Stereocontrolled Solid-Phase Synthesis of Phosphate/Phosphorothioate (PO/PS) Chimeric Oligodeoxyribonucleotides on an Automated Synthesizer Using an Oxazaphospholidine–Phosphoramidite Method

Abstract

Stereocontrolled solid-phase synthesis of phosphate/phosphorothioate chimeric oligodeoxyribonucleotides (PO/PS-ODNs) was achieved by integrating the conventional phosphoramidite method into a previously developed oxazaphospholidine method for the stereocontrolled synthesis of P-chiral oligonucleotides. P-Stereodefined PO/PS-ODNs with mixed sequences (up to 12-mers) were obtained in good yields and high stereoselectivities by reacting different combinations of monomers (conventional phosphoramidites/diastereopure nucleoside 3'-O-oxazaphospholidines), activators (ETT/CMPT), capping reagents (Pac2O/CF3COIm), and oxidizing/sulfurizing reagents (TBHP/POS) on an automated synthesizer. A thermal denaturation study examined the resultant diastereopure PO/PS-ODN 12-mers with three consecutive (Rp)- or (Sp)-PS-linkages at the internal or terminal regions of the molecules. We found that (Rp)-PO/PS-ODNs can only moderately destabilize duplexes with complementary oligoribonucleotides (ORNs) compared with their unmodified ODN counterparts (ΔTm = -0.4 °C per modification). In contrast, (Sp)-PO/PS-ODNs have larger destabilizing effects (ΔTm = -1.2 to -0.8 °C per modification). Although smaller destabilizing effects were observed when the (Sp)-PS-linkages were incorporated into the terminal regions of the molecule, there was a weaker correlation between the location of an incorporated (Rp)-PS-linkage and its destabilizing effect.

Stereocontrolled Synthesis of Boranophosphate DNA by an Oxazaphospholidine Approach and Evaluation of Its Properties

“…We previously reported an oxazaphospholidine approach for the stereocontrolled synthesis of PS-oligonucleotides. − This approach is a type of phosphoramidite method whose features include the use of oxazaphospholidine monomer units, , whose phosphorus atom is in a stereocontrolled manner, derived from optically pure aminoalcohols, and N -cyanomethylpyrrolidinium triflate (CMPT), an acidic activator with low nucleophilicity. Mainly on the basis of the oxazaphospholidine approach, a few groups clearly revealed that each stereoregular PS oligonucleotide is significantly different in its properties, including the affinity to its complementary DNA and RNA, nuclease resistance, and RNase H activity. ,,− Quite recently, another synthetic strategy has also been reported to construct stereoregular PS-DNA backbone using P­(V)-based reagents, which is different from conventional phosphoramidite chemistry . Some stereoregular PS nucleic acid therapeutics are still undergoing clinical trials, and therefore, stereoregular PS-oligonucleotides have the potential to be the next gold standard as nucleic acid therapeutics.…”

RNA Phosphorothioate Modification in Prokaryotes and Eukaryotes

“…Most of the aforementioned therapeutic oligonucleotides approved by the U.S. Food and Drug Administration (FDA) or currently under clinical trials utilize phosphorothioate modifications to improve the metabolic stability and cellular uptake. , The traditional synthesis of phosphorothioate oligonucleotides based on phosphoramidites affords a mixture of stereoisomers. Recently, the stereogenic synthesis of phosphorothioate oligonucleotides has been significantly improved, − which will have huge impacts on future therapeutic developments.…”

Synthesis and properties of DNA oligomers containing stereopure phosphorothioate linkages and C-5 modified deoxyuridine derivatives

“…These results were consistent with previous reports and the differences in the properties between the two diastereomers. 7,8,11,34,40 Moreover, the DT m values between the PO/(Rp)-PS-DNAs and the PO/(Sp)-PS-DNAs were different depending on the substituents on the C-5 position. In the case of dCG(U PS ) 8 CG (4a) and dCG(T PS ) 8 CG (4b), the DT m values between the PO/(Rp)-PS-DNAs and PO/(Sp)-PS-DNAs were relatively small, 2.8 C and 2.9 C, respectively.…”