Native Chemical Ligation of Hydrolysis‐Resistant 3′‐NH‐Cysteine‐Modified RNA

Abstract

Hydrolysis‐resistant RNA‐peptide conjugates that contain a 3′‐NH linkage between the adenosine ribose and the C‐terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid‐phase oligonucleotide synthesis and bioconjugation is described here. The key step is native chemical ligation (NCL) of 3′‐NH‐cysteine‐modified RNA to highly soluble peptide thioesters. These hydrolysis‐resistant 3′‐NH‐peptide‐modified RNAs, containing the universally conserved 3′‐CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle. © 2015 by John Wiley & Sons, Inc.

Efficient access to N-trifluoroacetylated 2′-amino-2′-deoxyadenosine phosphoramidite for RNA solid-phase synthesis

“…After work-up, the resulting amino group was protected by trifluoroacetylation applying a two-step procedure involving ethyltrifluoroacetate first, followed by trifluoroacetic anhydride to increase the overall yields of compound 3 . Protection of the exocyclic adenine 6-amino group was achieved using N , N -dibutylformamide dimethylacetal (prepared as described in “ Experimental ” [ 14 – 17 ]), and subsequently, the TIPDS moiety was deprotected using tetrabutylammonium fluoride (TBAF) and acetic acid to yield compound 4 . Finally, this nucleoside was transformed into the dimethoxytritylated derivative 5 under standard conditions.…”

“…The conversion of 5 into the corresponding phosphoramidite 6 was achieved in high yields by treatment with 2-cyanoethyl N , N -diisopropylchlorophosphoramidite under basic conditions. Starting with arabinoadenosine, our route provides 6 in 23% overall yield in six steps with seven chromatographic purifications; in total, 500 mg of phosphoramidite 6 was obtained in the course of this study.

Reaction conditions: a 1.3 equiv 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPSiCl 2 ) in anhydrous DMF and pyridine, room temperature, 14 h, 81%; b i) 1.5 equiv CF 3 SO 2 Cl, 3 equiv 4-(dimethylamino)pyridine (DMAP) in CH 2 Cl 2 , 0°C, 30 min; ii) 5 equiv NaN 3 in DMF, room temperature, 15 h, 75%; c i) palladium on carbon, H 2 (g) in THF, room temperature, overnight; ii) 10 equiv ethyltrifluoroacetate, 1.0 equiv trifluoroacetic anhydride in THF, room temperature, 48 h, 71%; d i) 3 equiv Bu 2 NCH(OCH 3 ) 2 [14–17] in THF, 60°C, overnight; ii) 1 M TBAF, 0.5 M acetic acid in THF, room temperature, 2 h, 78%; e 1.3 equiv 4,4'-dimethoxytrityl chloride (DMT-Cl), 0.3 equiv 4-(dimethylamino)pyridine (DMAP) in pyridine, room temperature, overnight, 81%; f 2 equiv N,N -diisopropylethylamine, 1.5 equiv 2-cyanoethyl N,N -diisopropylchlorophosphoramidite (CEP-Cl) in dichloromethane, room temperature, 2 h, 85%; total yield over six steps: 23%

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Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside phosphoramidite for RNA solid-phase synthesis

“…Our synthetic route to the functionalized 2-aminopurine riboside phosphoramidite 6 starts with the reduction of the commercially available 2-amino-6-chloropurine riboside using Pearlman′s catalyst (Pd(OH) 2 /C) and ammonium formate to yield compound 1 (Scheme 1). The exocyclic 2-amino function was selectively protected by treatment with N,N-dibutylformamide dimethyl acetal (DBFDMA) [25][26][27][28][29] producing nucleoside derivative 2. In the next step, the 5′ and 3′ hydroxyl groups were simultaneously protected by reaction with di-tert-butylsilyl bis(trifluoromethanesulfonate) ((tBu) 2 Si(OTf) 2 ) [30,31], followed by silylation of the 2′-hydroxyl group with tert-butyldimethylsilyl chloride (TBDMSCl) to give compound 3.…”

Facile synthesis of a 3-deazaadenosine phosphoramidite for RNA solid-phase synthesis

“…An authentic reference sample that was synthesized according to the previously established 12-step route was used for direct spectroscopic comparison (see Supporting Information File 1) and additionally confirmed its identity. Then, treatment with N , N -dibutylformamide dimethyl acetal [31] resulted in amidine protection of the exocyclic C6-NH 2 group. At the same time, the applied excess of the reagent allowed to transiently form the corresponding nucleoside 2’,3’- O -acetal [32], leaving the primary 5’-OH group available for selective tritylation with 4,4’-dimethoxytrityl chloride to give compound 6 .…”