Synthesis, NMR and structural studies of oligonucleotide phosphorodithioates

Yesun Cho, Purdue University

Abstract

Solid phase synthesis of phosphorodithioate oligonucleotides containing phosphorodithioate groups at different positions is described using thiophosphoramidite chemistry. The thiophosphoramidite method is at present the only developed method which allows introduction of phosphorodithioate groups at any position in an oligonucleotide. There are three variables--concentration of DNA, salt concentration and temperature--which affect the transition between the duplex and hairpin conformations. By changing three variables, four different phosphorodithioate oligonucleotides, duplex form of d(CGCTTpS$\sbsp{2}{-}$AAGCG)$\sb2,$ hairpin form of d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$AAGCG), hairpin decamer d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$ApS$\sbsp{2}{-}$GCG), and duplex decamer d(CGC TpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$ApS$\sbsp{2}{-}$ApS$\sbsp{2}{-}$GCG)$\sb2$ have been synthesized and studied. Assignments of the $\sp1$H and $\sp{31}$P NMR spectra of a phosphorodithioate oligonucleotides have been made by two-dimensional $\sp1$H-$\sp1$H and heteronuclear $\sp{31}$P-$\sp1$H correlated spectroscopy. A hybrid relaxation matrix methodology has been used in combination with restrained molecular dynamics to derive structures of four different phosphorodithioate DNAs. Importantly, the final refined structure of phosphorodithioate duplexes, d(CGCTTpS$\sbsp{2}{-}$AAGCG)$\sb2$ and d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$ApS$\sbsp{2}{-}$ApS$\sbsp{2}{-}$G CG)$\sb2$ show a significant deviation from the parent phosphoryl duplex, d(CGCTTAA GCG)$\sb2.$ An overall bend and unwinding in the phosphorodithioate duplex are observed. The local helical parameters for the final structure of phosphorodithioate duplex decamer d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$ApS$\sbsp {2}{-}$ApS$\sbsp{2}{-}$ GCG)$\sb2$ are compared with d(CGCTTAA GCG)$\sb2.$ The deviation of the roll angle of phosphorodithioate thymidine (residue 5) is very high between the two different duplexes. There is a significant decrease in the propeller twist of phosphorodithioate residues compared to that of d(CGCTTAAGCG)$\sb2.$ The minor groove distance of phosphorodithioate thymidine of d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$ApS$\sbsp{2}{-}$ApS$\sbsp {2}{-}$GCG)$\sb2$ is 10.38 A which is very much longer than that of d(CGCTTAAGCG)$\sb2$ (4.2 A). The analysis of torsional angles and helical parameters reveals a significant disruption of the backbone conformation because of the phosphorodithioates. Because of the structural perturbation of the duplex, phosphorodithioate DNA has a strong tendency to exist as a hairpin. Two phosphorodithioate decamers, d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$AAGCG) and d(CGCTpS$\sbsp{2}{-}$TpS$\sbsp{2}{-}$ApS$\sbsp {2}{-}$ApS$\sbsp{2}{-}$GCG), which adopt hairpin conformation have 3 Watson-Crick C-G base pairs which constitute the stem region, and a single stranded loop region of 4 bases which are not base paired. Because the hairpin structure can separate the dithiophosphates along the loop, hairpin conformation of dithiophosphate is more stable. For the investigation of the crosslinking of platinum(II) complexes to oligonucleotide phosphorodithioates, two new complexes of the formula (Pt(ethylenediamine) (diethyldithiophosphate)) NO$\sb3$ and (Pt(terpyridine)(diethyldithiophosphate)) Cl, have been synthesized. Since the phosphorodithioate group provides a strong and highly selective binding site for platinum, the crosslinking of platinum complexes to a phosphorodithioate oligonucleotide was attempted and the binding was confirmed by $\sp{31}$P NMR.

Degree

Ph.D.

Advisors

Gorenstein, Purdue University.

Subject Area

Biochemistry

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