Phosphate-ON Phosphoramidite

We report the catalytic stereocontrolled synthesis of dinucleotides. We have demonstrated, for the first time to our knowledge, that chiral phosphoric acid (CPA) catalysts control the formation of stereogenic phosphorous centers during phosphoramidite transfer. Unprecedented levels of diastereodivergence have also been demonstrated, enabling access to either phosphite diastereomer. Two different CPA scaffolds have proven to be essential for achieving stereodivergence: peptide-embedded phosphothreonine-derived CPAs, which reinforce and amplify the inherent substrate preference, and C2-symmetric BINOL-derived CPAs, which completely overturn this stereochemical preference. The presently reported catalytic method does not require stoichiometric activators or chiral auxiliaries and enables asymmetric catalysis with readily available phosphoramidites. The method was applied to the stereocontrolled synthesis of diastereomeric dinucleotides as well as cyclic dinucleotides, which are of broad interest in immuno-oncology as agonists of the stimulator of interferon genes (STING) pathway.

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 report describes the chemical synthesis of aminotroponyl-conjugated deoxyuridine analog (at-dU) and its difluoroboron complex (dfbat-dU) and their phosphoramidites by using the versatile phosphorylating reagent 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite. Tropolone is a non-benzenoid aromatic bioactive natural fluorescent molecule, possessing intramolecular charge transfer and metal chelating properties with transition metal ions such as Cu2+/ Zn2+/ Ni2+ . Its synthetic derivatives, 2-aminotropones also exhibit unique bioactivities and are considered potential therapeutic drug candidate. Recently, the fluorescence properties of aminotropone has improved by complexing with difluoroboron residue that generates aminotroponyl-BODIPY analog. These could be employed for the synthesis of at-dU/dfbat-dU containing DNA oligonucleotides for designing the 11 B/19 F-NMR/fluorescence-based DNA probes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N-propargyl-2-aminotropone (2) and difluoroboronyl N-propargyl-2-aminotropone (3) molecules. Basic Protocol 2: Synthesis of N-propargyl-2-aminotroponyl deoxyuridinyl (at-dU) phosphoramidites (7). Basic Protocol 3: Synthesis of difluoroboronyl N-propargyl-2-aminotroponyl deoxyuridinyl (dfbat-dU) phosphoramidites (10).