A new and potentially prebiotic α-cytidine derivative

A new α-cytidine derivative was synthesised from the prebiotic reaction of ribose aminooxazoline and dicyanoacetylene.


General methods
All reagents and solvents were purchased from Sigma-Aldrich and Acros Organics and were used without further purification. A Mettler Toledo SevenEasy pH Meter S20 with a ThermoFisher Scientific Orion 8103BN Ross combination semi-micro pH electrode was used to measure and adjust the pH. A Varian ProStar HPLC System was used for the reverse phase high-pressure liquid chromatography (RP-HPLC) linked with an Atlantis T3 C18 Prep Column OBD 10 μm (19×250 mm). A Grace Reveleris Prep purification system was used for direct phase chromatography with Reveleris Flash silica cartridges. 1

Synthesis of dicyanoacetylene 5
Dicyanoacetylene 5 was synthesised following a slightly modified literature method. 1 Acetylenedicarboxamide (2.0 g, 17.9 mmol) was mixed with dried and calcined sand (15.0 g) and phosphorus pentoxide (15.0 g, 52.8 mmol) using a S1 Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2017 mortar and pestle. The solid mixture was transferred into a 250 mL roundbottom flask and was heated at 215°C into preheated high temperature silicone oil. An inlet and outlet allowed argon to flow over the mixture, thus facilitating the transfer of the gaseous product into three consecutive connected flasks, which were cooled by acetone-dry ice baths for the collection of the distilled product. The argon flow was then led through a calcium chloride tube and finally through a trap containing sodium hypochlorite solution 6%. The reaction was completed after 15-20 min of heating and the flasks, containing 300 mg of the white crystalline solid 5 (yield 22%), were capped with glass stoppers, sealed with film and stored in the freezer at -30 o C for several days. The 13 C NMR spectrum was found to be in agreement with the one reported in the literature. 2

Reaction of ribose aminooxazoline 1 with dicyanoacetylene 5
Ribose aminooxazoline 1 (20 mg, 0.115 mmol) and NaH 2 PO 4 (16 mg, 0.100 mmol) were dissolved in water (0.5 mL, containing 10% D 2 O) and the pH of the mixture was adjusted to 6.9 with 1M HCl. Dicyanoacetylene 5 (60 mg, 0.789 mmol) was dissolved in water (0.5 mL, containing 10% D 2 O) and was immediately added to the reaction. The mixture was transferred into an NMR tube and was monitored by 1 H-NMR. After 3 d at rt, the 1 H-NMR spectrum showed that the reaction was not proceeding any further and that the final product 11 was formed in 32% yield. The mixture was separated by RP-HPLC using a water-acetonitrile gradient to give, after lyophilisation, the white crystalline amide acetal 11 (9 mg, 30%). 1   6.9 6.9 0.1M 32% 5 13.7 6.9 0.1M 30% Table 1 Conditions and yield for the reaction of ribose aminooxazoline 1 with dicyanoacetylene 5 Figure S1. 1

Synthesis of N-acetyl-α-cytidine 12
α-Cytidine 4 3 (167 mg, 0.69 mmol) was suspended in dry methanol (6 mL) and then acetic anhydride (0.67 mL, 7.10 mmol) was added. The mixture was refluxed for 1.5 h, until TLC indicated the consumption of the starting material. The mixture was evaporated and then suspended in diethylether, filtered and dried under vacuum. The white solid was pure enough in order to be used for the next step (180 mg, yield 92%), although an analytically pure sample of 12 was obtained by RP-HPLC using a water-acetonitrile gradient. 1

Synthesis of 2,3,5-tribenzoyl-α-cytidine 13
N-Acetyl-α-cytidine 12 (100 mg, 0.35 mmol) was dissolved in dry pyridine (10 mL) under nitrogen. Benzoyl chloride (0.20 mL, 1.75 mmol) was added and the mixture was stirred at rt overnight. Bulk pyridine was evaporated and then residual pyridine was co-evaporated with methanol. The crude product was dried under vacuum and used for the next step immediately. For this purpose, it was dissolved in dry methanol (15 mL), acetic acid was added (0.12 mL, 2.10 mmol) and the mixture was refluxed overnight. The solvent was removed by evaporation and the residue was dried under vacuum, separated by flash column chromatography using solvent mixtures of DCM:MeOH increasing the polarity from 99:1 to 90:10 to give, after evaporation, 144 mg of the pure product 13 (yield 74%, over two steps). 1

Synthesis of amide acetal 11
2′,3′,5′-Tribenzoyl-6-cyano-α-cytidine 15 (15 mg, 0.026 mmol) was dissolved in dry methanol (2 mL) and then sodium methoxide (NaOMe) (1.7 mg, 0.031 mmol) was added. This mixture was stirred at rt for 2 h and monitored by TLC, then it was evaporated and the residue was suspended in DCM and diethylether, filtered and then dried under vacuum. The white solid was dissolved in water and the pH of the solution was adjusted to 7. The solution was lyophilized to give the pure white product 11 (7 mg, yield 99%). All the analytical data were in agreement with those reported above.