Supporting Information. for. Angew. Chem. Int. Ed. Z Wiley-VCH 2003

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1 Supporting Information for Angew. Chem. Int. Ed. Z52089 Wiley-VCH Weinheim, Germany

2 DNA Topoisomerase Inhibitor Acutissimin A and Other Flavano- Ellagitannins in Red Wine Stéphane Quideau, * Michael Jourdes, Cédric Saucier, Yves Glories, Patrick Pardon and Christian Baudry [*] Dr. S. Quideau Institut Européen de Chimie et Biologie 16 avenue Pey-Berland, Pessac Cedex (France) Fax: (+33) s.quideau@iecb-polytechnique.u-bordeaux.fr Experimental Section General. ( )-Vescalagin (1a, 84% pure) and ( )-castalagin (1b, 86% pure) were extracted from Quercus robur and purified as previously described. [16] (+)-Catechin (3a) and ( )-epicatechin (3b) were purchased from Fluka. XAD 7 HP resin was purchased from Supelco and the TSK gel HW 40F was purchased from VWR International. Tetrahydrofuran (THF) was purified by distillation from sodium/benzophenone under N 2 immediately before use. Methanol was of HPLC quality and Milli-Q (Millipore) water was used for HPLC analyses and separations. HPLC analyses were carried out on a Thermo system using a Merck C-18 Lichrospher column (4.6 x 250 mm, 5 µm) with P1000 XR pumps, an autosampler AS3000 and a diode array detector UV 6000 LP, and on a Hewlett Packard serie 1100 system coupled to a Micromass Platform II for electrospray ionisation mass spectrometry (ESIMS) analysis. The mobile phase was composed of solvent A [H 2 O H 3 PO 4 (999:1)] and solvent B [Me H 3 PO 4 (999:1)]. The electrospray ionisation mass spectrometry detection was performed in negative ion mode with the following optimised parameters: source temperature 120 C, nebulizer gas flow 9 l/h, desolvation gas flow 220 l/h, and capillary voltage 3.5 kv. The cone voltage was 60 ev for pure compounds and 90 ev for wine samples. Semipreparative HPLC purifications were performed on a Varian Pro Star system using a Dinamax C-18 column (10 mm 250 mm, 5 µm). Column effluent was monitored by UV detection at 280 nm using a ProStar 320 UV-visible detector. NMR spectra of samples in the indicated solvent were run at 400 Mhz on a Bruker DPX 400 MHz spectrometer. Carbon multiplicities were determined by 1

3 DEPT135 experiments. Proton and carbon NMR signals were assigned on the basis of data comparison with published data on C-glycosidic ellagitannins, flavan-3-ols and flavanoellagitannins [11,12,17-21]. Connectivity between the flavanoid and the ellagic units in acutissimin and epiacutissimin regioisomers A and B was established via observation of diagnostic correlations in NMR HMBC spectra. Briefly, the glucose H-1 proton correlates with the flavonoid C-8 a carbon in the A regioisomers and with the flavonoid C-5 carbon in the B regioisomers. A similar analysis has been used to determine the regiochemistry of related complex tannins [22] and to assign interflavan connectivity in procyanidins [23, 24]. Relevant sections of HMBC spectra are shown below, and NMR signal assignments of the previously unknown epiacutissimins A and B are displayed in Tables 1 and 2. IR spectra were recorded with a FT-IR spectrometer Perkin Elmer Paragon 1000 PC spectrometer. Liquid secondary ion mass spectrometry low and high resolution (LSIMS, HRMS) were obtained from the mass spectrometry laboratory at the CESAMO, Université Bordeaux 1. Optical rotations were recorded using a polarimeter ADP 220 Bellingham Stanley Ltd. NB: It is often claimed that ellagitannins are not stable to air because the presence of so many phenolic hydroxyl groups per molecule must render them highly sensitive to oxygen-mediated autoxidation. Application potential of this class of natural products in drug development is thus considered very limited. We tested the stability of ( )-castalagin (1b) by exposing a 20 mg sample to air for 12 days in the dark. No degradation or structural changes was evidenced by 1 H NMR and HPLC analysis of the sample. Hemisynthesis of acutissimins A (4a) and B (4b). In organic solution. ( )-vescalagin (1a, 42 mg, mmol) and (+)-catechin (3a, 15 mg, mmol) were dissolved in THF (20 ml), and trifluoroacetic acid (300 µl, 3.9 mmol) was added. The reaction mixture was stirred at 60 C for 7 h, after which time HPLC monitoring indicated completion of the reaction. A gradient elution (0-20 min: 0% to 20% solvent B, min: 20% to 100% solvent B, min: 100% solvent B) was applied at a flow rate of 1.0 ml.min -1. The mixture was evaporated under vacuum and the residue was dissolved in water and freeze-dried. Semi-preparative HPLC separation of the resulting powder was carried out with a mobile phase composed of solvent C [H 2 O HCO (996:4)] and solvent D [Me HCO (996:4)]. A gradient elution (0-20 min: 0% to 20% solvent D, min: 20% to 100% solvent D, min: 100% solvent D) was applied at a flow rate of 16 ml.min -1 to furnish, after freeze-drying, 4a (35 mg, 65%) and 4b (12 mg, 22%). 2

4 In wine model solution. The wine model solution was an aqueous solution composed of 12% ethanol and 5g/l of tartaric acid. The ph of the solution was adjusted to a value of 3.2 by adding Na. ( )-Vescalagin (1a, 2 mg, mmol) and (+)-catechin (3a, 2 mg, mmol) were dissolved in the wine model solution (2 ml), and the mixture was stirred at room temperature for 25 days, after which time HPLC monitoring of the disappearance of 1a indicated that the reaction was complete. A gradient elution (0-20 min: 0% to 10% solvent B, min: 10% to 100% solvent B, min: 100% solvent B) was applied at a flow rate of 1.0 ml.min Acutissimin A (4a). Off-white amorphous powder; [α] D = [c = 0.2, acetone; lit. [12] 32 [α] D = (c = 1.2, acetone)]; IR (KBr): 3383, 1740, 1616 cm -1 ; 1 H NMR (400 MHz, acetone-d 6 ) δ 2.39 (d, J = 14.5 Hz, 1 H, Hβ-4 ), 2.86 (d, J = 16.5 Hz, 1 H, Hα-4 ), 4.08 (d, J = 12.0 Hz, 1 H, Hα- 6), 4.56 (s, 1 H, H-3 ), 4.68 (d, J = 12.1 Hz, 1 H, Hβ-6), 4.74 (d, J = 6.9 Hz, 1 H, H-3), 4.80 (s, 1 H, H-1), 5.18 (s, 1 H, H-2), 5.24 (t, J = 7.3 Hz, 1 H, H-4), 5.49 (s, 1 H, H-2 ), 5.63 (d, J = 7.5 Hz, 1 H, H-5), 6.04 (s, 1 H, H-6 ), 6.62 (s, 1 H, H V -2 ), 6.77 (s, 1 H, H III -2 ), 6.79 (d, J = 8.3 Hz, 1 H, H-5 ), 6.88 (s, 1 H, H-2 ), 6.94 (d, J = 7.7 Hz, 1 H, H-6 ), 7.07 (s, 1 H, H IV -2 ); 13 C NMR (100 MHz, acetone-d 6 ) δ (C V =O) (C I =O), (C IV =O), (C III =O), (C II =O), (C- 7 ), (C-5 ), (C-8 a), (C III -3 ), , , 144.7, , 144.0, 143.3(C II -3, C [I-V] -5 ), (C IV -3 ), (C V -3 ), (C-3 ), (C-4 ), (C IV -4 ), 136.2, (C [I-II] -4 ), (C III -4 ), (C V -4 ), (C-1 ), 127.6, 127.2, 125.1, (C [II-V] -1 ), (C I -1 ), (C I -2 ), (C-6 ), 116.5, , (C II -2 II, C [I-II] -6 ), (C V -6 ), (C IV -6 ), (C III -6 ), (C-5 ), (C-2 ), (C III -2 ), (C V -2 ), (C IV -2 ), (C-8 ), 97.9 (C-4 a), 96.5 (C-6 ), 79.6 (C-2 ), 77.2 (C-2), 71.9 (C-3), 70.8 (C-5), 70.7 (C-4), 67.3 (C-3 ), 65.4 (C-6), 37.5 (C-1), 23.4 (C-4 ); LSIMS m/z (relative intensity) 1229 (MNa +, 5), 1207 (MH +, 8), 447 (46), 391 (100); HRMS (LSIMS) calcd for C 56 H 39 O found NB: NMR spectra of acutissimin B (4b) and epiacutissimin A (5a) could not be run in deuterated acetone because of solubility problems. A deuterated acetone water solvent mixture was used. In this solvent system, two rotamers of the flavanol unit, here designated as ra and rb (unassigned), were observed as previously described for proanthocyanidins [2, 25]. 22 Acutissimin B (4b). Off-white amorphous powder; [α] D = -7.5 [c = 0.2, acetone; lit. [12] 32 [α] D = -5.5 (c = 0.84, acetone)]; IR (KBr): 3392, 1734, 1612 cm H NMR [400 MHz, acetone-d 6 /D 2 O (9:1)] δ 2.49 (dd, J = 8.4, 16.0 Hz, Hβ-4 ra), 2.61 (dd, J = 8.8, 15.2 Hz, Hα-4 rb and Hβ-4 rb), 2.88 (dd, J = 5.5, 16.0 Hz, Hα-4 ra), 3.99 (s, H-3 ra), 4.01 (s, H-3 H rb), 4.03 (d, J = 13.0 Hz, Hα- 3

5 6), 4.54 (s, H-2 ra), 4.57 (s, H-2 rb), 4.72 (s, H-1), 4.83 (d, J = 7.5 Hz, H-3), 4.87 (d, J = 13.3 Hz, Hβ-6), 5.11 (s, H-2), 5.25 (t, J = 7.4 Hz, H-4), 5.64 (d, J = 7.6 Hz, H-5), 5.87 (s, H-8 ra), 6.04 (s, H-8 rb), 6.64 (s, H IV -2 ), 6.76 (d, J = 1.8 Hz, H-5 rb), 6.80 (s, H III -2 ), 6.81 (d, J = 1.6 Hz, H-5 ra), 6.82 (s, H-2 rb), 6.83 (s, H-2 ra), 6.91 (d, J = 1.9 Hz, H-6 rb), 6.96 (d, J = 1.3 Hz, H-6 ra), 7.10 (s, H V -2 ); 13 C NMR [100 MHz, acetone-d 6 /D 2 O (9:1)] δ (C IV =O), (C I =O), (C III =O), (C V =O), (C II =O), (C-8 a rb), (C-8 a ra), (C-7 ), (C- 5 ), (C-3 ), (C-4 ), (C III -3 ), (C IV -3 ), (C V -3 ), , , 144.3, 144.2, 144.1, (C II -3, C [I-V] -5 ), (C V -4 ), 136.5, (C [I-II] -4 ), (C III -4 ), (C IV -4 ), (C-1 rb), (C-1 ra), 127.7, 126.7, 125.0, (C [II-V] -1 ), (C I - 1 ), (C I -2 ), (C-2 rb), (C-2 ra), (C V -6 ), , 113.7, (C II -2, C [I- II]-6 ), (C-5 rb), (C-5 ra), (C-6 rb), (C-6 ra), (C III -6 ), (C IV -6 ), (C V -2 ), (C III -2 ), (C IV -2 ), (C-6 ), (C-4 a rb), (C-4 a ra), 95.8 (C-8 rb), 94.8 (C-8 ra), 82.2 (C-2 rb), 82.1 (C-2 ra), 77.9 (C-2), 71.6 (C-3), 71.0 (C-5), 70.0 (C-4), 68.0 (C-3 rb), 67.8 (C-3 ra), 65.4 (C-6), 37.7 (C-1), 28.4 (C-4 rb), 26.7 (C-4 ra); LSIMS m/z (relative intensity) 1229 (MNa +, 46), 1207 (MH +, 11), 1055 (14); HRMS (LSIMS) calcd for C 56 H 38 O 31 Na found Hemisynthesis of epiacutissimins A (5a) and B (5b). In organic solution. ( )-vescalagin (1a, 42 mg, mmol) and ( )-epicatechin (3b, 16 mg, mmol) were dissolved in THF (20 ml), and trifluoroacetic acid (300 µl, 3.9 mmol) was added. The reaction mixture was stirred at 60 C for 9 h, after which time HPLC monitoring indicated completion of the reaction. The mixture was evaporated under vacuum and the residue was dissolved in water and freeze-dried. The resulting powder was analyzed and separated as described for the acutissimins 4a/4b to furnish, after freeze-drying, 5a (28 mg, 52%) and 5b (14 mg, 26%). 22 Epiacutissimin A (5a). Off-white amorphous powder; [α] D = (c = 0.53, acetone); IR (KBr): 3355, 1740, 1616 cm -1 ; 1 H NMR [400 MHz, acetone-d 6 /D 2 O (9:1)] δ 2.66 (dd, J = 3.2, 16.2 Hz, Hβ-4 ra ), 2.81 (dd, J = 4.4, 16.6 Hz, Hα-4 ra), 2.97 (bs, Hα-4 rb and Hβ-4 rb), 4.05 (d, J = 12.0 Hz, Hα-6), 4.21 (s, H-3 ra), 4.29.(s, H-3 rb), 4.69 (s, H-1), 4.81 (d, J = 12.1 Hz, Hβ-6), 4.86 (d, J = 4.1 Hz, H-3), 4.88 (s, H-2 ra and H-2 rb), 5.10 (s, H-2), 5.22 (t, J = 7.3, 7.4 Hz, H-4), 5.62 (d, J = 7.6 Hz, H-5), 5.92 (s, H-6 ra), 6.03 (s, H-6 rb), 6.63 (s, H IV -2 ), 6.79 (s, H III -2 ), 6.80 (bs, H-6 ra), 6.82 (bs, H-5 rb), 6.84 (bs, H-6 rb), 6.86 (bs, H-5 ra), 7.03 (bs, H-2 ra), 7.08 (bs, H-2 rb), 7.07(s, H V -2 ); 13 C NMR [100 MHz, acetone-d 6 /D 2 O (9:1)] δ (C III =O), (C I =O), (C V =O), (C IV =O),165.6 (C II =O), (C-5 ), (C-7 ), (C-8 a rb), (C- 8 a ra), (C III -3 ), (C IV -3 ), (C V -3 ), (C-4 ), (C-3 ), 144.6, 144.5, 4

6 144.38, , 144.1, (C II -3, C [I-V] -5 ), (C I -3 ), (C V -4 ), 136.6, (C [I-V] - 4 ), (C III -4 ), (C IV -4 ), (C-1 ra and C-1 rb), 127.6, 126.6, 125.0, (C [II-V] - 1 ), (C I -1 ), (C I -2 ), (C-6 rb), (C-6 ra), (C V -6 ), 116.4, 113.7, 113.2, (C II -2 and C [I-II] -6 ), (C-5 rb), (C-5 ra), (C-2 rb), (C-2 ra), (C IV -6 ), (C III -6 ), (C V -2 ), (C III -2 ), (C-8 ), (C III -2 ), 99.4 (C- 4 a ra and C-4 a rb), 95.8 (C-6 ra), 95.1 (C-6 rb), 78.9 (C-2 rb), 78.7 (C-2 ra), 77.8 (C-2), 71.6 (C-3), 70.9 (C-5), 70.0 (C-4), 66.4 (C-3 rb), 66.3 (C-3 ra), 65.5 (C-6), 37.8 (C-1), 29.1 (C-4 ra), 28.4 (C-4 rb); LSIMS m/z (relative intensity) 1245 (MK +, 21), 1229 (MNa +, 100), 1207 (MH +, 14), 1077 (10), 1055 (12); HRMS (LSIMS) calcd for C 56 H 38 O 31 Na found Table 1. NMR signal assignments of epiacutissimin A (5a). position δ H mult (J, Hz) δ C mult a HMQC b HMBC glucose [20,21] s 37.8 d C-1 C-7, C-8, C-8 a, C-1 I, C-2 I, C-3 I H-2, H s 77.8 d C-2 C-1, C-3, C-4, C-8, C-2 I, C I =O H-2, H d (J = 4.1 Hz) 71.6 d C-3 C-1, C-2, C II =O H-2, H-4, H t (J = 7.3, 7.4 Hz) 70.0 d C-4 C-2, C-3, C-5, C V =O H d (J = 7.6 Hz) 70.9 d C-5 C-3, C III =O H-4, H-6 6 α 4.05 d (J = 12.0 Hz) β 4.81 d (J = 12.1 Hz) 65.5 t C-6 C-6 aromatics [20,21] 1 I s H-1 1 [II-V] s, s, s, s 2 I s H-1, H-2 2 III 6.79 s d C-2 III C-3 III, C-4 III, C-6 III, C III =O 2 IV 6.63 s d C-2 IV C-3 IV, C-4 IV, C-6 IV, C IV =O 2 V 7.07 s d C-2 V C-3 V, C-4 V, C-6 V, C V =O 2 II and s, [I-II] s, s 3 I s H-1 3 III s H-2 III 3 IV s H-2 IV 3 V s H-2 V 3 II and s, [I-V] s, s, s, s, s, 5 C-5

7 4 [I-II] s, s 4 III s H-2 III 4 IV s H-2 IV 4 V s H-2 V 6 III s H-2 III 6 IV s H-2 IV 6 V s H-2 V carbonyls [20,21] C I =O s H-2 C II =O s H-3 C III =O s H-5, H-2 III C IV =O s H-2 IV C V =O s H-4, H-2 V epicatechin [18,24] 2 ra 4.88 s 78.7 d C-2 ra C-1 H4 ra 2 rb 4.88 s 78.9 d C-2 rb C-1 3 ra 4.21 s 66.3 d C-3 ra H4 ra 3 rb 4.29 s 66.4 d C-3 rb 4 ra α 2.66 dd (J = 3.2, t C-4 ra C-2 ra, C-3 ra, C-4 a, C-8 ara Hz) β 2.81 dd (J = 4.4, 16.6 Hz) 4 rb 2.97 bs 28.4 t C-4 rb 4 a* 99.4 s H4 ra, H6 ra, H6 rb 5 * s H6 ra, H6 rb 6 ra 5.92 s 95.8 d C-6 ra C-5, C-7, C-4 a 6 rb 6.03 s 95.1 d C-6 rb C-5, C-7, C-4 a 7 * s H-1, H6 ra, H6 rb 8 * s H-1, H-2 8 ara s H-1, H4 ra 8 arb s H-1 1 * s H-2,H-2, H5, H-6 2 ra 7.03 s d C-2 ra C-1 ra, C-3 ra, C-4 ra, C-6 ra H-6 ra 2 rb 7.08 s d C-2 rb C-1 rb, C-3 rb, C-4 rb, C-6 rb H-6 rb 3 * s H-2, H5 4 * s H-2, H5, H-6 5 ra 6.86 bs d C-5 ra C-1 ra, C-3 ra, C-4 ra 5 rb 6.82 bs d C-5 rb C-1 rb, C-3 rb, C-4 rb 6 ra 6.80 bs d C-6 ra C-1 ra, C-2 ra, C-4 ra H-2 ra 6 rb 6.84 bs d C-6 rb C-1 rb, C-2 rb, C-4 rb H-2 rb a Multiplicity inferred using the DEPT pulse sequence. b Carbons correlates to the proton resonance in the δ H column. *Proton and carbon NMR chemical shifts are the same for each rotamer. 6

8 22 Epiacutissimin B (5b). Off-white amorphous powder; [α] D = (c = 0.38, acetone); IR (KBr): 3338, 1734, 1617 cm -1 ; 1 H NMR NMR [400 MHz, acetone-d 6 ] δ 2.64 (d, J = 13.4 Hz, 1 H, Hβ-4 ), 2.99 (d, J = 13.7 Hz, 1 H, Hα-4 ), 4.02 (d, J = 12.4 Hz, 1 H, Hα-6), 4.47 (s, 1 H, H-3 ), 4.76 (d, J = 10.4 Hz, 1H, Hβ-6), 4.78 (bs, 1 H, H-3), 4.79 (s, 1 H, H-1), 5.13 (s, 1 H, H-2), 5.18 (t, J = 7.7 Hz, 1 H, H-4), 5.40 (s, 1 H, H-2 ), 5.62 (d, J = 7.6 Hz, 1 H, H-5), 6.06 (s, 1 H, H-8 ), 6.59 (s, 1 H, H IV -2 ), 6.64 (d, J = 8.6 Hz, 1 H, H-5 ), 6.75 (s, 2 H, H III -2, H V -2 ), 6.79 (bs, 1 H, H-6 ), 6.96 (s, 1 H, H-2 ); 13 C NMR [100 MHz, acetone-d 6 ] δ (C IV =O), (C I =O), (C III =O), (C II =O), (C V =O), (C-5 ), (C-8 a), (C-7 ), 145.3, , , 144.2, 144.0, (C II -3, C [I-V] -5 ), (C III -3 ), (C IV -3 ), (C V -3 ), (C- 4 ), (C-3 ), (C I -3 ), 137.1, (C [I-II] -4 ), (C V -4 ), (C III -4 ), (C IV - 4 ), (C-1 ), 127.5, 127.0, 126.4, (C [II-V] -1 ), (C I -1 ), (C I -2 ), (C-6 ), 116.5, 115.3, (C II -2, C [I-II] -6 ), (C-5 ), (C V -6 ), (C IV -6 ), (C III -6 ), (C-2 ), (C III -2 ), (C IV -2 ), (C V -2 ), (C-6 ), 99.3 (C-4 a), 96.4 (C-8 ), 79.2 (C-2 ), 77.7 (C-2), 71.8 (C-3), 70.8 (C-5), 69.5 (C-4), 66.2 (C-3 ), 65.6 (C-6), 37.2 (C-1), 26.7 (C-4 ); LSIMS m/z (relative intensity) 1245 (MK +, 12), 1229 (MNa +, 53), 1207 (MH +, 14), 1077 (5), 1055 (11); HRMS (LSIMS) calcd for C 56 H 38 O 31 Na found Table 2. NMR signal assignments of epiacutissimin B (5b). position δ H mult (J, Hz) δ C mult a HMQC b HMBC glucose [20,21] s 37.2 d C-1 C-5, C-6, C-7, C-1 I, C-2 I, C-3 I H-2, H s 77.7 d C-2 C-1, C-4, C-6, C-2 I, C I =O H bs 71.8 d C-3 C-1, C II =O H t (J = 7.7 Hz) 69.5 d C-4 C-2, C-5, C V =O H-2, H-5, H d (J = 7.6 Hz) 70.8 d C-5 C-3, C-4, C-6, C III =O H-4, H-6 6 α 4.02 d (J = 12.4 Hz) β 4.76 d (J = 10.4 Hz) 65.6 t C-6 C-6 C-5, C IV =O H-5 aromatics [20,21] 1 I s H-1 1 [II-V] s, s, s, s 2 I s H-1, H-2 2 III 6.75 s d C-2 III C-3 III, C-4 III, C-6 III, C III =O 2 IV 6.59 s d C-2 IV C-3 IV, C-4 IV, C-6 IV, C IV =O 2 V 6.75 s d C-2 V C-3 V, C-4 V, C-6 V, C V =O 2 II and 7

9 6 [I-II] s, s, s, 3 I H-1 3 III H-2 III 3 IV H-2 IV 3 V H-2 V 3 II and 5 [I-V] s s, s, s, s, s 4 [I-II] s, s 4 III s H-2 III 4 IV s H-2 IV 4 V s H-2 V 6 III s H-2 III 6 IV s H-2 IV 6 V s H-2 V carbonyls [20,21] C I =O s H-2 C II =O s H-3 C III =O s H-5, H-2 III C IV =O s H-6, H-2 IV C V =O s H-4, H-2 V epicatechin [18,24] s 79.2 d C s 66.2 d C-3 4 α 2.86 d (J = 16,5 Hz) β 2.39 d (J = 14,5 Hz) 26.7 t C-4 C-4 4 a 99.3 s H s H d H-1, H-2, H s H s 96.4 d C-8 C-4 a, C-6, C-8 a 8 a s H s H s d C-2 C-3, C-6 H s H s H-5, H d (J = 8.6 Hz) d C-5 C-1, C-4 H bs d C-6 C-2, C-4, C-5 H-2 a Multiplicity inferred using the DEPT pulse sequence. b Carbons correlates to the proton resonance in the δ H column. Identification of Acutissimins in Red Wine. A sample (100 ml) of a red wine aged for 18 months in an oak barrel was evaporated under vacuum and the resulting viscous dark red residue (3.83 g) 8

10 was dissolved in water (20 ml). This solution was loaded on column (150 mm 40 mm) that has been packed with some Amberlite XAD-7 HP resin, previously swelled in methanol overnight. An aqueous acidic solution [H 2 O HCO (996:4), 250 ml] was first used to wash out tartaric acid and sugars (2.82 g), and a 20% methanolic aqueous solution [H 2 O Me HCO (796:200:4), 250 ml] was then used to elute the ellagic fraction. This fraction (250 ml) was evaporated under vacuum to furnish a dark pink residue (372 mg), retaken in water (10 ml) and further purified by TSK HW 40F gel chromatography using a 120 mm 18 mm column. An aqueous acidic solution [H 2 O HCO (996:4), 50 ml] and a 70% methanolic aqueous solution [H 2 O Me HCO (296:700:4), 50 ml] were first used to separate further the sample mixture. A colorless fraction of 154 mg and a light red fraction of 155 mg were obtained after evaporation. The ellagitannins and flavano-ellegitannins of interest were then eluted using 50 ml of H 2 O acetone HCO (296:700:4). This fraction was evaporated under vacuum to furnish a reddish light brown residue (52 mg), which was dissolved in 400 µl of water for HPLC-ESIMS analysis (50 µl injection, Figure 3). NB: The UV-detected HPLC chromatogram is very complex (Figure 3A), but the presence of the oak ellagitannins vescalagin (1a), castalagin (1b), as well as other C-glycosidic ellagitannins (roburins and grandinin) [21,26], is clearly evidenced. The flavano-ellagitannins were more difficult to identify from this chromatogram, but their presence was determined from the m/z 1205 electrospray negative mode ion trace (Figure 3B). Four peaks were clearly distinguishable and relative comparison of their mass spectra and retention times with those of our hemisynthesized flavanoellagitannins permitted their unambiguous identification. The difference of about 48 s between retention times on the UV and ion trace chromatograms is due to connecting time between the UV and mass detectors. Additional References [17] K. Ishimaru, M. Ishimatsu, G. I. Nonaka, K. Mihashi, Y. Iwase, I. Nishioka, Chem. Pharm. Bull. 1988, 36, [18] A. L. Davis, Y. Cai, A. P. Davis, J. R. Lewis, Magn. Reson. Chem. 1996, 36, 887. [19] L. Balas, J. Vercauteren, Magn. Reson. Chem. 1994, 32, 386. [20] C. L. M. Hervé du Penhoat, V. M. F. Michon, A. Ohassan, S. Peng, A. Scalbert, D. Gage, Phytochemistry 1991, 30, 329. [21] C. L. M. Hervé du Penhoat, V. M. F. Michon, S. Peng, C. Viriot, A. Scalbert, D. Gage, J. Chem. Soc., Perkin Trans , [22] Z.-H. Jiang, T. Tanaka, I. Kouno, J. Nat. Prod. 1999, 62, 425. [23] L. Balas, J. Vercauteren, M. Laguerre, Magn. Reson. Chem. 1995, 33, 85. [24] M. L. Khan, E. Haslam, M. P. Williamson, Magn. Reson. Chem. 1997, 35, 854. [25] T. De Bruyne, L. Pieters, R. Dommisse, H. Kolodziej, V. Wray, D. Vanden Berghe, A. Vlietinck, in Plant Polyphenols 2: Chemistry, Biology, Pharmacology, Ecology (Eds.: G. G. Gross, R. W. Hemingway, T. Yoshida), Kluwer Academic/Plenum Publishers, New York, 1999, pp [26] G.-I. Nonaka, K. Ishimaru, R. Azuma, M. Ishimatsu, I. Nishioka, Chem. Pharm. Bull. 1989, 37, 20 9

11 HPLC Monitoring of Acutissimin Formation in the Acidic Organic Solution , , ,00 t= 0 h t= 7 h UV6000LP-280nm MJA 121 t0 UV6000LP-280nm MJA 121 6h 4a Elution gradient 0 to 20 min : 0 to 20 % B 1,8 1,6 1,4 m A U , mau 1, ,25 1a 4b 20 to 35 min : 20 to 100 % B 35 to 40 min : 100 % B 40 to 46 min : 100 to 0 % B 1,2 1, ,00 3a 0, ,75 0, ,50 0,4 0 0,25 0,2 0,00 0, ,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0 Minutes 1a: vescalagin, 3a: catechin, 4a: acutissimin A, 4b: acutissimin B 10

12 HPLC Monitoring of Acutissimin Formation in the Wine Model Solution t= 0 t= 6 jours t= 25 jours Elution gradient 0 to 20 min : 0 to 10 % B m a 20 to 30 min : 10 to 100 % B 30 to 40 min : 100 % B 40 to 45 min : 100 to 0 % B A U 150 1a ethylvescalagin 4a, 4b minutes 1a: vescalagin, 3a: catechin, 4a: acutissimin A, 4b: acutissimin B 11

13 12 Acutissimin A (4a) O O O O O O O O O O O V 8 a a I II III IV

14 Acutissimin A (4a) after HPLC Purification UV6000LP-280nm MJA Acutissimin 121 spf1 A after purification 2, ,00 1, , ,50 1,50 m ,25 1,25 A 800 mau 1,00 1,00 U 600 0,75 0, ,50 0, ,25 0,25 0 0,00 0,00-0, Minutes Minutes 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0-0,25 13

15 LC-MS of Acutissimin A (4a) -60eV % m/z 14

16 1 H/400 MHz/acetone-d 6 NMR of Acutissimin A (4a) 15 Integral (ppm)

17 1 H/400 MHz/acetone-d 6 NMR Peak Picking of Acutissimin A (4a) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

18 13 C/100 MHz/acetone-d 6 NMR of Acutissimin A (4a) (ppm)

19 13 C/100 MHz/acetone-d 6 NMR Peak Picking of Acutissimin A (4a) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

20 Dept 135/100 MHz/acetone-d 6 NMR of Acutissimin A (4a) (ppm)

21 Dept 135/100 MHz/acetone-d 6 NMR Peak Picking of Acutissimin A (4a) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

22 HMQC Spectrum of Acutissimin A (4a) (ppm) (ppm)

23 HMBC Spectrum of Acutissimin A (4a) (ppm) (ppm)

24 Portion of HMBC Spectrum of Acutissimin A (4a) Showing Relevant Connectivity Correlations between the Flavanoid and the C-Glycosidic Units H 1 -C 8 (ppm) H 1 -C 8a H 1 -C (ppm)

25 24 Acutissimin B (4b) 5 O O O O O O O O O O O 8 a a I II III V IV

26 Acutissimin B (4b) after HPLC Purification ,6 UV6000LP-280nm MJA Acutissimin 121 spf2 B after purification 1,6 1, , ,2 1,2 m A ,0 800 mau 0,8 1,0 0,8 U 600,6 0,6 400,4 0,4 200,2 0,2 0,0 0,0-0,2 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45, Minutes Minutes -0,2 25

27 LC-MS of Acutissimin B (4b) -60eV % m/z

28 1 H/400 MHz/(acetone-d 6/D2O 9 :1) NMR of Acutissimin B (4b) 27 Integral (ppm)

29 1 H/400 MHz/(acetone-d 6 /D 2 O 9 :1) NMR Peak Picking of Acutissimin B (4b) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

30 13 C/100 MHz/(acetone-d 6 /D 2 O 9 :1) NMR of Acutissimin B (4b) (ppm)

31 13 C/100 MHz/(acetone-d 6 /D 2 O 9 :1) NMR Peak Picking of Acutissimin B (4b) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

32 Dept 135/100 MHz/(acetone-d 6 /D 2 O 9 :1) NMR of Acutissimin B (4b) 31

33 Dept 135/100 MHz/(acetone-d 6 /D 2 O 9 :1) NMR Peak Picking of Acutissimin B (4b) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

34 HMQC Spectrum of Acutissimin B (4b) (ppm) (ppm)

35 HMBC Spectrum of Acutissimin B (4b) (ppm) (ppm)

36 Portion of HMBC Spectrum of Acutissimin B (4b) Showing Relevant Connectivity Correlations between the Flavanoid and the C-Glycosidic Units (ppm) H 1 -C H 1 -C 7 H 1 -C (ppm)

37 HPLC Monitoring of Epiacutissimin Formation in the Acidic Organic Solution m A U mau 2,0 1, , , ,2 1,0 UV6000LP-280nm UV6000LP-280nm t= MJA 0122 h t0 MJA t= hh 1a 5b 5a 2b Elution gradient 0 to 20 min : 0 to 20 %B 20 to 35 min : 20 to 100 %B 35 to 40 min : 100 %B 40 to 46 min : 100 to 0 % B 2,2 2,0 1,8 1,6 1,4 1,2 1,0 800,8 0, ,6 0, ,4 0,4 200,2 0,2 00,0 0,0 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0 0 5 Minutes 45 Minutes 1a: vescalagin, 2b: epicatechin, 5a: epiacutissimin A, 5b: epiacutissimin B 36

38 37 Epiacutissimin A (5a) O O O O O O O O O O O V 8 a a I II III IV

39 Epiacutissimin A (5a) after HPLC Purification Epiacutissimin A after purification UV6000LP-280nm MJA 122 spf ,00 2, ,75 1, ,50 1,50 m ,25 1,25 A U mau 800 1, ,75 1,00 0, ,50 0, ,25 0,25 0 0,00 0,00-0,25 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0 0 5 Minutes Minutes -0,25 38

40 LC-MS of Epiacutissimin A (5a) -60eV % m/z

41 1 H/400 MHz/(acetone-d 6/D2O 9 :1) NMR of Epiacutissimin A (5a) 40 Integral (ppm)

42 1 H/400 MHz/(acetone-d 6 /D 2 O 9:1) NMR Peak Picking of Epiacutissimin A (5a Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

43 13 C/100 MHz/(acetone-d 6 /D 2 O 9:1) NMR of Epiacutissimin A (5a) (ppm)

44 13 C/100 MHz/(acetone-d 6 /D 2 O 9:1) NMR Peak Picking of Epiacutissimin A (5a) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

45 Dept 135/100 MHz/(acetone-d 6 /D 2 O 9 :1) NMR of Epiacutissimin A (5a) (ppm)

46 Dept 135/100 MHz/(acetone-d 6 /D 2 O 9:1) NMR Peak Picking of Epiacutissimin A (5a) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

47 HMQC Spectrum of Epiacutissimin A (5a) (ppm) (ppm)

48 HMBC Spectrum of Epiacutissimin A (5a) (ppm) (ppm)

49 Portion of HMBC Spectrum of Epiacutissimin A (5a) Showing Relevant Connectivity Correlations between the Flavanoid and the C-Glycosidic Units H 1 -C 8 (ppm) H 1 -C 8a 160 (ppm)

50 49 Epiacutissimin B (5b) O O O O O O O O O O O 8 a a I II III V IV

51 Epiacutissimin B (5b) after HPLC Purification 2, ,8 UV6000LP-280nm MJA Epiacutissimin 122 spf1 B after purification 2,0 1, ,6 1, ,4 1,4 m A U 1200 mau 1, , , ,6 1,2 1,0 0,8 0, , ,2 0 0,0 0,4 0,2 0,0-0, ,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0 Minutes -0,2 50

52 LC-MS of Epiacutissimin B (5b) -60eV % m/z

53 1 H/400 MHz/acetone-d 6 NMR of Epiacutissimin B (5b) 52 Integral (ppm)

54 1 H/400 MHz/acetone-d 6 NMR Peak Picking of Epiacutissimin B (5b) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

55 13 C/100 MHz/acetone-d 6 NMR of Epiacutissimin B (5b) (ppm)

56 13 C/100 MHz/acetone-d 6 NMR Peak Picking of Epiacutissimin B (5b) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

57 Dept 135/100 MHz/acetone-d 6 NMR of Epiacutissimin B (5b) 56

58 Dept 135/100 MHz/acetone-d 6 NMR Peak Picking of Epiacutissimin B (5b) Peak Picking results Peak Nr. Data Point Frequency PPM Intensity %Int

59 HMQC Spectrum of Epiacutissimin B (5b) (ppm) (ppm)

60 HMBC Spectrum of Epiacutissimin B (5b) (ppm) (ppm)

61 Portion of HMBC Spectrum of Epiacutissimin B (5b) Showing Relevant Connectivity Correlations between the Flavanoid and the C-Glycosidic Units (ppm) H 1 -C H 1 -C 7 H 1 -C (ppm)

62 100 % B: m/z = 1205 HPLC-ESI-MS of a Partially Purified Red Wine Sample 4a 5b 5a 4b Α: λ = 280 nm X 1a 1b % 5b 4a Time Figure 3. A: UV detection at 280 nm, B: Ion trace chromatogram (m/z = 1205) 1a: vescalagin, 1b: castalagin, 4a: acutissimin A, 4b: acutissimin B, 5a: epiacutissimin A, 5b: epiacutissimin B, X: roburins A, B, C and grandinin 61

63 LC-MS of Acutissimin A (4a) from Red Wine -90eV % m/z