JP2002105095A - Atp derivative and method for assay using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To eliminate disadvantages in that operations are complicated due to the necessity of a conjugated enzyme, etc., and a considerable cost is required when the activity of a phosphoenzyme is assayed or a substance is assayed using the phosphoenzyme in clinical tests and to provide a method by which the assay can simply be carried out at a low cost. SOLUTION: When the activity of the phosphoenzyme is assayed or a substance is assayed using the phosphoenzyme, a compound in which a part of O atoms contained in the molecule of ATP are substituted with S [e.g. a compound represented by the formula (1)] can be used to produce SH groups when acting as a donor of phosphoric acid. Thereby, SH groups can be assayed to simply carry out the assay at a low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel phosphate donor for use in a method for measuring the activity of a phosphorylase contained in a biological sample or a method for measuring a substance using a phosphorylase, mainly used in the field of clinical testing. The present invention relates to a measuring method, a measuring reagent and a measuring reagent kit using the donor.

[0002]

2. Description of the Related Art Phosphorylase is also called a kinase or a phosphokinase, and is a general term for compounds that transfer a terminal phosphate group of a nucleoside triphosphate such as ATP to a compound other than water and catalyze a reaction to generate a phosphate compound. It is. Most of the phosphate group donors are ATP, and can be represented by the following reaction formula (reaction formula 1). Examples of phosphorylases include hexokinase, creatine kinase, glucokinase, pyruvate kinase, and many others.

[0003]

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For example, when the kinase is creatine kinase, the substrate S is creatine and SP is
It becomes creatine phosphate.

[0005] The activity of a kinase is measured by a method of measuring phosphoric acid released from a phosphorylated compound (corresponding to SP in the above reaction formula 1) which is a reaction product. ATP generated using the leftward reaction of the above reaction formula 1 was measured by using glucose, hexokinase, glucose-6-phosphate dehydrogenase, and NAD (P) (method). Taking creatine kinase as an example and explaining the above method, creatine and ATP are converted into creatine phosphate and ADP by the action of creatine kinase. The reaction scheme is shown in Reaction Scheme 2.

[0006]

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The method is carried out by measuring the amount of inorganic phosphorus released by heating the reaction product, creatine phosphate, in 0.1N HCl at 100 ° C. for 1 minute. In the method, creatine phosphate and ADP are converted to creatine and ATP by the action of creatine phosphate kinase in a reverse reaction to method 1. At this time, ATP which is a reaction product is measured by the following reaction scheme (reaction formula 3), and the activity of creatine phosphate kinase can be measured by measuring the generated NADPH at 340 nm.

[0008]

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As described above, the method of measuring the amount of inorganic phosphorus released from the phosphorylated compound as a reaction product and the method of using a conjugated enzyme are complicated in operation, require many conjugated enzymes, and are costly. Required.

[0010] Phosphorylating enzymes are also used for quantification of substances, for example, measurement of glucose. ATP and hexokinase are allowed to act on glucose. Glucose-6-phosphate is reacted with glucose-6-phosphate dehydrogenase, NADP, and NADP is produced.
The amount of glucose in the sample is determined from the amount of H (see reaction formula 3 and method). In addition, the produced ADP is allowed to act on phosphoenolpyruvate and pyruvate kinase,
The generated pyruvic acid can also be measured by using lactate dehydrogenase NADH (see reaction formula 4, method).

[0011]

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The method may be such that a phosphorylating compound dehydrogenase, such as glucose-6-phosphate dehydrogenase, is present, but the types thereof are not so many and the items that can be measured are limited. Is a decrease reaction. Further, a large amount of a conjugate enzyme is required, which requires a cost. As described above, there are many problems in the method for measuring the activity of a kinase and the measurement of a substance using a kinase.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to complicate the operation such as the necessity of a conjugate enzyme when measuring the activity of a phosphorylase or a substance using a phosphorylase in a clinical test. It is an object of the present invention to provide an inexpensive and easy-to-measure method which eliminates the drawbacks of cost and cost.

[0014]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventor has found that when measuring the activity of a phosphorylase or a substance using a phosphorylase, the ATP molecule is not used. When a compound in which a part of the contained O atom is replaced with S is used, when it acts as a donor of phosphoric acid, S
The present inventors have found that an H group is generated, and that the measurement of the SH group enables inexpensive and simple measurement, thereby completing the present invention.

That is, the present invention provides: An adenosine-5'-phosphate ester derivative, which is a phosphate donor and has a structure in which an SH group is generated. 2. 2. The adenosine-5'-phosphate ester derivative according to the above 1, wherein the adenosine-5'-phosphate derivative is used as a phosphate donor when activity of a kinase is measured or a substance is measured using the kinase. 3. Item 1 or 2 characterized by having the following structure:
Adenosine-5'-phosphate ester derivative according to,

Embedded image X1: S or O and at least one site is S, X2: hydrogen atom, halogen atom, other hetero atom, optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aryl, alkoxy or alkylthio group, nitro group 3. one or more groups selected from a group consisting of: a cyano group, an acyl group, an optionally protected hydroxyl group, an optionally protected or substituted amino group, n = 0 or 1, 4. The adenosine-5′-phosphate derivative according to any one of the above items 1 to 3, having the following structure. ,

Embedded image 5. Adenosine-5 'according to any one of the preceding items 1 to 4
5. a method for measuring a phosphorylase, which comprises using a phosphate ester derivative; 6. the measuring method according to the above item 5, wherein the phosphorylase is creatine kinase; Adenosine-5 'according to any one of the preceding items 1 to 4
7. a method for measuring a substance using a phosphoric ester derivative and a phosphorylating enzyme; 8. a measuring reagent used in the measuring method according to any one of the above items 5 to 7, 9. A measurement reagent kit comprising the measurement reagent according to the above item 8.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The phosphate donor of the present invention generates SH groups when it works. When acting as a phosphoric acid donor, the SH group may be formed by an equilibrium reaction in addition to the case where an SH group is generated. By measuring this SH group, it becomes possible to measure the activity of a kinase or a substance using a kinase.

The compound of the present invention is an adenosine-5'-phosphate derivative having the following structure.

[0018]

Embedded image X1: S or O and at least one site is S, X2: hydrogen atom, halogen atom, other hetero atom, optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aryl, alkoxy or alkylthio group, nitro group And at least one group selected from a group consisting of a cyano group, an acyl group, a hydroxyl group which may be protected, an amino group which may be protected or substituted, and n = 0 or 1.

The substituent is a halogen atom, a cyano group, a carboxyl group which may be protected, a hydroxyl group which may be protected, an amino group which may be protected, an alkyl group which may be protected. Examples include an amino group, an alkyl group, an alkoxy group, an alkoxycarbonyl group, an aryl group, an alkenyl group, a cycloalkyl group, and an alkyl group substituted with a halogen atom. The carboxyl protecting group includes all groups that can be used as a normal carboxyl protecting group, the amino group protecting group includes all groups that can be used as a normal amino group protecting group, and the hydroxyl group includes The protecting groups include all groups that can be used as ordinary hydroxyl protecting groups.

The most preferred compounds of the present invention are adenosine-5'-phosphate derivatives having the following structural formula:

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As the phosphorylase used in the present invention, those widely known can be used. Examples of the phosphorylase include hexokinase, creatine kinase, glucokinase, pyruvate kinase and the like. Is creatine kinase.

The SH group can be measured by a known method. Generally, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), 2,2'-dithiodipyridine,
6'-dithiodinicotine is used. These are NAD
The sensitivity is higher than when (P) H is used as a reaction indicator, and a trace amount of component can be detected. Among them, DTNB is preferably used.

Further, contaminating SH groups are present in the sample,
It is necessary to convert the SH group to one that does not affect the reaction. The method of converting the contaminated SH group into a group that does not affect the reaction may be performed according to a known method.
This can be done by using the method disclosed in US Pat.

The sample to be measured is not particularly limited, but examples include biological components such as saliva, pancreatic juice, blood, serum, and urine.

As the measurement conditions for carrying out the reaction according to the present invention, the reaction temperature is not particularly limited.
The reaction time is 5 to 40 ° C, and the reaction time can be freely selected depending on the purpose. The optimum pH is not particularly limited, but the pH is about 4
To 10 are preferred examples. The buffer for maintaining the optimum pH can be freely selected, and for example, phosphate, trishydroxymethylaminomethane-hydrochloric acid, Good's buffer and the like are arbitrarily selected.

[0026]

Embodiments of the present invention are described below.
As an example of the compound represented by Chemical Formula 2, a method of measuring the activity of creatine kinase as a method of measuring the activity of a phosphorylase using thioATP, and a method of measuring glucose using a hexokinase as a method of measuring a substance are taken as examples. The present invention will be described.

[0027]

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(Method of Measuring Creatine Kinase (CK) Activity) CK is a dimer that is widely distributed in living bodies such as skeletal muscle, myocardium, and brain, and is composed of two subunits of type M and type B. By combining two subunits, CK
There are three types of isozymes: -BB, CK-MB, and CK-MM. CK-BB mainly exists in the brain, and CK-MM mainly exists in skeletal muscle.

Since CK activity in blood is increased in diseases such as progressive muscular dystrophy, polymyositis, acute myocardial infarction and hypothyroidism, it is very important clinically to measure CK activity in blood. It is. In addition, since CK-MB is present at a high concentration in the myocardium, measuring the CK-MB activity in blood is an essential item for diagnosing the presence and degree of myocardial infarction. As described above, the conventional measurement of CK is complicated in operation, requires a large amount of conjugated enzyme, and is expensive.

Therefore, the use of the compound shown in Compound 2 made it possible to easily measure CK activity.
The reaction scheme of this reaction is shown below.

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Further, SH groups are generated by the following equilibrium reaction.

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The CK activity is measured by measuring the SH group formed there. DTNB can be used as a reagent for measuring the SH group, and DTNB can be converted to TNB (2-nitro-5-mercaptobenzoic acid) when the SH group is present.
Are generated and color is developed. The substrate of the present invention further comprises CK-M
It can also be used for CK-MB activity in the presence of a subunit inhibitor.

As the CK-M subunit inhibitor,
An example is an anti-CK-M subunit inhibitory antibody.

(Measurement method of glucose)
Energy sources, energy storage substances, glycoproteins,
It plays an important role in biological functions as a biological constituent such as glycolipid. Clinically, it is known to increase in diabetes, various shocks, and endocrine abnormalities, and to decrease in diseases such as insulinoma, hyperinsulinism, hypopituitarism, and hypothyroidism. As described above, conventional glucose measurement requires a large amount of a conjugate enzyme and is costly.

Thus, it has become possible to easily measure glucose by using the compound shown in Compound 2.

The reaction scheme of this reaction is shown below.

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Further, an SH group is generated by the following equilibrium reaction.

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The glucose in the sample is measured by measuring the SH group formed there.

In addition, not only the relationship between a kinase and a substrate such as hexokinase and glucose, but also a substance to be measured can be converted to react with the kinase, and then the kinase and adenosine-5'-phosphate are converted. The measurement object may be measured using an ester derivative.

The thioATP can be synthesized by the reaction scheme shown in FIG.

[0041]

The following examples are given, but the present invention is not limited to these examples.

[0042]

Example 1 (Synthesis) J. Biol. Chem, 243, 4671-4676, 1968
Methoxythiophosphoric acid was prepared based on the described synthesis method.
Commercially available Adenosine-5'-monophosphate (1.0 mmol) and methoxyphosphoric acid (10 mmol) were dissolved in 20 mL of pyridine, and when completely dissolved, N, N'-dicyclohexylcarbodiimide (DCC condensing agent, 50 mmol) was added. Stir at 24 ° C. for 24 hours.

The precipitated dicyclohexylurea is filtered and washed with 100 mL of purified water. The filtrate is concentrated at 30 ° C or lower. Two
Dissolve the concentrate in 0 mL purified water. Concentration is again performed to completely remove pyridine, and the finally obtained concentrate is dissolved in 100 mL of purified water. Biochem Prep 8 (1) 1-4 (1961)
Using d-washedcharcoal, processing was performed to obtain 40 mL (1 MN
Further, according to the same reference, Dowex2 (C
l ^-)) using anion-exchange column (7 cm × 2.2 cm in diameter) 0.0
Perform a gradient with 0.02-0.15MLiCl solution with 03M HCl,
Adenosine-5 '-(β-thio) diphosphate-β-O-metylester
Was obtained (0.25 mol). The yield was 25%.

This Adenosine-5 '-(β-thio) diphosphate-
β-O-metylester (0.25mol) and 85% orthophosphoric acide
_{H 3 PO 4 · H 2 O} (2.5mol) similarly pyridine above operation (5 mL)
And tri-n-butylamine (5.25 mmol), and the same treatment was performed.The gradient was Adenosine-5 '-(β-thio) trip
Hosphate-β-O-metylester was obtained (0.0875 mol). 35% yield
Met.

Elemental analysis of this final sample of this final synthesized product showed that C: 18.9 H: 4.50 N: 10.0 P: 13.3 S: 4.6
Actual value is C: 19.1 H: 4.3 N: 9.8 P: 1
3.7S: 4.7, confirming that this final product is the target product.

[0046]

[Example 2] (Confirmation of time course) 24 mmoL / L
Creatine, 7.2 mmol / L MgSO ₄ , 0.
100 mol / LGlycine buffer (pH 8.8) containing 3 mmol / L DTNB and 1.0% Triton X-100 was used as a first reagent, and 100 mol / LG was used.
Adenosine-5 '-(β) in lycine buffer (pH 8.8)
-thio) triphosphate-β-O-metylester concentration is 18mm
What was added so as to be oL / L was used as the second reagent.
Purified water and human serum were used as samples.
After adding 00 μL, the mixture was preliminarily heated at 37 ° C. for 5 minutes. Next, 60 μL of the second reagent was added. The change in absorbance at a measurement wavelength of 415 nm was measured.

The reaction proceeded linearly with almost no blank reaction, and no lag time was observed.

[0048]

[Example 3] (Confirmation of time course) 5 mmoL / L
MgSO ₄ , 0.3 mmol / L DTNB, 3 mmol
L / L Adenosine-5 '-(β-thio) triphosphate-β-O-met
1 containing ylester and 1.0% Triton X-100
00 mmol / L Glycine buffer (pH 8.
8) as the first reagent, 100 mmol / LGlycine buffer containing 15 U / mL hexokinase (pH 8.
8) was used as the second reagent. Purified water and human serum were used as samples, and 300 μL of the first reagent was added to 30 μL of the sample, and preliminarily heated at 37 ° C. for 5 minutes. Next, 60 μL of the second reagent was added.
The change in absorbance at a measurement wavelength of 415 nm was measured.

The reaction was completed in about 2 minutes with almost no blank reaction, after which the absorbance was stable.

[0050]

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for synthesizing thioATP.

Continuation of the front page (72) Inventor Tateo Honda 1-2-1 Muroya, Nishi-ku, Kobe-shi, Hyogo F-term in the International Reagents Research and Development Center 4B063 QA01 QA19 QQ03 QQ27 QQ68 QR07 QR42 QS28 QX01 4C057 AA20 BB02 CC03 DD03 LL27 LL41 LL46

Claims (9)

[Claims] 1. Adenosine-5′-phosphate donor, which has a structure in which an SH group is generated.
Phosphate derivatives. 2. The adenosine-5'-phosphate ester according to claim 1, wherein the adenosine 5'-phosphate ester is used as a phosphate donor when the activity of a kinase is measured or a substance is measured using the kinase. Derivatives. 3. The adenosine-5′-phosphate derivative according to claim 1, wherein the derivative has the following structure. Embedded image X1: S or O and at least one site is S, X2: hydrogen atom, halogen atom, other hetero atom, optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aryl, alkoxy or alkylthio group, nitro group And at least one group selected from a group consisting of: a cyano group, an acyl group, an optionally protected hydroxyl group, an optionally protected or substituted amino group, and n = 0 or 1. 4. The adenosine-5′-phosphate derivative according to claim 1, which has the following structure. Embedded image 5. A method for measuring a phosphorylase, comprising using the adenosine-5′-phosphate derivative according to any one of claims 1 to 4. 6. The method according to claim 5, wherein the kinase is creatine kinase. 7. A method for measuring a substance using the adenosine-5′-phosphate ester derivative and the kinase according to any one of claims 1 to 4. 8. A measuring reagent used in the measuring method according to claim 5. 9. A measurement reagent kit comprising the measurement reagent according to claim 8.