2D NMR Study of the DNA Duplex d(CTCTC*A*ACTTCC)‚d(GGAAGTTGAGAG) Cross-Linked by the Antitumor-Active Dirhodium(II,II) Unit at the Cytosine-Adenine Step ² Mijeong Kang, Helen T. Chifotides, and Kim R. Dunbar* Department of Chemistry, Texas A&M UniVersity, College Station, Texas 77843 ReceiVed September 17, 2007; ReVised Manuscript ReceiVed October 30, 2007 ABSTRACT: The 2D NMR analysis in solution of the DNA duplex d(CTCTC*A*ACTTCC)‚ d(GGAAGTTGAGAG) binding to the dirhodium unit cis-[Rh 2 (μ-O 2 CCH 3 ) 2 (η 1 -O 2 CCH 3 )] + showed that an unprecedented intrastrand adduct, dsII, is formed with the dirhodium unit cross-linking in the major groove residues C5 and A6 (indicated with asterisks), also corroborated by enzyme digestion studies. Formation of the dirhodium complex dsII destabilizes significantly the duplex as indicated by the substantial decrease in its melting temperature (ΔT m )-22.9 °C). The reduced thermal stability of dsII is attributed to the decreased stacking of the bases and the complete disruption and/or weakening of the hydrogen bonds within the base pairs in the immediate vicinity of the metalation site (C5‚G20 and A6‚T19), but the effects due to the metal binding are more severe for the base pairs in the 5′ direction to the lesion site. The NMR spectroscopic data indicate that Watson-Crick hydrogen bonding is completely disrupted for the C5‚G20 site and considerably weakened for A6‚T19. In dsII, the bases C5 and A6 bind to eq positions of the dirhodium unit cis-[Rh 2 (μ-O 2 CCH 3 ) 2 (η 1 -O 2 CCH 3 )] + , which retains one monodentate and two bridging acetate groups, presumably due to steric reasons. Binding of A6 takes place via N7, whereas binding of the C5 base takes place via the exocyclic N4 site, resulting in the anti-cytosine rotamer with respect to site N3 in its metal-stabilized rare iminooxo form. In spite of the phenomenal success of cisplatin and related platinum compounds as anticancer drugs (1-3), a need has risen for new types of antitumor agents due to the limitations of platinum drugs, i.e., natural and acquired resistance of tumor cells, in addition to the numerous deleterious side effects (4-7). In this vein, dirhodium(II) tetracarboxylate derivatives (8) emerged as one of the most promising classes of antitumor-active transition-metal compounds, with Rh 2 (O 2 - CCH 3 ) 4 being the first member in the series. Dirhodium tetraacetate exhibits a “paddlewheel” structure with four equatorial (eq) acetate groups bridging the dirhodium unit in a symmetrical fashion and two axial (ax) sites available to donor ligands (Chart 1) (8). Pioneering studies that emanated in the 1970s showed that dirhodium carboxylate compounds Rh 2 (O 2 CR) 4 (R ) Me, Et, Pr) exhibit significant in vivo antitumor activity against L1210 tumors (9, 10), Ehrlich ascites (11-13), and sarcoma 180 and P388 tumor lines (14). Although the exact mech- anism of action of dirhodium compounds has not yet been elucidated, the most probable targets are DNA (15), RNA (16, 17), and enzymes (18) involved in DNA and RNA synthesis. Studies of the binding affinity of Rh 2 (O 2 CCH 3 ) 4 to several biomolecules by employing 14 C-labeled Rh 2 (O 2 - CCH 3 ) 4 indicated that Rh 2 (O 2 CCH 3 ) 4 is only slightly reactive toward native calf thymus DNA and polyguanylic (poly-G) and polycytidylic (poly-C) acids, but binds fairly well to denatured DNA and polyadenylic acid (poly-A) (11, 12). The interactions of Rh 2 (O 2 CCH 3 ) 4 with adenine nucleos(t)ides (11, 12, 19, 20) are established by axial binding of the adenine bases (via N7 and N1 if available (19)) and formation of hydrogen bonds between the exocyclic adenine NH 2 groups and the oxygen atoms of carboxylate ligands (for binding via N7), as evidenced by the reported crystal structures of Rh 2 (O 2 CCH 3 ) 4 (1-MeAdo) 2 1 (21) and [Rh 2 (O 2 - CCH 3 ) 2 (NHCOCF 3 ) 2 (9-methyladeninium) 2 ](NO 3 ) 2 (22). In ² K.R.D. gratefully acknowledges the Welch Foundation (Grant A-1449) for financial support. The NMR instrumentation at the Biomolecular NMR Laboratory at Texas A&M University was sup- ported by a grant from the National Science Foundation (DBI-9970232) and the Texas A&M University System. * To whom correspondence should be addressed. E-mail: dunbar@ mail.chem.tamu.edu. Phone: (979)-845-5235. Fax: (979)-845-7177. 1 Abbreviations: Ado, adenosine; CD, circular dichroism; COSY, double-quantum-filtered correlation spectroscopy; dap, 1,12-diaza- perylene; dsI, native d(CTCTCAACTTCC)‚d(GGAAGTTGAGAG); dsII, metalated d(CTCTC*A*ACTTCC)‚d(GGAAGTTGAGAG) (dsD); DTolF, anion of N,N′-p-tolylformamidinate; ESI-MS, electrospray ionization mass spectrometry; HPLC, high-performance liquid chro- matography; HSQC, heteronuclear single-quantum correlation; MALDI- MS, matrix-assisted laser desorption ionization mass spectroscopy; NOESY, 2D nuclear Overhauser spectroscopy; ssI, native d(CTCT- CAACTTCC); ssII, native d(GGAAGTTGAGAG); ssIII, metalated d(CTCTC*A*ACTTCC); T m, melting temperature of a DNA duplex. Chart 1: Structure of Dirhodium Tetraacetate 2265 Biochemistry 2008, 47, 2265-2276 10.1021/bi701901c CCC: $40.75 © 2008 American Chemical Society Published on Web 01/31/2008 Downloaded by TEXAS A&M GROUP on July 2, 2009 Published on January 31, 2008 on http://pubs.acs.org | doi: 10.1021/bi701901c