Uridine 2′‐Carbamates: Facile Tools for Oligonucleotide 2′‐Functionalization

Abstract

A facile method for preparation of uridine 2'-carbamate derivatives based on reaction of 3',5'-disilyl-protected uridine with 1,1'-carbonyldiimidazole followed by treatment with an aliphatic amine is presented. A phosphoramidite monomer suitable for automated oligonucleotide synthesis is obtained in a few steps. The compounds are useful for the introduction of various labels and modifications into an oligonucleotide chain. Although 2'-carbamate modification is somewhat destabilizing for DNA-DNA and DNA-RNA duplexes, it is suitable for the direction of ligands into the minor groove of duplexes or at non-base-paired sites (e.g., loops and bulges) of oligonucleotides. Pyrene-modified oligonucleotide 2'-carbamates show a considerable increase in fluorescence intensity upon hybridization to a complementary RNA (but not DNA).

Postsynthetic On-Column 2′ Functionalization of RNA by Convenient Versatile Method

“…with the use of modifying phosphoramidite, since this is an attractive conjugation method to tailor RNA due to the simplicity of the procedure and a wide variety of commercially available reagents bearing various linkers or functional groups. Moreover, although pre-synthetic approaches to 2'carbamate derivatives of oligonucleotides are already known [50][51][52][53][54], post-synthetic 2' functionalization of RNA using 1,1'-carbonyldiimidazole coupling chemistry is, to our knowledge, not. In general, the following post-modification could be carried out whether on column or, if final deprotection is detrimental for desired functional group, in solution by known conjugation reactions (for instance, conventional N-acylation reactions, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) [55,56], copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) [57], inverseelectron-demand Diels-Alder (IEDDA) cycloadditions [58,59], etc.).…”

“…A particular attention has been paid to the 2′ modification with the use of modifying phosphoramidite, since this is an attractive conjugation method to tailor RNA due to the simplicity of the procedure and a wide variety of commercially available reagents bearing various linkers or functional groups. Moreover, although pre-synthetic approaches to 2′-carbamate derivatives of oligonucleotides are already known [ 50 , 51 , 52 , 53 , 54 ], post-synthetic 2′ functionalization of RNA using 1,1′-carbonyldiimidazole coupling chemistry is, to our knowledge, not.…”

“…A series of 2 -cholesterol-functionalized 21-mer oligonucleotides 8b-10b (Table 1 and Table S2) corresponding to the sense strand of a model siRNA-targeting GFP mRNA [70] was synthesized as described above (Table 3 and Supplementary Materials, Section 1.9, page 9). Concerning the introduction of 2'-O-carbamoyl modified nucleotides, this modification is known to decrease the thermostability of nucleic acids duplexes [50][51][52][53][54], most probably due to a steric clash and unfavorable electrostatics between the carbamoyl group and a nucleobase [64]. Interestingly, if a substituent with terminal cationic group is used, it may increase the duplex stability of the 2'-O-carbamoyl-modified oligonucleotides due to partial neutralization of the negatively charged internucleotide phosphates.…”