Supplementary Note. A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis

Transcription

1 Supplementary Note A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis Hitomi Nakamura, Erica E. Schultz, and Emily P. Balskus* Corresponding author: balskus@chemistry.harvard.edu S1

2 Table of Contents Synthesis of assay substrates and standards Synthesis of 6-Chlorodecanoic acid (16) Synthesis of 6-Chlorodecanoyl-CoA (22) Synthesis of chlorinated SNAC thioester substrates 18 and 12 (mixture of stereoisomers) Synthesis of stereochemically-defined thioester substrates for enzymatic resorcinol synthesis References Proton and carbon NMR spectra of synthetic compounds Chiral HPLC analysis of (oxiran-2-yl)pentan-1-ol (26) derivatized with 2-napthalenethiol S2

3 Synthesis of assay substrates and standards Synthesis of unfunctionalized resorcinol 14 and the unfunctionalized SNAC thioesters 17 and 19 has been reported previously. 1,2 All reactions were performed under an inert atmosphere. 1. Synthesis of 6-Chlorodecanoic acid (16) Supplementary Scheme 1. Synthesis of 6-chlorodecanoic acid Hydroxydecanoic acid was prepared following previously reported procedures. 3,4 6-Chlorodecanoic acid (16): 6-Hydroxydecanoic acid (415 mg, 2.2 mmol) was dissolved in dichloromethane (10 ml) and cooled to 0 ºC. Mesyl chloride (378 µl, 4.9 mmol) and triethylamine (1.54 ml, 11.0 mmol) were added, and the reaction mixture was stirred at 0 ºC for 30 min. The reaction was quenched with 1 M KOH (10 ml) and extracted with dichloromethane (3 10 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting mesylate was dissolved in THF (10 ml), and anhydrous lithium chloride (466 mg, 11.0 mmol) was added. The reaction mixture was heated to reflux overnight. The reaction was cooled to room temperature, diluted with water (20 ml), acidified with 1 M HCl (5 ml), and extracted with ethyl acetate (3 20 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography, eluting with 20-40% ethyl acetate in hexanes to afford 6-chlorodecanoic acid (16) (353 mg, 1.7 mmol, 78%). TLC: R f = 0.30 (4:1 hexanes/ethyl acetate). 1 H-NMR (500 MHz; CDCl 3 ): δ (s, 1H, COOH), 3.88 (m, 1H, CHCl), 2.37 (t, 2H, J = 7.3 Hz, COCH 2 ), (m, 8H, CH 2 ), (m, 2H, CH 2 ), (m, 2H, CH 2 ), 0.90 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C NMR (125 MHz; CDCl 3 ): δ 180.3, 64.0, 38.5, 38.3, 34.2, 28.9, 26.2, 24.4, 22.5, S3

4 2. Synthesis of 6-Chlorodecanoyl-CoA (22) Supplementary Scheme 2. Synthesis of 6-chlorodecanoyl-CoA Chlorodecanoyl-CoA (22): 6-Chlorodecanoic acid (16) (5.2 mg, mmol), CoA hydrate (21 mg, mmol), PyBOP (26 mg, 0.05 mmol), and potassium carbonate (14 mg, 0.1 mmol) were dissolved in a 1:1 solution of THF/water (1 ml). The reaction was stirred at room temperature for 2 h. The crude product was centrifuged at 16,100 g for 10 min and the supernatant was purified by preparative HPLC. The purification was performed using a Kromasil 100 C18 column (5 µm, 10 x 150 mm) at a flow rate of 3 ml/min. HPLC conditions were: a gradient increasing from 5% to 95% solvent B in solvent A over 30 min, 95% solvent B for 10 min, a gradient decreasing to 5% solvent B over 5 min, 5% solvent B for 10 min (solvent A = water with 0.1% formic acid, solvent B = acetonitrile with 0.1% formic acid). 6- Chlorodecanoyl-CoA (22) eluted at min, and the collected fractions were lyophilized overnight (10 mg, mmol, 42%). HRMS (ESI): calc d for C 31 H 52 ClN 7 O 17 P 3 S [M H], ; found, H-NMR (500 MHz; CD 3 OD): 8.69 (s, 1H, CH), 8.36 (s, 1H, CH), 6.11 (d, 1H, J = 5.2 Hz, NOCH), 4.98 (d, 1H, J = 8.6, 4.1 Hz, CHOH), 4.81 (m, 1H, CHOPO 2 3 ), (m, 1H, OCH), 4.40 (d, 1H, J = 12.0 Hz, O 2 3 POCHH), 4.33 (d, 1H, J = 12.0 Hz, O 2 3 POCHH), 4.09 (dd, 1H, J = 9.6, 4.9 Hz, OCH 2 C(CH 3 ) 2 ), 4.03 (s, 1H, CHOH), 3.90 (m, 1H, CHCl), 3.71 (dd, 1H, J = 9.5, 4.3 Hz, OCH 2 C(CH 3 ) 2 ), (m, 4H, NHCH 2 ), 2.98 (t, 2H, J = 6.8 Hz, SCOCH 2 ), 2.59 (t, 2H, J = 7.3 Hz, NHCOCH 2 ), 2.41 (t, 2H, J = 6.8 Hz, SCH 2 ), (m, 2H, CH 2 ), (m, 4H, CH 2 ), (m, 6H, CH 2 ), 1.07 (s, 3H, C(CH 3 ) 2 ), 0.92 (t, 3H, J = 7.2 Hz, CH 2 CH 3 ), 0.88 (s, 3H, C(CH 3 ) 2 ). S4

5 3. Synthesis of chlorinated SNAC thioester substrates 18 and 12 (mixture of stereoisomers) Supplementary Scheme 3. Synthesis of the SNAC thioester substrates 18 and 12. Weinreb amide (23): 6-Chlorodecanoic acid (16) (550 mg, 2.66 mmol) was dissolved in anhydrous dichloromethane (25 ml) under argon. EDC HCl (765 mg, 3.99 mmol), DMAP (488 mg, 3.99 mmol) and N,O-dimethylhydroxylamine hydrochloride (389 mg, 3.99 mmol) were added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with brine (30 ml), and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (3 30 ml). The combined organic layers were washed with 2 M HCl (30 ml) and brine (30 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain Weinreb amide (23) (589 mg, 2.36 mmol, 89%). The product was used for the next reaction without further purification. 1 H-NMR (500 MHz; CDCl 3 ): δ 3.90 (m, 1H, CHCl), 3.69 (s, 3H, OCH 3 ), 3.18 (s, 3H, NCH 3 ), 2.44 (t, 2H, J = 7.1 Hz, COCH 2 ), (m, 6H, CH 2 ), (m, 2H, CH 2 ), (m, 4H, CH 2 ), 0.91 (t, 3H, J = 7.2 Hz, CH 3 ). S5

6 Methyl 8-chlorododec-2-enoate (24): Weinreb amide (23) (589 mg, 2.36 mmol) was dissolved in anhydrous dichloromethane (8 ml), and the solution was cooled to 78 ºC. To the stirring solution, DIBAL (1 M solution in dichloromethane, 4.72 ml, 4.72 mmol) was added dropwise over 5 min and the reaction mixture was stirred at the same temperature for 1 h. The reaction mixture was quenched by addition of the saturated aqueous solution of potassium sodium tartrate (10 ml). The mixture was slowly warmed to room temperature and stirred for 30 min before diluting with brine (20 ml). The aqueous layer was extracted with ethyl acetate (3 25 ml), and the combined organic layers were washed with brine (30 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was further purified by flash chromatography, eluting with 5-15% ethyl acetate in hexanes to afford 6- chlorodecanal as a volatile colorless oil. The product was carried on to the next reaction without further purification. TLC: R f = 0.83 (silica gel, 1:1 hexanes/ethyl acetate). 1 H-NMR (600 MHz; CDCl 3 ): δ 9.72 (s, 1H, OCH), 3.84 (m, 1H, CHCl), 2.42 (td, 2H, J = 7.2, 1.6 Hz, COCH 2 ), (m, 12H, CH 2 ), 0.87 (t, 3H, J = 7.3 Hz, CH 3 ). Crude 6-chlorodecanal (2.36 mmol) was dissolved in anhydrous benzene (10 ml). Methyl (triphenylphosphoranylidene)acetate (0.946 g, 2.83 mmol) was added to the reaction flask and the reaction mixture was refluxed for 4 h. The reaction was concentrated in vacuo, and the crude product was purified by flash chromatography, eluting with 0-10% ethyl acetate in hexanes to yield methyl 8- chlorododec-2-enoate (24) as a colorless oil and a mixture of isomers in a Z:E = 1:20 ratio (423 mg, 1.71 mmol, 73%). TLC: R f = 0.48 (Z), 0.43 (E) (silica gel, 9:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 13 H 24 ClO + 2 [M+H] +, ; found, H-NMR (500 MHz; CDCl 3 ): δ 6.95 (dt, 1H, J = 15.6, 7.0 Hz, CHCH 2 ), 5.82 (dt, 1H, J = 15.6, 1.6 Hz, COCH), 3.86 (m, 1H, CHCl), 3.71 (s, 3H, OCH 3 ), (m, 2H, CHCH 2 ), (m, 4H, CHClCH 2 ), (m, 8H, CH 2 ), 0.90 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 167.0, 149.1, 121.1, 63.9, 51.3, 38.2(2), 32.0, 28.6, 27.6, 26.0, 22.2, S6

7 8-Chloro-3-methyldodecanoic acid (25): To a suspension of copper iodide (200 mg, 1.05 mmol) in anhydrous diethyl ether (5 ml) at 20 ºC, methylmagnesium bromide (3 M in diethyl ether, 1.73 ml, 5.19 mmol) was added dropwise over 15 min, and the reaction mixture was stirred at the same temperature for 1.5 h. Then, a solution of methyl 8-chlorododec-2-enoate (24) (256 mg, 1.04 mmol) in anhydrous ether (3 ml) was added dropwise over 5 min, and the reaction mixture was stirred at 20 ºC for an additional 1.5 h. The reaction mixture was quenched by slow addition of methanol (1 ml) followed by an aqueous solution of saturated sodium bicarbonate (40 ml). The aqueous layer was extracted with ethyl acetate (3 50 ml), and the combined organic layers were washed with brine (40 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude methyl 8-chloro3-methyldodecanoate was used directly in the next reaction without further purification. TLC: R f = 0.71 (silica gel, 9:1 hexanes/ethyl acetate). The crude mixture of methyl 8-chloro3-methyldodecanoate (1.05 mmol) was dissolved in 1:1:1 solution of THF/water/ethanol (15 ml), and lithium hydroxide (30.0 mg, 1.25 mmol) was added. The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was concentrated in vacuo, acidified with 1 M HCl (20 ml), and the aqueous layer was extracted with dichloromethane (3 25 ml). The combined organic layers were washed with brine (40 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography and the desired product was eluted with 20-50% ethyl acetate in hexanes to afford 8-chloro-3-methyldodecanoic acid (25) as a colorless oil (117 mg, 0.47 mmol, 39%). TLC: R f = 0.5 (silica gel, 1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 13 H 24 ClO 2 [M H], ; found H-NMR (500 MHz; CDCl 3 ): δ (bs, 1H, COOH), 3.88 (m, 1H, CHCl), 2.34 (dd, 1H, J = 15.0, 6.0 Hz, COCH 2 ), 2.15 (dd, 1H, J = 15.0, 8.1 Hz, COCH 2 ), (m, 1H, CHCH 3 ), (m, 4H, CHClCH 2 ), (m, 10H, CH 2 ), 0.96 (d, 3H, J = 6.7 Hz, CHCH 3 ), 0.90 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 179.8, 64.2, 41.5, 38.4, 38.2, 36.4, 30.1, 28.7, 26.6, 26.5, 22.3, 19.7, S7

8 8-Chloro-3-methyldodecanoyl-SNAC thioester (18): 8-Chloro-3-methyldodecanoic acid (25) (33 mg, 0.13 mmol) was dissolved in dichloromethane (2.5 ml), and the solution was cooled to 0 ºC. EDC HCl (51 mg, 0.27 mmol) was added to the reaction flask, and the reaction mixture was stirred at 0 ºC for 20 min. Then, N-acetylcysteamine (16 µl, 0.15 mmol) and DMAP (8 mg, 0.07 mmol) were added, and the reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was quenched with water (10 ml) and extracted with ethyl acetate (3 10 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography eluting at 40-50% ethyl acetate in hexanes to afford 8-chloro-3-methyldodecanoyl-SNAC thioester (18) as a white solid (25 mg, 53%). TLC: R f = 0.24 (silica gel, 1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 17 H 33 ClNO 2 S + [M+H] +, ; found H-NMR (500 MHz; CDCl 3 ): δ 5.90 (bs, 1H, NH), 3.87 (m, 1H, CHCl), 3.42 (q, 2H, J = 6.2 Hz, NHCH 2 ), 3.02 (t, 2H, J = 6.4 Hz, CH 2 CH 2 S), 2.55 (dd, 1H, J = 14.6, 6.0 Hz, SCOCH 2 ), 2.38 (dd, 1H, J = 14.6, 8.1 Hz, SCOCH 2 ), 2.01 (m, 1H, CH), 1.97 (s, 3H, NHCOCH 3 ), (m, 4H, CH 2 CHCl), (m, 2H, CH 2 ), (m, 8H, CH 2 ), 0.90 (m, 6H, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 199.7, 170.2, 64.2, 51.3, 39.8, 38.4, 38.2, 36.4, 31.0, 28.6, 28.5, 26.5, 26.4, 23.2, 22.3, 19.5, Chloro-5-methyl-3-oxotetradecanoyl-SNAC thioester 12: 8-Chloro-3-methyldodecanoic acid (18) (117 mg, 0.47 mmol) was dissolved in anhydrous dichloromethane (5 ml), and to this solution, Meldrum s acid (67.8 mg, 0.47 mmol) and DMAP (172 mg, 1.41 mmol) were added. The reaction mixture was cooled to 0 ºC, and a solution of EDC HCl (178 mg, 0.94 mmol) in dichloromethane (1 ml) was added dropwise over 5 min. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was diluted with dichloromethane (20 ml), and the organic layer was washed with 1 M HCl (3 30 ml) and brine (30 ml). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain crude 5-(8-chloro-3-methyldodecanoyl)-2,2- dimethyl-1,3-dioxane-4,6-dione in quantitative yield. The crude product was used directly in the next reaction without further purification. TLC: R f = 0.25 (silica gel, 1:1 hexanes/ethyl acetate). S8

9 5-(8-Chloro-3-methyldodecanoyl)-2,2-dimethyl-1,3-dioxane-4,6-dione (0.47 mmol) was dissolved in benzene (5 ml) and N-acetylcysteamine (101 µl, 0.93 mmol) was added. The reaction mixture was refluxed overnight. The reaction mixture was then cooled to room temperature, concentrated in vacuo, and purified by flash chromatography, eluting with 50-70% ethyl acetate in hexanes to afford 10-chloro- 5-methyl-3-oxotetradecanoyl-SNAC thioester (12) as a colorless oil and a mixture of tautomers (110 mg, 0.28 mmol, 59%). TLC: R f = 0.15 (silica gel, 1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 19 H 33 ClNO 3 S [M H], ; found H-NMR (500 MHz; CDCl 3 ): Keto form: δ 5.92 (s, 1H, NH), 3.89 (m, 1H, CHCl), (m, 2H, NHCH 2 ), (m, 2H, SCH 2 ), 2.51 (dd, 1H, J = 16.6, 5.7 Hz, COCH 2 ), 2.34 (dd, 1H, J = 16.6, 7.8 Hz, COCH 2 ), 1.98 (s, 3H, NHCOCH 3 ), 1.94 (m, 1H, CH), (m, 4H, CHClCH 2 ) (m, 10H, CH 2 ), 0.91 (m, 6H, CH 3 ). Enol form: δ (s, 1H, CHCOH), 5.92 (s, 1H, NH), 5.45 (s, 1H, CHCOH), 3.89 (m, 1H, CHCl), (m, 2H, NHCH 2 ), (m, 2H, SCH 2 ), 2.18 (dd, 1H, J = 7.8, 2.3 Hz, COCH 2 ), 2.02 (m, 1H, COCH 2 ), 1.98 (s, 3H, NHCOCH 3 ), 1.94 (m, 1H, CH), (m, 4H, CHClCH 2 ) (m, 10H, CH 2 ), 0.91 (m, 6H, CH 3 ). 13 C-NMR (125 MHz; CDCl3): δ 202.2, 194.4, 192.5, 177.0, 170.6, 170.5, , 64.5, 57.9, 51.0 (2), 42.8, 42.7, 40.1, 39.5, 38.7, 38.5, 36.8, 31.3, 29.9, 29.5, 29.1, 28.9, 28.1, 26.8, 26.7, 23.5, 23.4, 22.5, 19.9, 19.8, S9

10 4. Synthesis of stereochemically-defined thioester substrates for enzymatic resorcinol synthesis Supplementary Scheme 4. Synthesis of the stereochemically-defined SNAC thioester substrates for CylI-mediated resorcinol synthesis. a) Synthetic route for (S,R)-12. b) Synthesis of (S,S)-12 was performed following the same route as that of (S,R)-12, except starting with (S) (Oxiran-2-yl)pentan-1-ol (26): To a solution of mcpba (7.5 g, 33.5 mmol) in dichloromethane (110 ml) cooled to 0 ºC, 6-hepten-1-ol (3 ml, 22.3 mmol) was added dropwise over 5 min. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched with cold 1 M NaOH solution (60 ml) and extracted with diethyl ether (3 50 ml). The combined organic layers were washed with 1 M NaOH (100 ml), water (100 ml), and brine (150 ml). Then, the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 5-(oxiran-2- yl)pentan-1-ol (S13) as a clear liquid (2.69 g, 20.8 mmol, 93%). TLC: R f = 0.29 (1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 7 H 15 O + 2 [M+H] +, ; found H-NMR (500 MHz; CDCl 3 ): δ 3.63 (t, 2H, J = 6.6 Hz, CH 2 OH), 2.90 (m, 1H, OCH), 2.74 (dd, 1H, J = 5.0, 4.0 Hz, OCH 2 ), S10

11 2.46 (dd, 1H, J = 5.0, 2.8 Hz, OCH 2 ), (m, 8H, CH 2 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 62.9, 52.4, 47.2, 32.7, 32.5, 25.9, H and 13 C NMR spectra of 5-(oxiran-2-yl)pentan-1-ol (26) matched the previously reported spectra. 5 (R)-5-(Oxiran-2-yl)pentan-1-ol ((R)-26): (R,R)-[Co(salen)] complex (210 mg, 0.35 mmol) was dissolved in toluene (3 ml) and acetic acid (40 µl, 0.69 mmol) was added. The reaction mixture was stirred at room temperature for 30 min and then concentrated in vacuo. To the dried (R,R)- [Co(salen)(OAc)] catalyst, 5-(oxiran-2-yl)pentan-1-ol (26) (1.5 g, 11.5 mmol) was added. The reaction mixture was cooled to 0 ºC and water (125 µl, 6.9 mmol) was added. The reaction mixture was stirred for 14 h. The crude reaction mixture was dry loaded onto silica gel for purification by flash chromatography, eluting with 40-50% ethyl acetate in hexanes to afford (R)-5-(oxiran-2-yl)pentan-1-ol ((R)-26) (615 mg, 4.7 mmol, 41%). TLC: R f = 0.29 (1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 7 H 15 O + 2 [M+H] +, ; found H-NMR (500 MHz; CDCl 3 ): δ 3.63 (t, 2H, J = 6.6 Hz, CH 2 OH), 2.90 (m, 1H, OCH), 2.74 (dd, 1H, J = 5.0, 4.0 Hz, OCH 2 ), 2.46 (dd, 1H, J = 5.0, 2.8 Hz, OCH 2 ), (m, 8H, CH 2 ). 13 C NMR (125 MHz; CDCl 3 ): δ 62.9, 52.4, 47.2, 32.7, 32.5, 25.9, H and 13 C NMR spectra of (R)-26 are identical to those of the racemic mixture. (S)-5-(oxiran-2-yl)pentan-1-ol ((S)-26): (S,S)-[Co(salen)] complex (210 mg, 0.35 mmol) was dissolved in toluene (3 ml) and acetic acid (40 µl, 0.69 mmol) was added. The reaction mixture was stirred at room temperature for 30 min and then concentrated in vacuo. To the dried (S,S)-[Co(salen)(OAc)] catalyst, 5-(oxiran-2-yl)pentan-1-ol (26) (1.5 g, 11.5 mmol) was added. The reaction mixture was cooled to 0 ºC and water (125 µl, 6.9 mmol) was added. The reaction mixture was stirred for 14 h. The crude reaction mixture was dry loaded onto silica gel for purification by flash chromatography, eluting with 40-50% ethyl acetate in hexanes to afford (S)-5-(oxiran-2-yl)pentan-1-ol ((S)-26) (594 mg, 4.6 mmol, 40%). TLC: R f = 0.29 (1:1 hexanes/ethyl acetate). TLC: R f = 0.29 (1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 7 H 15 O + 2 [M+H] +, ; found H and 13 C NMR spectra of (S)-26 were identical to those of the racemic mixture. S11

12 Supplementary Scheme 5. Derivatization of (R)- and (S)-26 for chiral HPLC analysis. Analysis of the enantiomeric excess of (R)- and (S)-26: 5-(oxiran-2-yl)pentan-1-ol (26) (racemic, (R) or (S)) (5 mg, 0.04 mmol) and 2-naphthalenethiol (12 mg, 0.08 mmol) were dissolved in anhydrous methanol (1 ml) at room temperature. Triethylamine (10 µl, 0.07 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The derivatized epoxide was purified by flash chromatography, eluting with 10% ethyl acetate in hexanes, and then re-dissolved in 1:9 iproh/hexanes (1 mg/ml). The ee of the products were determined to be >99% by chiral HPLC analysis (ChiralPak AD- H, 5 µl injection volume, 10% iproh in hexanes, 1 ml/min, 254 nm). (R)-5-(Oxiran-2-yl)pentyl pivalate ((R)-27): To a solution of DMAP (1.4 g, 11.5 mmol) in dichloromethane (3 ml) cooled to 0 ºC, pivaloyl chloride (740 µl, 6.0 mmol) was added. To the reaction mixture, (R)-5-(oxiran-2-yl)pentan-1-ol ((R)-26) (600 mg, 4.6 mmol) in dichloromethane (3 ml) was added dropwise over 5 min. The reaction mixture was warmed to room temperature and stirred for 1.5 h. The reaction mixture was quenched with 1 M HCl (10 ml) and extracted with dichloromethane (3 10 ml). The combined organic layers were washed with 1 M HCl (20 ml) and brine (30 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography, eluting at 10% ethyl acetate in hexanes to afford (R)-5-(oxiran-2-yl)pentyl pivalate ((R)- 27) as a clear oil (886 mg, 4.1 mmol, 90%). TLC: R f = 0.83 (1:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 12 H 23 O + 3 [M+H] +, ; found H-NMR (500 MHz; CDCl 3 ): δ 3.98 (t, 2H, J = 6.6 Hz, COOCH 2 ), 2.82 (m, 1H, OCH), 2.66 (dd, 1H, J = 5.0, 4.0 Hz, OCH 2 ), 2.38 (dd, 1H, J = 5.1, 2.7 Hz, OCH 2 ), 1.57 (m, 2H, CH 2 ), (m, 6H, CH 2 ), 1.11 (s, 9H, CCH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 178.3, 64.0, 51.9, 46.7, 38.5, 32.1, 28.3, 27.0, 25.6, S12

13 (S)-6-Hydroxydecyl pivalate ((S)-28): (R)-5-(oxiran-2-yl)pentyl pivalate ((R)-27) (500 mg, 2.3 mmol) was dissolved in THF (6 ml) and cooled to 30 ºC. Then, Li 2 CuCl 4 solution (0.1 M in THF, 2.33 ml, 0.23 mmol) was added dropwise to the reaction mixture over 5 min and the reaction mixture was stirred at 30 ºC for 15 min. To the reaction mixture, n-propylmagnesium bromide solution (0.5 M in THF, 5.6 ml, 2.8 mmol) was added dropwise over 15 min. The reaction mixture was stirred at 30 ºC for 1 h. The reaction mixture was diluted with diethyl ether (20 ml) and quenched with a saturated aqueous solution of ammonium chloride (20 ml). The quenched reaction mixture was extracted with diethyl ether (3 20 ml). The combined organic layers were washed with water (40 ml) and brine (40 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography, eluting with 10-20% ethyl acetate in hexanes to afford (S)-6-hydroxydecyl pivalate ((S)- 28) as a colorless oil (393 mg, 1.5 mmol, 65%). TLC: R f = 0.23 (4:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 15 H 31 O + 3 [M+H] +, ; found H-NMR (500 MHz; CDCl 3 ): δ 4.02 (t, 2H, J = 6.6 Hz, COOCH 2 ), 3.55 (m, 1H, CHOH), 1.78 (s, 1H, OH), 1.61 (m, 2H, CH 2 ), (m, 12H, CH 2 ), 1.16 (s, 9H, CCH 3 ), 0.87 (t, 3H, J = 7.1 Hz, CH 3 ). 13 C NMR (125 MHz; CDCl 3 ): δ 178.6, 71.7, 64.2, 38.6, 37.2, 37.1, 28.5, 27.8, 27.1, 25.9, 25.2, 22.7, (R)-6-Chlorodecanol ((R)-20): (S)-6-hydroxydecyl pivalate ((S)-28) (353 mg, 1.37 mmol) was dissolved in anhydrous dichloromethane (7 ml). To the reaction mixture, pyridine (330 µl, 4.10 mmol), triphenylphosphine (537 mg, 2.05 mmol), and 2,2,2-trichloroacetamide (333 mg, 2.05 mmol) were added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with water (5 ml) and washed with brine (3 10-mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography, eluting with 10-15% ethyl acetate in hexanes to afford (R)-6-chlorodecyl pivalate. The crude product co-eluted with excess triphenylphosphine reagent and was carried onto the next reaction without further purification. TLC: R f = 0.77 (silica gel, 4:1 hexanes/ethyl acetate). 1 H-NMR (500 MHz; CDCl 3 ): δ 4.04 (t, 2H, J = 6.6 Hz, OCH 2 ), 3.86 (m, 1H, CHCl), (m, 14H, CH 2 ), 1.18 (s, 9 H, C(CH 3 ) 3 ), 0.90 (t, J = 7.2 Hz, 3H, CH 3 ). S13

14 (R)-6-chlorodecyl pivalate (1.37 mmol) was dissolved in methanol (10 ml) and NaOH solution (10 M, 3 ml) was added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched with a saturated aqueous solution of ammonium chloride (10 ml) and extracted with diethyl ether (3 10 ml). The combined organic layers were washed with a saturated aqueous solution of sodium bicarbonate (30 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was further purified by flash chromatography, eluting with 15-20% ethyl acetate in hexanes to afford (R)-6-chlorodecanol ((R)-20) (132 mg, 0.68 mmol, 50%). TLC: R f = 0.30 (silica gel, 4:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 10 H 22 ClO + [M+H] +, ; found H- NMR (500 MHz; CDCl 3 ): δ 3.89 (m, 1H, CHCl), 3.64 (t, 2H, J = 6.6 Hz, COCH 2 ), (m, 14H, CH 2 ), 0.90 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 64.2, 62.8, 38.4, 38.3, 32.6, 28.7, 26.3, 25.3, 22.3, Methyl (R)-8-chlorododec-2-enoate ((R)-24): (R)-6-Chlorodecanol ((R)-20) (173 mg, 0.90 mmol) was dissolved in anhydrous dichloromethane (9 ml) and cooled to 0 ºC. To the reaction mixture, Dess-Martin periodinane (685 mg, 1.61 mmol) was added, and the reaction mixture was slowly warmed to room temperature. After 30 min, the reaction mixture was cooled to 0 ºC and quenched with a saturated aqueous solution of sodium thiosulfate (10 ml). The reaction mixture was diluted with a saturated aqueous solution of sodium bicarbonate (10 ml) and stirred at room temperature for 30 min. The quenched reaction mixture was extracted with dichloromethane (3 10 ml). The combined organic layers were washed with brine (30 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude (R)-6-chlorodecanal was used directly in the next reaction without further purification. 1 H-NMR (500 MHz; CDCl 3 ): δ 9.74 (s, 1H, COH), 3.85 (m, 1H, CHCl), 2.43 (t, 2H, J = 7.2 Hz, COCH 2 ), (m, 12H, CH 2 ), 0.87 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ , 63.82, 43.76, 38.24, 38.20, 28.66, 26.06, 22.26, 21.58, To a solution of methyl(triphenylphosphoranylidine)acetate (360 mg, 1.08 mmol) in dichloromethane (1 ml) at 0 ºC, the solution of (R)-6-chlorodecanal (171 mg, mmol) in dichloromethane (2 ml) was added dropwise over 5 min. The reaction mixture was warmed to room temperature and reacted overnight. The reaction mixture was concentrated in vacuo and the crude product was purified by flash chromatography, eluting with 0-10% ethyl acetate in hexanes to afford methyl (R)-8-chlorododec-2- enoate ((R)-24) as a colorless oil (181 mg, 0.73 mmol, 82%). TLC: R f = 0.73 (silica gel, 4:1 S14

15 hexanes/ethyl acetate). HRMS (ESI): calc d for C 13 H 24 ClO + 2 [M+H] +, ; found, H- NMR (500 MHz; CDCl 3 ): δ 6.94 (dt, 1H, J = 15.6, 7.0 Hz, CHCH 2 ), 5.81 (dt, 1H, J = 15.6, 1.6 Hz, COCH), 3.86 (m, 1H, CHCl), 3.71 (s, 3H, OCH 3 ), 2.21 (q, 2H, J = 6.3 Hz, CHCH 2 ), (m, 4H, CHClCH 2 ), (m, 8H, CH 2 ), 0.89 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 167.0, 149.1, 121.1, 63.9, 51.3, 38.2 (2), 32.0, 28.6, 27.6, 26.0, 22.2, H and 13 C NMR spectra of methyl (R)-were identical to those of the racemic mixture (24). (3S,8R)-8-Chloro-3-methyldodecanoic acid ((S,R)-25): A suspension of copper iodide (2.3 mg, 0.01 mmol) and (S)-Tol-BINAP (12.4 mg, 0.02 mmol) in TBME (3 ml) was stirred at room temperature for 3 h until the solid turned yellow. The reaction mixture was cooled to 20 ºC and MeMgBr (3 M in ether, 1.01 ml, 3.04 mmol) was added dropwise over 5 min to the reaction mixture. After stirring for 15 min, a solution of (R)-methyl 8-chlorododec-2-enoate ((R)-24) (136 mg, 0.61 mmol) in TBME (4 ml) was added dropwise to the reaction mixture over 30 min. The reaction mixture was stirred at 25 ºC for 1 h and quenched with methanol (1 ml). The reaction mixture was diluted with a saturated aqueous solution of ammonium chloride (10 ml) and warmed to room temperature. The reaction mixture was extracted with ether (3 10 ml). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography, eluting with 10% diethyl ether in hexanes to afford methyl (3S,8R)-8-chloro-3-methyldodecanoate, which was used directly in the next reaction without further purification. TLC: R f = 0.64 (silica gel, 4:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 14 H 28 ClO + 2 [M+H] +, ; found, H-NMR (500 MHz; CDCl 3 ): δ 3.87 (m, 1H, CHCl), 3.66 (s, 3H, OCH 3 ), 2.29 (dd, 1H, J = 14.8, 6.1 Hz, COCH 2 ), 2.11 (dd, 1H, J = 14.8, 8.0 Hz, COCH 2 ), 1.95 (m, 1H, CHCH 3 ), (m, 4H, CHClCH 2 ), (m, 2H, CH 2 ), (m, 8H, CH 2 ), 0.92 (m, 6H, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 173.8, 64.3, 51.5, 41.7, 38.6, 38.4, 36.7, 30.4, 28.8, 26.8, 26.6, 22.4, 19.9, Methyl (3S,8R)-8-chloro-3-methyldodecanoate (0.61 mmol) was dissolved in 2:2:1 solution of THF/water/ethanol (10 ml). Lithium hydroxide (26.5 mg, 1.11 mmol) was added to the reaction mixture, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was acidified with 1 M HCl (15 ml) and extracted with dichloromethane (3 15 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography, eluting at 50% ethyl acetate in hexanes to afford (3S,8R)-8-chloro-3-methyldodecanoic S15

16 acid ((S,R)-25) as a colorless oil (86 mg, 0.35 mmol, 57%). TLC: R f = 0.17 (silica gel, 3:1 hexanes/ethyl acetate). HRMS (ESI): calc d for C 13 H 24 ClO 2 [M H], ; found, H-NMR (500 MHz; CDCl 3 ): δ (s, 1H, COOH), 3.88 (m, 1H, CHCl), 2.35 (dd, 1H, J = 15.0, 6.0 Hz, COCH 2 ), 2.16 (dd, 1 H, J = 15.0, 8.1 Hz, COCH 2 ), 1.97 (m, 1H, CHCH 3 ), (m, 4H, CHClCH 2 ), (m, 2H, CH 2 ), (m, 8H, CH 2 ), 0.97 (d, 3H, J = 6.7 Hz, CHCH 3 ), 0.91 (t, 3H, J = 7.2 Hz, CH 3 ). 13 C-NMR (125 MHz; CDCl 3 ): δ 179.8, 64.2, 41.5, 38.4, 38.2, 36.4, 30.1, 28.7, 26.6, 26.5, 22.3, 19.7, H and 13 C NMR spectra of (S,R)-25 were identical to those of the stereoisomeric mixture (25). (5S,10R)-10-Chloro-5-methyl-3-oxotetradecanoyl-SNAC thioester ((S,R)-12): The synthesis was performed following the same procedure as the preparation of the stereoisomeric mixture of 12, except using (3S,8R)-8-chloro-3-methyldodecanoic acid ((S,R)-25) (17 mg, mmol) as the starting material to obtain (5S,10R)-10-chloro-5-methyl-3-oxotetradecanoyl-SNAC thioester ((S,R)-12) (7 mg, mmol, 39%). The 1 H and 13 C NMR spectra of (S,R)-12 matched those of the stereoisomeric mixture (12). (5S,10S)-10-Chloro-5-methyl-3-oxotetradecanoyl-SNAC thioester ((S,S)-12): The synthesis was performed following the same procedure as the preparation of the stereoisomeric mixture of 12, except using (3S,8S)-8-chloro-3-methyldodecanoic acid ((S,S)-25) (99 mg, mmol) as the starting material to obtain (5S,10S)-10-chloro-5-methyl-3-oxotetradecanoyl-SNAC thioester ((S,S)-12) (55 mg, mmol, 53%). The 1 H and 13 C NMR spectra of (S,S)-10 matched those of the stereoisomeric mixture (12). S16

17 References 1. Nakamura, H., Hamer, H. A., Sirasani, G. & Balskus, E. P. Cylindrocyclophane Biosynthesis Involves Functionalization of an Unactivated Carbon Center. J. Am. Chem. Soc. 134, , (2012). 2. Nakamura, H., Wang, J. X. & Balskus, E. P. Assembly line termination in cylindrocyclophane biosynthesis: discovery of an editing type II thioesterase domain in a type I polyketide synthase. Chem. Sci. 6, , (2015). 3. Mino, T., Masuda, S., Nishio, M. & Yamashita, M. Synthesis of lactones by Baeyer-Villiger oxidation with magnesium monoperphthalate hexahydrate. J. Org. Chem. 62, , (1997). 4. Holmquist, H. E., Rothrock, H. S., Theobald, C. W. & Englund, B. E. Some Decomposition and Rearrangement Products of Decahydronaphthalene Hydroperoxide. J. Am. Chem. Soc. 78, , (1956). 5. Trenkle, J. D. & Jamison, T. F. Macrocyclization by Nickel-Catalyzed, Ester-Promoted, Epoxide- Alkyne Reductive Coupling: Total Synthesis of (-)-Gloeosporone. Angew. Chem. Int. Ed. 48, , (2009). S17

18 Proton and carbon NMR spectra of synthetic compounds S18

19 16 16 S19

20 18 18 S20

21 20 20 S21

22 22 23 S22

23 24 24 S23

24 25 25 S24

25 27 27 S25

26 28 28 S26

27 Chiral HPLC analysis of (oxiran-2-yl)pentan-1-ol (26) derivatized with 2-napthalenethiol S27