The Chemical and Biochemical Properties of Methylphosphotriester DNA and RNA in Comparison with Their Corresponding Methylphosphonates. A Dynamic Model Description

Abstract

Methylphosphotriester DNA and RNA are of great interest to investigate their hybridization affinity with natural DNA and RNA with respect to their physical and biological properties. The results are compared with related modified oligonucleotides. Specific attention will be given to the development of recent antiretroviral nucleosides focused on their molecular conformation and the mechanistic aspects based on the physical properties of phosphorus in a trigonal bipyramidal configuration corresponding with in vitro and in vivo kinetics.

Keywords:

• Methylphosphotriester DNA and RNA
• parallel RNA
• methylphosphonates
• kinetics

Notes

∗Related research has been shown that TAR-oligonucleotides inhibit the expression of a CAT gene that is regulated by the HTLV III HX10-LTR which contains the selected TAR region in comparison with an arbitrary sequence of the same number of bases.^{[ 20 ]}

^†For the preparation of partially and complete phosphate-methylated DNAs based on a solid-phase pro-cedure of Kuijpers et al. and of Alul et al.,^{[ 21 , 22 ]} the obtained results are in sharp contrast with our stepwise non-automated preparation.^{[ 1 , ,23 ]} If the reaction is carried out on a solid support the methyl group is more accessible than phosphorus in the oxidation with t-butyl hydroperoxide. After abstraction of a hydride anion of the methyl group accompanied with the formation of a three-membered ring, nucleophilic attack of OH⁻ on the secondary carbon results under ring opening in hydroxymethylphosphonate DNA. The driving force comes from the ring opening with the formation of a P═O bond. Since hydroxymethylphosphonate DNA shows resemblance with methylphosphonate DNA, the formation of duplexes on the DNA level is hindered by the unfavorable helix backbone in the phosphonate. In the case of the preparation of partially phosphate-methylated DNAs using a solid support a 400 MHz ¹H NMR spectrum is available.^{[ 21 ]} The interpretation was based on the intensity of the methyl-phosphate resonances relative to the other proton resonances as H₅′/H₅′′ of the 5′-end nucleotide. However this selection has been taken place arbitrarily. Moreover no duplex formation has been established.^{[ 22 ]}

^a The line width (Δ) of the methoxy doublets below the coalescence temperature was measured at half-height. The line broadening Δν was determined from a plot of ln Δ vs 1/T. From the plot of ln(Δν) versus 1/T, the E _A and the exchange rate, 1/τ (at 248 K), were determined, which were used to calculate the activation parameters according to the following: ΔH ^≠ = E _A—RT; ln (1/τ) = ln(RT/Nh) + ΔS ^≠/R—ΔH ^≠/RT; ΔG ^≠ = ΔH ^≠−TΔS ^≠.

^‡Epigenetic gene silencing is fundamental to cell determination and function. The epigenetic systems involved in repression of gene activity are based on histon methylation via the mono-, di-, or trimethylated cation state of lysine residues (DNA backbone interaction) and methylation of DNA bases. There are findings that methylation of histones and DNA bases are cooperative in gene-silencing networks in the cell.^{[ 44 ]} Such a cooperative model has been shown for naïve model systems with alternating CpG sequences. The significance of shielding of the phosphates in combination with methylation of the bases for conformational changes in DNA has been theoretically described by van Lier et al.^{[ 45 ]} Experimental evidence was given by Sugiyama et al.^{[ 46 ]}