We investigated the reactions of alpha -acetoxy-N-nitrosopyrrolidine (alpha
-acetoxyNPYR) with dGuo and DNA. alpha -AcetoxyNPYR is a stable precursor
to the major proximate carcinogen of NPYR, alpha -hydroxyNPYR (3). Our goal
was to develop appropriate conditions for the analysis of DNA adducts of N
PYR formed in vivo. Products of the alpha -acetoxyNPYR-dGuo reactions were
analyzed directly by HPLC or after treatment of the reaction mixtures with
NaBH3CN. Products of the alpha -acetoxyNPYR-DNA reactions were released by
enzymatic or neutral thermal hydrolysis of the DNA, then analyzed by HPLC.
Alternatively, the DNA was treated with NaBH3CN prior to hydrolysis and HPL
C analysis. The reactions of alpha -acetoxyNPYR with dGuo and DNA were comp
lex. We have identified 13 products of the dGuo reaction-6 of these were ch
aracterized in this reaction for the first time. They were four diastereome
rs of N-2-(3-hydroxybutylidene)dGuo (20, 21), 7-(N-nitrosopyrrolidin-2-yl)G
ua (2), and 2-(2-hydroxypyrrolidin-1-yl)deoxyinosine (12). Adducts 20 and 2
1 were identified by comparison to standards produced in the reaction of 3-
hydroxybutanal with dGuo. Adduct 2 was identified by its spectral propertie
s while adduct 12 was characterized by comparison to an independently synth
esized standard. With the exception of adduct 2, all products of the dGuo r
eactions were also observed in the DNA reactions. The major product in both
the dGuo and DNA reactions was N-2-(tetrahydrofuran2-yl)dGuo (10), consist
ent with previous studies. Several other previously identified adducts were
also observed in this study. HPLC analysis of reaction mixtures treated wi
th NaBH3CN provided improved conditions for adduct identification, which sh
ould be useful for in vivo studies of DNA adduct formation by NPYR.