PERACYLATION OF NUCLEOSIDES WITH METHIONINE - FOUNDATION FOR A METHODTO DETECT CARCINOGEN ADDUCTS

We report the chemical foundation for a new method to detect carcinoge n-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-methi onine N-hydroxysuccinimidyl ester (TBM-NHS). Acylation of 2'-deoxyguan osine (dGuo), used as a prototypical deoxynucleoside, and N-(deoxyguan osin-8-yl)-4-aminobiphenyl (dGuo-8-ABP), the major DNA adduct formed a fter in vivo exposure to 4-aminobiphenyl, a known human carcinogen, wi th TBM-NHS was optimized, and products were characterized by H-3 radio activity, UV absorbance, mass spectrometry, and H-1 and C-13 NMR. Deri vatives acylated on hydroxyl (5' or 3') and/or amine (N-2) groups were unambiguously determined to be mono-, bis-, and tris-TBM-acylated nuc leosides. Under optimal acylation conditions [TBM-NHS (greater than or equal to 4 x 10(5) molar equivalents), pyridine (50 mu L), THF (50 mu L), and diisopropylcarbodiimide (DIC) (1 mu L) 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-TBM-acylated nuc leoside representing the major product(88%). A linear correlation was obtained between the amount of [H-3]dGuo-8-ABP introduced into the rea ction and the total amount of TBM-acylated products formed. These resu lts support the validity of this strategy for adaptation as an analyti cal method for the detection of low levels of DNA adducts through the us e of (tert-butoxycarbonyl)-L-[S-35-methionine N-hydroxysuccinimidyl ester.