Methods
An equimolar gas mixture of carbon monoxide (350 Torr; UHP grade, Toho Sanso, Yokohama, Japan) and 15N-enriched nitrogen (350 Torr; 99.8% 15N, Shoko, Tokyo) was enclosed in a glass tube (400 ml) containing liquid water (5 ml). 15N2 was used to identify possible contamination. The gas mixture was irradiated with protons generated by a van de Graaff accelerator (Tokyo Institute of Technology, Tokyo) at 297 K for 3 h. Each proton has an energy of 2.5–3.0 MeV, which is much higher than the bond dissociation energy of CO (11 eV) and N2 (9.8 eV). The total deposited energy was 13 kJ. The flux was 1 mA•cm–2. A sample of the aqueous solution of the irradiated mixture was withdrawn and acid hydrolyzed with 6 M HCl at 100°C for 24 h. Acid hydrolysis was performed because the bioorganic compounds are expected to be formed as their precursors. Most amino acids synthesized with this method are detectable only after acid hydrolysis (25). The samples were separated into three fractions on Dowex 50 (H+) with 1.5, 2.5, and 6 M HCl as eluant. Uracil, orotic acid, and 5-hydroxyuracil eluted in the 1.5 M HCl fraction. Nicotinic acid and 4,5-dihydroxypyrimidine eluted in the 2.5 M HCl fraction. Adenine and guanine eluted in the 6 M HCl fraction. These fractions were then rechromatographed by reversed-phase HPLC (Beckman 110B pump, 6.0 x 250 mm YMC ODS-AQ column, Kratos UV spectrophotometer set at 260 nm, 0.1 M, pH 4.5, sodium phosphate buffer) for final purification. The peak corresponding to each compound was collected and rechromatographed by using 0.1 M, pH 3, sodium phosphate buffer, then again by using 0.02 M, pH 5, ammonium acetate buffer. Compounds were identified by UV absorbance spectrum and GC/MS analysis of their trimethylsilyl derivatives. The trimethylsilyl derivatives were obtained by heating the compounds in a solution of N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane and pyridine at 150°C for 30 min. The mass spectra can be seen in Figs. 2–6, which are published as supporting information on the PNAS web site, www.pnas.org. The quantitative data were obtained from the HPLC chromatograms with the ammonium acetate buffer by comparison with standard samples.
For calculation of production rates from cosmic rays and corona discharges, the value of 0.046 J•cm–2•yr–1 (26) and 0.105 J•cm–2•yr–1 (27) were taken as the energy flux, respectively.
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