Branching of poly(adp-ribose): Synthesis of the Core Motif

Transcription

1 Branching of poly(adp-ribose): Synthesis of the Core Motif Hans A. V. Kistemaker, Herman S. Overkleeft, Gijsbert A. van der Marel,* and Dmitri V. Filippov* Supporting information Table of contents Experimental procedures NMR Spectra S1- S5 S6- S29

2 General procedures All solvents used under anhydrous conditions were stored over 4Å molecular sieves except for methanol which was stored over 3Å molecular sieves. Solvents used for workup and column chromatography were of technical grade from Sigma Aldrich and used directly. Unless stated otherwise, solvents were removed by rotary evaporation under reduced pressure at 40 C. Reactions were monitored by TLC analysis using Merck 25 DC plastikfolien 60 F254 with detection by spraying with 20% H 2 SO 4 in EtOH, (NH 4 ) 6 Mo 7 O 24 4H 2 O (25 g/l) and (NH 4 ) 4 Ce(SO 4 ) 4 2H 2 O (10 g/l) in 10% sulfuric acid or by spraying with a solution of ninhydrin (3 g/l) in EtOH / AcOH (20/1 v/v), followed by charring at approx. 150 C. Column chromatography was performed by automation using a Biotage Isolera Spektra Four machine. For LC- MS analysis a JASCO HPLC system (detection simultaneously at 214 and 254 nm) equipped with an analytical C18 column (4.6 mmd 50 mml, 3μ particle size) in combination with buffers A: H 2 O, B: MeCN and C: 0.5% aq. TFA and coupled to a PE/SCIEX API 165 single quadruple mass spectrometer (Perkin-Elmer) was used, unless stated otherwise. Alternatively a Thermo Finnigan LCQ Advantage MAX ion-trap mass spectrometer with an electrospray ion source coupled to Surveyor HPLC system (Thermo Finnegan) was used with the same analytical column. High resolution mass spectra were recorded by direct injection (2 μl of a 2 μm solution in water/acetonitrile; 50/50; v/v and 0.1% formic acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electrospray ion source in positive mode (source voltage 3.5 kv, sheath gas flow 10, capillary temperature 250 C) with resolution R = at m/z 400 (mass range m/z = ) and dioctylpthalate (m/z = ) as a lock mass. The high resolution mass spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). 1 H and 13 C-NMR spectra were measured on a Brüker AV 400 (400 MHz), Brüker AV 500 (500 MHz) or a AVIII-Brüker DMX 600 (600 MHz) and all individual signal were assigned using 2D-NMR spectroscopy. Chemical shifts are given in ppm (δ) and directly referenced to TMS (0.00 ppm) in CDCl 3 or indirectly referenced to H 3 PO 4 (0.00 ppm) in D 2 O via the solvent residual signal and coupling constants are given in Hz. Infrared (IR) spectra were recorded on a Shimadzu FT-IR ,3,5-tri-O-benzoyl-2-O-(2,3,5-tri-O-benzyl-α-D-ribofuranosyl)-α-Dribofuranosyl (4) (N-Phenyl)-2,2,2-trifluoroacetimido-2,3,5-tri-O-benzyl-β-D-ribofuranose [1] (5.7 g, 9.6 mmol) and α-d-ribofuranose 1,3,5-tribenzoate (4.2 g, 9.0 mmol) were coevaporated with 1,4-dioxane (2x) and DCE (1x), dissolved in dry DCM (90 ml) and stirred with freshly activated 3 Å molecular sieves at room temperature for 1 hour under argon to remove traces of water. The solution was then cooled to -78 o C and TMSOTf (17 μl, 1 mol%) was added to the reaction mixture. The reaction was being stirred at the same temperature for 10 minutes after which it was quenched by the addition of triethylamine. The reaction mixture was concentrated in vacuo and purified by silica gel chromatography (Pentane/EA, 90/10 80/20) to afford the title compound as a white foam (6.56 g, 7.59 mmol, 84%). 1 H NMR (500 MHz, CDCl 3 ) δ (m, 4H, arom. Bz), (m, 2H, arom. Bz), (m, 5H, arom.), (m, 5H, arom.), (m, 6H, arom.), (m, 8H, arom.), 6.80 S1

3 (d, J = 4.2 Hz, 1H, H1 ), 5.67 (dd, J = 6.4, 1.9 Hz, 1H, H3 ), 5.29 (d, J = 3.5 Hz, 1H, H1 ), 4.75 (q, J = 3.8 Hz, 1H, H4 ), 4.71 (dd, J = 6.5, 4.3 Hz, 1H, H2 ), 4.66 (AB, J = 12.0, 3.4 Hz, 1H, H5 ), (m, 1H, H5 ), (m, 6H, CH 2 Bn), 4.22 (q, J = 3.6 Hz, 1H, H4 ), (m, 2H, H2, H3 ), 3.46 (AB, J = 10.8, 3.4 Hz, 1H, H5 ), 3.37 (AB, J = 10.8, 3.7 Hz, 1H, H5 ). 13 C NMR (126 MHz, CDCl 3 ) δ , , (CO Bz), , , (cq. arom.), , , (arom.), , (cq. arom.), (arom.), (cq. arom.), , , , , , , , , , , (arom.), (C1 ), (C1 ), (C4 ), (C4 ), (C2 ), (C3 ), (C2 ), 73.48, (CH 2 Bn), (C3 ), (CH 2 Bn), (C5 ), (C5 ). HRMS [C 52 H 48 O 12 + Na] + : found, calculated 1,3,5-tri-O-benzoyl-2-O-(3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3- diyl)-α-d-ribofuranosyl)-α-d-ribofuranosyl (5) Compound 4 (1.32 g, 7.3 mmol) was dissolved in tbuoh/dioxane/h 2 O (90 ml, 4/4/1; v/v/v) and Pd/C (600 mg, 10 wt. % Pd) was added. H 2 (g) was bubbled through the solution for 16 hours after which the reaction mixture was filtered over celite, concentrated in vacuo and co-evaporated with toluene (2x). Imidazole (1.49 g, 22 mmol) was added to the crude reaction mixture and coevaporated with toluene (2x). The mixture was dissolved in DCM (70 ml) and 1,3-dichloro-1,1,3,3- tetraisopropyldisiloxane (2.8 ml, 8.8 mmol) was added. The reaction was stirred at room temperature for 30 minutes and quenched upon the addition of H 2 O. The organic layer was washed with brine, dried over MgSO 4, concentrated under reduced pressure and purified by silica gel chromatography (eluting with Pentane/EA, 95/5 90/10) to obtain the product as a white foam (1.75 g, 2.09 mmol, 29%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.15 (dd, J = 8.3, 1.2 Hz, 2H, Bz), 8.11 (dd, J = 8.3, 1.2 Hz, 2H, Bz), 8.07 (dd, J = 8.3, 1.2 Hz, 2H, Bz), (m, 3H, Bz), (m, 2H, Bz), (m, 4H, Bz), 6.79 (d, J = 4.2 Hz, 1H, H1 ), 5.67 (dd, J = 6.3, 2.1 Hz, 1H, H3 ), 5.18 (d, J = 4.2 Hz, 1H, H1 ), 4.77 (q, J = 3.6 Hz, 1H, H4 ), 4.75 (dd, J = 6.3, 4.2 Hz, 1H, H2 ), 4.67 (AB, J = 12.1, 3.3 Hz, 1H, H5 ), 4.62 (AB, J = 12.1, 3.9 Hz, 1H, H5 ), (m, 1H, H3 ), (m, 1H, H2 ), 3.92 (ddd, J = 8.4, 4.9, 3.6 Hz, 1H, H4 ), 3.81 (AB, J = 11.7, 3.6 Hz, 1H, H5 ), 3.65 (AB, J = 11.7, 8.3 Hz, 1H, H5 ), 2.85 (d, J = 8.6 Hz, 1H, OH), 1.03 (t, J = 6.5 Hz, 12H, CH 3 TIPDS), 0.97 (d, J = 3.0 Hz, 6H, CH 3 TIPDS), 0.96 (d, J = 2.1 Hz, 6H, CH 3 TIPDS), 0.80 (d, J = 7.3 Hz, 2H, CH TIPDS), 0.73 (d, J = 7.3 Hz, 2H, CH TIPDS). 13 C NMR (126 MHz, CDCl 3 ) δ , (CO Bz), , , , , , (arom.), (cq. arom.), , , (arom.), (C1 ), (C1 ), (C4 ), (C4 ), (C2 ), (C3 ), (C2 ), (C3 ), (C5 ), (C5 ), 17.57, 17.51, 17.47, 17.43, 17.22, 17.19, 17.09, 16.85, 16.71, 13.48, 13.29, 13.19, 13.06, (CH, CH 3, TIPDS). HRMS [C 43 H 56 O 13 Si 2 + Na] + : found, calculated 1,3,5-tri-O-benzoyl-2 -O-(2 -O-(2,3-di-O-benzyl-5-Otriisopropylsilyl-α-D-ribofuranosyl)- 3,5-O-(1,1,3,3- tetraisopropyldisiloxane-1,3-diyl)- α-d-ribofuranosyl)-α-dribofuranosyl (6) (N-Phenyl)-2,2,2-trifluoroacetimido-5-triisopropylsilyl-2,3-O-benzyl-α,β- D-ribofuranose [2] (1.97 g, 3.0 mmol) and compound 5 (1.67 g, 2.0 mmol) were co-evaporated with 1,4-dioxane (2x), dissolved in dry DCM (40 ml) and left to stand with freshly activated 3 Å molecular sieves at room temperature for 16 hour under argon to remove traces of water. The solution was then cooled to -78 o C and TMSOTf (12 μl, 2 mol%) was added to the reaction mixture. The reaction was being stirred at the same temperature for 30 minutes after which it was quenched by the addition of triethylamine. The S2

4 reaction mixture was concentrated in vacuo and purified by silica gel chromatography (Pentane/EA, 95/5 90/10 85/15 ) to afford the title compound as a white foam (1.47 g, 1.13 mmol, 57%). 1 H NMR (500 MHz, CDCl 3 ) δ (m, 2H, Bz), (m, 4H, Bz), (m, 2H, Bz), (m, 3H, Bz), (m, 2H, arom.), (m, 7H, arom.), (m, 5H, arom.), 6.78 (d, J = 4.1 Hz, 1H, H1 ), 5.59 (dd, J = 6.2, 1.8 Hz, 1H, H3 ), 5.35 (d, J = 3.5 Hz, 1H, H1 ), 5.27 (d, J = 3.9 Hz, 1H, H1 ), 4.83 (dd, J = 6.2, 4.2 Hz, 1H, H2 ), 4.71 (d, J = 11.4 Hz, 1H, CH 2 Bn), (m, 1H, H4 ), 4.62 (AB, J = 12.0, 3.4 Hz, 1H, H5 ), 4.51 (AB, J = 12.0, 4.1 Hz, 1H, H5 ), 4.43 (d, J = 11.9 Hz, 1H, CH 2 Bn), 4.40 (dd, J = 5.1, 3.6 Hz, 1H, H2 ), 4.31 (d, J = 11.8 Hz, 1H, CH 2 Bn), (m, 2H, H3, H4 ), 4.04 (dt, J = 8.8, 2.2 Hz, 1H, H4 ), 3.96 (d, J = 11.4 Hz, 1H, CH 2 Bn), 3.81 (AB, J = 13.0, 2.0 Hz, 1H, H5 ), (m, 2H, H3, H5 ), 3.71 (AB, J = 10.9, 3.2 Hz, 1H, H5 ), 3.66 (AB, J = 11.0, 3.2 Hz, 1H, H5 ), 3.43 (dd, J = 6.4, 3.9 Hz, 1H, H2 ), (m, 45H, CH, CH 3, TIPDS, TIPS), (m, 4H, CH, TIPDS). 13 C NMR (126 MHz, CDCl 3 ) δ , , (CO Bz), , (cq. arom.), , , , (arom.), (cq. arom.), , (arom.), , (cq. arom.), , , , , , , , , (arom.), (C1 ), (C1 ), (C1 ), (C4 ), 81.24, (C4, C4 ), (C2 ), (C3 ), (C2 ), (C2 ), (C3 ), 72.35, (CH 2 Bn), (C3 ), (C5 ), (C5 ), (C5 ), 18.11, 17.55, 17.50, 17.44, 17.25, 17.21, 17.12, 16.99, 13.65, 13.18, 12.82, 12.64, (CH, CH 3, TIPS, TIPDS). HRMS [C 71 H 96 O 17 Si 3 + Na] + : found, calculated 1,3,5-tri-O-benzoyl-2 -O-(2 -O-(2,3-di-O-acetyl-5-Otriisopropylsilyl-α-D-ribofuranosyl)- 3-O-acety l-5-otertbutyldiphenylsilyl- α-d-ribofuranosyl)-α-d-ribofuranosyl (7) Compound 6 (1.43 g, 1.1 mmol) was dissolved in tbuoh/dioxane/h 2 O (20 ml, 4/4/1; v/v/v) and Pd/C (110 mg, 10 wt. % Pd) was added. H 2 (g) was bubbled through the solution for 16 hours. TLC and TLC-MS analysis indicated no complete conversion and additional Pd/C (250 mg) was added. The reaction was stirred for 2 days after which the reaction mixture was filtered over celite, concentrated in vacuo and co-evaporated with pyridine (2x). The mixture was dissolved in pyridine (10 ml) and HF.pyridine (1.3 ml) was added. The reaction was stirred at room temperature for 1.5 hour and quenched by the carefull addition of aq. NaHCO 3 (sat.). The mixture was extracted with DCM (2x), the organic layers were dried (MgSO 4 ) and concentrated under reduced pressure. The crude was coevaporated with pyridine (2x) and dissolved in pyridine (15 ml). Tert-butyldiphenylsilyl chloride (0.57 ml, 2.2 mmol) was added and the mixture was stirred at room temperature for 16 hours. Acetic anhydride (3 ml, 30 mmol) was added and the reaction was stirred for 6 hours. The reaction mixture was concentrated, dissolved in EtOAc and washed with aq. HCl (1M). The organic layer was washed with aq. NaHCO 3 (sat.), dried (MgSO 4 ) and concentrated in vacuo. Purification by silica gel chromatography (Pentane/EA, 90/10 85/15 80/20 ) afforded the title compound as a white foam (444 mg, 0.36 mmol, 33%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.20 (d, J = 7.3 Hz, 2H, Bz), 8.16 (d, J = 7.3 Hz, 2H, Bz), 8.07 (d, J = 7.4 Hz, 2H, Bz), (m, 7H, arom.), (m, 12H, arom.), 6.78 (d, J = 4.3 Hz, 1H, H1 ), 5.71 (dd, J = 6.3, 1.5 Hz, 1H, H3 ), 5.47 (dd, J = 6.5, 1.6 Hz, 1H, H3 ), (m, 3H, H1, H1, H3 ), 4.81 (dd, J = 6.9, 4.5 Hz, 1H, H2 ), (m, 1H, H4 ), 4.67 (AB, J = 12.0, 3.4 Hz, 1H, H5 ), 4.62 (AB, J = 12.1, 4.2 Hz, 1H, H5 ), 4.59 (dd, J = 6.2, 4.5 Hz, 1H, H2 ), 4.35 (dd, J = 6.5, 4.3 Hz, 1H, H2 ), (m, 2H, H4, H4 ), 3.88 (AB, J = 10.9, 2.5 Hz, 1H, H5 ), 3.75 (AB, J = 10.9, 2.8 Hz, 1H, H5 ), 3.65 (AB, J = 11.2, 2.6 Hz, 1H, H5 ), 3.61 (AB, J = 11.3, 3.1 Hz, 1H, H5 ), 2.01 (s, 3H, CH 3 Ac), 1.78 (s, 3H, CH 3 Ac), 1.66 (s, 3H, CH 3 Ac), (m, 21H, CH, CH 3, TIPS), 1.00 (s, 9H, CH 3 TBDPS). S3

5 13 C NMR (126 MHz, CDCl 3 ) δ , , (CO Ac), , , (CO Bz), , , , (arom.), , , (cq. arom.), , (arom.), (cq. arom.), , , (arom.), (cq. arom.), , , , (arom.), (C1 ), (C1 ), (C1 ), 83.83, 83.64, (C4, C4, C4 ), (C2 ), (C2 ), (C3 ), (C2 ), (C3 ), (C3 ), (C5 ), (C5 ), (C5 ), (CH 3 TBDPS), 20.71, 20.43, (CH 3 Ac), (cq. TBDPS), 18.07, 18.06, (CH, CH 3, TIPS). HRMS [C 67 H 82 O 19 Si 2 + Na] + : found, calculated N 6 -benzoyl-9-[3,5-di-o-benzoyl-2-o-(2 -O-(2,3-di-O-acetyl-5-Otriisopropylsilyl-α-D-ribofuranosyl)-3-O-acetyl-5-Otertbutyldiphenylsilyl-α-D-ribofuranosyl)- -D-ribofuranosyl]- adenine (8) Compound 7 (311 mg, 0.25 mmol) and N 6 -benzoyladenine (120 mg, 0.5 mmol) were co-evaporated with dioxane (3x) and MeCN (1x) and dissolved in MeCN (4 ml) under an argon atmosphere. N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) (0.4 ml, 1.5 mmol) was added and the mixture was stirred at room temperature until everything was dissolved. HClO 4 -SiO 2 (312 mg, 0.5 eq; 0.4 mmol/g) was added and the mixture was refluxed for 2 hours. TLC analysis showed no complete conversion and additional HClO 4 -SiO 2 (312 mg, 0.5 eq) was added. The mixture was refluxed for two more hours, filtered and concentrated in vacuo. Purification by silica gel chromatography (eluting with DCM/EA, 100/0 90/10 80/20 70/30) yielded the product as a white foam (215 mg, 0.16mmol, 63%). 1 H NMR (600 MHz, CDCl 3 ) δ 9.12 (s, 1H, NH), 8.67 (s, 1H, H2), 8.39 (s, 1H, H8), 8.07 (t, J = 8.2 Hz, 4H, arom.), 8.03 (d, J = 7.5 Hz, 2H, arom.), (m, 9H, arom.), (m, 10H, arom.), 6.31 (d, J = 4.6 Hz, 1H, H1 ), 5.93 (t, J = 5.4 Hz, 1H, H3 ), 5.66 (t, J = 5.0 Hz, 1H, H2 ), 5.45 (dd, J = 6.9, 2.2 Hz, 1H, H, H3 ), 5.30 (dd, J = 7.2, 3.2 Hz, 1H, H3 ), 5.24 (d, J = 4.4 Hz, 1H, H1 ), 5.13 (d, J = 4.4 Hz, 1H, H1 ), 4.88 (AB, J = 12.1, 3.7 Hz, 1H, H5 ), 4.84 (dd, J = 7.2, 4.4 Hz, 1H, H2 ), (m, 1H, H4 ), 4.69 (AB, J = 12.1, 4.9 Hz, 1H, H5 ), 4.31 (dd, J = 6.8, 4.4 Hz, 1H, H2 ), 4.08 (q, J = 2.8 Hz, 1H, H4 ), 3.99 (q, J = 2.5 Hz, 1H, H4 ), 3.86 (AB, J = 11.1, 2.5 Hz, 1H, H5 ), 3.75 (AB, J = 11.1, 3.0 Hz, 1H, H5 ), 3.57 (AB, J = 11.2, 2.6 Hz, 1H, H5 ), 3.43 (AB, J = 11.3, 3.2 Hz, 1H, H5 ), 2.11 (s, 3H, CH 3 Ac), 2.06 (s, 3H, CH 3 Ac), 1.69 (s, 3H, CH 3 Ac), (m, 30H, CH, CH 3, TIPS, TBDPS). 13 C NMR (151 MHz, CDCl 3 ) δ , , (CO Ac), , , (CO Bz), (CH C2), (C4), (C6), (CH C8), , (arom.), (cq. arom.), , (arom.), (cq. arom.), (arom.), (cq. arom.), , , (arom.), , (cq. arom.), , , , , , (arom.), (C5), (C1 ), (C1 ), (C1 ), (C4 ), (C4 ), (C4 ), (C2 ), (C2 ), (C3 ), (C2 ), (C3 ), (C3 ), (C5 ), (C5 ), C5 ), (CH 3 TBDPS), 20.80, 20.68, (CH 3 Ac), (cq. TBDPS), 18.01, 17.99, (CH, CH 3, TIPS). HRMS [C 72 H 85 N 5 O 18 Si 2 + H] + : found, calculated N 6 -benzoyl-9-[3,5-di-o-benzoyl-2-o-(2 -O-(2,3-di-O-acetyl-5- O-triisopropylsilyl-α-D-ribofuranosyl)-3-O-acetyl-α-Dribofuranosyl)- -D-ribofuranosyl]-adenine (9) Compound 8 (30 mg, 22 mol) was dissolved in pyridine (1 ml) and HF.pyridine (50 L) was added. The mixture was stirred at room temperature for 1 hour after which it was quenched by the carefull addition of aq. NaHCO 3 (sat.). The mixture was extracted with DCM (2x) and the organic layers dried over MgSO 4. After concentration under reduced pressure, the crude was purified by silica gel S4

6 chromatography (DCM/EA, 70/30 50/50) to afford the title compound as a white foam (18 mg, 16 mol, 73%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.66 (s, 1H, H2), 8.38 (s, 1H, H8), 8.11 (d, J = 7.7 Hz, 2H, Bz), 8.06 (d, J = 7.5 Hz, 2H, Bz), 8.03 (d, J = 7.7 Hz, 2H, Bz), (m, 2H, Bz), (m, 3H, Bz), (m, 4H, Bz), 6.31 (d, J = 4.4 Hz, 1H, H1 ), (m, 1H, H3 ), 5.71 (t, J = 4.8 Hz, 1H, H2 ), 5.27 (dd, J = 7.1, 3.0 Hz, 1H, H3 ), (m, 1H, H1 ), 5.21 (dd, J = 7.1, 3.0 Hz, 1H, H3 ), 5.12 (d, J = 4.3 Hz, 1H, H1 ), (m, 2H, H2, H5 ), 4.76 (q, J = 4.5 Hz, 1H, H4 ), 4.69 (AB, J = 12.0, 4.7 Hz, 1H, H5 ), 4.15 (dd, J = 6.8, 3.7 Hz, 1H, H2 ), 4.06 (d, J = 2.6 Hz, 1H, H4 ), 4.01 (d, J = 2.7 Hz, 1H, H4 ), 3.83 (AB, J = 10.9, 2.5 Hz, 1H, H5 ), 3.73 (AB, J = 10.9, 2.6 Hz, 1H, H5 ), (m, 2H, H5 ), 2.08 (s, 3H, CH 3 Ac), 2.05 (s, 3H, CH 3 Ac), 1.68 (s, 3H, CH 3 Ac), (m, 21H, CH, CH 3, TIPS). 13 C NMR (126 MHz, CDCl 3 ) δ , , (CO Ac), , (CO Bz), (C4), (C6), (arom.), (cq. arom.), , , , (arom.), (cq. arom.), , , , (arom.), (C5), (C1 ), (C1 ), (C1 ), (C4, C4 ), (C4 ), (C2 ), (C2 ), (C3 ), (C2 ), (C3 ), (C3 ), (C5 ), (C5 ), (C5 ), 29.83, 20.77, 20.70, (CH 3 Ac), 18.02, 18.00, (CH, CH 3, TIPS). HRMS [C 56 H 67 N 5 O 18 Si + H] + : found, calculated 9-[2 -O-((2 -O-(α-D-ribofuranosyl)-α-D-ribofuranosyl)-β-Dribofuranosyl]-adenine (10) Compound 8 (136 mg, 100 mol) was dissolved in THF (2 ml) and tetrabutylammonium fluoride (TBAF) (0.3 ml; 1M in THF) was added. After 2 hours additional TBAF (0.6 ml) was added and the reaction was stirred for 16 hours at room temperature. The reaction mixture was transferred to a microwave vial, aq. NH 4 OH (30%) (10 ml) was added and the vial was sealed with a septum. The mixture was stirred at 50 o C for 16 hours, concentrated in vacuo and purified over HW-40 gel filtration (0.15 M, ammonium acetate). Repeated lyophilization yielded the title compound as a white solid (41 mg, 76 mol, 76%). 1 H NMR (500 MHz, D 2 O) δ 8.35 (s, 1H, H8), 8.16 (s, 1H, H2), 6.20 (d, J = 6.6 Hz, 1H, H1 ), 5.25 (d, J = 3.6 Hz, 1H, H1 ), 4.96 (d, J = 4.0 Hz, 1H, H1 ), 4.90 (dd, J = 6.5, 5.1 Hz, 1H, H2 ), 4.56 (dd, J = 5.1, 2.7 Hz, 1H, H3 ), 4.35 (q, J = 3.0 Hz, 1H, H4 ), (m, 1H, H4 ), (m, 2H, H2, H3 ), (m, 1H, H4 )), (m, 2H, H2, H3 ), 3.96 (AB, J = 12.9, 2.8 Hz, 1H, H5 ), 3.88 (AB, J = 12.9, 3.5 Hz, 1H, H5 ), 3.73 (AB, J = 12.4, 3.4 Hz, 2H, H5, H5 ), 3.65 (dt, J = 12.3, 5.1 Hz, 2H, H5, H5 ). 13 C NMR (126 MHz, D 2 O) δ (C6), (C2), (C4), (C8), (C5), (C1 ), (C1 ), (C1 ), (C4 ), (C4 ), (C4 ), (C2 ), (C2 ), (C2 ), (C3 ), (C3 ), (C3 ), 61.55, 61.37, (C5, C5, C5 ). HRMS [C 20 H 29 N 5 O 12 + Na] + : found, calculated [1] G. J. van der Heden van Noort, H. S. Overkleeft, G. A. van der Marel, D. V. Filippov, Org. Lett. 2011, 13, [2] H. A. V. Kistemaker, G. J. van der Heden van Noort, H. S. Overkleeft, G. A. van der Marel, D. V. Filippov, Org. Lett. 2013, 15, S5

7 Compound 4, 1 H NMR 500 MHz, CDCl 3 S6

8 Compound 4, 13 C NMR 126 MHz, CDCl 3 S7

9 Compound 4, HSQC-HECADE S8

10 Compound 4, HSQC-HECADE S9

11 Compound 5, 1 H NMR 500 MHz, CDCl 3 S10

12 Compound 5, 13 C NMR 126 MHz, CDCl 3 S11

13 Compound 6, 1 H NMR 500 MHz, CDCl 3 S12

14 Compound 6, 13 C NMR 126 MHz, CDCl 3 S13

15 Compound 6, HSQC-HECADE S14

16 Compound 6, HSQC-HECADE S15

17 Compound 7, 1 H NMR 500 MHz, CDCl 3 S16

18 Compound 7, 13 C NMR 126 MHz, CDCl 3 S17

19 Compound 8, 1 H NMR 600 MHz, CDCl 3 S18

20 Compound 8, 13 C NMR 151 MHz, CDCl 3 S19

21 Compound 8, HSQC-HECADE S20

22 Compound 8, HSQC-HECADE S21

23 Compound 9, 1 H NMR 500 MHz, CDCl 3 S22

24 Compound 9, 13 C NMR 126 MHz, CDCl 3 S23

25 Compound 10, 1 H NMR 500 MHz, D 2 O S24

26 Compound 10, 1 H NMR 500 MHz, D 2 O, H 2 O suppression S25

27 Compound 10, 13 C NMR 126 MHz, D 2 O S26

28 Compound 10, 1 H NMR 400 MHz, D 2 O, COSY S27

29 Compound 10, 1 H NMR 400 MHz, D 2 O, HSQC S28

30 Compound 10, 1 H NMR 400 MHz, D 2 O, HMBC S29