The structures of the DNA adducts formed in the metabolism of cyclic N-nitrosamines are not known. To approach this problem, we studied the reactions with deoxyguanosine, catalyzed by porcine liver esterase, of α-acetoxy-N-nitrosopyrrolidine and 4-(carbethoxynitrosamino)butanal which are stable precursors to the reactive intermediates, α-hydroxy-N-nitrosopyrrolidine and 3-formyl-1-propanediazohydroxide, formed in the α-hydroxylation of the cyclic nitrosamine, N-nitrosopyrrolidine. The same two major deoxyguanosine adducts were produced in each reaction. They were isolated by high-performance liquid chromatography and characterized by their ultraviolet spectra, mass spectra, and proton magnetic resonance spectra. On the basis of these spectral data, the structures of the two major adducts were assigned as 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-methylpyrimido [1,2-a]purine-10(3H)one and 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-methylpyrimido[1,2-a]purine-10(3H)one, in which a new saturated six-membered ring is formed by reaction at the 1- and N2-positions of deoxyguanosine. The same two adducts were formed in the reaction of crotonaldehyde (2-butenal) with deoxyguanosine. Mild acid hydrolysis of each of the two diastereomeric deoxyguanosine adducts yielded a pair of enantiomeric tricyclic 1,N2-guanine derivatives which had spectral properties in accordance with the assigned structures. Treatment of these tricyclic 1,N2-guanine derivatives with NaOH and NaBH4 yielded N2-(3-hydroxy-1-methylpropyl)guanine, confirming the structural assignments. The results of this study demonstrate that, unlike saturated dialkylnitrosamines which are metabolized to intermediates that give mainly 7-alkylation of deoxyguanosine, the intermediates formed in the α-hydroxylation of a cyclic nitrosamine, N-nitrosopyrrolidine, modify deoxyguanosine by forming cyclic 1,N2-adducts.


This study was supported by National Cancer Institute Grant CA 23901. PMR spectra (300-MHz) were obtained using the 7T spectrometer at the Rockefeller University, purchased in part with funds from the National Science Foundation (PCM-7912083) and from the Camille and Henry Dreyfus Foundation. This is Paper 49 of the series, “A Study of Chemical Carcinogenesis.” This publication is dedicated to the founder of the American Health Foundation, Dr. Ernst L. Wynder, on the occasion of the 10th anniversary of the Naylor Dana Institute for Disease Prevention.

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