Interstrand Cross-Link Formation in Duplex and Triplex DNA by Modified Pyrimidines

Abstract

DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2′-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO 4 . The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10× faster than the thymidine derivative when incorporated at the 5′-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.DNA alkylation is a common chemical process that is useful as a chemical tool but can also have biological consequences. For instance, alkylating agents are employed in the laboratory for determining DNA structure. However, alkylated DNA can be genotoxic and/or cytotoxic. 1 In addition, a number of molecules, including some antitumor agents, react with DNA by forming covalent bonds to the opposing strands. 2 The cross-linked strands are strong blocks to replication and are cytotoxic if unrepaired. 3 Modified oligonucleotides containing alkylating agents have also been used to form interstrand cross-links in duplex and triplex DNA. 4-6 We recently described a modified thymidine (1) that forms stable base pairs with dA and produces interstrand cross-links (ICLs) (4a, 4b) in duplex DNA under mild oxidative conditions (Scheme 1). 7-9 The cross-linking reaction is unusual in that like the antitumor agent N,N′-bis E-mail: mgreenberg@jhu.edu. † Johns Hopkins University. ‡ N...

Synthesis and incorporation of a furan-modified adenosine building block for DNA interstrand crosslinking

“…10,11 However such ICLs are highly dependent on the proper positioning of thiol groups and, more importantly, require modification of both strands. More recent studies on site-specific ICL formation involve modified phenylselenyl derivatives of thymidine or 5-methyl-2 0 -deoxycytidine, 12 1,4-dioxobutane abasic lesion, 13 alkyl-connected 2-amino-6-vinylpurine 14 and 4-amino-6-oxo-2-vinylpyrimidine. 15 We have earlier developed a complementary methodology incorporating furan-modified 2 0 -amido-uridine and an acyclic building block to give a site-specific ICL upon oxidation with Nbromosuccinimide (NBS).…”

5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells

“…PhSe-T has been shown to produce an ICL with the opposing dA upon UV photolysis through a 5-(2 0 -deoxyuridinyl) methyl radical intermediate or upon oxidation through a methide intermediate [10][11][12][13][14]. MeSe-T, which was recently synthesized using a similar synthetic method to that of PhSe-T, triggered similar apoptotic signaling pathways as PhSe-T and showed more toxicity than PhSe-T in all cell lines used in this study [15].…”

“…Previous studies have shown that phenylselenide derivative 5-phenylselenyl-methyl-2 0 -deoxyuridine (PhSe-T) can be incorporated into DNA by DNA polymerases and can produce ICLs with the opposing strand 2 0 -deoxyadenosine (dA) [10][11][12][13][14]. The nucleoside derivative 5-methylselenylmethyl-25-phenylselenyl-methyl-2 0 -deoxyuridine-deoxyuridine (MeSe-T) was recently synthesized following a similar synthetic method as PhSe-T, and its molecular structure is identical to that of PhSe-T, except for replacement of the phenyl group with a methyl group [15] (Fig.…”

DNA Radiosensitization: The Search for Repair Refractive Lesions Including Double Strand Breaks and Interstrand Crosslinks

“…The chemical structures of several DNA interstrand crosslinks have been reported only recently. Several groups have focused on the identification of ICL structures synthesized in model systems using light-and/or oxidationsensitive precursors (Hong et al, 2006;Weng et al, 2007;Peng et al, 2008;Kim & Hong, 2008;Op de Beeck & Madder, 2011). A few structures have been definitively identified in natural conditions and, to our knowledge, only one in cellular DNA (Regulus et al, 2007) (Fig.…”