JP3409337B2 - A novel sugar derivative and a method for measuring the activity of a physiologically active substance using the same as a substrate - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention includes biological components,
For example, the present invention relates to a sugar derivative useful as a substrate for measuring the activity of a physiologically active substance such as a sugar-modifying enzyme, and a method for measuring a physiologically active substance using the sugar derivative.

[0002]

BACKGROUND OF THE INVENTION It is well known that various physiologically active substances exist in the living body, and examples of physiologically active substances acting on sugars include glycosyltransferases, sugar hydrolases, and sugar isomerases. Sugar-modifying enzymes such as are known.

These sugar-modifying enzymes are enzymes that play an important role in the biosynthesis and metabolism of carbohydrates in the living body. Therefore, it is considered that by measuring the activity of these enzymes, important physiological knowledge, such as clinical significance as a tumor marker, can be obtained.

Since these contents are small, it is necessary to use a substrate labeled with a substance having high detection sensitivity for accurate measurement. Substrate labeling methods include radioactive labeling methods and non-radioactive labeling methods, but non-radioactive labeling methods are preferable and measurement sensitivity is taken into consideration when considering the convenience of measurement, restrictions on equipment, safety to the human body, etc. Among them, the fluorescent labeling method is more preferable.

However, conventionally used fluorescent labeling reagents, that is, fluorescent labeling reagents such as fluorescein isothiocyanate (FITC), eosin, and coumarin isothiocyanate have various problems due to their low water solubility. Had. That is, for example, since the fluorescent labeling reagent as described above must be dissolved in a polar organic solvent once and used, the fluorescent labeling operation is complicated and the fluorescence prepared using the fluorescent labeling reagent as described above. Since the labeled substance has a reduced water solubility, it is necessary to prepare an aqueous solution of the required concentration when other substances are detected using the fluorescent labeling substrate prepared by using the above-mentioned fluorescent labeling reagent. May not be possible, etc.

For the purpose of solving these problems, water-soluble fluorescent labeling reagents such as dansyl chloride, lucifer yellow, o-phthalaldehyde and the like, which have introduced water-soluble groups such as sulfo group and carboxyl group, have been developed. It was
However, these reagents themselves have poor storage stability, and the fluorescent-labeled substances prepared using these reagents also have poor storage stability, although they have improved water solubility. Therefore, it has a problem that it cannot be stored after preparation.

On the other hand, aminopyridines such as 2-aminopyridine and 3-aminopyridine are themselves water-soluble and very stable fluorescent substances, and thus labeled substances are also excellent in storage stability, and When the substance to be labeled is water-soluble, the water-solubility is maintained as it is, and thus it is a compound that has recently been widely used as a fluorescent labeling substance for sugars (Biochemical and Biophysical Research Commu
nications 85, 257, 1978 etc.).

However, there is a problem in using aminopyridine itself as a labeling substance such as sugar.
That is, as a fluorescent labeling method using aminopyridine, a method of binding to an aldehyde group of a substance to be labeled via a Schiff base is generally used, but using this method, aminopyridine is bound to a sugar. In the case of the saccharide, the ring of the sugar residue on the reducing end side of the sugar will be opened, so that the target structure can be That is the problem that one extra sugar residue must be attached to the reducing end side of the saccharide having the amino acid (Biochem. Biophys. Res. C).
ommun., vol.152, 107-112, 1988).

Therefore, a fluorescent labeling reagent capable of easily introducing a fluorescent group such as a pyridylamino group into a sugar or the like under mild conditions and without opening the ring of the sugar residue on the reducing terminal side. And the emergence of a novel fluorescent labeling method using the same have been awaited.

In order to solve the above problems, Ikenaka et al. Developed a method in which a substituted aminoalkyl group having fluorescence is bonded to the hydroxyl group at the 1-position of the reducing end of the sugar by a glycosidic bond (JP-A-1-42496). Gazette).

This method comprises a substituted aminoalkyl group having fluorescence at the 1-position hydroxyl group of the reducing end of the sugar under mild conditions and without opening the ring of the sugar residue on the reducing end side of the sugar. Is a method that can be introduced, which is a clear advantage compared with the conventional method.

However, even when the activity of a physiologically active substance is measured using the fluorescently labeled substrate prepared by this method, for example, in measuring the activity of a glycosyltransferase,
There is a case where an amino acid residue such as an asparagine residue, a serine residue, a threonine residue or the like or a peptide residue containing these bound to the reducing end of the sugar acceptor sugar chain influences the activity measurement (International Immunology, vol.2, 105-112,
(1989, etc.), there is a problem that the activity of a target physiologically active substance may not be sufficiently measured.

Further, since a glycopeptide in which an amino acid residue or a peptide residue is bound to a sugar chain is extremely hydrophilic and does not dissolve in a polar organic solvent, the conventional fluorescent labeling reagent can be used for the desired fluorescent labeling. There is also a problem that it is difficult to implement,
It has been desired to develop a carbohydrate that is fluorescently labeled and has an amino acid residue or a peptide residue.

[0014]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and for example, a fluorescently labeled sugar derivative useful as a substrate for measuring the activity of a physiologically active substance such as a sugar-modifying enzyme and a high-performance product using the same It is intended to provide a sensitive method for measuring a physiologically active substance.

[0015]

The present invention includes the following general formula (1) R1-A-NH-R2 (1) [wherein R1 is glucose, mannose or galactose].
Su, fucose, sialic acid, N-acetylglucosamine, N
2 to 2 sugar residues selected from acetylgalactosamine
It has 10 sugar chains and can be a substrate for sugar-modifying enzyme
It represents what forms a sugar chain structure that, A is aspartic
Residues, serine residues, threonine residues, or
Represents a peptide residue which is made, R2 is _{R3- (CH 2) m-NH} -CO- (CH 2) n-CO- (A) R3-CO- (CH 2) n-CO-NH- (CH _{2) m-CO- (B)} R3- (CH 2) m-CO-NH- (CH 2) n-CO- (C) ( wherein, R3 is 2-pyridylamino or 3 Pirijirua
Represents a mino group, m and n are each independently an integer of 1 to 4
Represents ) Represents a group represented by. ] The sugar derivative represented by the above formula and the sugar derivative represented by the above general formula (1) are used as substrates, and the invention is a method for measuring the activity of a sugar modifying enzyme .

That is, the inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and the sugar derivative represented by the above general formula (1) is used to measure the activity of a physiologically active substance such as a sugar-modifying enzyme. It has been found to be extremely useful as a substrate for use, and by using this as a substrate, the activity of a sugar-modifying enzyme or the like can be measured with high accuracy and high sensitivity, and the present invention has been completed.

R1 of the sugar derivative represented by the general formula (1) of the present invention has 2 to 2 sugar residues selected from glucose, mannose, galactose, fucose, sialic acid, N-acetylglucosamine and N-acetylgalactosamine. Have 10
Sugar chains . There is no particular limitation on the type of linkage between sugar residues of the sugar chain, and for example, α1 → 4, β1 → 4, α
1 → 6, α2 → 3, β1 → 2, β1 → 3, α2 → 6, α
It may be any of 1 → 3, α1 → 2, β1 → 6 and the like. However,
It goes without saying that R1 has a sugar chain structure that can serve as a substrate for a target physiologically active substance, for example, a sugar-modifying enzyme such as a glycosyltransferase, a sugar hydrolase, or a sugar isomerase.

Further, A of the sugar derivative represented by the general formula (1) of the present invention includes an asparagine residue, a serine residue, a threonine residue or a peptide residue composed of these. From the viewpoint of separation and analysis when measuring activity, amino acid residues are desirable. When A is also used as a substrate for a target physiologically active substance, for example, a sugar-modifying enzyme such as a glycosyltransferase, a sugar hydrolase, or a sugar isomerase, it has a structure capable of sufficiently performing the target activity measurement. Needless to say. For R2,
Following general formula (A), (B), or _{(C) R3- (CH 2)} m-NH-CO- (CH 2) n-CO- (A) R3-CO- (CH 2) n-CO- _{NH- (CH 2) m-CO-} (B) R3- (CH 2) m-CO-NH- (CH 2) n-CO- (C) ( wherein, R3 is 2-pyridylamino or 3 Pirijirua
Represents a mino group, m and n are each independently an integer of 1 to 4
Represents ) . Among them, a 2-pyridylaminoethylsuccinamoyl group, N- (2-pyridyl) -succinamoylaminopropionyl group, N- (2-
Pyridyl) -β-alanylaminopropionyl group and the like are preferable.

The sugar derivative represented by the general formula (1) of the present invention can be easily synthesized, for example, as follows. That is, 1 mol of the sugar derivative is added to a sugar derivative having an amino acid residue or a peptide residue represented by the general formula (2) R 1 -A-NH ₂ (2) (wherein R 1 and A are the same as above). With respect to usually 1 to 10 mol, preferably 1 to 5 mol, more preferably 2 to 4 mol, for example the general formula (3)
(4) or (5) R3 chromatography _{(CH 2) m-NH-} CO- (CH 2) n-COO-R4 (3) R3 over _{CO- (CH 2) n-CO} -NH- (CH 2) m -COO-R4 (4) R3- ( CH 2) m-CO-NH- (CH 2) n-COO-R4 (5) ( wherein, R3 represents a 2-pyridylamino group or 3-pyridylamino group, R4 Represents a group derived from an active esterifying agent, and m and n each independently represent an integer of 1 to 4). A group having reactivity with an amino group at the terminal (eg, succinimideoxycarbonyl group, phthalic acid imidooxycarbonyl group, 5-norbornene-2,3-dicarboximidooxycarbonyl group, p-nitrophenyloxycarbonyl group , Formyl group, tresylalkyl group, tosylalkyl group, etc.) and water or, for example, methano Alcohols such as toluene and ethanol, polar organic solvents such as acetone, acetonitrile and dimethylformamide, or mixed solvents of these polar organic solvents and water (hereinafter, these are collectively referred to as "water solvent and the like"). The reaction can be carried out in the medium and, if necessary, reduction treatment (when a compound having a formyl group at the terminal is used, etc.) is performed, and then purification can be performed by a conventional method to easily synthesize the compound.

In carrying out the above reaction, p of water solvent or the like is used.
H is not particularly limited as long as it does not affect the stability of the sugar derivative represented by the general formula (2), but in consideration of the reactivity of the compounds represented by the general formulas (3) to (5), for example. ,
A pH of about 6 to 10 (weakly acidic to weakly alkaline) is desirable.
Further, in the reaction solution, a sugar derivative represented by the general formula (2) and a compound represented by the general formula (3), (4), (5) or the like, a pyridylamino group at one end is represented. Does not have a primary amino group such as phosphate or bicarbonate as long as it does not interfere with the reaction of the compound with a compound having a group reactive with an amino group at the other end. A buffer, an antiseptic, a surfactant, a stabilizer and the like may coexist.

The sugar derivative represented by the general formula (2) can be easily obtained by linking a target amino acid residue or peptide residue to a sugar chain having a target structure synthesized by a standard method by a standard method. It can be prepared from natural glycoproteins by enzymatic treatment (eg, protease treatment, glycosidase treatment, etc.) or chemical treatment (eg, hydrolysis treatment with acid or alkali). It can also be obtained by further chemically and / or enzymatically modifying a sugar derivative having an amino acid residue or a peptide residue obtained by a chemical treatment to convert it into a sugar derivative having a desired structure. If it is desired to obtain a large amount of the compound represented by the general formula (2), a method of enzymatically or chemically treating a natural glycoprotein as a raw material is more specifically described, for example, immunoglobulin, ovalbumin, transferrin. , Thyroid stimulating hormone (TSH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), luteinizing hormone (LH)
Glycoproteins such as trypsin, chymotrypsin, pepsin, papain, V8 protease and the like, and β-galactosidase, neuraminidase, N-
Preference is given to treatment with a glycosidase such as acetyl-β-glucosaminidase, α-mannosidase I, α-mannosidase II, chemical treatment, and if necessary further chemical and / or enzymatic modification.

As the compounds represented by the general formulas (3), (4) and (5), those synthesized as follows may be used. First, the compound represented by the general formula (3) can be easily synthesized, for example, according to the following synthetic route.

(In the above reaction scheme, X represents a halogen atom, and m, n and R4 are the same as above.) That is, first, for example, 2-chloropyridine, 2-bromopyridine, 3-chloropyridine, 3-bromo. 2- or 3-halopyridine such as pyridine,
10 to 20 times mol of alkylenediamine (for example, ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, etc.) with respect to the 2- or 3-halopyridine at 80 to 120 ° C.
Allow to react for ~ 24 hours with stirring. After the reaction, the reaction solution is concentrated under reduced pressure, the target product is extracted with chloroform, dichloroethane or the like, the solvent is distilled off, and the residue is crystallized using a suitable solvent (e.g. benzene-petroleum ether mixed solvent). An aminoalkylaminopyridine represented by the above general formula (3a) is obtained. Then, the aminoalkylaminopyridine and 1 to 3 times the molar amount of a dicarboxylic acid anhydride (for example, succinic anhydride, glutaric anhydride, maleic anhydride, etc.)
Or a dicarboxylic acid (eg succinic acid, glutaric acid,
Intermolecular dehydration condensation product of maleic acid) {[HOOC- (CH
₂ ) n-CO] ₂ O} (wherein n is the same as above) in a solvent such as methanol, ethanol or isopropanol,
The reaction is carried out at 0-30 ° C for 2-6 hours with stirring. After the reaction, the precipitated crystals are collected by filtration and dried to obtain the compound represented by the above general formula (3b) (hereinafter abbreviated as compound (3b)). Then, the compound (3b) is added to a 1- to 3-fold molar amount of the active esterifying agent [for example, N-hydroxy-5-norbornene-2,3-dicarboximide (hereinafter abbreviated as HONB). , N-hydroxysuccinimide (hereinafter referred to as HOS
Abbreviated as u. Etc.] and, for example, in a solvent such as dimethylformamide (DMF), tetrahydrofuran (THF), dioxane, etc., for example, 1 to 3 moles of triphenylphosphine with respect to compound (3b), and a disulfide compound in an equimolar amount to this. [For example, 2,2'-dithiodipyridine (hereinafter,
It is abbreviated as 2-PDS. ), 2,2′-dithiobis (5-nitropyridine), etc.] at 0-40 ° C. for 2-8 hours with stirring. After the reaction, the reaction solution is concentrated under reduced pressure, and the obtained residue is crystallized using an appropriate solvent (eg, ethyl acetate-diethyl ether mixed solvent) to give the compound of the general formula (3)
A compound represented by is obtained. In the above reaction, instead of using a combination reagent of triphenylphosphine and a disulfide compound, for example, dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC Even if a condensation reagent such as) is used, the compound represented by the general formula (3) can be similarly obtained.

The compound represented by the general formula (4) can be easily synthesized according to the following synthetic route.

[Formula 2] (In the above reaction scheme, R represents an alkyl group, m,
n and R4 are the same as above. That is, first, for example, aminopyridine such as 2-aminopyridine and 3-aminopyridine, and 1 to 3 moles of dicarboxylic acid anhydride (for example, succinic anhydride, glutaric anhydride, maleic anhydride) relative to the aminopyridine. Acid or the like) or a dicarboxylic acid (eg, succinic acid, glutaric acid, maleic acid, etc.) intermolecular dehydration condensate {[HO
OC- (CH2) n-CO] 2O} (wherein n is the same as above) in a solvent such as ethanol, THF, toluene or benzene at 60 to 100 ° C. for 2 to 4 hours and then 10 React with stirring at -30 ° C for 2-4 hours. After the reaction, the precipitated crystals are collected by filtration and dried to obtain the compound represented by the general formula (4a) (hereinafter, abbreviated as compound (4a)).

Then, the obtained compound (4a) and 1 to 3 moles of the amino acid ester with respect to the compound (eg, glycine ethyl ester, β-alanine methyl ester, etc.)
And in a solvent such as DMF or dioxane, the compound (4
1 to 3 times the molar amount of triphenylphosphine with respect to a),
An equimolar disulfide compound (eg 2-PDS,
2,2'-dithiobis (5-nitropyridine) etc.)
React with stirring at -40 ° C for 2-8 hours. After the reaction, insoluble materials were removed by filtration, the solvent was distilled off, and the resulting residue was extracted with a solvent such as chloroform or dichloromethane. The extract was saturated with sodium bicarbonate water, an alkaline solution such as an aqueous solution of sodium carbonate, Then, it is washed successively with water. Thereafter, the extract is dried with, for example, anhydrous sodium sulfate, anhydrous magnesium sulfate, etc., and the solvent is distilled off to obtain a residue, which is then crystallized using a suitable solvent (eg, ethyl acetate-diethyl ether mixed solvent). From the above, the compound represented by the above general formula (4b) is obtained. When this is saponified by a conventional method and the ester is hydrolyzed, a compound represented by the general formula (4c) (hereinafter abbreviated as compound (4c)) is obtained. The above compound (4
The amino acid ester to be reacted with a) may be a mineral acid salt such as hydrochloride or sulfate, but in this case, it is usually an equimolar base (eg triethylamine, N-methylmorpholine etc.). Are used together. In addition, the dehydration condensation reaction between the compound (4a) and the amino acid ester is carried out by using, for example, DCC, WS instead of the method using a combined reagent of triphenylphosphine and a disulfide compound.
Needless to say, it may be carried out by a conventional method used in peptide synthesis such as a method using a condensation reagent such as C, a mixed acid anhydride method and an azide method.

Then, the compound (4c) thus obtained is treated with 1 to 3 moles of the active esterifying agent (eg, HO).
NB, HOSu, etc.) in a solvent such as DMF, THF, dioxane and the like, and 1 to 3 moles of triphenylphosphine with respect to the compound (4c), and a disulfide compound (eg, 2-PDS, 2 2,2'-dithiobis (5-nitropyridine) and the like) and reacted at 0-40 ° C for 2-8 hours with stirring. After the reaction, the reaction solution is concentrated under reduced pressure, and the obtained residue is crystallized using an appropriate solvent (eg, ethyl acetate-diethyl ether mixed solvent) to give a compound of the general formula
The compound represented by (4) is obtained. In the above reaction, the compound represented by the general formula (4) can be similarly obtained by using a condensing reagent such as DCC or WSC instead of the combined reagent of triphenylphosphine and disulfide compound.

Further, among the compounds represented by the general formula (5),
The compound in which m is 2 can be easily synthesized in good yield, for example, according to the following synthetic route.

[Formula 3] (In the above reaction scheme, R ′ represents an alkyl group, and n
And R4 are the same as above. ) That is, for example, aminopyridine such as 2-aminopyridine and 3-aminopyridine, and unsaturated fatty acid ester (for example, methyl acrylate, ethyl acrylate, etc.) in 1 to 3 moles relative to the aminopyridine, The reaction is carried out in the presence of a polymerization inhibitor such as 2,5-di-tert-butylhydroquinone at 60 to 100 ° C for 12 to 36 hours with stirring. After the reaction, the reaction solution is poured into a solvent such as benzene or toluene, the precipitated crystals are filtered off, and the solvent is distilled off to obtain a residue, which is then mixed with a suitable solvent (such as benzene-
The compound represented by the above general formula (5a) is obtained by recrystallization from a hexane mixed solvent). Then,
If the ester is hydrolyzed by saponification by a conventional method, the general formula
A compound represented by (5b) (hereinafter abbreviated as compound (5b)) is obtained.

The compound (5b) thus obtained and 1 to 3 moles of the amino acid ester (eg, glycine ethyl ester, β-alanine methyl ester, etc.) relative to the compound are mixed in a solvent such as DMF or dioxane. For example, the reaction is carried out at 0 to 30 ° C. for 12 to 36 hours with stirring in the presence of 1 to 3 times the molar amount of a condensation reagent such as DCC or WSC. After the reaction, insoluble matter was removed by filtration, the residue obtained by concentrating the filtrate under reduced pressure was extracted with a solvent such as chloroform or dichloromethane, and the extract was extracted with saturated sodium bicarbonate water, an alkaline solution such as an aqueous solution of sodium carbonate, and then extracted. Wash sequentially with water. The extract is dried with, for example, anhydrous sodium sulfate, anhydrous magnesium sulfate, etc., the solvent is distilled off, and the obtained residue is crystallized using a suitable solvent (eg ethyl acetate-diethyl ether mixed solvent). According to the general formula (5c)
A compound represented by is obtained. Next, this is saponified by an ordinary method to hydrolyze the ester to obtain a compound represented by the general formula (5d) (hereinafter, abbreviated as compound (5d)). The amino acid ester to be reacted with the compound (5b) may be a mineral acid salt such as a hydrochloride or a sulfate, but in that case, it is usually an equimolar base (eg triethylamine, N -Methylmorpholine, etc.) is also used. Further, the dehydration condensation reaction between the compound (5b) and the amino acid ester is carried out by a method using a condensation reagent such as DCC, WSC, a mixed acid anhydride method instead of a method using a combination reagent of triphenylphosphine and a disulfide compound. Needless to say, it may be carried out by a conventional method used in peptide synthesis such as the azide method.

Then, the compound (5d) thus obtained is treated with 1 to 3 moles of the active esterifying agent (for example, HO).
NB, HOSu, etc.) in a solvent such as DMF, THF, dioxane and the like, and 1 to 3 times the molar amount of triphenylphosphine to the compound (5d) and an equimolar disulfide compound (eg 2-PDS, 2 2,2'-dithiobis (5-nitropyridine) and the like) and reacted at 0-40 ° C for 2-8 hours with stirring. After the reaction, the reaction solution is concentrated under reduced pressure, and the obtained residue is purified by silica gel column chromatography or the like to give a compound represented by the general formula (5e) (of the compounds represented by the general formula (5), m = 2). A compound) is obtained.
In the above reaction, the compound represented by the general formula (5e) can also be easily obtained by using a condensing reagent such as DCC or WSC instead of using a combination reagent of triphenylphosphine and a disulfide compound. Can be obtained.

As a general method for producing the compound represented by the general formula (5), for example, the following synthetic route can be mentioned.

[Formula 4] (In the above reaction scheme, R'represents an alkyl group, X
Represents a halogen atom, and m, n and R4 are the same as defined above. ) That is, for example, aminopyridine such as 2-aminopyridine and 3-aminopyridine, and 1 for the aminopyridine.
~ 3 times mol of monohalocarboxylic acid ester (eg, ethyl monobromoacetate, methyl 4-chlorobutyrate, ethyl 5-chlorovalerate, etc.) in a solvent such as DMF, chloroform, benzene, etc. Sodium chloride, N, N′-diisopropylethylamine, triethylamine, etc.) at 0-40 ° C. for 3-8 hours with stirring. After completion of the reaction, the reaction solution is concentrated under reduced pressure, and the desired product is extracted from the obtained residue using a solvent such as chloroform or dichloromethane. The extract obtained is, for example, 0.
It is washed successively with an acid solution such as 1 to 2 N hydrochloric acid and a 10% citric acid aqueous solution, for example, saturated sodium bicarbonate water, an alkaline solution such as a sodium carbonate aqueous solution, and water. Then, for example, dried over anhydrous sodium sulfate, anhydrous magnesium sulfate, etc., the solvent is distilled off,
The compound represented by the above general formula (5f) is obtained by purifying the obtained residue by silica gel chromatography or the like. Next, this is saponified by a conventional method to hydrolyze the ester to obtain a compound represented by the general formula (5g) (hereinafter, abbreviated as compound (5g)).

The compound (5 g) thus obtained and 1 to 3 moles of the amino acid ester (eg, glycine ethyl ester, β-alanine methyl ester, etc.) with respect to the compound are dissolved in a solvent such as DMF or dioxane. For example, the reaction is carried out at 0 to 30 ° C. for 12 to 36 hours with stirring in the presence of 1 to 3 times the molar amount of a condensation reagent such as DCC or WSC. After the reaction, insoluble matter was removed by filtration, the residue obtained by concentrating the filtrate under reduced pressure was extracted with a solvent such as chloroform or dichloromethane, and the extract was extracted with saturated sodium bicarbonate water, an alkaline solution such as an aqueous solution of sodium carbonate, and then extracted. Wash sequentially with water. The extract is dried with, for example, anhydrous sodium sulfate, anhydrous magnesium sulfate, etc., the solvent is distilled off, and the obtained residue is crystallized using a suitable solvent (eg ethyl acetate-diethyl ether mixed solvent). According to the general formula (5h)
A compound represented by is obtained. Next, this is saponified by an ordinary method to hydrolyze the ester to obtain a compound represented by the general formula (5i) (hereinafter abbreviated as compound (5i)). The amino acid ester to be reacted with the compound (5 g) may be a mineral acid salt such as a hydrochloride or a sulfate, but in that case, it is usually an equimolar base (eg triethylamine, N -Methylmorpholine, etc.) is also used. In addition, the dehydration condensation reaction between the compound (5 g) and the amino acid ester is carried out by a method using a condensation reagent such as DCC, WSC, a mixed acid anhydride method instead of a method using a combination reagent of triphenylphosphine and a disulfide compound. Needless to say, it may be carried out by a conventional method used in peptide synthesis such as the azide method.

Then, the obtained compound (5i) is treated with 1 to 3 moles of the active esterifying agent (for example, HO).
NB, HOSu, etc.) in a solvent such as DMF, THF, dioxane and the like, and 1 to 3 moles of triphenylphosphine with respect to the compound (5i) and a disulfide compound (eg, 2-PDS, 2 2,2'-dithiobis (5-nitropyridine) and the like) and reacted at 0-40 ° C for 2-8 hours with stirring. After the reaction, the reaction solution is concentrated under reduced pressure, and the obtained residue is purified by silica gel column chromatography or the like to obtain the compound represented by the general formula (5). In the above reaction, instead of using a combination reagent of triphenylphosphine and a disulfide compound, for example, DC
Even if a condensation reagent such as C or WSC is used, the same formula (5)
The compound represented by is easily obtained.

The group derived from the active esterifying agent of R4 in the compounds represented by the above general formulas (3) to (5) is a group derived from the carboxyl group activating agent.
It may be any as long as it has reactivity with a primary amino group, and specifically, for example, a succinimide group, a phthalic acid imide group, a 5-norbornene-2,3-dicarboximide group, p -Nitrophenyl group and the like are preferable.

The physiologically active substance that can be measured by using the sugar derivative of the present invention is not particularly limited as long as it is a physiologically active substance using the sugar derivative of the present invention as a substrate. For example, fucose (α1 → 2) transferase, fucose (α1 → 3) transferase, fucose (α
1 → 4) transferase, fucose (α1 → 6) transferase, sialic acid (α2 → 3) transferase, sialic acid (α2 → 6) transferase, galactose (β1 → 3) transferase, galactose (β1 → 4) ) Transferase, galactose (α1 → 3)
Transferase, N-acetylglucosamine (β1 → 2) transferase, N-acetylglucosamine (β1 → 4) transferase, N-acetylglucosamine (β1 → 6) transferase,
Mannose (α1 → 2) transferase, mannose (α1 →
3) transferase, mannose (α1 → 6) transferase, mannose (β1 → 4) transferase, xylose (β1 → 2)
Glycosyltransferases such as transferases, such as sialidase, β (or α) -galactosidase, β-N-acetylglucosaminidase, α (or β) -mannosidase, α-fucosidase, α (or β) -N-acetylgalactosaminidase Preferable examples thereof include sugar hydrolases such as xylosidase (both endo-type and exo-type), and sugar-modifying enzymes such as sugar isomerases such as glucose isomerase.

The origin of physiologically active substances such as sugar-modifying enzymes is not limited, and examples thereof include serum, plasma, urine of animals including humans,
Examples thereof include those derived from body fluids such as cerebrospinal fluid, biological tissues, animal (or plant) cell extracts, cell culture fluids, and the like, which are thought to have physiologically active substances.

The method for measuring the activity of a physiologically active substance of the present invention is the same as that of the literature (for example, J. Biol. Chem., Vol. 246, 5154-5161, 1971, Biochem. Bio, except that the sugar derivative of the present invention is used as a substrate.
phys.Res.Commun., vol.136, 563-569, 1986) and the like, which are used in a conventional method known per se, and this can be carried out in accordance with an operation in a conventional method known per se.

The method for measuring the activity of the present invention will be described more specifically by taking the case of measuring the activity of a glycosyltransferase as an example. That is, first, for example, from sugar nucleotides such as cytidine monophosphate neuraminic acid, uridine diphosphate galactose, uridine diphosphate glucose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose A sugar reagent as an appropriately selected sugar nucleotide and a reagent solution for measurement containing a sugar derivative of the present invention prepared according to the specificity of the glycosyltransferase to be measured as a sugar acceptor are prepared, and the reagent solution And a sample containing the glycosyltransferase to be measured are mixed to start the reaction, and after a certain period of time, the amount of the enzyme reaction product (or the amount of the remaining sugar acceptor) is measured by the fluorescence method. By easily applying the obtained measured values to a calibration curve showing the relationship between the amount of enzyme to be measured and the amount of enzyme reaction product (or the amount of residual sugar acceptor), which was created in advance by performing the same operation method, It can be carried out. In the above measuring method, the concentration of the sugar nucleotide as the sugar donor or the sugar derivative of the present invention as the sugar acceptor is not particularly limited as long as it is a concentration at which the intended activity measurement can be carried out. The concentration of the enzyme is usually about 0.1 to 20 mM during the enzyme reaction.
In addition, the concentration of the sugar derivative of the present invention is appropriately selected from the range of usually about 1 to 1,000 μM as the concentration during the enzyme reaction.

The reaction temperature for carrying out the measuring method of the present invention is not particularly limited, but it may be appropriately selected from the range of about 25 to 40 ° C., and the reaction time may be appropriately selected depending on the purpose. Not limited. The pH of the reaction varies slightly depending on the properties of the target physiologically active substance, but is usually about 4 to
9, preferably selected from the range of 6 to 8. Further, the buffer used for the purpose of maintaining the pH can be appropriately selected from those which do not interfere with the intended enzymatic reaction, and for example, trishydroxymethylaminomethane-
Preferable examples include hydrochloric acid, cacodylic acid-sodium hydroxide, Good's buffer and the like.

The method for separating and quantifying the reaction product generated by the action of the physiologically active substance is not particularly limited, but gel permeation by high performance liquid chromatography (HPLC) has advantages such as easy operation and short measurement time. Method, reverse phase method, ion exchange method, affinity chromatography method and the like are preferably mentioned.

As an example of HPLC conditions, there is a method in which reversed-phase chromatography uses a chemically bonded silica gel having an octadecylsilane group or an amide group introduced as a packing material. As the eluent in the method, an appropriate buffer may be appropriately selected and used according to the purpose, and for example, 1-butanol, acetonitrile or methanol can be used in an amount of 0.
0.1 to 80% 0.1M ammonium acetate buffer (pH3.0
~ 8.0) and the like, and the flow rate is appropriately selected within the range of 0.1 to 3.0 ml / min. According to the diameter of the separation column used, but is not particularly limited thereto. Further, the detection is naturally performed by the fluorescence method. When the product has a charge, such as when measuring the activity of a sialic acid-related modifying enzyme, the HPLC method using an ion exchange column or the separation / quantification method by electrophoresis is also effective.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

【Example】

Reference Example 1. Synthesis of 2-pyridylaminoethylsuccinamic acid 5-norbornene-2,3-dicarboximide ester (PAE-Suc-ONB) 1) 2- (2-aminoethyl) aminopyridine (PAEA) ) Of 2-chloropyridine 85g and ethylenediamine 450g, 120
The mixture was reacted under reflux at 8 ° C for 8 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the target product was extracted with chloroform. Chloroform was distilled off from the extract, and the residue was recrystallized with benzene-petroleum ether (1: 5) to obtain 86 g of PAEA (yield 83.7%). Melting point: 36-40 ° C. IRνmax (neat) cm ^-1 : 3500-3100 (NH), 3000-2800 (-C
H ₂ -), 780 (pyridine ring). ¹ H-NMR (60 MHz, CDCl ₃ ) δppm: 1.64 (bs, 2H, N
_{H 2), 2.82~3.58 (m,} 4H, -CH 2 CH 2 -), 6.40~8.24 (m, 4H, pyridine ring). MS: m / z 137 (M ⁺ ). 2) 2-pyridylaminoethylsuccinamic acid (PAE-S
Synthesis of uc-OH) 10 g of PAEA obtained in 1) above and 7.3 g of succinic anhydride were dissolved in 300 ml of ethanol and reacted at room temperature with stirring for 2 hours. After the reaction was completed, the precipitated crystals were collected by filtration, dried and PAE.
13.6 g of -Suc-OH was obtained (yield 78.6%). Melting point: 128-130 ° C. IR (nu) max (KBr) cm < ^-1 >: 3400-3200 (NH), 1700 (carboxylic acid), 1660,1560 (amide), 780 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.32 (t, 2H, Suc
-CH _2- , J = 6.48Hz), 2.34 (t, 2H, Suc-CH _2- , J = 6.48Hz), 3.2
0~3.30 (m, 4H, AEA- CH 2 -CH 2 -), 6.43~7.95 (m, 4H, pyridine ring). MS: m / z 237 (M ⁺ ). 3) 2-Pyridylaminoethyl succinamic acid 5-norbornene-2,3-dicarboximide ester (PAE-Su
Synthesis of c-ONB) 2.37 g of PAE-Suc-OH obtained in 2) above and N-hydroxy-5-norbornene-2,3-dicarboximide (HON
B) 1.8 g was dissolved in DMF 50 ml, and DMF solution 20 ml containing 2,2′-dithiodipyridine (2-PDS) 2.2 g and triphenylphosphine 2.6 g was added thereto, and then the mixture was stirred at room temperature for 4 hours. The reaction was allowed to stir for hours. After completion of the reaction, DMF was distilled off under reduced pressure and the obtained residue was crystallized with ethyl acetate-ether to give PAE-Suc-ONB2.
5 g was obtained (yield 62.5%).

[Chemical 1] Melting point: 122 ° C- (dec.). IRνmax (KBr) cm ^-1 : 3300-3100 (NH), 3100-2800 (-CH
_2- ), 1780 (ester), 1660, 1600 (amide), 780 (pyridine ring). ¹ H-NMR (60 MHz, DMSO-d6) δppm: 1.60 (m, 2H, N
B), 2.50 ~ 2.88 (m, 4H, AEA-CH ₂ CH _2- ), 3.33 ~ 3.60 (m, 8H,
Suc-CH ₂ CH ₂ -and NB), 6.23 (s, 2H, NB), 6.50 ~ 8.16 (m, 4
H, pyridine ring). MS: m / z 398 (M ⁺ ).

Reference Example 2. N- (2-pyridyl) succinamoylaminopropionic acid succinimide ester (Py-
Synthesis of Suc-Ala-OSu) 1) Synthesis of 2-pyridylsuccinamic acid (Py-Suc-OH) 131 g of 2-aminopyridine and 139 g of succinic anhydride were suspended in 2 l of toluene and then 90 ° C. The mixture was reacted for 1 hour at room temperature and then for 1 hour at room temperature with stirring. After the reaction was completed, the precipitated crystals were collected by filtration,
It was dried to obtain 195 g of Py-Suc-OH (yield 72.2
%). Melting point: 171-172 ° C. IR ν max (KBr) cm ⁻¹ : 3300 to 3000 (NH), 1700 (carboxylic acid), 1620,1580 (amide), 780 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 1.84 (dd, 2H, -C
H _2- , J = 6.21), 1.98 (dd, 2H, -CH _2- , J = 6.21), 6.35 ~ 7.62
(m, 4H, pyridine ring), 9.72 (s, 1H, NH), 11.36 (s, 1H, carboxylic acid). MS: m / z 194 (M ⁺ ). 2) N- (2-pyridyl) succinamoylaminopropionic acid
Synthesis of methyl ester (Py-Suc-Ala-OMe) 9.7 g of Py-Suc-OH obtained in 1) above and β-alanine
Dissolve 7.0 g of methyl ester hydrochloride in 250 ml of DMF,
After adding 7.0 ml of triethylamine to this, 2-PDS11.
DMF solution containing 0g and 13.1g triphenylphosphine 100
ml was added, and the mixture was reacted at room temperature for 6 hours with stirring. After completion of the reaction, the reaction solution was concentrated under reduced pressure, the obtained residue was extracted with chloroform, and the extract was washed successively with saturated aqueous sodium bicarbonate solution and saturated brine. The chloroform layer was dried over anhydrous sodium sulfate, chloroform was distilled off, and the obtained residue was recrystallized from ethyl acetate-ether to obtain Py-Suc-Ala-OM.
e11.7g was obtained (yield 84.0%). Melting point: 135-139 ° C. IRνmax (KBr) cm ^-1 : 3400-3100 (NH), 3100-2900 (-CH
_2- ), 1740 (ester), 1660,1580 (amide), 780 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.36 to 2.51 (m,
4H, Suc-CH ₂ CH _2- , J = 7.02), 2.61 (t, 2H, Ala-CH ₂ CO, J = 6.2
1), 3.26 (t, 2H, AlaN-CH _2- , J = 6.21), 3.60 (s, 3H, CH ₃ ),
7.02 ~ 8.28 (m, 5H, pyridine ring and NH), 10.41 (s, 1H, N
H). MS: m / z 279 (M ⁺ ). 3) Synthesis of N- (2-pyridyl) succinamoylaminopropionic acid (Py-Suc-Ala-OH) 15.4 g of Py-Suc-Ala-OMe obtained in 2) above and 2.5 g of sodium hydroxide, It was dissolved in a mixed solvent of 200 ml of water and 100 ml of methanol and reacted at room temperature for 8 hours with stirring.
After the reaction was completed, it was neutralized with 62 ml of 1N-hydrochloric acid and concentrated under reduced pressure until the liquid volume became 100 ml. The precipitated crystals were collected by filtration, washed successively with water and acetone and then dried to give Py-Suc-Ala-.
12.1 g of OH was obtained (yield 82.5%). Melting point: 186-189 ° C. IRνmax (KBr) cm ^-1 : 3500-3200 (NH), 3100-2900 (-CH
_2- ), 1700 (carboxylic acid), 1680 to 1550 (amide), 800 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.35 to 2.42 (m,
4H, Suc-CH ₂ CH-, J = 7.02), 2.61 (t, 2H, Ala-CH ₂ CO, J = 6.4
8), 3.24 (dd, 2H, AlaN-CH ₂ -J = 6.48), 7.03 ~ 8.08 (m, 5H,
Pyridine ring and NH), 10.42 (s, 1H, NH). MS: m / z 265 (M ⁺ ). 4) N- (2-pyridyl) succinamoylaminopropionic acid
Succinimide ester (Py-Suc-Ala-OS
Synthesis of u) Py-Suc-Ala-OH (2.7 g) obtained in the above 3) and N-hydroxysuccinimide (1.2 g) were dissolved in DMF (50 ml), and 2-PDS (2.2 g) and triphenylphosphine (2.
After adding 20 ml of a DMF solution containing 6 g, the mixture was reacted at room temperature for 4 hours with stirring. After the reaction was completed, the reaction solution was concentrated under reduced pressure,
The obtained residue was crystallized with ethyl acetate-ether to give 3.4 g of Py-Suc-Ala-OSu represented by the following chemical formula.
Was obtained (yield 93.1%).

[Chemical 2] Melting point: 172-176 ° C. IRνmax (KBr) cm ^-1 : 3400-3200 (NH), 3100-2900 (-CH
_2- ), 1790 (ester), 1660, 1580 (amide), 790 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.42 to 2.59 (m,
4H, Suc-CH ₂ CH _2- , J = 7.02), 2.83 (s, 4H, Su), 2.83 to 2.97
(m, 2H, Ala-CH ₂ CO), 3.24 to 3.42 (dd, 2H, AlaN-CH _2- , J = 6.2
1), 7.00 to 8.45 (m, 4H, pyridine ring), 10.47 (b, 1H, NH). MS: m / z 362 (M ⁺ ).

Reference Example 3. Synthesis of N- (2-pyridyl) -β-alanylaminopropionic acid 5-norbornene-2,3-dicarboximide ester (Py-Ala-Ala-ONB) 1) N- (2-pyridyl) -β-alanine Methyl ester (Py
-Ala-OMe) Synthesis of 2-aminopyridine (94 g) with methyl acrylate (100 ml) and 2,5-diamine
1.1 g of -tert-butylhydroquinone was added, and the mixture was refluxed for 24 hours with stirring. After completion of the reaction, the reaction solution was poured into 1 liter of benzene and left at 0 ° C. for 5 hours. The precipitated crystals were filtered off, the filtrate was concentrated, and the resulting residue was benzene-hexane (1:
Crystallization was performed in 5) to obtain 64.5 g of Py-Ala-OMe (yield 35.8%). Melting point: 48-49 ° C. IRνmax (KBr) cm ^-1 : 3300-3200 (NH), 3100-2800 (-CH
_2- ), 1760 (ester), 1610,1580 (amide), 780 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.57 (t, 2H, -CH
₂ CO-, J = 7.02), 3.48 (dd, 2H, NCH _2- , J = 7.02), 3.60 (s, 3H,
_{CH 3), 6.44~7.98 (m,} 4H, pyridine ring). MS: m / z 180 (M ⁺ ). 2) N- (2-pyridyl) -β-alanine (Py-Ala-OH)
Synthesis of Py-Ala-0Me (62.1 g) obtained in 1) above was suspended in 620 ml of water and refluxed for 24 hours while stirring. After completion of the reaction, water was distilled off under reduced pressure, and the obtained residue was recrystallized with ethanol to obtain 43.1 g of Py-Ala-OH (yield 75.2%). Melting point: 136-138 ° C. IRνmax (KBr) cm ^-1 : 3300 ~ 3000 (NH), 3000 ~ 2800 (-CH
_2- ), 1700 (carboxylic acid), 1640,1550 (amide), 760 (pyridine ring). ¹ H-NMR (270 MHz, DMSO-d6) δppm: 2.50 (t, 2H, -CH ₂
CO, J = 6.75), 3.45 (m, 2H, NCH _2- , J = 6.75), 6.44 ~ 7.98 (m,
4H, pyridine ring). MS: m / z 166 (M ⁺ ). 3) Synthesis of N- (2-pyridyl) -β-alanylaminopropionic acid methyl ester (Py-Ala-Ala-OMe) In 60 ml of DMF, 3.3 g of Py-Ala-OH obtained in the above 2) was added.
And β-alanine methyl ester hydrochloride 2.8g and DCC
4.1 g was added, and triethylamine (2.8 ml) was further added thereto, and the mixture was reacted at room temperature for 24 hours with stirring. After completion of the reaction, the insoluble matter was filtered off, the filtrate was concentrated under reduced pressure, the resulting residue was extracted with chloroform, and the extract was washed successively with saturated sodium bicarbonate water and saturated saline. The chloroform layer was dried over anhydrous sodium sulfate, chloroform was distilled off, and the obtained residue was recrystallized from ethyl acetate-ether to obtain Py-Ala-Ala.
-OMe 4.3g was obtained (yield 86.5%). Melting point: 68-70 ° C. IRνmax (KBr) cm ^-1 : 3400-3200 (NH), 3100-2800 (-CH
_2- ), 1730 (ester), 1650, 1600 (amide), 780 (pyridine ring). ¹ H-NMR (60 MHz, DMSO-d6) δppm: 2.35 to 2.69 (m, 4
H, 2 × Ala-CH ₂ CO), 3.30 to 3.57 (m, 4H, 2 × AlaN-CH _2- ), 3.
59 (s, 3H, CH ₃ ), 6.33-8.02 (m, 6H, pyridine ring and 2 × N
H). MS: m / z 251 (M ⁺ ). 4) N- (2-pyridyl) -β-alanylaminopropionic acid (P
y-Ala-Ala-OH) 16.0 g of Py-Ala-Ala-OMe obtained in 3) above and 2.6 g of sodium hydroxide were dissolved in a mixed solvent of 200 ml of water and 100 ml of methanol, and then room temperature. And reacted for 24 hours under stirring. After completion of the reaction, the mixture was neutralized with 1N-hydrochloric acid 64 ml and concentrated under reduced pressure. The obtained residue was recrystallized from water-ethanol (1: 5) to obtain 14.7 g of Py-Ala-Ala-OH (yield 9
7.4%). Melting point: 146 to 149 ° C IRνmax (KBr) cm ^-1 : 3300 (NH), 3000 to 2800 (-CH _2- ), 1
680 (carboxylic acid), 1630,1550 (amide), 760 (pyridine ring). ¹ H-NMR (60 MHz, DMSO-d6) δppm: 2.20 to 2.59 (m, 4
H, 2 × Ala-CH ₂ CO), 3.09 to 3.64 (m, 4H, 2 × AlaN-CH _2- ), 6.
38-8.09 (pyridine ring and 2xNH). MS: m / z 237 (M ⁺ ). 5) N- (2-pyridyl) -β-alanylaminopropionic acid 5-
Norbornene-2,3-dicarboximide ester (Py-
Synthesis of Ala-Ala-0NB) 3.6 g of Py-Ala-Ala-OH obtained in 4) above and HO
2.7 g of NB was dissolved in 75 ml of DMF, and then 30 ml of a DMF solution containing 3.3 g of 2-PDS and 3.9 g of triphenylphosphine was added thereto, and the mixture was reacted at room temperature for 6 hours with stirring. After completion of the reaction, the reaction solution was concentrated under reduced pressure, the obtained residue was purified by silica gel chromatography (eluent; chloroform-acetone = 1: 1 mixed solvent), and Py-Ala-Ala-ONB2 represented by the following chemical formula: 0.4 g was obtained as an oily substance (yield 40.5%).

[Chemical 3] IRνmax (neat) cm ^-1 : 3600-3200 (NH), 3100-2900 (-C
H ₂ -), 1780 (ester), 1700,1580 (amide), 760 (pyridine ring). ¹ H-NMR (60 MHz, DMSO-d6) δppm: 1.29 (s, 2H, N
B), 2.41 to 2.85 (m, 4H, 2 x Ala-CH ₂ CO), 3.09 to 3.68 (m, 8
H, 2 × AlaNCH ₂ -and NB), 6.15 (s, 2H, NB), 6.44 ~ 7.97
(m, 6H, pyridine ring and 2xNH). MS: m / z 398 (M ⁺ ).

Example 1. Synthesis of fluorescent labeled sugar derivative (1) Preparation of glycopeptide from human serum-derived transferrin 1 g of human serum transferrin (manufactured by Sigma) was dissolved in 40 ml of 50 mM Tris-hydrochloric acid (pH 8.0, containing 1 mM calcium chloride), 20 mg of pronase (Boehringer Mannheim) was added and reacted at 30 ° C. for 24 hours. After 24 hours, 10 mg of pronase was added, and the mixture was further reacted at 30 ° C. for 24 hours. After completion of the reaction, the reaction solution was purified by chromatography (eluent: 0.1N-acetic acid) using a Toyopearl 40S column (Tosoh Corporation, 2.5 × 140 cm.). Of the collected fractions, orange-colored fractions were collected by the phenol-sulfuric acid method and freeze-dried to obtain glycopeptide standards. 2 mg of the glycopeptide preparation obtained above was dissolved in an appropriate amount of 6N-hydrochloric acid and hydrolyzed at 110 ° C. for 24 hours, and the resulting amino acid was analyzed by the PTC amino acid analysis method. As a result, peaks of asparagine, lysine and glucosamine were detected in this glycopeptide preparation. This result and the results of comparing a primary structure of human serum transferrin, the structure of the glycopeptide Asn ⁴¹³ - was estimated (sugar) -Lys. (2) Fluorescent labeling of glycopeptide 10 mg of the glycopeptide obtained in (1) above was dissolved in 2 ml of 0.1 M sodium hydrogen carbonate solution, and 250 mg of PAE-Suc-ONB obtained in Reference Example 1 was added. The reaction was carried out at 0 ° C for 3 hours. After completion of the reaction, the reaction solution was fractionated and purified by reverse phase column chromatography using a Wakopak 5C18 column (4.6 × 150 mm, manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a target fluorescently labeled glycopeptide. . The measurement conditions for column chromatography are as follows. -Device: SHIMADZU LC-6A (manufactured by Shimadzu Corporation). Eluent A: 0.1M ammonium acetate buffer (pH 4.
0).・ Eluent B: Eluent containing 12% acetonitrile A ・ Gradient condition: A → B 0 to 10 minutes B 10 to 12 minutes ・ Flow rate: 1.5 ml / min ・ Column temperature: 55 ° C ・ Detection: Excitation wavelength: 320 nm And measurement wavelength: 400 nm for fluorescence detection. (3) Modification of sugar chain by enzymatic digestion a) Digestion of neuraminidase 10 μg of the fluorescently labeled glycopeptide obtained in (2) above was added to 100 μl of 0.1 M phosphate buffer (pH 6.0, containing 20 mM of γ-galactolactone). To the dissolved product, 0.1 international unit of neuraminidase (derived from Streptococcus, manufactured by Toyobo Co., Ltd.) was added and reacted at 37 ° C. for 18 hours. After the reaction, Wako
Fractionation and purification were carried out by reverse phase column chromatography using a pak 5C18 column (4.6 × 150 mm, manufactured by Wako Pure Chemical Industries, Ltd.) to obtain the target fluorescently labeled glycopeptide. The measurement conditions for column chromatography are the same as in (2) above. b) β-galactosidase digestion The neuraminidase-digested fluorescent-labeled glycopeptide obtained in the above a) was dissolved in 100 μl of 0.1 M acetate buffer (pH 3.5), and β-galactosidase (nama bean-derived, Seikagaku Corp. )) 0.1 international unit was added, and the reaction was carried out at 37 ° C for 18 hours. After the reaction was completed, Wakopak 5C18 column (4.6 x 150
mm, manufactured by Wako Pure Chemical Industries, Ltd.) was used for fractionation and purification by reverse phase column chromatography to obtain a target fluorescently labeled sugar derivative. The measurement conditions for column chromatography are the same as in (2) above. From the glycopeptide structure confirmed in (1) above, the sugar chain structure in the fluorescence-labeled glycopeptide obtained by digestion of these two types of exoglycosidase is considered to be as shown in FIG. Further, the results of mass spectrometry of the fluorescently labeled sugar derivative (recrystallized from ammonium acetate buffer) are as follows. MS: m / Z 1794.7 (M + NH 4). * _{Calculated: C 71 H 115 N 11 O} 41 = 1777. In the reaction of (2) above, PAE-Suc-ONB
In place of Py-Suc-Ala- obtained in Reference Example 2.
OSu or Py-Ala-Ala- obtained in Reference Example 3
ONB was used as a fluorescent labeling reagent, otherwise PA
Reaction and post-treatment were carried out in exactly the same manner as in the case of E-Suc-ONB, and a fluorescence-labeled glycopeptide having the same glycopeptide structure as obtained above was obtained.

Example 2. α1 → 6 fucosyltransferase (F
T16) Activity measurement (FT16 enzyme source) Serum from hepatocellular carcinoma patients was used. (Sugar donor solution) Guanosine diphosphate-β-fucose (Sumitomo Seika, molecular weight
A solution of 589.3) dissolved in water to 1 mM was used as a sugar donor solution. (Sugar acceptor solution) The fluorescence-labeled sugar derivative obtained in Example 1 (hereinafter referred to as FT16A)
Is abbreviated. Was dissolved in 50 mM phosphate buffer (pH 7.0, containing 150 mM sodium chloride and 0.2% bovine serum albumin) to give a sugar receptor solution. (Reaction buffer) Adenosine 5'-trisodium triphosphate trihydrate (manufactured by Oriental Yeast Co., Ltd.) 605 mg, sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.) 125 mg, magnesium chloride hexahydrate (sum Koujunyaku Kogyo Co., Ltd.) 1-g and 3- (N-morpholino) propanesulfonic acid (MOPS) 2g were dissolved in water to adjust the pH to 7.5, and then the final volume was 100 ml. And (HPLC conditions) -Device: SHIMADZU LC-9A (manufactured by Shimadzu Corporation). -Column: TSKgel Amide 80 (4.6 x 250 mm, manufactured by Tosoh Corporation). Eluent A: 0.1 M ammonium acetate buffer (pH 4.0) containing 60% acetonitrile. Eluent B: 0.1 M ammonium acetate buffer (pH 4.0) containing 54% acetonitrile. -Gradient: A-> B 0-10 minutes. B 10-12 minutes. -Flow rate: 1 ml / min. -Column temperature: 45 ° C. -Detection: It was performed by fluorescence detection with an excitation wavelength of 320 nm and a measurement wavelength of 400 nm. (Procedure) 5 μl of the sample having the composition shown in Table 1 below was added to 2 μl of the sugar donor solution, 5 μl of the sugar acceptor solution, and 8 μl of the reaction buffer, and 37
The reaction was carried out at 16 ° C for 16 hours. After completion of the reaction, eluent A is added to the reaction solution.
200 μl was added and stirred well, and 100 μl was analyzed by HPLC.

[Table 1] (Results) As a result of analysis by HPLC, unreacted FT16A was eluted after 10.9 minutes, and fucose was converted into FT16A by the action of FT16.
It was found that the product added to the N-acetylglucosamine residue at the reducing end of was eluted after 11.3 minutes. A calibration curve showing the relationship between the peak height of the product and the amount of FT16 enzyme source solution added is shown in FIG. As is clear from FIG. 2, the product is produced in proportion to the amount of FT16 enzyme source solution, that is, in proportion to the amount of FT16 enzyme. In addition, it has been confirmed that a substance in which fucose is added to a sugar residue other than the reducing terminal N-acetylglucosamine residue of FT16A elutes faster than FT16A under the analysis conditions of this time of HPLC. It is understood that the FT16 activity can be measured by the measuring method. In this Example, instead of using FT16 as a substrate, instead of PAE-Suc-ONB in the reaction of (2) of Example 1 above, the Py-Suc-obtained in Reference Example 2 was used. When Ala-OSu or Py-Ala-Ala-ONB obtained in Reference Example 3 was used as a fluorescent labeling reagent and a fluorescent labeled glycopeptide obtained in the same manner as FT16 was used as a substrate, the same results as above were obtained. Results were obtained.

[0046]

INDUSTRIAL APPLICABILITY As described above, the present invention has an important function in sugar chain biosynthesis and acts on sugars typified by glycosyltransferases, which have recently attracted attention as tumor markers. The present invention provides a novel sugar derivative that can be a substrate for a physiologically active substance, and a method for measuring the activity of a physiologically active substance that uses the substrate as a substrate, which is simple and excellent in quantification. Is.

[Brief description of drawings]

FIG. 1 shows the sugar chain structure in the fluorescently labeled glycopeptide obtained in Example 1.

FIG. 2 shows a calibration curve obtained in Example 2 showing the relationship between the amount of FT16 enzyme source solution in a sample and the peak height of the product of the reaction of FT16 enzyme.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI C12Q 1/533 C12Q 1/533

Claims (5)

(57) [Claims] 1. A compound represented by the following general formula (1): R1-A-NH-R2 (1) [wherein R1 is glucose, mannose or galactose.
Su, fucose, sialic acid, N-acetylglucosamine, N
2 to 2 sugar residues selected from acetylgalactosamine
It has 10 sugar chains and can be a substrate for sugar-modifying enzyme
It represents what forms a sugar chain structure that, A is aspartic
Residues, serine residues, threonine residues, or
Represents a peptide residue which is made, R2 is _{R3- (CH 2) m-NH} -CO- (CH 2) n-CO- (A) R3-CO- (CH 2) n-CO-NH- (CH _{2) m-CO- (B)} R3- (CH 2) m-CO-NH- (CH 2) n-CO- (C) ( wherein, R3 is 2-pyridylamino or 3 Pirijirua
Represents a mino group, m and n are each independently an integer of 1 to 4
Represents ) Represents a group represented by. ] The sugar derivative shown by these. 2. R2 is 2-pyridylaminoethylsuccina
Moyl group, N- (2-pyridyl) -succinamoylaminop
Ropionyl group, N- (2-pyridyl) -β-alanylamino
2. A group selected from a propionyl group.
Sugar derivative. 3. The following general formula (1) R1-A-NH-R2 (1) [wherein R1 is glucose, mannose or galactose].
Su, fucose, sialic acid, N-acetylglucosamine, N
2 to 2 sugar residues selected from acetylgalactosamine
It has 10 sugar chains and can be a substrate for sugar-modifying enzyme
It represents what forms a sugar chain structure that, A is aspartic
Residues, serine residues, threonine residues, or
Represents a peptide residue which is made, R2 is _{R3- (CH 2) m-NH} -CO- (CH 2) n-CO- (A) R3-CO- (CH 2) n-CO-NH- (CH _{2) m-CO- (B)} R3- (CH 2) m-CO-NH- (CH 2) n-CO- (C) ( wherein, R3 is 2-pyridylamino or 3 Pirijirua
Represents a mino group, m and n are each independently an integer of 1 to 4
Represents ) Represents a group represented by. ] The sugar derivative shown by these is used as a substrate, The activity measuring method of the sugar modification enzyme characterized by the above-mentioned. 4. R2 is 2-pyridylaminoethylsuccina
Moyl group, N- (2-pyridyl) -succinamoylaminop
Ropionyl group, N- (2-pyridyl) -β-alanylamino
4. A group selected from a propionyl group.
Measurement method. 5. A sugar modification enzymes, measurement method described in claim 3 or 4 is a glycosyltransferase, a sugar hydrolase or sugar isomerase.