Apparatus and UV irradiation experiments

The photolysis of interstellar ice analogues was performed using a setup for analysis of molecular and radical reactions of astrochemical interest (SAMRAI)25,26. SAMRAI includes an ultra-high vacuum reaction chamber, a Fourier transform infrared spectrometer (FTIR), a quadrupole mass spectrometer and an aluminium (Al) substrate that can be cooled to 10 K using a helium (He) refrigerator. The base pressure is of the order of 10−7 Pa. Two deuterium (D 2 ) discharge lamps (L12098, Hamamatsu Photonics), whose photon fluxes are in the range 1013–1014 photons cm−2 s−1, are attached to the reaction chamber. Photon fluxes are estimated using the electric current measured by a photodiode (AXUV199G, Opto Diode Corp.) placed in front of the substrate. Gaseous samples of H 2 O, CO, NH 3 , and CH 3 OH in a mixing ratio of 5:2:2:2, respectively, were supplied via continuous vapour deposition (gas deposition rate of ~5 × 1012 molecules cm−2 s−1) with photoirradiation onto the Al substrate for ~200 h at 10 K. The 200-h-long deposition yields the ice with the thickness of ~3600 monolayers (ML; 1 ML = 1 × 1015 molecules cm−2) if no photolysis of molecules occurred under the conditions utilised in this study. The photon fluence for 200 h corresponds to ~2 × 107−8 years in molecular clouds when the photon flux is 104 photons cm−2 s−1, which is a mean photon flux in those environments37. The number of photons per molecule is in the range of 2–20. In a separate experiment, a mixture of deuterated methanol isotopologues including CH 3 OH, CH 2 DOH, CHD 2 OH, CD 3 OH, and CH 3 OD was used instead of pure CH 3 OH in a mixing ratio of 100:30:6:1:2, respectively, which is similar to the ratio observed in the low-mass protostar IRAS16293-2422 (ref. 38). CH 3 OD was introduced via a separate gas line to avoid hydrogen isotopic exchange of the –OD group with other polar molecules (i.e., H 2 O and NH 3 ) before vapour deposition. This experiment would be helpful to estimate the deuterium enrichment of nucleobases which could be present in that environment. In addition, we performed an additional 15N-isotope probing experiment under the same conditions with the use of 15NH 3 gas (15N purity = 98%) instead of 14NH 3 . A similar procedure was repeated without using gaseous samples to check a potential contribution from inside the chamber. The recovered sample was analysed as an entire procedural blank in this experiment (Supplementary Fig. 7).

Molecular identification of the experimental products

After simultaneous gas deposition and photon irradiation, the substrate was warmed up to room temperature to remove volatile species at a ramping rate of 0.2 K min−1, and the formation of solid organic residues was confirmed using FTIR19. The organic residues were dissolved in several tens of microlitres of a water/methanol mixture (1/1 by vol./vol.) and extracted from the reaction substrate using a small amount of quartz wool. The quartz wool with the samples was further transferred into a separate glass vial, and 0.5 ml of H 2 O was added to the vial. Subsequently, the aqueous solution was collected using a microsyringe and transferred into another glass vial. For an accurate chromatographic baseline resolution when focusing on the target molecules, especially those with nitrogen-containing functional groups (i.e., amide, amino, imino, and N-heterocyclic species; please see the Supplementary Fig. 34), a purification procedure was performed using cation-exchange column chromatography (AG-50W-X8 resin; 200–400 mesh, Bio-Rad Laboratories)39. This pretreatment advantageously eliminates uncharacterised matrix effects to assist with the evaluation of the irradiation products. The purified solution was dried under a gentle nitrogen gas (N 2 ) flow and subsequently dissolved in 50 μl of ultrapure H 2 O (QToFMS grade from Wako Chemical, Ltd.) before analysis. All glassware and the quartz wool were heated in air at 450 °C for 3 h to prevent contamination by organic compounds.

The sample solution was injected into an orbitrap mass spectrometer (Q Exactive Plus, Thermo Fischer Scientific) with a mass resolution of m/Δm = ~ 140,000 at a mass-to-charge ratio (m/z) of 200 via a high-performance liquid chromatograph (HPLC) system (UltiMate 3000, Thermo Fischer Scientific) equipped with a reversed-phase C18 separation column (1.5 × 250 mm, particle size of 3 μm, InertSustain C18, GL Science) at 40 °C. The eluent program for this HPLC setup is as follows: solvent A (H 2 O + 0.1% formic acid by volume), solvent B (acetonitrile + 0.1% formic acid by volume) = 100:0 for the initial 5 min, followed by a linear gradient of A:B = 40:60 at 35 min, and it was kept at this ratio for 10 min. The flow rate was 70 μl min−1.

Nucleobases were also analysed using the same HPLC/HRMS equipped with a HypercarbTM separation column (4.6 × 150 mm, particle size of 5 μm, Thermo Fischer Scientific) at 10 °C to verify their presence in the sample. The eluent program is as follows: at t = 0, solvent A (20 mM nonafluoropentanoic acid in distilled water + 0.1% formic acid (dissolved)), solvent B (acetonitrile + 0.1% formic acid (dissolved)) = 100:0, followed by a linear gradient of A:B = 40:60 at t = 60 min and it was kept at this ratio for 10 min. The flow rate was 0.2 ml min−1. The detailed analytical conditions of the HPLC system have been described previously25,40.

The identification of nitrogen-bearing molecules was based on a co-injection analysis in which the analyte sample and standard reagent solutions were analysed as part of the same HPLC/HRMS run.

For sugar molecules, we analysed the organic residues without the cation-exchange chromatography purification procedure (cf. this section for amide, amino, imino, and N-heterocyclic species). The mass spectra were recorded in the positive electrospray ionisation (ESI) mode for the nucleobases, other nitrogen heterocycles, dipeptides, and amino acids with an m/z range of 50–400 and a spray voltage of 3.5 kV. To analyse the sugars, the mass spectra were recorded in the negative ESI mode with an m/z range of 50−215 and a spray voltage of 3.0 kV. The capillary temperature of the ion transfer was 300 °C. The injected samples were vaporised at 300 °C. We set up an inverse gradient program to maintain the ionisation efficiency during the ESI. To minimise analytical noise and the background signals in the liquid chromatography (LC) and orbitrap mass spectrometry (MS), we used high purity grade water and acetonitrile (LC/MS grade from Wako Chemical, Ltd.). Under these experimental conditions, the mass precision is always better than 3 ppm for each chromatogram (e.g. 113.0348 ± 0.0003 for protonated uracil). The same volume of distilled water was measured to validate the contamination level of the MS; no prebiotic molecules were detected during the analytical blank measurement. For another sample (processed as described earlier in this paragraph), when no gases were deposited (i.e., UV photons only) on the substrate, we found that no prebiotic molecules formed (Supplementary Fig. 7), further confirming the blank control. Standard reagent grade target molecules were used and amino acids were purchased from Wako Chemicals, Ltd. and Sigma-Aldrich, Ltd. Pyrimidine and purine nucleobases and other nitrogen heterocycles were purchased from Tokyo Chemical Industry, Ltd. and Sigma-Aldrich, Ltd., and dipeptides were from AnaSpec, Inc. and PH Japan, Ltd. Dihydrouracil was synthesised in our laboratory from reagent grade β-alanine and potassium cyanate (both from Wako Chemicals, Ltd.), according to the method reported by Dakin41. The target molecules (standards) were dissolved in distilled H 2 O, and a mixture of the solution was analysed using the aforementioned procedure. To prevent misidentification, structural isomers were not analysed during the same run; e.g., to identify pyrimidine and its isomers (pyridazine and pyrazine), we performed three independent analyses.

After the analyses of the standard reagents and the sample in a separate run when the C18 column was used, 1 μl (5 μl when the HypercarbTM was used) of the sample solution was co-injected with 1 μl (2 μl for the HypercarbTM) of a mixture of the standard reagents. The concentration of the co-injected solution was adjusted to not significantly exceed the sample peak heights. Eddhif et al.27 identified various prebiotic molecules based on a comparison of the retention times of their target molecules on a separation column. Although this method is appropriate when the number of peaks in the mass chromatogram is not high, it becomes unsuitable when a number of peaks appear very close to each other. Moreover, the co-injection analysis is not always able to identify molecules in a mass chromatogram due to an insufficient separation of peaks and/or the absence of an appropriate chemical reagent. In this case, analysis of the sample using a different separation column on the HPLC/HRMS would be ideal to firmly identify a target molecule in a mixture of complex organic molecules. In the present study, we identified pyrimidine nucleobases by using two different separation columns (C18 and HypercarbTM) and the yields that were estimated under the two different conditions were consistent with each other, which strongly supports our conclusion that pyrimidine nucleobases are actually present in the sample. In contrast, under the present analytical conditions, purine nucleobases, except for hypoxanthine, were not eluted from the HypercarbTM separation column probably due to the strong interaction between nucleobases and the stationary phase of the column; their detection was successful when the C18 column was used as a separation column. Hence, the cross-validation analysis is a promising approach to better understand the molecular compositions in complex organic residues.