Syntheses of 4'-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4'-thiocytosine

Abstract

A facile synthetic route for the 4′-thioribonucleoside building block 4′SN (N = U, C, A and G) with the ribose O4′ replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case. Incorporation of 4′-thiocytidines into oligoribonucleotides improved the thermal stability of the corresponding duplexes by ∼1°C per modification, irrespective of whether the strand contained a single modification or a consecutive stretch of 4′SC residues. The gain in thermodynamic stability is comparable to that observed with oligoribonucleotides containing 2′-O-methylated residues. To establish potential conformational changes in RNA as a result of the 4′-thio modification and to better understand the origins of the observed stability changes, the crystal structure of the oligonucleotide 5′-r(CC4′SCCGGGG) was determined and analyzed using the previously solved structure of the native RNA octamer as a reference. The two 4′-thioriboses adopt conformations that are very similar to the C3′-endo pucker observed for the corresponding sugars in the native duplex. Subtle changes in the local geometry of the modified duplex are mostly due to the larger radius of sulfur compared to oxygen or appear to be lattice-induced. The significantly increased RNA affinity of 4′-thio-modified RNA relative to RNA, and the relatively minor conformational changes caused by the modification render this nucleic acid analog an interesting candidate for in vitro and in vivo applications, including use in RNA interference (RNAi), antisense, ribozyme, decoy and aptamer technologies.

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

“…Selenium Derivatization at the 4'-Position. The replacement of the sugar-ring Oatom (at the 4'-position) of nucleosides and nucleic acids by the S-atom has been applied to the modification of aptamers and siRNA, as well as gene regulation [79] [80]. The synthesis of the corresponding Se counterparts has been quite challenging.…”

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

“…Although this puts limitations on the use of this modification in antisense applications, where RNase H-mediated degradation of the target mRNA is considered important for efficacy, the properties of the s 2 U, m 5 s 2 U and m 5 s 2 Umoe modifications may render them of interest for use in siRNAs. Together with DNA phosphorothioates (8) and 4′-thio-RNA (3,48,49), they constitute the third class of sulfur-modified nucleic acid building blocks with promising features for the generation of nucleic acid therapeutics.…”

Carbohydrate Chemistry for RNA Interference: Synthesis and Properties of RNA Analogues Modified in Sugar-Phosphate Backbone

“…The 4'-thio RNA exhibits very useful biophysical properties [57,58,60,63]. The 4'-thio RNA/4'-thio RNA homoduplexes (next to the LNA/LNA duplexes, see the next chapter) are among the most stable modified RNA duplexes [57].…”

“…Very recently, Haeberli et al [63] reported a very efficient synthesis of 18 starting from the pyranoside 22 (Fig. 9, Route 3) available from L-lyxose in a simple two step "one pot" procedure.…”

“…Another "one pot" procedure installs the sulfur functionality via an S N 2 displacement of 4-O-triflate and after exchange of protecting groups gives the pyranoside 23. Exposure of 23 to acetic anhydride in the presence of sulfuric acid initiates a ring contraction that gives the key thioglycosyl donor 24, which is further converted into all four 4'-thionucleosides bearing the natural heterocycles [63]. Synthesis of 18 following Route 3 is accomplished in only five straightforward steps and excellent yields (32% for 4'-thiouridine).…”