Supporting Information

for

Angew. Chem. Int. Ed. Z52089

© Wiley-VCH 200369451 Weinheim, Germany

1

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) 540-00-64-30

E-mail: 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 N2 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 [H2O–H3PO4

(999:1)] and solvent B [MeOH–H3PO4 (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. Semi-

preparative 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

2

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 flavano-

ellagitannins[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 1H NMR and

HPLC analysis of the sample.

Hemisynthesis of acutissimins A (4a) and B (4b).

In organic solution. (–)-vescalagin (1a, 42 mg, 0.045 mmol) and (+)-catechin (3a, 15 mg, 0.052

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, 20-35 min: 20% to

100% solvent B, 35-40 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 [H2O–HCOOH (996:4)] and solvent D [MeOH–HCOOH (996:4)]. A

gradient elution (0-20 min: 0% to 20% solvent D, 20-35 min: 20% to 100% solvent D, 35-40 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%).

3

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

NaOH. (–)-Vescalagin (1a, 2 mg, 0.0018 mmol) and (+)-catechin (3a, 2 mg, 0.0068 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, 20-30 min: 10% to 100% solvent B,

30-40 min: 100% solvent B) was applied at a flow rate of 1.0 ml.min-1.

Acutissimin A (4a). Off-white amorphous powder; [α]D22 = -80.0° [c = 0.2, acetone; lit.[12] [α]D

32

= -74.0° (c = 1.2, acetone)]; IR (KBr): 3383, 1740, 1616 cm-1; 1H NMR (400 MHz, acetone-d6) 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, HV-2’), 6.77 (s, 1 H, HIII-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, HIV-2’); 13C NMR (100 MHz,

acetone-d6) δ 169.1 (CV=O) 167.4 (CI=O), 167.0 (CIV=O), 166.9 (CIII=O), 165.6 (CII=O), 156.9 (C-

7’), 155.7 (C-5’), 152.6 (C-8’a), 145.2 (CIII-3’), 145.12, 144.80, 144.7, 144.58, 144.0, 143.3(CII-3’,

C[I-V]-5’), 145.0 (CIV-3’), 144.83 (CV-3’), 144.50 (C-3”), 144.4 (C-4”), 136.6 (CIV-4’), 136.2, 135.0

(C[I-II]-4’), 136.1 (CIII-4’), 135.3 (CV-4’), 131.2 (C-1”), 127.6, 127.2, 125.1, 124.4 (C[II-V]-1’), 126.6

(CI-1’), 119.9 (CI-2’), 117.9 (C-6”), 116.5, 114.19, 113.10 (CII-2’II, C[I-II]-6’), 114.7 (CV-6’), 114.13

(CIV-6’), 113.17 (CIII-6’), 115.9 (C-5”), 112.6 (C-2”), 108.5 (CIII-2’), 107.0 (CV-2’), 106.6 (CIV-2’),

104.5 (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 C56H39O31 1207.1475 found

1207.1408.

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].

Acutissimin B (4b). Off-white amorphous powder; [α]D22 = -7.5° [c = 0.2, acetone; lit.[12] [α]D

32 =

-5.5° (c = 0.84, acetone)]; IR (KBr): 3392, 1734, 1612 cm-1. 1H NMR [400 MHz, acetone-d6/D2O

(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-

4

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, HIV-2’), 6.76 (d, J = 1.8 Hz, H-5” rb), 6.80 (s, HIII-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, HV-2’); 13C NMR [100 MHz, acetone-d6/D2O (9:1)] δ 169.0 (CIV=O), 167.1 (CI=O), 167.0

(CIII=O), 166.8 (CV=O), 165.6 (CII=O), 157.2 (C-8’a rb), 156.8 (C-8’a ra), 156.3 (C-7’), 154.8 (C-

5’), 145.5 (C-3”), 145.3 (C-4”), 145.28 (CIII-3’), 145.21 (CIV-3’), 144.8 (CV-3’), 144.51, 144.50,

144.3, 144.2, 144.1, 143.2 (CII-3’, C[I-V]-5’), 137.0 (CV-4’), 136.5, 135.0 (C[I-II]-4’), 136.2 (CIII-4’),

135.4 (CIV-4’), 131.5 (C-1” rb), 131.2 (C-1” ra), 127.7, 126.7, 125.0, 124.7 (C[II-V]-1’), 126.3 (CI-

1’), 120.3 (CI-2’), 119.9 (C-2” rb), 119.5 (C-2” ra), 116.49 (CV-6’), 116.45, 113.7, 113.2 (CII-2’, C[I-

II]-6’), 115.49 (C-5” rb), 115.46 (C-5” ra), 115.3 (C-6” rb), 115.0 (C-6” ra),114.38 (CIII-6’), 114.31

(CIV-6’), 109.7 (CV-2’), 108.4 (CIII-2’), 107.2 (CIV-2’), 106.9 (C-6’), 100.9 (C-4’a rb), 100.1 (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 C56H38O31Na 1229.1294 found 1229.1306.

Hemisynthesis of epiacutissimins A (5a) and B (5b).

In organic solution. (–)-vescalagin (1a, 42 mg, 0.045 mmol) and (–)-epicatechin (3b, 16 mg,

0.055 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%).

Epiacutissimin A (5a). Off-white amorphous powder; [α]D22 = -65.4° (c = 0.53, acetone); IR

(KBr): 3355, 1740, 1616 cm-1; 1H NMR [400 MHz, acetone-d6/D2O (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, HIV-2’), 6.79 (s, HIII-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, HV-2’); 13C NMR [100 MHz, acetone-d6/D2O (9:1)] δ 169.1 (CIII=O), 167.3 (CI=O),

167.0 (CV=O), 166.9 (CIV=O),165.6 (CII=O), 157.1 (C-5’), 157.0 (C-7’), 156.5 (C-8’a rb), 155.0 (C-

8’a ra), 145.28 (CIII-3’), 145.21 (CIV-3’), 145.0 (CV-3’), 144.9 (C-4”), 145.8 (C-3”), 144.6, 144.5,

5

144.38, 144.31, 144.1, 143.2 (CII-3’, C[I-V]-5’), 143.3 2 (CI-3’), 137.0 (CV-4’), 136.6, 135.0 (C[I-V]-

4’), 136.2 (CIII-4’), 135.4 (CIV-4’), 131.5 (C-1” ra and C-1” rb), 127.6, 126.6, 125.0, 124.7 (C[II-V]-

1’), 126.3 (CI-1’), 120.3 (CI-2’), 118.9 (C-6” rb), 118.8 (C-6” ra), 116.5 (CV-6’), 116.4, 113.7,

113.2, (CII-2’ and C[I-II]-6’), 115.2 (C-5” rb), 115.1 (C-5” ra), 114.9 (C-2” rb), 114.8 (C-2” ra),

114.4 (CIV-6’), 114.3 (CIII-6’), 109.4 (CV-2’), 108.4 (CIII-2’), 107.2 (C-8’), 106.9 (CIII-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 C56H38O31Na 1229.1294 found 1229.1288.

Table 1. NMR signal assignments of epiacutissimin A (5a). position δH mult (J, Hz) δC multa HMQCb HMBC

glucose[20,21] 1 4.69 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-3

2 5.10 s 77.8 d C-2 C-1, C-3, C-4, C-8’, C-2’I, CI=O

H-2, H-4 3 4.86 d (J = 4.1 Hz) 71.6 d C-3 C-1, C-2, CII=O

H-2, H-4, H-5 4 5.22 t (J = 7.3, 7.4 Hz) 70.0 d C-4 C-2, C-3, C-5, CV=O

H-2 5 5.62 d (J = 7.6 Hz) 70.9 d C-5 C-3, CIII=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 C-5

aromatics[20,21] 1’I 126.3 s H-1

1’[II-V]

124.7 s, 125.0 s, 126.6 s,

127.6 s

2’I 120.3 s H-1, H-2 2’III 6.79 s 108.4 d C-2’III C-3’III, C-4’III, C-6’III, CIII=O 2’IV 6.63 s 106.9 d C-2’IV C-3’IV, C-4’IV, C-6’IV, CIV=O 2’V 7.07 s 109.4 d C-2’V C-3’V, C-4’V, C-6’V, CV=O

2’II and 6’[I-II]

113.2 s, 113.7 s, 116.4 s

3’I 143.3 s H-1 3’III 145.28 s H-2’III 3’IV 145.21 s H-2’IV 3’V 145.0 s H-2’V

3’II and 5’[I-V]

143.2 s, 144.1 s, 144.31 s,

144.38 s, 144.5 s, 144.6 s,

6

4’[I-II] 135.0 s, 136.6 s 4’III 136.2 s H-2’III 4’IV 135.4 s H-2’IV 4’V 137.0 s H-2’V 6’III 114.3 s H-2’III 6’IV 114.4 s H-2’IV 6’V 116.5 s H-2’V

carbonyls[20,21] CI=O 167.3 s H-2 CII=O 165.6 s H-3 CIII=O 169.1 s H-5, H-2’III CIV=O 166.9 s H-2’IV CV=O 167.0 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, 16.2

Hz) β 2.81 dd (J = 4.4, 16.6

Hz)

29.1 t C-4’ra C-2’ra, C-3’ra, C-4’a, C-8’ara

4’rb 2.97 bs 28.4 t C-4’rb 4’a* 99.4 s H4’ra, H6’ra, H6’rb 5’* 157.1 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’* 157.0 s H-1, H6’ra, H6’rb 8’* 107.2 s H-1, H-2

8’ara 155.0 s H-1, H4’ra 8’arb 156.5 s H-1 1”* 131.5 s H-2’,H-2”, H5”, H-6” 2”ra 7.03 s 114.8 d C-2”ra C-1”ra, C-3”ra, C-4”ra, C-6”ra

H-6”ra 2”rb 7.08 s 114.9 d C-2”rb C-1”rb, C-3”rb, C-4”rb, C-6”rb

H-6”rb 3”* 144.8 s H-2”, H5” 4”* 144.9 s H-2”, H5”, H-6” 5”ra 6.86 bs 115.1 d C-5”ra C-1”ra, C-3”ra, C-4”ra 5”rb 6.82 bs 115.2 d C-5”rb C-1”rb, C-3”rb, C-4”rb 6”ra 6.80 bs 118.8 d C-6”ra C-1”ra, C-2”ra, C-4”ra

H-2”ra 6”rb 6.84 bs 118.9 d C-6”rb C-1”rb, C-2”rb, C-4”rb

H-2”rb aMultiplicity inferred using the DEPT pulse sequence. bCarbons correlates to the proton resonance in the δH column. *Proton and carbon NMR chemical shifts are the same for each rotamer.

7

Epiacutissimin B (5b). Off-white amorphous powder; [α]D22 = -90.7° (c = 0.38, acetone); IR

(KBr): 3338, 1734, 1617 cm-1; 1H NMR NMR [400 MHz, acetone-d6] 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, HIV-2’), 6.64 (d, J = 8.6 Hz, 1 H, H-5”), 6.75 (s, 2 H, HIII-2’, HV-2’), 6.79 (bs, 1 H, H-6”), 6.96

(s, 1 H, H-2”); 13C NMR [100 MHz, acetone-d6] δ 169.1 (CIV=O), 167.1 (CI=O), 167.0 (CIII=O),

166.0 (CII=O), 165.6 (CV=O), 156.7 (C-5’), 155.6 (C-8’a), 153.2 (C-7’), 145.3, 145.27, 144.53,

144.2, 144.0, 143.3 (CII-3’, C[I-V]-5’), 145.25 (CIII-3’), 145.21 (CIV-3’), 144.8 (CV-3’), 144.51 (C-

4”), 144.4 (C-3”), 142.7 (CI-3’), 137.1, 136.9 (C[I-II]-4’), 136.1 (CV-4’), 135.2 (CIII-4’), 135.0 (CIV-

4’), 130.9 (C-1”), 127.5, 127.0, 126.4, 124.5 (C[II-V]-1’), 125.0 (CI-1’), 120.8 (CI-2’), 119.1 (C-6”),

116.5, 115.3, 113.1 (CII-2’, C[I-II]-6’), 114.8 (C-5”), 114.38 (CV-6’), 114.31 (CIV-6’), 114.2 (CIII-6’),

113.9 (C-2”), 109.1 (CIII-2’), 108.3 (CIV-2’), 106.8 (CV-2’), 105.1 (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 C56H38O31Na 1229.1294 found 1229.1292.

Table 2. NMR signal assignments of epiacutissimin B (5b).

position δH mult (J, Hz) δC multa HMQCb HMBC glucose[20,21]

1 4.79 s 37.2 d C-1 C-5’, C-6’, C-7’, C-1’I, C-2’I, C-3’I

H-2, H-3 2 5.13 s 77.7 d C-2 C-1, C-4, C-6’, C-2’I, CI=O

H-4 3 4.78 bs 71.8 d C-3 C-1, CII=O

H-5 4 5.18 t (J = 7.7 Hz) 69.5 d C-4 C-2, C-5, CV=O

H-2, H-5, H-6 5 5.62 d (J = 7.6 Hz) 70.8 d C-5 C-3, C-4, C-6, CIII=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, CIV=O

H-5

aromatics[20,21] 1’I 125.0 s H-1

1’[II-V]

124.4 s, 126.4 s, 127.0 s, 127.5 s

2’I 120.8 s H-1, H-2 2’III 6.75 s 109.1 d C-2’III C-3’III, C-4’III, C-6’III, CIII=O 2’IV 6.59 s 108.3 d C-2’IV C-3’IV, C-4’IV, C-6’IV, CIV=O 2’V 6.75 s 106.8 d C-2’V C-3’V, C-4’V, C-6’V, CV=O

2’II and

8

6’[I-II] 113.1 s, 115.3 s, 116.5 s,

3’I 142.7 H-1 3’III 145.25 H-2’III 3’IV 145.21 H-2’IV 3’V 144.8 H-2’V

3’II and 5’[I-V]

143.3 s 144.0 s, 144.2 s, 144.53

s, 145.27 s, 145.3 s

4’[I-II] 136.9 s, 137.8 s 4’III 135.2 s H-2’III 4’IV 135.0 s H-2’IV 4’V 136.1 s H-2’V 6’III 114.2 s H-2’III 6’IV 114.31 s H-2’IV 6’V 114.38 s H-2’V

carbonyls[20,21] CI=O 167.1 s H-2 CII=O 166.0 s H-3 CIII=O 167.0 s H-5, H-2’III CIV=O 169.1 s H-6, H-2’IV CV=O 165.6 s H-4, H-2’V

epicatechin[18,24] 2’ 5.49 s 79.2 d C-2’ 3’ 4.56 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-8’ 5’ 156.7 s H-1 6’ 105.1 d H-1, H-2, H-8’ 7’ 153.2 s H-1 8’ 6.06 s 96.4 d C-8’ C-4’a, C-6’, C-8’a 8’a 155.6 s H-8’ 1” 130.9 s H-5” 2” 6.96 s 113.9 d C-2” C-3”, C-6”

H-6” 3” 144.4 s H-2” 4” 144.5 s H-5”, H-6” 5” 6.64 d (J = 8.6 Hz) 114.8 d C-5” C-1”, C-4”

H-6”

6” 6.79 bs 119.1 d C-6” C-2”, C-4”, C-5” H-2”

aMultiplicity inferred using the DEPT pulse sequence. bCarbons correlates to the proton resonance in the δH 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)

9

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 [H2O–HCOOH (996:4), 250 ml] was first used to wash out tartaric acid and

sugars (2.82 g), and a 20% methanolic aqueous solution [H2O–MeOH–HCOOH (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 [H2O–

HCOOH (996:4), 50 ml] and a 70% methanolic aqueous solution [H2O–MeOH–HCOOH

(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 H2O–acetone–HCOOH

(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 flavano-

ellagitannins 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,

3319. [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. 1 1991, 1653. [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. 193.

[26] G.-I. Nonaka, K. Ishimaru, R. Azuma, M. Ishimatsu, I. Nishioka, Chem. Pharm. Bull. 1989, 37, 20

10

HPLC Monitoring of Acutissimin Formation in the Acidic Organic Solution

1a: vescalagin, 3a: catechin, 4a: acutissimin A, 4b: acutissimin B

Minutes0,0 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

mAU

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

2,25

2,50

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8UV6000LP-280nmMJA 121 t0

UV6000LP-280nmMJA 121 6h

0

200

400

600

800

1000

1200

0 5 10 15 20 25 30 35 40 45 Minutes

m

A

U

1400

1600

1800

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

3a

4a

4b 1a

t= 0 h t= 7 h

11

HPLC Monitoring of Acutissimin Formation in the Wine Model Solution

1a: vescalagin, 3a: catechin, 4a: acutissimin A, 4b: acutissimin B

5 10 15 20 25 30 45 40 35

200

150

100

50

0

0

250

t= 6 jours t= 25 jours

1a

3a

minutes

t= 0

300

350

4a, 4b

ethylvescalagin

m

A

U

Elution gradient

0 to 20 min : 0 to 10 % B

20 to 30 min : 10 to 100 % B

30 to 40 min : 100 % B

40 to 45 min : 100 to 0 % B

12

Acutissimin A (4a)

OOO

O

O O O

HOOH

OH OH

HO

HO

OH

OH

HOOH

HO

HO

HO

HO

HO

OOO

O

HO

HO

OH

HO

OH 1’

6’

2’

3’

4’5’

V

8’a

2’

8’

7’ 6’

5’

4’

3’

1”

6”

2”

3”

4” 5”

4’a

I 1’

6’

2’3’

4’

5’

1’

6’ 2’

3’ 4’

5’II

III 1’

6’

2’ 3’

4’

5’

1’

6’

2’

3’

4’ 5’

IV

2341

56

13

Acutissimin A (4a) after HPLC Purification

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

mAU

-0,25

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

-0,25

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

UV6000LP-280nmMJA 121 spF1

0 5 10 15 20 25 30 35 40 45 Minutes

0

200

400

600

800

1000

1200

m

A

U

1400

1600

Acutissimin A after purification

14

LC-MS of Acutissimin A (4a) -60eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

602

301

275493

915

603

1205916

15

1H/400 MHz/acetone-d6 NMR of Acutissimin A (4a)

1.05

640.

9439

0.99

741.

0516

1.06

661.

1437

1.00

00

1.15

871.

0833

1.22

301.

1695

1.16

931.

2066

1.26

461.

5739

1.10

51

0.96

02

Inte

gral

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

16

1H/400 MHz/acetone-d6 NMR Peak Picking of Acutissimin A (4a)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 74322 2832.79 7.0797 102051920 51.8 2 77459 2784.84 6.9598 27103112 13.8 3 77966 2777.10 6.9405 34005736 17.3 4 79412 2755.00 6.8853 58257348 29.6 5 81356 2725.29 6.8110 60696440 30.8 6 81898 2717.00 6.7903 57603624 29.2 7 82235 2711.85 6.7774 197051056 100.0 8 86292 2649.85 6.6225 127171136 64.5 9 101328 2420.05 6.0482 58343896 29.6 10 111731 2261.06 5.6508 48943616 24.8 11 112226 2253.49 5.6319 54190048 27.5 12 115709 2200.26 5.4989 44971672 22.8 13 121323 2114.46 5.2844 41491084 21.1 14 121798 2107.20 5.2663 70016848 35.5 15 122277 2099.88 5.2480 42342740 21.5 16 123845 2075.92 5.1881 77268944 39.2 17 133751 1924.52 4.8097 61813204 31.4 18 135557 1903.78 4.7558 40899859 20.8 19 135565 1896.80 4.7404 45137148 22.9 20 136266 1886.08 4.7137 36920728 18.7 21 137068 1873.83 4.6830 36958092 18.8 22 140118 1827.21 4.5665 44000248 22.3 23 151830 1648.21 4.1192 110595064 56.1 24 152610 1636.29 4.0894 125753112 63.8 25 183650 1161.90 2.9038 20485690 10.4 26 184728 1145.43 2.8626 24388788 12.4 27 196098 971.66 2.4283 22444878 11.4 28 197046 957.17 2.3921 19748068 10.0

17

13C/100 MHz/acetone-d6 NMR of Acutissimin A (4a)

(ppm)0102030405060708090100110120130140150160170180190

18

13C/100 MHz/acetone-d6 NMR Peak Picking of Acutissimin A (4a)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 4623 17016.92 169.1329 8017559 1.6 2 4893 16851.46 167.4884 3083714 0.6 3 4958 16811.63 167.0925 7453146 1.5 4 4988 16793.24 166.9097 5945136 1.2 5 5198 16664.55 165.6307 5966738 1.2 6 6629 15787.63 156.9149 2856200 0.6 7 6828 15665.69 155.7028 3745649 0.7 8 7330 15358.06 152.6453 2777487 0.6 9 8539 14617.18 145.2816 9858008 2.0 10 8565 14601.25 145.1233 4825386 1.0 11 8569 14598.80 145.0989 6841277 1.4 12 8598 14575.67 144.8372 6154896 1.3 13 8618 14568.77 144.8005 7895723 1.6 14 8627 14563.25 144.7457 6280194 1.2 15 8653 14547.32 144.5873 351046 0.1 16 8667 14538.74 144.5020 7964203 1.6 17 8679 14531.39 144.4289 7981656 1.6 18 8739 14494.62 144.0635 6451136 1.3 19 8860 14420.47 143.3265 2808412 0.6 20 8875 14411.28 143.2352 2972085 0.6 21 9961 13745.77 136.6207 3179056 0.6 22 10020 13709.62 136.2613 2963626 0.6 23 10042 13696.14 136.1273 6852798 1.4 24 10170 13617.70 135.3477 7482492 1.5 25 10213 13591.35 135.0858 2947912 0.6 26 10842 13205.90 131.2547 3232074 0.6 27 11434 12843.12 127.6490 2906086 0.6 28 11494 12806.35 127.2836 2767561 0.5

29 11599 12742.00 126.6441 7112457 1.4 30 11846 12590.64 125.1397 4872781 1.0 31 11957 12522.62 124.4636 3693001 0.7 32 12696 12069.76 119.9626 2621857 0.5 33 13026 11867.53 117.9526 2779701 0.6 34 13249 11730.88 116.5944 6233405 1.2 35 13347 11670.82 115.9975 5739544 1.1 36 13548 11547.65 114.7733 3013063 0.6 37 13628 11421.48 114.1985 3059782 0.5 38 13652 11483.92 114.1398 3597860 0.7 39 13811 11386.48 113.1714 7616961 1.5 40 13822 11379.74 113.1044 2710368 0.5 41 13898 11333.17 112.6415 2848664 0.6 42 14569 10921.98 108.5547 4069466 0.8 43 14824 10765.71 107.0015 2831273 0.6 44 14884 10728.95 106.6361 3674757 0.7 45 15219 10523.66 104.5957 3098965 0.6 46 16312 9853.86 97.9386 3158893 0.6 47 16537 9715.98 96.5682 2771735 0.5 48 19309 8017.29 79.6847 3231145 0.6 49 19716 7767.88 77.2058 3477279 0.7 50 20582 7237.20 71.9312 5080002 1.0 51 20765 7125.05 70.8166 5119727 1.0 52 20780 7115.86 70.7253 3941411 0.8 53 21332 6777.59 67.3632 5359608 1.1 54 21649 6583.34 65.4324 3958005 0.8 55 26225 3779.15 37.5614 3264983 0.6 56 28537 2362.35 23.4796 2671931 0.5

19

Dept 135/100 MHz/acetone-d6 NMR of Acutissimin A (4a)

(ppm)0102030405060708090100110120130140150160170180190

20

Dept 135/100 MHz/acetone-d6 NMR Peak Picking of Acutissimin A (4a)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 6808 11862.54 117.9030 89184192 49.1 2 6955 11665.78 115.9474 100383232 55.2 3 7207 11328.48 112.5949 116005632 63.8 4 7514 10917.57 108.5108 161741024 89.0 5 7589 10845.73 107.0154 156352893 70.3 6 7659 10723.49 106.5818 146721520 80.7 7 8416 9710.25 96.5111 95938432 52.8 8 9685 8011.70 79.6291 106847064 58.8 9 9871 7762.74 77.1547 123366896 67.9 10 10268 7231.36 71.8733 155781184 85.7 11 10343 7130.95 70.8125 146899685 80.5 11 10351 7120.27 70.7691 161948160 89.1 12 10611 6772.26 67.3102 181744336 100.0 13 10756 6578.18 65.3812 -78717136 -43.3 14 12851 3774.04 37.5106 97649336 53.7 15 13910 2356.58 23.4223 -43463604 -23.9

21

HMQC Spectrum of Acutissimin A (4a)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

140

120

100

80

60

40

(ppm)

22

HMBC Spectrum of Acutissimin A (4a)

(ppm) 6.4 5.6 4.8 4.0 3.2 2.4

160

140

120

100

80

60

40

(ppm)

23

Portion of HMBC Spectrum of Acutissimin A (4a) Showing Relevant Connectivity Correlations

between the Flavanoid and the C-Glycosidic Units

(ppm) 5.20 5.00 4.80 4.60

160

140

120

(ppm)

H1-C7’ H1-C8a’

H1-C8’

24

Acutissimin B (4b)

5

OOO

O

O O O

HOOH

OH OH

HO

HO

OH

OH

HOOH

HO

HO

HO

HO

HO

OOO

O

OH

HO

OH

OH

OH

8’a8’

7’

6’5’ 4’

3’

2’1”

6”

2”3”

4”

5”

4’a12346

I1’

6’

2’3’

4’

5’

1’

6’ 2’

3’ 4’

5’II

III 1’

6’

2’ 3’

4’

5’

1’

6’

2’

3’

4’5’

V6’

1’ 2’

3’

4’ 5’

IV

25

Acutissimin B (4b) after HPLC Purification

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

mA

U

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

UV6000LP-280nmMJA 121 spF2

0 5 10 15 20 25 30 35 40 45 Minutes

0

200

400

600

800

1000

1200

m

A

U

1400

1600 Acutissimin B after purification

26

LC-MS of Acutissimin B (4b) -60eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

602

301

275493

915

603

1205916

27

1H/400 MHz/(acetone-d6/D2O 9 :1) NMR of Acutissimin B (4b)

0.87

281.

5173

3.57

080.

9743

0.87

89

1.00

00

0.99

310.

9970

1.00

640.

9754

1.07

451.

5896

0.58

66

1.09

240.

5469

Inte

gral

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

28

1H/400 MHz/(acetone-d6/D2O 9 :1) NMR Peak Picking of Acutissimin B (4b)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 73592 2843.94 7.1076 45406920 42.5 2 77137 2789.37 6.9721 47286424 44.3 3 77250 2788.04 6.9678 45022632 42.2 4 78582 2767.48 6.9170 23414068 21.9 5 78724 2765.51 6.9115 24433312 22.9 6 80760 2734.39 6.8338 77330168 72.4 7 80921 2731.93 6.8276 42554304 39.9 8 81157 2728.03 6.8186 54390884 51.0 9 81280 2726.45 6.8139 52578428 49.3 10 81559 2722.18 6.8032 106740056 100.0 11 82546 2706.60 6.7655 19856712 18.6 12 82678 2705.08 6.7605 18245904 17.1 13 85636 2659.87 6.6475 95203920 89.2 14 101529 2416.98 6.0405 24745428 23.2 15 105840 2351.09 5.8758 19247440 18.0 16 111343 2266.99 5.6656 24326340 22.8 17 111834 2259.48 5.6469 23667168 22.2 18 121177 2116.69 5.2900 23530202 22.0 19 121640 2109.62 5.2723 35199440 33.0 20 122132 2102.10 5.2535 20228080 19.0 21 125668 2048.05 5.1185 53126024 49.8 22 131158 1965.25 4.9088 16779080 15.7

23 131952 1952.01 4.8785 20363852 19.1 24 132623 1941.76 4.8528 30266716 28.4 25 133114 1934.26 4.8341 25468224 23.9 26 136057 1889.28 4.7217 44173640 41.4 27 140021 1828.69 4.5702 47753712 44.7 28 140569 1820.32 4.5493 52250140 49.0 29 153196 1627.34 4.0670 44002360 41.2 30 154046 1614.35 4.0346 42675388 40.0 31 154641 1605.25 4.0118 32229762 30.2 32 155152 1597.44 3.9923 33289880 31.2 33 182809 1174.75 2.9359 10228059 9.6 34 183187 1169.18 2.9215 9771426 9.2 35 183858 1158.62 2.8959 12637497 11.8 36 184226 1153.10 2.8818 10821135 10.1 37 189555 1071.65 2.6783 10071731 9.4 38 190150 1062.56 2.6555 11660541 10.9 39 190557 1056.34 2.6400 10511490 9.8 40 191133 1047.54 2.6180 8474667 7.9 41 192681 1023.48 2.5589 11587140 10.9 42 193258 1015.06 2.5368 11468445 10.7 43 193740 1007.49 2.5184 9541440 8.9 44 194306 999.04 2.4968 8722278 8.2

29

13C/100 MHz/(acetone-d6/D2O 9 :1) NMR of Acutissimin B (4b)

(ppm)0102030405060708090100110120130140150160170180190

30

13C/100 MHz/(acetone-d6/D2O 9 :1) NMR Peak Picking of Acutissimin B (4b)

�Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 4635 17009.56 169.0598 3507570 52.4 2 4953 16814.69 167.1229 3251449 48.6 3 4968 16805.50 167.0316 2906773 43.4 4 5001 16785.28 166.8306 2863687 42.8 5 5200 16663.33 165.6185 3262360 48.8 6 6573 15821.95 157.2560 2174489 32.5 7 6639 15781.51 156.8540 2000405 29.9 8 6722 15730.64 156.3485 2055353 30.7 9 6976 15574.99 154.8014 3323483 49.7 10 8501 14640.47 145.5131 3669581 54.8 11 8525 14628.34 145.3286 4861558 70.2 12 8539 14617.18 145.2816 6690265 100.0 13 8550 14610.44 145.2146 3452197 51.6 14 8616 14569.99 144.8127 3503764 52.4 15 8648 14553.85 144.5154 3506354 52.9 16 8653 14547.32 144.5073 3421097 51.1 17 8696 14520.97 144.3254 3183703 47.6 18 8707 14514.23 144.2584 3078065 46.0 19 8726 14502.59 144.1427 3017617 45.1 20 8863 14418.63 143.3083 3435581 51.4 21 8872 14413.12 143.2534 3126874 46.7 22 9898 13784.38 137.0044 3428733 51.2 23 9976 13736.58 136.5293 3157851 47.2 24 10029 13704.10 136.2065 3136531 46.9 25 10158 13625.05 135.4208 3220181 48.1 26 10222 13585.83 135.0310 3426972 51.2 27 10801 13231.02 131.5045 2482591 37.1 28 10835 13210.19 131.2974 3479993 52.0 29 11424 12849.24 127.7099 3146920 47.0 30 11576 12756.10 126.7842 3074267 46.0 31 11653 12708.91 126.3152 2963896 44.3 32 11855 12585.13 125.0849 3258719 48.7 33 11904 12555.10 124.7864 3471454 51.9

34 12636 12106.53 120.3280 1629463 24.4 35 12692 12072.21 119.9869 2117668 31.7 36 12756 12032.99 119.5971 3379649 50.5 37 13265 11721.07 116.4970 2988105 44.7 38 13272 11716.78 116.4543 3129336 46.8 39 13430 11619.96 115.4920 4233002 63.3 40 13435 11616.90 115.4615 4100241 61.3 41 13466 11597.90 115.3227 2529894 37.8 42 13503 11575.23 115.0474 3490928 52.2 43 13611 11509.04 114.3896 3017345 45.1 44 13623 11501.69 114.3165 2953122 44.1 45 13722 11441.02 113.7135 2881668 43.1 46 13805 11390.16 113.2080 3313318 49.5 47 14374 11041.48 109.7424 943455 14.1 48 14582 10914.01 108.4755 2504623 37.4 49 14781 10792.07 107.2634 2755094 41.2 50 14830 10762.04 106.9650 2415439 36.1 51 15821 10154.75 100.9291 2320406 34.7 52 15949 10076.31 100.1495 2386054 35.7 53 16658 9641.83 95.8312 2749781 41.1 54 16813 9546.85 94.8871 3220480 48.1 55 18897 8269.77 82.1941 3873849 57.9 56 18910 8261.80 82.1149 2488859 37.2 57 19595 7842.03 77.9428 2218022 33.2 58 20629 7208.39 71.6450 3351113 50.1 59 20734 7144.05 71.0054 2723310 40.7 60 20899 7042.94 70.0005 1918448 28.7 61 21224 6843.78 68.0210 2573313 38.5 62 21252 6826.62 67.8505 3145032 47.0 63 21638 6590.08 65.4994 2041481 30.5 64 26188 3801.82 37.7867 2234294 33.4 65 27718 2864.24 28.4679 3103965 46.4 66 28007 2687.14 26.7077 1106384 16.5

31

Dept 135/100 MHz/(acetone-d6 /D2O 9 :1) NMR of Acutissimin B (4b)

32

Dept 135/100 MHz/(acetone-d6 /D2O 9 :1) NMR Peak Picking of Acutissimin B (4b)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 6647 12067.97 119.9448 17159916 23.9 2 6677 12027.82 119.5457 43093344 60.2 3 6982 11619.58 115.4881 71685784 100.0 4 6984 11616.90 115.4615 47736044 66.6 5 6994 11603.52 115.3285 64028728 89.7 6 7015 11575.41 115.0491 33537512 46.9 7 7411 11045.37 109.7810 29671620 41.3 8 7513 10908.84 108.4240 56410248 78.2 9 7602 10789.71 107.2400 15866219 22.4 10 8459 9642.63 95.8390 23408910 32.2 11 8534 9542.24 94.8413 40200728 56.5 12 9482 8273.35 82.2297 46858880 65.6 13 9486 8268.00 82.1765 63382376 88.0 14 9804 7842.36 77.9460 45420064 63.2 15 10280 7205.24 71.6136 40768560 56.0 16 10325 7145.00 71.0149 23586586 32.8 17 10398 7047.29 70.0438 46597760 65.4 18 10550 6843.84 68.0217 26282592 36.9 19 10566 6822.43 67.8088 26525496 37.2 20 10739 6590.87 65.5073 -24037818 -33.2 21 12828 3794.76 37.7165 47030596 65.4 22 13525 2861.84 28.4441 -23857895 -33.0 23 13655 2687.83 26.7146 -7827385 -10.4

33

HMQC Spectrum of Acutissimin B (4b)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

140

120

100

80

60

40

(ppm)

34

HMBC Spectrum of Acutissimin B (4b)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

160

140

120

100

80

60

40

(ppm)

35

Portion of HMBC Spectrum of Acutissimin B (4b) Showing Relevant Connectivity Correlations

between the Flavanoid and the C-Glycosidic Units

(ppm) 5.20 5.00 4.80 4.60 4.40

160

152

144

136

128

120

112

(ppm)

H1-C7’

H1-C5’

H1-C6’

36

HPLC Monitoring of Epiacutissimin Formation in the Acidic Organic Solution

1a: vescalagin, 2b: epicatechin, 5a: epiacutissimin A, 5b: epiacutissimin B

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

Minutes0,0 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

mA

U

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

2,2UV6000LP-280nmMJA 122 t0

UV6000LP-280nmMJA 122 4h

0

200

400

600

800

1000

1200

0 5 10 15 20 25 30 35 40 45 Minutes

m

A

U

1400

1600

1800

2000

2b

5a

5b

1a

t= 0 h t= 7 h

37

Epiacutissimin A (5a)

OOO

O

O O O

HOOH

OH OH

HO

HO

OH

OH

HOOH

HO

HO

HO

HO

HO

OOO

O

HO

HO

OH

HO

OH 1’

6’

2’

3’

4’5’

V

8’a

2’

8’

7’ 6’

5’

4’

3’

1”

6”

2”

3”

4” 5”

4’a

I 1’

6’

2’3’

4’

5’

1’

6’ 2’

3’ 4’

5’II

III 1’

6’

2’ 3’

4’

5’

1’

6’

2’

3’

4’ 5’

IV

2341

56

38

Epiacutissimin A (5a) after HPLC Purification

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

mA

U

-0,25

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

-0,25

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

UV6000LP-280nmMJA 122 spF2

0 5 10 15 20 25 30 35 40 45 Minutes

0

200

400

600

800

1000

1200

m

A

U

1400

1600

Epiacutissimin A after purification

39

LC-MS of Epiacutissimin A (5a) -60eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

602

457

289

601

603

625915 1205

40

1H/400 MHz/(acetone-d6/D2O 9 :1) NMR of Epiacutissimin A (5a)

2.11

56

4.61

441.

2390

0.60

96

1.00

00

0.99

621.

0894

3.82

231.

1415

1.24

84

1.16

73

Inte

gral

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

41

1H/400 MHz/(acetone-d6/D2O 9:1) NMR Peak Picking of Epiacutissimin A (5a

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 74117 2835.92 7.0875 42091624 47.3 2 74450 2830.83 7.0748 81047496 91.1 3 75619 2812.97 7.0301 19535792 21.9 4 79862 2748.12 6.8681 30075214 33.8 5 80593 2736.95 6.8401 48531000 54.5 6 81119 2728.91 6.8201 28919228 32.5 7 81417 2724.35 6.8087 39394880 44.3 8 81850 2717.74 6.7921 89013040 100.0 9 85845 2656.68 6.6395 88110328 99.0 10 101656 2415.04 6.0356 17799936 20.0 11 104579 2370.36 5.9240 15474340 17.4 12 111906 2258.38 5.6441 18441668 20.7 13 112403 2250.79 5.6251 20770404 23.3 14 121870 2106.10 5.2635 17923110 20.1 15 122353 2098.72 5.2451 30841252 34.6 16 122829 2091.44 5.2269 18320112 20.6 17 125895 2044.58 5.1098 47910104 53.8 18 131839 1953.74 4.8828 50474276 56.7 19 132098 1949.78 4.8729 23666564 26.6 20 132367 1945.67 4.8626 27221524 30.6 21 133028 1939.97 4.8374 36452680 41.0 22 133536 1927.81 4.8179 24144852 27.1 23 136748 1878.72 4.6953 44783324 50.3 24 147183 1719.24 4.2967 25029984 28.1 25 149357 1686.01 4.2137 22262852 25.0 26 181705 1191.63 2.9781 10113781 11.4 27 184615 1147.15 2.8670 6703223 7.5 28 184927 1142.38 2.8550 6451478 7.2 29 185725 1130.19 2.8246 9311739 10.5 30 186016 1125.74 2.8134 8580901 9.6 31 188494 1087.87 2.7188 8042188 9.0 32 188711 1084.55 2.7105 8090490 9.1 33 189586 1071.18 2.6771 4813693 5.4 34 189798 1067.94 2.6690 4777029 5.4

42

13C/100 MHz/(acetone-d6/D2O 9:1) NMR of Epiacutissimin A (5a)

(ppm)0102030405060708090100110120130140150160170180190

43

13C/100 MHz/(acetone-d6/D2O 9:1) NMR Peak Picking of Epiacutissimin A (5a)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 4624 17016.30 169.1268 1732880 16.1 2 4919 16835.53 167.3300 1547955 14.3 3 4959 16811.01 167.0864 1489794 13.8 4 4982 16796.92 166.9463 1624577 15.1 5 5192 16668.23 165.6672 1472563 13.6 6 6587 15813.37 157.1707 745336 6.9 7 6614 15796.83 157.0063 847638 7.9 8 6690 15750.25 156.5434 815959 7.6 9 6938 15598.28 155.0329 1483639 13.7 10 8538 14617.79 145.2877 1922452 17.8 11 8551 14609.83 145.2086 1824199 16.9 12 8584 14589.60 145.0076 1515732 14.0 13 8593 14584.09 144.9527 2056815 19.1 14 8604 14577.35 144.8857 1755253 16.3 15 8642 14554.06 144.6543 1688686 15.7 16 8655 14546.10 144.5751 1526914 14.2 17 8687 14526.49 144.3802 1644080 15.2 18 8697 14520.36 144.3193 1733338 16.1 19 8721 14505.65 144.1731 1392237 12.9 20 8850 14426.60 143.3874 1320819 12.2 21 8872 14413.12 143.2534 1535835 14.2 22 9897 13784.99 137.0105 1930931 17.9 23 9963 13744.55 136.6085 1328255 12.3 24 10028 13704.72 136.2126 1955753 18.1 25 10156 13626.28 135.4330 1462238 13.6 26 10221 13586.45 135.0371 1394096 12.9 27 10789 13238.37 131.5775 2397750 22.2 28 11434 12843.12 127.6490 1413775 13.1 29 11597 12743.23 126.6563 1519910 14.1 30 11652 12709.53 126.3213 1266767 11.7 31 11864 12579.61 125.0300 1605263 14.9 32 11914 12548.97 124.7255 1833521 17.0

33 12627 12112.04 120.3828 979621 9.1 34 12861 11968.65 118.9576 1786121 16.6 35 12873 11961.29 118.8845 1628760 15.1 36 13257 11725.98 116.5457 1484887 13.8 37 13274 11715.56 116.4421 1428877 13.2 38 13451 11607.09 115.2641 2152494 19.9 39 13464 11599.13 115.1249 1426759 13.2 40 13517 11566.65 114.9621 2308420 21.4 41 13522 11563.58 114.8316 1657491 15.4 42 13598 11517.01 114.4687 1161875 10.8 43 13627 11499.24 114.2921 1262133 11.7 44 13727 11437.96 113.7130 1313007 12.2 45 13809 11387.71 113.2136 1837821 17.0 46 14421 11012.67 109.4561 838348 7.8 47 14584 10912.79 108.4633 1244569 11.5 48 14789 10787.16 107.2147 1465995 13.6 49 14840 10755.91 106.9041 1187845 11.0 50 16072 10000.94 99.4003 780377 7.2 51 16648 9647.96 95.8921 1040715 9.6 52 16768 9574.43 95.1612 1094218 10.1 53 19426 7945.60 78.9721 1471142 13.6 54 19466 7921.08 78.7285 1452238 13.5 55 19604 7836.52 77.8879 1198384 11.1 56 20632 7206.56 71.6267 1411032 13.1 57 20743 7138.54 70.9506 1527374 14.2 58 20885 7051.52 70.0857 946192 8.8 59 21476 6689.35 66.4861 1493655 13.8 60 21491 6680.16 66.3948 1192103 11.0 61 21636 6591.30 65.5116 831957 7.7 62 26184 3804.28 37.8111 876323 8.1 63 27607 2932.26 29.1440 10790246 100.0 64 27725 2859.95 28.4253 1445169 13.4

44

Dept 135/100 MHz/(acetone-d6/D2O 9 :1) NMR of Epiacutissimin A (5a)

(ppm)0102030405060708090100110120130140150160170180190

45

Dept 135/100 MHz/(acetone-d6/D2O 9:1) NMR Peak Picking of Epiacutissimin A (5a)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 6733 11966.78 118.9391 96126712 70.6 2 6738 11960.09 118.8725 47939204 35.2 3 7003 11605.39 115.2371 136066192 100.0 4 7008 11598.70 115.1206 47736044 35.1 5 7033 11565.23 114.9480 117644568 86.5 6 7039 11556.35 114.8369 57628463 45.1 7 7446 11012.44 109.4537 53031532 39.0 8 7522 10910.71 108.4427 80688656 59.3 9 7639 10754.11 106.8862 80524624 59.2 10 8466 9647.18 95.8843 44286368 32.5 11 8521 9573.56 95.1526 35905076 26.4 12 9738 7944.62 78.9624 47745048 35.1 13 9757 7919.19 78.7096 87514160 64.3 14 9820 7834.86 77.8715 88173416 64.8 15 10290 7205.77 71.6189 79985032 58.8 16 10341 7137.51 70.9404 80992520 59.5 17 10406 7050.51 70.0757 62257824 45.8 18 10677 6687.78 66.4705 48828204 35.9 19 10684 6678.41 66.3774 68184040 50.1 20 10750 6590.07 65.4993 -63596408 -46.7 21 12833 3801.99 37.7884 51693944 38.0 22 13488 2925.28 29.0747 2720645 2.0 23 13538 2858.36 28.4095 -47030596 -34.6

46

HMQC Spectrum of Epiacutissimin A (5a)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

140

120

100

80

60

40

(ppm)

47

HMBC Spectrum of Epiacutissimin A (5a)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

160

140

120

100

80

60

40

(ppm)

48

Portion of HMBC Spectrum of Epiacutissimin A (5a) Showing Relevant Connectivity Correlations

between the Flavanoid and the C-Glycosidic Units

(ppm) 5.20 5.00 4.80 4.60 4.40

160

140

120

(ppm)

H1-C8a’

H1-C8’

49

Epiacutissimin B (5b)

OOO

O

O O O

HOOH

OH OH

HO

HO

OH

OH

HOOH

HO

HO

HO

HO

HO

OOO

O

OH

HO

OH

OH

OH

8’a8’

7’

6’5’ 4’

3’

2’1”

6”

2”3”

4”

5”

4’a

I1’

6’

2’3’

4’

5’

1’

6’ 2’

3’ 4’

5’II

III 1’

6’

2’3’

4’

5’

1’

6’

2’

3’

4’5’

V6’

1’ 2’

3’

4’ 5’

IV

12346 5

50

Epiacutissimin B (5b) after HPLC Purification

Minutes0,0 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

mAU

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0UV6000LP-280nmMJA 122 spF1

0 5 10 15 20 25 30 35 40 45 Minutes

0

200

400

600

800

1000

1200 m

A

U

1400

1600

1800

Epiacutissimin B after purification

51

LC-MS of Epiacutissimin B (5b) -60eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

m/z0

100

%

602

301

275601

467 483

603

915

1205

52

1H/400 MHz/acetone-d6 NMR of Epiacutissimin B (5b)

0.83

933.

0761

1.24

680.

9808

0.79

95

1.12

170.

8329

1.35

180.

9172

2.95

90

1.00

73

1.00

00

1.09

77

Inte

gral

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

53

1H/400 MHz/acetone-d6 NMR Peak Picking of Epiacutissimin B (5b)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 77391 2785.88 6.9624 14906994 21.2 2 81866 2717.49 6.7915 19001308 27.0 3 82854 2702.39 6.7538 67148040 95.5 4 82868 2702.18 6.7532 70333376 100.0 5 85161 2667.13 6.6657 13838021 19.7 6 85722 2658.56 6.6442 11017860 15.7 7 86994 2639.12 6.5957 22695722 32.3 8 100930 2426.13 6.0634 15304663 21.8 9 112020 2256.64 5.6398 18463364 26.3 10 112518 2249.03 5.6207 20146082 28.6 11 118210 2162.04 5.4033 13208805 18.8 12 122837 2091.32 5.2266 9692892 13.8 13 123311 2084.08 5.2085 13135934 18.7 14 123815 2076.37 5.1892 7845317 11.2 15 125142 2056.09 5.1386 16620267 23.6 16 134157 1918.31 4.7942 33448220 47.6 17 134339 1915.53 4.7873 28856124 41.0 18 134832 1908.00 4.7684 30726168 43.7 19 142387 1792.53 4.4799 9566231 13.6 20 153505 1622.61 4.0552 19454938 27.7 21 154313 1610.27 4.0244 20447492 29.1 22 180239 1214.03 3.0341 5860023 8.3 23 181140 1200.26 2.9997 7073372 10.1 24 189786 1068.12 2.6694 6218422 8.8 25 190790 1054.78 2.6411 5453231 7.8

54

13C/100 MHz/acetone-d6 NMR of Epiacutissimin B (5b)

(ppm)0102030405060708090100110120130140150160170180190

55

13C/100 MHz/acetone-d6 NMR Peak Picking of Epiacutissimin B (5b)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 4626 17015.08 169.1146 4660042 75.3 2 4935 16825.72 167.1326 1205033 19.5 3 4965 16807.34 167.0498 2037964 32.9 4 5131 16705.61 166.0388 930097 15.0 5 5204 16660.88 165.5942 4245596 68.6 6 6656 15771.09 156.7504 981030 15.9 7 6843 15656.49 155.6115 2173864 35.1 8 7227 15421.18 153.2727 1151814 18.6 9 8430 15625.65 145.3092 5276453 70.2 10 8437 14620.45 145.2754 5836583 80.3 11 8544 14614.12 145.2512 5749308 92.9 12 8550 14610.44 145.2146 5584579 90.3 13 8605 14576.74 144.8797 3022036 48.8 14 8661 14542.42 144.5386 6186991 100.0 15 8665 14539.97 144.5142 6098060 98.6 16 8677 14532.61 144.4411 2651140 42.9 17 8717 14508.10 144.1975 1438955 23.3 18 8741 14493.39 144.0513 2433439 39.3 19 8866 14416.79 143.2900 921678 14.9 20 8951 14364.71 142.7723 855007 13.8 21 9869 13802.15 137.1810 1544242 25.0 22 9906 13779.48 136.9556 1412437 22.8 23 10035 13700.43 136.1699 3408550 55.1 24 10176 13614.02 135.2112 3148777 50.9 25 10224 13584.61 135.0188 1659468 26.8 26 10889 13177.09 130.9685 1038259 16.8 27 11451 12832.70 127.5455 1917309 31.0 28 11546 12774.48 126.9669 1423799 23.0 29 11637 12718.72 126.4126 1009201 16.3 30 11867 12577.77 125.0118 3879262 62.7 31 11941 12532.43 124.4110 1768358 28.6 32 12544 12162.91 120.8884 1165312 18.8 33 12824 11991.32 119.1830 1577248 25.5 34 13250 11730.27 116.5883 4321735 69.9 35 13456 11604.03 115.3336 2523917 40.8 36 13534 11556.23 114.8585 1398641 22.6

37 13590 11535.10 114.3865 1594278 30.6 38 13601 11521.28 114.3156 3876519 60.1 39 13626 11499.85 114.2982 5729538 92.6 40 13684 11464.31 113.9449 912806 14.8 41 13813 11385.26 113.1592 1431497 23.1 42 14468 10983.87 109.1698 1257904 20.3 43 14611 10896.24 108.2989 3335620 53.9 44 14852 10748.56 106.8310 3027712 48.9 45 15137 10573.91 105.0951 1185484 19.2 46 16084 9993.58 99.3272 1188914 19.2 47 16564 9699.44 96.4037 1248570 20.2 48 19378 7975.01 79.2644 3078033 49.8 49 19625 7823.65 77.7600 2166498 35.0 50 20593 7230.46 71.8642 4076543 65.9 51 20765 7125.05 70.8166 2853657 46.1 52 20974 6996.98 69.5437 1188777 19.2 53 21522 6661.16 66.2060 3650870 59.0 54 21610 6607.24 65.6700 1993545 32.2 55 26273 3749.74 37.2690 1364911 22.1 56 28007 2687.14 26.7077 1477399 23.9

56

Dept 135/100 MHz/acetone-d6 NMR of Epiacutissimin B (5b)

57

Dept 135/100 MHz/acetone-d6 NMR Peak Picking of Epiacutissimin B (5b)

Peak Picking results -------------------- Peak Nr. Data Point Frequency PPM Intensity %Int. ---------------------------------------------------------------- 1 6707 11987.66 119.1466 40710460 37.7 2 7031 11553.99 114.8363 68989384 60.5 3 7100 11461.64 113.9183 47721944 41.8 4 7460 10979.78 109.1291 113184048 99.5 5 7519 10900.81 108.3442 67607904 59.9 6 7634 10746.88 106.8143 81478176 71.4 7 8411 9706.87 96.4776 54214496 47.4 8 9706 7973.53 79.2497 52161836 45.3 9 9822 7818.26 77.7065 100124184 87.5 10 10263 7227.99 71.8397 99311960 87.5 11 10341 7123.59 70.8021 111742768 98.1 12 10434 6999.11 69.5648 94394600 82.4 13 10683 6665.82 66.2523 114056616 100.0 14 10731 6601.58 65.6137 -38434100 -33.4 15 12862 3749.25 37.2642 71820272 63.1 16 13649 2695.86 26.7945 -36822560 -32.8

58

HMQC Spectrum of Epiacutissimin B (5b)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

140

120

100

80

60

40

(ppm)

59

HMBC Spectrum of Epiacutissimin B (5b)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

160

140

120

100

80

60

40

(ppm)

60

Portion of HMBC Spectrum of Epiacutissimin B (5b) Showing Relevant Connectivity Correlations

between the Flavanoid and the C-Glycosidic Units

(ppm) 6.8 6.4 6.0 5.6 5.2 4.8 4.4

160

140

120

100

(ppm)

H1-C7’ H1-C5’

H1-C6’

61

HPLC-ESI-MS of a Partially Purified Red Wine Sample

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

5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0 4 5 .0 0T im e5

1 0 0

%

0

1 0 0

%

4a

Α: λ = 280 nm

B: m/z = 1205

5b 5a

4b

1b 1a

X

5b 4a

62

LC-MS of Acutissimin A (4a) from Red Wine -90eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

301

249

245

275

915

593305

493

467351 425

369517

575

518

631 897683 787701 871

975

916

976

9771205

10531083 12061265 1344 1457 1499

63

LC-MS of Acutissimin B (4b) from Red Wine -90eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

289

287

245

577

575

407

301

405315

425

449

465493

915

593

729

613728

785

730

863

786

1017

916

1015

1153

1018

1152

130511541205 1307 1441 1457

64

LC-MS of Epiacutissimin A (5a) from Red Wine -90eV

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

577

287

245

407

301407

303369

575425

425451

465569

865578

865

695633 739 863755

915

8671017916

91711691153

10561205

14411249 1312 1473

65

LC-MS of Epiacutissimin B (5b) from Red Wine -90eV

200 400 600 800 1000 1200 1400 1600m/z0

100

%

915

301

287

275897

303

493467

407369

341

602

591

575

533

613

746745

699871754

975

917

976

100511691083 1205

1207 14931399 1511 1591