Synthesis of Oligoribonucleotides Containing a 2'-Amino-5'- S-phosphorothiolate Linkage

Abstract

Oligoribonucleotides containing a photocaged 2'-amino-5'-S-phophorothiolate linkage have potential applications as therapeutic agents and biological probes to investigate the RNA structure and function. We envisioned that oligoribonucleotides containing a 2'-amino-5'-S-phosphorothiolate linkage could provide an approach to identify the general base within catalytic RNAs by chemogenetic suppression. To enable preliminary tests of this idea, we developed synthetic approaches to a dinucleotide, trinucleotide, and oligoribonucleotide containing a photocaged 2'-amino-5'-S-phosphorothiolate linkage. We incorporated the photocaged 2'-amino-5'-S-phosphorothiolate linkage into an oligoribonucleotide substrate for the hepatitis delta virus (HDV) ribozyme and investigated the pH dependence of its cleavage following UV irradiation both in the presence and absence of the ribozyme. The substrate exhibited a pH-rate profile characteristic of the modified linkage but reacted slower when bound to the ribozyme. Cleavage inhibition by the HDV ribozyme could reflect a non-productive ground-state interaction with the modified substrate's nucleophilic 2'-NH₂ or a poor fit of the modified transition state at the ribozyme's active site.

Figure 1

Oligoribonucleotides containing 2′-aminonucleotide (A), 5′-S-phosphorothiolate linkage (B), and 2′-amino-5′-S-phosphorothiolate linkage (C).

Figure 2

Possible mechanism of ribozyme-catalyzed cleavage of the RNA substrate containing a 2′-NH₂/5′-S linkage at the cleavage site at pH > 7.

Scheme 1

Scheme 2

Scheme 3

Figure 3

Construction of RNA-containing 5′-C_2′-NHX-ps-G-3′ by a consecutive ligation approach.

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8

Scheme 9

Figure 4

Characterization of 3′-radiolabeled RNAs (11 mer) containing 2′-amino-5′-O- (26b) or 2′-amino-5′-S-linkage (24b). The figure was depicted from the right side of a large gel, so the oligo on lane 9 moved a little bit slower than the same oligo on lanes 1 and 5.

Figure 5

pH rate profiles of cleavages of 5′-radiolabeled 24b in the presence/absence of HDV ribozyme and 10 mM MgCl₂. k(HDV) (○): cleavage rate in the presence of HDV ribozyme. k(OH) (▲): cleavage rate in the absence of HDV ribozyme.