CS255146B1 - Process for preparative isolation of (methyl-3)thymidine and (6-3h)thymidine - Google Patents

Abstract

(Methyl-1H) -thymidine, (6-II) -thymidine are used as tracers in biochemical, biological and medical research. The aim of the solution is to increase the yields of the above-mentioned preparations, to reduce the separation cycle and to improve the quality of the product. Higher performance is due to the use of high performance liquid chromatography, which makes it possible to obtain radiochemically pure thymidines with a higher yield in a shorter separation cycle. This procedure yielded a 90% yield of radioactive thymidine with radioohemical purity greater than 97%.

Description

The invention relates to the preparative isolation of (methyl-H) -thymidine and (6- H) -thymidine from the reaction mixtures after their preparation by enzymatic synthesis.

The usual methods for isolating the above compounds from the reaction mixtures after their enzymatic synthesis are preparative paper chromatography methods. Especially in the preparation of (Me 3 -yl-H) -thymidine and (6-H) -thymidine with high molar activities, this method of isolation has a number of drawbacks. In particular, insulation losses (up to 30%) occur and the isolated compound is not chemically pure. With today's requirements for high molar activity of radionuclide-labeled compounds, the insulation losses when using standard procedures are no longer negligible economically.

The low degree of chemical purity of the preparations isolated by preparative paper chromatography is mainly due to the presence of extracts from chromatographic paper or impurities from solvents. These impurities interfere with the application of these preparations in biochemical, biological and medical research. Another shortcoming of paper chromatography falls within the field of safety of work with radioactive compounds. Handling with radioactive substances when applied to chromatographic papers, autoradiography and elution significantly increases the possibility of contamination of persons and the environment. It should be noted that preparative paper chromatography is a time-consuming method.

By way of illustration, we present the technological procedure used so far to isolate (methyl-H) -thyadien from the reaction mixture after its enzymatic preparation. Isolation of (methyl-H) -thymidine from the reaction mixture was performed by paper chromatography method on chromatographic paper W-3 in water-saturated ethyl acetate system, the thymidine in this system was 0.45. The identification of radioactive compounds in the chromatograms is carried out by comparing them with inactive standards. The band corresponding to radioactive thymidine is excised and eluted with redistilled water. Since the desired radiochemical purity is not achieved, the paper chromatography needs to be repeated in another development system, thus increasing the losses of the isolated preparation again.

The method for preparative isolation (methyl- ³ H) thymidine of 1.5-2 mole TBG activity / mmol, respectively, (6 -?) - thymidine about 0.060 to 1.00 mole of TBG activity / mmol elution HPLC according to the invention consists in that the mixture of compounds resulting from the preparation of radioactive thymidines is separated on a column of a hydrophobic specific adsorbent. The surface of the sorbent is covered with octadecyl groups, preferably the domestic sorbent SEPARON SIX S 18 can be used. A 0.6% aqueous solution of pure 3 3 ethanol is used for separation. The eluted radioactive (methyl-H) -thymidine and (6- H) -thymidine, respectively, after dosimetry and radiochemical purity determination, are diluted to expedition concentration and shipped directly.

The advantages of the novel process are that the yield of radioactive thymidines is 20% higher in the long-term average than when recovered by the above-mentioned preparative paper chromatography method.

A major advantage of the isolation of the thymidines of the present invention is the possibility of obtaining products of high radiochemical purity (min. 97-98%) and without the presence of chemical impurities. Furthermore, the corresponding unreacted are recovered. radioactive thymines, which can be used again for the preparation of (methyl-H) -thymidine and (6- H) -thymidine, respectively. Another advantage is the reduction of the separation time, instead of 24 hours only 8 hours. Separation by liquid chromatography takes place in a liquid medium in a closed semi-automated system. There is no need for autoradiography and drying of chromatographic papers where contamination can easily occur.

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This considerably increases the safety of work in the preparation of (methyl-H) -thymidine or (6- H) -thymidine.

To illustrate further example of the separation of radioactive (mety1- ³ H) thymidine from the reaction mixture after the enzymatic synthesis.

Q The composition of the reaction mixture prior to the start of the reaction was as follows:

mg of 2-Deoxy-D-Ribosa-1-phosphate

0.5 mg of (methyl-H) -thymine, i.e. 6.600 MBq

0.4 ml of redistilled water

0.2 ml of the enzyme Echerichia coli

The reaction was carried out at 37 ° C in an aqueous medium at pH 7.4 to 7.8. The course of the reaction was controlled by sampling and immediate analysis on an analytical liquid chromatograph.

The reaction was complete when the composition of the reaction mixture was:

71.8% (methyl- ³ H) thymidine

21.2% (methyl- ³ H) thymine% substances whose chemical nature is not identified.

The flask with the reaction mixture was immersed in hot water to flocculate the enzyme, which was removed from the reaction mixture by centrifugation. The grounds were washed 3 times and the individual supernatants were combined. The total volume of the reaction mixture thus treated was 8 ml. For separation separation a 350 x 15 mm glass column was used, which was filled with a domestic sorbent SEPARON

SIXC 18. The column was washed with a 0.6% aqueous solution of C ₂ H ₅ OH for 120 minutes before loading the sample.

The eluent flow rate was 100 ml / h. After loading the sample, the column was washed with the same speed eluent. At this flow rate, unreacted (methyl-H) -thymine began to elute 87 minutes after the beginning of the wash. The time required to elute the radioactive thymine was 20 minutes and the resulting material was found in 33 ml _of 0.6% C h _E 0H.

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Radioactive (methyl-H) -thymidine began to elute after 214 minutes (from the beginning of the wash) its elution time was 45 minutes, representing an elution volume of 74 ml. 4.375 MBq of (methyl-H) -thymidine of 97% radioactive purity was obtained. Further, 1,490 MBq of unreacted 3 (methyl-H) -thymine was obtained.

As previously mentioned, the enzymatic reaction was 71.8%. That is, in the reaction mixture 4738 MBq (methyl- ³ H) thymidine (71.8% of 6,600 MBq). 4.375 MBq of radioactive thymidine was obtained, which represents a yield of 92.3%.

Claims (1)

A process for the isolation of (methyl-H) -thymidine and (6-H) -thymidine from the reaction mixture by elution high performance liquid chromatography, characterized in that the mixture of compounds resulting from the enzymatic preparation of (methyl-H) -thymidine and ( 6- H-thymidine is separated on a column of a porous hydrophobic ion exchange resin based on silica gel, the surface of which is coated with octadecyl groups using a 0.6% aqueous solution of pure ethanol as eluent.