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1 DOI: /NCHEM.1746 Component-based syntheses of trioxacarcin A, DC-45-A1 and structural analogues Thomas Magauer, Daniel J. Smaltz, and Andrew G. Myers* Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts Supplementary Information Table of Contents General Experimental Procedures Materials Instrumentation Large-Scale Synthesis of Synthetic Precursor 1 Synthesis of DC-45-A1 and Trioxacarcinose B Monoglycoside 9 Synthesis of Trioxacarcin A Preparation of Fully Synthetic Trioxacarcin Analogs Additional Experiments Measurement of GI50 values 1H and 13 C NMR Spectra Comparison of Natural and Synthetic DC-45-A1 Comparison of Natural and Synthetic Trioxacarcin A S1 S2 S2 S3 S9 S17 S21 S46 S48 S49 S82 S87 General Experimental Procedures All reactions were performed in round-bottom flasks fitted with rubber septa under a positive pressure of argon, unless otherwise noted. Air- and moisture-sensitive liquids were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation (house vacuum, ca Torr) at ambient temperature, unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed using glass plates precoated with silica gel (0.25 mm, 60 Å pore-size, mesh, Merck KGA) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet light, then were stained with either an aqueous sulfuric acid solution of ceric ammonium molybdate (CAM), an acidic solution of p-anisaldehyde in ethanol (Anis), or an aqueous sodium hydroxide potassium carbonate solution of potassium permanganate (KMnO 4 ) then briefly heated with a flameless heat gun. Flash-column chromatography was performed as described by Still et al., 1 employing silica gel (60 Å, μm, 1 Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, NATURE CHEMISTRY 1

2 standard grade, Dynamic Adsorbents, Inc. and 60 Å, μm, standard grade, Agela Technologies). Materials Commercial solvents and reagents were used as received with the following exceptions: Tetrahydrofuran, dichloromethane, benzene, and ether were purified by the method of Pangborn et al. 2 Instrumentation Proton magnetic resonance ( 1 H NMR) spectra were recorded on Varian INOVA 500 (500 MHz) or 600 (600 MHz) NMR spectrometers at 23 C. Proton chemical shifts are expressed in parts per million (ppm, scale) and are referenced to residual protium in the NMR solvent (CHCl 3, 7.26). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and/or multiple resonances, br = broad, app = apparent), integration, and coupling constant (J) in Hertz. Carbon nuclear magnetic resonance spectra ( 13 C NMR) were recorded on Varian INOVA 500 (125 MHz) NMR spectrometers at 23 C. Carbon chemical shifts are expressed in parts per million (ppm, scale) and are referenced to the carbon resonances of the NMR solvent (CDCl 3, 77.0). Infrared (IR) spectra were obtained using a Shimadzu 8400S FT-IR spectrometer. Data are represented as follows: frequency of absorption (cm 1 ), intensity of absorption (s = strong, m = medium, w = weak, br = broad). Optical rotations were measured on a Jasco DIP-0181 digital polarimeter with a sodium lamp and are reported as follows: [ ] T[ C] λ (c = g/100 ml, solvent). Circular dichroism spectra were obtained using a Jasco J-710 spectropolarimeter. High-resolution mass spectra were obtained at the Harvard University Mass Spectrometry Facility. High performance liquid chromatography purifications were performed using an Agilent Technologies 1200 Series preparative HPLC system. 2 Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, NATURE CHEMISTRY 2

3 (For clarity, intermediates that have not been assigned numbers in the text are numbered sequentially in the Supporting Information beginning with 40.) Large-Scale Synthesis of Synthetic Precursor 1 (E)-7-Propenyl-3,4-dihydroanthracen-1-one (19). 3 Lithium tert-butoxide (1.0 M solution in tetrahydrofuran, 47.5 ml, 47.5 mmol, 3.0 equiv) was added to a solution of (E)-4-(methoxymethoxy)-6-methyl-3-oxo-5-(prop-1-enyl)-1,3- dihydroisobenzofuran-1-carbonitrile 17 (4.33 g, 15.8 mmol, 1 equiv) in tetrahydrofuran (86 ml) at 78 C. After 5 min, a solution of (4S,6S)-6-(tert-butyldimethylsilyloxy)-4-(4- methoxybenzyloxy)cyclohex-2-enone 18 (5.74 g, mmol, 1.0 equiv) in tetrahydrofuran (86 ml) was added by cannula. The reaction flask was allowed to warm to 20 C over 3 h, then dimethylsulfate (13.6 ml, 142 mmol, 9.0 equiv) was added. The reaction flask was allowed to warm to 23 C over 2 h. After an additional 2 h, the reaction mixture was partitioned between saturated aqueous ammonium chloride solution (500 ml) and ethyl acetate (1 L). The layers were separated. The organic layer was washed sequentially with water (500 ml) then saturated aqueous sodium chloride solution (500 ml) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (hexanes initially, grading to 5% ethyl acetate hexanes) to provide the product, (E)- 7-propenyl-3,4-dihydroanthracen-1-one (19), as an orange foam (6.87 g, 70%). 3 TLC: (17% ethyl acetate hexanes) R f = 0.36 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 7.63 (s, 1H), 7.28 (d, 2H, J = 8.5 Hz), 6.87 (d, 2H, J = 8.2 Hz), 6.53 (d, 1H, J = 16.3 Hz), 6.11 (dq, 1H, J = 16.2, 6.4 Hz), 5.2 (m, 1H), 5.09 (d, 1H, J = 6.2 Hz), 5.04 (d, 1H, J = 6.2 Hz), 4.99 (dd, 1H, J = 12.6, 5.3 Hz), 5.78 (d, 1H, J = 11.0 Hz), 4.58 (d, 1H, J = 11.0 Hz), 3.85 (s, 3H), 3.79 (s, 3H), 3.59 (s, 3H), 2.72 (ddd, 1H, J = 13.3, 4.8, 3.2 Hz), 2.50 (s, 3H), 2.18 (m, 1H), 1.97 (d, 3H, J = 6.4 Hz), 0.98 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 203.7, 161.4, 159.2, 153.8, 144.8, 141.2, 3 Švenda, J.; Hill, N.; Myers, A. G. P. Natl. Acad. Sci. USA 2011, 108, NATURE CHEMISTRY 3

4 133.1, 132.4, 131.7, 130.2, 129.5, 125.4, 125.1, 119.1, 188.8, 113.8, 108.4, 101.3, 70.8, 69.4, 68.8, 62.8, 57.9, 55.3, 36.6, 25.9, 22.2, 19.3, 18.6, 4.4, 5.3; FTIR (neat), cm 1 : 2951 (w), 2930 (w), 1611 (m), 1514 (w), 1443 (w), 1381 (w), 1362 (m), 1248 (m), 1155 (m), 1124 (m), 1040 (s), 1003 (m), 928 (m), 872 (m), 835 (m), 779 (m); HRMS (ESI): Calcd for (C 35 H 46 O 8 Si+Na) , found Formyl-3,4-dihydroanthracen-1-one (40) 3 2,6-Lutidine (2.57 ml, 22.1 mmol, 2.0 equiv) was added to an ice-cooled solution of (E)-7- propenyl-3,4-dihydroanthracen-1-one 19 (6.87 g, 11.0 mmol, 1 equiv), potassium osmate dihydrate (203 mg, mmol, 0.05 equiv), and sodium periodate (9.44 g, 44.1 mmol, 4.0 equiv) in a mixture of tetrahydrofuran (160 ml) and water (80 ml). After 5 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 C. After 2.5 h, the reaction mixture was partitioned between water (600 ml), ethyl acetate (1.2 L), and hexanes (600 ml). The layers were separated. The organic layer was washed with water (600 ml) then saturated aqueous sodium chloride solution (600 ml) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (10% ethyl acetate hexanes initially, grading to 15% ethyl acetate hexanes) to provide the product, 7-formyl- 3,4-dihydroanthracen-1-one (40), as an orange foam (4.52 g, 67%). 3 TLC: (10% ethyl acetate hexanes) R f = 0.18 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 9.64 (s, 1H), 7.64 (s, 3H), 7.28 (d, 2H, J = 8.5 Hz), 6.87 (d, 2H, J = 8.5 Hz), (m, 3H), 5.00 (dd, 1H, J = 12.2, 5.1 Hz), 4.70 (d, 1H, J = 11.0 Hz), 4.60 (d, 1H, J = 11.0 Hz), 3.86 (s, 3H), 3.80 (s, 3H), 3.61 (s, 3H), 2.74 (s, 3H), 2.73 (m, 1H), 2.19 (m, 1H), 0.98 (s, 9H), 0.26 (s, 3H), 0.18 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 204.2, 193.3, 163.2, 161.6, 159.4, 144.7, 141.8, 137.0, 129.9, 129.5, 127.6, 120.7, 118.1, 113.9, 109.2, 102.8, 71.1, 69.3, 68.6, 62.9, 58.4, 55.3, 36.3, 25.8, 22.3, 18.5, 4.5, 5.3; FTIR (neat), cm 1 : 2953 (w), 2930 (w), 2857 (w), 1686 (m), 1611 (s), 1514 (m), 1385 (m), 1364 (m), 1246 (s), 1153 (s), 1042 (s), 1011 (m), 930 (m), 872 (m), 837 (m), 779 (m); HRMS (ESI): Calcd for (C 33 H 42 O 9 Si+Na) , found NATURE CHEMISTRY 4

5 8,9-Dihydroxy-7-formyl-3,4-dihydroanthracen-1-one (41). 3 A solution of B-bromocatecholborane (2.54 g, 12.8 mmol, 2.0 equiv) in dichloromethane (94 ml) was added to a solution of 7-formyl-3,4-dihydroanthracen-1-one 40 (3.90 g, 6.39 mmol, 1 equiv) in dichloromethane (94 ml) at 78 C. After 50 min, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (300 ml) and dichloromethane (500 ml). The cooling bath was removed, and the partially frozen mixture was allowed to warm to 23 C. The biphasic mixture was diluted with 0.2 M aqueous sodium hydroxide solution (1.25 L). The layers were separated. The dark-purple aqueous layer was extracted with dichloromethane (2 1 L). The organic layers were combined. The combined solution was washed sequentially with 0.1 M aqueous hydrochloric acid solution (500 ml), water (3 500 ml), then saturated aqueous sodium chloride solution (500 ml) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the product, 8,9-dihydroxy-7-formyl-3,4- dihydroanthracen-1-one (41), as a yellow foam. 3 Attempts to purify 41 by flash-column chromatography led to decomposition. TLC: (30% ethyl acetate hexanes) R f = 0.22 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : (br s, 1H), (s, 1H), 7.28 (d, 2H, J = 8.7 Hz), 7.25 (s, 1H), 6.87 (d, 2H, J = 8.7 Hz), 5.16 (m, 1H), 4.99 (dd, 1H, J = 12.3, 5.2 Hz), 4.68 (d, 1H, J = 11.1 Hz), 4.59 (d, 1H, J = 10.7 Hz), 3.84 (s, 3H), 3.80 (s, 3H), 2.73 (s, 3H), 2.71 (m, 1H), 2.18 (m, 1H), 0.97 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 203.9, 192.7, 166.5, 163.6, 159.4, 144.8, 142.7, 137.7, 130.7, 129.9, 129.5, 114.8, 114.2, 113.9, 108.5, 71.1, 69.0, 68.6, 62.8, 55.3, 36.5, 25.8, 21.0, 18.5, 4.5, 5.3; FTIR (neat), cm 1 : 3329 (w, br), 2953 (m), 2930 (m), 2857 (m), 1682 (m), 1614 (s), 1514 (m) 1391 (m) 1246 (s), 1153 (s), 1049 (m), 1034 (m) 870 (m), 837 (s); HRMS (ESI): Calcd for (C 31 H 38 O 8 Si+H) , found NATURE CHEMISTRY 5

6 Differentially Protected Aldehyde Di-tert-butyldichlorosilane (2.43 ml, 11.5 mmol, 1.8 equiv) was added to a solution of 8,9- dihydroxy-7-formyl-3,4-dihydroanthracen-1-one 41 (1 equiv, see paragraph above), N,Ndiisopropylethylamine (5.58 ml, 31.9 mmol, 5.0 equiv), and anhydrous 1-hydroxybenzotriazole (432 mg, 3.19 mmol, 0.5 equiv) in dimethylformamide (128 ml) at 23 C. The reaction flask was heated in an oil bath at 55 C. After 80 min, the heating bath was removed and the reaction flask was allowed to cool to 23 C. The reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution (300 ml) and ether (1.5 L). The layers were separated. The organic layer was washed with water (2 500 ml) then saturated aqueous sodium chloride solution (300 ml) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (10% ethyl acetate hexanes) to provide of the product 20 as a yellow foam (2.50 g, 55% over steps). 3 TLC: (40% ethyl acetate hexanes) R f = 0.87 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : 10.8 (s, 1H), 7.34 (s, 1H), 7.31 (d, 2H, J = 6.5 Hz), 6.88 (d, 2H, J = 7.0 Hz), 5.20 (dd, 1H, J = 3.0, 3.0 Hz), 4.88 (dd, 1H, J = 12.5, 5.0 Hz), 4.73 (d, 1H, J = 11.0 Hz), 4.63 (d, 1H, J = 10.5 Hz), 3.90 (s, 3H), 3.79 (s, 3H), (m, 1H), 2.72 (s, 3H), (m, 1H), 1.15 (s, 9H), 1.12 (s, 9H), 0.96 (s, 9H), 0.24 (s, 3H), 0.14 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 194.2, 190.9, 160.4, 159.4, 150.2, 146.3, 140.3, 134.0, 133.2, 130.0, 129.8, 199.4, 116.5, 115.6, 115.3, 114.0, 71.4, 71.2, 69.8, 62.7, 55.3, 36.3, 26.1, 26.0, 26.0, 22.5, 21.4, 21.0, 18.7, 4.3, 5.4; FTIR (neat), cm 1 : 2936 (s), 1786 (s), 1684 (s), 1607 (s), 1373 (s), 1247 (s), 1157 (s), 1034 (s); HRMS (ESI): Calcd for (C 39 H 54 O 8 Si 2 +H) , found NATURE CHEMISTRY 6

7 Gram-Scale Endo-Selective Carbonyl Ylide Aldehyde Cycloaddition: A solution of (S)-1-diazo-3-epoxy-5,5-dimethoxypenta-2,4-dione 21 3 (2.27 g, 10.6 mmol, 3.0 equiv) in dichloromethane (3.9 ml) was added by a motor-driven syringe pump over 6 h to a suspension of differentially protected aldehyde 20 (2.50 g, 3.54 mmol, 1 equiv), rhodium(ii) acetate (31 mg, 71 mol, 0.02 equiv), and powdered 4-Å molecular sieves (500 mg) in dichloromethane (3.9 ml) at 23 C. After 30 min, the reaction mixture was filtered through a short pad of silica gel (length: 10 cm; diameter: 1.5 cm), eluting with 40% ethyl acetate hexanes to remove the rhodium(ii) acetate and powdered 4-Å molecular sieves. The filtrate was concentrated. The residue was purified by flash-column chromatography (10% ethyl acetate hexanes initially, grading to 40% ethyl acetate hexanes) to provide, together, 2.59 g of a mixture of cycloadducts 42 (44%), 43 (37%), 44 (16%), and 45 (3%), as depicted above (82% yield). 3 In addition, 380 mg (15%) of the starting material, the differentially protected aldehyde 20, was recovered. NATURE CHEMISTRY 7

8 Synthetic Precursor 1. Triethylamine trihydrofluoride (1.42 ml, 8.70 mmol, 3.0 equiv) was added to a solution of a mixture of cycloadducts 42, 43, 44, and 45 (2.59 g, 2.90 mmol, 1 equiv, as described above) in acetonitrile (58 ml) at 23 C. After 15 min, the bright yellow solution was diluted with dichloromethane (500 ml). The diluted solution was washed sequentially with ph 7 aqueous phosphate buffer solution (100 ml) then saturated aqueous sodium chloride solution (100 ml) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel deactivated with triethylamine (30% ethyl acetate hexanes initially, grading to 40% ethyl acetate hexanes; two purifications) to provide synthetic precursor 1 as a yellow-green foam (690 mg, 32%). 3 TLC: (30% ethyl acetate hexanes) R f = 0.46 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : 7.37 (s, 1H), 7.30 (d, J = 9.0 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 5.74 (d, J = 3.0 Hz, 1H), 5.48 (d, J = 3.5 Hz, 1H), 5.15 (dd, J = 2.5, 3.0 Hz, 1 H), 4.85 (dd, J = 12.0, 4.5 Hz, 1H), 4.77 (s, 1H), 4.70 (d, J = 11.0 Hz, 1H), 4.60 (d, J = 11.0 Hz, 1H), 3.87 (s, 3H), 3.79 (s, 3H), 3.63 (d, J = 6.0 Hz, 1H), 3.61 (s, 3H), 3.61 (s, 3H), 3.07 (d, J = 7.0 Hz, 1H), 2.75 (s, 3H), (m, 1H), (m, 1H), 1.19 (s, 9H), 1.07 (s, 9H), 0.95 (s, 9H), 0.23 (s, 3H), 0.13 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 201.0, 194.4, 159.3, 150.1, 149.1, 146.2, 139.6, 131.2, 130.3, 129.7, 129.4, 118.2, 116.8, 115.8, 114.5, 113.8, 107.0, 101.6, 83.4, 79.6, 71.3, 71.2, 69.6, 64.3, 62.6, 56.6, 55.9, 55.3, 50.9, 36.7, 26.4, 26.1, 26.0, 23.6, 21.2, 21.2, 18.7, 4.3, 5.3; FTIR (neat), cm 1 : 2928 (s), 2857 (s), 1784 (s), 1699 (s), 1611 (s), 1371 (s), 1250 (s); HRMS (ESI): Calcd for (C 47 H 64 O 13 Si 2 +H) , found NATURE CHEMISTRY 8

9 Synthesis of DC-45-A1 and Trioxacarcinose B Monoglycoside 9 Benzylic Alcohol 2. 2,3-Dichloro-5,6-dicyanobenzoquinone (16 mg, 70 mmol, 1.1 equiv) was added to a vigorously stirring, biphasic solution of synthetic precursor 1 (48 mg, 64 mmol, 1 equiv) in dichloromethane (1.0 ml) and ph 7 aqueous phosphate buffer solution (100 μl) at 23 C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 2 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 ml) and dichloromethane (40 ml). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure benzylic alcohol 2 as a yellow-green powder (33.6 mg, 83%). TLC: (60% ethyl acetate hexanes) R f = 0.39 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 7.40 (s, 1H), 5.41 (t, J = 3.4 Hz, 1H), 5.25 (d, J = 4.1 Hz, 1H), 4.88 (dd, J = 11.7, 4.8 Hz, 1H), 4.85 (d, J = 4.1 Hz, 1H), 4.70 (s, 1H), 4.55 (br, 1H), 3.90 (s, 3H), 3.61 (s, 3H), 3.46 (s, 3H), 3.10 (d, J = 5.5 Hz, 1H), 3.02 (d, J = 5.5 Hz, 1H), 2.58 (s, 3H), (m, 1H), (m, 1H), 0.94 (s, 9H), 0.21 (s, 3H), 0.15 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 202.9, 162.4, 151.5, 143.8, 142.0, 135.1, 129.5, 116.1, 114.7, 114.7, 107.9, 103.9, 100.1, 98.5, 73.2, 69.6, 69.4, 69.2, 62.6, 62.5, 57.0, 56.6, 50.4, 38.7, 25.8, 20.4, 18.5, 4.5, 5.3; FTIR (neat), cm 1 : 3466 (br), 2953 (w), 1622 (m), 1389 (m), 1123 (s), 1069 (s), 945 (s), 729 (s); HRMS (ESI): Calcd for (C 31 H 41 O 12 Si+H) , found NATURE CHEMISTRY 9

10 -Glycoside 4. Boron trifluoride etherate (4.6 μl, 36 μmol, 1.0 equiv) was added to a suspension of benzylic alcohol 2 (23 mg, 36 μmol, 1 equiv), 1-O-acetyltrioxacarcinose A (3) 4 (18 mg, 73 μmol, 2.0 equiv, a 1:12 mixture of - and -anomers, respectively), and powdered 4-Å molecular sieves (~50 mg) in dichloromethane (720 μl) at 40 C. After 5 min, saturated aqueous sodium bicarbonate solution (1 ml) was added rapidly, and the reaction flask was allowed to warm to 23 C. The mixture was partitioned between dichloromethane (40 ml) and saturated aqueous sodium chloride solution (5 ml). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide the pure -glycoside 4 as a yellowgreen powder (23.5 mg, 79%). TLC: (5% methanol dichloromethane) R f = 0.40 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 7.47 (s, 1H), 5.38 (d, J = 3.6 Hz, 1H), 5.35 (app s, 1H), 5.26 (d, J = 4.0 Hz, 1H), 4.84 (d, J = 4.0 Hz, 1H), 4.78 (dd, J = 12.3, 5.2 Hz, 1H), 4.75 (s, 1H), 4.71 (s, 1H), 4.52 (q, J = 6.6 Hz, 1H), 4.35 (s, 1H), 3.83 (s, 3H), 3.81 (s, 1H), 3.62 (s, 3H), 3.47 (s, 3H), 3.15 (d, J = 5.3 Hz, 1H), 3.05 (d, J = 5.3 Hz, 1H), 2.60 (s, 3H), 2.58 (m, 1H), 2.35 (dt, J = 12.5, 2.6 Hz, 1H), 2.14 (s, 3H), 1.96 (dd, J = 14.6, 4.1 Hz, 1H), 1.62 (d, J = 14.6 Hz, 1H), 1.23 (d, J = 6.6 Hz, 3H), 1.08 (s, 3H), 0.95 (s, 9H), 0.24 (s, 3H), 0.16 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 202.8, 170.5, 163.2, 151.8, 144.4, 142.4, 135.2, 126.6, 116.8, 115.2, 115.1, 108.3, 104.0, 100.3, 98.6, 98.3, 74.6, 73.4, 69.8, 69.5, 69.5, 68.9, 68.4, 62.9, 62.7, 57.2, 56.8, 50.7, 38.8, 36.8, 26.0, 25.9, 21.1, 20.6, 18.6, 17.0, 4.2, 5.3; FTIR (neat), cm 1 : 3516 (br), 2934 (w), 1749 (m), 1622 (m), 1391 (m), 1229 (s), 1121 (s), 995 (s), 943 (s), 868 (m), 837 (m); HRMS (ESI): Calcd for (C 40 H 54 O 16 Si+Na) , found Smaltz, D. J.; Švenda, J.; Myers, A. G. Org. Lett. 2012, 14, NATURE CHEMISTRY 10

11 DC-45-A1. Triethylamine trihydrofluoride (42 μl, 0.26 mmol, 30 equiv) was added to a solution of - glycoside 4 (7.0 mg, 8.6 μmol, 1 equiv) in acetonitrile (290 μl) at 23 C. The reaction flask was covered with aluminum foil to exclude light. After 15 h, the product solution was partitioned between dichloromethane (50 ml) and saturated aqueous sodium chloride solution (10 ml). The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 20 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration pure DC-45-A1 as a yellow-orange powder (4.6 mg, 76%). TLC: (5% methanol dichloromethane) R f = 0.30 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 7.49 (s, 1H), (m, 2H), 5.26 (d, J = 4.0 Hz, 1H), 4.84 (d, J = 3.7 Hz, 1H), (m, 2H), 4.71 (s, 1H), 4.54 (q, J = 6.2 Hz, 1H), 4.43 (br, 1H), 3.84 (s, 3H), 3.62 (s, 3H), 3.58 (br, 1H), 3.47 (s, 3H), 3.16 (d, J = 5.1 Hz, 1H), 3.04 (d, J = 5.5 Hz, 1H), (m, 1H), 2.62 (s, 3H), 2.22 (dt, J = 13.2, 2.6 Hz, 1H), 2.14 (s, 3H), 1.96 (dd, J = 14.3, 4.0 Hz, 1H), 1.61 (d, J = 14.3 Hz, 1H), 1.24 (d, J = 6.2 Hz, 1H), 1.07 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 202.8, 170.3, 163.1, 151.7, 144.8, 143, 135.5, 126.5, 116.8, 115.3, 114.8, 107.3, 103.9, 100.1, 98.6, 98.1, 74.4, 73.2, 69.5, 69.3, 68.8, 67.9, 67.7, 62.9, 62.7, 57.1, 56.6, 50.5, 36.7, 36.6, 25.7, 20.9, 20.5, 16.9; FTIR (neat), cm 1 : 3476 (br), 2934 (m), 1736 (s), 1622 (m), 1389 (s), 1229 (s), 1144 (s), 1082 (s), 997 (s); HRMS (ESI): Calcd for (C 34 H 40 O 16 +Na) , found NATURE CHEMISTRY 11

12 -Glycoside 6. Example 1: (20 equiv TMSOTf, 30 equiv 5) To a solution of the synthetic precursor 1 (30.0 mg, mmol, 1 equiv) in benzene (3 ml) at 23 C was added 1-O- -acetyl glycoside 5 5 (328 mg, 1.19 mmol, 30.0 equiv). The bright yellow solution was concentrated and the residue was blanketed with argon. Dichloromethane (1.9 ml) and ether (0.27 ml) were added, followed by crushed 4-Å molecular sieves (600 mg). The bright yellow suspension was stirred for 90 min at 23 C, then was cooled to 78 C. Trimethylsilyl trifluoromethanesulfonate (72 L, mmol, 10.0 equiv) was added dropwise by syringe over 10 min. After 2 h, a second portion of trimethylsilyl trifluoromethanesulfonate (36 L, mmol, 5.0 equiv) was added over 10 min. After 1 h, a third portion of trimethylsilyl trifluoromethanesulfonate (36 L, mmol, 5.0 equiv) was added over 10 min and the internal temperature of the reaction mixture was maintained at 78 C for 1 h. Triethylamine (111 L, mmol, 20.0 equiv) was added at 78 C. The resulting yellow-orange solution was diluted with dichloromethane (50 ml) and the product mixture was filtered through a plug of Celite. The filtrate was partitioned between saturated aqueous sodium bicarbonate solution (50 ml) and dichloromethane (150 ml). The aqueous layer was extracted with dichloromethane (3 30 ml) and the combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. To remove the bulk of unreacted 1-O- -acetyl glycoside 5, the residue was passed through a short column of silica gel deactivated with triethylamine (20% ethyl acetate hexanes initially, grading to 60% ethyl acetate hexanes). A colorless fraction containing unreacted 1-O- -acetyl glycoside 5 was collected. A later-eluting, bright yellow fraction was concentrated. This residue was further purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure -glycoside 6 (30 mg, 78%, in fractions eluting at min) as a bright yellow powder. TLC (50% ethyl acetate hexanes): R f = 0.30 (UV, CAM). [ ] 23 D (c 0.90, CH 2 Cl 2 ). 1 H NMR (600 MHz, CDCl 3 ) : (s, OH), 7.46 (s, 1 H), 7.28 (d, J = 8.4 Hz, 2H), Magauer, T.; Myers, A.G. Org. Lett. 2011, 13, NATURE CHEMISTRY 12

13 (d, J = 8.4 Hz, 2 H), 5.83 (dd, J = 3.6, 1.8 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.19 (d, J = 4.2 Hz, 1H), 5.19 (m, 1H), 5.00 (q, J = 6.0 Hz, 1H), 4.96 (dd, J = 12.0, 4.8 Hz, 1H), 4.75 (t, J = 3.6 Hz, 1H), 4.74 (s, 1H), 4.70 (d, J = 10.8 Hz, 1H), 4.59 (d, J = 10.8 Hz, 1H), 3.86 (s, OH), 3.83 (s, 3H), 3.80 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 2.81 (d, J = 6.0 Hz, 1H), (m, 1H), 2.70 (d, J = 6.0 Hz, 1H), 2.59 (s, 3H), 2.35 (s, 3H), (m, 2H), 2.22 (s, 3H), (m, 1H), 1.08 (d, J = 6.0 Hz, 3H), 0.97 (s, 9H), 0.25 (s, 3H), 0.17 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ), : 208.8, 203.4, 170.3, 162.9, 159.3, 151.7, 144.1, 141.8, 135.1, 130.1, 129.5, 127.7, 116.3, 114.9, 114.2, 113.9, 108.6, 104.5, 101.5, 99.4, 92.3, 78.1, 71.0, 70.6, 69.3, 69.1, 68.9, 68.2, 64.4, 62.7, 56.6, 55.8, 55.3, 47.3, 36.3, 29.7, 28.9, 26.6, 25.9, 21.3, 20.3, 18.6, 14.5, 4.4, 5.4; FTIR (neat), cm 1 : 3470 (w), 2928 (s), 1740 (s), 1719 (m), 1620 (s), 1514 (m), 1389 (s), 1248 (s), 1082 (m). HRMS (ESI): Calcd for (C 49 H 62 O 18 Si+H) , found ; HRMS (ESI): Calcd for (C 49 H 62 O 18 Si+Na) , found Example 2: (5.7 equiv TMSNTf 2, 8.5 equiv 5) To a solution of synthetic precursor 1 (160 mg, mmol, 1 equiv) in benzene (10 ml) at 23 C was added 1-O- -acetyl glycoside 5 5 (495 mg, 1.81 mmol, 8.5 equiv). The bright yellow solution was concentrated and the residue was blanketed with argon. Dichloromethane (800 L) and ether (250 L) were added, followed by crushed 4-Å molecular sieves (250 mg). The bright yellow suspension was stirred for 60 min at 23 C, then was cooled to 78 C. A freshly prepared solution of N-(trimethylsilyl)-bis(trifluoromethanesulfonyl)imide in dichloromethane (1.0 M, 1.21 ml, 1.21 mmol, 5.7 equiv) was added dropwise by syringe over 1.5 h. The internal temperature of the reaction mixture was maintained at 78 C for 1 h. Triethylamine (296 L, 2.13 mmol, 10 equiv) was added at 78 C. The resulting yellow-orange solution was diluted with a 10:1 mixture of dichloromethanemethanol (50 ml), and the product mixture was filtered through a plug of Celite. The filtrate was partitioned between saturated aqueous sodium bicarbonate solution (50 ml) and dichloromethane (150 ml). The aqueous layer was extracted with dichloromethane (3 20 ml) and the combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure -glycoside 6 (120 mg, 58%, in fractions eluting at min) as a bright yellow powder. 6 6 In Example 2 only, 39 mg (22 %) of the trimethylsilylated hemiketal 46, shown below, was isolated separately in fractions eluting at min. NATURE CHEMISTRY 13

14 Example 3: (3.3 equiv TMSNTf 2, 5 equiv 5) To a solution of synthetic precursor 1 (80.0 mg, mmol, 1 equiv) in benzene (5 ml) at 23 C was added 1-O- -acetyl glycoside 5 5 (146 mg, mmol, 5 equiv). The bright yellow solution was concentrated and the residue was blanketed with argon. Dichloromethane (800 L) and ether (250 L) were added, followed by crushed 4-Å molecular sieves (250 mg). The bright yellow suspension was stirred for 60 min at 23 C, then was cooled to 78 C. A freshly prepared solution of N-(trimethylsilyl)-bis(trifluoromethanesulfonyl)imide in dichloromethane (1.0 M, 354 L, mmol, 3.3 equiv) was added dropwise by syringe over 2.5 h. The internal temperature of the reaction mixture was maintained at 78 C for 1 h. Triethylamine (49.3 L, mmol, 3.3 equiv) was added at 78 C. The resulting yellow-orange solution was diluted with a 10:1 mixture of dichloromethane-methanol (10 ml), and the product mixture was filtered through a plug of Celite. The filtrate was partitioned between saturated aqueous sodium bicarbonate solution (50 ml) and dichloromethane (100 ml). The aqueous layer was extracted with dichloromethane (3 20 ml) and the combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure -glycoside 6 (65.0 mg, 63%, in fractions eluting at min) as a bright yellow powder. Alcohol 7. Potassium carbonate (2.6 mg, 0.5 equiv) was added to a solution of -glycoside 6 (37 mg, 38 mol, 1 equiv) in methanol (7.7 ml) at 0 C. After 3 h, the mixture was partitioned between chloroform (100 ml) and saturated aqueous sodium chloride solution (20 ml). The layers were separated. The aqueous layer was extracted with chloroform (2 20 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the alcohol 7 (32 mg, 90%). NATURE CHEMISTRY 14

15 Benzylic Alcohol 8. 2,3-dichloro-5,6-dicyanobenzoquinone (4.7 mg, 21 mol, 1.2 equiv) was added to a vigorously stirring, biphasic solution of alcohol 7 (16 mg, 17 mol, 1 equiv) in dichloromethane (2.2 ml) and water (440 μl) at 23 C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 2.5 h, the color of the reaction mixture changed from myrtle green to yellow. The product solution was partitioned between water (10 ml) and dichloromethane (40 ml). The layers were separated. The aqueous layer was extracted with dichloromethane (20 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure benzylic alcohol 8 (12.7 mg, 91%) as a yellow-green oil. Trioxacarcinose B Monoglycoside 9. Triethylamine trihydrofluoride (18 L, 110 mol, 30 equiv) was added to a solution of benzylic alcohol 8 (3.0 mg, 3.7 mol, 1 equiv) in acetonitrile (0.5 ml) at 23 C. The reaction flask was covered with aluminum foil to exclude light. After 21 h, the product solution was partitioned between dichloromethane (50 ml) and saturated aqueous sodium chloride solution (20 ml). The layers were separated. The aqueous layer was extracted with dichloromethane (3 10 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 20 50% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure trioxacarcinose B monoglycoside 9 (1.7 mg, 66%). TLC: (ethyl acetate) R f = 0.18 (UV, CAM); 1 H NATURE CHEMISTRY 15

16 NMR (500 MHz, CDCl 3 ) : (br, 1H), 7.48 (s, 1H), 5.85 (d, 1H, J = 2.6 Hz), 5.46 (t, 1H, J = 2.9 Hz), 5.37 (d, 1H, J = 4.0 Hz), 5.24 (d, 1H, J = 4.0 Hz), 5.02 (q, 1H, J = 6.6 Hz), 4.93 (dd, 1H, J = 12.8, 5.5 Hz), 4.77 (s, 1H), 4.25 (d, 1H, J = 9.5 Hz), 4.16 (br, 1H), 3.92 (s, 3H), 3.71 (brd, 1H, J = 8.8 Hz), 3.63 (s, 3H), 3.49 (s, 3H), 3.00 (br, 1H), 2.98 (d, 1H, J = 5.9 Hz), 2.89 (d, 1H, J = 5.9 Hz), 2.74 (ddd, 1H, J = 13.2, 5.5, 2.9 Hz), 2.62 (s, 3H), 2.49 (s, 3H), 2.44 (dt, 1H, J = 14.7, 3.7 Hz), (dt, 1H, J = 13.2, 3.3 Hz), 2.11 (ddd, 1H, J = 14.7, 2.6, 1.5 Hz), 1.09 (d, 3H, J = 6.6 Hz); 13 C NMR (125 MHz, CDCl 3 ) : 210.4, 203.3, 162.4, 151.8, 144.4, 142.6, 135.7, 129.6, 116.6, 114.6, 107.3, 104.7, 101.5, 99.7, 94.9, 79.6, 71.4, 70.2, 69.2, 68.3, 67.7, 63.8, 62.9, 61.9, 56.9, 56.1, 48.2, 45.7, 37.0, 31.6, 27.8, 20.4, 14.5; FTIR (neat), cm 1 : 3431 (br w), 2926 (m), 1713 (m), 1622 (s), 1256 (s), 1103 (s); HRMS (ESI): Calcd for (C 33 H 38 O 16 + Na) + : Found: NATURE CHEMISTRY 16

17 Synthesis of Trioxacarcin A Benzylic Alcohol 22. To a vigorously stirring, biphasic solution of -glycoside 6 (120 mg, mmol, 1 equiv) in dichloromethane (1.9 ml) and ph 7 phosphate buffer (380 μl) at 23 C was added 2,3-dichloro-5,6- dicyanobenzoquinone (33.8 mg, mmol, 1.1 equiv). The reaction flask was covered with aluminum foil to exclude light. Over the course of 2 h, the color of the reaction mixture changed from myrtle green to yellow. The product solution was partitioned between water (10 ml) and dichloromethane (60 ml). The layers were separated. The aqueous layer was extracted with dichloromethane (3 20 ml). The combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure benzylic alcohol 22 (100 mg, 95%, in fractions eluting at min) as a bright yellow solid. TLC: (60% ethyl acetate hexanes) R f = 0.31 (UV, CAM); [ ] 23 D +2.9 (c 0.614, CHCl 3 ). 1 H NMR (600 MHz, CDCl 3 ) : (s, OH) (s, 1 H), 5.82 (t, J = 2.4 Hz, 1H), 5.43 (app d, J = 1.8 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.30 (s, 1H), 5.19 (d, J = 3.6 Hz, 1H), 5.01 (q, J = 6.6 Hz, 1H), 4.90 (dd, J = 12.0, 4.8 Hz, 1H), 4.75 (app t, J = 3.6 Hz, 1H), 4.74 (s, 1H), 3.90 (s, 3H), 3.89 (s, OH), 3.63 (s, 3H), 3.47 (s, 3H), 2.83 (d, J = 6.0 Hz, 1H), 2.74 (d, J = 5.4 Hz, 1H), 2.60 (s, 3H), (m, 1H), 2.42 (br s, 1H), 2.36 (s, 3H), (m, 2H), 2.23 (s, 3H), 1.08 (d, J = 6.6 Hz, 3H), 0.95 (s, 9H), 0.23 (s, 3H), 0.16 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 208.7, 202.9, 170.3, 162.6, 151.8, 143.7, 142.1, 135.2, 129.6, 116.0, 114.8, 114.3, 107.9, 104.5, 101.6, 99.4, 92.4, 78.1, 72.10, 70.6, 69.3, 69.1, 68.2, 64.4, 62.7, 62.6, 56.6, 55.8, 47.4, 38.8, 28.9, 27.0, 25.8, 21.3, 20.4, 18.5, 14.5, 4.5, 5.4. FTIR (neat), cm 1 : 3475 (m), 2934 (s), 2857 (m), 1738 (s), 1721 (s), 1620 (s), 1570 (m), 1445 (m), 1389 (s), 1240 (s), 1082 (s). HRMS (ESI): Calcd for (C 41 H 54 O 17 Si + Na) + : Found: NATURE CHEMISTRY 17

18 1-Phenylthiotrioxacarcinoside A (23). Boron trifluoride etherate (72 μl, 0.57 mmol, 1.4 equiv) was added to a solution of 1-Oacetyltrioxacarcinoside A (3) 4 (100 mg, 0.41 mmol, 1 equiv, a 1:12 mixture of - and -anomers, respectively) and thiophenol (50 μl, 0.49 mmol, 1.2 equiv) in dichloromethane (8.1 ml) at 40 C. After 5 min, saturated aqueous sodium bicarbonate solution (2 ml) was added rapidly. The cooling bath was removed and the reaction flask was allowed to warm to 23 C. The mixture was partitioned between dichloromethane (50 ml) and saturated aqueous sodium bicarbonate solution (5 ml). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (25% ethyl acetate hexanes) to provide 1-phenylthiotrioxacarcinoside A (23), as a 2.4:1 mixture of - and -anomers, respectively (103 mg, 86%). TLC: (50% ethyl acetate hexanes) R f = 0.71 (CAM); 1 H NMR (500 MHz, CDCl 3 ) : -anomer (minor): 7.49 (d, J = 7.8 Hz, 2H), (m, 3H), 5.64 (d, J = 6.4 Hz, 1H), 4.81 (q, J = 6.4 Hz, 1H), 4.74 (s, 1H), 2.58 (br, 1H), 2.35 (dd, J = 14.7, 6.4 Hz, 1H), 2.15 (s, 3H), 1.95 (m, 1H), 1.21 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H); -anomer (major): 7.51 (d, J = 7.8 Hz, 2H), (m, 3H), 5.19 (d, J = 11.7 Hz, 1H), 4.62 (s, 1H), 4.21 (q, J = 6.4 Hz, 1H), 2.15 (s, 3H), 1.93 (m, 1H), 1.82 (d, J = 13.7 Hz, 1H), 1.20 (s, 3H), 1.17 (d, J = 6.4 Hz, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 170.8, 170.5, 136.4, 134.7, 130.7, 130.6, 128.8, 128.7, 126.9, 126.8, 83.4, 80.7, 74.4, 73.7, 70.6, 70.4, 69.1, 62.8, 39.5, 38.3, 27.0, 26.8, 20.8, 20.8, 17.0, 16.6; FTIR (neat), cm 1 : 3493 (br), 2980 (m), 1743 (s), 1373 (m), 1233 (s), 1067 (s), 1007 (s); HRMS (ESI): Calcd for (C 15 H 20 O 4 S+Na) , found NATURE CHEMISTRY 18

19 3''-O-Acetyl-2-O-(tert-butyldimethylsilyl)-trioxacarcin A (24). Silver hexafluorophosphate (116 mg, mmol, 6.0 equiv) was added to a suspension of benzylic alcohol 22 (65.0 mg, mmol, 1 equiv), 1-phenylthiotrioxacarcinoside A 23 (68 mg, 0.23 mmol, 3.0 equiv, a ~1:2.4 mixture of and anomers, respectively), 2,6-di-tert-butyl-4- methylpyridine (126 mg, mmol, 8.0 equiv), and powdered 4-Å molecular sieves (~100 mg) in dichloromethane (3.8 ml) at 0 C. After 1 h, pyridine (310 μl, 3.84 mmol, 50 equiv) was added, and the reaction flask was allowed to warm to 23 C. After 30 min, the product mixture was filtered through a short pad of Celite, washing with dichloromethane (100 ml). The filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with % acetonitrile in water, flow rate: 15 ml/min) to provide after concentration pure 3''-O-acetyl-2-O-(tert-butyldimethylsilyl)- trioxacarcin A (24) as a yellow-green foam (58.3 mg, 74%). TLC (60% ethyl acetate hexanes): R f = 0.39 (UV, CAM); [ ] 23 D 33.5 (c 0.71, CH 2 Cl 2 ); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 7.46 (s, 1H), 5.81 (t, J = 2.7 Hz, 1H), (m, 2H), 5.31 (d, J = 4.4 Hz, 1H), 5.19 (d, J = 4.0 Hz, 1H), 5.01 (q, J = 6.2 Hz, 1H), 4.78 (dd, J = 12.5, 5.1 Hz, 1H), (m, 3H), 4.52 (q, 6.6 Hz, 1H), 3.89 (br, 1H), 3.82 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 2.84 (d, J = 5.9 Hz, 1H), 2.76 (d, J = 5.9 Hz, 1H), 2.59 (s, 3H), (m, 1H), 2.36 (s, 3H), (m, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.95 (dd, J = 14.7, 4.0 Hz, 1H), 1.63 (d, J = 14.7 Hz, 1H), 1.23 (d, J = 6.6 Hz, 3H), (m, 3H), 1.08 (s, 3H), 0.96 (s, 9H), 0.25 (s, 3H), 0.16 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ), : 208.6, 202.6, 170.3, 170.2, 163.1, 151.8, 144.1, 142.2, 134.8, 126.4, 116.4, 114.9, 114.6, 108.1, 104.4, 101.5, 99.3, 98.0, 92.3, 78.0, 74.3, 71.9, 70.5, 69.2, 68.9, 68.7, 68.1, 68.0, 64.4, 62.6, 62.5, 56.4, 55.8, 47.3, 38.7, 36.5, 29.6, 28.8, 26.8, 25.7, 21.2, 20.8, 20.2, 18.3, 16.8, 14.4, 4.4, 5.6; FTIR (neat), cm 1 : 3482 (br), 2934 (w), 1740 (m), 1620 (m), 1389 (m), 1227 (s), 1117 (s), 1082 (s), 995 (s), 943 (s); HRMS (ESI): Calcd for (C 50 H 68 O 21 Si+H) , found NATURE CHEMISTRY 19

20 Trioxacarcin A. Potassium carbonate (13 mg, 95 μmol, 2.0 equiv) was added to a solution of 3''-O-acetyl-2- O-(tert-butyldimethylsilyl)-trioxacarcin A (24) (49 mg, 47 μmol, 1 equiv) in methanol (9.5 ml) at 0 C. After 60 min, ammonium chloride (10 mg, 0.19 mmol, 4.0 equiv) was added. After 1 min, the mixture was filtered through a short pad of Celite, washing with methanol (50 ml). The filtrate was concentrated. Analysis of the residue by 1 H NMR spectroscopy (500 MHz, CDCl 3 ) established that the 3''-O-acetyl protecting group ( 2.22, (s, 3H)) had been cleaved and that the 4'-O-acetyl group was retained ( 2.13 (s, 3H)). The residue (1 equiv, see above) was dissolved in acetonitrile (1.6 ml), and triethylamine trihydrofluoride (232 μl, 1.4 mmol, 30 equiv) was added at 23 C. The reaction flask was covered with aluminum foil to exclude light. After 14 h, saturated aqueous sodium bicarbonate solution (10 ml) was added. The mixture was extracted with dichloromethane (2 50 ml). The organic layers were combined. The combined solution was dried over sodium sulfate. The dried solution was filtered and filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 20 80% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration pure trioxacarcin A as an orange-red powder (23 mg, 55% yield over two steps). TLC (80% ethyl acetate hexanes): R f = 0.25 (UV, CAM); 1 H NMR (500 MHz, CDCl 3 ) : (s, 1H), 7.51 (s, 1H), 5.85 (d, J = 2.6 Hz, 1H), 5.39 (m, 1H), 5.37 (d, J = 4.0 Hz, 1H), 5.24 (d, J = 4.0 Hz, 1H), 5.02 (q, J = 6.6 Hz, 1H), 4.78 (m, 1H), 4.77 (s, 1H), 4.75 (app s), 4.54 (q, J = 6.6 Hz, 1H), 4.24 (d, J = 9.2 Hz, 1H), 4.16 (br, 1H), 3.84 (s, 3H), 3.72 (br d, J = 8.8 Hz, 1H), 3.63 (s, 3H), 3.56 (s, 1H), 3.49 (s, 3H), 2.99 (d, J = 5.9 Hz, 1H), 2.91 (d, J = 5.5 Hz, 1H), 2.81 (m, 1H), 2.62 (s, 3H), 2.50 (s, 3H), 2.45 (dt, J = 14.3, 3.3 Hz, 1H), 2.21 (dt, J = 13.2, 2.2 Hz, 1H), 2.14 (s, 3H), 2.11 (m, 1H), 1.97 (dd, J = 14.7, 4.0 Hz, 1H), 1.62 (d, J = 14.7 Hz, 1H), 1.24 (d, J = 6.6 Hz, 3H), 1.10 (d, J = 6.2 Hz, 3H), 1.07 (s, 3H); 13 C NMR (125 MHz, CDCl 3 ), : 14.5, 16.8, 20.3, 20.9, 25.7, 27.8, 31.5, 36.6, 48.1, 56.2, 56.8, 62.7, 62.9, 63.8, 67.4, 67.9, 68.3, 68.8, 69.1, 70.2, 71.4, 74.4, 79.5, 94.8, 97.9, 99.7, 101.5, 104.6, 107.4, 114.8, 116.9, 126.6, 135.4, 142.8, 144.8, 151.7, 163.1, 170.3, 202.9, 210.3; FTIR (neat), cm 1 : 3514 (br), 2976 (w), 2940 (w), 1748 (m), 1620 (m), 1387 (m), 1225 (s), 1086 (s), 997 (s); HRMS (ESI): Calcd for (C 42 H 52 O 20 H) , found NATURE CHEMISTRY 20

21 Preparation of Fully Synthetic Trioxacarcin Analogs -Glycoside 47. To a solution of dideoxy-dc-45-a2 3 (20.0 mg, mmol, 1 equiv) in benzene (2.0 ml) at 23 C was added 1-O- -acetyl glycoside 5 5 (56.4 mg, mmol, 5.0 equiv). The bright yellow solution was concentrated and the residue was blanketed with argon. Dichloromethane (1.5 ml) and ether (500 L) were added followed by crushed 4-Å molecular sieves (500 mg). The bright yellow suspension was stirred for 30 min at 23 C, then was cooled to 78 C. N-(trimethylsilyl)- bis(trifluoromethanesulfonyl)imide (20.2 L, mmol, 2.0 equiv) was added dropwise by syringe over 10 min. After 20 min, a second portion of N-(trimethylsilyl)- bis(trifluoromethanesulfonyl)imide (20.2 L, mmol, 2.0 equiv) was added over 10 min. After 2.5 h, the cold mixture was diluted with 15% methanol dichloromethane (25 ml). Saturated aqueous sodium bicarbonate solution (10 ml) was added. The layers were separated. The aqueous layer was extracted with dichloromethane (25 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure -glycoside 47 (19.7 mg, 68%, in fractions eluting at min) as a bright yellow solid. TLC: (50% ethyl acetate hexane) R f = 0.58 (UV, CAM). [ ] 23 D (c 0.23, CHCl 3 ). 1 H NMR (600 MHz, CDCl 3 ) : (s, OH) (s, 1 H), 5.84 (t, J = 3.6 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.19 (d, J = 4.2 Hz, 1H), 5.01 (q, J = 6.6 Hz, 1H), 4.75 (t, J = 3.6 Hz, 1H), 4.73 (s, 1H), 3.88 (s, OH), 3.77 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 3.03 (app q, J = 5.4 Hz, 2H), 2.84 (d, J = 6.0 Hz, 1H), 2.77 (d, J = 6.0 Hz, 1H), 2.72 (app t, J = 6.6 Hz, 2H), 2.58 (s, 3H), 2.36 (s, 3H), 2.33 (app t, J = 3.0 Hz, 2H), 2.23 (s, 3H), (m, 2H), 1.08 (d, J = 6.0 Hz, 3H), 13 C NMR (125 MHz, CDCl 3 ) : 208.7, 204.3, 170.3, 162.9, 151.8, 142.3, 141.7, 135.2, 130.3, 115.6, 113.2, 113.1, 111.0, 104.5, 101.5, 99.5, 92.3, 78.2, 72.1, 70.7, 69.2, 68.3, 64.4, 60.9, 56.5, 55.8, 47.4, 38.8, 28.9, 27.0, 23.6, 22.1, 21.3, 20.3, FTIR (neat), cm 1 : 3482 (w), 2928 NATURE CHEMISTRY 21

22 (m), 2854 (w), 1740 (s), 1717 (s), 1622 (s), 1449 (m), 1389 (m), 1238 (s), 1096 (s). HRMS (ESI): Calcd for (C 35 H 40 O 15 + Na) + : Found: Alcohol 29. Potassium carbonate (1 mg, 7.24 mol, 0.34 equiv) was added to a solution of the - glycoside 47 (15 mg, mmol, 1 equiv) in methanol (1 ml) at 0 C. After 30 min, the cooling bath was removed and the reaction mixture was allowed to warm to 23 C. After 10 h, the reaction mixture was partitioned between saturated aqueous sodium chloride solution (20 ml) and chloroform (100 ml). The layers were separated. The aqueous layer was extracted with chloroform (3 30 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure alcohol 29 (12.0 mg, 85%, in fractions eluting at min) as a yelloworange solid. TLC: (50% ethyl acetate hexane) R f = 0.23 (UV, CAM); [ ] 23 D (c 0.60, CHCl 3 ). 1 H NMR (600 MHz, CDCl 3 ) : (s, OH) (s, 1 H), 5.86 (d, J = 2.4 Hz, 1H), 5.34 (d, J = 4.2 Hz, 1H), 5.24 (d, J = 3.6 Hz, 1H), 5.02 (q, J = 6.6 Hz, 1H), 4.77 (s, 1H), 4.29 (d, J = 9.6 Hz, OH), 4.15 (s, OH), 3.77 (s, 3H), 3.71 (dt, J = 9.6,3 Hz, 1H), 3.63 (s, 3H), 3.49 (s, 3H), 3.04 (app q, J = 5.4 Hz, 2H), 2.98 (d, J = 5.4 Hz, 1H), 2.92 (d, J = 5.4 Hz, 1H), 2.73 (app t, J = 7.2 Hz, 2H), 2.59 (s, 3H), 2.50 (s, 3H), 2.43 (t, J = 14.4, 3.6 Hz, 2H), (m, 2H), 1.09 (d, J = 6.6 Hz, 3H); 13 C NMR (125 MHz, CDCl 3 ) : 210.5, 204.5, 162.9, 151.6, 142.4, 141.6, 135.2, 130.5, 115.9, 113.1, 113.0, 111.1, 104.6, 101.4, 99.7, 94.8, 79.6, 77.2, 71.5, 70.3, 69.4, 68.4, 63.8, 60.9, 56.8, 56.1, 48.2, 38.8, 27.8, 23.6, 22.1, 20.3, FTIR (neat), cm 1 : 3522 (w), 2930 (s), 2857 (m), 1746 (s), 1717 (s), 1622 (s), 1498 (s), 1456 (s), 1389 (s), 1256 (s), 1095 (s). HRMS (ESI): Calcd for (C 33 H 38 O 14 + Na) + : Found: ; HRMS (ESI): Calcd for (C 33 H 38 O 14 + NH 4 ) + : Found: NATURE CHEMISTRY 22

23 -Glycoside 49 1-O-acetyl glycoside 48 7 (27.3 mg, mmol, 16.0 equiv) was added to a solution of synthetic precursor 1 (5.0 mg, 6.6 mol, 1 equiv) in benzene (0.5 ml). The solution was concentrated and the residue was blanketed with argon. Dichloromethane (100 L) and ether (30 L) were added, followed by crushed 4-Å molecular sieves (20 mg). The suspension was stirred for 30 min at 23 C then was cooled to 78 C. A freshly prepared solution of N-(trimethylsilyl)- bis(trifluoromethanesulfonyl)imide in dichloromethane (1.0 M, 70.4 L, mmol, 10.6 equiv) was added dropwise by syringe over 1.5 h. After 4 h, triethylamine (0.05 ml) was added. The mixture was diluted with 15% methanol dichloromethane (50 ml) and saturated aqueous sodium bicarbonate solution (10 ml). The layers were separated. The aqueous layer was extracted with dichloromethane (2 20 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 40 90% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure -glycoside 49 (4.5 mg, 64%). TLC: (50% ethyl acetate hexanes) R f = 0.43 (UV, CAM); 1 H NMR (600 MHz, CDCl 3 ) : (br, 1H), 7.48 (s, 1H), 7.28 (d, 2H, J = 8.5 Hz), 6.87 (d, 2H, J = 8.7 Hz), 5.91 (t, 1H, J = 2.5 Hz), 5.23 (d, 1H, J = 2.3 Hz), 5.22 (d, 1H, J = 4.1 Hz), 5.19 (m, 2H), 4.97 (dd, 1H, J = 12.6, 5.3 Hz), 4.74 (s, 1H), 4.70 (d, 1H, J = 10.8 Hz), 4.60 (d, 1H, J = 11.0 Hz), 4.40 (q, 1H, J = 6.6 Hz), 4.09 (ddd, 1H, J = 11.2, 6.4, 3.0 Hz), 3.83 (s, 3H), 3.80 (s, 3H), 3.63 (s, 3H), 3.46 (s, 3H), 2.85 (d, 1H, J = 6.0 Hz), 2.74 (d, 1H, J = 6.0 Hz), 2.72 (m, 1H), 2.59 (s, 3H), 2.19 (s, 3H), (m, 2H), 1.15 (d, 3H, J = 6.4 Hz), 0.98 (s, 9H), 0.25 (s, 3H), 0.17 (s, 3H); FTIR (neat), cm 1 : 2926 (s), 2854 (m), 2104 (m), 1746 (m), 1620 (s), 1516 (m), 1250 (s), 1107 (s); HRMS (ESI): Calcd for (C 47 H 60 O 16 N 3 Si + H) + : , Found: O-Acetyl glycoside 48 was prepared from daunosamine hydrochloride by a two-step sequence which has been described previously: Zhang, G.; Fang, L.; Zhu, L.; Aimiuwu, J. E.; Shen, J.; Cheng, H.; Muller, M. T.; Lee, G. E.; Sun, D.; Wang, P. G. J. Med. Chem. 2005, 48, NATURE CHEMISTRY 23

24 Azido Diol 31. 2,3-dichloro-5,6-dicyanobenzoquinone (1.1 mg, 4.7 mol, 1.0 equiv) was added to a solution of -glycoside 49 (4.5 mg, 4.7 mol, 1 equiv) in a mixture of dichloromethane (833 L) and water (167 L) at 23 C. The flask was covered with aluminum foil to exclude light. Over the course of 6.5 h, the color of the reaction mixture changed from myrtle green to yellow. The product solution was partitioned between water (10 ml) and dichloromethane (50 ml). The layers were separated. The aqueous layer was extracted with dichloromethane (3 20 ml). The organic layers were combined and the combined solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was dissolved in acetonitrile (0.5 ml), and triethylamine trihydrofluoride (23 L, 0.14 mmol, 30 equiv) was added at 23 C. The reaction flask was covered with aluminum foil to exclude light. After 14 h, saturated aqueous sodium bicarbonate solution (10 ml) was added. The mixture was extracted with dichloromethane (2 50 ml). The organic layers were combined. The combined solution was dried over sodium sulfate. The dried solution was filtered and filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep- C18 column, 10 μm, mm, UV detection at 270 nm, gradient elution with 20 80% acetonitrile in water, flow rate: 15 ml/min) to provide after concentration the pure azido diol 31 (1.6 mg, 47% over 2 steps). 1 H NMR (600 MHz, CDCl 3 ) : (s, 1H), 7.47 (s, 1H), 5.88 (t, 1H, J = 2.5 Hz), 5.47 (br, 1H), 5.24 (app d, 2H, J = 4.1 Hz), 5.20 (d, 1H, J = 4.1 Hz), 4.94 (dd, 1H, J = 12.8, 5.5 Hz), 4.75 (s, 1H), 4.41 (q, 1H, J = 6.4 Hz), 4.11 (ddd, 1H, J = 11.7, 5.7, 3.0 Hz), 3.93 (s, 3H), 3.63 (s, 3H), 3.56 (br s, 1H), 3.47 (s, 3H), 2.89 (d, 1H, J = 6.0 Hz), 2.79 (d, 1H, J = 6.0 Hz), 2.75 (ddd, 1H, J = 13.3, 5.5, 2.8 Hz), 2.61 (s, 3H), 2.21 (br, 1H), (m, 2H), 2.19 (s, 3H), 1.16 (d, 3H, J = 6.4 Hz); HRMS (ESI): Calcd for (C 33 H 37 O 15 N 3 + H) + : , Found: NATURE CHEMISTRY 24