JP2000143690A - Substrate for assaying glycosyltransferase activity and method of assaying the activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject substrate, a fluorescence-labeled sugar acceptor or fluorescence-labeled sugar donor, for use in assaying the activity of a glycosyltransferase, esp. galactose transferase, a method for assaying galactose transferase activity using the substrate easily, in high sensitivity in a short time, and a reagent for the assay. SOLUTION: This substrate, e.g. a sugar nucleotide derivative, specifically uridine-5'-diphosphate naphthylated galactose, is such a sugar donor for glycosyltransferase enzyme reaction that a fluorescent substance is bound to a sixteto-hydroxyl group of a sugar nucleotide. The other objective method for assaying the activity of a glycosyltransferase, esp. β-1,4-galactose transferase comprises conducting a transglucosylation using the above substrate to form a compound bearing two kinds of fluorescent substances, exciting either one of the fluorescent substances, exciting the other fluorescence wavelength by the fluorescence wavelength emitted by the former excitation, and measuring the fluorescence intensity for the latter fluorescence wavelength.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel substrate to be used as a substrate in the activity of glycosyltransferases, particularly galactosyltransferases, a method for measuring glycosyltransferase activity using the substrates, and a reagent for the measurement. More specifically, the present invention relates to a substrate for measuring a glycosyltransferase activity having a better signal-to-noise ratio, high sensitivity, and simplicity, a measuring method using the substrate, and a reagent composition for measuring the same.

[0002]

2. Description of the Related Art Glycosyltransferases such as glucose transferase, galactose transferase, acetylgalactosyltransferase, acetylglucosamine transferase, acetylneuraminic acid transferase, and fucose transferase are used as sugar residues in sugar compounds containing sugar. It is an enzyme that has the action of transferring a sugar residue using a specific sugar nucleotide as a donor.
yococonjugate). In recent years, many cell-derived glycosyltransferases have been isolated from humans, pigs, rabbits, microorganisms, and the like.

In particular, β1,4 galactosyltransferase is 1
It has been thought that there are only different types, but in addition to the conventionally known β1,4-galactosyltransferase I, β
1,4-galactosyltransferases II, III, IV have been found. It is known that the behavior of the expression levels of 1,4-galactosyltransferase I and IV differs during the lactation period of mice.
Also, in the adult brain, the expression levels of the mRNAs of the four enzymes were compared with those of the mRNA of 1,4-galactosyltransferase IV.
Exists, but mRNAs of the other three enzymes have not been detected. The expression of mRNA in the brain differs during the ontogenesis of mice, and it is said to be involved in neural tissue formation.

In fertilization, the reaction of the body (acrosome) at the front end of the sperm head is the first step of fertilization, and the sperm recognizes and enters the zona pellucida of the egg. Then, when the sperm arrives at the plasma membrane of the egg, the zona pellucida denatures and prevents other sperm from entering. The sperm plasma membrane then fuses with that of the egg and the nucleus enters the egg. At this time, β1,4-galactosyltransferase is considered to play an important role.

As described above, β1,4-galactosyltransferase is thought to play an important role in vivo, but it is not easy to measure a trace amount of enzyme activity simply and quickly. The level of mRNA expression has been confirmed by such methods.

Heretofore, the activity of β1,4-galactosyltransferase has been measured by isolating a sugar donor or a sugar donor obtained by a method of separating a reaction product formed from the enzyme, for example, ion chromatography or high performance liquid chromatography. Alternatively, labeling has been performed with fluorescent materials or the like. Further, in the galactose transfer reaction by the enzyme, uridine-5'-diphosphate generated from uridine-5'-diphosphate galactose as a sugar donor is converted to pyruvate kinase (pyruva kinase).
te kinase) and lactate dehydrogenase
The reaction is carried out according to the following formula. There is a method in which the reaction from β-NADH to NAD is followed by the absorption at a wavelength of 340 μm to determine the amount of galactose transfer per unit time to measure the enzyme activity.

[0007]

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However, these methods have the disadvantage that the isotope-labeled sugar nucleotide as a donor substrate is dangerous to the human body and usually requires a long time. On the other hand, pyridylaminated sugar chains (Biochemical., Vol. 95, pp. 197-203, 198)
4), ANTS (8-aminonaphthalene-1,3,6-trisulfoni
sugar chains labeled with a fluorescent dye having a primary amino group and two sulfonic acid groups such as c-acid) and AMAC (2-aminoacridone) (J. Biochm., Vol. 270, Vol. 705-713).
P. 1990).

However, when enzyme activity is measured with these labeled sugar substrates, it is necessary to separate the substances produced by the enzymatic reaction in both the donor substrate and the acceptor substrate by high performance liquid chromatography and measure the amount produced. There is. Therefore, since the product t of the reaction solution cannot be directly measured by an instrument as in many ordinary enzyme activity measurements, the operation is very complicated and time-consuming at present. In addition, there is a method of enzymatically measuring UDP generated by the above reaction, but there is a problem in sensitivity.

As a method for measuring enzyme activity using a fluorescently labeled sugar chain, a method using a measurement resonance energy transfer method has been developed. In this method, a substrate derivative having a double fluorescent probe is prepared, and the enzymatic activity is measured by utilizing the effectiveness of an activity evaluation method called a fluorescence energy transfer method. The effectiveness of the activity evaluation method called the fluorescence energy transfer method means that the S / N sensitivity is improved by using the extinction field of the fluorescence characteristics by the resonance transfer method between the energy donor fluorophore and the acceptor fluorophore, respectively. It is something to try. The present inventors have disclosed Japanese Patent Application Laid-Open
No. 173096 has already reported a method for measuring the activity of glycosyltransferases utilizing this principle.

However, as a specific example, it has been shown that α2,6-sialyltransferase is measured by an oligosaccharide derivative having a fluorescent substance bound to Gal β1 → 4GlcNAc. No effective and suitable fluorescently labeled substrate for measuring enzyme activity has been reported. In particular, the enzyme activity of β1,4-galactosyltransferase could not be measured based on the fluorescence energy transfer method because an appropriate fluorescently labeled sugar donor could not be synthesized.

[0012]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a fluorescently labeled sugar acceptor and a fluorescently labeled sugar donor for use in measuring the activity of galactosyltransferase. Another object of the present invention is to provide a method for measuring galactosyltransferase activity which is highly sensitive, simple, and in a short time by using these substrates, and to provide a reagent for the measurement.

[0013]

As described above, the present inventors have conducted intensive studies to solve the problems in measuring glycosyltransferases, particularly galactosyltransferases. We succeeded in synthesizing novel fluorescently labeled sugar acceptor substrates and fluorescently labeled sugar donor substrates used for activity measurement. Then, two types of fluorophores generated by the galactosyltransferase reaction using these substrates (ie,
The present inventors have found that the above problems can be solved by utilizing the change in the fluorescence properties of compounds having a donor fluorophore and an acceptor fluorophore) due to resonance transfer between the fluorophores, and have completed the present invention.

That is, the present invention has the following configuration. (1) A sugar nucleotide derivative, which is a sugar donor for a glycosyltransferase reaction in which a fluorescent substance is bonded to a quaternary hydroxyl group of a sugar of a sugar nucleotide. (2) The sugar donor of the glycosyltransferase reaction in which a fluorescent substance is bonded to the quaternary hydroxyl group of the sugar of the sugar nucleotide is uridine-5 '
-The sugar nucleotide derivative according to (1), which is naphthylated galactose diphosphate. (3) An oligosaccharide derivative in which a fluorescent substance is bonded to a reducing end of a monosaccharide or an oligosaccharide via a spacer, or a sugar receptor of a glycosyltransferase reaction in which a fluorescent substance is bonded to an amino acid at a peptide or lipid site of a sugar complex. A sugar conjugate derivative and a sugar nucleotide derivative that is a sugar donor of a glycosyltransferase reaction in which a fluorescent substance is bonded to a quaternary hydroxyl group of the sugar of the sugar nucleotide are used as a substrate to carry out a glycosyltransfer reaction. A method for measuring glycosyltransferase activity, comprising exciting one fluorescent substance of a compound having a fluorescent substance, exciting the other fluorescent wavelength by a fluorescent wavelength emitted by the excitation, and measuring the fluorescence intensity. . (4) The method for measuring glycosyltransferase activity according to (3), wherein the glycosyltransferase is β1,4-galactosyltransferase. (5) Fluorescent substance bound to amino group of peptide or lipid moiety of saccharide derivative or saccharide complex which is a saccharide acceptor of glycosyltransferase reaction in which a reducing substance is bound to reducing terminal of monosaccharide or oligosaccharide via a spacer. And a saccharide comprising a saccharide derivative which is a saccharide acceptor for a glucosyltransferase reaction and a saccharide nucleotide derivative which is a saccharide donor for a glucosyltransferase reaction in which a fluorescent substance is bonded to a quaternary hydroxyl group of a saccharide of a saccharide nucleotide. Reagent for measuring transferase activity.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The basic principle of the present invention is to apply the resonance energy transfer method to the measurement of galactosyltransferase activity. That is, in a reaction product generated from a sugar donor that binds a fluorescent substance and a sugar acceptor that binds a different fluorescent substance by a galactose transferase reaction, two kinds of fluorescent substances are excited by an appropriate light beam and released by the excitation. The glycosyltransferase activity is measured by measuring the fluorescence intensity of the other at the determined fluorescence wavelength.

The sugar acceptor in the present invention refers to a sugar derivative (FIG. 1) in which a fluorescent substance is bonded to the reducing end of acetylglucosamine via a spacer or acetylglucosamine in the side chain via a spacer. An acrylamide copolymer having a fluorescent substance in a side chain via a spacer (FIG. 2), that is, a sugar having a fluorophore group that accepts energy having an emission wavelength excited from the wavelength emitted from the sugar donor (for example, , Galactose)
Refers to a sugar derivative that accepts In addition, the relative relationship between energy donation and acceptance may be reversed for a sugar acceptor and a sugar donor.

As the sugar derivative, for example, a derivative in which the sugar has a terminal sugar chain bonded to a fluorescent substance via acetylglucosamine via a spacer (FIG. 1) or acetylglucosamine in a side chain via a spacer, and It is an acrylamide copolymer having a fluorescent substance in a side chain via a spacer (FIG. 2).

Examples of such a sugar acceptor include 3- [N- (5- (N, N-dimethylamino) -1] shown below.
-Naphthalenesulfonyl) amino] propyl 2-acetamido-3,4,6-tri-o-acetyl-2-deoxy-β-D-glucopyranoside.

[0019]

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Further, acrylamide shown below, 3
-(N-acryloylamino) propyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
β-D-glucopyranoside and N- [5- (N, N-
Dimethylamino) -1-naphthalenesulfonyl] -β-
An acrylamide copolymer synthesized by reacting alanine allylamide is exemplified.

[0021]

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The sugar donor for the glycosyltransferase reaction in the present invention is a sugar nucleotide derivative in which a fluorescent substance is bonded to the quaternary hydroxyl group of the sugar of sugar nucleotide (FIG. 2). That is, it refers to a sugar nucleotide derivative that is a substrate specific to a glycosyltransferase having a fluorophore (eg, galactose transferase).

Examples of sugar nucleotide derivatives include, for example,
Uridine-5 '-[6-fluoresceinylthioureido) deoxy-6-d-D-galactopyranosyl] diphosphate (UDP- (6-fluoresceinyl) -Ga
l) and so on. Fluoresceinyl
) Group is a dansyl group, naphtyl
) Group, pyrenyl group and the like.

An example of such a fluorescently labeled uridine deoxygalactopyranosyl diphosphate (UDP-galactose) is uridine having the following structural formula:
5 '-[6-deoxy-6-N- (1-naphthyl) -d
-D-galactopyranosyl] diphosphate disodium salt.

[0025]

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Examples of the fluorophore group of the fluorescent substance F2 which binds to the sugar derivative as the sugar acceptor in the present invention include a naphthyl group, a pyrenyl group, a dansyl group, a fluorescamine group and the like.

Further, examples of the fluorophore group of the fluorescent substance F1 which binds to the sugar nucleotide as the sugar donor in the present invention include a naphthyl group, a pyrenyl group, a dansyl group, a fluorescamine group and the like.

In the present invention, the fluorescent substance F2 of the sugar receptor is used.
Is a dansyl group, the fluorescent substance F1 of the sugar donor is preferably a naphthyl group.

In the method for producing a substrate of the present invention, for example, when the fluorescent substance F2 is a dansyl group and the fluorescent substance F1 is a naphthyl group, an amino group is attached to the reducing end of N-acetylglucosamine via a spacer as a sugar acceptor. The desired substance is obtained by reacting a fluorescent substance having a dansyl group with a phosphatase group, and a naphthyl group is introduced as a sugar donor into the tertiary hydroxyl group of galactose, and the uridine-5 ′ as the desired substance is obtained.
-[6-deoxy-6-N- (1-naphthyl) -dD
-Galactopyranosyl] diphosphate disodium salt can be obtained.

The method for measuring a glycosyltransferase in the present invention means that one of the compounds having two different fluorescent substances produced by reacting the above sugar donor and the sugar acceptor with the glycosyltransferase is used. Is excited by ultraviolet light, and the glycosyltransferase activity is measured by measuring the fluorescence intensity of the other from the fluorescence wavelength emitted by the excitation. The transfer enzyme reaction conditions follow the optimal reaction conditions of the enzyme.

The conditions for exciting the fluorescent substance and the method for measuring the fluorescence are described, for example, in Luminescence spectrometer LS50B (Perkin
Elmer), the excitation wavelength is 280 nm, and the fluorescence measurement is performed from 310 to 600 nm. Fluorescence measurement is 50 nm
Scanspee at 37 ° C in HEPES buffer (pH 7.0)
d 500 nm / min, Slit size 5 nm. The glycosyltransfer reaction is performed, for example, by adding 0.05 units of β1,4-galactosyltransferase and 10 nmo of a sugar donor to 4.2 ml of the above buffer solution.
1, 300 nmol of sugar acceptor, and α-lactosamine. The ratio of sugar donor to acceptor is 1:10
1: 100 is desirable, but it is not particularly limited to this range.

As an embodiment of the reagent composition for measuring a glycosyltransferase activity of the present invention, for example, one containing the above-mentioned sugar donor and the above-mentioned sugar acceptor, a buffer, and an enzyme activity activator can be mentioned. The buffer is not particularly limited as long as it can be used for the reaction of glycosyltransferase.

Although the phenomenon of resonance energy transfer was observed by Perrin in the early 20th century, Forster proposed a theory describing intermolecular interactions by resonance energy transfer in the late 1940's, and explained the distance between chromogenic acids and the optics of chromophores. Derived a velocity equation that relates to dynamic properties (Sinanoglu, O., E
d., Modern Quantum Chemistry 93-137).

The above method has been improved so that the average intermolecular distance can be obtained with reliable data by measuring the resonance energy transfer. Usually, the measurement of energy transfer is
Since the detection is based on fluorescence, there is an advantage that high sensitivity can be secured. The Forster distance between the energy donor and the acceptor is basically determined by (a) the energy donor quantum yield,
It can be measured by (b) the fluorescence emission spectrum of the energy donor and (c) the intermolecular extinction coefficient of the energy acceptor, but it is necessary to consider the solution state of the fluorescent substance.

In the present invention, since a glycosyltransferase is an enzyme that transfers a sugar donor, a sugar nucleotide, to a sugar acceptor, two suitable fluorescent substances can be added to both of these substrates without inhibiting the enzyme activity. By labeling, the enzyme activity can be measured by a change in fluorescence emission due to a change in the distance between the two fluorophores before and after the change during the enzymatic reaction. That is, a fluorescent energy transfer pair consists of an energy donor molecule and an energy acceptor molecule, the energy donor molecule having a first emission wavelength, and the energy acceptor molecule can be excited by the first emission wavelength. Having a fluorescent chromophore having a second emission wavelength that can be made and detected separately from the first emission wavelength;
Further, these sugar donors and sugar acceptors do not inhibit the enzyme transferase reaction.

Under the most preferable conditions from the principle and purpose of the present invention, under the most preferable conditions, the above sugar donor and sugar acceptor are used, and if the enzymatic reaction proceeds, the sugar having a fluorophore is transferred to the acceptor and the fluorescent energy is The energy-donor and energy-acceptor molecules of a transfer pair are in close proximity on the same molecule, which can cause energy transfer according to Forster's principle. In addition, it may be observed that the intensity of the transferred fluorescent substance changes significantly as the sugar having the fluorophore is transferred to the receptor and the physical properties of the product proceeds to the reaction. The enzyme activity can be measured by directly monitoring the intensity of the enzyme.

As described above, these enzymatic reactions are excited, specifically the energy-donating fluorophore is excited at its excitation wavelength and the emission wavelength of the acceptor is measured, or the transferred donor is measured. By measuring the fluorescence intensity of the enzyme, the enzyme activity in the sample can be measured qualitatively or quantitatively.

[0038]

The present invention will be described below in detail with reference to examples.

Embodiment 1 1. Synthesis of fluorescent substance-labeled sugar receptor Synthesis of intermediate (I) 156.6 g of D-glucosamine hydrochloride (0.73 mol)
l) was suspended in 500 ml of pyridine, and 102 ml (0.73 mol) of triethylamine was added to neutralize the hydrochloride, and then 500 ml (about 1.1 mol) of acetic anhydride was added, followed by stirring overnight. After confirming that the solution became transparent, the completion of the reaction was confirmed by TLC (thin layer chromatography), and the solution was concentrated. After about 0.5 ml of chloroform was added to the residue to dissolve it, it was washed with deionized water, aqueous sodium bicarbonate and brine. The organic layer was dried over magnesium sulfate, filtered through celite and concentrated, and the residue was dissolved in ethanol and recrystallized.
The crystals were filtered and dried under reduced pressure. The ethanol solution was concentrated, dissolved again in ethanol, and recrystallized. As described above, 256 g of 2-acetamido-1,3,4,6-tetra-o-acetyl-2-deoxy-D-glucopyranose was obtained in a yield of 90%.

Next, this 2-acetamido-1,3,3
5.0 g (13 mmol) of 4,6-tetra-o-acetyl-2-deoxy-D-glucopyranose was dissolved in 50 ml of 1,2-dichloroethane, and TMSOTf was added at 60 ° C.
2.6 ml (13 mmol) of (trimethylsilyltrifluoromethanesulfonate) was added, and the mixture was stirred for 2 hours. The completion of the reaction was confirmed by TLC, and 5.4 ml (38 mmol) of triethylamine was added to quench TMSOTf. Thereafter, the solvent was completely distilled off once, and the residue was dissolved again in 50 ml of dichloromethane.

To this solution, 8.0 g (38 mmol) of 3- (N-benzyloxycarbonylamino) propanol was added.
, And CSA ((±) -camphor-10-sulfonic acid) is added little by little until the pH becomes 2-3, and 7
The mixture was stirred at 0 ° C under reflux for 4 hours. After confirming the completion of the reaction by TLC, the reaction solution was cooled to room temperature, and washed with aqueous sodium hydrogen carbonate and brine. The organic layer was dried over magnesium sulfate, filtered through celite and concentrated. Finally, the residue was purified by silica gel chromatography (developing solvent: chloroform: methanol = 50: 1) to give 3- (N-benzyloxycarbonylamino) propyl-2-acetamide-3,4,6-
Tri-o-acetyl-2-deoxy-β-D-glucopyranoside was obtained in a yield of 2.8 g and a yield of 40%.

[0042]

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3- [N- (5- (N, N-dimethylamino) -1-naphthalenesulfonyl) amino] propyl 2
-Acetamide 3,4,6-tri-o-acetyl-2-
Synthesis of deoxy-β-D-glucopyranoside 300 mg (0.56 mmol) of the above intermediate (I) was dissolved in 8 ml of dry methanol, and sodium methoxide 1
0 mg was added, and the mixture was stirred at room temperature overnight. The completion of the reaction was confirmed by TLC, and neutralized with an ion exchange resin (Dowex H ⁺ type). The ion exchange resin is removed by cotton plug filtration and concentrated.

Next, the residue was dissolved in 8 ml of methanol,
100 mg of Pd / C (palladium carbon) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1 hour. After confirming the completion of the reaction by TLC, the reaction mixture was filtered through celite and further concentrated. The residue was dissolved in 5 ml of dry methanol, and 93 μl (0.67 mmol) of triethylamine and 150 mg (0.56 mmol) of dansyl chloride were added.
Stirred for hours. The completion of the reaction was confirmed by TLC, and the mixture was concentrated. Finally, purification was performed by gel filtration (developing solvent was deionized water) using Sephadex G-10 (manufactured by Pharmacia Biotech). The yield was 267 mg and the yield was 100%.

NMR analysis value (proton NMR) H-1: 4.10 ppm (d, 1H, J1,2 = 8.8 Hz) H-2: 3.45 ppm (t, 1H, J2,3 = 10.3 Hz) H-3: 3.33ppm (t, 1H, J3,4 = 8.8Hz) H-5: 3.56ppm (m, 1H, J4,5 = 11.0Hz, J5,6a = 0.0Hz,
J5,6b = 5.1Hz) 3.21-3.28ppm (m, 2H, H-4, H-6b) 1.65ppm (s, 3H, Ac) 2.68ppm (s, 6H, Dan's Me × 2) 2.68-3.56ppm ( m, 6H, CH ₂ × 3) 7.21ppm (d, 1H, NH) 7.49-8.33ppm (d × 3, dd × 1,6H, Naph)

Embodiment 2 2. Synthesis of fluorescent substance-labeled sugar receptor Synthesis of intermediate (II) 2-acetamido-1,3,4,6-tetra-o-acetyl-2-deoxy-D-glucopyranose 50 g
(0.13 mol) was dissolved in 300 ml of dry chloroform, 5 ml of acetic anhydride and 115 ml (0.45 mol) of 30% hydrogen bromide-acetic acid were added at 0 ° C., and the mixture was stirred for 2 hours while gradually returning to room temperature. When the completion of the reaction was confirmed by TLC, the reaction solution was cooled to 0 ° C., and pyridine (60 ml, 0.
64 mol), and the mixture was further stirred overnight while gradually returning to room temperature. After confirming the completion of the reaction by TLC, the reaction solution was washed with deionized water, aqueous sodium bicarbonate, and brine. The organic layer was dried over magnesium sulfate, filtered through celite, and concentrated.

Next, the residue was treated with 1,2-dichloroethane 20
0-ml, and 3- (N-benzyloxycarbonylamino) propanol 13.4 g (64 mm
ol), and further add CSA ((±)-camphor-1
0-sulfonic acid) was added little by little until the pH became 2-3, and the mixture was stirred at 70 ° C under reflux overnight. After confirming the completion of the reaction by TLC, the reaction solution was cooled to room temperature, and washed with aqueous sodium hydrogen carbonate and brine. The organic layer is dried over magnesium sulfate,
Celite filtration and concentration. Finally, the residue is purified by silica gel chromatography (developing solvent: chloroform: methanol = 50: 1), and further recrystallized from ethanol to give 3- (N-benzyloxycarbonylamino) propyl-2-acetamide-3,4,6. -Tri-o-acetyl-2-deoxy-β-D-glucopyranoside was synthesized in a yield of 16.0 g and a yield of 46%.

[0048]

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Next, 3- (N-acryloylamino) propyl 2-acetamido-3,4,6-tri-o-acetyl-2-deoxy-β-D-glucopyranoside 1.9.
g (3.5 mmol) in 20 ml of methanol,
200 mg of Pd / C (palladium carbon) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1 hour. After confirming the completion of the reaction by TLC, the mixture was filtered through celite and further concentrated. The residue was dissolved in a mixed solvent of 60 ml of THF (tetrahydrofuran) -25 ml of methanol, and 0.59 ml (4.2 mmol) of triethylamine and 0.34 ml (4.2 mmol) of acryloyl chloride were added at 0 ° C. Stir for 2 hours. At this time, triethylamine was added little by little so that the pH was not biased toward the acidic side.

The progress of the reaction was confirmed to be poor by TLC.
0 ml (4.9 mmol) was added, and the mixture was stirred for 2 hours. T
After confirming the completion of the reaction by LC, the mixture was concentrated, the residue was dissolved in chloroform, and washed with aqueous sodium bicarbonate and brine. The organic layer was dried over magnesium sulfate, filtered through celite, further concentrated, and purified by silica gel chromatography (developing solvent: chloroform: methanol = 30: 1).
To give 3- (N-acryloylamino) propyl-2-acetamido-3,4,6-tri-o-acetyl-2-deoxy-β-D-glucopyranoside in a yield of 491 m.
g, 31% yield.

Next, 3- (N-acryloylamino) propyl 2-acetamido-3,4,6-tri-o-acetyl-2-deoxy-β-D-glucopyranoside 491
mg (1.07 mmol) was dissolved in methanol (10 ml), sodium methoxide (10 mg) was added, and the mixture was stirred overnight. After confirming the completion of the reaction by TLC, the mixture was neutralized with an ion exchange resin (Dowex H ⁺ type). Finally,
The ion exchange resin was removed by cotton plug filtration, concentrated, and the yield was 1
3- (N-acryloylamino) propyl-2 at 00%
-Acetamide-2-deoxy-β-D-glucopyranoside was obtained.

Synthesis of Intermediate (III) Nt-butyloxycarbonyl-β-synthesized above
5.0 g (26.4 mmol) of alanine and 2.0 ml (26.7 mmol) of allylamine are dissolved in 50 ml of ethanol-benzene (1: 1), and EEDQ (1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) is dissolved. 7.2 g (29.1 mmol) was added, and the mixture was stirred at room temperature overnight. After confirming the completion of the reaction by TLC, the mixture was further concentrated and purified by silica gel chromatography (developing solvent).
Chloroform: methanol = 50: 1) to obtain 5.5 g of Nt-butyloxycarbonyl-β-alanine allylamide in a yield of 92%.

5.5 g of Nt-butyloxycarbonyl-β-alanine allylamide is dissolved in 150 ml of a 4N hydrogen chloride dioxane solution, and the mixture is stirred at room temperature overnight. TLC
After the completion of the reaction was confirmed by, the mixture was further concentrated and recrystallized from ethanol. 3.0 g, 81% yield and β-
Alanine acrylamide hydrochloride was obtained.

The above synthesized Nt-butyloxycarbonyl-β-alanine hydrochloride 114.6 mg (0.74
was dissolved in 5 ml of dry methanol, and 0.24 ml (1.72 mmol) of triethylamine and 200 mg (0.74 mmol) of dansyl chloride were added at 0 ° C., followed by stirring for 4 hours. Confirm the completion of the reaction by TLC,
It was concentrated and purified by silica gel chromatography (developing solvent: chloroform: methanol = 50: 1), and N- [5
-(N, N-dimethylamino) -1-naphthalenesulfonyl] -β-alanine acrylamide in a yield of 133 m
g, 51% yield.

[0055]

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Synthesis of Acrylamide Copolymer 3- (N-acryloylamino) propyl-2-acetamido-2-deoxy-β-D-glucopyranoside 1
0.0 mg (0.030 mmol) of N- [5- (N, N-dimethylamino)-dissolved in a small amount of methanol
1.04 mg (0.0030 mmol) of 1-naphthalenesulfonyl] -β-alanine acrylamide and 21.3 mg (0.30 mmol) of acrylamide were dissolved in deionized water, and degassed while applying ultrasonic waves under reduced pressure. In addition, add degassed methanol until the solution is clear,
TEMED (N, N, N, N-tetramethylethylenediamine) 2.00 μl (0.013 mmol) and APS
1.22 mg of (ammonium peroxodisulfate) (5.
3 mmol) and stirred overnight under a nitrogen atmosphere. The reaction solution is directly used as a gel filtration column (Sephadex G-5).
Purified by subjecting the acrylamide copolymer to 19.)
6 mg was obtained with a yield of 61%.

NMR analysis value (proton NMR) H-1: 4.41 ppm (d, 1H) H-6a: 3.35 ppm (s, 1H) 3.45-3.66 ppm (m, 3h, H-2, H-3, H -4) 3.82ppm (m, 2H, H-5, H-6b) 1.95ppm (s, 3H, Ac) 1.55-2.33ppm (m, CH 2, CH) 7.61-8.42ppm (m, Naph)

From the integrated value, glucosamine residue: acrylamide residue: dansyl residue = 7.47: 92.37:
0.16 was calculated.

¹³ C NMR C-1 101.97 ppm C-2 56.40 ppm C-3 74.61 ppm C-4 70.78 ppm C-5 76.82 ppm C-6 61.61 ppm 35.27-37.04 ppm (CH ₂ ) 42.21-42.99 ppm (CH) 180.28ppm (CO)

Embodiment 3 1. Synthesis of Fluorescent Substance Labeled Sugar Donor Synthesis of Intermediate (IV) Galactose (500 g) was suspended in a mixed solvent of pyridine (1.5 ml) / acetic anhydride (1.5 ml) and stirred overnight. After confirming that the solution was clear, TLC confirmed the completion of the reaction and concentrated. About 1.5 chloroform in the residue
After adding and dissolving the solution, the solution was washed with deionized water, aqueous sodium bicarbonate and brine. The organic layer was dried over magnesium sulfate, filtered through celite and concentrated, and the residue was dissolved in ethanol and recrystallized. The crystals were filtered and dried under reduced pressure. The ethanol solution was concentrated and stored as it was. Yield 1.08
kg, 100% yield, 1,2,3,4,6-hexa-
o-Acetyl-D-caractopyranose was obtained.

Next, 1,2,3,4,6-hexa-o-
12.6 g of acetyl-D-caractopyranose (32.
3 mmol) was dissolved in 130 ml of THF, 5.30 ml (48.5 mmol) of benzylamine was added, and the mixture was stirred at room temperature overnight. The completion of the reaction was confirmed by TLC, and the reaction solution was concentrated to about 30 ml. About 100 chloroform in the residue
ml, and concentrated again to 30 ml. About 300 ml of chloroform was added to the residue, and the mixture was washed with 1N hydrochloric acid, aqueous sodium bicarbonate and brine. The organic layer was dried over magnesium sulfate, filtered through celite, concentrated, and then purified by silica gel chromatography (developing solvent: toluene: ethyl acetate =
5: 1 to 2: 1), and 2,3,4,6-
Tetra-o-acetyl-D-calactopyranose was obtained.

Next, 1.29 g of dibenzyl phosphite
(4.9 mmol) was dissolved in 20 ml of toluene.
0.66 g (4.9 mmol) of chlorosuccinimide is added, and the mixture is stirred at room temperature for 1 hour. After confirming the completion of the reaction by TLC, the reaction mixture was filtered through a glass filter and concentrated. 0.57 g (1.6 mmol) of the residue and 2,3,4,6-tetra-o-acetyl-D-caractopyranose were added to THF2
The solution was dissolved in 0 ml, and 1.0 ml (1.6 mmol) of a 1.6 M n-butyllithium hexane solution was added at 70 ° C. Raise the reaction solution gradually to 40 ° C over about 2 hours,
The completion of the reaction was confirmed by TLC. The reaction solution was diluted with about 100 ml of chloroform, and washed with aqueous sodium bicarbonate and brine. The organic layer was dried over magnesium sulfate, filtered through celite, concentrated, and purified by silica gel chromatography (developing solvent: toluene: ethyl acetate = 5: 1) to give diphenyl-
2,3,4,6-tetra-o-acetyl-α-D-calactopyranosyl phosphate was obtained in a yield of 0.53 g and a yield of 50%.

Next, 0.30 mg (0.49 mmol) of diphenyl-2,3,4,6-tetra-o-acetyl-α-D-caractopyranosyl phosphate was dissolved in 5 ml of methanol, and Pd / C ( Palladium carbon) 100m
g was added, and the mixture was stirred at room temperature for 1 hour under a hydrogen atmosphere. TLC
After confirming the completion of the reaction by filtration, the mixture was filtered through celite and further concentrated. The residue was dissolved in a mixed solvent of triethylamine: methanol: deionized water = 1.5 ml: 10 ml: 4.6 ml and stirred at room temperature for 1 hour. After confirming the completion of the reaction by TLC, the reaction mixture was concentrated to obtain α-D-galactopyranosyl phosphate-di-n-butylammonium salt in a yield of 10%.
It was synthesized at 0%.

[0064]

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Synthesis of Intermediate (V) 117 mg (0.253 mmol) of Intermediate (IV), 1 mg of catalase and 20 mg of galactose oxidase were dissolved in 20 ml of 0.01 M phosphate buffer (pH 7.0), and the mixture was dissolved for 30 minutes. Shake. Subsequently, 70.9 mg (0.51 mmol) of α-naphthylamine, acetonitrile 6
ml was added and shaken for 30 minutes. Next, acetonitrile was added until all the α-naphthylamine was dissolved.
Shake time. Sodium cyanoborohydride 16.
Add 4 mg (0.25 mmol), shake for 5 minutes,
Diluted with 100 ml of deionized water and washed with diethyl ether and toluene. The aqueous layer was freeze-dried, and the crystals were purified by gel filtration (developing solvent deionized water) using Sephadex G-10. Furthermore, after passing through a cation exchange resin (Dowex 50W-X8, pyridinium ion type) column (diameter 0.5 cm × length 5.0 cm), 14.7 μl (62 μmo) of n-butylamine was added.
l) Added, concentrated and azeotroped with pyridine three times to give 6-deoxy-6-N- (1-naphthyl) -α-D-galactopyranosyl phosphate in a yield of 21 mg and 15.3.
%.

[0066]

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Uridine-5 '-[6-deoxy-6-N
Synthesis of-(1-naphthyl) -α-D-galactopyranosyl] diphosphate disodium salt Uridine-5′-monophosphomorpholidate-4-morpholine-N, N-dicyclohexylcarboxamidine salt 34 mg (50 μmol) Was azeotroped three times with pyridine. Residue and 6-deoxy-6-N- (1-naphthyl)-
21 mg (39 μmol) of α-D-galactopyranosylphosphate-di-n-butylammonium salt is dissolved in about 5 ml of pyridine, concentrated and concentrated again.
and stirred overnight. The mixture was diluted with about 50 ml of deionized water, washed with diethyl ether, and the aqueous layer was freeze-dried. The crystals are converted to an anion exchange resin (DEAE-Sephacel,
Purification using hydrogencarbonate ion type (developing solvent 0.05)
０．５0.5 M aqueous solution of ammonium hydrogen carbonate) and further purified using Sephadex G-10. Finally, a cation exchange resin (Dowex 50W-X8, Na
⁺ Form), freeze-dry the filtrate and uridine-5'-
[6-Deoxy-6-N- (1-naphthyl) -α-d-
[Galactopyranosyl] diphosphate disodium salt was obtained in a yield of 9.5 mg and a yield of 33.6%.

NMR analysis value H-1 ': 5.47 ppm (d, 1H, J1, P = 8.0 Hz, J1, 2 = 0.0 Hz) H-4': 4.99 ppm (s, 1H, J3, 4 = 0.0 Hz) , J4,5 = 0.0Hz) 3.73-4.77ppm (m, 11H, H-2 ', H-3', H = 5 ', H-6a', H-6b ', H-
1, H-2, H-3, H-4, H-5a, H-5b) 5.57-5.83ppm (m, 2H, uridine) 6.98ppm (d, 1H, NH-Naph) 7.33-7.93ppm (m , 8H, uridine)

Intermediate (IV) 107 mg (0.23 mmol
l), 1 mg of catalase and 10 mg of galactose oxidase were added to a 0.01 M phosphate buffer (pH 7.0) 20
The mixture was dissolved in ml and shaken for 30 minutes. Subsequently, 68 μl (0.46 mmol) of 1-naphthylmethylamine and 6 ml of acetonitrile were added, and the mixture was shaken for 30 minutes. Next, 1-
Acetonitrile was added until all the naphthylmethylamine was dissolved, and the mixture was shaken for another hour. 15 mg (0.23 mmol) of sodium cyanoborohydride was added, shaken for 5 minutes, diluted with 100 ml of deionized water, and washed with diethyl ether and toluene. The aqueous layer was freeze-dried, and the crystals were purified by gel filtration (developing solvent deionized water) using Sephadex G-10. Furthermore, after passing through a cation exchange resin (Dowex 50W-X8, pyridinium ion type) column (diameter 0.5 cm × length 5.0 cm), 14.7 μl (62 μmo) of n-butylamine was added.
1) Add and concentrate, azeotrope three times with pyridine,
Deoxy-6-N- (1-naphthylmethyl) -α-D-
Galactopyranosyl phosphate was obtained in a yield of 55 mg and a yield of 46%.

[0070]

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Uridine-5 '-[6-deoxy-6-N
Synthesis of-(1-naphthylmethyl) -α-D-galactopyranosyl] diphosphate disodium salt 20 mg of uridine-5′-monophosphomorpholidate-4-morpholine-N, N-dicyclohexylcarboxyamidine salt ( (37 μmol) was azeotroped with pyridine three times. The residue and 6-deoxy-6-N- (1-naphthylmethyl) -α-D-galactopyranosyl phosphate-di-
16 mg (29 μmol) of n-butylammonium salt was dissolved in about 5 ml of pyridine, concentrated,
and stirred overnight. Diluted with about 50 ml of deionized water, washed with diethyl ether, and lyophilized the aqueous layer. The crystals were purified twice using an anion exchange resin (DEAE-Sephacel, bicarbonate ion type) (developing solvent 0. 1).
(0.5-0.5 M aqueous solution of ammonium bicarbonate) and further purified twice with Sephadex G-10. Finally, cation exchange resin (Dowex 50W-X8, Na ⁺ type)
And the filtrate was freeze-dried to obtain uridine-5 '-[6-deoxy-6-N- (1-naphthylmethyl) -α-D-galactopyranosyl] diphosphate disodium salt.

H-1 ': 5.55 ppm (d, 1H, J1, P = 8.0 Hz, J1, 2
= 0.0Hz) H-3 ': 4.26ppm (dd, 1H) H-4': 5.08ppm (d, 1H, J3,4 = 2.9Hz, J4,5 = 0.0Hz) H-5 ': 3.78ppm ( m, 1H) 4.06-4.13ppm (m, 6H, H-2 ', H-6a', H-6b ', ribose) 3.51ppm, 4.72ppm (eachs, 3H, ribose) 5.63-5.66ppm (m, 2H , Uridine) 7.46-8.02ppm (m, 8H, Naph, uridine)

Example 4 Uridine-5 '-[6-deoxy-6-
N- (1-naphthyl) -d-D-galactopyranosyl]
2.38 μM diphosphate disodium salt and 3- [N- (5- (N, N-dimethylamino) -1-naphthalenesulfonyl) amino] propyl 2 which is a sugar acceptor
-Acetamide 3,4,6-tri-o-acetyl-2-
Deoxy-β-D-glucopyranoside at 71.42 μM
And 4.2 ml of a reaction solution consisting of 9.52 μg / ml of α-lactalbumin and 0.05 unit of β1,4-galactose transferase (50 mM HEPES buffer; pH
Under the condition of (7.0), the reaction was carried out at 37 ° C., and the reaction solution was excited with a fluorescent substance over time to measure the fluorescence intensity.

The conditions for exciting the fluorescent substance and the measurement of the fluorescence intensity are described in Luminescence spectorphotometer L.
Excitation with S50B (Perkin Elmer).
It went to 10-570 nm. As shown in FIG. 3, the naphthyl group was emitted at 430 nm, and as a result, the dansyl group was excited and the emitted fluorescence was detected at 530 nm. Further, a change in the fluorescence intensity at 430 nm due to the naphthyl group was observed.

[0075]

As described above, in the measurement method using the substrate according to the present invention, the non-specific background lowers the S / N ratio, but an appropriate combination of buffer, reagent and fluorescent substance is selected. Can be reduced or eliminated. Further, according to the measurement method of the present invention, the enzyme activity can be measured with high sensitivity without separating the product of galactose transferase by high performance liquid chromatography or the like, so that the measurement can be performed simply and in a short time.

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of the sugar receptor derivative of the present invention.

FIG. 2 shows a schematic diagram of the sugar donor derivative of the present invention.

FIG. 3 shows a fluorescence spectrum in a galactose transferase reaction solution.

Claims (5)

[Claims] 1. A sugar nucleotide derivative, which is a sugar donor for a glycosyltransferase reaction in which a fluorescent substance is bonded to a quaternary hydroxyl group of a sugar of a sugar nucleotide. 2. The sugar nucleotide derivative according to claim 1, wherein the sugar donor in the glycosyltransferase reaction in which a fluorescent substance is bonded to the carboxy tertiary hydroxyl group of the sugar nucleotide is uridine-5′-diphosphate naphthylated galactose. . 3. A sugar acceptor in a glycosyltransferase reaction in which a fluorescent substance is bonded to an oligosaccharide derivative or a peptide of a sugar complex or an amino acid at a lipid site, wherein the fluorescent substance is bonded to the reducing end of a monosaccharide or oligosaccharide via a spacer. A sugar conjugate derivative that is a body and a sugar nucleotide derivative that is a sugar donor of a glycosyltransferase reaction in which a fluorescent substance is bonded to a quaternary hydroxyl group of the sugar of the sugar nucleotide to perform a sugar transfer reaction as a substrate,
A saccharide characterized in that one of the two compounds having two kinds of fluorescent substances is excited, the other fluorescent wavelength is excited by the fluorescent wavelength emitted by the excitation, and the fluorescence intensity is measured. Method for measuring transferase activity. 4. The method according to claim 3, wherein the glycosyltransferase is β1,4-galactosyltransferase. 5. A fluorescent substance is attached to an amino group of a peptide or a lipid of a saccharide derivative or a saccharide complex which is a saccharide receptor for a glycosyltransferase reaction in which a fluorescent substance is bonded to a reducing end of a monosaccharide or an oligosaccharide via a spacer via a spacer. Containing a glycoconjugate derivative which is a sugar acceptor of a glycosyltransferase reaction and a sugar nucleotide derivative which is a sugar donor of a glycosyltransferase reaction in which a fluorescent substance is bound to a quaternary hydroxyl group of a sugar of a sugar nucleotide. For measuring glycosyltransferase activity.