A prebiotically plausible synthesis of pyrimidine β-ribonucleosides and their phosphate derivatives involving photoanomerization

Abstract

Previous research has identified ribose aminooxazoline as a potential intermediate in the prebiotic synthesis of the pyrimidine nucleotides with remarkable properties. It crystallizes spontaneously from reaction mixtures, with an enhanced enantiomeric excess if initially enantioenriched, which suggests that reservoirs of this compound might have accumulated on the early Earth in an optically pure form. Ribose aminooxazoline can be converted efficiently into α-ribocytidine by way of 2,2'-anhydroribocytidine, although anomerization to β-ribocytidine by ultraviolet irradiation is extremely inefficient. Our previous work demonstrated the synthesis of pyrimidine β-ribonucleotides, but at the cost of ignoring ribose aminooxazoline, using arabinose aminooxazoline instead. Here we describe a long-sought route through ribose aminooxazoline to the pyrimidine β-ribonucleosides and their phosphate derivatives that involves an extraordinarily efficient photoanomerization of α-2-thioribocytidine. In addition to the canonical nucleosides, our synthesis accesses β-2-thioribouridine, a modified nucleoside found in transfer RNA that enables both faster and more-accurate nucleic acid template-copying chemistry.

Figure 1. Previous prebiotic syntheses of pyrimidine β-ribonucleotides via 2,2'-anhydronucleoside intermediates.

The route initially described by Orgel is indicated by the blue arrows. Thus treatment of ribose with cyanamide gave a beautifully crystalline aminooxazoline 1 which underwent reaction with cyanoacetylene 2 to give the anhydronucleoside 3 and thence, by hydrolysis, α-ribocytidine 4. However, anomerization to β-ribocytidine 5 by irradiation of 4 with UV light was very inefficient and this, along with doubts about the prebiotic provenance of ribose, lessened the attractiveness of the route. We determined that one of the reasons for the low photoanomerization yield was competing formation of the oxazolidinone 6 and then found an alternate route to the pyrimidine ribonucleotides via the arabinose aminooxazoline 7 and anhydronucleoside 8. The key steps were a regioselective phosphorylation and 2'-inversion reaction of 8 giving β-ribocytidine-2',3'-cyclic phosphate 9 and the partial photochemical conversion of 9 to the corrsponding uridine analogue 10. We also found that pentose aminooxazolines, predominantly 1 and 7, can be produced in an indirect way from glyceraldehyde 11 and 2-aminooxazole 12. Although our route marked the first high-yielding synthesis of the pyrimidine ribonucleotides under prebiotically plausible conditions, it did not exploit the crystallinity of 1.

Figure 2. Synthesis of pyrimidine β-ribonucleosides and β-ribonucleotides involving photoanomerization of α-2-thioribocytidine 13.

The reaction scheme incorporates the new synthesis and the known hydrolysis of β-ribocytidine 5 to β-ribouridine 19. Thiolysis of the ribose anhydronucleoside 3 gives α-2-thioribocytidine 13 in high yield along with a small amount of α-ribocytidine 4. In marked contrast to 4, 13 undergoes remarkably efficient photoanomerization, giving β-2-thioribocytidine 14 in excellent yield. The canonical pyrimidine ribonucleosides 5 and 19 can be produced from 14 by hydrolysis. Phosphorylation of 14 is accompanied by conversion of the 2-thiocytosine nucleobase to cytosine providing a direct route to the canonical pyrimidine ribonucleoside-2',3'-cyclic phosphates 9 and thence 10 and an alternative route to the ribonucleoside 5. 2-Thioribouridines 16 and 17 can also be produced by hydrolysis of 13 and 14 respectively under acidic conditions.

Figure 3. ¹H NMR spectra demonstrating the inherently favoured nature of the individual reactions of the newly discovered synthetic sequence.

¹H NMR spectra were recorded at 400 MHz with samples of reaction products (after removal of solvents) and synthetic standard dissolved in H₂O-D₂O a, Spectrum of the starting material, 2,2'-anhydro-ribocytidine 3. b, Spectrum of the products of thiolysis of 3 by HS^– in wet formamide (singlet at δ ~ 8.0 ppm is due to residual formamide). Note the quantitative transformation of 3 and the predominance of α-2-thioribocytidine 13 over α-ribocytidine 4 in the products. c, Spectrum of an authentic sample of α-2-thioribocytidine 13. d, Spectrum of the products of irradiation of 13 in aqueous solution (singlet at δ ~ 7.9 ppm is due to residual dimethylformamide from the conventional synthesis of 13). Note the remarkably clean conversion of 13 to the anomer β-2-thioribocytidine 14. e, Spectrum of an authentic sample of β-2-thioribocytidine 14. f, Spectrum of the products of hydrolysis of 14 in pH = 7 phosphate buffer after ~ one half life. Despite the conversion of 14 being only partial, note the clean production of β-ribocytidine 5 and the appearance of its hydrolysis product, β-ribouridine 19 as well as β-2-thioribouridine 17, the product of alternate hydrolysis of 14. g, Spectrum of an authentic sample of β-ribocytidine 5.

Figure 4. Reaction mechanisms.

a, Mechanisms of phosphorylation and the degradation of β-2-thioribocytidine-2',3'-cyclic phosphate 22. The generation of the active phosphorylating agent, formidoyl phosphate 21, in the phosphorylation of β-2-thioribocytidine 14 is shown in the box – the phosphorylation of 14 itself involves a hydroxyl group thereof attacking the imidoyl phosphate in a manner analogous to the reverse of the electron movements indicated by the arrows. Outside of the box, the mechanisms of the reactions taking place during the phosphorylation of β-2-thioribocytidine 14 are shown. b–e, ¹H NMR spectra (400 MHz, H₂O-D₂O) demonstrating incorporation of deuterium at at C-1' of α/β-2-thioribocytidine 13/14 during photoanomerization. b, Expansion of the spectrum of α-2-thioribocytidine 13 showing signals for H-6, H-1' and H-5. c, Spectrum of the products of irradiation of α-2-thioribocytidine 13 in D₂O for 12 hours showing signals for H-6 and H-5 of both 13 and β-2-thioribocytidine 14 but lacking signals for H-1' of either anomer. d, Spectrum of 14 prior to irradiation. e, Spectrum of the products of irradiation of 14 in D₂O for 36 hours. The deuteration data shown indicate a mechanism of photoanomerization involving exchange of H-1' with solvent.

Figure 5. Mechanism of the key photoanomerization reaction.

a, Conventional organic chemical depiction of the mechanism of the photoanomerization of α/β-2-thioribocytidine 13/14 via the diradical 25. b, The same mechanism but analysed at a deeper level to explain the niceties of the reaction. Potential energy (PE) profiles presenting the photoanomerization reaction of α-2-thioribocytidine. The PE cuts on the left-hand side were obtained by interpolations between the Franck-Condon region, S₁ minimum, S₁/T₂ minimum energy crossing point and the T₁ minimum. The PE profile on the right was obtained from a relaxed scan along the S–H distance. The inset box on the right corresponds to the relaxed scan along the S–H distance of α-2-thioribouridine, for which the T₁(ππ*) and S₀ states do not cross. The energies were calculated using the ADC(2)/cc-pVTZ method and the computations of the SOC matrix elements were performed at the CASPT2/CASSCF level (employing cc-pVTZ-DK basis set). Details of the S₁/T₂ intersystem crossing rate constant computations can be found in the Supplementary Information.