A new biocompatible strategy for photoinduced DNA interstrand cross-linking is presented. Methylene blue induced (1)O(2) formation triggers furan oxidation; the resulting aldehyde then rapidly reacts with complementary A or C with formation of stable adducts. Easily accessible furan modified nucleosides, a commercially available photosensitizer, and visible light irradiation constitute the necessary tools to achieve selective duplex interstrand cross-linking.

Chemical reagents that form interstrand cross-links have been used for a long time in cancer therapy. They covalently link two strands of DNA, thereby blocking transcription. Cross-link repair enzymes, however, can restore the transcription processes, causing resistance to certain anti-cancer drugs. The mechanism of these cross-link repair processes has not yet been fully revealed. One of the obstacles in this study is the lack of sufficient amounts of well-defined, stable, cross-linked duplexes to study the pathways of cross-link repair enzymes. Our group has developed a cross-link strategy where a furan moiety is incorporated into oligodeoxynucleotides (ODNs). These furan-modified nucleic acids can form interstrand crosslinks upon selective furan oxidation with N-bromosuccinimide. We here report on the incorporation of the furan moiety at the 2 0 -position of a uridine through an amido or ureido linker. The resulting modified ODNs display an unprecedented selectivity for cross-linking toward a cytidine opposite the modified residue, forming one specific cross-linked duplex, which could be isolated in good yield. Furthermore, the structure of the formed cross-linked duplexes could be unambiguously characterized. ' INTRODUCTIONThe high specificity with which oligonucleotides recognize nucleic acids has led to considerable interest in the design of modified oligonucleotides. 1 These customized nucleic acid derivatives can be used for a range of therapeutic and analytical purposes, including the treatment of diseases (antisense, 2-4 siRNA 5-8 ), the regulation of gene expression (antigene, 9-11 decoy DNA 12,13 ), the investigation of RNA tertiary structures, 14-16 and the study of DNA damage and the resulting repair processes. [17][18][19][20][21] DNA is not indefinitely stable, and numerous sources of DNAdamaging agents of endogenous and exogenous origin contribute further to the instability of DNA. 22 DNA interstrand cross-links (ICLs) are among the most cytotoxic lesions known, the covalent bond between the two DNA strands prohibiting strand separation and thereby blocking vital aspects of DNA metabolism. ICLs are mostly formed by reaction of cellular DNA with bifunctional electrophilic compounds that are either formed endogenously (e.g., lipid peroxidation) or present through exogenous exposure. 23 Additionally, generation of an abasic site in the DNA upon depurination can also give rise to ICL formation. 20,24 The cytotoxic properties of such ICL-forming agents are exploited by cancer chemotherapeutics, where their toxicity ultimately leads to apoptosis of the malignant cells. 25 Development of resistance of the tumor cells to treatment with antitumor drugs can often be attributed to enhanced ICL repair. 23 These repair pathways find their evolutionary origin in the necessity to counteract the threat posed by endogenous and exogenous crosslinking compounds, and failure to repair these lesions can lead to inherited disorders such as Fanconi anemia. 23 Thus, although DNA repair is essential for a heal...

2 0 -O-(3-(Furan-2-yl)propyl)adenosine was synthesized and evaluated for interstrand crosslink (ICL) formation in DNA duplexes. In situ oxidation of the furan moiety with NIS showed rapid crosslink formation to dA and dC, while dT and dG were inactive.Oligonucleotides that form interstrand crosslinks (ICLs) have found widespread applications in chemical biology research areas. Therefore, chemical and enzymatic methods have been developed to incorporate crosslinks into helical regions of DNA and RNA. 1 Examples include the synthesis of a duplex incorporating a preformed crosslinked dinucleotide 2,3 or postsynthetic modification of duplexes by bifunctional crosslinking reagents. 4,5 However, in some cases, the site-specific introduction of a crosslink is problematic due to formation of a mixture of monoadducts, intrastrand and interstrand crosslinks. Introduction of reactive moieties at a specific position within the duplex can circumvent the selectivity issue as is shown for photocrosslinking with 4-thiouridine, 6 5-bromouridine, 7 5-methyleneaminouridine 8 or 8-azidoadenosine. 9 Furthermore, a thio-modified oligonucleotide (ON) can form disulfide bonds post-synthetically. 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). 16,17 First crosslinking results obtained for the furan-modified 2 0 -amido-uridine with complementary adenine (A) were further extended into a more detailed selectivity study against all canonicals using a more synthetically accessible acyclic furanmodified building block. In the latter case, strong selectivity for crosslinking to its opposite canonical A or cytidine (C) was observed, without formation of crosslinks to neighboring or distant bases. 17 In this context, furan-modification on other canonicals than uridine and the potential impact on the crosslinking process have not been evaluated yet. From this point of view, we became interested in evaluating the scope of furan-modified purines for ICL. We here wish to report on the synthesis and incorporation of 2 0 -O-(3-(furan-2-yl)propyl)adenosine as a building block for crosslinking of DNA duplexes.From a synthetic point of view, 2 0 -modification was an obvious choice as nucleobase modification will in most cases be unfavourable for hybridization. Taking into account that 2,2 0 -anhydrointermediates are only feasible for pyrimidines, a 2 0 -amido modification of adenosine is difficult to achieve. Therefore, we decided to take advantage of our previously developed 2 0 -O-alkylation strategy ...

The furan modified adenosine building block (I) is synthesized and evaluated for interstrand cross-link formation in DNA duplexes. -(JAWALEKAR, A. M.; DE BEECK, M. O.; VAN DELFT*, F. L.; MADDER, A.; Chem. Commun. (Cambridge) 47 (2011) 10, 2796-2798, http://dx.doi.org/10.1039/c0cc04667a ; Inst. Mol. Mater., Radboud Univ. Nijmegen, NL-6525 ED Nijmegen, Neth.; Eng.) -M. Paetzel

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