Purification of chemically synthesised dinucleoside(5',5') polyphosphates by displacement chromatography

Dinucleoside(5',5') polyphosphates (ApnA, ApnG, GpnG, n=3-6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5',5') polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5',5') polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1-0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5')-polyphospho-(5')guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of ApnA, ApnG and GpnG (with n=3-6 in all cases). The reaction mixture was concentrated on a preparative C18 reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.