Synthesis of a Peptidoyl RNA Hairpin via a Combination of Solid‐Phase and Template‐Directed Chain Assembly

Abstract Peptidoyl RNAs are the products of ribosome‐free, single‐nucleotide translation. They contain a peptide in the backbone of the oligoribonucleotide and are interesting from a synthetic and a bioorganic point of view. A synthesis of a stabilized version of peptidoyl RNA, with an amide bond between the C‐terminus of a peptide and a 3′‐amino‐2′,3′‐dideoxynucleoside in the RNA chain was developed. The preferred synthetic route used an N‐Teoc‐protected aminonucleoside support and involved a solution‐phase coupling of the amino‐terminal oligonucleotide to a dipeptido dinucleotide. Exploratory UV‐melting and NMR analysis of the hairpin 5′‐UUGGCGAAAGCdC‐LeuLeu‐AA‐3′ indicated that the peptide‐linked RNA segments do not fold in a cooperative fashion. The synthetic access to doubly RNA‐linked peptides on a scale sufficient for structural biology opens the door to the exploration of their structural and biochemical properties.

The reaction mixture was stirred for 20 h at room temperature. The progress of the reaction was monitored by TLC (silica, DCM/MeOH, 20:1, v/v, Rf = 0.10). The reaction mixture was dried in vacuo, dissolved in DCM and subjected to column chromatography (silica, de-activated with DCM containing 0.5% NEt3 prior to applying of the crude) using a gradient of methanol in dichloromethane (0-10%).
The product eluted at 5% methanol in dichloromethane. Product-containing fractions were combined and evaporated in vacuo to yield the title compound (10) as a colourless glass (132 mg, 0.20 mmol, 46%).
Succinic anhydride (87 mg, 0.87 mmol, 20 eq.) was coevaporated twice from dry pyridine, dissolved in dry pyridine (2 mL) and subsequently added to the solid support. Then DMAP (17 mg, 0.14 mmol, 3.2 eq.) was added, and the reaction vessel was placed on an orbital shaker for 20 h at room temperature.
The support was washed with pyridine, dichloromethane and diethyl ether (20 mL each) and dried in vacuo. Then, nucleoside 10 (65 mg, 0.07 mmol, 1.6 eq.), previously coevaporated twice from dry pyridine and hydroxybenzotriazole (70 mg, 0.45 mmol, 10.4 eq), previously coevaporated three times from dry pyridine were dissolved in dry pyridine (1.5 mL). The solution was mixed with DIC (0.05 mL, 0.39 mmol, 9 eq), followed by addition to the solid support. The reaction mixture was put on an orbital shaker for 22 h at room temperature. Pentachlorophenol (63 mg, 0.07 mmol, 1.6 eq.) was added and the mixture shaken for three more hours at room temperature. The support with the immobilized nucleoside was washed with pyridine, dichloromethane and diethyl ether (20 mL each) and dried in vacuo. A solution of piperidine in DMF (3 mL, 10% v/v) was added and the mixture was shaken for 4 min. Then, the support was washed with dichloromethane and diethyl ether (20 mL each) and dried in vacuo. A mixture of Cap A solution for DNA synthesis (acetic anhydride/ 2-6-lutidine/THF 1:1:8) and Cap B solution (DMAP in THF, 6.5 % w/v) (6 mL, 1:1 v/v) was added to the solid support, and the reaction mixture was put on a shaker for 1 h. Then, the solid support was washed with pyridine, dichloromethane and diethyl ether (20 mL each) and dried in vacuo. The loading with the immobilized nucleoside was determined to be 16.5 µmol/g, indicating a yield of 19% in terms of the loading of the amino groups of the lcaa-cpg with the nucleoside.

Solid-phase synthesis of hairpin 6
The solid phase synthesis was performed on a H-2 Synthesizer (K&A, Schaafheim, Germany) on a 1 µmol scale using the posphoramidite method according to the recommendations of the manufacturer.
The following protocol is for the hairpin with a terminal amino group (6)  After conditioning, a cation exchange resin in the ammonium form (DOWEX, 50W X4 H+-Form 50-100 mesh, changed into ammonia form, 660 mg) was put directly on the cartridge. Water (1 mL) was added to the cartridge and it was let sit for 5 min. After that, the crude product was diluted with demineralized water (6 mL) to have a lesser concentration of TBA cations and remaining fluoride ions.
The dilution did not alter the purification process. The crude, diluted product was applied to the cartridge followed by demineralized water (10 mL). Then the cation exchange resin was removed from the cartridge with a spatula to prevent elution of the TBA cations and a gradient of ammonium carbonate and water (50 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 500 mM) was used to purify the sample. Product elutes with a concentration between 350 and 375 mM ammonium carbonate. Productcontaining fractions were pooled, the pH value was adjusted to 7.6, and the combined fractions were lyophilized. The product was freed of residual ammonium ions by ion exchange to the sodium form using a C18-cartridge, following a protocol published earlier. The protocol for the synthesis of the dipeptido dinucleotide 13 was similar to a literature protocol, [S5] except that the organocatalyst/leaving group was changed from 4-dimethylaminopyridine (DMAP) to hydroxybenzotriazole (HOBt). A solution of HOBt (35 mg, 335 mM, 149 eq.) in water (0.5 mL) was prepared and the pH-value was adjusted to 6.0. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 49 mg, 511 mM, 228 eq.) was added to the HOBt solution. In a polypropylene cup, dimer pAA (5,224 nmol, 0.15 mg) was dissolved in 100 µL of the HOBt and EDC solution. The reaction mixture was allowed to react for 2 h at room temperature. The following precipitation, coupling and purification were performed according to the literature in C. Richert et al. [S5] The product was isolated in a yield of 0.72 mg (107 nmol, 48%).

Full-Length Reference RNA Hairpin 5'-UUGGCGAAAGCCAA-3' (14)
without NaCl     .   NOESY Spectrum in H2O/D2O Figure S18. Section of the NOESY spectrum of compound 1 with crosspeaks from the two interior, shielded G:C base pairs of the hairpin stem highlighted in color. We note that a broadened peak of lower intensity is observable at approx. 12 ppm, which is likely a third imino resonance, mostly probably the one of NH1 of residue G3, whose base pair with dC12 is partially solvent exposed, explaining why it is of too low an intensity to be readily assigned. Conditions: phosphate buffer (50 mM), NaCl (100 mM), pH = 7, H2O: D2O 9:1, 700 MHz, mixing time: 300 ms, T=25 °C.