 6-position is an aminoalkyl group obtained in the above-mentioned reactions can be purified by any method, High yield (about 95% or more) by HPLC (high performance liquid chromatography), preferably as an aqueous solution
Can be highly purified and isolated. Since the method of the present invention is an extremely mild method, it achieves the above-mentioned high yield.

Purification using HPLC is carried out, for example, by Cos.
Reverse phase HPLC using mosil 5C18-AR or ion exchange HPLC using TSK gel Amide-80 can be used. Since this sugar residue-containing compound 1r has a reactive amino group at the 6-position, it has the function of a fluorescent labeling agent for labeling any other organic compound and also makes an artificial glycoconjugate. It has the function of a reactant for

The conditions for producing the artificial glycoconjugate are not particularly limited, but the following methods can be specifically exemplified. a. A functional group that reacts with an amino group of a sugar residue-containing compound 1r having a desired sugar residue structure (for example, a carboxyl group,
A method for obtaining a desired artificial glycoconjugate by binding to a desired organic compound having a formyl group). b. a. As the organic compound of (1), a compound (eg, a spacer) having a functional group that reacts with other organic compounds in addition to the functional group that reacts with the amino group of the sugar residue-containing compound 1r is selected,
A method for obtaining an artificial glycoconjugate by reacting the obtained sugar residue-containing compound 1r derivative (eg, sugar residue-containing compound 1r-spacer conjugate) with a desired organic compound. The sugar residue-containing compound 1r derivative may have a plurality of functional groups, and a plurality of organic compounds that react with this may also be used.

As the spacer having a plurality of functional groups, glyoxal, disuccinimidyl suberate,
Examples thereof include those derived from succinimidyl 3- (2-pyridyldithio) propionate, succinimidyl 6-maleimidohexanoate, and the like. c. A method of incorporating a desired organic compound into a functional group which is introduced or previously exists in the artificial glycoconjugate obtained in b.

Examples of the reaction between the sugar residue-containing compound 1r and the organic compound in a include an amide bond reaction, a Schiff base formation reaction, a urethane bond reaction and an alkylation reaction. The sugar residue-containing compound 1r derivative in b functions as a reagent for preparing one kind of artificial glycoconjugate or other artificial glycoconjugate. The organic compound for obtaining this reaction agent may be one having a function only as a spacer for binding another organic compound or a compound having another useful action. Also,
In this sugar residue-containing compound 1r derivative, by selecting an organic compound to be introduced, it is possible to synthesize a compound which itself can be a useful reagent. For example, if a biotin derivative is selected as the organic compound, analysis of lectins using labeled avidin or the like (ELISA
Etc.) useful reagents are obtained.

The functional group introduced into the sugar residue-containing compound 1r derivative by the reaction between the sugar residue-containing compound 1r and the organic compound is a functional group capable of causing a binding reaction between the organic compound and the above-mentioned a. In addition, functional groups in which a disulfide bond reaction, a disulfide exchange reaction, an amidine bond reaction, a carbon-carbon bond reaction and the like occur, for example, a carboxyl group, an amino group, a hydroxyl group, a disulfide group, an acryloyl group and the like can be mentioned.

In addition, in the above reactions a to c, the functional group can be previously activated and provided for the reaction. For example,
When the functional group is a carboxyl group, it can be activated by a succinimide group, and in the case of a mercapto group,
It can be activated by the 3-nitro-2-pyridylsulfenyl group. The above-mentioned organic compounds in a to c are arbitrary, but particularly preferably sugar, protein, peptide, amino acid, lipid, nucleic acid, nucleotide, nucleoside, biotin, or synthetic polymer (including its monomer), and various A useful artificial glycoconjugate can be easily obtained.

The sugar compound used in the present invention is not limited as long as it has at least a reducing sugar at its terminal, and specific examples thereof include monosaccharides, oligosaccharides, polysaccharides and glycosaminoglycans. it can. The bond by the reductive amination reaction in the present invention is carried out by reacting compound 1 with the reducing end of a sugar compound to form a Schiff base, and then reducing it to form a —CH ₂ NH— bond to form a sugar residue-containing compound 1 The desired sugar residue-containing compound 1 can be detected and separated and purified by performing separation by means of a separation means such as high performance liquid chromatography (HPLC), if necessary, and detecting by UV or fluorescence spectrum. .

The Schiff base forming reaction and the subsequent reduction reaction are known methods (for example, Japanese Patent Laid-Open No. 64-1).
0177, JP-A-1-141356, J. Bioc
hem. , Vol. 95, pp. 197-203 (198
4), "Protein Nucleic Acid Enzymes", Vol. 36, No. 1, No. 6
3 to 68 (1991)). That is, in an inorganic acid such as hydrochloric acid or hydrofluoric acid, or in an organic acid such as acetic acid or trifluoroacetic acid and an organic solvent such as pyridine, room temperature to 100 ° C., several minutes to several hours (preferably about 90 ° C., 1 to The Schiff base can be produced by reacting the saccharide with about 20 to 100 equivalents of compound 1 under a reaction condition of pH 3 to 6.4) for 3 hours. For the reduction of the Schiff base, a reducing agent usually used for the reduction of the Schiff base can be used, and particularly, a volatile borane complex (for example,
Borane dimethylamine complex (BH ₃ · Me ₂
NH), borane triethylamine complex, borane pyridine complex, etc.), sodium borohydride, sodium cyanoborohydride (NaBH ₃ C
N) and the like are preferable. The reduction reaction is completed at room temperature to 100 ° C for 1 hour to 10 hours (preferably about 80 to 90 ° C for about 1 hour).

[0024]

EXAMPLES Examples of the present invention will be specifically described below, but the present invention is not limited thereto. Synthesis Example 1 Synthesis of Compound 1-1 (n = 3, R = Boc) 2-Tritylamino-6- (3-) obtained by reducing 2-tritylamino-6- (2-cyanoethyl) pyridine with LiAlH _4. Aminopropyl) pyridine (Bo
c) ₂ -tritylamino-6-obtained by reacting with ₂ O
Acetic acid-distilled Me of (3-t-butoxycarbonylaminopropyl) pyridine (2.00 g, 4.05 mmol).
OH [1: 1 (v / v)], 14 ml] solution at 50 ° C
It was stirred for 5 hours and a half. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography [silica gel 50 g, chloroform-methanol (15: 1)]. Since part of the salt was an acetate, ethyl acetate was added for dissolution for desalting, and the mixture was washed successively with a saturated aqueous sodium hydrogen carbonate solution and a saturated saline solution, and then dried over magnesium sulfate. After the desiccant was filtered off, it was concentrated under reduced pressure to obtain a pale yellow oil. The oil is left in the refrigerator for 2 weeks and the title compound of needle crystals 1-1
Got Yield 931 mg (2-tritylamino-6-
(Yield from 3-t-butoxycarbonylaminopropyl) pyridine 91.6%), mp 72-74 ° C. ¹ H
Compound -NMR (270MHz, CDCl ₃₎ 1-1
The structure of was confirmed. Also, the calculated value of C ₁₃ H ₂₁ O ₂ N ₃ ,
Found for C 62.13; H 8.42; N 16.72, C 61.90; H 8.48; N 16.
It was 59.

Synthesis Example 2 Compound 1-2 (n = 3, R =
Synthesis of Z) 2-tritylamino-6- (2-cyanoethyl) pyridine is reduced by LiAlH ₄ to obtain 2-tritylamino-6- (3-aminopropyl) pyridine by ZOS.
2-tritylamino-6- (3-benzyloxycarbonylaminopropyl) pyridine (697 mg, obtained by reacting with u (Su is N-hydroxysuccinimide))
1.32 mmol) acetic acid-MeOH [1: 1 (v /
v), 10 ml] of the solution was stirred at 50 ° C. for 18 hours and a half. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography [silica gel 10 g, chloroform-methanol (15: 1)]. Since a part thereof was an acetate, the title compound 1-2 as a pale yellow oil was obtained in the same manner as in Synthesis Example 1. Yield 361 mg (2-tritylamino-
6- (3-benzyloxycarbonylaminopropyl)
Yield from pyridine 96.2%). ¹ H-NMR (27
The structure of Compound 1-2 was confirmed at 0 MHz, CDCl ₃ ). Also, the calculated value of C ₁₆ H ₁₉ O ₂ N ₃ , C 65.8
3; H 6.81; N 14.38, found, C
65.83; H 6.72; N 14.38.

Synthesis Example 3 Compound 1-3 (n = 3, R = T
Synthesis of fa) 2-tritylamino-6- (2-cyanoethyl) pyridine was reduced with LiAlH ₄ to give 2-tritylamino-6- (3-aminopropyl) pyridine (Tf
a) ₂ -Tritylamino-6-obtained by reacting with ₂ O
Acetic acid-MeOH of (3-trifluoroacetylaminopropyl) pyridine (140 mg, 285 μmol)
[1: 1 (v / v)], 4 ml] solution at 22 ° C for 22
Stir for hours. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography [silica gel 8 g, dichloromethane-methanol (20: 1)]. Since a part thereof was an acetate, the title compound 1-3 was obtained as pale yellow needle crystals in the same manner as in Synthesis Example 1. Yield 61.4 mg (yield from 2-tritylamino-6- (3-trifluoroacetylaminopropyl) pyridine 96.2%). ¹ H-NMR
The structure of Compound 1-3 was confirmed at (270 MHz, CDCl ₃ ). Also, the calculated value of C ₁₀ H ₁₂ ON ₃ F ₃ , C 4
Found at 8.58; H 4.89; N 17.00, C 48.62; H 4.81; N 16.87.
Met.

Examples 1-1 to 3 Dried sugar compounds (listed in Table 1) (5.55 μm)
1) to Compounds 1-1 to 3 (27.8 μmol, described in Table 1) and acetic acid-pyridine [1:17 (v / v), 48.0).
μL] was added, the tube was sealed, heated at 90 ° C. for 3 hours and allowed to cool, and then the reduction reaction solution [BH ₃ · Me ₂ NH (6.55) was added.
mg, 111 μmol) in acetic acid (33.5 μL) solution]
(Me: methyl group) was added, and the tube was sealed and heated at 80 ° C. for 1 hour. After allowing to cool, water was added to the mixture to perform HPLC [column: Cosmo Seal 5C18AR 20 mm ID × 250 mm L;
Elution solvent: acetonitrile-0.1 M ammonium formate (pH 4.5); concentration gradient: [0-60% acetonitrile (2% / min)]; flow rate: 8 mL / min; detection: UV3
00 nm], each sugar residue-containing compound 1 was purified and then lyophilized. The respective yields are shown in Table 1.

[0028]

[Table 1]

Next, the following three types of sugar residue-containing compounds 1 (ac) obtained in Example 1-3, that is,

[0030]

[Chemical 2]

The protecting group of was removed as follows. (1) Boc Removal of Compound a A solution of compound a (1.18 mg, 1.60 μmol) in trifluoroacetic acid (200 μL) was stirred at room temperature for 30 minutes. Then, nitrogen gas was blown to remove trifluoroacetic acid, water was added to the residue to dissolve it, and after separation and purification by HPLC, lyophilization was performed to obtain a compound ar represented by the following formula in which Boc was replaced with H. The yield was quantitative and almost 100% by HPLC analysis.

[0032]

[Chemical 3]

(2) Removal of Z from compound b Compound b (1.31 mg, 1.69 μmol) was added with water (5
(00 μL) was added and dissolved, and palladium black (11.2 m
g) was added, and the mixture was stirred at room temperature for 90 minutes under a hydrogen atmosphere of 4 atm. Then, after separation and purification by HPLC, lyophilization was performed to obtain a compound ar in which Z was replaced with H. HPLC yield
By analysis, it was quantitative and almost 100%. (3) Removal of Tfa from Compound c A solution of compound c (0.15 mg, 0.2 μmol) in 1M aqueous piperidine solution (250 μL) was stirred at 0 ° C. for 1 hour. Then, after separation and purification by HPLC, freeze-drying is performed to
A compound ar in which fa was replaced by H was obtained. The yield is HPL
It was quantitative and almost 100% by C analysis.

Comparative Example In the compound ar, 32 μmol of a compound having a cyanoethyl group at the 6-position of the pyridine ring was added to 25% aqueous ammonia (14 mL) in the presence of palladium black (50 mg), and hydrogen was supplied at 9 kg / cm ² at room temperature. Hydrogenated for 66 hours,
Compound ar was synthesized. The catalyst was filtered off and the filtrate was concentrated. The concentrated residue was dissolved in water (10 mL) and HP
LC [Column: Cosmosil 5C ₁₈ AR, 20 mm x 25
0 mm; _{solvent: MeCN-0.1M CH 3 CO 2} NH
₄ (pH 6.9); gradient: 5% MeCN-50% MeC
N (1.5% / min); flow rate: 8 mL / min; detection: UV 307 nm]. The fraction containing the compound ar was lyophilized and the resulting powder was added to water (10 mL).
Dissolved in. To remove ammonium salts mixed in this solution, HPLC [column: Cosmocil 5C ₁₈ AR, 20
mm × 250 mm; solvent: 15% MeCN-0.01%
TFA; Flow rate: 8 mL / min; Detection: UV307n
m]. The yield was 77%.

Example 2 Example 2-1 Synthesis of artificial glycoconjugate composed of bovine serum albumin (BSA) and compound ar The title artificial glycoconjugate was synthesized by the synthetic route shown in the following chemical formula 4.

[0036]

[Chemical 4]

Compound ar and succinimidyl 3- (3
-Nitro-2-pyridyldithio) propionate (1)
Tetrahydrofuran (THF) -water (2: 1)
Binding in A (triethylamine) solution (pH 9),
Reversed phase HPLC (column: Cosmocil 5C18 AR,
The compound (2) was obtained with a solvent: MeCN-0.1% TFA aqueous solution. Compound (2) was obtained as 2 trifluoroacetic acid salt.

Next, BSA having 6 cysteine residues
(3) and compound (2) in various molar ratios [compound (2): BSA = 1 to 6: 1] with 0.5 M Tris-0.
It was dissolved together in 005 MEDTA sodium salt buffer (pH 7.5) and subjected to an exchange reaction between the -SH group of BSA and the disulfide group of compound (2) at 4 ° C. to give an artificial glycoconjugate (4).
Got The obtained artificial glycoconjugate (4) is gel filtered HP.
LC [column: Asahi Pack GS-510, solvent: 30
% MeCM-0.2M sodium phosphate buffer (pH
7.0)] to separate the compound (2).

Example 2-2 Synthesis of artificial glycoconjugate composed of biotin and compound ar The title artificial glycoconjugate was synthesized by the synthetic route shown in the following chemical formula 5.

[0040]

[Chemical 5]

Compound ar (15.0 mg, 16.6 μm
ol) 0.5% aqueous NaHCO ₃ solution-DMF [1: 1
(V / v), 500 μL] aqueous solution of N-hydroxysuccinimide biotin (5) (40.0 mg, 117 μm
ol) was added and the mixture was stirred at room temperature for 3.5 hours. Then, the compound (5) (24.0 mg, 70.4 m)
mol) was added, the mixture was filtered, and the filtrate was analyzed by HPLC [column: Cosmocil 5C ₁₈ AR, 20 mm
× 250 mm; solvent: MeCN-0.1M HCO ₂ N
H ₄ (pH6.3); Gradient: 5% MeCN-50% Me
CN (1.5% / min); flow rate: 8 mL / min; detection: UV 300 nm]. Fractions eluted after 20.9 minutes were collected and freeze-dried to obtain compound (6) as a colorless powder (yield 9.3 mg (yield 65%)). Compound (6) was confirmed by ¹ H-NMR (D ₂ O), and m / z 866.7 [(M +
H) ⁺ ].

Example 2-3 Synthesis of artificial glycoconjugate composed of acrylic acid and compound ar The title artificial glycoconjugate was synthesized by the synthetic route shown in the following chemical formula 6.

[0043]

[Chemical 6]

Compound ar (15 mg, 16.6 μmo
l) and 4-acryloyloxyphenyldimethylsulfonium methylsulfate (7) (20.0 mg,
62.5 μmol) to a saturated NaHCO ₃ solution (300 μm
L), and the solution was stirred at room temperature for 5.5 hours to give 15.0 mg of the compound (7) in water (100 μL) as an aqueous solution 1
Added 6.6 μL. The mixture was stirred for another night,
It was then filtered. The filtrate was subjected to HPLC [column: Cosmosil 5C ₁₈ AR, 20 mm × 250 mm; solvent: Me.
_{CN-0.1M HCO 2 NH 4 (} pH6.3); Gradient: 5% MeCN-50% MeCN (1.5% / mi
n); Flow rate: 8 mL / min; Detection: UV 300n
m]. Fractions eluted after 18.7 minutes were collected and freeze-dried to obtain compound (8) as colorless powder (yield 9.2 mg (yield 80%)). The structure of compound (8) is
Confirmed by ¹ H-NMR (D ₂ O) and positive F
AB-MS showed a quasi-molecular ion peak at m / z 694.4 [(M + H) ⁺ ]. The compound (8) can be easily copolymerized with acrylamide to give a polymer compound.

[0045]

INDUSTRIAL APPLICABILITY According to the method for fluorescent labeling of sugars of the present invention, a pyridine derivative of a sugar having an alkylamino group at the 6-position bonded to a reducing sugar residue at the 2-position, that is, a fluorescent labeling agent is collected under extremely mild conditions. Identification of various substances is possible because they can be produced efficiently and the fluorescently labeled artificial glycoconjugate can be easily produced by binding the amino group at the 6-position to a biological substance such as a desired protein, lipid, sugar or nucleic acid. It is possible to provide a very useful means for quantification, preparation, purification and the like.

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[Procedure amendment]

[Submission date] March 16, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

[0024]

EXAMPLES Examples of the present invention will be specifically described below, but the present invention is not limited thereto. Synthesis Example 1 Synthesis of Compound 1-1 (n = 3, R = Boc) 2-Tritylamino-6- (3-) obtained by reducing 2-tritylamino-6- (2-cyanoethyl) pyridine with LiAlH _4. Aminopropyl) pyridine (Bo
c) ₂ -tritylamino-6- (3-t-butoxycarbonylaminopropyl) pyridine (2.00 g, obtained by reacting with ₂ O (di -tert- butyl dicarbonate))
4.05 mmol) acetic acid-distilled MeOH [1: 1 (v
/ V)], 14 ml] of the solution was stirred at 50 ° C. for 5 hours and a half. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography [silica gel 50 g, chloroform-methanol (15: 1)]. Since part of the salt was an acetate, ethyl acetate was added for dissolution for desalting, and the mixture was washed successively with a saturated aqueous sodium hydrogen carbonate solution and a saturated saline solution, and then dried over magnesium sulfate. After the desiccant was filtered off, it was concentrated under reduced pressure to obtain a pale yellow oil. The oil was left in the refrigerator for 2 weeks to give the title compound 1-1 as needle crystals. Yield 931 mg (yield from 2-tritylamino-6- (3-t-butoxycarbonylaminopropyl) pyridine 9
1.6%), mp 72-74 ° C. ¹ H-NMR (27
The structure of compound 1-1 was confirmed at 0 MHz, CDCl ₃ ). Also, the calculated value of C ₁₃ H ₂₁ O ₂ N ₃ , C 62.1
3; H 8.42; N 16.72, found,
C 61.90; H 8.48; N 16.59.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

Synthesis Example 2 Compound 1-2 (n = 3, R =
Synthesis of Z) 2-tritylamino-6- (2-cyanoethyl) pyridine is reduced by LiAlH ₄ to obtain 2-tritylamino-6- (3-aminopropyl) pyridine by ZOS.
u (benzyl N-succinimidyl carbonate)
2-tritylamino-6- (3-benzyloxycarbonylaminopropyl) pyridine (6
97 mg, 1.32 mmol) acetic acid-MeOH [1:
1 (v / v), 10 ml] solution was stirred at 50 ° C. for 18 hours and a half. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography [silica gel 10 g, chloroform-methanol (15: 1)]. Since a part thereof was an acetate, the title compound 1-2 as a pale yellow oil was obtained in the same manner as in Synthesis Example 1. Yield 361 mg (yield from 2-tritylamino-6- (3-benzyloxycarbonylaminopropyl) pyridine 96.2%). ¹ H-NMR
The structure of Compound 1-2 was confirmed at (270 MHz, CDCl ₃ ). Also, the calculated value of C ₁₆ H ₁₉ O ₂ N ₃ , C 6
Found at C5.83; H6.81; N14.38 was C65.83; H6.72; N14.38.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

Synthesis Example 3 Compound 1-3 (n = 3, R = T
Synthesis of fa) 2-tritylamino-6- (2-cyanoethyl) pyridine was reduced with LiAlH ₄ to give 2-tritylamino-6- (3-aminopropyl) pyridine (Tf
a) ₂ -Tritylamino-6- (3-trifluoroacetylaminopropyl) pyridine (140 mg, 285 μm) obtained by reacting with ₂ O (trifluoroacetic anhydride)
ol) acetic acid-MeOH [1: 1 (v / v)], 4 m
The solution of 1] was stirred at 50 ° C. for 22 hours. Concentrated under reduced pressure,
The residue was purified by medium pressure silica gel chromatography [silica gel 8 g, dichloromethane-methanol (20: 1)]. Since a part thereof was an acetate, the title compound 1-3 was obtained as pale yellow needle crystals in the same manner as in Synthesis Example 1. Yield 61.4 mg (Yield 9 from 2-tritylamino-6- (3-trifluoroacetylaminopropyl) pyridine 9
6.2%). ¹ H-NMR (270 MHz, CDC
l ₃ ) confirmed the structure of compound 1-3. Also, C ₁₀ H
Calculated for ₁₂ ON ₃ F ₃ , C 48.58; H 4.8.
9; Found at Cn 17.00, C 48.6.
2; H 4.81; N 16.87.

Claims (10)

[Claims] 1. A 2-aminopyridine derivative having an aminoalkyl group having an amino-protecting group at the 6-position and a sugar compound having at least a reducing sugar at the terminal are linked by a reductive amination reaction, and then the amino-protecting group is eliminated. A method for fluorescently labeling sugars, which comprises: 2. The method for fluorescent labeling of sugar according to claim 1, wherein the amino protecting group is a urethane type amino protecting group or a trihaloacetyl group. 3. The method for fluorescent labeling of sugar according to claim 1, wherein the amino protecting group is a t-butoxycarbonyl group, a benzyloxycarbonyl group or a trifluoroacetyl group. 4. The method for fluorescent labeling of sugar according to claim 1, wherein the elimination reaction of the amino protecting group is carried out by treatment under acidic conditions or basic conditions, or by reduction reaction. 5. The method for fluorescent labeling of sugar according to claim 4, wherein the treatment under acidic conditions is performed using trifluoroacetic acid. 6. The method for fluorescent labeling of sugar according to claim 4, wherein the treatment under basic conditions is performed using an aqueous piperidine solution. 7. The method for fluorescent labeling of sugar according to claim 4, wherein the reduction reaction is carried out by catalytic reduction. 8. A 2-aminopyridine derivative having an aminoalkyl group having an amino-protecting group at the 6-position and a sugar compound having a terminal at least a reducing sugar are bound by a reductive amination reaction, and then the amino-protecting group is eliminated. To obtain a conjugate of a 2-aminopyridine derivative having an aminoalkyl group at the 6-position and a sugar compound, and then the amino group of the aminoalkyl group at the 6-position of pyridine of the conjugate can be bound to the amino group. A method for producing an artificial glycoconjugate, which comprises directly reacting with an organic compound having a functional group to bind, or reacting with an organic compound through a spacer having a functional group capable of binding with an amino group to cause binding. . 9. The organic compound is sugar, protein, peptide,
Amino acids, lipids, nucleic acids, nucleotides, nucleosides,
The method for producing an artificial glycoconjugate according to claim 8, which is biotin or a synthetic polymer compound. 10. The artificial complex sugar according to claim 8, wherein the organic compound or the spacer is a compound having a carboxyl group, and the carboxyl group is activated to react with the amino group of the aminoalkyl group at the 6-position of pyridine. Quality manufacturing method.