Selective covalent binding of a positively charged water-soluble benzoheterocycle triosmium cluster to single- and double-stranded DNA

Edward Rosenberg, Fabrizio Spada, Kent Sugden, Brooke Martin, Roberto Gobetto, Luciano Milone, Alessandra Viale

Research output: Contribution to journalArticlepeer-review

Abstract

The water-soluble triosmium cluster [Os3(CO)9(μ- η2-(4-CHO)C9H5N)(μ-H)(P(OCH 2CH2N(CH3)3I)3)] (4) was tested for its reactivity with plasmid DNA. In contrast to the band retardation previously observed with a related series of positively charged clusters, an intensification and retardation of three discrete bands was observed with increasing cluster concentration. In order to further investigate the apparent modification of DNA by 4, its interaction with a 22-oligomer (sequence 5′-AGT TGT GGT GAC TTT CCC AGG C-3′) was examined. Incubation with this oligonucleotide (pH 7.4 in Tris-HCl buffer and 100 mM NaCl) followed by HPLC analysis revealed the formation of three dose dependent products assigned as covalent modifications at three sites of the oligonucleotide. Incubation of 4 with 32P-ATP labeled oligonucleotide at the 5′-end followed by treatment with piperidine and comparison with the standard Maxam-Gilbert sequencing protocol products revealed only general background cleavage, indicating that the modification products are piperidine labile and suggesting that the modification involved formation of a Schiff base. An alternative approach was then pursued which involved annealing the 4-oligonucleotide products with their complementary strand and treatment of the resulting duplex DNAwith the exonuclease, Exo III. This assay indicated three exonuclease stops, consistent with the three products observed by HPLC whose electrophoretic mobility approximately matched guanine containing fragments when compared with the Maxam-Gilbert sequencing lanes. Reduction of the 4-oligonucleotide products with borohydride reducing agents, followed by treatment with piperidine, resulted in the formation of one product (by HPLC) with the same electrophoretic mobility as the AGTT fragment based on comparison with the Maxam-Gilbert sequencing lanes. This product most likely results from reduction of an initially formed Schiff base adduct (to the corresponding amine) with the guanine of the TGT fragment of the oligonucleotide, and corresponds to the most stable of the three Schiff base adducts detected by HPLC and by incubation with the exonuclease. The other two products are less stable and competitive reduction of the free aldehyde functionality on the cluster in equilibrium with these adducts precludes their detection after treatment with the reducing agents. The formation of the Schiff base adduct is further corroborated by the model reaction of [Os3(CO)10(μ-η2-(4- CHO)C9H5N)(μ-H)] (4′) with acetylated guanine in nonaqueous solvents where disappearance of the aldehyde resonance and the appearance of several new resonances in the 6-9 ppm region of the 1H NMR of the reaction mixture is noted.

Original languageEnglish
Pages (from-to)4729-4738
Number of pages10
JournalJournal of Organometallic Chemistry
Volume689
Issue number25 SPEC. ISS.
DOIs
StatePublished - Dec 6 2004

Keywords

  • Bio-markers
  • Covalent binding
  • DNA
  • Osmium clusters
  • Schiff base

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