JPH0631292B2 - N-acetyl-β-D-hexosamine derivative and N-acetyl-β-D-hexosaminidase activity measuring reagent using the derivative as a substrate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an N-acetyl-β-D-hexosamine derivative and an N-acetyl-β-D-hexosaminidase (hereinafter referred to as NAGase) using the derivative as a substrate. The present invention relates to an activity measuring reagent.

NAGase is one of the enzymes present in lysosome, which is abundant in renal tubular epithelium, and is involved in the degradation of mucopolysaccharides and glycoproteins. It is recognized that urinary NAGase is elevated after various renal diseases and renal surgery, and that not only urine but also serum NAGase is elevated in diabetes. The detection of such various renal diseases is used as an index for observing the course of treatment, studying drug nephrotoxicity, and clinically and in animal experiments.
Atase measurement is drawing attention.

[Problems to be Solved by Conventional Techniques and Inventions]

Conventionally, p-nitrophenyl-N-acetyl-β-D-glucosaminide [Biochemi was used for the measurement of NAGase activity.
cal Preparations, 10 , 118 (1963)] and Δ-methylumbelliferyl-N-acetyl-β-D-glucosaminide [Clinica Chimica Acta, 69 (1), 85 (1976)] are widely used. However, the former is easily affected by biological components such as bilirubin and hemoglobin at the measurement wavelength, it is necessary to measure a blank for each sample, and the operation for measurement is complicated.
Since the optimum pH of AGase (pH 4-5.5) and the color development pH of p-nitrophenol (pH 9 or higher) are different, it is necessary to separately perform the enzyme reaction and the color reaction when measuring NAGase activity. Since the number of reagents and the number of operation steps increase, it is not possible to apply the rate analysis method, which is the most appropriate method for determining enzyme activity.
The latter also has the drawback of requiring special equipment such as a fluorometer.

Further, m-cresolsulfonephthaleinyl-N-acetyl-β-D-glucosaminide is known as an improved substrate which does not require a blank test (JP-A-58-994). However, in this case, since the endpoint method is used in which a reaction terminator reagent is added and the color is developed in alkalinity, the rate analysis method cannot be applied and it is necessary to perform the measurement with the two-reagent method, which requires a long time and is automated. There is a problem that the model for is limited.

Further, a chemical synthesis method (JP-A-61-11092) is known as a method for producing the compound (I) of the present invention, but these methods produce various by-products or require complicated reaction steps. However, it is hard to say that it has been completed to the extent that it can be put to practical use. Moreover, the specifically described 2-chloro-4-nitrophenyl-N-acetyl-β-
When D-glucosaminide is used as a substrate, the water solubility is extremely poor, so that it is difficult to dissolve the mass of the substrate required for NAGase activity measurement, and accurate measurement cannot be performed. Furthermore, in the NAGase activity measurement range pH 4.0 to 6.0, the chromogenic group 2-
There was a problem that chloro-4-nitrophenol could not show sufficient sensitivity.

[Means for solving problems]

It is an object of the present invention to provide a compound which is excellent in water solubility and can be used as a substrate for NAGase activity measurement with high sensitivity and rate assay, and a NAGase activity measurement reagent using the compound as a substrate.

The present inventors have conducted extensive studies to achieve such an object, and as a result, the compound represented by the general formula (I) has excellent water solubility, and by using this compound as a substrate, NAGase activity in body fluid can be reduced in a short time. The present invention has been achieved by finding that the rate assay can be performed accurately and easily with.

That is, the present invention has the general formula (I) (In the formula, X represents a hydrogen atom, a halogen atom or a methoxy group, and n is 2 to 4.) The N-acetyl-β-D-hexosamine derivative represented by the formula (I) as a substrate. N-acetyl-containing a compound represented by
N-acetyl-β-D-containing a cyclodextrin together with a reagent for measuring β-D-hexosamidase activity and the compound represented by the general formula (I).
The present invention relates to a reagent for measuring hexosaminidase activity.

The compound represented by the general formula (I) is N-acetylhexosamine or a derivative thereof which is β-bonded with a 4-nitrophenol group which is fluorine-substituted at the reducing end thereof. This compound is, for example, acetylated N-acetylhexosamine, and after coupling the acetylated N-acetylhexosamine with a fluorine-substituted 4-nitrophenol, for example, 2,3-difluoro-4-nitrophenol, it is deprotected. Acetylation method (Journal of Experimental Chemistry, Vol. 24, p. 304, 1958), halogenation of acetylated N-acetylhexosamine, and then its halide and fluorine-substituted 4-nitrophenol, for example, 2,3. -Method of deacetylating ether-bonded difluoro-4-nitrophenol (Methods in Carbohydrate Chemistry, II, No. 334)
Page).

In the fluorine-substituted 4-nitrophenol group of the compound represented by the above general formula (I), when the fluorine atom is replaced with another halogen atom, the solubility in water is poor and the present invention intends. It is inappropriate to use it as a substrate.

Examples of the fluorine-substituted 4-nitrophenol used in the present invention include 2,3-difluoro-4-nitrophenol, 2,5-difluoro-4-nitrophenol,
2,6-difluoro-4-nitrophenol, 3,5-
Difluoro-4-nitrophenol, 2,3,5-trifluoro-4-nitrophenol, 2,3,6-trifluoro-4-nitrophenol, 2,3,5,6-tetrafluoro-4-nitrophenol There is.

When the compound represented by the general formula (I) of the present invention is used as a reagent for measuring NAGase activity, it is dissolved in a buffering agent capable of maintaining a value of pH 4.0 to 6.0 which is the optimum pH of NAGase in body fluid. There are various buffers that satisfy this requirement, and examples thereof include buffers for organic acids such as citric acid, acetic acid, succinic acid, and phthalic acid. Furthermore, the measurement sensitivity of NAGase activity can be improved by adding cyclodextrin. Here, as cyclodextrin, α-cyclodextrin, β
-Cyclodextrin, γ-cyclodextrin and the like can be mentioned, and α-cyclodextrin is preferable.
There is no particular limitation on the substrate concentration, and the concentration showing the maximum NAGase activity is appropriate, usually 1 mM or more,
It is preferably 3 to 10 mM.

If necessary, a surfactant, a preservative, sodium chloride, a stabilizer and the like can be appropriately added to the reagent of the present invention.

To measure NAGase activity using the reagent of the present invention, the reagent is reacted with a sample to hydrolyze the substrate, and the absorbance change due to the color development of the aglycone produced, that is, fluorine-substituted 4-nitrophenol, is directly measured. Colorimetrically quantitate using a spectrophotometer. That is, a test tube containing a reagent containing a substrate and a test tube containing a buffer solution without the substrate in place of the reagent are prepared and heated at 37 ° C. for 5 minutes. Then, the sample is added to the reagent and reacted at 37 ° C. for 15 minutes, and then a reaction stop solution is added to stop the reaction. On the other hand, the purified water is put into another test tube, the same reaction is performed, and then the reaction is stopped. For the absorbance of the sample, use the liquid excluding the substrate as a control,
The sample blank absorbance is measured at the maximum absorption wavelength (400 nm) of aglycone by the rate assay method using water as a control.

〔Example〕

 Next, the present invention will be described in detail with reference to Examples.

Example 1 50 g of N-acetyl-tetra-O-acetyl-D-glucosamine, 50 g of 2,3-difluoro-4-nitrophenol and 12.5 g of zinc chloride were taken and heated at 130 ° C. Then, it was extracted with benzene, evaporated to dryness, and recrystallized with anhydrous alcohol to give 2,3-difluoro-4-
Nitrophenyl-N-acetyl-β-D-glucosaminide was obtained. The physicochemical properties of this compound are shown below.

Melting point: 146-148 ° C. [α] ²⁴ _D ▼: −26.1 (water) UV: λ _max 299 nm (water) Example 2 The amount of NAGase activity in a test solution was measured by the following method using the following reagents. did.

Reagent A 2,3-difluoro-4-nitrophenyl-N-acetyl-β-D-glucosaminide 2 mM α-cyclodextrin 0.2% Sodium chloride 200 mM Citrate buffer 0.05M pH5.0 Reagent B 2,3-difluoro-4 -Nitrophenyl-N-acetyl-β-D-Glucosaminide 10 mM α-Cyclodextrin 0.2% Sodium chloride 200 mM Citrate buffer 0.05M pH5.0 Reagent C 2,3-Difluoro-4-nitrophenyl-N-acetyl-β -D-glucosaminide 2mM sodium chloride 200mM citrate buffer 0.05M pH5.0 Reagent D 2,3-difluoro-4-nitrophenyl-N-acetyl-β-D-glucosaminide 10mM sodium chloride 200mM citrate buffer 0.05M pH5 .0 Reagent E 2-chloro-4-nitrophenyl-N- Acetyl-β-
D-Glucosaminide 2 mM Sodium chloride 200 mM Citrate buffer 0.05 M pH 5.0 Reagent F 2-Chloro-4-nitrophenyl-N-acetyl-β-
D-Glucosaminide 10 mM Sodium chloride 200 mM Citrate buffer 0.05 M pH5.0 NAGase-containing test solution 50 μM was added with 3 ml of the above reagent and heated at 37 ° C. for 3 minutes, and then the change in absorbance was 400 nm.
And the change in absorbance for 1 minute was determined (blank is N
Water was used instead of the test solution containing AGase). The results are shown in Table 1.

Reagents A, B, C and D of the present invention have excellent substrate solubility as compared with Comparative Examples E and F. Furthermore, the reagent A of the present invention,
B has a higher measurement sensitivity than reagents C and D.

〔The invention's effect〕

The compound of the present invention represented by the general formula (I) is excellent in water solubility and can be used as a substrate for NAGase activity measurement which enables rate assay. further,
High sensitivity N by adding cyclodextrin
It is possible to measure AGase activity. Therefore, by using the reagent of the present invention, NAGase activity in body fluid can be measured accurately and easily in a short time.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshio Nishikawa 514-2 Ogawa, Yaizu City, Shizuoka Prefecture (72) Inventor Minoru Uemura 514-2 Ogawa, Yaizu City, Shizuoka Prefecture (56) Reference JP-A-61-112092 (JP) , A) JP-A-1-19090 (JP, A)

Claims (3)

[Claims] 1. A general formula (I) (In formula, X shows a hydrogen atom, a halogen atom, or a methoxy group, and n is 2-4.) The N-acetyl- (beta) -D-hexosamine derivative represented by these. 2. A substrate of the general formula (I) (In the formula, X represents a hydrogen atom, a halogen atom or a methoxy group, and n is 2 to 4.) N-acetyl-β-D-hexosamine derivative represented by the formula: -Β-D-
Hexosaminidase activity measurement reagent. 3. General formula (I) (In the formula, X represents a hydrogen atom, a halogen atom or a methoxy group, and n is 2 to 4.) An N-acetyl-β-D-hexosamine derivative and cyclodextrin are contained. Reagent for measuring N-acetyl-β-D-hexosaminidase activity.