Chemical transformation/derivatization of O6-methyl- and O6-(hydroxyethyl)guanine for detection by GC-EC/MS

Abstract

In this project we set out to make an important class of DNA adducts, comprising O6-alkyl and O6-(hydroxyalkyl)guanines, susceptible to sensitive detection by GC-EC/MS. While existing literature indicated that pentafluorobenzylation would be useful for the ring NH site on these compounds, how to best overcome the polarity of the exocyclic NH2 and OH groups, without losing the O6-alkyl moiety, was less clear. Working with O6-methylguanine and O6-(2'-hydroxyethyl)guanine as representative analytes, we found that the NH2 group could be converted into fluoro without loss of the O6 substituent. For the OH group, a comparison of several derivatives (OR') led to R' = tert-butyl as the best choice at this stage. The latter work, especially via NMR, also allowed exact structural assignments to be made for the N7 and N9 pentafluorobenzyl isomeric derivatives that formed. Of these R' derivatives, the N7 isomers migrated slower on silica-TLC, had higher GC retention times, had lower responses by GC-EC/MS, and were preferentially destroyed as the GC column aged. However, the N9 isomer was slower on TLC when the OH was not derivatized. This behavior was rationalized using a concept of "polar footprint" for the derivatives. The concept also seemed to explain the puzzling GC-EC/MS behavior of some related compounds in our laboratory. Apparently the polar footprint should be minimized in designing derivatives for trace detection by GC-EC/MS.

Detection of DNA Adducts by Electron Capture Mass Spectrometry

“…Methyl O 6 -G (23,29,30,56). This analyte was derivatized in the same way as 2′-(hydroxyethyl)-O 6 -G, which is discussed above, except that the last step, derivatization of the hydroxyethyl group, was not needed.…”

High-Performance Liquid Chromatography/Electrospray Mass Spectrometry for the Analysis of Modified Bases in DNA: 7-(2-Hydroxyethyl)Guanine, the Major Ethylene Oxide−DNA Adduct

“…Mass spectrometry, despite its lower sensitivity compared to the previously mentioned methods, offers molecular weight identification and sometimes structural specificity . The use of MS for the characterization of DNA adducts has followed technical evolutions; early derivatization (permethylation or persilylation , or field desorption methods have been replaced by techniques like fast atom bombardment, − matrix-assisted laser desorption, and derivatization using electrophoric groups. − Electrospray ionization and, more specifically, its interface with liquid chromatography or capillary electrophoresis provides the possibility of both ionization and liquid-to-gas transition for a wide variety of thermally labile compounds, including many biomolecules. Electrospray mass spectrometry (ESMS) has been used in the study of normal or chemically modified synthetic oligonucleotides , and also for the analysis of unmodified nucleotides and nucleosides. , Characterization of DNA adducts has been carried out recently at the nucleoside level by coupling electrospray to HPLC (after DNA exposure at a high single dose of xenobiotic) and also at the nucleotide level by coupling ESMS to capillary electrophoresis and analyzing standard adduct samples, , with a detection limit of approximately 3 × 10 -9 M using selected ion recording (SIR).…”

4-(4′-Methyltetrafluorophenyl)-2,3,5,6-tetrafluorobenzyl bromide: A new electrophoric derivatizing reagent

“…The latter compound is known from prior work in our laboratory as an intermediate in the conversion of O 6 -methylguanine, a DNA adduct, to 2-fluoro-O 6 -(2′-hydroxyethyl)-N-(7 and 9)-(pentafluorobenzyl)hypoxanthines [6]. In this prior work, where pentafluorobenzyl bromide was employed as the electrophoric derivatizing reagent, two isomers were formed (N7 and N9) in nearly equal yields.…”

“…The N9 isomer also eluted earlier (3.3 min) than the N7 isomer (3.7 min) by GC-EC-MS. The difference in the retention and responses of these compounds was hypothesized to arise from the N7 compound having a polar footprint, making it not only less volatile by GC, but more susceptible to adsorption losses or decomposition in the GC-EC-MS system [5,6].…”