DD225696A1 - METHOD FOR PRODUCING NUCLEOSIDE 5'-DI AND TRIPHOSPHATES OF HIGH SPECIFIC ACTIVITY - Google Patents

Abstract

A process for the preparation of nucleoside 5- (α-32P di- or triphosphates with high specific activity. The aim of the invention is to develop a technically simple, rapid process which, in good yields, gives products with maximum specific activity at a high activity level The P-32-labeled nucleoside-5-monophosphate produced by reacting carrier-free H332PO4 with nucleosides is purified according to the invention on anion exchangers and phosphorylated directly in the elution buffer with substrates and kinase to give nucleoside 5-di- or triphosphate by column chromatography on an anion exchanger whose capacity parameters differ from that of the intermediate purification.

Description

Field of application of the invention

The invention relates to a process for the preparation of nucleoside 5 '[a- ³² P] di- and triphosphates of the formula I.

x = phosphate or diphosphate

rest B = adenine, thymine, guanine, cytosine, uracil and their derivatives

R ₁ = OH ₁ R ₂ = OH or HR ₁ = H ₁ R ₂ = OH or H

Application is the radiochemical synthesis, nucleotide chemistry

Characteristic of the known technical solutions

Symons described a simple chemical-enzymatic process in 1978, in which the chemical and enzymatic steps of the synthesis were carried out in succession in a reaction vessel. In this synthesis, labeled and unlabeled H ₃ ³² PO _{4 were} reacted with nucleosides. Impurities and radiolysis products lead to losses of yield and, above all, to low specific product activity. From WP C 07 H / 2468398 a one-pot process for the production of [a- ³² P] nucleoside 5'-phosphates of high specific activity is known. However, this method has the disadvantage that the H ₃ ³² PO ₄ used as the starting compound for enzymatic synthesis steps must be of high purity, which requires a high technical complexity of purification processes and is economically unfavorable.

Object of the invention

The aim of the invention is to develop a technically simple process which enables the preparation of nmol amounts of specifically highly labeled nucleoside 5 '[α- ³² P] di- or triphosphates in short reaction times and good yields.

Explanation of the essence of the invention

The compounds of formula I are prepared by reacting carrier-free H ₃ ³² PO ₄ with a nucleoside to the P-32-labeled nucleoside 5'-monophosphate. According to the invention, the [a- ³² P] -labeled nucleoside B'-monophosphate of impurities and Radiolyseprodukten on a Anionenaustauschergel, z. B. DEAE-cellulose, by Eiuticn with a salt solution, eg. As triethylammonium bicarbonate, purified.

Subsequently, the enzymatic phosphorylation is carried out in the elution buffer to di- or triphosphate. Within 4-2Oh yields are obtained which, depending on the type of enzyme 3O ⁰ Zo-80%. The nucleoside 5 '- [a- ³² P] di- or triphosphate is from the reaction mixture by column chromatography on an anion exchanger whose capacity parametersichich different from that for purifying the monophosphate, z. B. DEAE-Sephadex A25, separated. Elution takes place according to a gradient program.

The process according to the invention has the advantage that it gives a technically simple process for preparing the di- and triphosphates with high specific radioactivity. It has proved to be much more economical to purify the resulting monophosphates than the H ₃ ³² PO ₄ used as the starting compound.

The following embodiments illustrate the invention in more detail.

embodiment

1st example

27mCi carrier-free H ₃ ³² PO ₄ in 25μί 0.1 NHCI are iyophilized with 0.5 μmol Chelaplex II in a small tube.

The lyophilizate is taken up in 50μΐ dimethylsulfoxide containing 5mg 2'-deoxyadenosine, 2μΙ triethylamine, 2μί trichloroacetonitrile.

After 60 minutes the reaction is stopped with 0.5 ml of water. The mixture is then placed on a column of DEAE-Ceüulose-Gei in bicarbonate form (0.3 ml bed volume). The column is then washed with 5 ml of water, 0.5 ml of 0.02M triethylammonium bicarbonate (TAC) and the product eluted with 4 ml of 0.15M TAC. To the eluate of 4 ml are added 4nmol ATP, 9.8μ.ΓηοΙ phosphoenolpyruvate, Ο.δμ, ΓηοΙ KCl, 32.5μΐηοΙ Mg (ac) ₂ , 100 / xg myokinase and 200μ9 pyruvate kinase.

After reaching a yield of 60% of 2'-deoxyadenosine-5 '- [a- ³² P] -triphosphate, the mixture is placed on a column of DEAE-Sephadex A25 in bicarbonate form and washed with 20 ml of water and 20 ml of 0.3% TAC. Subsequently, in a continuous gradient, 25 ml of 0.3 M TAC against 25 ml of 0.7 M TAC, with an elution rate of 1 ml / min, the desired triphosphate after about 10ml as 8ml fraction isolated.

The radiochemical purity of the fraction is> 99% [a- ³² P] dATP. After lyophilization and taking up the product in 5 mM mercaptoethanol / water, a yield of product activity of 25-30% results.

The product characterization is carried out by thin-layer chromatography with inactive dATP comparison:

R _F = 0.31 on PEI cellulose from Merck

Eluent 0.5 M KH ₂ PO ₄ buffer, pH = 3.5.

2nd example

27mCi of carrier-free H ₃ ³² PO ₄ in 20μ.Ι 0.1 N HCI are lyophilized with 0.5μ.ιηοΙ Chelaplex II in a slide tube. The lyophilisate is taken up in 50% dimethylsulfoxide containing 5 mg of 2'-deoxycytidine, 2 μl of trichloroacetonitrile and 2 μl of triethylamine. After 60 minutes, stop the reaction by adding 0.5 ml H ₂ O. The mixture is placed on a column filled with 0.3 ml of DEAE cellulose gel. The anion exchange gel is washed with 5 ml of water, 0.02 M TAC and with 1.5 ml 0.12 m TAC the [a- ³² P] dCMP Ge! eluted. Directly in the elution buffer, the enzymatic phosphorylation to triphosphate by addition of 10.7 / xmol Phosphoenolpyruvat, 8.1 / xmol MgSO ₄ , 0.4μΓηοΙ KCI, 5μΙ Mercaptoethanoi, 2.2 / * mol ATP, 200 / Xg pyruvate kinase and 740 / ng nucleotide kinase from Escherichia coli started.

After 18 hours, a yield of [a- ³² P] dCTP of 30% is achieved. The mixture is applied to a column of DEAE-Sephadex A25 (3 ml bed volume) and washed with 20 ml of water, 20 ml of 0.3M TAC and in a continuous gradient, 20 ml 0.3 M TAC, at an elution rate of 1 ml / min, the desired 2'- Deoxycytidine-5 '[a- ³² P] triphosphate isolated as a 9 ml fraction. The radiochemical purity of the product is> 95%, the activity yield of the synthesis is 10%.

The product characterization is carried out by thin-layer chromatography with inactive dCTP comparison R _F = 0.5 on PEI-cellulose from Merck

Eluent 0.5M KH ₂ PO ₄ buffer, pH = 3.5.

Claims (2)

Invention claims: 1. A process for the preparation of nucleoside 5 '- [a ³² P] di- and triphosphates of high specific activity of the formula I, \ <~> in the χ = phosphate or diphosphate
B = adenine, thymine, guanine, cytosine, uracil and their derivatives R ₁ = OH, R ₂ = OH or HR ₁ = H, R ₂ = OH or H mean, by reacting carrier-free H ₃ ³² PO ₄ with a nucleoside to [a- ³² P] -labeled nucleoside 5'-monophosphate, characterized in that [a- ³² P] -labeled nucleoside 5'-monophosphate at an anion exchanger of impurities and Radiolyseprodukten, then phosphorylated in the elution buffer directly after addition of appropriate kinases and substrates for di- or triphosphate and this column chromatography. 2. The method according to item 1, characterized in that the contamination of the [a- ³² P] -labeled nucleoside ö'-monophosphate are separated on a DEAE-cellulose gel in bicarbonate form with triethylammonium bicarbonate.