Synthesis, properties, and applications of oligonucleotides containing an RNA dinucleotide phosphorothiolate linkage

Abstract

RNA represents a prominent class of biomolecules. Present in all living systems, RNA plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level. In this context, RNA molecules bearing 3'- or 5'-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. The resulting models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered an experimental strategy to circumvent the classic problem of kinetic ambiguity; mechanistic enzymologists have used this tool to assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. In this Account, we review the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage. Phosphorothioate linkages are structurally very similar to phosphorothiolate linkages, as reflected in the single letter of difference in nomenclature. Phosphorothioate substitutions, in which sulfur replaces one or both nonbridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. Indeed, synthetic phosphorothioate linkages can be introduced readily via a sulfurization step programmed into automated solid-phase oligonucleotide synthesizers. In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 3'- or 5'-bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. Here we begin by outlining the synthetic strategies used to access these phosphorothiolate RNA analogues. The Arbuzov reaction and phosphoramidite chemistry are often brought to bear in creating either 3'- or 5'-S-phosphorothiolate dinucleotides. We then summarize the responses of the phosphorothiolate derivatives to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered. They demonstrate particular utility as probes of metal-ion-dependent phosphotransesterification, general acid-base-catalyzed phosphotransesterification, and rate-limiting chemistry. The 3'- and 5'-S-phosphorothiolates have proven invaluable in elucidating the mechanisms of enzymatic and nonenzymatic phosphoryl transfer reactions. Considering that RNA cleavage represents a fundamental step in the maturation, degradation, and regulation of this important macromolecule, the significant synthetic challenges that remain offer rich research opportunities.

Figure 1

Structures of oligonucleotides containing bridging or non-bridging sulfur atoms.

Figure 2

Summary of approaches used for the preparation of dinucleotides or oligonucleotides containing phosphorothiolate linkages.

Figure 3

Solid-phase synthesis of d(ACGGTCT)rC-ps-d(ACGAGC), a substrate for the hammerhead ribozyme reaction.

Figure 4

Solid-phase synthesis of r[UUC_2′-O-NBn-ps-(dG)GGUCGGC] and r(UUC_2′-O-NBn-ps-GGGUCGGC), substrates for the HDV ribozyme reaction.

Figure 5

Construction of an ODN containing rCps-dC via enzymatic ligation.

Figure 6

Summary of the unique properties of r(B₁)-sp-r(B₂).

Figure 7

The pH-dependent first-order rate constants for cleavage (k₁, left) and isomerization (k₂, right) of r(UpU) (red dots) and r(IspU) (blue dots) at 90 °C (0.1 M NaCl).^,

Figure 8

Model compounds^– simulating 2′-O-transphosphorylation, with measured nucleophile and leaving group kinetic isotope effects (ND, not determined).

Figure 9

Model of the group I intron transition state showing metal ion-ligand interactions. Hashed lines represent hydrogen bonds, black dotted lines represent experimentally confirmed metal ion interactions, purple and blue dotted lines represent metal ion interactions still under investigation, and dashed lines represent the bonds broken and formed during the reaction. M_A, M_B, and M_C represent the three distinct metal ions implicated from functional data. M_B is not observed in the crystal structures of group I ribozymes, and it has been proposed, based on spatial proximity, that M_C forms an additional interaction with the 3′ oxygen of the nucleophilic guanosine in the transition state.

Figure 10

Equilibrium between C3′-endo and C2′-endo conformations in RNA.

Figure 11

Mechanism of the general acid-catalyzed ribozyme (HDV and VS) reactions. A 5′-sulfur substitution at the cleavage site provides a ‘hyperactivated’ substrate that is less dependent on leaving group protonation.

Scheme 1

Synthesis of dinucleotides r(UspU) (3) and r(IspU) (6).

Scheme 2

Synthesis of 3′-S-thioribonucleoside phosphoramidites 8a–d and 2′-O-methyl-3′-S-thioguanosine phosphoramidite 11.

Scheme 3

Synthesis of dinucleotide r(UpsU) 14 via Arbusov reaction.

Scheme 4

Synthesis of r(UpsU) (14) and U_2′-NH2 - ps - rU (17) via phosphoramidite chemistry.

Scheme 5

Synthesis of poly 5′-S-thiouridylyl phosphate (19).

Scheme 6

Synthesis of 5′-S-thioadenosine 3′-O-phosphoramidites 21 and 23.

Scheme 7

Synthesis of dinucleotide r(G_2′-O-NBn-ps-A) 29.

Scheme 8

Ligation scheme for constructing the 29-nucleotide VS ribozyme substrate 34.

Scheme 9

Pathways for cleavage and isomerization of r(IspU).^,

Scheme 10

Pathways for cleavage and isomerization of r(UpsU) (14) under aqueous conditions.