Peracylation of nucleosides with methionine: foundation for a method to detect carcinogen adducts

We report the chemical foundation for a new method to detect carcinogen-DNA adducts, which we have designated adduct detection by acylation with methionine (ADAM). The method is based on reaction of DNA adducts with a protected methionine derivative, (tert-butoxycarbonyl)-L-methionine N-hydroxysuccinimidyl ester (TMB-NHS). Acylation of 2'-deoxyguanosine (dGuo), used as a prototypical deoxynucleoside, and N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dGuo-8-ABP), the major DNA adduct formed after in vivo exposure to 4-aminobiphenyl, a known human carcinogen, with TBM-NHS was optimized, and products were characterized by 3H radioactivity, UV absorbance, mass spectrometry, and 1H and 13C NMR. Derivatives acylated on hydroxyl (5' or 3') and/or amine (N2) groups were unambiguously determined to be mono-, bis-, and tris-TBM-acylated nucleosides. Under optimal acylation conditions [TBM-NHS (> or = 4 x 10(5) molar equivalents), pyridine (50 microL), THF (50 microL), and diisopropylcarbodiimide (DIC) (1 microL) and incubation for 2 h at 37 degrees C], the efficiency of acylation for picomole or smaller quantities of dGuo-8-ABP exceeded 95%, with the tris-TMB-acylated nucleoside representing the major product (88%). A linear correlation was obtained between the amount of [3H]dGuo-8-ABP introduced into the reaction and the total amount of TBM-acylated products formed. These results support the validity of this strategy for adaptation as an analytical method for the detection of low levels of DNA adducts through the use of (tert-butoxycarbonyl)-L-[35S]-methionine N-hydroxysuccinimidyl ester.