CS236751B2 - Processing of substrates for determining of alpha-amylase - Google Patents

Abstract

A substrate for the determination of alpha -amylase of the formula I is prepared by reacting the particular phenyl glucoside or nitrated phenyl glucoside with alpha -cyclodextrin, amylase or soluble starch in the presence of Bacillus macerans amylase. The radical R in the formula I has the meaning stated in the claim. <IMAGE>

Description

The invention relates to a process for the preparation of novel compounds which are substrates for .alpha.-amylase and are particularly suitable for its determination.

Determination of serum α-amylase levels is an important clinical indicator of pancreatic function. Commercial reagents for the determination of α-amylase are predominantly based on a process in which starch is degraded by α-amylase and the resulting cleavage products are determined in the visible or ultraviolet range, depending on whether colored or natural starch is used as the amylase substrate. . An important disadvantage of these methods or reagents is that starch as a macromolecule can only be poorly characterized and standardized, so that the degree of conversion of the individual batches is very different and the standard must always be used when determining the measurements. To achieve better results, a more uniform substrate would be required, which yields reliable results in cleavage.

A step forward towards a more uniform substrate was brought about by the use of maltopentose. Maltopentose is cleaved by α-amylase into maltotriose and maltose, maltotriose and maltose are converted by treatment with α-glucosidase in glucose, which can then be determined by any method, for example by the known hexokinase method.

In addition to maltopentosis, mialtopentosis and maltohexose have already been proposed as substrates (U.S. Patents 3,879,263 and 4,000,042). However, tetrose results in significantly worse results than pentose and hexose even worse results than tetrose. Thus, a stoichiometric reaction is reported for maltotetrose and maltopentosis, while deviations from the stoichiometric reaction, which are still within the permissibility limits, have already been found for the · hexose.

However, a disadvantage of maltopentosis, which is also a disadvantage of tetrose, is that a significant blank value of the reagent is formed, i.e. the measurement reaction is already taking place before the sample to be determined is added. In addition, even this blind reagent value is not constant at higher substrate concentrations, but varies for more than 25 minutes before the side reaction is constant. It has also been shown that the distinct cleavage of maltopentosis by the pancreatic α-amylase and the salivary α-amylase, which would allow the differentiation of the two amylases, does not actually occur (J. BC. 1970, 245, pp. 3917-3927). J. Biochem. 51, XVIII (1952). ...........

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The object of the present invention was therefore to provide a method for producing a substrate for the determination of amylase, which substrate is characterized by better purity and uniformity than known substrates, is readily available and meets the required requirements for serum-free blanking. the duration of the lag phase and the achievable maximum activity. Furthermore, a simple assay procedure without expensive and complex apparatus, as well as suitability for immediate determination, for example in the form of test strips, should be made possible.

The invention solves this task process for producing a compound of formula I ^1,

OH

(l) (i) where

R represents a phenylglucoside, a minonitrophenylglucoside or a dinitrophenylglucoside group, which is. The method is characterized by being competent. the phenylglucoside, optionally nitrated phenylglucoside, is reacted with? -cyclodextrin, amylase or soluble starch in the presence of an amylase from Bacillus macerans.

The process according to the invention for the enzymatic production of phenyl derivatives is carried out by transglucosidation of the phenylglucoside or the corresponding nitrated phenylglucosides with α-cyclodextrin, amylase or soluble starch in the presence of a specific microbial transferase. Preferably, transferases from Bacillus macerans are used. The known Bacillus amylases may be used for this transglucosidation. Macerans (EC 2.4.1.19 DSM 24; isolation: JA de Pinto, LL Campbell, Biochemistry 7 [1968] p. 114; Reaction: Methods in Carbohydrate Chemistry, Vol. II, p. 347], which, in addition to its hydrolytic and cyclizing effect, is apparently also characterized by glucoseyl transfer efficiency.

The compounds which can be prepared by the process according to the invention are particularly suitable for the method for the determination of α-amylase according to U.S. Pat. No. 236,758.

The compounds produced by the process of the invention are readily available and are obtained in high purity. They are suitable for the determination of γ-amylase in biological fluids such as serum, heparin plasma, urine and the like, or in other liquid or solids.

The invention is illustrated by the following examples.

Example 1

Preparation of α-Phenylmaltoheptoside α] Tride-Acetyl-3-D-Maltoheptose

57.6 g (50 mmol) of maltoheptose and 41 g (500 mmol) of sodium acetate (anhydrous) are suspended in 543 ml (5.75 mol) of acetic anhydride and the mixture is stirred vigorously at 1- for 4 hours in the absence of moisture. QO ^e · C. After cooling to about 60 · · ° C, the reaction mixture is stirred into about 1 liter of ice water ·. Stirring was continued overnight at 4 ° C, leaving a solid semicrystalline mass. After the liquid has been poured over the mass, the residue is again stirred in ice water. The product crystallizes completely. After isolation, it is washed. · And dried.

Yield: 80.7 g (76% of theory) of colorless crystals.

[.alpha.] D @ ²⁰ = + 137.5 DEG (c = 1.15, chloroform), m.p. 150 DEG C. (not sharp).

The mother liquor (decantate) is evaporated to dryness under reduced pressure, and the residue is also triturated with ice water and crystallized.

Yield 22.3 g.

[.Alpha.] D @ ²⁰ = 136 DEG (c = 1, chloroform), m.p. 150 DEG. The total yield is 103 g, i.e. approximately 97% of theory.

The product obtained can be recrystallized from ethanol without changing the melting point or rotation even after repeated recrystallization. The product is thus optically uniformly [-3] configured on the first carbon atom.

b) Do (decosacetyl-1-phenyl-D-methylheptoside)

A mixture of 9.54 g (4.5 mmol) of tridecosacetyl-3-D-maaloheiptose, 0.61 g (4.5 mmol) of freshly molten ZnCl2 zinc chloride and 4.23 g (45 mmol) of distilled phenol was added. the mixture is stirred for 2.5 hours at 100 DEG C. in the absence of moisture. The mixture is then dissolved in ethyl acetate while still warm and washed twice with 100 ml ^. ml of water, in each case 100 ml of 1 N sodium hydroxide solution, 100 ml of 1 N acetic acid and 100 ml of saturated sodium chloride solution. The brown solution initially becomes discolored in pale yellow. After drying over magnesium sulfate, the reaction mixture is evaporated to dryness and the residue is dissolved in 30 ml of warm methanol. Upon standing overnight, a syrupy substance precipitated which was crystallized from ethanol.

Yield: 8.7 g (90% of theory).

Mp 155-165 ^° C (dec.). [.alpha.] D @ ²⁰ = + 147 DEG (c = 8, in chloroform).

(c) Phenylla-D-maltohoroside

10.8 g (5 mmol) of peracetyl-1-phenyl-α-D-maltoheptoside are dissolved in 200 ml of anhydrous methanol while warm and 30 ml of 0.1 N sodium methoxide are added under stirring at room temperature. The mixture was stirred at room temperature for 16 hours. After 20 minutes, the semi-crystalline product began to precipitate. Finally, 200 ml of acetone are added, the mixture is cooled to 4 ° C and the product is filtered off with suction. This is then dissolved in 160 ml of water, the solution is decolourised with charcoal and freed from salts in a cation exchange resin (Dowex 50 · H +).

Yield: 5.6 g (91% of theory) of the colorless lyophilisate.

[.alpha.] D @ ²⁰ = + 176 DEG (c = 10, in water); free maltoheptoses; These are separated by chromatography on cross-linked dextran (Sephadex LH 20) with water as the eluent.

Example 2

Preparation of p-nitrophenyl-.alpha.-D-amino-heptoside

a) Peracetyl-p-nitrophenyl-αa-D-maltoheptoside

13.6 g ^(20: mmoles) peracetylmaltoheptózy, prepared as described in Example · Ia), together with 11.9 g (100 mmol) of p-nitrophenol dissolved in 90 ml of anhydrous benzene under stirring and exclusion of moisture was added a 10 4 g = 4.5 ml (40 mmol) of tin (II) chloride SnCl2. This results in a bulky mass which, however, dissolves again by heating. The reaction mass was heated to reflux for 1 hour. A solid was formed by cooling, which was redissolved after addition of 80 ml of ethyl acetate. After mixing the solution into 180 ml of a 2 N sodium carbonate solution of Na 2 CO 3, the tin oxide hydrate precipitates, which is separated by the addition of a small amount of activated carbon. The aqueous phase is separated and the organic phase is washed well, finally with a saturated sodium chloride solution. After drying and evaporating the solution, a resin product is obtained which crystallizes from ethanol.

Yield: 6.2 g (41% of theory).

Melting point 100 ° C (dec.).

«2] _D ² ° = + 131 ° (c = 1, chloroform).

This product is also obtained when the feed is carried out in chloroform or with TiCl4 instead of SnCl4.

b) p-nitrophenyl-z-D-malloheptoside

5.5 g (7 mmol) of peracetyl-p-nitrophenynyl maltoheptoside are suspended in 100 ml of anhydrous methanol and 5 ml of approximately 1N sodium methoxide solution are added to the suspension. The starting material becomes honey-like and slowly dissolves. After some time, crystalline deposition of the desacetylated product begins, which is complete after stirring overnight. The product is filtered off with suction, washed with ethyl acetate

Yield: 2.8 g (86% of theory). = + 124 ° (c = 0.6, in water).

The product was purified on cross-linked dextran (Sephadex LH 20) with water as eluent. 1.2 g (45% of theory) of the compound described above, which is active in the enzymatic test, are obtained. In addition, this compound is also obtained by nitration of the α-phenyl maltoheptoside of Example 1 with a nitration acid according to the procedure described in Bull. Chem. Soc. Japan, 34, (1961) 718.

By using dinitrophenol instead of mononitrophenol, the corresponding dinitrophenyl compounds are obtained.

Example 3

P-nitrophenyl-α'-aaltOΌllgosaccharides by enzymatic synthesis using amylase from Bacillus maaerans (E. C. 2. 4. 1. 19. from Bac. Mac. DSM 24)

Bacillus macerans amylase has, in addition to its hydrolytic and cyclizing effects, glycyltransferring properties which can be used to synthesize oligosaccharides and their derivatives (Methods in Cyclobacterium, Vol. II, p. 347). For the synthesis of p-nitrophenyloligosaccharides, this process has been optimized as follows:

680 · mg amylase from Bacillus macerans (EC 2.4.1.19 from Bac. Mac. DSM 24) (ly-offill) (0.4-6 U / mg · batches, protein content in batch 28 5% (without p-nitrophenyl-αD-glucoside cleavage activities) is mixed with 400'-mg of α-β-nitrophenylglucoside, 3.5 g of α-cyclodextrin and 70 ml of Soorensen phosphate buffer pH 6.2; The batch is kept in a 37 ° C incubator for 24 hours. For purification, the α- and β-cyclodextrin formed is then separated, first through trtrachlorethylene complex compound. Chromatography on cross-linked dextran (Sephadex LH 20) yields 50 mg of p-nitrophenylmalloheploetide lyophilisate, highly active in the amylase assay.

Example 4

Preparation of maltoheptite g Maltoheptose is dissolved in 50 ml of water, 2 g of sodium borohydride NaBH 3 are added in portions and the mixture is stirred at room temperature for 75 minutes (reduced sugar test negative). Chromatographically, sodium Na + is removed on a cation exchange resin (Dowex 50 H +) (pH of the solution after passing through the ion exchanger equals 3.5). The solution was evaporated to dryness under reduced pressure and redissolved several times with a mixture of methanol and water (addition of water to the product solution) and evaporated to remove the boronic acid methyl ester. The solution, which is neutral at the end of this procedure, is lyophilized.

The yield was 9 g (90%) of the sugar-free product. Content (determined enzymatically) of glucose content 86.1 percent, sorbitol 89.0% water and 8.5% water.

Example 5

Production of maltoheptonic acid

To 150 ml of water 11.5 g (0.01 mol) of malheptose are added 6 g of barium benzoate. With stirring and cooling, 1 ml of bromine was added and stirring was continued for 36 hours. After stripping off the excess bromine with nitrogen, 4 ml of 4 N sulfuric acid and a small amount of activated carbon are added to the reaction mixture, the mixture is filtered and the filtrate is extracted with chloroform to remove benzoic acid. To the aqueous solution was added 3.2 g of silver carbonate Ag 2 CO 3 and the mixture was stirred at neutral pH. The insoluble salts were filtered off and the clear solution was passed through 20 ml of Amberlite JRH +. The acidic eluate was immediately neutralized with sodium hydroxide and lyophilized.

Yield: 8 g (72% of theory).

Content (determined enzymatically) of 85% glucose, 80% gluconic acid, 9.3% water.

Example 6

Detection of α-amylases using phenyl-α-maltoheptoside as substrate

Phenyl-α-maltoheptoside is cleaved by α-amylase into phenyl-α-maltotriide or phenyl-α-maltotetraid, which is converted to phenol and _Z glucose by α-glucosidase. The liberated phenol is oxidatively coupled with the monophenol oxidase to the nucleophilic reagent, 3-methyl-6-sulfonylbenzthiazolone hydrozone (2) (HSK) to form a red dye whose rate of formation is proportional to the amylase activity in the sample and can be monitored photometrically.

Test principle:

1.

a) phenyl-α-maltoheptoside + α-amylase + 1HO --------> phenyl-α-D-tri- (tetra) glucopyranoside + maltotetra- (tri) -ose

b) phenyl-α-D-trii (tetra) glucopyranoside + α-glucosidase + 3 (4) H2O ----------> phenol +

4- (optionally 4) glucose

c) phenol + HSK 4-monophenol oxidase

4- O 2 -----------> dye · + + 2 H 2 O.

Measurement conditions: 25 ^C U, wave length

492 nm, 1 cm cuvettes; the composition of the reagent is shown in the following table:

Agent

Phosphate buffer concentration, pH 7.4 0.05 mol / l sodium chloride 0.05 mol / l phenynyl-α-D-maltoheptoside 10 mmol / l

HSK 1 mmol / l monophenol oxidase 1 J / ml α-glucosidase 10 J / ml

Test volume: 2.0 ml.

Glycine, glycylglycine, hepes, tris, tra- and other buffers may also be used in place of phosphate buffer.

Example 7

Determination of α-amylase by ptnitiophenylt maltoheptoside

Test principle:

p-nitrophenyl-α-maltoheptoa-amylase zid —------- (glucose) n + p-nitrophenyl-glucosidase α- (glucose) m -------- ► n glucose +

4- p-nitrophenol.

After cleavage of maltoheptoside, the cleavage products are degraded to glucose and p-nitrophenol. The resulting p-nitrophenol anion in the alkaline solution is yellow in color and can therefore be measured directly by optometry.

Test system:

Reaction mixture:

mmol / l phosphate buffer, pH 7.4

50 ¹ mmol / 1 sodium chloride and 50 j./ml -glucosidase or 10 mmol / 1 p-nitrofenylta maltoheptozidu.

Test Handle:

ml of reagent mixture -h- 50 μ1 of sample (serum).

Temperature 30 ° C, wavelength: 405 nm.

Initiate the test by adding serum after the measurement temperature has been reached (preincubation with 10 niinrite).

Example 8

Determination of α-amylase using maltoheptite

Test principle:

a-amylase maltoheptit 4-H2O -----> m.artotg iit 44- maltotetrose

10

α-glucosidase diaphorase maltotriit + H2O -------> sorbitol 4 NADH + INT + H + -----> formazan + maltose + NAD H SDH SDH = sorbitol dehydrogenase; sorbtitol + NAD + -----> fructose + NAD == ; oxidized mkofinamidadeniιn- + NADH + H + dinucleotide; NADH - reduced nicotinamideadenine- dinucleotide. Test system: Re-agitation handle ml Test concentration Na- (K-PO), - 0.02 mol / l buffer + triton x 100 1.92 12.8 mmol / 1 POi 0 ml / 100 ml + NaCl 10 mmol / L, pH 6.9 6.4 mmol / l MgCl ² 0.3 mol / L 0.01 1.0 mmol / l maltoheptite 100 mmol / l 0,10 · 3.3 mmol / l NAD c = 40 mg / ml 0.05 1.0 mmol / l INT c = 0.6 mg / ml 0.20 0.09 mmol / l diaphorase (ClostT. Kluyveri) 5 mg / ml *) 0.05 167 j / 1 ATP c = 50 mg / ml 0.10 3,29 mmo.lu/1 hexokinase 280 j / ml *) 0,05 ' 4,666 j / 1 SDH 180 J / ml *) 0.30 18,000 j / l α-glucosidase 1120 j / ml * 0.20 · 74,670 j / 1 sample (serum) 0.02

) in assay buffer

Initiate the test by adding a sample: the measurement is made at a wave length of 492 nm; temperature 25 ° C.

Example 9

Determination of wamylase using maltoheptagluconic acid

Test principle:

maltoheptagluconic acid + α-amylase + H2O -----> maltotriogluconic acid + maltotetrose maltotriogluconic acid + α-glucosidase + H2O -------> gluconic acid + maltose gluconic acid + α-gluconate kinase

ATP ----------> 6 - Gluconic acid + adenosine diphosphate

Gluconic acid 6-phosphate + acid dehydrogenase + NAD ------------->

6-phosphouluconate

- ribulose-5-phosphate + · NADPH + CO2 + + H +.

ATP = adenosine triphosphate

NADP = oxidized nicotinamide adenine dinucleotide phosphate

NADPH = reduced nicotinamide adenine dinucleotide phosphate.

Test system:

Reagent Handle ml Ends rntrace during the test Na- / K-POi-buffer 0.02 mol / l 2.19 14.6 mmol / l phosphate + NaCl 10 mmol / L, pH 6.9 7.3 mmol / l NaCl MgCl2 1 mol / l 0.01 3.3 mmol / l maltoheptagluconic acid 150 mmol / l 0.10 5 mmol / l NADP c = 10 mg / ml 0.10 0.38 mmol / l ATP c = 50 mg / ml 0,10: 2.7 mmol / l Gluconate kinase 40 J / ml 0.03 1066 j / 1 6-phosphogluconic acid dehydrogenase j / 1 24 J / ml 0.10 800 α-glucosidase 1120 j / ml 0.30 11.2 x 104 J / L Sample (sulphate) 0.02

Start the test by adding a sample; the measurement shall be made at a wavelength of 340 · nm · or 365 nm; temperature: 25 ° C; test volume

3,00 ml.

Claims (1)

OBJECT OF THE INVENTION A process for the preparation of a substrate for the determination of .alpha.-amylase comprising a compound of formula (I), wherein R is a phenylglucoside, monomitrophenylglucoside or dinitrophenylglucoside group, characterized in that the corresponding phenylglucoside or optionally nitrated phenylglucoside is reacted with α-cyclodextrin, amylose or soluble starch in the presence of Bacillus macerans amylase.