Azido Functionalized Nucleosides Linked to Controlled Pore Glass as Suitable Starting Materials for Oligonucleotide Synthesis by the Phosphoramidite Approach

Abstract

In memory of Klaus HafnerIt has long been debated whether easily reducible azide groups can withstand the conditions of oligonucleotide synthesis by phosphoramidite chemistry. We have synthesized various 2'and 3'-azido modified nucleosides and immobilized them on controlled pore glass (CPG) to be used as starting material for the synthesis of oligonucleotides (ONs) with 3'-terminal azide (attached to C2' or C3'). In a model study, immobilized 3'azidoadenosine was used as a starting block for the synthesis of a series of oligodeoxynucleotides (ODNs) of increasing length. Upon synthesis, the ODNs were enzymatically digested into monomers and analyzed by RP-HPLC. A peak corresponding to 3'-azidoadenosine was clearly identified in all samples. Quantitative analysis showed that 3'-azidoadenosine was present in nearly the expected ratio to deoxycytidine, which was used as an internal standard. Most importantly, the ratio remained the same for all three ODNs regardless of their length, demonstrating that a higher number of coupling cycles does not lead to higher degradation of the azide. Thus, 2'-or 3'-azido nucleosides attached to a solid support are excellent starting materials for the synthesis of oligonucleotides with 3'-terminal azide.

Azide‐Modified Nucleosides as Versatile Tools for Bioorthogonal Labeling and Functionalization

“…[28] We have used the epoxide approach in the past for the successful synthesis of 3'azide-functionalized uridine and adenosine derivatives. [27,29] Furthermore, the strategy has also been described for the preparation of 3'-azidocytidine and ribothymidine. [30] For the synthesis of 2'-azide-modified nucleosides a different approach is needed.…”

“…We recently synthesized a 2'-azide-functionalized adenosine derivative originating from arabinofuranosyl adenine utilizing this approach. [29] Alternatively, when the reaction is started from a ribonucleoside, epimerization of the 2'-position is required. This can be achieved by a procedure described by General strategy for the synthesis of 5'-azide-modified ribonucleosides: i: PPh 3 (1.2 eq.…”

“…We recently demonstrated the excellent stability of 3'-azide-modified adenosine in ON synthesis. [29] The modified nucleoside was linked to solid support, and various 3'-azide-modified oligodeoxynucleotides (ODNs) of increasing length were synthesized. There was virtually no degradation of the azide observed during iterative phosphoramidite treatments.…”

“…However, this preference can be diminished in the absence of electronegative substituents in the 5′‐position [28] . We have used the epoxide approach in the past for the successful synthesis of 3′‐azide‐functionalized uridine and adenosine derivatives [27,29] . Furthermore, the strategy has also been described for the preparation of 3′‐azidocytidine and ribothymidine [30] …”

“…Thus, for 3′‐terminal functionalization of an ON with an azido group, the azide‐modified nucleoside can be directly connected to the solid support and used as start nucleoside for synthesis. We recently demonstrated the excellent stability of 3′‐azide‐modified adenosine in ON synthesis [29] . The modified nucleoside was linked to solid support, and various 3′‐azide‐modified oligodeoxynucleotides (ODNs) of increasing length were synthesized.…”