Speaker: Serge L. Beaucage, Center for Biologics Evaluation and Research, FDA, Bethesda, MD

Topic: Deoxyribonucleoside Cyclic N-Acylphosphoramidites as a New Class of Monomers for the Stereocontrolled Synthesis of Oligonucleoside Phosphorothioates and the Potential Synthesis of Oligonucleotides on Arrayable Surfaces

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Friday, May 19, 2000, at 2:00 PM

Abstract: Oligodeoxyribonucleoside phosphorothioates (PS-ODNs) have been and continue to be extensively studied as potential therapeutic agents against various types of cancer and infectious diseases in humans. Given that these oligonucleotide analogues are P-chiral, each of the n internucleotidic phosphorothioate linkages adopts either a R_P or a S_P configuration. Stereopure R_P-PS-ODNs were shown to possess a lower stability to nucleases endogenous to human serum than the parent PS-ODNs with undefined P-chirality. Conversely, stereopure S_P-PS-ODNs have demonstrated superior stability to these nucleases than P-diastereomeric PS-ODNs. Thus, further investigation into the biological, pharmacokinetic, and toxicological properties of P-stereopure PS-ODNs requires improved chemical methods to synthesize these biomolecules and increase their availability for clinical studies. We now wish to report deoxyribonucleoside cyclic N-acylphosphoramidites as a promising new class of monomers in the P-stereospecific synthesis of PS-ODNs. A straightforward synthesis of deoxyribonucleoside cyclic N-acylphosphoramidites begins with N-acylation of (±)-2-amino-1-phenylethanol followed by reaction of the resulting amido alcohol with hexaethylphosphorous triamide to generate a cyclic N-acylphosphoramidite intermediate. Then, condensation of this intermediate with a properly protected deoxyribonucleoside gave, after chromatography, the corresponding nucleoside cyclic N-acylphosphoramidite derivative isolated as pure R_P and S_P diastereomers. Mixing the R_P (or S_P) diastereomer with 3'-O-acetylthymidine and N,N,N',N'-tetramethylguanidine led to near quantitative formation of a dinucleoside phosphite triester with complete P-stereospecificity as judged by ³¹P NMR spectroscopy. Sulfurization of this triester produced, as expected, a P-stereodefined dinucleoside phosphorothioate. The solid-phase synthesis of [R_P,R_P]- and [S_P,S_P]-d(C_PSC_PSC), and [R_P,S_P,R_P]-d(C_PSC_PSC_PSC) was then attempted using P-stereodefined deoxyribonucleoside cyclic N-acylphosphoramidites. HPLC analysis of these oligonucleoside phosphorothioates showed a single peak, indicative of P-stereopurity, for each oligomer. Thus, the application of deoxyribonucleoside cyclic N-acyl phosphoramidites to P-stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates may offer a compelling alternative to the methods currently used for such syntheses. Furthermore, deoxyribonucleoside cyclic N-acylphosphoramidites can produce oligonucleotides under relatively wet conditions. This property is particularly indicated for parallel syntheses of oligonucleotide arrays using robotic equipment under atmospheric conditions.