JPH0541636B2 - - Google Patents

Description

[Detailed description of the invention]

[0001] The present invention relates to β-(2-chloro-4-nitrophenyl)-maltopentaoside.

[0002] The β-(2-chloro-4-nitrophenyl)-maltopentaoside of the present invention can be used as an α-amylase measurement reagent for measuring α-amylase contained in serum or other biological body fluids. Useful.

[0003] Among the reagents for measuring α-amylase known so far, oligosaccharide glycosides include, for example, oligosaccharides in which paranitrophenol is bound to the α position (JP-A-53-12831, JP-A-54 -51892) or an oligosaccharide with a halogenated phenyl group (Japanese Patent Application Laid-open No. 56-35998). In addition, JP-A-53
-12831 shows a phenyl group substituted at the reducing end of maltopentaoside, but according to the detailed explanation, the bonding state of the phenyl group is α at the reducing end of maltopentaoside. - bond, and only para-nitrophenyl group is shown as the substituted phenyl group. When α-amylase was measured using these known substrates, it was found that with the former substrate, the action of α-amylase was slow when the oligosaccharide was a short chain of 4 or less, and when the oligosaccharide was 5 and the substituted phenyl For substrates with α-coordinated groups and oligosaccharides with six or more groups, two or more α-glucoside bonds are cleaved in the substrate molecule. This means that the product produced by the reaction between α-amylase and the substrate further acts as a substrate for the enzyme, and therefore the stoichiometry of the reaction does not hold, and the late assay method It cannot be said that it is a preferable substrate. Furthermore, when the latter substrate is used, there are drawbacks such as the fact that measured values are easily affected by therapeutic drugs such as phenol derivatives administered into body fluids, and late assays are also extremely difficult.

[0004] Therefore, the present inventors conducted research to find a substrate suitable for amylase measurement without the above-mentioned drawbacks, and found that only a substrate with 5 oligosaccharides and a substituted phenyl group in the β-coordination was found to be α-coordinated. -Amylase mainly cleaves one α-1,4-glucoside bond, and 2-chloro- is stable around pH 7.0 and has a maximum molecular extinction coefficient.
It has been found that particularly excellent measurement results can be obtained when a 4-nitrophenyl group is used.

[0005] The present invention is based on the following formula

[Case 2]

[Chemical formula] β-
(2-chloro-4-nitrophenyl)-maltopentaoside.

[0006] The compound of the present invention can be produced by the following method. The following formula

[Chemical formula 3]

Maltopentaose represented by [Chemical formula] is reacted with an organic acid anhydride represented by the following formula (RCO) ₂ O () (in the formula, R means an alkyl group) to obtain the following formula:

[C4]

Compound (heptadecaacylmaltopentaose) represented by [Formula R has the above meaning]
is halogenated to form the following formula:

[C5]

Compound (1-halogeno-1-deoxyhexadecaacylmaltopentaose, also known as hexadecaacylmaltopentaosyl halide) represented by:
, and then the following formula

[C6]

The following formula obtained by reacting 2-chloro-4-nitrophenol represented by [chemical formula] in the form of its organic salt or in the presence of an organic base

[C7]

[Chemical formula] (wherein R has the meaning described above)
The compound of formula (1) is obtained by deacylating (2-chloro-4-nitrophenyl)-hexadecaacyl maltopentaoside.

[0007] The reducing terminal of the malto-oligosaccharide shown in JP-A-56-35998 is an anomeric carbon, and conventionally the substituent on this carbon has been obtained only as a mixture of α and β coordination, and its isolation has been difficult. Purification was considered almost impossible. However, by employing the method of the present invention, it has become possible to isolate and purify maltopentaoside in which a 2-chloro-4-nitrophenol group is β-bonded to the reducing end.

[0008] Each reaction of the present invention will be explained below. Acylation reaction of hydroxyl group: Acylation of maltopentaose (2) is carried out by a known method, for example, heat treatment in an organic acid anhydride as a reactant, preferably in the presence of a catalyst such as an alkali metal salt of an organic acid anhydride. Implemented by doing. Examples of the organic acid anhydride represented by (RCO) ₂ O include acetic anhydride, propionic anhydride, butyric anhydride, and the like. As a catalyst, sodium salt of anhydrous organic acid,
Alkali metal salts such as potassium salts, pyridine, collidine, etc. are used. In order to facilitate reaction control or purification of the target product after the reaction, a non-aqueous solvent such as chloroform, dichloromethane, etc. may be added to the reaction solution. The amount of organic acid anhydride used in the above reaction is 5 to 50 times the weight of maltopentaose, preferably 7 to 15 times, and when an alkali metal salt of an anhydrous organic acid is used as a catalyst, The amount is 0.5 to 3 times the weight of maltopentaose, preferably 0.5 to 3 times the weight of maltopentaose.
It is 1.5 times.

[0009] The reaction temperature is usually about 90-140°C, preferably 100-110°C. The reaction time is influenced by the reaction temperature, but is about 2 to 4 hours under preferred reaction temperature conditions. The reaction mixture was adjusted to 0 to 0 by a conventional method.
Cool to 5°C, separate the precipitated solids, wash with water, and then dry. The obtained solid product (heptadecaacylmaltopentaose IV) can be prepared by using solvents such as alcohols such as ethanol and methanol, ketones such as methyl ethyl ketone and acetone, and ethers such as dimethyl ether and diethyl ether alone or in combination. Although the solid product can be used for recrystallization, the solid product can also be sufficiently dried and used as it is for the next reaction.

[0010] Terminal halogenation: Halogenation of heptadecaacylmaltopentaose () is carried out with anhydrous hydrogen halide, aluminum chloride and phosphorous pentachloride, or titanium tetrachloride, stannic chloride, etc., but the product In view of the yield, suppression of related side reactions, and ease of purification of the target product, a method of treatment using anhydrous titanium tetrahalide in a low polar nonaqueous solvent such as chloroform or dichloromethane is particularly preferred. . As the anhydrous titanium tetrahalide, titanium tetrachloride, titanium tetrabromide, titanium tetraiodide, etc. can be used, and the amount of anhydrous titanium tetrahalide relative to heptadecaacyl maltopentaose is usually 1 to 20%. It may be twice the molar amount, preferably 3 to 8 times the molar amount.

[0011] This halogenation reaction is carried out at normal pressure between room temperature and the boiling point of the solvent used, but it is particularly preferably carried out at the boiling point of the solvent while refluxing. The reaction time is affected by the reaction temperature, but when reacting near the boiling point of the solvent, it usually takes 30 minutes or more.
It takes about 1.0 hours. The reaction mixture is cooled in a conventional manner, an organic solvent such as chloroform, dichloromethane, ethyl acetate, etc. is added thereto, the organic solvent layer is separated, washed several times with water, a saturated aqueous solution of sodium bicarbonate, etc., and then dried to dryness.

[0012] The obtained solid product () is separated and purified by conventional methods such as silica gel chromatography, and then separated into alcohols such as ethanol and methanol, ketones such as methyl ethyl ketone and acetone, and ethers such as dimethyl ether and diethyl ether. It can be recrystallized by using similar solvents alone or in combination, or it can be used in the next reaction after being sufficiently dried as a dry solid.

[0013] Substitution reaction: The anomeric halogen group of the 1-halogeno-1-deoxyhexadecaacylmaltopentaose () is substituted with a 2-chloro-4-nitrophenoxy group to form β-(2-chloro- 4-Nitrophenyl)-hexadecaacylmaltopentaoside () is obtained. The amount of 2-chloro-4-nitrophenol used in this reaction is 1 to 20 times the molar amount, preferably 1.2 to 6.0 times the molar amount.
It is twice the mole.

[0014] 2-chloro-4-nitrophenol needs to be dissociated as a salt in the reaction solvent in order to promote this reaction, and for this reason, an organic salt of 2-chloro-4-nitrophenol, For example, triethylamine salt, tributylamine salt, pyridine salt, picoline salt, etc. are used. Two or more of these salts can be used together, and
-An organic base may be added to the reaction solution without preparing the chloro-4-nitrophenol salt, or the organic base may be used directly as a reaction solvent. The amount of base added is preferably the amount necessary to maintain the liquid in a neutral or alkaline state until the reaction is completed.

[0015] This reaction is usually preferably carried out in the presence of a solvent. The solvent is not particularly limited as long as it does not participate in this reaction, but a solvent that has a high solubility for hexadecaacyl maltopentaosyl halide and 2-chloro-4-nitrophenol or a salt thereof and that increases the reactivity is used. Preferably, for example, the following solvents are used. Amides such as methylformamide, dimethylacetamide, etc., nitriles such as acetonitrile, benzonitrile, etc., dimethylsulfoxide, organic bases such as trialkylamines, pyridine, lutidine, etc., aromatic hydrocarbons such as benzene, toluene, etc., and mixtures of two or more of these. liquid.

[0016] This reaction generally proceeds at about -5 to 100°C, but a reaction temperature of 10 to 50°C is usually preferred. The reaction time varies depending on the type of base used as a reaction aid and the reaction temperature, but is usually 5 to 20 hours. After completion of the reaction, the reaction mixture is poured into ice water and the precipitated solid is collected by filtration, or the target product is extracted with an appropriate organic solvent, dried and then evaporated to dryness to obtain a solid. The compound is obtained as a solid. This can be purified by a conventional method, for example, by applying an appropriate combination of column chromatography using alumina, silica gel, etc., crystallization method using an organic solvent, and the like.

Deacylation Reaction: Removal of acyl groups from compounds can be carried out in known manner, for example in the presence of a catalyst such as an alkali metal alkoxide in dehydrated methanol or a methanolic solution of anhydrous ammonia. As the alkali metal alkoxide, for example, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, potassium t-butoxide, etc. can be used.

[0018] In order to facilitate the purification of the target product after the reaction is completed, it is preferable to add a low polar non-aqueous solvent such as chloroform or dichloromethane to dehydrated methanol for the reaction. The low polar non-aqueous solution added does not inhibit the deacylation reaction and the generated 2-chloro-
Since it is necessary for 4-nitrophenyl maltopentaoside to be precipitated from the reaction system, the amount thereof varies depending on the solvent, but is preferably 0.5 to 2 times the amount of dehydrated methanol used.

[0019] The deacylation reaction is completed within 6 to 24 hours at a temperature of 0 to 30°C. In a reaction system using dehydrated methanol as a sole solvent, methanol is distilled off under reduced pressure after the reaction is completed, and the resulting solid is treated with an acidic ion exchange resin or an inorganic acid to neutralize the mixed basic substances. The compound is purified by layer chromatography, column chromatography, etc. In the case of a reaction system in which a low polar solvent is added, the target product will precipitate from the reaction solution, so this should be collected by filtration.
It can be subjected to a separation and purification process.

[0020] When measuring α-amylase activity using the substrate obtained as described above, there are the following advantages. (1) The substrate has five oligosaccharides, and the bonding state of the 2-chloro-4-nitrophenyl group, which is a substituted phenyl group, is β coordination, so it is cleaved by α-amylase in the substrate molecule. α-1,4-
There is only one glucoside bond, and this cleavage site is the same in pancreatic amylase and salivary amylase, which account for the majority of α-amylase in human body fluids, so it is not possible to detect the α-amylase reaction stoichiometrically. can. When α-amylase is measured using this substrate, the theoretical value and the measured value match, and the reliability of the measurement system is significantly improved compared to conventional methods.

(2) The substrate is hydrolyzed under optimal conditions by the action of α-amylase at a specific and rapid reaction rate. Chromophore 2 is also quantified colorimetrically.
-Chloro-4-nitrophenol has a large molecular extinction coefficient at its absorption peak and can be measured with extremely high sensitivity.

[0022] The β-(2-chloro-4-nitrophenyl)-maltopentaoside of the present invention is extremely useful as a reagent for measuring α-amylase contained in serum or other biological fluids.

[0023]

[Example 1] (A) Production of heptadecaacylmaltopentaose Maltopentaose 20g (24mmol), acetic anhydride
A mixture of 262 ml and 19.8 g of anhydrous sodium acetate
The mixture was stirred at 103°C for 4 hours, then poured into ice water and stirred overnight. The sticky substance was ground in ice water and filtered. The obtained crystals are recrystallized from ethanol to obtain 32.6 g of heptadecaacetyl maltopentaose (21 mmol, 87.5%).

[0024] Melting point: 125-130°C Infrared spectrum cm ^-1 : 1740, 1370, 1230, 1030 Thin layer chromatography (silica gel, developing solvent:
Benzene/ethyl acetate = 2:3): Rf = 0.47 Elemental analysis value: as C ₆₄ H ₈₆ O ₄₃ C H Theoretical value (%) 49.81 5.62 Measured value (%) 49.30 5.72

(B) Production of hexadecaacetylmaltopentaosyl chloride A mixture of 5g (3.2mmol) of heptadecaacetylmaltopentaose obtained in (A), 25ml of chloroform, and titanium tetrachloride was refluxed for 1 hour. Stir and
Add 300ml of chloroform to the reaction solution and dilute with 100ml of water.
After washing twice, anhydrous sodium sulfate is added to the chloroform layer to dehydrate it, and then concentrated to dryness. 4.8 g of the obtained crude product was purified by silica gel column chromatography, and the fraction eluted with a benzene-ethyl acetate mixture (volume ratio 4:3) was recrystallized from methanol to yield 3.2 g of hexadecaacyl maltopentae. Sil chloride is obtained (2.1 mmol,
65%).

[0026] Melting point: 175-132°C Infrared absorption spectrum cm ^-1 : 1750, 1370, 1250,
1040, 760 Thin layer chromatography (silica gel, developing solvent:
Benzene/ethyl acetate = 2:3): Rf = 0.50 Elemental analysis value: C ₆₂ H ₈₃ O ₄₁ As Cl C H Theoretical value (%) 49.00 5.56 Measured value (%) 48.56 5.58 (C) β-(2-chloro -4-Nitrophenyl)-hexadecaacetyl maltopentaoside 3 g (2 mmol) of the compound obtained in (B) and 1.8 g (10 mmol) of 2-chloro-4-nitrophenol were dissolved in 30 ml of dehydrated benzene. , triethylamine 2.5ml
and heated to reflux with stirring for 2 hours. The mixture is then poured into approximately 100 ml of ice water and extracted with 200 ml of ethyl acetate. The extract is washed with saturated aqueous sodium bicarbonate solution and water, and the organic solvent layer is dried over anhydrous sodium sulfate and then dried under reduced pressure to yield 3.1 g of crude product. This product was purified by silica gel column chromatography, and the fraction eluted with a benzene-ethyl acetate mixture (volume ratio 4:3) was recrystallized from methanol.
-(2-chloro-4-nitrophenyl)-hexadecaacetyl maltopentaoside 1.4 g (0.8 mmol,
40%).

[0028] Melting point: 123-128°C Ultraviolet absorption spectrum: Maximum absorption wavelength [λ _nax ] =
283nm Molecular extinction coefficient (ε) = 8900 (CHCl ₃ ) Infrared absorption spectrum cm ^-1 : 1740, 1580, 1520,
1480, 1360, 1200, 1020 Thin layer chromatography (silica gel, developing solvent:
Benzene/ethyl acetate = 2:3) Rf = 0.50 Elemental analysis value: C ₆₈ H ₈₆ O ₄₄ as NCl C H Theoretical value (%) 49.30 5.23 Measured value (%) 48.71 5.35

(D) Process for producing β-(2-chloro-4-nitrophenyl)-maltopentaoside Dissolve 1 g (0.6 mmol) of the compound obtained in (C) in a mixture of 7 ml of dehydrated methanol and 7 ml of dichloromethane. Then, 1.0 ml of 0.5N sodium methoxide is added while stirring at room temperature, and the mixture is allowed to react for 16 hours. After the reaction, the precipitate was collected by filtration, washed with a mixture of dehydrated methanol and dichloromethane (1:1), and dried under reduced pressure to give crude β-(2-chloro-
4-Nitrophenyl)-maltopentaoside 0.55g
(0.56 mmol, 93%) of this crude product
0.55g is purified by column chromatography on Biogel P2 using water, and 0.41g of β-(2-chloro-4-nitrophenyl)-maltopentaoside having the following physicochemical properties is obtained from the middle distillate. (0.42 mmol, 70%).

[0030] Melting point: 198-201°C Ultraviolet absorption spectrum: Maximum absorption wavelength [λ _nax ] =
295nm Molecular extinction coefficient (ε) = 8100 (H ₂ O) Infrared absorption spectrum cm ^-1 : 3400, 2920, 1580,
1520, 1480, 1350, 1270, 1020 Nuclear magnetic resonance spectrum (250MHz) ppm 8.31 (d, J, = 3Hz, 1H) 8.18 (dd, J = 3Hz, J = 9Hz, 1H) 7.43 (d, J = 9Hz, 1H) 5.34-5.57 (m, 9H) 3.92-3.03 (m, 26H) The fact that the 2-chloro-4-nitrophenyl group of this substance is in the β-position means that both α-glucosidase and β-glucosidase enzymes It was confirmed using

【0031】

[Experimental Example 1] The reactivity of human pancreatic amylase (hereinafter referred to as P-amylase) was measured using the following reagent. Reagent A (substrate solution): β-(2-chloro-4-nitrophenyl)-maltopentaoside (hereinafter referred to as G5β-
CNP) and α-(4-nitrophenyl)-
Maltopentaoside (hereinafter referred to as G5α-PNP)
Prepare each substrate to 6mM in 0.1M phosphate buffer (PH7.0). Reagent B (reaction stop solution): 1M phosphoric acid and acetonitrile Sample: Prepare P-amylase to 500 IU/1. HPLC measurement conditions Mobile phase: 10% acetonitrile Column: TSK-gel NH ₂ -60 (manufactured by Tosoh Corporation) Flow rate: 0.7ml/min Detection: UV meter

[0032] Measurement procedure: Prewarm 0.6 ml of reagent A at 37°C for 5 minutes. Next, add 0.02ml of P-amylase and after 60 minutes
Add 0.1 ml of 1M phosphoric acid and 0.6 ml of acetrinitrile to stop the reaction. The reactivity of amylase was measured by HPLC using 15 μl of this reaction solution as a sample.
Amount of remaining substrate, generated G2α-PNP, G3α-PNP,
The amounts of G2β-CNP and G3β-CNP are shown in the table below.

[0033] G5β-CNP of the present invention and G5α of the comparative example
- Comparing the action sites of P-amylase on PNP, P-amylase is
The second glucosidic bond from the reducing end (G2−G3
(between). On the other hand, P-amylase has low specificity for G2-G3 in G5α-PNP, and has high reactivity for G3-G4, which has a longer sugar chain.

【0034】

[Table 1] ■■■ Turtle shell [0066] ■■■

【0035】

Example 2 The reactivity of α-glucosidase was measured using the following reagents. Reagent A: Glucose oxidase 50U/ml, N-ethyl-N- in 0.1M phosphate buffer (PH7.0)
(2-hydroxy-3-propyl)-3,5-dimethoxyaniline sodium (DAOS) 1mM, 4
-Aminoantipyrine mM, baroxidase
Prepare by adding 3U/ml. Reagent B (substrate): p-nitrophenyl-α・D・
Dissolve 20mM each of G1 to G5 or G2 to G5 in 0.1M phosphate buffer (PH7.0). (G: glucose unit). Reagent C: 0.5M citric acid Sample: α-glucosidase 0.01-2U/ml

[0036] Measurement method: Mix 1.0 ml of sample A and 0.5 ml of reagent B, and preheat at 87° C. for 5 minutes. Then the sample
After 15 minutes, 2.0 ml of reagent C was added to stop the reaction, the absorbance was measured at 590 nm, and the value obtained for G2 (maltose) was taken as 100%, and each maltooligosaccharide and p-nitro The values of phenyl maltooligo etc. were calculated.

[0037] The results are shown in FIG. Figure 1 is a graph showing the relationship between the reactivity of α-glucosidase to various substrates and the degree of substrate polymerization. be.

[0038] One of the important conditions for amylase measurement substrate
One of the reasons is that amylase has only one site of action, and even if there are two or more sites of action, the conjugate enzyme (α-glucosidase) performs the same reaction on all of the reaction products produced. It is necessary to be able to show the characteristics and be able to completely guide the measurement system (2nd Clinical Chemistry Summer Seminar Program Material Collection). In the substrate of the present invention, the amylase reaction product is almost exclusively G2β-CNP, whereas in the substrate of the comparative example (G5α-PNP), it is G2α-CNP.
It becomes a mixture of PNP and G3α-PNP.

[0039] As shown in FIG. 1, the reactivity of α-glucosidase to G3α-PNP and G2α-PNP differs by about 4 times, and when G3α-PNP is produced in large amounts, it becomes an obstacle to completely introduce it into the measurement system.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the reactivity of α-glucosidase to various substrates and the degree of substrate polymerization.

[Chemical formula 8]

[ka]

Claims (1)

[Claims] Claim 1: A β- compound represented by the following formula [Chemical 1]
(2-chloro-4-nitrophenyl)-maltopentaoside.