5'-Cholesterol-CE Phosphoramidite - CAS 143723-64-2

Automated chemical synthesis of oligonucleotides is of fundamental importance for the production of primers for the polymerase chain reaction (PCR), for oligonucleotide-based drugs, and for numerous other medical and biotechnological applications. The highly optimised automised chemical oligonucleotide synthesis relies upon phosphoramidites as the phosphate precursors and one of the drawbacks of this technology is the poor bench stability of phosphoramidites. Here, we report on the development of an on-demand flow synthesis of phosphoramidites from their corresponding alcohols, which is accomplished with short reaction times, near-quantitative yields and without the need of purification before being submitted directly to automated oligonucleotide synthesis. Sterically hindered as well as redox unstable phosphoramidites are synthesised using this methodology and the subsequent couplings are near-quantitative for all substrates. The vision for this technology is direct integration into DNA synthesisers thereby omitting manual synthesis and storage of phosphoramidites.

This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pAm pA, pAm pG, and pAm pU that utilizes phosphoramidite chemistry. The overall synthetic strategy involves three steps. The first step involves the coupling reaction between 5'-O-MMT protected nucleoside-3'-O-phosphoramidite and a protected nucleoside containing a free 5'-OH group in the presence of tetrazole, followed by the oxidation of phosphite triester using tert-butyl hydroperoxide to give the corresponding protected Nm pN. Next, the 5'-O-MMT is cleaved under 3% trichloroacetic acid in dichloromethane conditions. Finally, the 5'-hydroxyl group is phosphorylated by the use of an activated bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite using tetrazole, followed by the oxidation of trivalent to pentavalent phosphorus using tert-butyl hydroperoxide and subsequent deprotection using ammonium hydroxide to afford the corresponding RNA dinucleotide, pNm pN, in good yields with high purity (>99.5%).

Introduction of cationic modifications into an oligonucleotide can increase its nuclease resistance and duplex- or triplex-forming abilities. In a recent study, we found that the nuclease resistance and RNA binding selectivity of an oligonucleotide containing a 5'-(R)-amino-5'-deoxy-5'-(R)-hydroxymethylthymidine residue were greater than those of the unmodified oligonucleotide. In this article, we describe the synthesis of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-alkene derivative using either of two commercial AD (asymmetric dehydroxylation) mixes or via epoxidation and ring opening. We also provide detailed protocols for the syntheses of oligonucleotides containing 5'-amino-5'-deoxy-5'-hydroxymethylthymidine residues. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine phosphoramidites 9a and 9b Basic Protocol 2: Synthesis of oligonucleotides 1 and 2 containing 5'-amino-5'-deoxy-5'-hydoxymethylthymidine residues (R T and S T).