摘要：

Methods are described for the routine chemical preparation of α- 32P-labelled ribonucleoside 5′-di- and triphosphates starting with 32P i and 2′,3′- O-isopropylidene ribonucleosides. After preparation of the nucleoside 5′-[ 32P]monophosphate, the α- 32P-labelled di- or triphosphate was formed by reaction of the monophosphate in situ with 2-cyanoethylphosphoryl imidazolidate (Ib) or 2-cyanoethylpyrophosphoryl imidazolidate (II), respectively, in dimethyl formamide as solvent. After removal of the cyanoethyl group by alkaline hydrolysis, the required nucleotide was purified by adsorption on and elution from charcoal followed by paper chromatography. Labelled nucleotides were obtained in a yield of 35–50% relative to starting 32P i and with a specific activity of 1–5 mC/μmole. Nucleoside 5′-[ β- 32P]diphosphates were prepared by the reaction of excess 32P i with the appropriate nucleoside 5′-phosphoromorpholidate using dimethyl sulphoxide as solvent. Labelled nucleotides were purified by adsorption on and elution from charcoal and by paper chromatography; they were obtained in a yield of about 25% relative to 32P i and with a specific activity of 0.1–0.5 mC/μmole. In a similar way, [ 32P]pyrophosphate was prepared by reaction in pyridine of excess 32P i with 2-cyanoethylphosphoryl 2-hydroxypyridine (Ia). After alkaline hydrolysis of the cyanoethyl group, the labelled pyrophosphate was purified by paper chromatography and obtained in about 25% yield.

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