New monomers, 5‘-O-DMT-deoxyribonucleoside 3‘-O-(2-thio-“spiro”-4,4-pentamethylene-1,3,2-oxathiaphospholane)s, were prepared and used for the stereocontrolled synthesis of PS−Oligos via the oxathiaphospholane approach. These monomers and their 2-oxo analogues were used for the synthesis of “chimeric” constructs (PS/PO−Oligos) possessing phosphate and P-stereodefined phosphorothioate internucleotide linkages. The yield of a single coupling step is approximately 92−95%, and resulting oligomers are free of nucleobase- and sugar-phosphorothioate backbone modifications. Thermal dissociation studies showed that for heteroduplexes formed by [R P]-, [S P]-, or [mix]-PS/PO-T10 with dA12, dA30, or poly(dA), for each template, the melting temperatures, as well as free Gibbs' energies of dissociation process, are virtually equal. Stereochemical evidence derived from crystallographic analysis of one of the oxathiaphospholane monomers strongly supports the participation of pentacoordinate intermediates in the mechanism of the oxathiaphospholane ring-opening condensation.

No abstract

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3 0 -phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns.