Identification of 3,N4-propanodeoxycytidine 5'-monophosphate formed by the reaction of acrolein with deoxycytidine 5'-monophosphate

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Abstract
Acrolein reacts with deoxycytidine 5''-monophosphate at physiological pH to form one major adduct. A second minor adduct can be detected when a 3-fold excess of acrolein is present in the reaction mixture. The products were separated by ion-pair HPLC on two reverse-phase columns connected in series using triethylammonium formate as ion-pair reagent. The major adduct was characterized as 3-(5''-monophospho-2''-deoxyribosyl)-7,8,9-trihydro-7-hydroxy-pyrimido[3,4-c]pyrimidin-2-one (3,N4-propanodeoxycytidine 5''-monophosphate). This mixture of diastereomers was formed by addition of C1 of acrolein to the exocyclic amino group at the 4-position of cytidine, followed by ring closure between C3 of acrolein and N3 of the heterocyclic ring. In order to address the utility of 32P postlabeling for the detection of this exocyclic adduct in arolein-modified nucleic acids, an acrolein-deoxycytidine 3''-monophosphate reaction mixture was subjected to 32P postlabeling. 3''-Dephosphorylation with nuclease P1 and the 3''-phosphatase activity of T4 polynucleotide kinase yields a nucleotide 5''-[32P]monophosphate which cochromatographs with 3,N4-propanodeoxycytidine 5''-monophosphate. These data indicate that 32P postlabeling and 3''-dephosphorylation can be used in conjunction with ion-pair HPLC for the detection and quantitation of acrolein-modified nucleotides.