Characterization of aziridinylbenzoquinone DNA cross-links by liquid chromatography-infrared multiphoton dissociation-mass spectrometry

Abstract

DNA cross-linking was evaluated by liquid chromatography-tandem mass spectrometry to determine the relative cross-linking abilities of two aziridinylbenzoquinones. Reactivities of RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone), a clinically studied antitumor cross-linking agent, and an analogue containing a phenyl group (2,5-diaziridinyl-3-[hydroxymethyl]-6-phenyl-1,4-benzoquinone, PhRH1) rather than a methyl group were compared. The bulky phenyl substituent was added to determine the impact of steric hindrance on the formation of cross-links within a double helical structure. Cross-links formed by RH1 and PhRH1 were observed at 5'-dGNC sites as well as 5'-dGAAC/dGTTC sites. RH1 was more effective at forming cross-links than PhRH1 for a variety of duplexes. Infrared multiphoton dissociation (IRMPD) and collision-induced dissociation results confirmed the presence and the location of the cross-links within the duplexes, and IRMPD was used to identify the dissociation pathways of the cross-linked duplexes.

Figure 1

Aziridinyl benzoquinones.

Figure 2

Selected mass spectra from LC-MS experiments for RH1 (a–d) and PhRH1 (e–h) after reaction with DS4. Elution times were as follows: a. 19.66–20.4 min., b. 28.54–29.76 min, c. 33.57–34.43, d. 16.73–17.49 min., e. 19.17–19.73 min., f. 28.88–30.53 min., g. 40.54–41.55, and h. 15.84–16.33 min. RH1 monoadducts are noted by ▲ and crosslinks are noted by ■ and PhRH1 monoadducts are noted by △ and crosslinks by □.

Figure 3

Relative percentages of crosslink formation for RH1 and PhRH1.

Figure 4

CID (a) vs. IRMPD (b) spectra for [DS3+RH1]⁶⁻. Ssa and ssb refer to the single strands as described in Table 1. The irradiation time for IRMPD was 5 ms. Fragments from ssa are in bold italics.

Figure 5

IRMPD spectra for selected crosslinked duplexes. Dissociation spectrum from [DS4+RH1]⁶⁻ after 5 ms irradiation time and dissociation spectrum from [DS5+RH1]⁶⁻ after 3 ms irradiation time. Fragments from ssa are in bold italics. Fragments containing the crosslink are noted by ■.

Figure 6

Time-variable IRMPD of [DS4+RH1]⁶⁻.

Figure 7

Major fragmentation pathways for duplexes crosslinked with RH1 or PhRH1.

Scheme 1

Reduction mechanism and possible crosslink structure for RH1. Adapted from ref. .

Scheme 2

Fragmentation process of a duplex with an interstrand crosslink at two guanine bases after IR irradiation.