5-Cyanoimino-4-Oxomethylene-4,5-Dihydroimidazole and 5-Cyanoamino-4-Imidazolecarboxylic Acid Intermediates in Nitrosative Guanosine Deamination: Evidence from ¹⁸O-Labeling Experiments

The nitrosative deaminations (37 °C, NaNO ₂ , NaAc buffer, pH 3.7) of guanosine 1r in ( ¹⁸ O)-water (97.6%) and of [6- ¹⁸ O]- 1r in normal water were studied. [6- ¹⁸ O]- 1r was prepared from 2-amino-6-chloropurine riboside using adenosine deaminase. The reaction products xanthosine 3r and oxanosine 4r were separated by HPLC and characterized by LC/MS analysis and ¹³ C NMR spectroscopy. The ¹⁸ O-isotopic shifts on the ¹³ C NMR signals were measured and allowed the identification of all isotopomers formed. The results show that oxanosine is formed via 5-cyanoimino-4-oxomethylene-4,5-dihydroimidazole, 5 , and its 1,4-addition product 5-cyanoamino-4-imidazolecarboxylic acid, 6 . This hydration of 5 to 6 leads to aromatization and greatly dominates over water addition to the cyanoimino group of 5 to form 5-guanidinyliden-4-oxomethylene-4, 5-dihydroimidazole, 7 . 5-Guanidinyl-4-imidazolecarboxylic acid, 8 , the product of water addition to 6 , is not involved.