Supporting Information for: Using a Lipase as a High Throughput Screening Method for Measuring the Enantiomeric. Excess of Allylic Acetates

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1 Supporting Information for: Using a Lipase as a High Throughput Screening Method for Measuring the Enantiomeric Excess of Allylic Acetates M. Burak Onaran and Christopher T. Seto* Department of Chemistry, Brown University, Providence, RI Christopher_Seto@Brown.edu Table of Contents Page General Methods S2 Figure S-1. Replication of analysis of 88 crude samples of compound 2. S3 Scheme S-1. Synthesis of racemic 4. S3 Scheme S-2. Synthesis of (R)-3, (R)-4 and (S)-4. S4 Figure S-2. 1 H NMR of compound 1. S6 Figure S C NMR of compound 1. S7 Figure S-4. HPLC analysis of compound (R)-1. S7 Figure S-5. HPLC analysis of compound (S)-1. S8 Figure S-6. 1 H NMR of compound 2. S8 Figure S C NMR of compound 2. S9 Figure S-8. HPLC analysis of compound (R)-2. S9 Figure S-9. HPLC analysis of compound (S)-2. S10 Figure S-10. HPLC analysis of compound (R)-3. S10 Figure S H NMR of compound 4. S11 Figure S C NMR of compound 4. S11 Figure S-13. HPLC analysis of compound (R)-4. S12 Figure S-14. HPLC analysis of compound (S)-4. S12 S1

2 General Methods. Enzyme assays were performed in 96-well plates using an UV plate reader. Data analyses were performed using the commercial graphing package GraFit (version 3.0) from Erithacus Software. Chiral HPLC analyses were performed using a Chiralcel OD-H column (0.46 cm i.d. x 25 cm) with UV detection at 254 nm. The 1 H and 13 C spectra were recorded at 400 MHz and 100 MHz, respectively. All NMR spectra were recorded in CDCl 3 using TMS as the internal standard. Racemic alcohol 3 (inden-1-ol) was prepared according to the procedure by Hoppe and coworkers. 1 All solvents used were of reagent grade. Solvent removal was performed by rotary evaporation at water aspirator pressure. All reactions were performed at room temperature under an atmosphere of N 2 unless otherwise noted. S2

3 1 0.8 Relative rate % Enantiomer excess Figure S-1. Lipase-based screen of 88 samples of compound 2 that range in % ee from 100% (S) to 100% (R). These samples are crude products from the acylation reaction shown in Figure 4, and were analyzed without isolation or purification. This is a replication the assays that were performed to generate the data in Figure 5, and are provided to demonstrate the reproducability of the assay. Scheme S-1. Synthesis of racemic 4. OH Ac 2 O, DMAP OAc THF, NEt % 4 Racemic 4. Compound 4 was prepared from inden-1-ol 3 following the general procedure provided in the experimental section. 1 H NMR (CDCl 3, 400 MHz) δ 2.12 (s, 3H), 6.22 (s, 1H), 6.35 (dd, J = 5.7, 1.9 Hz, 1H), 6.79 (d, J = 6.2 Hz, 1H), (m, 3H), 7.43 (d, J = 7.3 Hz, 1H); 13 C NMR (CDCl 3, 100 MHz) δ 171.8, 143.4, 142.4, 135.2, 133.8, 129.4, 126.7, 124.7, 122.0, 77.9, 21.5; HRMS (ESI): Calcd for C 11 H 10 O 2 : , found: S3

4 Scheme S-2. Synthesis of (R)-3, (R)-4 and (S)-4. OH Ac 2 O, DMAP OAc THF, NEt 3 OAc Immobilized Lipase from Candida antarctica 45% conversion (R)-3 99% ee + OAc 100% (R)-4 99% ee OAc 4 Additional 10% (S)-4 conversion with the lipase (S)-4 82% ee 99% ee Alcohol (R)-3. (R)-3 was synthesized from racemic 4 by enantioselective hydrolysis using an immobilized lipase from Candida antarctica. To the racemic acetate 4 (910 mg, 5.23 mmol) was added acetonitrile (50 ml), water (50 ml) and the immobilized lipase (400 mg) and the reaction mixture was gently shaken at room temperature for 24 h under ambient atmosphere. After the reaction had reached 45% completion as determined by the 1 H NMR spectrum of an aliquot of the reaction mixture, the immobilized enzyme was removed by filtration and the acetonitrile was removed by rotary evaporation. Water (50 ml) was added, the product was extracted into ethyl acetate and the organic phase was dried over MgSO 4. Purification by flash column chromatography (EtOAc:hexanes 1:4) yielded (R)-3 (264 mg, 2.0 mmol, 85% of the theoretical yield) in 99% ee as judged by HPLC analysis (Chiralcel OD-H column, 90:10 hexane:2-propanol, 1 ml/min, 254 nm UV detection), t R = 6.8 min for (R)-3 and t R = 6.2 min for (S)-3. 1 H NMR (CDCl 3, 400 MHz) δ 1.58 (s, 1H), 5.19 (s, 1H), 6.41 (dd, J = 5.6, 2.0 Hz, 1H), 6.74 (d, J = 5.7 Hz, 1H), (m, 3H), 7.52 (d, J = 7.0 Hz, 1H); 13 C NMR (CDCl 3, 100 MHz) δ 146.0, 142.8, 138.2, 132.9, 128.8, 126.5, 123.9, 121.8, The chromatographic purification also yielded (S)-4 in 82% ee. S4

5 Acetate (R)-4. (R)-4 was prepared from (R)-3 following the general procedure provided in the experimental section. 99% ee as judged by HPLC analysis (Chiralcel OD-H column, 90:10 hexane:2-propanol, 1 ml/min, 254 nm UV detection), t R = 8.6 min for (R)-4 and t R = 10.7 min for (S)-4. Acetate (S)-4. The (S)-4 (82% ee) that was obtained during the synthesis of alcohol (R)-3 was subjected to an additional 10% conversion with the immobilized lipase from Candida antarctica. After the desired conversion was attained as determined by the 1 H NMR spectrum of an aliquot of the reaction mixture, the immobilized enzyme was filtered and the acetonitrile was removed by rotary evaporation. Water (50 ml) was added, the product was extracted into ethyl acetate and the organic phase was dried over MgSO 4. Purification by flash column chromatography (EtOAc:hexanes 1:4) yielded acetate (S)-4 (414 mg, 2.38 mmol, 91% of theoretical). 99% ee as judged by HPLC analysis (Chiralcel OD-H column, 99.7:0.3 hexane:2- propanol, 1 ml/min, 254 nm UV detection), t R = 8.6 min for (R)-4 and t R = 10.7 min for (S)-4. S5

6 Figure S-2. 1 H NMR of compound 1. S6

7 Figure S C NMR of compound 1. Figure S-4. HPLC analysis of compound (R)-1 (t R = 8.3 min for (R) and t R = 12.5 min for (S)). S7

8 Figure S-5. HPLC analysis of compound (S)-1 (t R =8.3 min for (R) and t R = 12.5 min for (S)). Figure S-6. 1 H NMR of compound 2. S8

9 Figure S C NMR of compound 2. Figure S-8. HPLC analysis of compound (R)-2 (t R = 22.7 min for (R) and t R = 26.5 min for (S)). S9

10 Figure S-9. HPLC analysis of compound (S)-2 (t R = 22.7 min for (R) and t R = 26.5 min for (S)). Figure S-10. HPLC analysis of compound (R)-3 (t R = 6.8 min for (R) and t R = 6.2 min for (S)). S10

11 Figure S H NMR of compound 4. Figure S C NMR of compound 4. S11

12 Figure S-13. HPLC analysis of compound (R)-4 (t R = 8.6 min for (R) and t R = 10.7 min for (S)). Figure S-14. HPLC analysis of compound (S)-4 (t R = 8.6 min for (R) and t R = 10.7 min for (S)). S12

13 References 1 Hoppe, I.; Marsch, M.; Harms, K.; Boche, G.; Hoppe, D. Angew. Chem. Int. Ed. 1995, 34, WARNING: This procedure for the synthesis of compound 3 (inden-1-ol) involves the preparation of indene-1-peroxide as an intermediate. We have performed this procedure a number of times, using appropriate safety precautions including the use of a blast shield, without mishap. However, on one occasion while the reaction mixture was being quenched with a solution of NaI, a serious explosion occurred that resulted in a small fire and injury from flying glass. We have not determined the exact cause of this explosion, but recommend that all appropriate safety precautions be used when carrying out this procedure. S13