Efficient synthesis, liquid chromatography purification, and tandem mass spectrometric characterization of estrogen-modified DNA bases

Qiang Zhang, Michael L. Gross

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


Estrogens are metabolized to active quinones that modify DNA and may lead to various cancers. To extend the analytical methodology for estrogen-modified purine bases, we report here a simple modification to existing synthetic procedures that use 2-iodoxybenzoic acid (IBX) as the oxidizing agent for the reference material and putative biomarker, 4-hydroxyestrone-1-N3adenine (4-OH-E1-1-N3Ade). The reaction leads to two catechol estrogen quinones, CE1-2,3-Q and CE13,4-Q, both of which react via Michael additions to afford 4-OH-E1-N3Ade and other DNA adducts. Liquid chromatography separation permits the isolation of high-purity 4-OH-E1-1-N3Ade. With this method, we also prepared single 13C and uniformly 15N (U-15N) labeled 4-OH-E1-1-N3Ade with 8-13C-labeled Ade and U- 15N-labeled adenosine 5′-monophosphate (AMP). The approach is also effective for the synthesis of 4-hydroxyestradiol-1-N3adenine, 4-OH-E 2-1-N3Ade, and 4-hydroxyestrone(estradiol)-1-N7guanine, 4-OH-E 1 (E2)-1-N7Gua. The tandem mass spectra (MS2 and MS3) of 4-OH-E1-(unlabeled, 8-13C-, and U-15N-labeled)1-N3Ade and accurate mass measurements for MS 2 product ions allow us to assign unambiguously the formula of fragments and delineate the fragmentation pathways. One important reaction is dehydration, which occurs at the ketone oxygen in the C-17 position of estrone. Another is loss of NH3, an ubiquitous process for purines and modified purines, which is affected by the steroid modification. Evidence from MS/MS supports the migration of H-atom(s) from estrone in the loss of NH 3. An interesting interaction occurs between the steroid and the Ade in the modified base, promoting loss of CH2NH, a loss that distinguishes modified Ade from unmodified Ade. The synthesis of a stable isotope-labeled 4-OH-E1-1-NSAde and the understanding of the fragmentation processes will enable studies aimed at the detection of naturally occurring 4-OH-E1-1-N3Ade in biological samples.

Original languageEnglish
Pages (from-to)1244-1252
Number of pages9
JournalChemical Research in Toxicology
Issue number6
StatePublished - Jun 2008


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