Synthesis of 5-Hydroxymethylcytidine- and 5-Hydroxymethyl­uridine-Modified RNA

Abstract

We report on the syntheses of 5-hydroxymethyl-uridine [5hm(rU)] and -cytidine [5hm(rC)] phosphoramidites and their incorporation into RNA by solid-phase synthesis. Deprotection of the oligonucleotides is accomplished in a straightforward manner using standard conditions, confirming the appropriateness of the acetyl protection used for the pseudobenzylic alcohol moieties. The approach provides robust access to 5hm(rC/U)-modified RNAs that await applications in pull-down experiments to identify potential modification enzymes. They will also serve as synthetic probes for the development of highthroughput-sequencing methods in native RNAs.

Synthesis and Multiple Incorporations of 2′‐O‐Methyl‐5‐hydroxymethylcytidine, 5‐Hydroxymethylcytidine and 5‐Formylcytidine Monomers into RNA Oligonucleotides

“…[10, 15] We set out to develop a synthesis of a hm 5 Cm monomer that would be compatible with solid-phase RNA ONs synthesis, which has not yet been described. In addition, the reported monomer for the synthesis of f 5 C-containing RNA ONs requires a multistep synthesis with 2′-bis(2-acetoxyethoxy)methyl (ACE) orthoester and 5′- O -benzhydroxy-bis(trimethylsiloxy)silyl (BZH) protection.…”

“…To overcome these practical limitations, we designed an alternative f 5 C monomer in which the reactive formyl group is appropriately masked. Riml and Micura reported the synthesis and incorporation of a hm 5 C monomer into RNA ONs; their route provides this monomer in a 3% overall yield in eight steps from 5-hydroxymethyluridine (hm 5 U),[15] which itself is obtained in a three-step synthesis from uridine, and this brings the total number of steps to 11. [17] We therefore, at the same time, considered the development of a faster and more efficient route to this monomer.…”

“…The presence of the 2′-hydroxy group makes the synthesis and incorporation of functionally modified ribonucleoside phosphoramidites particularly challenging compared to that of the analogous DNA phosphoramidite. [10, 15, 18] Moreover, an orthogonal protection strategy developed for the synthesis of DNA ONs does not necessarily work for RNA due to its inherently more labile nature. [18a] Herein, we describe the development of phosphoramidite building blocks of hm 5 Cm, f 5 C and hm 5 C (Scheme 1) and demonstrate the incorporation of all these cytosine modifications into RNA ONs at several positions and in several combinations by solid-phase RNA synthesis.…”

“…[15] Starting with commercially available 2′ -O -methyl-5-methyluridine (4), we protected the 5′-hydroxy group with a 4,4′-dimethoxytrityl (DMT) and the 3′-hydroxy group with a tert -butyldimethylsilyl (TBS) group (Scheme 2). [19] The DMT- and TBS-protected uridine, 6, was then subjected to azobisisobutyronitrile (AIBN)-catalysed bromination of the 5-methyl group by using N -bromosuccinimide (NBS).…”

“…[19] From here onwards we followed the route reported by Riml et al and obtained monomer 3 in eight steps with 6.4% overall yield (Scheme 4). [15, 19] During the preparation of this manuscript, the Micura group improved their initial synthesis and reported an alternative method, starting from cytidine, to obtained this monomer in eight steps with 9.2% overall yield. [24]…”