Supporting Information

Supporting Information First Total Synthesis of Neoantimycin Hikaru Ogawa, Hideo Iio, and Yoshinosuke Usuki* Division of Molecular MaterialsScience, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Received May 25, 2015; CL-150509; E-mail: usuki@sci.osaka-cu.ac.jp) Copyright The Chemical Society of Japan

Table of Contents 1. General Information 2. Experimental procedures for 9, 10, 12, 13, 15, 16, S1, S2, 17, 18, 7, 6, 5, 19, 20, 4, 22, 1 3. Copies of 1 H and 13 C NMR spectra of 12, 13, 15, 16, 18, 7, 6, 5, 19, 20, 4, 22, 1

General Methods 1 H and 13 C NMR spectra were recorded on either JEOL JNM-LA 300 (300 and 75 MHz), JEOL JNM-LA 400 (400 and 100 MHz), Bruker AVANCE 300 (300 and 75 MHz) or Bruker AVANCE III 600 (600 and 150 MHz) instruments. Chemical shifts of 1 H NMR were reported in parts per million (ppm, δ) relative to Me 4 Si (0 ppm) and CDCl 3 (7.26 ppm for 1 H NMR and 77.0 ppm for 13 C NMR) as the internal reference. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and br = broad), coupling constant in Hz, integration. Coupling constants were determined directly from 1 H and 13 C NMR spectra. Mass spectra were obtained on a JEOL JMS-700T (EI, CI, FAB) or a Bruker solarix (ESI) spectrometer. Optical rotations were measured on a PERKIN-ELMER 241 polarimeter with path length of 1 dm or JASCO P-1030 with path length of 0.1 dm at ambient temperature; the concentrations are reported in g/dl. Melting points (mp) were determined on a Yanaco MP-I3 micro melting point apparatus and the thermometer was used without correction. All air- and moisture-sensitive reactions were carried out in flame-dried, Ar-flushed, two-necked flasks sealed with rubber septa, and dry solvents and reagents were introduced with a syringe. THF was fleshly distilled from sodium benzophenone ketyl. CH 2 Cl 2 was fleshly distilled from phosphorus oxide. Flash column chromatography was carried out on KANTO CHEMICAL silica gel 60 N (spherical, neutral, 40-50 µm). Analytical and preparative thin-layer chromatography (TLC) were performed on Merck precoated silica gel (#5715 Kieselgel 60F 254 0.25 mm) and (#5744 Kieselgel 60F 254 0.5 mm), respectively. 1

tert-butyl (S)-2-acetoxy-3-methylbutanoate (9) a NaNO 2 (5.52 g, 80 mmol) was added portionwise to a solution of L-valine (4.68 g, 40 mmol) in acetic acid (60 ml). The solution was cooled intermittently with an ice bath to keep the reaction mixture 15-20 ºC. After the addition was completed, the resulting mixture was left to be stirred at rt for 24 h. Then, acetic acid was removed in vacuo, the residue was dissolved in Et 2 O, and washed with water. The organic layer was back-extracted with sat aq NaHCO 3. The combined aqueous extracts was then acidified with 3 M HCl and extracted with Et 2 O. The combined organic layers was washed with brine, dried over MgSO 4, and concentrated in vacuo. Azeotropic evapolation with cyclohexane afforded α-acetyloxy carbonic acid (6.09 g). α-acetyloxy carbonic acid, DMAP (1.46 g, 12 mmol) and tert-butyl alcohol (5.93 g, 80 mmol) were dissolved in CH 2 Cl 2 (70 ml). Then, a solution of DCC (12.4 g, 60 mmol) in CH 2 Cl 2 (70 ml) was added dropwise at 0 ºC. The reaction mixture was stirred at room temperature for overnight. The reaction mixture was diluted with n-hexane and filtered through a pad of Celite and concentrated in vacuo. The crude residue was purified by flash column chromatography (10% EtOAc in n-hexane) to give tert-butyl ester 9 as colorless oil (6.92 g, 32 mmol, 80%). [α] D = -30.0 (c 1.10, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 4.72 (1H, d, J = 4.5 Hz), 2.25 2.14 (1H, m), 2.13 (3H, s), 1.47 (9H, s), 1.00 (3H, d, J = 5.0 Hz), 0.98 (3H, d, J = 5.0 Hz); LR-ESI-MS m/z 217 [M+H] +. Ref a) M. Kitihata, K. Uehara, PCT Int. Appl. 2013, WO2013118823 A120130815. tert-butyl (S)-2-hydroxy-3-methylbutanoate (10) To a solution of ester 9 (1.32 g, 7.58 mmol) in MeOH (2 ml) was added a solution of K 2 CO 3 (2.90 g, 21.0 mmol) in H2O/MeOH (20 ml, 7:5) dropwise at 0 ºC. After 2 h, the reaction mixture was diluted with H 2 O and extracted with CH2Cl2 (x 5). The combined organic layers was dried over MgSO 4. Concentrated in vacuo afforded 10 as colorless oil (970 mg, 5.57 mmol, 73%). [α] D = +1.2 (c 2.12, MeOH), (lit a, [α] D = +1.6 (c 0.01, MeOH)); [α] D = +7.5 (c 1.13, CHCl 3 ), (R)-isomer : (lit b, [α] D = -4.4 (c 0.31, CHCl 3 )); 2

1 H NMR (300 MHz, CDCl 3 ) δ 3.91 (1H, dd, J = 5.9, 3.3 Hz), 2.75 (1H, d, J = 5.9 Hz), 2.10 1.95 (1H, m), 1.50 (9H, s), 1.02 (3H, d, J = 6.9 Hz), 0.86 (3H, d, J = 6.9 Hz); LR-ESI-MS m/z 175 [M+H] +. Ref a) T. Tuccinardi, A. Martinelli, E. Nuti, P. Carelli, F. Balzano, G. Uccello-Barretta, G. Murphy, A. Rossello, Bioorg. Med. Chem. 2006, 14, 4260. b) N. Kurono, K. Ohtsuga, M. Wakabayashi, T. Kondo, H. Ooka, T. Ohkuma, J. Org. Chem. 2011, 76, 10312. (S)-1-(tert-Butoxy)-3-methyl-1-oxobutan-2-yl N-((benzyloxy)carbonyl)-O-(tert-butyl dimethylsilyl)-l-threoninate (12) To a mixture of alcohol 10 (799 mg, 4.59 mmol), 11 (2.02 g, 5.51 mmol) and DMAP (167 mg, 1.38 mmol) in CH 2 Cl 2 (18 ml) was added a solution of DCC (1.25 g, 6.06 mmol) in CH 2 Cl 2 (18 ml) dropwise at 0 ºC. The reaction mixture was stirred overnight at rt. The reaction mixture was diluted with n-hexane. The resulting mixture was filtered through a pad of Celite and concentrated in vacuo. The crude residue was purified by flash column chromatography (7% EtOAc in n-hexane) to give compound 12 as colorless oil (2.24 g, 4.28 mmol, 93%). [α] D = -23.4 (c 1.1, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.45 7.30 (5H, m), 5.45 (1H, d, J = 9.6 Hz), 5.14 (2H, s), 4.82 (1H, d, J = 4.2Hz), 4.50 (1H, qd, J = 6.2, 2.0 Hz), 4.31 (1H, dd, J = 9.6, 2.0 Hz), 2.25 2.12 (1H, m), 1.46 (9H, s), 1.25 (3H, d, J = 6.2 Hz), 0.98 (3H, d, J = 7.0 Hz), 0.96 (3H, d, J = 7.0 Hz), 0.83 (9H, s), 0.06 (3H, s), 0.03 (3H, s); 13 C NMR (75 MHz, CDCl 3 ) δ 170.53, 167.85, 156.49, 136.24, 128.45, 128.13, 128.09, 81.85, 77.24, 68.40, 66.96, 59.70, 30.33, 27.92, 25.65, 21.24, 18.60, 17.78, 17.04, -4.55, -5.24; HR-ESI-MS calcd. for C 27 H 46 NO 7 Si 524.3038: found 524.3038 [M+H] +. (S)-1-(tert-Butoxy)-3-methyl-1-oxobutan-2-yl((benzyloxy)carbonyl)-L-threoninate (13) 3

12 (2.32 g, 4.43 mmol) was weighted into a Teflon test tube. THF (19 ml) and pyridine (2.5 ml) were added, and then HF Pyr (2.5 ml) was added to the solution. The mixture was stirred for 4 h. The reaction was quenched with sat aq NaHCO 3 and extracted with EtOAc. The combined organic layers was washed with brine, dried over Na 2 SO4 and concentrated in vacuo. Purification by flash column chromatography (25 % EtOAc in n-hexane) afforded alcohol 13 as colorless oil (1.60 g, 3.91 mmol, 88%). [α] D = -22.1 (c 1.8, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 7.27 (5H, m), 5.53 (1H, d, J = 9.6 Hz), 5.13 (2H, s), 4.90 (1H, d, J = 3.4 Hz), 4.64 4.54 (1H, m), 4.50 (1H, dd, J = 9.6, 2.2 Hz), 3.60 (1H, d, J = 4.4 Hz), 2.34 2.22 (1H, m), 1.46 (9H, s), 1.26 (3H, d, J = 6.4 Hz), 1.03 (3H, d, J = 6.9 Hz), 0.98 (3H, d, J = 6.9 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 170.71, 169.50, 156.56, 136.13, 128.20, 127.77, 127.61, 83.13, 77.29, 67.88, 66.68, 59.40, 29.52, 27.65, 18.60, 18.38, 16.43; HR-ESI-MS calcd. for C 21 H 32 NO 7 410.2173: found 410.2173 [M+H] +. (2R,3S)-3-(((Benzyloxy)carbonyl)amino)-4-(((S)-1-(tert-butoxy)-3-methyl-1-oxobuta n-2-yl)oxy)-4-oxobutan-2-yl (2S,3S)-2-((tert-butyldimethylsilyl)oxy)-3-methylpent anoate (15) To a mixture of freshly prepared 14 (660 mg, 1.83 mmol) and DMF (28 µl, 0.37 mmol) in CH2Cl2 (10 ml) at 0 ºC was added oxalyl chloride (0.19 ml, 2.2 mmol) dropwise. The mixture was stirred at 0 ºC for 1.5 h then warmed to rt and stirred for an additional 1 h. Then, the volatiles were removed in vacuo. To this crude carboxylic acid chloride residue was then added a solution of 13 (164 mg, 0.40 mmol) in CH 2 Cl 2 (0.5 ml) and pyridine (3 ml) dropwise at room temperature. After 14 h, the reaction mixture was diluted with THF and filtered through a pad of Celite. The filtrate was concentrated in vacuo. The crude residue was dissolved in EtOAc and then washed with water, sat aq NaHCO3 and brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by flash column chromatography (8 % EtOAc in n-hexane) afforded 15 as colorless oil (208 mg, 0.327 mmol, 82%). [α] D = -19.9 (c 1.2, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 7.28 (5H, m), 5.49 5.37 (2H, m), 5.14 (2H, s), 4.71 (1H, d, J = 4.1 Hz), 4.56 (1H, dd, J 4

= 9.6, 3.4 Hz), 4.09 (1H, d, J = 4.2 Hz), 2.28 2.12 (1H, m), 1.87 1.68 (1H, m), 1.50 1.30 (1H, m), 1.45 (9H, s), 1.37 (3H, d, J = 6.4 Hz), 1.15 1.05 (1H, m), 1.00 0.82 (21H, m), 0.04 (3H, s), 0.01 (3H, s); 13 C NMR (75 MHz, CDCl 3 ) δ 172.15, 169.35, 167.32, 156.41, 136.03, 128.47, 128.16, 81.91, 78.09, 75.98, 70.65, 67.22, 57.73, 39.21, 30.05, 27.91, 25.69, 25.60, 23.79, 18.65, 18.13, 17.00, 16.91, 15.72, 11.63, -4.83, -5.51; HR-ESI-MS calcd. for C 33 H 56 NO 9 Si 638.3719: found 638.3718 [M+H] +. (5S,8R,9S,12S)-9-(((Benzyloxy)carbonyl)amino)-5-((S)-sec-butyl)-12-isopropyl-2,2, 3,3,8-pentamethyl-6,10-dioxo-4,7,11-trioxa-3-silatridecan-13-oic acid (16) To a stirring solution of 15 (227 mg, 0.356 mmol) in CH 2 Cl 2 (4 ml) at 0 ºC were added 2,6-lutidine (0.25 ml, 2.14 mmol) and TESOTf (0.24 ml, 1.07 mmol) successively. The reaction mixture was stirred at rt for 16 h, then diluted with phosphate buffer (ph 7) and acidified by 1 M aq KHSO 4, and extracted with EtOAc. The combined organic layers was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by flash column chromatography (20% to 50% EtOAc in n-hexane) afforded carboxylic acid 16 as brown oil (166 mg, 80%). [α] D = -17.5 (c 1.2, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 7.30 (5H, m), 5.53 5.44 (1H, m), 5.43 (1H, d, J = 9.5 Hz), 5.15 (2H, s), 5.01 4.97 (1H, m), 4.71 (1H, d, J = 4.1 Hz), 4.56 (1H, dd, J = 9.5, 3.0 Hz), 4.02 (1H, d, J = 4.3 Hz), 2.36 2.24 (1H, m), 1.80 1.69 (1H, m), 1.45 1.10 (2H, m), 1.37 (3H, d, J = 6.4 Hz), 1.01 (3H, d, J = 7.7 Hz), 0.99 (3H, d, J = 7.7 Hz), 0.90 0.82 (6H, m), 0.89 (9H, s), 0.04 (3H, s), 0.01 (3H, s); 13 C NMR (75 MHz, CDCl 3 ) δ 172.96, 172.27, 169.24, 156.42, 135.82, 128.44, 128.17, 128.01, 77.41, 76.17, 70.36, 67.31, 57.65, 39.23, 30.00, 25.59, 23.68, 18.68, 18.04, 16.91, 16.85, 15.40, 11.47, -5.00, -5.59; HR-ESI-MS calcd. for C 29 H 48 NO 9 Si 582.3093: found 582.3092 [M+H] +. 5

Ethyl (4R)-4-(benzyloxy)-3-hydroxy-5-phenylpentanoate (S1) To a stirred solution of (R)-2-benzyloxy-3-phenylpropanal a (1.99 g, 8.3 mmol) in CH 2 Cl 2 (36 ml) at -78 º C was added dropwise SnCl 4 (1.0 M solution in CH 2 Cl 2, 9.1 ml, 9.1 mmol). The mixture was stirred at -78 º C for 15min, and then ketene silyl acetal 15 (2.32 g, 12.4 mmol) in CH 2 Cl 2 (16 ml) was added dropwise. After 15 h, the reaction was quenched by sat aq NaHCO 3. The mixture was allowed to warm to rt, then the aq layer was extracted with Et 2 O. The combined organic layers was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by flash column chromatography (10 % EtOAc in n-hexane) afforded alcohol S1 as colorless oil (2.09 g, 6.36 mmol, 77%). [α] D = +19.1 (c 1.3, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.45-7.17 (10H, m), 4.49 (2H, q, J = 11.3 Hz), 4.15 (2H, q, J = 7.1 Hz), 4.15-4.00 (1H, m), 3.63 (1H, td, J = 6.7, 3.2 Hz), 3.06 (1H, dd, J = 13.5, 6.7 Hz), 2.96 (1H, dd, J = 13.5, 6.7 Hz), 2.65 (1H, dd, J = 15.9, 8.6 Hz), 2.52 (1H, dd, J = 15.9, 4.3 Hz), 1.27 (3H, t, J =7.1 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 172.31, 138.33, 137.83, 129.48, 128.38, 128.29, 127.98, 127.72, 126.25, 81.85, 72.58, 68.46, 60.55, 38.28, 36.46, 14.05; HRFABMS m/z Calcd. For C 20 H 25 O 4 329.1753 [M+H] + : found 329.1749. Ref a) C. Dubost, B. Leroy, I. E. Markó, B. Tinant, J. P. Declercq, J. Bryans, Tetrahedron 2004, 60, 7693 7704; H. Lubin, A. Tessier, G. Chaume, J. Pytkowicz, T. Brigaud Org. Lett. 2010, 12, 1496 1499. Ethyl (R)-4-(benzyloxy)-3-oxo-5-phenylpentanoate (S2) S1 (636 mg, 1.95 mmol) and freshly prepared IBX (1.09 g, 3.90 mmol) were dissolved in EtOAc (25 ml). After being refluxed for 2 h, the mixture was cooled to rt. Then, precipitate was removed by filtration through a pad of Celite. The filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (10% EtOAc in n-hexane) to give ketone S2 as pale yellow oil (568 mg, 1.74 mmol, 89%). [α] D = +56.2 (c 1.3, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40-7.00 (10H, m), 4.51-4.34 (2H, ABq, J = 11.5 Hz, ν = 38.3 Hz), 4.15-4.06 (1H, m), 4.12 (2H, q, J = 7.2 Hz), 3.58-3.38 (2H, ABq, J = 16.2 Hz, ν = 43.1 Hz), 3.08 (1H, dd, J =14.1, 4.4 Hz), 6

2.95 (1H, dd, J = 14.1, 7.9 Hz), 1.22 (3H, t, J = 7.2 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 205.01, 167.19, 136.98, 136.66, 129.55, 128.38, 127.90, 127.76, 127.56, 126.74, 85.32, 72.98, 61.26, 45.44, 38.16, 14.03; HRESIMS m/z Calcd. For C 20 H 22 NaO 4 [M+Na] + 349.1410: found 349.1410. Ethyl (R)-4-hydroxy-3-oxo-5-phenylpentanoate (17) To a solution of S2 (568 mg, 1.74 mmol) in ethanol (10 ml) was added 10% Pd/C (51 mg). The resulting suspension was stirred under H 2 for overnight. Then the mixture was filtered through a pad of Celite. The filtrate was concentrated to give alcohol 17 (408 mg, quantitative yield). The crude product was used in the next step without further purification. [α] D = +1.6 (c 1.1, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.37-7.08 (5H, m), 4.46 (1H, dd, J = 8.0, 4.4 Hz), 4.17 (2H, q, J = 7.1 Hz), 3.66-3.38 (2H, m), 3.14 (1H, dd, J = 14.1, 4.4 Hz), 2.87 (1H, dd, J = 14.1, 8.0 Hz), 1.26 (3H, t, J = 7.1 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 204.75, 167.01, 136.23, 129.30, 128.59, 126.96, 77.58, 61.59, 45.55, 39.64, 13.97; HRESIMS m/z Calcd. For C 13 H 16 NaO 4 [M+Na] + 259.0941: found 259.0941. Ethyl (R)-4-(((5S,8R,9S,12S)-9-(((benzyloxy)carbonyl)amino)-5-((S)-sec-butyl)-12 -isopropyl-2,2,3,3,8-pentamethyl-6,10-dioxo-4,7,11-trioxa-3-silatridecan-13-oyl)oxy )-3-oxo-5-phenylpentanoate (18) To a stirred mixture of 16 (282 mg, 0.485 mmol), 17 (114 mg, 0.485 mmol) and DMAP (17.7 mg, 0.146 mmol) in CH 2 Cl 2 (2.5 ml) was added a solution of DCC (120 mg, 0.582 mmol) in CH 2 Cl 2 (2.5 ml) was added dropwise at 0 ºC. After being stirred at rt for overnight, the reaction mixture was diluted with n-hexane and filtered through a pad of Celite. The filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (15% EtOAc in n-hexane) to give compound 18 as 7

colorless oil (309 mg, 0.386 mmol, 80%). [α] D = +1.3 (c 0.83, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.38 7.12 (10H, m), 5.50 5.34 (3H, m), 5.12 (2H, s), 4.76 (1H, d, J = 3.7 Hz), 4.54 (1H, dd, J = 9.7, 3.2 Hz), 4.19 (2H, q, J = 7.2 Hz), 4.05 (1H, d, J = 4.0 Hz), 3.61 3.41 (2H, ABq, J = 16.5 Hz, ν = 48.9 Hz), 3.27 (1H, dd, J = 14.4, 4.0 Hz), 3.01 (1H, dd, J = 14.4, 9.7 Hz), 2.02 1.86 (1H, m), 1.81 1.67 (1H, m), 1.40 1.07 (2H, m), 1.32 (3H, d, J = 6.3 Hz), 1.27 (3H, t, J = 7.2 Hz), 0.89 (9H, s), 0.91 0.79 (9H, m), 0.59 (3H, d, J = 6.8 Hz), 0.00 (6H, s); 13 C NMR (75 MHz, CDCl 3 ) δ 200.09, 172.01, 169.46, 167.92, 166.45, 156.32, 135.86, 135.31, 129.05, 128.54, 128.43, 128.16, 127.98, 127.05, 79.13, 75.79, 70.40, 67.25, 61.36, 57.51, 45.84, 39.23, 36.67, 29.67, 25.64, 23.69, 18.57, 18.08, 16.75, 16.30, 15.65, 14.01, 11.59, -4.91, -5.57; HR-ESI-MS calcd. for C 42 H 61 NNaO 12 822.3855: found 822.3854 [M+Na] +. Ethyl (R)-4-(((S)-2-((N-((benzyloxy)carbonyl)-O-((2S,3S)-2-hydroxy-3-methylpenta noyl)-l-threonyl)oxy)-3-methylbutanoyl)oxy)-3-oxo-5-phenylpentanoate (7) To a solution of 18 (130 mg, 0.162 mmol) in acetonitrile (3 ml) at 0 ºC was added 46.0~48.0% HF aq (0.4 ml). The reaction mixture was stirred at rt for 20 h, and then quenched with sat NaHCO 3, extracted with EtOAc. The combined organic layers was washed with brine, dried over Na 2 SO 4. Concentration in vacuo provided 7 as colorless oil (108 mg, 0.157 mmol, 97%), which was used in the next step without further purification. [α] D = +18.2 (c 1.1, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.41 7.10 (10H, m), 5.53 (1H, qd, J = 6.4, 2.9 Hz), 5.45 5.38 (2H, m), 5.14 (2H, s), 4.80 (1H, d, J = 4.0 Hz), 4.62 (1H, dd, J = 9.6, 2.9 Hz), 4.20 (2H, q, J = 7.1 Hz), 3.97 (1H, d, J = 4.3 Hz), 3.62 3.44 (2H, ABq, J = 16.5 Hz, ν = 39.3 Hz), 3.28 (1H, dd, J = 14.5, 4.0 Hz), 2.98 (1H, dd, J = 14.5, 10.0 Hz), 2.81 (1H, brs), 1.98 1.86 (1H, m), 1.82 1.68 (1H, m), 1.40 1.05 (2H, m), 1.33 (3H, d, J = 6.4 Hz), 1.28 (3H, t, J = 7.1 Hz), 0.95 (3H, d, J = 6.8 Hz), 0.88 (3H, t, J = 7.5 Hz), 0.83 (3H, d, J = 7.0 Hz), 0.59 (3H, d, J = 7.0 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 199.57, 173.65, 169.26, 168.34, 166.54, 156.44, 135.77, 135.21, 129.05, 128.56, 128.49, 128.25, 128.05, 127.12, 79.26, 77.45, 74.28, 71.21, 67.38, 61.48, 57.19, 45.89, 38.49, 36.45, 29.57, 23.76, 18.50, 16.52, 16.30, 15.17, 14.00, 8

11.65; HR-ESI-MS calcd. for C 36 H 47 NNaO 12 708.2990: found 708.2986 [M+Na] +. Benzyl ((2S,5R,6S,9S,12R)-12-benzyl-2-((S)-sec-butyl)-9-isopropyl-5-methyl- 3,7,10,13,15-pentaoxo-1,4,8,11-tetraoxacyclopentadecan-6-yl)carbamate (6) A mixture of anhydrous CuSO 4 (503 mg, 3.15 mmol) and 7 (108 mg, 0.158 mmol) in toluene (160 ml) was refluxed at 120 ºC for 5 h. After cooling to rt, Cu salt was removed by filtration. The filtrate was concentrated and purified by flash column chromatography (20% EtOAc in n-hexane) to give 6 as colorless oil (89 mg, 0.139 mmol 88%). [α] D = +8.6 (c 0.51, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.41 7.10 (10H, m), 5.56 (1H, d, J = 9.5 Hz), 5.49 (1H, dd, J = 8.8, 4.5 Hz), 5.43 (1H, qd, J = 6.3, 2.3 Hz), 5.13 (2H, s), 5.07 (1H, d, J = 6.0 Hz), 4.87 (1H, d, J = 7.3 Hz), 4.61 (1H, dd, J = 9.5, 2.3 Hz), 3.73 3.22 (2H, ABq, J = 14.4 Hz, ν = 140.3 Hz), 3.21 (1H, dd, J = 14.5, 4.5 Hz), 3.01 (1H, dd, J = 14.5, 8.8 Hz), 2.15 1.89 (2H, m), 1.60 1.44 (1H, m), 1.36 (3H, d, J = 6.3 Hz), 1.21 1.05 (1H, m), 0.93 0.75 (12H, m); 13 C NMR (75 MHz, CDCl 3 ) δ 199.91, 169.12, 168.60, 167.21, 165.12, 156.21, 135.77, 134.98, 129.18, 128.53, 128.46, 128.20, 128.03, 127.12, 79.58, 77.48, 76.36, 71.88, 67.30, 56.96, 44.96, 37.07, 35.27, 30.09, 24.05, 17.90, 17.30, 16.00, 14.43, 10.87; HR-ESI-MS calcd. for C 34 H 41 NNaO 11 662.2572: found 662.2566 [M+Na] +. Benzyl ((3S,6S,7R,10S,15R)-15-benzyl-10-((S)-sec-butyl)-3-isopropyl-7,13,13-trime thyl-2,5,9,12,14-pentaoxo-1,4,8,11-tetraoxacyclopentadecan-6-yl)carbamate (5) To a solution of Na 2 CO 3 (158 mg, 1.49 mmol) and 6 (95 mg, 0.149 mmol) in DMSO 9

(4.2 ml) at rt was added MeI (50 µl, 0.59 mmol). The reaction mixture was stirred at rt for 4 h, diluted with water, and extract with hexane/etoac (1:1, x 3). The combined organic layers was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by flash column chromatography (20% EtOAc in n-hexane) afforded 5 as colorless oil (70 mg, 0.105 mmol, 70%). [α] D = +29.2 (c 0.89, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 7.10 (10H, m), 5.77 (1H, dd, J = 6.5 Hz), 5.74 (1H, qd, J = 6.5, 2.3Hz), 5.58 (1H, d, J = 9.6 Hz), 5.23 (1H, d, J = 5.4 Hz), 5.14 (2H, s), 4.64 (1H, dd, J = 9.6, 2.3 Hz), 4.63 (1H, d, J = 7.9 Hz), 3.26 3.13 (2H, m), 2.14 2.00 (1H, m), 1.98 1.82 (1H, m), 1.52 1.37 (1H, m), 1.30 (3H, d, J = 6.5 Hz), 1.22 (3H, s), 1.21 (3H, s), 1.20 1.05 (1H, m), 0.92 (3H, d, J = 6.9 Hz), 0.87 0.81 (6H, m), 0.78 (3H, d, J = 6.9 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 202.33, 171.65, 168.34, 168.10, 167.61, 156.44, 135.83, 135.01, 129.76, 128.48, 128.39, 128.19, 128.00, 127.06, 77.03, 76.88, 76.20, 71.70, 67.28, 57.45, 54.68, 37.43, 35.83, 30.50, 24.48, 21.41, 21.00, 18.12, 16.97, 16.30, 14.16, 10.43; HR-ESI-MS calcd. for C 36 H 45 NNaO 11 690.2885: found 690.2884[M+Na] +. Benzyl ((3S,6S,7R,10S,14R,15R)-15-benzyl-10-((S)-sec-butyl)-14-hydroxy-3-isopro pyl-7,13,13-trimethyl-2,5,9,12-tetraoxo-1,4,8,11-tetraoxacyclopentadecan-6-yl)carb amate (19) To a solution of 5 (19.0 mg, 28.5 µmol) in MeOH (0.9 ml) at 0 ºC was added NaBH 4 (3.2 mg, 85.5 µmol). The reaction mixture was stirred at 0 ºC for 1 h, and then quenched with 1 M HCl, extracted with Et 2 O. The combined organic layers was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by flash column chromatography (20% EtOAc in n-hexane) afforded 19 as a white solid (14.8 mg, 22.1 µmol, 78%). mp 61 62 ºC; [α] D = +13.8 (c 0.81, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 7.15 (10H, m), 5.64 (1H, qd, J = 6.4, 2.5 Hz), 5.54 (1H, d, J = 9.5 Hz), 5.50 (1H, dd, J = 9.6, 5.8 Hz), 5.40 (1H, d, J = 3.6 Hz), 5.14 (2H, s), 4.64 (1H, dd, J = 9.5, 2.5 Hz), 4.55 (1H, d, J = 8.5 Hz), 3.53 (1H, d, J = 12.4 Hz), 3.17 (1H, d, J = 12.4 Hz), 3.14 (1H, dd, J = 13.9, 9.6 Hz), 2.92 (1H, dd, J = 13.9, 5.8 Hz), 1.99 1.86 (1H, m), 1.84 1.73 (1H, 10

m), 1.55 1.40 (1H, m), 1.38 (3H, s), 1.32 (3H, d, J = 6.4 Hz), 1.28 (3H, s), 1.25 1.05 (1H, m), 0.88 0.83 (6H, m), 0.80 (3H, d, J = 6.9 Hz), 0.43 (3H, d, J = 6.9 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 176.68, 168.55, 168.41, 168.15, 156.54, 136.77, 135.87, 129.19, 128.57, 128.31, 128.15, 126.78, 78.99, 76.31, 75.10, 72.23, 71.58, 67.41, 57.35, 45.32, 40.24, 35.85, 30.68, 26.78, 24.71, 21.85, 18.66, 16.11, 15.99, 14.19, 10.43; HR-ESI-MS calcd. for C 36 H 48 NO 11 670.3222: found 670.3220 [M+H] +. (4R,5R)-5-Benzyl-4-hydroxy-3,3-dimethyldihydrofran-2-one (20) From 19: To a solution of crude 19, prepared from 5 (6.6 mg, 10.3 µmol), in MeOH (0.4 ml) was added 1M aq. NaOH (1 ml) at rt. The reaction mixture was stirred overnight, and then quenched with 1 M HCl, extracted with Et 2 O. The combined organic layers was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by preparative TLC (40% EtOAc in n-hexane) afforded 20 (1.3 mg, 5.9 µmol, 53%). From 21: To a solution of 21 (25.0 mg, 50.2 µmol) in EtOH (1 ml) was added 20% Pd(OH) 2 (5 mg). The resulting suspension was stirred under H 2 for overnight. Then the mixture was filtered through a pad of Celite. The filtrate was concentrated to give a crude mixture, which was treated with conc. HCl (1 drop) in CH 2 Cl 2 (1 ml). After being stirred at 0 ºC for 1 h, and then concentrated in vacuo. Purification by flash column chromatography (20% EtOAc in n-hexane) afforded 20 (10.2 mg, 46.2 µmol, 92%). [α] D = +70.5 (c 0.12, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.40 7.20 (5H, m), 4.71 (1H, td, J = 7.2, 3.3 Hz), 3.91 (1H, brd, J = 3.3 Hz), 3.21 (1H, dd, J = 13.9, 7.2 Hz), 3.10 (1H, dd, J = 13.9, 7.5 Hz), 1.82 (1H, brs), 1.28 (3H, s), 1.23 (3H, s); 13 C NMR (75 MHz, CDCl 3 ) δ 180.24, 136.49, 129.27, 128.72, 126.90, 81.23, 77.18, 45.78, 34.62, 22.55, 17.77; HR-ESI-MS calcd. for C 13 H 16 NaO 3 243.0997: found 243.0959 [M+Na] +. 11

(3S,6S,7R,10S,14R,15R)-6-Amino-15-benzyl-10-((S)-sec-butyl)-14-hydroxy-3-isopro pyl-7,13,13-trimethyl-1,4,8,11-tetraoxacyclopentadecane-2,5,9,12-tetraone (4) To a solution of 19 (14.8 mg, 22.1 µmol) in EtOAc (1.5 ml) was added 20% Pd(OH) 2 (5 mg). The resulting suspension was stirred under H 2 for overnight. Then the mixture was filtered through a pad of Celite. The filtrate was concentrated to give 4 as clear oil (11.7 mg, quantitative yield). The product was used in the next step without further purification. [α] D = -3.1 (c 1.1, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 7.35 7.15 (5H, m), 5.62 (1H, qd, J = 6.5, 2.5 Hz), 5.51 (1H, dd, J = 9.3, 6.0 Hz), 5.41 (1H, d, J = 3.8 Hz), 4.59 (1H, d, J = 8.4 Hz), 3.57 (1H, d, J = 2.5 Hz), 3.17 (1H, brs), 3.14 (1H, dd, J = 14.0, 9.3 Hz), 2.93 (1H, dd, J = 14.0, 6.0 Hz), 1.99 1.88 (1H, m), 1.84 1.74 (1H, m), 1.57 1.05 (2H, m), 1.40 (3H, d, J = 6.5 Hz), 1.39 (3H, s), 1.28 (3H, s), 0.89 0.84 (6H, m), 0.80 (3H, d, J = 6.9 Hz), 0.45 (3H, d, J = 6.9 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 176.66, 172.40, 168.60, 168.39, 136.84, 129.22, 128.55, 126.74, 78.94, 75.84, 75.23, 72.56, 71.47, 57.87, 45.28, 40.21, 35.84, 30.72, 26.79, 24.76, 21.85, 18.65, 16.31, 16.21, 14.20, 10.50; HR-ESI-MS calcd. for C 28 H 42 NO 9 536.2854: found 536.2852 [M+H] +. N-((3S,6S,7R,10S,14R,15R)-15-Benzyl-10-((S)-sec-butyl)-14-hydroxy-3-isopropyl-7, 13,13-trimethyl-2,5,9,12-tetraoxo-1,4,8,11-tetraoxacyclopentadecan-6-yl)-2-(benzyl oxy)-3-formamidobenzamide (22) To a solution of 4 (6.9 mg, 12.9 µmol) in DMF (0.7 ml) were added 3 (7.0 mg, 25.8 µmol), HOBt (3.5 mg, 25.8 µmol), EDCI HCl (4.9 mg, 25.8 µmol) and NMM (4.3 µl, 39 µmol) successively. The reaction mixture was stirred at room temperature for 17 h, deluted with H 2 O, and extracted with EtOAc (x 3). The combined organic layers was 12

washed with sat NaHCO 3 and brine, dried over Na 2 SO 4, and concentrated in vacuo. Purification by flash column chromatography (40% EtOAc in n-hexane) afforded 22 as a white solid (8.6 mg, 10.9 µmol, 79%). mp 94 95 ºC; [α] D = -2.1 (c 0.35, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 8.45 (1H, dd, J = 8.1, 1.7 Hz), 8.31 (1H, d, J = 8.8 Hz), 8.14 (1H, d, J = 1.7 Hz), 7.81 (1H, dd, J = 7.9, 1.7 Hz), 7.45 7.15 (12H, m), 5.79 (1H, qd, J = 6.4, 2.6 Hz), 5.54 (1H, dd, J = 9.5, 5.8 Hz), 5.49 (1H, d, J = 3.6 Hz), 5.34 4.85 (2H, ABq, J = 11.2 Hz, ν = 136.5 Hz), 5.27 (1H, dd, J = 8.9, 2.6 Hz), 4.50 (1H, d, J = 8.5 Hz), 3.60 (1H, d, J = 12.4 Hz), 3.21(1H, d, J = 12.4 Hz), 3.17 (1H, dd, J = 13.9, 9.5 Hz), 2.94 (1H, dd, J = 13.9, 5.8 Hz), 1.90 1.77 (2H, m), 1.50 0.90 (2H, m), 1.43 (1H, s), 1.34 (3H, d, J = 6.4 Hz), 1.30 (3H, s), 0.91 0.78 (6H, m), 0.73 (3H, d, J = 6.8 Hz), 0.46 (3H, d, J = 6.8 Hz); 13 C NMR (75 MHz, CDCl 3 ) δ 176.83, 168.64, 168.41, 168.15, 165.49, 158.45, 146.18, 136.77, 135.39, 131.39, 129.42, 129.20, 129.03, 128.62, 126.84, 126.53, 126.02, 125.44, 124.75, 79.05, 78.76, 76.44, 75.17, 72.60, 71.66, 55.65, 45.38, 40.26, 35.91, 30.80, 26.91, 24.65, 21.87, 18.67, 16.36, 16.16, 14.10, 10.53; HR-ESI-MS calcd. for C 43 H 53 N 2 O 12 789.3593: found 789.3592 [M+H] +. Neoantimycin (1) To a solution of 22 (4.3 mg, 5.5 µmol) in EtOAc (1 ml) was added 20% Pd(OH) 2 (1 mg). The resulting suspension was stirred under H 2 for overnight. Then the mixture was filtered through a pad of Celite. The filtrate was concentrated in vacuo. Purification by preparative TLC (50% EtOAc in n-hexane) afforded neoantimycin (1) as a white solid (3.0 mg, 4.3 µmol, 78%). mp 120 121 ºC, natural 2 : mp 121 122 ºC; [α] D = +53.3 (c 0.30, CHCl 3 ), natural 2 : [α] D = +58.3 (c 1, CHCl 3 ); 1 H NMR (600 MHz, CDCl 3 ) δ 12.63 (1H, s), 8.56 (1H, d, J = 8.1 Hz), 8.50 (1H, d, J = 1.7 Hz), 7.92 (1H, s), 7.32 (1H, dd, J = 8.1, 1.5 Hz), 7.29 7.19 (5H, m), 7.14 (1H, d, J = 8.7 Hz), 6.94 (1H, t, J = 8.1 Hz), 5.73 (1H, qd, J = 6.5, 2.6 Hz), 5.52 (1H, dd, J = 9.6, 5.8 Hz), 5.44 (1H, d, J = 3.5 Hz), 5.12 (1H, dd, J = 8.7, 2.6 Hz), 4.66 (1H, d, J = 8.3 Hz), 3.54 (1H, d, J = 12.4 Hz), 3.18 (1H, d, J = 12.4 Hz), 3.16 (1H, dd, J = 14.0, 9.6 Hz), 2.94 (1H, dd, J = 14.0, 5.8 Hz), 1.99 1.93 (1H, m), 1.83 1.78 (1H, m), 1.55 1.48 (1H, m), 1.41 (3H, s), 1.33 (1H, d, J = 13

6.5 Hz), 1.30 (3H, s), 1.25 1.16 (1H, m), 0.89 (3H, d, J = 6.9 Hz), 0.88 (3H, t, J = 7.5 Hz), 0.81 (3H, d, J = 6.9 Hz), 0.45 (3H, d, J = 6.9 Hz); 13 C NMR (150 MHz, CDCl 3 ) δ 176.90, 170.20, 168.27, 168.27, 168.10, 158.93, 150.53, 136.74, 129.20, 128.62, 127.34, 126.85, 124.73, 120.25, 118.96, 112.85, 79.05, 77.00, 76.70, 75.10, 72.38, 71.79, 55.16, 45.39, 40.26, 35.97, 30.68, 26.89, 24.73, 21.87, 18.70, 16.27, 16.09, 14.30, 10.52; HR-ESI-MS calcd. for C 36 H 47 N 2 O 12 699.3124: found 699.3224 [M+H] +. 14