Synthesis of chiral 3-alkyl-3,4-dihydroisocoumarins by dynamic kinetic resolutions catalyzed by a Baeyer-Villiger monooxygenase.

Transcription

1 Synthesis of chiral 3-alkyl-3,4-dihydroisocoumarins by dynamic kinetic resolutions catalyzed by a Baeyer-Villiger monooxygenase. Ana Rioz-Martínez, a Gonzalo de Gonzalo a, Daniel E. Torres Pazmiño, b Marco W. Fraaije b and Vicente Gotor *,a a Departamento de Química rgánica e Inorgánica, Instituto de Biotecnología de Asturias, Universidad de viedo, c/ Julián Clavería 8, 33006, viedo, Spain. HUvgs@fq.uniovi.esU b Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. USupporting Information Contents 1. General S2 2. Experimental procedures and characterization data of compounds S3 3. Determination of the absolute configurations..s15 4. GC and HPLC analyses S16 5. Supporting Information References.S17 6. NMR Spectra S18 S1

2 1. General. Recombinant histidine-tagged phenylacetone mononoxygenase mutant (M446G PAM) and recombinant 4-hydroxyacetophenone monooxygenase (HAPM) were overexpressed and purified as previously described Unit of BVM will oxidize 1.0 µmol of phenylacetone to benzyl acetate per minute at ph 9.0 and room temperature in presence of NADPH. Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides, starting racemic ketones (±)-1-3a and (±)-5-6a were purchased from commercial sources. All other reagents and solvents were of the highest quality grade available. Cell-free extract from overexpressed M446G PAM on E. coli TP10 has been obtained following a similar procedure as previously described. 2 Terrific Broth (TB), containing 50 µg ml -1 ampicillin and 0.02% L-arabinose, was inoculated with 1% of an overnight preculture of recombinant E. coli TP10 overexpressing M446G PAM. The culture was incubated at 200 rpm at 28ºC in an orbital shaker for 24 hours. Cells were harvested by centrifugation (6000 rpm for 10 minutes, 4ºC, A614 rotor), washed and resuspended in 10 ml of 50 mm Tris/HCl ph 9.0. A crude extract was prepared by ultrasonication (70% amplitude, 5 min, 2 sec on/off, 4ºC). Cell debris were removed by centrifugation (10000 rpm for 30 min, 4ºC) resulting in the cell-free extract. The latter was stored at -20ºC before use. Protein concentration of cell free extract (15 mg ml -1 ) was determined by Bradford method using bovine serum albumin (BSA) as standard for the calibration curve. 3 Chemical reactions were monitored by analytical TLC, performed on silica gel 60 F 254 plates and visualized by UV irradiation. Flash chromatography was carried out with silica gel 60 ( mesh). Melting points were taken on samples in open capillary tubes and are uncorrected. IR spectra were recorded on infrared spectrophotometer using KBr pellets. 1 H-NMR, 13 C-NMR and DEPT spectra were recorded with tetramethylsilane (TMS) as the internal standard with a DPX ( 1 H: MHz; 13 C: 75.5 MHz) spectrometer. The chemical shift values (δ) are given in ppm. ptical rotations were measured using a polarimeter and are quoted in units of 10-1 deg cm 2 g -1. APCI + and ESI + using a chromatograph mass detector or EI + with a mass spectrometer were used to record mass spectra (MS). S2

3 2. Experimental procedures and characterization data of compounds Synthesis of racemic (±)-2-isopropyl-1-indanone (±)-4a. General procedure for the cross coupling of isopropylzinc chloride with (±)-2-chloro-1-indanone. Isopropylzinc chloride was prepared by adding a 2.0 M isopropylmagnesium chloride in Et 2 (2.3 ml, 4.60 mmoles) and a solution 1.0 M of ZnCl 2 in Et 2 (4.5 ml, 4.50 mmoles) to 15.8 ml Et 2 at 0 o C. The resulting suspension was stirred vigorously for 3 hours at 0 o C. The RZnCl MgCl 2 complex (22.6 ml, 4.52 mmoles) was added via syringe to a suspension of 2-chloro-1-indanone (500 mg,, 3.01 mmoles) and Cu(acac) 2 (39.0 mg, 0.15 mmoles) in Et 2 (6.1 ml) at room temperature. Stirring was maintained for 72 h and then the reaction mixture was diluted with Et2 (300 ml) and washed with an aqueous saturated solution of NH 4 Cl (1 200 ml). The organic layer was separated, dried over Na 2 S 4, filtered, concentrated under reduced pressure and purified using silica gel chromatography with hexane/ethyl acetate 9:1 to afford (±)-2-isopropyl-1- indanone (±)-4a (143.3 mg, 0.82 mmoles, 29% yield). 4 (±)-2-Isopropyl-1-indanone, (±)-4a. UR fu (8:2 CH 2 Cl 2 -hexane) UMpU: Yellow oil. UIRU (KBr): υ 3015, 2960, 1712, 1608, 1465, 1385 and 1370 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 0.78 (d, 3H, 3 J HH 6.8 Hz), 1.05 (d, 3H, 3 J HH 6.8 Hz), (m, 1H), (m, 1H), 2.94 (dd, 1H, 2 J HH 17.5 Hz, 3 J HH 3.9 Hz), 3.14 (dd, 1H, 2 J HH 17.5 Hz, 3 J HH 8.1Hz), 7.35 (t, 1H, 3 J HH 7.7 Hz), 7.46 (d, 1H, 3 J HH 7.7 Hz ), 7.56 (t, 1H, 3 J HH 7.7 Hz ) and 7.74 (d, 1H, 3 J HH 7.7 Hz). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 17.2 (CH 3 ), 20.8 (CH 3 ), 28.1 (CH), 28.9 (CH 2 ), 53.0 (CH), (CH), (CH), (CH), (CH), (C), (C) and (C=). UMSU (ESI +, m/z): 197 [(M+Na) +, 56%] General method for the preparation of racemic lactones (±)-1-6b and (±)-5c. Lactones (±)-1-6b and (±)-5c were synthesized by chemical Baeyer-Villiger oxidation of ketones (±)-1-6a. In a typical procedure, to a solution of racemic ketone (200 mg, 1.25 mmoles 1a, 1.37 mmoles 2a, 1.25 mmoles 3a, 1.48 mmoles 4a, 0.99 mmoles 5a and 1.37 mmoles 6a) in CH 2 Cl 2 (5.0 ml), m-cpba (312 mg, 1.50 mmoles for 1a, 365 mg, 1.64 mmoles for 2a, 312 mg, 1.50 mmoles for 3a, 368 mg, 1.77 mmoles for 4a, 247 mg, 1.19 mmoles for 5a, 365 mg, 1.64 mmoles for 6a,) was added at 0ºC. Reactions S3

4 were followed by TLC (CH 2 Cl 2 /hexane 8:2). nce finished, the crude mixture was washed with a saturated solution of NaHC 3 (4 10 ml). The organic layer was dried over Na 2 S 4, filtered and the solvent evaporated under reduced pressure. The crude residues were purified by flash chromatography on silica gel with CH 2 Cl 2 /hexane 8:2 to afford lactones (±)-1b (132.0 mg, 0.75 mmoles, 60% yield), (±)-2b (122.0 mg, 0.75 mmoles, 55% yield), (±)-3b (94.6 mg, 0.54 mmoles, 43% yield), (±)-4b (21.8 mg, 0.11 mmoles, 10% yield) and (±)-6b (88.7 mg, 0.53 mmoles, 40% yield). The oxidation of 2- butyl-1-indanone (±)-5a using this methodology led to a mixture of two regioisomeric lactones (±)-5b (41.2 mg, 0.20 mmoles, 19% yield) and (±)-5c (10.8 mg, 0.05 mmoles, 5% yield). (±)-3-Methyl-4,5-dihydrobenzo[b]oxepin-2(3H)-one, (±)-1b. UR fu (8:2 CH 2 Cl 2 -hexane) Mp: Yellow oil. UIRU (KBr): υ 3040, 2976, 1765, 1608, 1487 and 1389 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.16 (d, 3H, 3 J HH 6.3 Hz), (m, 1H), (m, 1H), (m, 2H), (m, 1H) and (m, 4H). U13 C- NMRU (CDCl 3 -d 1, 75.4 MHz): δ 16.0 (CH 3 ), 28.1 (CH 2 ), 35.1 (CH), 36.0 (CH 2 ), (CH), (CH), (CH), (CH), (C), (C) and (C=). UMSU (EI +, m/z): 176 (M +, 100%), 161 (31), 133 (39), 107 (76) and 91 (26). HRMS (ESI + ) calcd for C 11 H 12 Na 2 (M+Na) + : ; found: (±)-3-Methyl-3,4-dihydrocoumarin, (±)-2b. UR fu (8:2 CH 2 Cl 2 -hexane) Mp: Yellow pale oil. UIRU (KBr): υ 3056, 2978, 1765, 1489 and 1389 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.37 (d, 3H, 3 J HH 6.5 Hz), (m, 2H), (m, 1H), (m, 2H) and (m, 2H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 15.3 (CH 3 ), 31.6 (CH 2 ), 34.2 (CH), (CH), (C), (CH), (CH), (CH), (C) and (C=). UMSU (EI +, m/z): 162 (M +, 100%), 134 (64), 119 (69) and 91 (51). HRMS (ESI + ) calcd for C 10 H 10 Na 2 (M+Na) + : ; found: (±)-3-Ethyl-3,4-dihydrocoumarin, (±)-3b. UR fu (8:2 S4 CH 2 Cl 2 -hexane) UMp:U Yellow pale oil. UIRU (KBr): υ 3044, 2968, 1767, 1489 and 1383 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.06 (t, 3H, 3 J HH 7.5 Hz), (m, 1H), (m, 1H), (m, 1H), (m, 1H) 3.05 (dd, 1H, 2 J HH 15.6, 3 J HH 5.7 Hz), (m, 2H) and (m, 2H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 11.2 (CH 3 ), 22.7 (CH 2 ), 28.6 (CH 2 ), 40.4 (CH), (CH), (C), (CH), (2CH), (C) and (C=). UMSU (EI +, m/z): 176 (M +, 83%), 148 (28), 133 (100), 119

5 (49) and 91 (38). HRMS (ESI + ) calcd for C 11 H 13 2 (M+H) + : ; found: (±)-3-Isopropyl-3,4-dihydrocoumarin, (±)-4b. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow oil. UIRU (KBr): υ 3043, 2965, 1768, 1489 and 1459 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.01 (d, 3H, 3 J HH 6.8 Hz), 1.06 (d, 3H, 3 J HH 6.9 Hz), (m, 1H), (m, 1H), (m, 2H), (m, 2H) and (m, 2H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 18.6 (CH 3 ), 20.5 (CH 3 ), 25.4 (CH 2 ), 26.9 (CH), 45.4 (CH), (CH), (C), (CH), (2CH), (C) and (C=). UMSU (EI +, m/z): 190 (M +, 75%), 175 (18), 147 (86), 107 (100) 91 (40) and 77 (28). HRMS (ESI + ) calcd for C 12 H 14 Na 2 (M+Na) + : ; found: (±)-3-Butyl-3,4-dihydrocoumarin, (±)-5b. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow pale solid. Mp: 30-32ºC. UIRU (KBr): υ 3045, 2957, 1769, 1489 and 1380 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 0.92 (t, 3H, 3 J HH 8.1 Hz), (m, 5H), (m, 1H), (m, 2H), 3.03 (dd, 1H, 2 J HH 15.5, 3 J HH 5.7 Hz) and (m, 4H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 13.8 (CH 3 ), 22.5 (CH 2 ), 28.9 (CH 2 ), 29.1 (CH 2 ), 29.3 (CH 2 ), 39.0 (CH), (CH), (C), (CH), (2CH), (C) and (C=). UMSU (EI +, m/z): 204 (M +, 60%), 148 (69), 133 (97), 107 (100) and 77 (28). HRMS (ESI + ) calcd for C 13 H 16 Na 2 (M+Na) + : ; found: (±)-4-Methyl-3,4-dihydrocoumarin, (±)-6b. The physical and spectral properties are in accord with those described. 5 UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow oil. UIRU (KBr): υ 3043, 2968, 1771, 1489 and 1380 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.36 (d, 3H, 3 J HH 7.0 Hz), 2.60 (dd, 1H, 2 J HH 15.8, 3 J HH 8.6 Hz), 2.89 (dd, 1H, 2 J HH 15.8, 3 J HH 5.5 Hz), (m, 1H), (m, 4H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 19.8 (CH 3 ), 29.4 (CH), 36.7 (CH 2 ), (CH), (CH), (CH), (C), (CH), (C) and (C=). UMSU (EI +, m/z): 162 (M +, 100%), 147 (75), 120 (44) and 91 (63). (±)-3-Butyl-3,4-dihydroisocoumarin, (±)-5c. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow pale oil. UIRU (KBr): υ 3071, 2970, 1725, 1466 and 1383 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 0.93 (t, 3H, 3 J HH 8.1 Hz), (m, 6H), (m, 2H), (m, 1H), 7.23(d, 1H, 3 J HH 7.6 Hz), 7.38 (t, 1H, 3 J HH 7.6 Hz), 7.52 (t, 1H, 3 J HH 7.6 Hz) and 8.09 (d, 1H, 3 13 J HH U 7.6 Hz). C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 13.9 S5

6 (CH 3 ), 22.4 (CH 2 ), 26.9 (CH 2 ), 33.2 (CH 2 ), 34.6 (CH 2 ), 78.7 (CH), (C), (CH), (CH), (CH), (CH), (C) and (C=). UMSU (EI +, m/z): 204 (M +, 60%), 148 (69), 133 (97), 107 (100) and 77 (28). HRMS (ESI + ) calcd for C 13 H 16 Na 2 (M+Na) + : ; found: (R)-5c: [α] 25 D = (c 0.74, CHCl 3 ), ee 92% General procedure for the synthesis of the racemic lactones (±)-2-4c and (±)- 6c. For the preparation of racemic lactones (±)-2-4c and (±)-6c, a three-step procedure starting from commercially avaliable 3-phenylpropan-2-ol, 1-phenylbutan-2-ol and 2- phenylpropan-1-ol, respectively, was employed. For the preparation of (±)-4c, it was necessary one step more since (±)-3-methyl-1-phenylbutan-2-ol (±)-4d was not commercial. Thus, this compound was prepared by the reduction of 3-methyl-1- phenylbutan-2-one (1.0 g, 6.16 mmoles) with NaBH 4 (352 mg, 9.25 mmoles) in dry MeH (20.0 ml). The final product (±)-4d was obtained without any further purification (630.0 mg, 3.84 mmoles, 62% yield). 6 (±)-3-methyl-1-phenylbutan-2-ol, (±)-4d. UR fu (8:2 hexane-ch 2 Cl 2 ) Mp: Yellow oil. UIRU (KBr): υ 3427, 2028, 2960, 1495, 1385 and 1176 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.02 (d, 6H, 3 J HH 6.8 Hz), (m, 1H), 2.61 (dd, 1H, 2 J HH 13.6, 3 J HH 9.4 Hz), 2.74 (dd, 1H, 2 J HH 13.6, 3 J HH 6.3 Hz), (m, 1H) and (m, 5H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 17.2 (CH 3 ), 18.8 (CH 3 ) 33.0 (CH), 40.7 (CH 2 ), 77.4 (CH), (CH), (2CH), (2CH) and (C). UMSU (EI +, m/z): 164 (M +, 4%), 121 (16), 103 (22), 92 (100%) and 77 (14). UStep 1: R 1 R 2 MEMCl / N,N-DIEA R 1 R 2 H CH 2 Cl 2 rt (±)-2e R 1 : H R 2 : Me (±)-3e R 1 : H R 2 : Et (±)-4e R 1 : H R 2 : i Pr (±)-6e R 1 : Me R 2 : H To a mixture of the corresponding alcohol (500 mg, 2.23 mmoles for 2e, 2.10 mmoles for 3e, 1.98 mmoles for 4e, 2.23 mmoles for 6e ) and N,N-diisopropylethylamine (580 S6

7 µl, 3.35 mmoles for 2e, 545 µl, 3.15 mmoles for 3e, 514 µl, 2.97 mmoles for 4e, 580 µl, 3.35 mmoles for 6e) in dry CH 2 Cl 2 (6.0 ml), methoxyethoxymethyl chloride ( 381 µl, 3.35 mmoles for 2e, 359 µl, 3.15 mmoles for 3e, 338 µl, 2.97 mmoles for 4e, 381 µl, 3.35 mmoles for 6e) was added dropwise at rt under nitrogen atmosphere. Reactions were followed by TLC using hexane/ethyl acetate 9:1. nce finished, the crude mixture was washed successively with water (2 10 ml), a 1.0 N solution of NaH (2 10 ml) and water (2 10 ml). The organic layer was dried over Na 2 S 4, filtered and evaporated under reduced pressure. The residues were purified by flash chromatography on silica gel with hexane/ethyl acetate 9:1 to afford compounds (±)-2e (730.1 mg, 3.25 mmoles, 88% yield), (±)-3e (587.1 mg, 2.46 mmoles, 74%), (±)-4e (360.3 mg, 1.43 mmoles, 47%) and (±)-6e (696.9 mg, 3.11 mmoles, 84%). (±)-[2-[(2-Methoxyethoxy)methoxy]propyl]benzene, (±)-2e. 7 S7 UR fu (8:2 hexane-ethyl acetate) Mp: White oil. UIRU (KBr): υ 3028, 2969, 1496 and 1377 cm -1. U1 H- NMRU (CD 3 D-d 4, MHz): δ 1.28 (d, 3H, 3 J HH 6.3 Hz), 2.81 (dd, 1H, 2 J HH 13.6, 3 J HH 5.7 Hz), 2.94 (dd, 1H, 2 J HH 13.6, 3 J HH 8.7), 3.45 (s, 3H), (m, 4H), (m, 1H), 4.68 (d, 1H, 2 J HH 7.1 Hz), 4.81 (d, 1H, 2 J HH 7.1 Hz) and (m, 5H). U13 C-NMRU (CD 3 D-d 4, 75.4 MHz): δ 20.8 (CH 3 ), 45.0 (CH 2 ), 59.5 (CH 3 ), 68.1 (CH 2 ), 73.3 (CH 2 ), 75.9 (CH), 95.0 (CH 2 ), (CH), (2CH), (2CH) and (C). UMSU (APCI +, m/z): 225 [(M+H) +, 100%]. (±)-[2-[(2-Methoxyethoxy)methoxy]butyl]benzene, (±)-3e. UR fu (8:2 hexane-ethyl acetate) Mp: White oil. UIRU (KBr): υ 3028, 2963, 1495 and 1372 cm -1. U1 H- NMRU (CD 3 D-d 4, MHz): δ 1.01 (t, 3H, 3 J HH 7.4 Hz), (m, 2H), (m, 2H), 3.43 (s, 3H), (m, 2H), (m, 2H), (m, 1H), 4.62 (d, 1H, 2 J HH = 7.2 Hz), 4.72 (d, 1H, 2 J HH = 7.1 Hz) and (m, 5H). U13 C- NMRU (CD 3 D-d 4, 75.4 MHz): δ 10.2 (CH 3 ), 28.1 (CH 2 ), 41.9 (CH 2 ), 59.3 (CH 3 ), 68.1 (CH 2 ), 73.3 (CH 2 ), 81.1 (CH), 95.3 (CH 2 ), (CH), (2CH) (2CH) and (C). UMSU (ESI +, m/z): 261 [(M+Na) +, 100%]. HRMS (ESI + ) calcd for C 14 H 22 Na 3 (M+Na) + : ; found: (±)-1-[2-[(2-Methoxyethoxy)methoxy]-3-methylbutyl]benzene, (±)-4e. UR fu (8:2 hexane-ethyl acetate) UMp:U White oil. UIRU (KBr): υ 3028, 2963, 1495, 1386, 1366 and 1166 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 0.93 (d, 3H, 3 J HH 5.3 Hz), 0.99 (d, 3H, 3 J HH 5.3 Hz), (m, 1H), (m, 2H), (m, 7H), (m, 1H), 4.49 (d, 1H, 2 J HH 7.2 Hz), 4.65 (d, 1H, 2 J HH 7.2 Hz) and (m,

8 5H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 17.6 (CH 3 ), 18.0 (CH 3 ), 30.5 (CH), 37.2 (CH 2 ), 58.8 (CH 3 ), 66.7 (CH 2 ), 71.6 (CH 2 ), 83.0 (CH), 95.4 (CH 2 ), (CH), (2CH) (2CH) and (C). UMSU (ESI +, m/z): 253 [(M+H) +, 42%]. HRMS (ESI + ) calcd for C 15 H 24 Na 3 (M+Na) + : ; found: (±)-[1-[(2-Methoxyethoxy)methoxy]propan-2-yl]benzene, (±)-6e. UR fu (8:2 hexaneethyl acetate) UMp:U White oil. UIRU (KBr): υ 3029, 2961, 1495 and 1386 cm -1. U1 H-NMRU (CD 3 D-d 4, MHz): δ 1.43 (d, 3H, 3 J HH 6.7 Hz), (m, 1H), 3.51 (m, 3H), (m, 5H), 4.79 (s, 3H), and (m, 5H). U13 C-NMRU (CD 3 D-d 4, 75.4 MHz): δ 19.2 (CH 3 ), 41.6 (CH), 59.3 (CH 3 ), 68.1 (CH 2 ), 73.2 (CH 2 ), 74.9 (CH 2 ), 96.7 (CH 2 ), (CH), (2CH), (2CH) and (1C). UMSU (ESI +, m/z): 247 [(M+Na) +, 100%]. HRMS (ESI + ) calcd for C 13 H 20 Na 3 (M+Na) + : ; found: UStep 2: R 1 R 2 TiCl 4 (1.0 M CH 2 Cl 2) CH 2 Cl 2 rt R 1 R 2 (±)-2e R 1 : H R 2 : Me (±)-3e R 1 : H R 2 : Et (±)-4e R 1 : H R 2 : i Pr (±)-6e R 1 : Me R 2 : H (±)-2f R 1 : H R 2 : Me (±)-3f R 1 : H R 2 : Et (±)-4f R 1 : H R 2 : i Pr (±)-6f R 1 : Me R 2 : H To a solution of the corresponding -protected alcohols (±)-2-4e or (±)-6e (500 mg, 2.23 mmoles 2e, 2.10 mmoles 3e, 1.98 mmoles 4e and 2.23 mmoles 6e) ) in dry CH 2 Cl 2 (6.0 ml) was added dropwise a 1.0 M solution of titanium tetrachloride in CH 2 Cl 2 (1.05 equiv.) at rt. The mixture was stirred under nitrogen atmosphere until consumption of the starting alcohol (followed by TLC using hexane/ethyl acetate 9:1), filtered and the solvent evaporated under reduced pressure. The residues were purified by flash chromatography on silica gel with hexane/ethyl acetate 9:1 to afford 3- methylisochroman (±)-2f (237.8 mg, 1.60 mmoles, 72% yield), 3-ethylisochroman (±)- 3f (303.6 mg, 1.87 mmoles, 92%), 3-isopropylisochroman (±)-4f (174.3 mg, 0.99 mmoles, 50% yield) and 4-methylisochroman (±)-6f (175.0 mg, 1.18 mmoles, 53%). (±)-3-Methylisochroman, (±)-2f. 7 UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow pale oil. UIRU (KBr): υ 3065, 2971, 1585, 1499 and 1384 cm -1. U1 H-NMRU (CD 3 D-d 4, MHz): δ 1.60 (d, 3H, 3 J HH 6.1 Hz), (m, 2H), (m, 1H), 5.07 S8

9 (s, 2H) and (m, 4H). U13 C-NMRU (CD 3 D-d 4, 75.4 MHz): δ 22.1 (CH 3 ), 37.1 (CH 2 ), 69.4 (CH 2 ), 72.9 (CH), (CH), (CH), (CH), (CH), (C) and (C). UMSU (EI +, m/z): 148 (M +, 17%), 104 (100) and 91 (7). (±)-3-Ethylisochroman, (±)-3f. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U White oil. UIRU (KBr): υ 3065, 2963, 1496, 1456 and 1371 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.14 (t, 3H, 3 J HH 7.4 Hz), (m, 2H), 2.79 (d, 2H, 3 J HH 7.0 Hz), (m, 1H), 4.86 (d, 1H, 2 J HH 15.2 Hz), 4.95 (d, 1H, 2 J HH 15.2 Hz), (m, 1H) and (m, 3H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 9.7 (CH 3 ), 28.7 (CH 2 ), 33.5 (CH 2 ), 68.0 (CH 2 ), 76.0 (CH), (CH), (CH) (CH), (CH), (C) and (C). UMSU (EI +, m/z): 162 (M +, 17%), 104 (100) and 91 (10). HRMS (APCI + ) calcd for C 11 H 14 (M) + : ; found: (±)-3-Isopropylisochroman, (±)-4f. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow oil. UIRU (KBr): υ 3024, 2961, 1465, 1386 and 1368 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.05 (d, 6H, 3 J HH 6.8 Hz), (m, 1H), 2.77 (dd, 1H, 2 J HH 14.0 Hz, 3 J HH 9.9 Hz), 2.96 (dd, 1H, 2 J HH 14.0 Hz, 3 J HH 2.8 Hz), (m, 1H), 4.62 (d, 1H, 2 J HH 11.4 Hz), 4.77 (d, 1H, 2 J HH 11.4 Hz ) and (m, 4H). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 17.5 (CH 3 ), 18.7 (CH 3 ) 33.8 (CH), 36.8 (CH 2 ), 44.3 (CH 2 ), 77.2 (CH), (CH), (CH) (CH), (CH), (C) and (C). UMSU (EI +, m/z): 176 (M +, 25%) and 91 (85). HRMS (APCI + ) calcd for C 12 H 16 (M) + : ; found: (±)-4-Methylisochroman, (±)-6f. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow pale oil. UIRU (KBr): υ 3021, 2960, 1584, 1492 and 1386 cm -1. U1 H-NMRU (CD 3 D-d 4, MHz): δ 1.37 (d, 3H, 3 J HH 7.0 Hz), (m, 1H), 3.74 (dd, 1H, 2 J HH 12.0, 3 J HH 5.0 Hz), 4.08 (dd, 1H, 2 J HH 12.0, 3 J HH 4.4 Hz), 5.54 (s, 2H) 7.11 (d, 1H, 3 J HH 6.8 Hz) and (m, 3H). U13 C-NMRU (CD 3 D-d 4, 75.4 MHz): δ 20.1 (CH 3 ), 33.4 (CH), 69.5 (CH 2 ), 72.7 (CH 2 ), (CH), (CH) (CH), (CH) (C), (C). UMSU (EI +, m/z): 148 (M +, 30%), 118 (100), 105 (21), 91 (20) and 77 (11). HRMS (APCI + ) calcd for C 10 H 12 (M) + : ; found: UStep 3: S9

10 Mp:U Mp:U R 1 R 2 + N H NaCl 2 CH 3 CN / H 2 reflux R 1 R 2 (±)-2f R 1 : H R 2 : Me (±)-3f R 1 : H R 2 : Et (±)-4f R 1 : H R 2 : i Pr (±)-6f R 1 : Me R 2 : H (±)-2c R 1 : H R 2 : Me (±)-3c R 1 : H R 2 : Et (±)-4c R 1 : H R 2 : i Pr (±)-6c R 1 : Me R 2 : H Finally, to a mixture of compounds (±)-2-4f or (±)-6f (125 mg, 0.84 mmoles 2f, 0.75 mmoles 3f, 0.71 mmoles 4f and 0.84 mmoles 6f) ) and N-hydroxyphtalimide (0.1 equiv) in 8.0 ml of CH 3 CN/water 3:1, sodium hypochlorite was added carefully. The reaction was refluxed for 18 hours, allowed to rt and poured into a 10% solution of Na 2 S 3. The crude mixture was then extracted with Et 2 (5 10 ml) and the combined organic layers were washed with a saturated solution of NaHC 3 (2 15 ml), dried over Na 2 S 4, filtered and the solvent evaporated under reduced pressure. In most of cases, racemic lactones were obtained without any further purification. (±)-2c was obtained with 68% yield (93.0 mg, 0.57 mmoles), (±)-3c with 75% yield (101.8 mg, 0.58 mmoles) and 82.1 mg (0.51 mmoles) of (±)-6c were achieved (60% yield). nly in the case of the synthesis of (±)-4c, further purification was necessary. The corresponding crude was purified by flash chromatography on silica gel with hexane/ethyl acetate 8:2 to afford (±)-3-isopropylisochroman-1-one (±)-4c (270 mg, 1.42 mmoles, 20% yield). (±)-3-Methyl-3,4-dihydroisocoumarin, (±)-2c. The physical and spectral properties were in accord to those reported. 8 UR fu (8:2 CH 2 Cl 2 -hexane) U S10 White oil. UIRU (KBr): υ 3055, 2986, 1724, 1462 and 1266 cm -1. U1 H-NMRU (CD 3 D-d 4, MHz): δ 1.67 (d, 3H, 3 J HH 6.3 Hz), (m, 2H), (m, 1H), 7.53 (d, 1H, 3 J HH 7.8 Hz), 7.60 (t, 1H, 3 J HH 7.6 Hz), 7.76 (t, 1H, 3 J HH 7.6 Hz) and 8.18 (, 1H, 3 J HH 7.7 Hz). U13 C-NMRU (CD 3 D-d 4, 75.4 MHz): δ 21.3 (CH 3 ), 35.8 (CH 2 ), 77.3 (CH), (C), (CH), (CH), (CH), (CH), (C) and (C=). UMSU (EI +, m/z): 162 (M +, 26%), 118 (100) and 90 (75). (R)-2c: [α] D 25 = (c 1.41, CHCl 3 ), ee 85% (described [α] D 25 = (c 0.55, CH 2 Cl 2 ) for (S)-2c in lit.] 9 (±)-3-Ethyl-3,4-dihydroisocoumarin, (±)-3c. UR fu (8:2 CH 2 Cl 2 -hexane) U White oil. UIRU (KBr): υ 3065, 2963, 1767, 1416 and 1371 cm -1. U1 H-NMRU (CDCl 3 -d 1, MHz): δ 1.09 (t, 3H, 3 J HH 7.3 Hz), (m, 2H), (m, 2H), (m, 1H), 7.25 (d, 1H, 3 J HH 7.6 Hz), 7.38 (t, 1H, 3 J HH 7.6 Hz), 7.51 (t, 1H, 3 J HH 7.6

11 Hz) and 8.07 (d, 1H, 3 J HH 7.8 Hz). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 9.2 (CH 3 ), 27.8 (CH 2 ), 32.5 (CH 2 ), 79.8 (CH), (C), (CH), (CH), (CH), (CH), (C) and (C=). UMSU (EI +, m/z): 176 (M +, 26%), 118 (100), 133 (100) and 90 (61). HRMS (ESI + ) calcd for C 11 H 12 Na 2 (M+Na) + : ; found: (R)-3c: [α] D 25 = (c 0.90, CHCl 3 ), ee 91%. (±)-3-Isopropyl-3,4-dihydroisocoumarin, (±)-4c. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow oil. UIRU (KBr): υ 3033, 2965, 1760, 1580, 1465, 1385 and 1368 cm -1. U1 H- NMRU (CDCl 3 -d 1, MHz): δ 1.09 (d, 3H, 3 J HH 6.9 Hz), 1.11 (d, 3H, 3 J HH 6.9 Hz), (m, 1H), 2.85 (dd, 1H, 2 J HH 16.1, 3 J HH 3 Hz ), 3.01 (dd, 1H, 2 J HH 16.1, 3 J HH 11.8 Hz), (m, 1H), 7.36 (d, 1H, 3 J HH 6.6 Hz), 7.50 (t, 1H, 3 J HH 6.4 Hz), 7.55 (t, 1H, 3 J HH 6.4 Hz) and 8.09 (d, 1H, 3 J HH 7.7 Hz). U13 C-NMRU (CDCl 3 -d 1, 75.4 MHz): δ 18.2 (2CH 3 ), 30.1 (CH 2 ), 32.7 (CH), 83.5 (CH), (C), (CH), (CH), (CH), (CH), (C) and (C=). UMSU (ESI +, m/z): 213 [(M+Na) +, 100%]. HRMS (ESI + ) calcd for C 12 H 14 Na 2 (M+Na) + : ; found: (R)- 4c: [α] D 25 = (c 1.15, CHCl 3 ), ee 97%. (±)-4-Methyl-3,4-dihydroisocoumarin, (±)-6c. UR fu (8:2 CH 2 Cl 2 -hexane) UMp:U Yellow oil. UIRU (KBr): υ 3072, 2968, 1730, 1606, 1465 and 1379 cm -1. U1 H-NMRU (CD 3 D-d 4, MHz): δ 1.53 (d, 3H, 3 J HH 6.0 Hz), (m, 1H), 4.44 (dd, 1H, 2 J HH 9.6, 3 J HH 6.1 Hz), 4.73 (dd, 1H, 2 J HH 9.6, 3 J HH 4.2 Hz), (m, 2H), (m, 1H) and (m, 1H). U13 C-NMRU (CD 3 D-d 4, 75.4 MHz): δ 17.1 (CH 3 ), 33.2 (CH), 74.1 (CH 2 ), (C), (CH), (CH), (CH), (CH), (C) and (C=). UMSU (EI +, m/z): 162 (M +, 56%), 132 (100), 104 (72) and 91 (11). HRMS (ESI + ) calcd for C 10 H 10 Na 2 (M+Na) + : ; found: [α] D 25 = (c 0.75, CHCl 3 ), ee 97% General procedure for the BVM-biocatalyzed oxidation of racemic 2-methyl- 1-tetralone, (±)-1a. (±)-2-Methyl-1-tetralone 1a (14 mm) was dissolved in a 50 mm Tris/HCl buffer at ph 10.0 containing 5% hexane if stated (1.0 ml), glucose-6-phosphate (28 mm), glucose-6- phosphate dehydrogenase (10.0 units), NADPH (0.2 mm) and the corresponding Baeyer-Villiger monooxygenase (1.0 unit). Reactions are shaken at 250 rpm and the temperature established in a rotatory shaker for 72 hours. nce finished, the crude S11

12 reactions were extracted with EtAc (2 500 µl). The organic layer was dried onto Na 2 S 4 and analyzed directly by GC in order to determine the conversion of the oxidations and the enantiomeric excesses of the lactone (R)-1b and the remaining ketone (S)-1a. Table S1. Baeyer-Villiger biocatalyzed oxidation of racemic 2-methyl-1-tetralone, 1a. Entry BVM Cosolvent T (ºC) ee (S)-1a (%) a ee (R)-1b (%) a c (%) a 1 HAPM None HAPM 5% hexane M446G PAM None M446G PAM 5% hexane a Determined by GC M446G PAM-biocatalyzed oxidation of racemic 2-methyl-1-indanone, (±)-2a employing different buffer solutions. (±)-2-Methyl-1-indanone 2a (14 mm) was dissolved in a 50 mm buffer at ph 10.0 containing 5% hexane (1.0 ml), glucose-6-phosphate (28 mm), glucose-6-phosphate dehydrogenase (10.0 units), NADPH (0.2 mm), M446G PAM (1.0 unit). Reactions are shaken at 250 rpm and 40ºC in a rotatory shaker for 48 hours. nce finished, the crude mixtures were extracted with EtAc (2 x 500 µl). The organic layer were separated by centrifugation, dried onto Na 2 S 4 and analyzed directly by GC in order to determine the conversion of the oxidations and the enantiomeric excesses of the lactone (R)-2c and the remaining ketone (S)-2a. Table S2. Baeyer-Villiger biocatalyzed oxidation of (±)-2a in different reaction media. Entry Buffer ee (S)-1a (%) a ee (R)-1b (%) a c (%) a 1 Gly/NaH NaHC 3 /NaH Borax/NaH 3 n.d. 3 a Determined by GC. n.d. not determined Time study of dynamic kinetic resolution of racemic 2-methyl-1-indanone, (±)- 2a, catalyzed by M446G PAM. Ketone (±)-2a (14 mm) was dissolved in a 50mM Tris/HCl ph 10.0 buffer containing 5% hexane (1.0 ml). Then, NADPH (0.2 mm), glucose-6-phosphate (28 mm), glucose-6- S12

13 phosphate dehydrogenase (10.0 units) and M446G PAM (1.0 unit) were added. The mixture was shaken at 250 rpm at 40 ºC for the times selected. Then, the reactions were stopped by extraction with ethyl acetate (2 0.5 ml) and the organic layer was dried onto Na 2 S 4. Conversions and enantiomeric excesses of compounds 2a and 2c were determined by GC analysis. Table S3. Time study of the M446G PAM-catalyzed oxidation of (±)-2-methyl-1-indanone when working in 50 mm Tris/HCl ph 10.0 containing 5% hexane. time (h) ee (S)-2a (%) a ee (R)-2c (%) a c (%) a a Determined by GC M446G PAM-biocatalyzed oxidation of racemic 2-methyl-1-indanone, (±)-2a in presence of anion exchange resins. (±)-2-Methyl-1-indanone 2a (14 mm) was dissolved in a 50 mm Tris/HCl buffer at ph 10.0 containing 5% hexane (1.0 ml), glucose-6-phosphate (28 mm), glucose-6- phosphate dehydrogenase (10.0 units), NADPH (0.2 mm), M446G PAM (1.0 unit) and the corresponding anion exchange resin (5.0 resin equiv., previously washed with distilled water). Reactions are shaken at 250 rpm and 40ºC in a rotatory shaker for 48 hours. nce finished, the crude reactions were extracted with EtAc (2 x 500 µl). The organic layer were separated by centrifugation, dried onto Na 2 S 4 and analyzed directly by GC in order to determine the conversion of the oxidations and the enantiomeric excesses of the lactone (R)-2c and the remaining ketone (S)-2a. S13

14 Table S4. M446G PAM-catalyzed oxidation of (±)-2a in presence of different anion exchange resins. Entry Resin Strength ee (S)-2a (%) a ee (R)-2c (%) a c (%) a 1 Lewatit MP62 Weak Dowex MWA-1 Weak Amberlite IRA 67 Weak Amberlite IRA 440C Strong Amberlyst A26 Strong Dowex 1 Strong Amberlite IRA 910 Strong a Determined by GC Enzymatic Baeyer-Villiger oxidation of racemic 3-methyl-1-indanone, (±)-6a. (±)-3-Methyl-1-indanone, (±)-6a (14 mm) was dissolved in a 50 mm Tris/HCl buffer at ph 10.0 containing 5% hexane or methanol when stated (1.0 ml), glucose-6-phosphate (2 equiv.), glucose-6-phosphate dehydrogenase (5.0 or 10.0 units), NADPH (0.2 mm) and the corresponding BVM (1.0 or 2.0 units). Reactions are shaken at 250 rpm and the temperatures established in a rotatory shaker for 120 h. nce finished, the crude reactions were extracted with EtAc (2 500 µl). The organic layer were separated by centrifugation, dried onto Na 2 S 4 and analyzed directly by GC in order to determine the conversion and the enantiomeric excesses of remaining ketone (S)-6a and lactone (R)- 6b when HAPM was employed or (R)-6c when using M446G PAM. Table S5. Baeyer-Villiger biocatalyzed oxidation of racemic 3-methyl-1-indanone, (±)-6a. Entry BVM Cosolvent T (ºC) ee (S)-6a (%) a ee (R)-6bc (%) a c (%) a E b 1 HAPM None HAPM 5% hexane c HAPM None M446G PAM None M446G PAM 5% hexane d M446G PAM 5% MeH a Determined by GC. b Enantiomeric ratio, E = ln{(1-ee s )/[1+(ee s /ee p )]}/ln{(1+ee s )/[1+(ee s /ee p )]}. c Reaction performed with double amount of enzyme. d In this case, another reaction was performed by using double amount of M446G PAM, but no improvement in the conversion was achieved. S14

15 3. Determination of the absolute configurations. Absolute configurations of ketones 1-3a 10 were determined by comparison of elution order on HPLC with published data, meanwhile the absolute configuration of compound 6a was obtained by comparison of elution order on GC. 11 Absolute configuration of 2- isopropyl-1-indanone 4a and 2-butyl-1-indanone 5a was established by analogy with 2- methyl- and 2-ethyl-1-indanone (2a and 3a, respectively). S15

16 4. GC and HPLC Analyses. The following column was used for the determination of conversions and enantiomeric excesses: Restek RT-BetaDEXse (30 m x 0.25 mm x 0.25 µm, 12 psi N 2 ). For all the analyses, the injector temperature was 225ºC and the FID temperature was 250ºC Table S5. Determination of conversion values and enantiomeric excesses by GC. Compound Program a Retention times (min) 1a 70/5//3/ (R); 37.8 (S) 1b 70/5//3/ (R); 41.8 (S) 2a 70/5/1/ (S); 64.4 (R) 2b 70/5/1/ (R); 80.8 (S) 2c 70/5/1/ (S); 91.8 (R) 3a 70/5/1/ (S); 71.9 (R) 3c 70/5/1/ a 70/5/3/200/ c 70/5/3/200/ a 70/5/3/200/ c 70/5/3/200/ a 70/5/1/ (S); 70.9 (R) 6b 70/5/1/ (S); 87.2 (R) 6c 70/5/1/ (S); 97.5 (R) a Program: initial T (ºC)/ time (min)/ slope (ºC/min)/T (ºC)/ time (min). Enantiomeric excess of lactone 3c was determined by HPLC using a Chiralcel B-H (Daicel, 4.5 x 250 mm) column, n-hexane/iprh, 9:1; 30ºC, flow rate 0.7 ml min -1 ; t R (S) 24.3, t R (R) Enantiomeric excesses of starting ketone 4a and lactone 4c were determined by chiral HPLC analysis HPLC using also a Chiralcel J-H (Daicel, 4.5 x 250 mm) column, n- hexane/iprh, 98:2; 30ºC, flow rate 0.8 ml min -1. For 4a t R (S) 6.8 min, t R (R) 7.5 min, while for 4c t R (S) 13.9 min, t R (R) 14.9 min. Enantiomeric excesses of starting ketone 5a and lactone 5c were determined by chiral HPLC analysis HPLC using a Chiralcel J-H (Daicel, 4.5 x 250 mm) column, n- hexane/iprh, 99:1; 30ºC, flow rate 1.0 ml min -1. For 5a t R (S) 5.7 min, t R (R) 6.4 min, while for 5c t R (R) 14.2 min, t R (S) 16.6 min. S16

17 5. Supporting information references. 1. a) Fraaije, M. W.; Wu, J.; Heuts, D. P. H. M.; van Hellemond, E. W.; Lutje Spelberg, J. H.; Janssen, D. B. Appl. Microbiol. Biotechnol. 2005, 66, ; b) Kamerbeek, N. M.; Moonem, M. J. H.; van der Ven, J. G. M.; van Berkel, W. J. H.; Fraaije, M. W. Janssen, D. B. Eur. J. Biochem. 2001, 268, ; c) Torres Pazmiño, D. E.; Snajdrova, R.; Rial, D. V.; Mihovilovic, M. D.; Fraaije, M. D. Adv. Synth. Catal. 2007, 349, Torres Pazmiño, D. E.; Snajdrova, R.; Baas, B.-J.; Ghobrial, M.; Mihovilovic M. D. Fraaije, M. W. Angew. Chem. Int. Ed., 2008, 47, Bradford, M. M. Anal. Biochem., 1976, 72, Malosh, C. F.; Ready, J. M. J. Am. Chem. Soc. 2004, 126, Matsuda, T.; Shigeno, S.; Murakami, M. J. Am. Chem. Soc. 2007, 129, Guijarro, D.; Mancheno, B.; Yus, M. Tetrahedron, 1992, 48, Compound 2e described in: Mohler, D. L.; Thompson, D. W. Tetrahedron Lett. 1987, 28, Shinkaruk, S.; Bennetau, B.; Babin, P.; Schmitter, J.-M.; Lamothe, V.; Bennetau- Pelissero, C.; Urdaci, M. C. Bioorg. Med. Chem. 2008, 16, Kerti, G.; Kurtán, T.; Illyés, T.-Z.; Kövér, K. E.; Sólyom, S.; Pescitelli, G.; Fujioka, N.; Berova, N.; Antus, S. Eur. J. rg. Chem. 2007, Poisson, T.; Dalla, V.; Marsais, F.; Dupas, G.; udeyer, S.; Levacher, V. Angew. Chem. Int. Ed. 2007, 46, Yun, J.; Buchwald, S. L. J. rg. Chem. 2000, 65, S17

18 5. NMR Spectra 4a S18

19 4a S19

20 4a S20

21 1b S21

22 1b S22

23 1b S23

24 2b S24

25 2b S25

26 2b S26

27 3b S27

28 3b S28

29 3b S29

30 4b S30

31 4b S31

32 4b S32

33 5b S33

34 5b S34

35 5b S35

36 6b S36

37 6b S37

38 6b S38

39 5c S39

40 5c S40

41 5c S41

42 4d H S42

43 4d H S43

44 4d H S44

45 2e S45

46 2e S46

47 2e S47

48 3e S48

49 3e S49

50 3e S50

51 4e S51

52 4e S52

53 4e S53

54 6e S54

55 6e S55

56 6e S56

57 2f S57

58 2f S58

59 2f S59

60 3f S60

61 3f S61

62 3f S62

63 4f S63

64 4f S64

65 4f S65

66 6f S66

67 6f S67

68 6f S68

69 2c S69

70 2c S70

71 2c S71

72 3c S72

73 3c S73

74 3c S74

75 4c S75

76 4c S76

77 4c S77

78 6c S78

79 6c S79

80 6c S80