1. Daniel Glavin and Jason Dworkin NASA Goddard Space Flight Center, Greenbelt MD GCA Team Meeting March 23, 2006 Investigating amino acid enantiomeric excesses in CM meteorites with liquid chromatography time of flight mass spectrometry NASA Astrobiology Institute Goddard Center for Astrobiology

2. Background Over 80 amino acids identified in the CM carbonaceous meteorite Murchison Most amino acids nearly racemic (D/L ~ 1) indicating abiotic origin (Kvenvolden et al. 1970; 1971) Several  -dialkyl amino acids in Murchison have small L-enantiomeric excesses (ee’s) (Cronin and Pizzarello, 1997; Pizzarello and Cronin, 2000) L-isovaline excesses in Murchison range from 0 to 15% (Pizzarello et al. 2003) EE’s inconsistent with Strecker synthesis (Peltzer et al. 1984; Bada 1997) Meteoritic contribution to origin of biologic homochirality on Earth? Left handed (L) Right handed (D) D,L-Isovaline (Iva)

3. Goals of this Study Demonstrate that UV fluorescence liquid chromatography-time of flight-mass spectrometry (LC-ToF-MS) technique can be used for amino acid analyses of meteorites Measure D- and L-isovaline abundances in CM meteorites Murchison and LEW 90500 using LC-ToF-MS Investigate all possibilities for reported L-isovaline enantiomeric excesses (ee’s) Determine D/L ratios for both free and bound isovaline

4. Protocol Meteorite powder (~5-6 g) Water extraction (100ºC 24 h) Acid hydrolysis (6 M HCl 150ºC 3 h) Desalting (AG50W-X8 resin) Derivatization (OPA/NAC primary amines) HPLC with UV fluorescence + ToF-MS detection LC-ToF-MS Instrument at Goddard 50%

5. UV Fluorescence LC-ToF-MS C2C2 C3C3 C4C4 C6C6 C5C5 m/z=379.13 iva val

6. C 5 Amino Acid Isomers  -amino isomer  -amino isomer  -amino isomer  -amino isomer 2-Aminopentanoic acid (norvaline) 3-Aminopentanoic acid (3-apa) 4-Aminopentanoic acid (4-apa) 5-Aminopentanoic acid (5-apa) 2-Amino-2-methylbutanoic acid (isovaline) 3-Amino-2-methylbutanoic acid (3-a-2-mba) and Allo-3-a-2-mba 4-Amino-2-methylbutanoic acid (4-a-2-mba) 2-Amino-3-methylbutanoic acid (valine) 3-Amino-3-methylbutanoic acid (3-a-3-mba) 4-Amino-3-methylbutanoic acid (4-a-3-mba) 3-Amino-2,2-dimethylpropanoic acid (3-a-2,2-dmpa) 3-Amino-2-ethylpropanoic acid (3-a-2-epa) * = chiral carbon 13 possible amino acid isomers of isovaline (C 5 H 11 NO 2 )

7. LC-ToF-MS Data D-iva L-iva 3-a,2,2-dmpa 5-apa 3-a-3-mba L-val D-val D,L-Nor m/z = 379.13 ± 0.02 (ES+ single ion traces) Murchison LEW 90500 Serpentine Blank % L ee = [(L-D)/(L+D)] x 100

8. Enantiomeric Excess Data SampleIsovaline L ee (%) # Valine L ee (%) # Murchison (total) 20.8 ± 1.2 14 35.9 ± 6.2 6 Murchison (free) 18.4 ± 0.9 12 32.3 ± 4.6 6 LEW 90500 (total) 3.3 ± 1.1 15 35.6 ± 6.6 6 LEW 90500 (free) -0.9 ± 1.7 12 36.5 ± 5.2 6 Standard -2.3 ± 1.3 14 0.0 ± 0.4 14 Total = free + bound amino acids in sample hydrolyzed by 6 M HCl Free = amino acids in unhydrolyzed water extract Free isovaline has lower L ee than bound isovaline

9. Other Explanations….. 1.Co-eluting C 5 amino acid that is not L-isovaline? –D/L ratios after 1 and 15 min derivatization are identical –Only two  -dialkyl C 5 amino acids increase in area after 15 min (3-a-3-mba and isovaline) –3-a-3-mba does not interfere 15 min. 1 min. 3-a-3-mba D/L = 0.66 ± 0.02 D/L = 0.65 ± 0.04 3.Co-eluting  -dialkyl C 5 primary amine compound that is not an acid? –Need to test for this possibility Iva 2.Interfering non OPA/NAC labeled compound with m/z = 379.13? –No peaks observed at this mass in unlabeled Murchison extract e.g. 3-Amino-4-hydroxy-3-methyl-2-butanone (C 5 H 11 NO 2 : m/z = 379.13)

10. L-Isovaline Excess in Meteorites Isovaline resistant to racemization –  -Hydrogen analogs (e.g. norvaline) in Murchison racemic (Cronin and Pizzarello, 1997) –No significant racemization at 100ºC in 6M NaOH (Pollock et al. 1975) –Up to 6% radioracemization by  -irradiation (Bonner et al. 1979) –Photostability of bound amino acids greater than free amino acids (Takano et al. 2004) L-isovaline D-isovaline Asymmetric decomposition due to UV CPL –Bonner-Rubenstein Hypothesis: Degradation of amino acids in molecular cloud –Excesses up to 2.5% achieved for UV CPL photolysis of racemic leucine (Flores et al. 1977; Nishino et al. 2002) Strecker synthesis of isovaline on CM parent body? –Formation of racemic isovaline from 2-butanone, HCN, NH 3 and H 2 O –Mechanism inconsistent with ~20% L-isovaline excess found in Murchison slow

11. Amplification of Isovaline EE’s? E.g. amplification of small ee of 5-pyrimidyl alkanol via enantioselective reactions with an asymmetric catalyst has been demonstrated (Soai et al. 1995) Soai autocatalytic reaction + Low ee (~2%) Asymmetric autocatalysis High ee (~90%)

12. Implications for Homochirality Contribution of meteoritic  -dialkyl amino acids with L- enantiomeric enrichment to prebiotic Earth Amplification of amino acid ee’s via asymmetric autocatalysis to generate homochirality?  -Dialkyl amino acids readily form helical structures Transfer  -dialkyl asymmetry to  -hydrogen amino acids common to modern terrestrial life If extinct or extant life in the solar system is based on same handedness, then search for evidence of independent origin of life could be difficult (Bada, 1997)

13. Summary Largest L-enantiomeric excesses for isovaline in Murchison reported to date (+18 to +20%) Smaller ee’s detected in LEW 90500 (-1 to +3%) These ee’s are not due to interfering C 5 amino acid isomers Lower ee of free compared to bound isovaline is consistent with photolysis and radioracemization stability Mechanism(s) for the formation of large ee’s of  - dialkyl amino acids in Murchison remains unclear. Future work: synthesis of isovaline from irradiation of interstellar ice analogs?

14. Acknowledgments This research was supported by the NASA Astrobiology Institute and the Goddard Center for Astrobiology. We thank K. Righter, T. McCoy, and L. Welzenbach for providing the meteorites used in this study, and S. Pizzarello for providing several amino acid standards. Meteorite extracts were obtained with the support of A. Aubrey and J. L. Bada at the Scripps Institution of Oceanography.