Evolution of Solid Phase Homochirality for a. Proteinogenic Amino Acid

Transcription

1 Supporting Information Evolution of Solid Phase Homochirality for a Proteinogenic Amino Acid Cristobal Viedma, 1 * José E. Ortiz, 2 Trinidad de Torres, 2 Toshiko Izumi, 3 Donna G. Blackmond 3.4 * 1 Departamento Cristalografia-Mineralogia, Facultad Geologia, Universidad Complutense, 28040, Madrid, Spain 2 Laboratorio de Estratigrafía Biomolecular, Escuela Técnica Superior de Ingenieros de Minas de Madrid, Madrid, Spain; 3 Department of Chemical Engineering and 4 Department of Chemistry, Imperial College, London SW7 2AZ, UK viedma@eucmax.sim.ucm.es, d.blackmond@imperial.ac.uk Materials All chemicals were obtained from Fluka and Aldrich. CR(+) column was purchased from VWR International Ltd. Hypersil BDS C18 reverse-phase column was purchased from xxxxxx. Experiments with attrition enhancement Low temperature (90 o C) experiments. Both L- & D-aspartic acid (0.5 g in total: initial imbalance + 5% ee) were grounded in a mortar. The solid was placed in a screw caped bottle and an oval PTFEcoated magnetic stirring bar (L 20 mm, ø 10 mm, Aldrich), 8 g of glass balls (ø mm, Aldrich) and acetic acid (5 ml) were added. The mixture was stirred at 1200 rpm and was heated to o C using an oil bath. To this suspension, salicylaldehyde (0.3 ml, 6 vol%) was added to start racemization and samples were taken every 2-3 days. For sampling procedure, 0.5 ml solution with crystals were taken 1

2 and filtered using a syringe filter (PTFE membrane, 0.45 μm, 25 mm). Solid was washed with acetic acid (2 ml x 4 times) and dried in a desiccator for 3 days to be used in HPLC analysis. High temperature (160 o C) experiments. Both L- & D-aspartic acid (0.5 g in total: initial imbalance + 9% ee (L) & - 20% ee) were grounded in a mortar and was placed in a screw caped bottle with an oval PTFE-coated magnetic stirring bar (L 20 mm, ø 5 mm, Aldrich), 8 g of glass balls (ø mm, Aldrich) and acetic acid (5 ml). The mixture was stirred at 900 rpm and was heated on a hot stirring plate set to 160 o C. To this suspension, salicylaldehyde (0.3 ml, 6 vol%) was added to start racemization and samples were taken every 2 days. For sampling procedure, 0.5 ml solution with crystals was taken and was transferred into a centrifuge tube containing 3 ml acetic acid. This tube was centrifuged at 4000 rpm for 1 minute for 4-5 times. This sample was dried in a desiccator to be used in HPLC analysis. Experiment with no attrition enhancement Low temperature (105 o C) experiments. Both L- & D-aspartic acid ( g in total: initial imbalance + 9% ee (L)) were grounded in a mortar. The solid was placed in a screw caped bottle and an oval PTFE-coated magnetic stirring bar (L 20 mm, ø 10 mm, Aldrich) and acetic acid (5 ml) were added. The mixture was stirred at 600 rpm and was heated to 105 o C using an oil bath. To this suspension, salicylaldehyde (0.1 ml, 2 vol%) was added to start racemization and samples were taken every 5-10 days. For sampling procedure, 0.5 ml solution with crystals were taken and filtered using a syringe filter (PTFE membrane, 0.45 μm, 25 mm). Solid was washed with acetic acid (2 ml x 4 times) and dried in a desiccator for 3 days to be used in HPLC analysis. Reflux experiments at 125 o C. Both L- & D-aspartic acid (4.125 g in total: initial imbalance + 9% ee (L)) were grounded in a mortar and was placed in a two neck 100 ml round bottom flask with an oval PTFE-coated magnetic stirring bar (L 20 mm, ø 10 mm, Aldrich) and acetic acid (20 ml). The mixture was stirred at 600 rpm and was set under reflux at 125 o C using an oil bath. To this suspension, 2

3 salicylaldehyde (0.2 ml, 1 vol%) was added to start racemization and samples were taken every 2 days. For sampling procedure, 0.5 ml solution with crystals was taken and was transferred into a centrifuge tube containing 3 ml acetic acid. This tube was centrifuged at 4000 rpm for 1 minute for 4-5 times. This sample was dried by purging N 2 and placed in a desiccator overnight to be used in HPLC analysis. High temperature (160 o C) experiments. Both L- & D-aspartic acid (0.5 g in total: initial imbalance + 4.5% ee (L)) were grounded in a mortar. The solid was placed in a screw caped bottle with an oval PTFE-coated magnetic stirring bar (L 20 mm, ø 5 mm, Aldrich) and acetic acid (5 ml). The mixture was stirred at 600 rpm and was heated on a hot stirring plate set to 160 o C. To this suspension, salicylaldehyde (0.1 ml, 2 vol%) was added to start racemization and samples were taken every 2 days. For sampling procedure, 0.5 ml solution with crystals was taken and was transferred into a centrifuge tube containing 3 ml acetic acid. This tube was centrifuged at 4000 rpm for 1 minute for 4-5 times. This sample was dried in a desiccator to be used in HPLC analysis. Determination of ee by RP-HPLC analysis Low temperature (105 o C) and reflux experiments (125 o C). Samples were prepared so that the optimum sample concentration would become 1-2 mg/ml. Solid samples were dissolved in 1.5 ml eluent and injected into CR(+) column (150 x 4 mm ID), mobile phase: 100 % ph1.5 HClO4, detection λ = 200 nm, flow rate = 0.4 ml/min, temp = 5 o C, retention time: D-Asp 5 min, L-Asp 7 min). High temperature (160 o C) experiment. Amino acid concentrations and ratios were quantified using HPLC following the sample preparation protocol described in Kaufman and Manley 1 and Kaufman 2. Samples were injected into an Agilent HPLC-1100, equipped with a fluorescence detector. Excitation and emission wavelengths were programmed at 230 nm and 445 nm, respectively. A Hypersil BDS C18 reverse-phase column (5 μm; 250 x 4 mm ID) was used for the analysis. 3

4 The derivatization takes place before injection by mixing the sample (2 μl) with the pre-column derivatization reagent (2.2 μl), which comprised 260 mm isobutyryl-l-cysteine (chiral thiol) and 170 mm o-phtaldialdehyde, dissolved in 1.0 M potassium borate buffer solution at ph Eluent A consisted of 23 mm sodium acetate with 1.5 mm sodium azide and 1.3 mm EDTA, adjusted to ph 6.00 with 10 M sodium hydroxide and 10% acetic acid. Eluent B was HPLC-grade methanol and eluent C consisted of HPLC-grade acetonitrile. At the time of injection, a linear gradient was performed at a flow rate of 1.0 ml/min at 25ºC with 95% A and 5% B. These percentages were gradually changed over 31 minutes duration to 76.6% A, 23% B, and 0.4% C. Retention time: L-Asp 14.5 min, D-Asp 15.5 min. Representative HPLC chromatograms; 4

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6 References [1] D.S. Kaufman, W. F. Manley, A new procedure for determining DL amino acid ratios in fossils using reverse phase liquid chromatography, Quaternary Geochronology, 1998, 17, 987. [2] D. S. Kaufman, 2000, Amino acid racemization in ostracodes, in Perspectives in Amino Acid and Protein Geochemistry (eds. G. Goodfriend, M. Collins, M. Fogel, S. Macko, J. Wehmiller), , Oxford University Press, New York. 6