A Transient Product of DNA Alkylation Can Be Stabilized by Binding Localization

Abstract

A 9-aminoacridine conjugate of a silyl-protected bis(acetoxymethyl)phenol (bisQMP) was synthesized and evaluated as an inducible cross-linking agent of DNA to test our ability to harness the chemistry of reactive quinone methide intermediates (QM). The acridine component was chosen for its ability to delivery an appendage to the major groove of DNA, and the silyl-protected component was chosen for its ability to generate two quinone methide equivalents in tandem upon addition of fluoride. This design created competition between reaction of (1) the 2-amino group of guanine that reacts irreversibly to form a stable QM adduct and (2) the more nucleophilic N7 group of guanine that reacts more efficiently but reversibly to form a labile QM adduct. This lability was apparently compensated by co-localization of the N7 group and QM in the major groove since the N7 adduct appeared to dominate the profile of products formed by duplex DNA. The controlling influence of acridine was also expressed in the sensitivity of the conjugate to ionic strength. High salt concentration inhibited covalent reaction just as it inhibits intercalation of the cationic acridine. As expected for QM formation, the presence of fluoride was indeed necessary for initiating reaction, and no direct benzylic substitution was observed. The conjugate also cross-linked DNA with high efficiency, forming one cross-link for every four alkylation events. Both alkylation and cross-linking products formed by duplex DNA were labile to hot piperidine treatment which led to approximately 40% strand scission and approximately 50% reversion to a material with an electrophoretic mobility equivalent to the parent DNA. All guanines exhibited at least some reactivity including those which were recalcitrant to cross-linking by an oligonucleotide-bisQMP conjugate designed for triplex formation [Zhou, G.; Pande, P.; Johnson, A. E.; Rokita, S. E. Bioorg. Med. Chem. 2001, 9, 2347-2354].

A walk along DNA using bipedal migration of a dynamic and covalent crosslinker

“…Most relevant to the current study was the design of bisfunctional precursors capable of generating two interrelated QM intermediates (bisQM) in series. These derivatives act as a potent crosslinker of DNA with an enviable ratio of crosslinking to monoalkylation 32,[48][49][50] . The reversible nature of reaction additionally prolongs the effective lifetime of the electrophile many-fold and can maintain interstrand crosslinking while still permitting strand exchange (Fig.…”

“…Second, the time required for reorientation could have been exacerbated by slow exchange of acridine between sites of intercalation. Acridine was originally attached to the bisQM to increase its local concentration proximal to duplex DNA and consequently minimize the molar equivalents required for efficient crosslinking 32,53 . The role of intercalation was similarly examined with the help of an additional derivative lacking the acridine component (bisQMP3, 3).…”

“…Reversible alkylation of G N7 had previously dominated the reaction of bisQMP1 (1) with duplex DNA and dynamic reaction at the C N3 and A N1 were also detected with related QM intermediates 32,43,47 . All of these nucleophiles likely participate in crosslinking, particularly since OD2 contains only a single G and this G is far from the region that could crosslink with OD5.…”

“…The use of DNA was initially suggested by the dynamics of its alkylation and crosslinking with a series of quinone methide (QM) derivatives [31][32][33] . QMs represent a readily available class of electrophiles capable of both capture and release of various nucleophiles 34 .…”

Synthesis of Reactive Oligonucleotides for Gene Targeting and Their Application to Gene Expression Regulation

“…This conformational change subsequently promoted the intermolecular transfer of quinone methide, causing the alkylation of the DNA target during duplex hybridization ( Figure 10). 56,57 This strategy may provide a general approach for the target-promoted alkylation of target genes in biological systems.…”