Depurinating acylfulvene-DNA adducts: characterizing cellular chemical reactions of a selective antitumor agent

J Am Chem Soc. 2007 Feb 21;129(7):2101-11. doi: 10.1021/ja0665951. Epub 2007 Jan 27.


Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF-DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / metabolism
  • Alkylation
  • Animals
  • Antineoplastic Agents, Alkylating / chemistry*
  • Antineoplastic Agents, Alkylating / pharmacology
  • Cattle
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • DNA / chemistry*
  • DNA / drug effects
  • DNA / metabolism
  • DNA Adducts / chemistry*
  • DNA Adducts / metabolism
  • Deoxyadenosines / chemistry
  • Deoxyadenosines / metabolism
  • Deoxyguanosine / chemistry
  • Deoxyguanosine / metabolism
  • Humans
  • NADP / chemistry
  • NADP / metabolism
  • Nucleic Acid Conformation
  • Sesquiterpenes / chemistry
  • Sesquiterpenes / pharmacology
  • Spectrometry, Mass, Electrospray Ionization
  • Spiro Compounds / chemistry
  • Spiro Compounds / pharmacology


  • Antineoplastic Agents, Alkylating
  • DNA Adducts
  • Deoxyadenosines
  • Sesquiterpenes
  • Spiro Compounds
  • acylfulvene
  • NADP
  • DNA
  • calf thymus DNA
  • Alcohol Oxidoreductases
  • Deoxyguanosine