CS233134B1 - A method of preparing U-14C / D-fructose for high molar activity - Google Patents

Abstract

The invention relates to the field of biochemistry, carbohydrate chemistry, as well as medical and food chemistry. The invention solves the preparation of /U-^C/ D-fructose with high molar activity, for the study of carbohydrate metabolism, as well as for theoretical studies in the field of echemia and biochemistry of carbohydrates. The stated purpose is achieved by the fact that /U-14C/ D-glucose is transformed by the action of the enzyme xylose isomerase in a soluble or immobilized state or as part of immobilized cells and the reaction mixture is separated chromatographically. The invention is used primarily in the preparation of /U-C/ fructose as a pure chemical, suitable for the study of carbohydrate metabolism, as well as for theoretical studies in the field of echemistry and biochemistry of carbohydrates, as well as in medical research and the pharmaceutical industry.

Description

Correction in printed descriptions of inventions (PV 5254-83)

In the description of the invention for the author's certificate no. The name of the second inventor is misprinted.

Correct: KUČÁR ŠTEFAN RNDr., CSc., BRATISLAVA

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The invention relates to a process for the preparation of D-fructose of high molar activity.

A common method for the preparation of Aβ-C / D-fructose is the inversion of radioactive sucrose in a weakly acidic medium (U.S. Pat. No. 121,808) or the transformation of [Ui] C / D-glucose with a saturated solution of calcium hydroxide. ) or [.alpha.] - .DELTA.-D-glucose transformation by treatment with sodium aluminate solution (U.S. Pat. No. A0 187 109). The preparation of (U-C) D-fructose from (U-C) sucrose is uneconomical due to the low content of sucrose in the algae, as the starting biological material for the preparation of labeled carbohydrates. In chemical transformations of aldoses and ketoses, the capture of the labeled carbohydrates by the radionuclide in the precipitates is relatively high, thereby reducing the yield of AJ-C / D-fructose. Washing of the precipitates with water until radioactivity enters the filtrate is also a technical difficulty. In view of the yield, further loss of radionuclide-labeled carbohydrates results from desalination of the reaction mixture on ion exchangers.

The above-mentioned disadvantages are substantially eliminated by the process for the preparation of the high molar activity of N-C / D-fructose according to the invention, characterized in that the N-C / D-glucose is transformed by the action of xylose isomerase in soluble or immobilized state. or as part of immobilized cells and the reaction mixture is separated by chromatography.

Upon completion of the catalysed xylose isomerase (EC 5.3.1.5) reaction, the reaction mixture contains N 1 -c (D) -glucose and (N 1 -c) C / D-fructose in a molar ratio of about 1: 1. The advantage of the proposed procedure is its simplicity, possibility of preparation of radioactive preparation with high specific activity and possibility of reverse quantitative isolation of unreacted AJ-C / D-glucose.

- 2 Example 1

233 134 mg / U-q / D-glucose having an activity of 248 MBq (6.7 mCi), molar activity 4440 MBq (120 mCi) nmol was dissolved in 0.2 ml of Tris- (hydroxymethyl) -aminomethane buffer hydrochloride 20 mmol / l, pH 8.0, containing 0.1 mol magnesium sulfate heptahydrate and 1 mmol cobalt chloride hexahydrate and xylose isomerase (ex Wingea robertsii) 60 nkat. Incubation was carried out for 12 h at 70 ° C. The reaction mixture was then loaded onto a chromatographic paper (Whatman 3MM) and eluted in descending order in a 2-butanone saturated with water and water, respectively. ethyl acetate-2-propanol-water (6: 2: 1). After partitioning of the reaction mixture, the individual zones pertaining to pure (U-C) D-glucose and [U-C] D-fructose were eluted with distilled water. 4.8 mg of D-fructose with a total activity of 120 MBq and 5.2 mg / U- ¹⁴ C / D-fructose with a total activity of 128 MBq were obtained. The radiochemical purity of the preparations was greater than 98%, controlled by paper chromatography in 1-butanol-acetic acid-water systems (4: 1: 5) and 1-butanol-1-propanol-ammonia-water (7: 5: 7: 2). ).

Example 2

The procedure is as in Example 1 except that immobilized xylose isomerase (ex Streptomyces violaceus) immobilized in cross-linked polyethyleneimine with a total activity of 63 nkal (per 85 mg) is used instead of water-soluble enzyme (ex Wingea robertsii). The yield of (U- ¹ ) D-fructose was 50% at a reaction time of 6 hours.

Example 3

The procedure is as in Example 1 except that whole Wingea robertsii cells, permeabilized for 5 min, are eluted at 70 ° C for transformation. Xylose isomerase activity of cells immobilized in cross-linked cellulose and impregnated with cross-linked polyethylenimine was 0.71 nkat / mg. The total volume of the reaction mixture was 0.7 ml with 100 mg immobilized Wingea robertsii cells. The reaction mixture contained 46% (U- ¹ ) of D-fructose and 54% (U-1%) of D-glucose and immobilized Wingea robertsii cells were useful for catalyzing the isomerase reaction by 45-fold.

- 3 233 134

The present invention can find wide application in the production of (N-C) D-fructose and in the study of carbohydrate metabolism, as well as in theoretical studies of carbohydrate chemistry and biochemistry, as well as in medical research and the pharmaceutical industry.

Claims (1)

SUMMARY OF THE INVENTION A process for the preparation of [U-1] (D-fructose) of high molar activity, characterized in that [U-c] D-glucose is transformed by the addition of the enzyme xylose isamerase in a soluble or immobilized state or as part of the immobilized cells, and the reaction mixture is separated by chromatography. 7