Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices

Abstract

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.

P-Stereodefined phosphorothioate analogs of glycol nucleic acids—synthesis and structural properties

“…Our efforts to obtain crystals of 5a–d for X-ray analysis were unsuccessful. Following the previous studies on crystallization of the oxathiaphospholane derivatives in DNA 11 and LNA 12 series, we tried to remove the DMT group in 5a–d to obtain 3′-OH OTP- G N′ derivatives 5*a–d (shown in square brackets in Scheme 1 ) using earlier successful p -toluenesulfonic acid monohydrate, yet with no success. Another protic acid (dichloroacetic acid), Lewis' acids (ZnCl 2 , SnCl 4 ), tetra- n -butylammonium peroxydisulfate 30 and NaHSO 4 /SiO 2 ( ref.…”

“…To prevent the DBU-promoted cleavage of the standard lcaa linker, the first nucleoside was attached to the solid support via a DBU-resistant sarcosine linker. 36 Even with double coupling used at each condensation step (earlier effectively applied in synthesis of PS-LNA 12 ), only ca. 75% repetitive yield was achieved (based on a DMT cation assay), so synthesis of octamers and decamers would provide only ca.…”

“…§ § Theoretical studies on the PS-DNA energy alteration suggest that R P -phosphorothioate units destabilize the B-helical structure by 2.7 ± 3.4 kcal mol −1 , while S P -counterparts stabilize it by −1.4 ± 2.4 kcal mol −1 , compared to the reference B-DNA helix. 40 Over several years P -stereodefined oligonucleotides (up to 15–18 nt in length) of PS-DNA 11 and PS-LNA 12 series, were obtained using an oxathiaphospholane method. 13 In several instances these oligomers showed P -stereodependent biological properties.…”

“…Because interesting P-stereodependent effects were observed in PS-DNA (e.g., different thermodynamic stability of antiparallel duplexes (PS-DNA : DNA) 22 or different propensity for the B / Z conversion 23 ), as well as in conformationally more restricted "locked" PS-LNA, 12 we have turned our efforts toward synthesis of P-stereodened phosphorothioate analogs of conformationally exible, glycol-based GNA (PS-GNA). Taking into account poor hybridization properties of GNA and the fact that the phosphorothioate modication usually decreases thermal stability of the modied duplexes{ we assumed that PS-GNA oligomers would not be able to act as antisense or antigene probes specically interacting with mRNA or genomic DNA strands.…”

P-stereocontrolled synthesis of oligo(nucleoside N3′→O5′ phosphoramidothioate)s – opportunities and limitations

“…4a,19 This relationship was later conrmed for the Pm-OTP derivatives of LNA nucleosides. 24,25 Unfortunately, although NPO-oligos were hydrolyzed by svPDE to a measurable extent (aer 4.5 h incubation $50% of NPO-T 10 remained intact; 13a aer 24 h incubation phosphoramidate T NP T was hydrolyzed in 22% yield, data not shown), phosphoramidothioates 3 when treated with svPDE or Nuclease P1 remained intact. A few years ago, chromatographically separated 5Tf and 5Ts (R ¼ Me, B 0 ¼ Thy) and 3 0 -O-acetylated thymidine were used in the synthesis of P-diastereomeric DMT T NPS T OAc (5 0 -O-DMT and 3 0 -O-Ac protected precursors of 3, Chart 3).…”