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

Abstract

Access to 3-deazaadenosine (c3A) building blocks for RNA solid-phase synthesis represents a severe bottleneck in modern RNA research, in particular for atomic mutagenesis experiments to explore mechanistic aspects of ribozyme catalysis. Here, we report the 5-step synthesis of a c3A phosphoramidite from cost-affordable starting materials. The key reaction is a silyl-Hilbert–Johnson nucleosidation using unprotected 6-amino-3-deazapurine and benzoyl-protected 1-O-acetylribose. The novel path is superior to previously described syntheses in terms of efficacy and ease of laboratory handling.

Atom‐Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active‐Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes

“…The above mentioned deazaadenosine mutations require phosphoramidite building blocks for solid‐phase RNA synthesis that are not commercially available. Therefore, the c 1 A phosphoramidite was synthesized in eleven steps in analogy to previously published procedures (see the Supporting Information), and c 3 A phosphoramidite was synthesized in five steps according to our recent method . With these phosphoramidites in hand, we synthesized the corresponding two‐stranded pistol ribozymes with c 1 A and c 3 A mutations in position 32.…”

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

“…Mass spectrometry experiments were performed with aF innigan LCQ Advantage MAX ion trap instrument. 1 H, 13 C, 31 PNMR spectra were recorded with aB ruker DRX 300 MHz spectrometer; 19 was added, followed by 4,4'-dimethoxytriphenylmethyl chloride (880 mg, 2.62 mmol) in two portions over aperiod of 1h.T he solution was stirred for 13 hb efore more 4,4'-dimethoxytriphenylmethyl chloride (100 mg, 0.295 mmol) was added and stirring was continued for 3h.T he reaction was ended by the addition of methanol (0.5 mL), and the mixture was evaporated, and subjected to column chromatographic purification on SiO 2 (1-6 %, CH 3 O 6 -tert-Butyl-N,N-bis(tert-butyloxycarbonyl)-2'-O-tert-butyl dimethyl silyl-5'-O-(4,4'-dimethoxy triphenylmethyl) guanosine (7a):I dentical to 1b,s ee the Supporting Information). Compound 7 (1.0 g, 1.53 mmol) was coevaporated with anhydrous pyridine and dissolved in anhydrous pyridine (5 mL).…”

“…1 H, 13 C, 31 P NMR spectra were recorded with a Bruker DRX 300 MHz spectrometer; 19 F NMR spectra were measured with a Bruker Avance II + 600 MHz spectrometer equipped with a TCI Prodigy Cryo-Probe. The chemical shifts are referenced to the residual proton signal of the deuterated solvents: CDCl 3 ( 1 H: δ = 7.26 ppm, 13 C: δ = 77.1 ppm), [D 6 ]DMSO ( 1 H: δ = 2.5 ppm, 13 C: δ = 39.5 ppm), 31 P shifts are relative to external 85% phosphoric acid, 19 F shifts are relative to external CCl 3 F. 1 Hand 13 C-assignments were based on COSY and HSQC experiments. Compound 3 (1.0 g, 2.28 mmol) was coevaporated with pyridine (6 mL) and subsequently dissolved in anhydrous pyridine (7 mL).…”

“…FNMR spectra were measured with aB ruker Avance II + 600 MHz spectrometer equipped with aT CI Prodigy Cryo-Probe. The chemical shifts are referenced to the residual proton signal of the deuterated solvents: CDCl 3 ( 1 H: d = 7.26 ppm, 13 C: d = 77.1 ppm), [D 6 ]DMSO ( 1 H: d = 2.5 ppm, 13 C: d = 39.5 ppm),31 Ps hifts are relative to external 85 % phosphoric acid,19 Fs hifts are relative to external CCl 3 F. 1 H-and13 C-assignments were based on COSY and HSQC experiments.…”

“…With respect to modified nucleosides, the situation can be quite different. [18,19] For instance, although ribose 2′-modified pyrimidine nucleoside phosphoramidites are well accessible, the corresponding purine counterparts-in particular guanosine derivatives-are much more challenging and time-consuming in terms of synthesis. This is true for simple 2′-Oaminoalkyl modified building blocks, and even more for 2′-SeCH 3 , 2′-SCF 3 or 2′-N 3 modifications, to name a few.…”