JPH03215492A - N-acetyl-d-hexosamine derivative, its production, n-acetyl-beta-d-hexosaminidase activity-determining substrate and determining method - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (one of A1 and A2 is H and the other is OH; G is derivative of sugar or sugar excluding glucose). EXAMPLE:A compound expressed by formula II. USE:Used as a substrate for determination of N-acetyl-beta-D-hexosaminidase activity. PREPARATION:A D-hexosamine is acetylated and allowed to react with a thiol- based compound to obtain a N-acetyl-D-hexosamine-1-thio derivative. Next, said derivative is made to an oxazoline compound, then a resultant compound is subjected to a reaction with a derivative of a sugar or a sugar except glucose at a condition of 20-90 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to N-acetyl-D-hexosamine derivatives and methods for producing the same, as well as substrates and methods for measuring N-acetyl-β-D-hexosaminidase activity.

[Prior art] N-acetyl-β-D-hexosaminidase (
Hereinafter, it may be abbreviated as rNAGJ. ) Activity measurement has high clinical significance as it provides information on the early diagnosis of renal diseases such as acute renal failure and glomerulonephritis, and the nephrotoxicity of drugs, and is widely practiced. Several standard methods for measuring NAG activity have been proposed and used for actual diagnosis. Specifically, the following ■
The basic purchase of ~■ is proposed. ■ Substrate with β-bond of p-nitrophenol at the 1st position of N-acetyl-D-glucosamine (Methods E
nxya+ol. , 28, 702, (1972)
)． ■ m at the 1st position of N-acetyl-D-glucosamine
-Substrate with β-linked cresolsulfophthalein (C
lin. Chea+. , 29. 1713 (198
3)) , ■ Substrate with β-bond of phenol at the 1st position of N-acetyl-D-glucosamine (Japanese Patent Application Laid-Open No. 1983-60997)
．． ■ Substrate (Cll
n. Chew. ．． 34. (1G), 2140(
198B)). By the way, as a method for synthesizing β-glycoside of N-acetylhexosamine, a method via an oxazoline form of N-acetyl-D-hexosamine is known.
Several methods have been reported to synthesize the oxazoline compound, as described below. ■ A method of ring-closing α-methyl glycoside of N-acetylglucosamine under acetolysis conditions to form an oxazoline compound (Carbohydr. Res., 6, 18
4-198 (196B)) ■ A method of reacting β-acetate of N-acetylglucosamine with ferric chloride in methylene chloride to form an oxazoline compound (Carbohydr
．． Res. , 21, 460-464 (1972))
, O A method of starting from an acetohalogen form of N-acetylglucosamine and treating it with sodium hydrogen carbonate in the presence of halide ions to form an oxazoline form (J. ^ta.
Chew. Soc. ,97. 4063-4068
(1975)).

■ A method of isomerizing the allyl group of allyl β-glycoside of N-acetylglucosamine to a propenyl group and treating it with mercuric chloride (Carbohydr. Res., 8
2. 237-252 (1980)). [Problems to be Solved by the Invention] Among the conventional substrates (■) to (■) above, N-acetyl-D (■)
- When using a substrate in which p-nitrophenol is β-linked to the 1st position of glucosamine as a substrate for measuring NAG activity,
Since p-nitrophenol does not develop color at the optimal pH of NAG, the reaction with NAG and the color development (detection) reaction must be carried out separately. For this reason, the measurement operation was complicated and there were problems with measurement accuracy. As a result, rate assays (
It was also difficult to adapt to kinetic analysis). Such problems have not been solved with the substrates (1) and (2) above.

In the substrate in which 2-chloro-4-ditrophenol is β-bonded to the 1st position of N-acetyl-D-glucosamine, p
-By introducing a chloro group into nitrophenol, the pKa
Although the pK was lowered to allow color development in the lower pti region, the pK was not satisfactory for measuring β-NAG activity.
It has not yet been possible to reduce a. Therefore, a little pH
There is also the drawback that the degree of color development changes significantly. In addition, the chromogenic groups of the phenolic conductors (2) to (2) have a narrow spectral band width, and have the disadvantage that accurate absorbance measurement is difficult unless a highly accurate spectrometer is used. Furthermore,
For example, hemoglobin and bilirubin (
The absorption wavelength overlaps with that of phenolic conductors, and they are easily decomposed by light. ) and surfactants (which can change the molar extinction coefficient of phenolic conductors), it is a well-known fact that the absorbance tends to fluctuate and there are problems with measurement accuracy. The problem is not resolved.

By the way, in testing for urine components, the mainstream method is to soak a filter paper on a test paper with chemicals, soak it in urine, and make a determination based on the presence or absence of color (test paper method).
In AG measurement, not only methods using automatic analyzers for clinical chemistry but also simple methods such as the test strip method are desired. However, it has been impossible or extremely difficult to apply conventional substrates to the test strip method. As mentioned above, all of the conventional methods using NAG activity measurement substrates are difficult to apply to rate assays (kinetic analysis), have many problems with operability, measurement accuracy, etc., and are not widely desired. It had many drawbacks such as difficulty in applying it to the test strip method. On the other hand, the conventional methods for synthesizing oxazolines described in (1) to (2) above all have extremely poor yields, so improvements are required for practical use. Moreover, when coupling the oxazoline obtained by these methods with other sugars (β-glycosidation), conventionally the reaction temperature was 100°C.
Since the methods described above (for example, in the document ① above) were carried out, there was still the problem that the yield was low due to the occurrence of side reactions. The present invention solves the above-mentioned conventional problems. 1. NAG rate assay with excellent quantitative properties is possible. 2. Measurement accuracy is improved because it is almost unaffected by measurement conditions such as endogenous substances, pH changes, and temperature changes. N-acetyl-D-hexosamine conductor that can provide an excellent substrate that can be used for measurements using a simple test strip method, a method for producing this conductor in high yield, and a method for producing this conductor in high yield. An object of the present invention is to provide a substrate for measuring N-acetyl-β-D-hexosaminidase activity and a measuring method.

[Means and effects for solving the problem] The N-acetyl-D-hexosamine conductor of claim (1) is represented by the following general formula [11].

1 C=0 1 CH3 The N-acetyl-D-hexosamine derivative of claim (2) has the general formula [G in N is fructose, glucose-6-phosphate, sucrose or galactose] in claim (1). characterized by something.

The method for producing an N-acetyl-D-hexosamine derivative according to claim (3) includes, in producing the N-acetyl-D-hexosamine derivative according to claim (1) or (2), after acetylating D-hexosamine. , a compound obtained by reacting with a thiol compound to form an N-acetyl-D-hexosamine-1-thioconductor and then converting it into an oxazoline, and a sugar conductor or a sugar other than glucose at a concentration of 20 to 90%
It is characterized by being reacted under conditions of ℃. Claim (4
) is the N-acetyl-β-D-pekinsaminidase activity measurement substrate of claim (1) or (2).
It is characterized by containing a D-hexosamine derivative.

The method for measuring N-acetyl-β-D-hexosaminidase activity according to claim (5) is a method for measuring N-acetyl-β-D-hexosaminidase activity according to claim (4).
The method is characterized in that the N-acetyl-β101 hexosaminidase activity in the sample is measured by contacting the sample with a substrate for measuring D-hexosaminidase activity and measuring the released sugar or sugar derivative. do. The present invention will be explained in detail below.

The N-acetyl-D-hexosamine derivative (hereinafter abbreviated as rNAcH derivative) of the present invention has the general formula [11
A structure represented by N-acetyl-D-glucosamine or N-acetyl-D-galactosamine bound to a saccharide or a saccharide derivative, preferably fructose, glycol-6-phosphate, sucrose or galactose through a β bond It is. As a result of repeated investigations into a method for obtaining this strong NAcH9 conductor in high yield, we found that when synthesizing an oxazoline compound, the yield was significantly improved by lightening the 1-thio derivative, and the β-glycosidation reaction In this case, they discovered that by keeping the reaction temperature to a certain constant temperature, there were almost no side reactions and the yield improved, and as a result, it was possible to synthesize an NAcH conductor with a significantly high yield, and completed the production method of the present invention. Ta.

The method for producing the NAcH derivative of the present invention includes the following one-shell formula [
D-hexosamine represented by the following general formula [II1] is acetylated to obtain acetyl-D-hexosamine represented by the following general formula [II1], and then converted to the α-octalogeno form by a conventional method (
(not shown here), and by reacting this with a thiol compound, N-acetyl-D-
A derivative of hexosamine-1-1 is converted into an oxazoline compound, which is then converted into an oxazoline compound represented by the following general formula [V], and then mixed with a sugar conductor or a sugar other than glucose at 20-90°C, preferably at 50-90°C. The reaction is carried out at a temperature of 70°C, and the protecting group is finally removed to obtain an NAcH derivative. The method for producing the NAcH derivative of the present invention will be explained in more detail below.

In addition, in the following, AI, A2, BI82. AC,R
are the same as above, and furthermore, Bn is a benzyl group and ph is a phenyl group.

(+) Synthesis of oxazoline compound represented by the above general formula [V] 110 Commercially available glucosamine hydrochloride or galactosamine hydrochloride is acetylated by a conventional method (for example, reacting with an acetic anhydride/pyridine system). i-■ Acetylated form C III] was dissolved in acetyl chloride and HCj2 gas was passed through it to form α-halogen form [
]. i-■ The α-octarogeno form [Vl] was dissolved in HSCSN (CH3)2. in acetone. HSCSN (C2 H
5)2, HSCSOC2 H5, HSCOCH3
, HSCN or its alkali metal salt or SC (N
H2)2 to react with 1-thiotsuba conductor [JV
]. i-1 Oxazoline compound [V] is obtained in high yield by reacting 1-thio derivative [[V] with HgCf2, HgO in acetonitrile. (if) Synthesis of a sugar derivative to be coupled with the oxazoline compound [V] (Leave only the OH group to be subjected to the coupling reaction, and protect other OH groups by replacing them with protective groups.) [Synthesis of fructose flexiconductor] It-■ D-fructose in acetone, concentrated H2SO4
isopropylidene using a catalyst. +1 ■ The isopropylidene compound is reacted with dilute H2SO4 in MoH to remove the isobropylidene groups at the 4 and 5 positions, and acetylated with pyridine/acetic anhydride. fi-■ The acetyl compound is reacted with benzyl chloride in dioxane using xoH as a catalyst to benzylate. if-■ Heat the benzylic compound in an acetic acid aqueous solution to remove the isopropylidene group and obtain a fructose conductor [■]. [Synthesis of glucose-6-phosphate conductor] ii-■ D
Glucose is reacted in benzyl alcohol containing HCIL gas to form pendyl glycosidation.

il-■ Penzyl glycoxide is reacted with benzaldehyde under an acid catalyst to form 4,6-penzylidene. ii-■ 4.6-Pendylidene is acetylated with pyridine/acetic anhydride, debenzylidened in dilute H2SO4, and then phosphoric acid esterified to form glucose-6-phosphate conductor [■] if -■ Shea[ React with benzaldehyde under acid catalyst at low temperature to form penzylidene. ii-■ Acetylate the free 10H group of the penzylidene body. ■1■ The acetyl compound was dissolved in methanol under Pd catalyst with H2
Catalytic reduction is performed with gas to obtain 5181 conductor [1]. Punishment 5'y. ．． -Xm4 bodies. 8] [■] 1i-■ D-
Galactose is reacted in benzyl alcohol containing Hci gas to form pendyl glycosidation. if-■ The pendyl glucoside is reacted with benzaldehyde under an acid catalyst to form 4,6-penzylidene. The galactose derivative [X] is obtained by acetylating the il-■4.6-penzylidene compound with pyridine/acetic anhydride and debenzylidene in dilute H2SO4.

(i i i) Oquinazoline body [V] and sugar bladder conductor [
■] [■] [DC] Synthesis of various groups by coupling with [X] [Coupling between oxazoline [V] and fructose derivative [■]] Oxazoline [V] and fructose derivative [■] is dissolved in dry methylene chloride and reacted by heating at 50 to 70°C in the presence of p-toluenesulfonic acid for coupling. Next, it is deacetylated by reacting with Na O C H 3 in methanol, and the benzyl group is removed by catalytic reduction using a Pd catalyst to obtain the desired NAcH derivative [)a]. [1] [Coupling of oxazoline compound [V] and glucose-6-phosphate derivative [■]] Oxazoline compound [V] and glucose-6-phosphate derivative [■] were dissolved in dry methylene chloride. Coupling is carried out by heating reaction at 50 to 70 tons in the presence of P-toluenesulfonic acid. Next, the desired NAcH conductor [
X [I] (III) [Coupling of oxazoline compound [V] and sucrose derivative [IX]] Oxazoline compound [V] and sucrose derivative [IX] were dissolved in dry methylene chloride, and p-toluenesulfonic acid was added. Coupling is carried out by heating the reaction at 50 to 70°C in the presence of Next, it is deacetylated by reacting with NaOCH2 in methanol to obtain the desired NAcH conductor [chicken]. [Coupling of oxazoline compound [V] and galactose derivative [X]] Oxazoline compound [V] and galactose derivative [X] are dissolved in dry methylene chloride and heated at 50 to 70°C in the presence of p-toluenesulfonic acid. React and couple. Next, it is deacetylated by reacting with NaOCHz in methanol, and then the benzyl group is removed by catalytic reduction using a Pd catalyst to obtain the desired NAcH conductor [XIV]. AC CITYI The substrate for measuring NAG activity of the present invention is produced in this way [)a] to [Xllll! ] and other NACH-approved conductors.

The method for measuring NAG activity of the present invention can be applied to such NAG activity.
This is a method for measuring NAG activity in a sample by contacting a sample with a substrate for measuring NAG activity containing an AcH conductor and measuring the released sugar or sugar derivative.

Below, the measurement mechanism of each substrate according to the present invention will be specifically explained.

■ NA in which G is fructose in the general formula [11]
When using a cH conductor (abbreviated as rNAG-FJ) as a substrate. When NAG in the sample acts on NAG-F, fructose (F) is released. The free fructose is reacted with, for example, mannitol dehydrogenase (MDH) or sorbitol dehydrogenase (SDH) in the presence of NADH to generate NAD, and the amount of fructose is quantitatively measured by measuring the decrease in absorbance. can do.

NAD}I NAD NAI1}l NAfl In addition, by reacting the liberated fructose with, for example, fructose dehydrogenase, the change in absorbance of NAD-NADH or via an electron carrier such as PMS (phenazine methosulfate) can be achieved as shown in the scheme below. , nitro blue tetrazolium (NBT), etc.
It is possible to quantitatively measure the amount of fructose by measuring the formazan color system. Nelmazan NBT ■ NAcH conductor (abbreviated as rNAG-G6PJ) in which G is glucose-6-phosphate in the general formula [I].
) is used as a substrate. When NAG in the sample acts on NAG-06P, glucose-6-phosphate (06P) is formed.
is liberated. When the liberated 06P is reacted with, for example, glucose-6-phosphate dehydrogenase in the presence of NAD (P), NAD (P) is generated in the same manner as described above.
By measuring the increase in H absorbance, the amount of 06P can be quantitatively determined. (2) When using the NAcH derivative of the general formula [11, in which G is sucrose (abbreviated as rNAG-Suc)] as a substrate. NAG in the sample acts on NAG-Suc to produce sicosugar (S
uc) is released. When the liberated sucrose is reacted with α-glucosidase, it dissociates into glucose and fructose, and this fructose is converted into the NADH-N
It can be quantified using an AD or NAD-NADH reaction system and a formazan coloring system using NBT or the like. ?
AG NAG-Suc■Yellow* Suc α-Glucosida 1-Suc--1-----1-1-→G+FNADH NAD Alternatively, when the released sucrose is reacted with phosphate in the presence of sucrose phosphorylase, glucose- 1-phosphoric acid (GIP) and fructose are produced, and this fructose can be measured using a formazan coloring system using NADH-NAD or NAD-NADH and NBT as described above. ,am+! C/Acid Si Ku a-my, Horilla'
GIP+F■ NAcH derivative (rNAG-Gall) in which G is galactose in the general formula [1]
”. ) is used as a substrate.

NAG-Gaj! Galactose (Gaj2) is released by the action of NAG in the sample. The liberated galactose reacts with dissolved oxygen in the presence of galactose oxidase, producing galactose and H202
occurs. Furthermore, H2O2 is reacted with a hydrogen donor (abbreviated as rHDJ) in the presence of peroxidase to produce a pigment. By measuring the absorbance of the dye produced here, the amount of liberated galactose can be quantitatively measured. The HD used here includes, for example, aniline conductors such as dimethylaniline and diethylaniline, phenolic conductors such as 4-chlorophenol and 2,4-dibromophenol, and antipyrolyte conductors such as 4-aminoantivirine. Single conductors, penzothiazolinone derivatives such as 3-methyl-2-benzothiazolinone, naphthol derivatives, hydroxyquinoline conductors, and those commonly used as substrates for peroxidase such as leuco-type dyes. Alternatively, two or more types can be used in combination.

NAG NAG-GajZ-Ichiyu@Gall Gau+02 (Dissolved, i51h-)Prshi9-f Gal outside-, Ki rib lung Ichiyu Ya■202H2
02 + HD Toser dye + H2 0As above, NAG-F%HAG-Suc is NADH-NA
D or NAD-NADH reaction system and formazan coloring system, NAG-G6P is NAD (P) → NAD (
The P)H reaction system is used to measure NAG activity, and in the case of NAG-GafL, color development using a peroxidase substrate is used to measure NAG activity. Therefore, according to the present invention, compared to the conventional method of measuring the absorbance of phenol derivatives, highly accurate measurement is possible with almost no influence from coexisting substances in the sample or measurement conditions. It is. By the way, it was difficult to apply the conventional system for measuring the absorbance of phenol derivatives to the test strip method, but since the substrate of the present invention can direct the reaction system to color development, it can be applied to the test strip method. It is possible.

As mentioned above, NAG-F, NAG-G6P, and NAG-Sus are NADH-NAD or NAD(P)-NAD.
It is detected using a (P)H reaction system. By coexisting diaphorase (or PMS), nitro blue tetrazolium (NBT), etc. in this reaction system, a formazan coloring system can be obtained, and by measuring the coloration due to reduction of NBT, etc., NAG activity can be determined by the test strip method. can be measured. In addition, as mentioned above, NAG-Gau can
G activity is measured.

Note that the measurement conditions are such that the sample and this substrate are reacted at the normal optimal pH for NAG, and there is no need to change the pH, and the detection can be easily performed.

[Examples] The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to the following Examples unless it exceeds the gist thereof.

Example 1 (Manufacture of NAcH conductor) First, an oxazoline form of N-acetylglucosamine was synthesized without going through a 1-thio derivative, and was coupled with a fructose derivative at high temperature (β-glucosylation) to produce the product according to the present invention. NAcHi conductor NAG-F [X[]
Here is an example of synthesizing.

(i) Synthesis of oxazoline compound of N-acetyl-D-glucosamine First, 3.3 g of glucosamine hydrochloride was refluxed with 3.3 g of anhydrous sodium acetate and acetic anhydride at 65 m DEG C. while stirring for 3 hours. The reaction solution was poured into 2JZ ice water and stirred for 6 hours, and the precipitate was filtered through a suction port, thoroughly washed with water, and then dried in vacuum.

Next, perilla oven column (chloroform/ether)
was purified to obtain tetraacetyl-N-acetyl-D-glucosamine [a] in a yield of 95%.

Next, 3.2 g of [a] was dissolved in 100 mL of methylene chloride, 1.6 g of ferric chloride was added, and the mixture was reacted at room temperature for 3 hours. The reaction solution was washed with water, dried over sodium sulfate, and then evaporated to obtain a syrup-like product. It was purified using a silica open column (chloroform/Koichitel) to obtain the oxazoline form of D-glucosamine [b] in a yield of 16% (yield from [a]). (i i) Synthesis of fructose derivative 14.4 g of D-fructose, 280 ml of acetone, 1.4 ml of concentrated sulfuric acid
and stirred at room temperature for 2 hours. The mixture was neutralized by adding an ice-cooled aqueous sodium hydroxide solution, the acetone was distilled off using an evaporator, the mixture was extracted with methylene chloride, and the extract was washed twice with water and dried over anhydrous sodium sulfate. The filtrate was concentrated using an evaporator and then recrystallized from ether/pentane to obtain diisopyridenefructose [C] as a white powder in a yield of 45%.

[c] 0.5g methanol 16mλ, 10% H2
Add 1.75 mj2 of SO4, seal it and let it cool to 1.5 mj2 at room temperature.
I left it for a while. After neutralization by adding K2CO3, the mixture was filtered and concentrated to dryness. After completely drying in vacuum, pyridine 10mj2
and 10 ml of acetic anhydride were added, and after stirring at room temperature overnight, the mixture was concentrated to dryness. The extract was extracted three times with 25 ml of methylene chloride, washed twice with water, dehydrated with calcium chloride, and then concentrated to dryness. Recrystallized from water/methanol to produce isobropylidene triacetyl-D-fructose [d] as a white powder in a yield of 83%.
(Yield from [C]). 84.2 g of KO was pulverized, 4 mjl of dioxane and 5 ml of benzyl chloride were added thereto, and dl was added thereto and refluxed with stirring in an oil bath at 115°C. After 2 hours, the mixture was cooled to room temperature, water was added, and the mixture was stirred overnight. Extract with methylene chloride,
It was washed with water three times, dried with magnesium sulfate, and then evaporated. Furthermore, the benzyl alcohol is removed by vacuum distillation.
After purification using a silica oven column (benzene/ether), isobropylidene-tribenzyl-D-fructose [e] was obtained as a syrup in a yield of 70% (yield from [d]). [80% acetic acid was added to 9.7 g of e-coat and heated at 95° C. for 2 hours. It was concentrated using an evaporator, dried, and purified using a silica oven column (benzene/ether) to obtain tribenzy-D-fructose [■] as a syrup in a yield of 44% (yield from [e]).

(i i i) Coupling of oxazoline compound and fructose derivative (β-glycosylation) 2.1 g of [b] and 2.0 g of [■] were dissolved in 50 ml of anhydrous toluene and nitromethane 1:1 mixture, and 110 to 12
Heated at 0°C for 50 minutes. After cooling, chloroform 200m
jZ was added, washed with a cooled aqueous sodium bicarbonate solution and water, and the organic layer was dried and concentrated using an evaporator. The syrup was recrystallized from chloroform/ether to obtain [f] in a yield of 12% ([ b] yield).

[f] 0.2g methanol 4mJZ10.5NNa
0.08 mL of OMe was added, the mixture was tightly stoppered, and the mixture was stirred at room temperature for 30 minutes. Ion exchange resin Amberlite IR-
Neutralized with 120 (H”) and concentrated to dryness to obtain [g] with a yield of 78
% (yield from [f]).

Next is PdCJ! 21.4g dist. methanol 60
Stir in mIL for 1.5 hours under H2 gas flow, and
acked. Using this catalyst, dist. methanol 6
0mj! 2.0 g of [g] was added thereto, and the mixture was stirred at room temperature for 37 hours under a stream of H2 gas. Pdblack as t[jl
After concentrating with an evaporator, silica oven column (
Purification with 70% isobrobanol/ethyl acetate = 2/1) gave [XI] as a white powder with the following physical properties in a yield of 90% (
[yield from [g]].

Therefore, the yield of [X[] from N-acetyl-D-glucosamine was 1.3%.

Engineering L Yu J Ro 1ij 1. NMR spectrum 1-1 'H-NMR spectrum' H-NMR δ"4: : 2.07" (38
, s, NAc) 1-2 "C-NMR spectrum C-t 72.95 13C-NMR chemical shift [δ (ppm) from
TMS"C-2 (:-3 G-4
C-5 C-698.68 69.21
70.85 70.33 64.84] C-1'C-2' C-3゜ C-4゜ C-5
゜ C-6' NCOCH3 NCOCIs102.
88 56.80 74.95 71.11
77.16 61.93 178.15 23.
502. Elemental analysis (calculated value) C: 43.86, H: 8.57, N
:3.65(*)(Actual IJ{a) C:43.78,
H: 8.62, N: 3.81 (*) 3. Light intensity [
a ] I! 0-58.7° (C-0.89, H2
0) Example 2 (Production example of NAcH derivative via 1-chi 8 conductor) (i) Synthesis of oxazoline form of N-acetyl-D-glucosamine The synthesis of [a] was carried out in the same manner as in Example 1. ．． Next [a]
2.0 g was dissolved in 20 mA of acetyl chloride, and while cooling with dry ice-methanol, HCJZ gas was passed through the solution for 20 minutes. After the mixture was sealed and left for one day, it was concentrated to dryness. Next, 3.69 g of sodium dimethyldithiocarbamate and 40 mj2 of acetone were added and centrifuged for 15 minutes. After concentration, it was extracted with methylene chloride, washed with water, dehydrated, and dried to obtain [h] as a white powder in a yield of 95%. [h] HgCILz 3.9 1 g to 1.2 g, H
After adding 3.92 g of g0 and stirring for 20 minutes in 50 mj2 of acetonitrile, the mixture was filtered and the filtrate was concentrated to dryness. Add methylene chloride and a saturated KI aqueous solution to this, separate the methylene chloride layer using a separating funnel, wash with water, dehydrate, and concentrate to obtain [b] as a white powder with a yield of 97% (yield from [h] ). (ii) Synthesis of fructose derivative [■] was obtained in the same manner as in Example 1. (tit) Coupling of oxazoline and fructose derivative (β
-Glycosidation) [b] 1.38g and [■Co2.40g] of p-toluenesulfonic acid. The mixture was dissolved in OIMdry methylene chloride solution, tightly capped, and stirred at 60°C for 22 hours. An appropriate amount of pyridine was added to this, concentrated, and purified using a silica open force column (chloroform/acetone) to obtain [f] in a yield of 85% (yield from [b]). Next, in the same manner as in Comparative Example 1, [XI]
I got it. The physical properties of this product were similar to those obtained in Comparative Example 1.

The method of this example had a better yield than that of Example 1, and the yield of [X[] from N-acetyl-D-glucosamine was 52.5%.

Example 3 (Measurement of NAG activity using the substrate of the present invention) NAG activity in a test solution was measured using the following reagents and procedure.

Reagent NAG-F 4mL IoIL/fl Sodium chloride 200 mmon/ILMTT”
3.9 1Imail/ItTr
iton X-100 0.5%1-m
ethoxy PMS O. 15 a.m.
ojl/j2 (*: 3-(4.5-dimethyl-2thiazolyl)-2.5-diphenyl-2H tetrazolium bromide) Procedure: 2.7 mJ of the above reagent! Prewarmed at 37℃ and heated to 570n
After confirming that there is no change in absorbance at 0.3 m
A was added and stirred thoroughly, and the change in absorbance was measured at 570 nm. The reaction curve when 200 mU/mj2-NAG was added using this method is shown in FIG. Moreover, the relationship with the reaction rate when various active samples were measured is shown in FIG.

Example 4 (Measurement of NAG activity using the substrate of the present invention) NAG activity in a test solution was measured using the following reagents and procedure.

Reagent NAG-F 4m mob/12Trito
n X-100 0.5% sodium chloride
200 tm moIL/ItNT B
3.9 IIltroll/II1-methoxy PMS O. 15 ra IIlaIL/
11 Procedure The above reagents were infiltrated into a filter paper and freeze-dried. After adding a solution of known NAG activity to the filter paper prepared in this way, the temperature was 23°C.
The reaction time course was determined using a reflection intensity meter (center wavelength: 535 nm), and the results are shown in FIG.

Example 5 (Measurement of NAG activity using the substrate of the present invention) NAG activity in a test solution was measured using the following reagents and procedure.

Reagent NAG-06 P 4 m mail/fL Sodium chloride 200 m moi/fLNA
D 4mmojZ/j! Procedure: Prewarm 2.4 ml of the above reagent at 37°C, and
After confirming that there is no change in absorbance at 340 m, stir
The rate of increase in absorbance was measured at nm, 200 mU/m
The reaction curve when j2-NAG was added by this method is shown in FIG.

From the above results, it is clear that NAG activity can be measured with high accuracy using the substrate of the present invention, and that it is also effective in the test strip method.

[Effects of the Invention] As detailed above, the NAcH derivatives of claims (1) and (2) provide novel compounds useful as substrates for measuring NAG activity. Such NAcH derivatives can be easily and efficiently produced in high yield by the method of claim (3). Therefore, according to the substrate for measuring NAG activity of claim (4) containing the NAcHi conductor of claims (1) and (2) and the method of measuring NAG activity of claim (5) using this substrate, ■ the conventional NAG rate assay, which has been difficult with substrates, is now possible.

■ High measurement accuracy due to the following reasons. ■ With conventional substrates, the half-width of the absorbance measured is narrow, requiring a highly sensitive and accurate analytical device, and the values vary depending on the device, but with this substrate, it is almost unaffected by the device.

■ Not affected by endogenous substances (hemoglobin, bilirubin, surfactants, etc.).

■ Unaffected by pH and temperature changes.

■ It can be applied to the test strip method, which was difficult to use with conventional substrates.

These effects are achieved, and it becomes possible to measure NAG activity easily and with high accuracy.

[Brief explanation of drawings]

Figures 1 and 2 are graphs showing the results of Example 3;
The figure is a graph showing the results of Example 4, and FIG. 4 is a graph showing the results of Example 5.

Claims (5)

[Claims] (1) N-acetyl-D represented by the following general formula [I]
-Hexosamine derivatives. ▲There are mathematical formulas, chemical formulas, tables, etc.▼... [I] [I] In the formula, one of A_1 and A_2 is hydrogen and the other is OH, and G is a sugar derivative or a sugar other than glucose. shows. (2) The N-acetyl-D-hexosamine derivative according to claim 1, wherein G is fructose, glucose-6-phosphate, sucrose or galactose. (3) After acetylating D-hexosamine, it is reacted with a thiol compound to form an N-acetyl-D-hexosamine-1-thio derivative, and then the compound obtained by converting it into an oxazoline, excluding sugar derivatives or glucose A method for producing an N-acetyl-D-hexosamine derivative according to claim 1 or 2, characterized in that the reaction is carried out with sugar under conditions of 20 to 90°C. (4) N-acetyl- containing the N-acetyl-D-hexosamine derivative according to claim 1 or 2.
Substrate for measuring β-D-hexosaminidase activity. (5) N-acetyl-β according to claim 4
-Measure the N-acetyl-β-D-hexosaminidase activity in the sample by contacting the sample with a substrate for measuring D-hexosaminidase activity and measuring the liberated sugar or sugar derivative. Characteristic method for measuring N-acetyl-β-D-hexosaminidase activity.