Shock synthesis of amino acids from impacting cometary and icy planet surface analogues

SUPPLEMENTARY INFORMATION DOI: 10.1038/NGEO1930 Shock synthesis of amino acids from impacting cometary and icy planet surface analogues 2 3 Includes supplementary Method, supplementary Text, supplementary Tables S1 and S2, supplementary Figures S1, S2, S3 and S4, and supplementary References. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Supplementary Method Chemicals and materials All the glassware and ceramics used in the preparation of the ices and amino acid analysis, and the stainless steel containers and projectiles used in the impact shock experiments were sterilized by wrapping in aluminium foil and baked at 500 ºC for six hours. For the preparation of the ice mixture, bags of CO 2 granules were obtained from BOC Special Gases, ammonium hydroxide solution ( 99.99% purity) and ammonium bicarbonate ( 99.5%) from Sigma-Aldrich, methanol and water were high-performance liquid chromatography (HPLC) grade sourced from Fisher Chemicals. All amino acid standards (>99% purity) except isovaline and pyrene in methyl chloride (200 µl/ml, analytical standard), were purchased from Sigma-Aldrich. D- and L-isovaline (>99% purity) and ammonium hydroxide (NH 4 OH) (28 30 wt %, puriss. p.a.) used in the amino acid analysis (desalting step) were purchased from Acros Organics. Sodium hydroxide (NaOH, >99% purity) and hydrochloric acid (HCl) (37%, 5 ppm extractable organic substances) were acquired from Boom. AG 50W-X8 resin (100-200 mesh) was purchased from Bio-Rad. HPLC-grade dichloromethane (99.8+% purity) was obtained from Fisher Scientific. The trifluoroacetic anhydride/isopropanol (TFAA-IPA) derivatization kit, including acetyl chloride, was bought from Grace Davison Discovery Sciences. NATURE GEOSCIENCE www.nature.com/naturegeoscience 1

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Amino acid analysis The ice residues were analysed for amino acids based on the experimental method for detection of amino acids in extra-terrestrial samples S9-S12. Each ice residue was brought up in 1.5 ml HPLC grade water (3 0.5 ml, stirred each time), transferred to a small test tube (3 ml) and dried under vacuum. An additional small test tube containing HPLC grade water was also dried under vacuum, subjected to the same amino acid extraction experimental procedure as the ice samples and used as an extraction procedural blank. Each sample was then hydrolysed by placing each small test tube inside a Pyrex test tube containing 1 ml 6 M HCl, flame sealing each Pyrex test tube and placing them in an oven at 150 ºC for three hours. Hydrolysis under these conditions leads to the bonds between amino acids being cleaved, and allowing the determination of the total (free + bound) amino acid content of the sample. This method does not destroy or create organic molecules. The outside of the Pyrex tubes were rinsed after the hydrolysis step with HPLC grade water before being opened, the small test tubes were taken out and the extracts dried under vacuum. The dried extracts were brought up in 3 ml water (3 1 ml, stirred each time), desalted on a cation exchange resin, and amino acids eluted with 5 ml of 2 M ammonium hydroxide. The eluates were dried under vacuum, dissolved in 100 μl of water, transferred to 1 ml V-vials and dried with a flow of N 2 gas before the amino acid derivatization step S10,S11. The amino acid derivatization consisted of adding 100 μl of an acetylchloride:isopropanol mixture (30:70 V/V) to each vial, which were then tightly capped with Teflon-lined screw caps and placed in heating blocks for one hour at 110 ºC. The vials were then cooled to room temperature, and the samples slowly dried with a flow of N 2 gas. 100 µl of methylene chloride and 50 µl of TFAA were added, the vials were tightly capped and heated at 100 ºC for 10 minutes. The vials were then cooled to room temperature, and the samples slowly dried with a flow of N 2 gas. 65 μl of DCM and 10 μl of pyrene in methylene chloride (200 2

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 µg/µl), which was used as internal standard were added to the samples and stirred. 1 µl of sample was then injected into the GC-MS. The set of samples #1 (target ice, control ice and blank) were run in a different, and independent, GC-MS than the set of samples #2 (target ice, control ice and blank) in order to test reproducibility of the experiment and results. Each residue was run on the GC-MS six times. The GC-MS analyses for the set of samples #1 were performed using an Agilent 7673 series injector (splitless injection), 6890 network GC system, 5973 mass selective detector, MSD transfer line set to 220 ºC, MS quad set to 150 ºC, MS source to 230 ºC, and helium as the carrier gas with a flow of 1 ml/min. Separation of the D, L-amino acid enantiomers was achieved using two Chirasil-L-Val columns from Varian (2 times 25 m 0.25 mm ID 12 µm film thickness, connected by a Valco zero dead volume union from Alltech). The oven program was held for 5 minutes at 65 ºC, increased by 2 ºC/min to 80 ºC, held for 5 minutes, increased to 100 ºC by 1 ºC/min, increased to 200 ºC by 2 ºC/min held for 10 minutes, and finally increased by 10 ºC/min to 220 ºC and held for 5 minutes. The GC-MS analyses for the set of samples #2 were performed using a Perkin Elmer Turbo Mass Spectrometer and AutoSystem XL GC system with splitless injection, transfer line set to 220 ºC, MS quad set to 150 ºC, MS source to 230 ºC, and helium as the carrier gas with a flow of 1 ml/min. Separation of the D, L-amino acid enantiomers was achieved using two new Chirasil-L-Val columns from Varian (2 times 25 m 0.25 mm ID 12 µm film thickness, connected by a Valco zero dead volume union from Alltech). The oven program was held for 10 minutes at 45 ºC, increased by 2 ºC/min to 80 ºC, held for 5 minutes, increased to 100 ºC by 1 ºC/min, and increased to 200 ºC by 2 ºC/min, held for 10 minutes. Note that the retention time for amino acids in the set of samples #1 is different than for the set of samples #2 due to a different GC-MS temperature programme (as described above). D- and L-isovaline enantiomers could not be separated under any of the chromatographic conditions. 3

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Enantiomeric identification of D- and L-α-ABA was not possible because optically pure standards were not available. All the detected amino acids were identified by comparison of the retention time and mass spectrum (and corresponding mass fragmentation pattern) with known amino acid standard mixtures ran each day. The Supplementary Text explains the mass fragmentation for each of the derivatized (N-TFA, O-isopropyl) amino acid standards, and Fig. S4 shows the amino acid standard mixture ran with the method used to analyse the set of samples #1). The amino acid content of the samples was quantified by the response factor of each amino acid relative to an internal standard added to the samples. The peak area integration of the corresponding ion fragment was converted to abundances by using calibration curves. Single ions of the amino acids that are used to identify and quantify the amino acid content of samples are shown in Supporting Fig. S1 and Table S2. The calibration curves were created by plotting the amino acid standard target ion peak area/internal standard (pyrene) target ion peak area ratio versus the mass of amino acid standard injected into the GC-MS column. The internal standard (pyrene) does not co-elute with any amino acid (retention time 90 min, m/z 202). 91 92 93 94 95 96 97 98 99 100 Supplementary Text When a sample is run on the GC-MS, the molecules are fragmented into charged particles. The fragmentation pattern is unique for each molecule and works as diagnostic to the compound present in the sample. The mass and charge of the particles are represented as mass/charge ratio (m/z). The supplementary Fig. S1a) shows the GC-MS mass spectrum for the peak assigned as the derivatized (N-TFA, O-isopropyl) amino acid standard glycine, its corresponding chemical formula and mass fragmentation pattern. Its fragmentation will lead to peaks at m/z 69 (fragment F 3 C), m/z 126 (fragment F 3 C-CO-HN-CH 2 ) and 87 (fragment COO-CH-(CH 3 ) 2 ), 4

101 102 103 104 105 106 107 108 109 110 111 112 113 and m/z 154 (fragment F 3 C-CO-HN-CH 2 -CO) and 59 (fragment O-CH-(CH 3 ) 2 ). The same fragmentation pattern is observed in the mass spectrum for the derivatized (N-TFA, O- isopropyl) amino acid glycine present in the target ice sample #1 (Fig. S1b)), confirming the definitive detection of this amino acid in the target ice. The main fragments of derivatized (N- TFA, O-isopropyl) amino acid glycine will appear at m/z 126 (the highest peak) and 154. Supplementary Fig. S4 shows all the single ion GC-MS traces (at m/z 126, 140, 154, 168, 180, 182 and 184) of the derivatized (N-TFA, O-isopropyl) amino acid standard mixture ran with the conditions used to analyse the set of sample #1, while the main molecular ion (m/z) used for identification and quantification of the derivatized (N-TFA, O-isopropyl) amino acids present in the target ice sample is described in Table S2. All the detected amino acids were identified by comparison of the retention time and also comparison of the mass spectrum (and corresponding fragmentation pattern) with known amino acid standard mixtures ran each day. 114 115 116 117 118 119 120 121 122 123 124 125 5

126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Supplementary References S1. Festou M., Uwe-Keller, H and Weaver, H. A (editors). Comets-II. University of Arizona Press (2005). S2. DiSanti M. A., Bonev, B. P., Villanueva, G. L. and Mumma M. J. Highly depleted ethane and mildly depleted methanol in Comet 21P/Giacobini-Zinner: application of a new empirical υ2-band model for CH3OH near 50K. The Astrophysical Journal 763, 1 (2013). S3. Crovisier, J. and Bockelée-Morvan, D. Remote observations of the composition of cometary volatiles. Space Science Reviews 90, 19 32 (1999). S4. Ehrenfreund, P. et al. Astrophysical and astrochemical insights into the origin of life. Reports on Progress in Physics 65, 1427 1487 (2002). S5. Ehrenfreund, P. and Charnley, S.B. Organic molecules in the interstellar medium, comets, and meteorites: a voyage from dark clouds to the early Earth. Annual Review of Astronomy and Astrophysics 38, 427 483 (2000). S6. Mumma, M.J. et al. Remote infrared observations of parent volatiles in comets: a window on the early solar system. Advances in Space Research 31, 2563 2575 (2003). S7. Bockelée-Morvan, D. et al. New molecules found in cometc/1995 O1 (Hale Bopp). Investigating the link between cometary and interstellar material. Astronomy and Astrophysics 353, 1101 1114 (2000). S8. Burchell M. J., Cole M. J., McDonnell J. A. M. and Zarnecki, J. C. Hypervelocity impact studies using the 2 MV Van de Graaff accelerator and two-stage light gas gun of the University of Kent at Canterbury. Measurement Science & Technology 10, 41 50 (1999). S9. Kvenvolden et al. Evidence for extraterrestrial amino-acids and hydrocarbons in the Murchison meteorite. Nature 228, 923-926 (1970). S10. Silfer, J. A., Engel, M. H., Macko, S. A. and Jumeau, E.J. Stable carbon isotope analysis of amino acid enantiomers by conventional isotope ratio mass spectrometry and 6

151 152 153 154 155 156 157 158 combined gas chromatography/isotope ratio mass spectrometry. Analytical Chemistry 63, 370-374 (1991). S11. Macko, S. A., Uhle, M. E., Engel, M. H. and Andrusevich, V. Stable nitrogen isotope analysis of amino acid enantiomers by gas chromatography combustion-isotope ratio mass spectrometry. Analytical Chemistry 69, 926 929 (1997). S12. Martins, Z., Alexander, C.M.O D., Orzechowska, G.E., Fogel, M.L., and Ehrenfreund, P. Indigenous amino acids in primitive CR meteorites. Meteoritics & Planetary Science 42, 2125 2136 (2007). 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 7

176 177 178 179 180 181 182 183 184 Supplementary Tables Supplementary Table 1 List of ice mixtures and shot parameters and results of amino acid detection results. The projectile was a sterilised 1 mm diameter stainless steel sphere in all cases. Shot ID Ice mix Impact velocity Amino acids detected? (km s -1 ) G090212#2 NH 4 OH:CO 2 :CH 3 OH 7.15 a Yes (see main text) G090113#2 NH 4 OH:CO 2 :CH 3 OH 7.00 Yes (see main text) G170113#1 NH 4 OH:CO 2 :CH 3 OH 5.79 No G200112#2 NH 4 OH:CO 2 :CH 3 OH 3.66 No G130112#1 NH 4 OH:CO 2 :CH 3 OH N/A b No G09013#1 NH 4 OH:CO 2 7.12 No G130112#2 NH 4 OH:CO 2 6.50 No a Hydrocode modelling indicates that at 7.15 km s -1, the peak shock pressure experienced by the ice mixture was ~60 GPa. b Misfire. No projectile impacted the target ice, but the shotgun cartridge fired. Acts as a double blank control shot. 185 186 187 188 189 190 191 Supplementary Table 2 Molecular ions (m/z) used for identification and quantification (first ion) of the amino acids. Single ion m/z 202 was selected to identify and quantify pyrene (used as an internal standard). Amino acid Single ion Amino acid Single ion Amino acid Single ion α-aib 154 D,L-β-AIB a,b 182/153 L-Norleucine 182/114 D, L-Isovaline a 168 D-Norvaline 168 γ-aba 182/154 D-Alanine 140 L-Norvaline 168 D-Aspartic acid 184/212 L-Alanine 140 β-alanine 168/185 L-Aspartic acid 184/212 D,L-α-ABA b 154 D,L-β-ABA b 140/182/153 D-Glutamic acid 180/198 D-Valine 168 D-Leucine 140/182 L-Glutamic acid 180/198 L-Valine 168 L-Leucine 140/182 Glycine 126/154 D-Norleucine 182/114 a Enantiomers could not be separated under the chromatographic conditions. b Optically pure standard, not available for enantiomeric identification. 8

192 193 194 195 196 197 Supplementary Figure 1 - (a) GC-MS mass spectrum for the peak assigned to derivatized (N-TFA, O-isopropyl) amino acid standard glycine, its chemical formula and correspond mass fragmentation. (b) GC-MS mass spectrum for the peak assigned to derivatized (N-TFA, O-isopropyl) amino acid glycine present in the target ice sample #1. 9

198 199 200 201 202 203 204 205 206 Supplementary Figure 2 Chromatogram of extracts from Control (unshocked) ice sample #1. The 10 to 70 min region of the single ion GC-MS traces (m/z 126, 140, 154, 168, 180, 182 and 184) of the derivatized (N-TFA, O-isopropyl) HCl-hydrolysed extracts of the control ice sample #1. (a) (identical starting materials as the ice sample #1, but did not experience the impact-shock) and the procedural blank #1 (b) (same extraction procedure as the ice sample #1). Samples were run on an Agilent GC-MS (see Methods section). No amino acid was detected above the detection limit of the GC-MS (10 pg of amino acid in a sample). 10

207 208 209 210 211 212 213 214 Supplementary Figure 3 - Chromatogram of extracts from Control (unshocked) ice sample #2. The 20 to 90 min region of the single ion GC-MS traces (m/z 126, 140, 154, 168, 180, 182 and 184) of the derivatized (N-TFA, O-isopropyl) HCl-hydrolysed extracts of the control ice sample #2. (a) (identical starting materials as the ice sample #2, but did not experience the impact-shock) and the procedural blank #2. (b) (same extraction procedure as the ice sample #2). Samples were run on a Perkin Elmer GC-MS (see Method Section for details). No amino acid was detected above the detection limit of the GC-MS (10 pg of amino acid in a sample). 11

215 216 217 218 219 220 221 222 223 224 225 Supplementary Figure 4 - Chromatogram of extracts from amino acid standards. The 10 to 70 min region of the single ion GC-MS traces (m/z 126, 140, 154, 168, 180, 182 and 184) of the derivatized (N-TFA, O-isopropyl) amino acid standards (conditions as set of sample #1) 1. α-aminoisobutyric acid (α-aib); 2. D,L-Isovaline; 3. D-Alanine; 4. L-Alanine; 5. D, L-αaminobutyric acid (α-aba); 6. D-Valine; 7. L-Valine; 8. Glycine; 9. D-Norvaline; 10. L- Norvaline; 11. β-alanine; 12. D,L-β-aminoisobutyric acid (β-aib); 13. D, L-β-aminobutyric acid (D,L-β-ABA); 14. D-Leucine; 15. L-Leucine; 16. D-Norleucine; 17; L-Norleucine; 18. γ- aminobutyric acid (γ-aba); 19. D-Aspartic acid; 20. L-Aspartic acid; 21. D-Glutamic acid; 22. L-Glutamic acid. 12