Roburin A, A dimeric ellagitannin from heartwood of Quercus robur

Phytochemistry, Vol. 30,No. 1,pp. 329-332,1991 Printedin GreatBritain.

ROBURIN

A, A DIMERIC

00319422/91 %3.00+0.00 Q 1990PergamonPressplc

ELLAGITANNIN FROM HEARTWOOD QUERCUS ROBUR

OF

CATHERINE L. M. HERVE DU PENHOAT, VERONIQUE M. F. MICHON, ABDELHAMID OHASSAN, SHUYUN PENG,* AUGUSTIN SCALBERT* and DOUGLAS GAGE? Laboratoire de Chimie, Ecole Normale Supkrieure, 24 rue Lhomond, Paris 75231, France; * Laboratoire de Chimie Biologique (INRA) LN.A.-P.G., Thiverval-G&non 78850, France; TDepartment of Biochemistry, Biochemistry Building, Michigan State University, East Lansing, MI 488241319, U.S.A. (Received in revised form 23 April 1990)

Key

Word

Index-Quercus

robur; Fagaceae; heartwood, ellagitannin; vescalagin; castalagin; dimer; NMR.

Abstract-Three ellagitannins from Quercus robur wood have been studied by high resolution ‘H and 13C NMR. Two are the epimeric 1,2,3,5-nonahydroxytriphenoyl-4,6-hexahydroxydiphenoyl-glucoses, castalagin and vescalagin. The third is a dimeric compound, roburin A, composed of two vescalagin subunits probably linked through an ether bond between the diphenoyl group of one subunit and the triphenoyl moiety of the other one.

INTUODUCTION

Recently, correlation of tannin biological activity with structure has been made possible due to isolation by reverse-phase HPLC methods and structural determination by high resolution NMR and FAB mass spectrometric techniques. Heartwood of Quercus robur is known to contain ca 10% by wt of ellagitannins [l] which are responsible for the high durability of this wood [Z]. These hexahydroxydiphenoyl (HHDP) esters also contribute to the taste and the colour of brandies and wines aged in oak barrels [3-53. Castalagin (1) and vescalagin (2), the two main HHDP esters, were originally characterized by Mayer [6] but the presence of at least six other ellagitannins of unknown structure was reported recently [l]. These compounds have now been purified by a combination of chromatography on Sephadex LH-20 and reverse-phase HPLC [7]. The structural elucidation of the fastest-eluted component of these unknown tannins, roburin A (3) is reported here. RESULTS AND DISCUSSION

Several structural studies of 1 and 2 have been reported. ‘H NMR data for 1 [8] in methanol-d,-benzened, (60”) and 2 [9] in methanol-d, and the 13C spectrum of 1 [lo] in acetone-d, have been described. The absolute configuration of the triphenoyl group of 1 and 2 has been determined by circular dichroism [ 111. However, structural analysis of unknown tannins using NMR techniques often relies on comparison of their data with those of various related monomeric units [12,13]. Thus, ‘H NMR spectra in both acetone-d, (data not given) and DMSO-d, were recorded for 1,2 and the unknown polar tannin (3) under comparable conditions. The proton coupling networks for the glucose units were extracted from homonuclear correlation 2D COSY (6-6, ‘H-‘H) experiments and are collected in Table 1. The data for 1 and 2 agree well with those in the above studies. Both the

‘H chemical shifts and the 3J, ,.,coupling constants of the glycosyl residues of open-chaih C-glucosidic ellagitannins are very different from those of compounds containing glucopyranose moieties in either the 4C, [ 143 or ‘C, [ 15, 161 conformations. Moreover, epimers 1 and 2 are readily distinguished by their 3J, 2 coupling constants (4.5 and <2 Hz, respectively). The ‘lH NMR spectrum of 3 showed signals for two glucose units (14 protons) between 2.7 and 5.7 ppm and the total coupling networks for both of these residues were extracted from the COSY spectrum. The glucose units with H-5 resonating at low and high fields, respectively, will be referred to as Glcl and Glc2 and numbering of the aromatic rings (I-V and I’-V’) is indicated on the formulae. The chemical shifts, with the exception of the primary hydroxyl protons of Glc2, as well as the 3J, a coupling constants were very similar to the data obtained for 2 suggesting that this tannin is a dimer composed of two vescalagin subunits. T’he high field shifts of H-6 and H-6’ of Glc2 (-0.73 and - 1.39 ppm, respectively) as compared to those of Glcl and 2 arc striking. A possible explanation for these high field shifts would be that the OH-6 group of Glc2 is not acylated. It is known that glucose protons show characteristic low-field shifts (+ 1.3 ppm for galloylation of the OH-5 in casuarinin [17]) upon acylation of the adjacent hydroxyl group with a polyphenolic carboxyl group. However, C-glucosidic tannins with a free primary hydroxyl group show a small chemical shift difference A(&_, -a,_,.) for the primary hydroxy protons (e.g. an AB system for punicacortein A [18]) in contrast to the large chemical shift difference, 1.523 ppm, observed for Glc2. In order to visualize the environment of the primary hydroxyl groups a molecular model of 2 was constructed. Karplus curves for the various vicinal coupling constants, 3J~.H of 2 were established using Altona’s empirical equations [ 191 and applying the observed coupling constants to these curves led to values of CQ55” for both the H-5/C-5/C-6/H-6 and H-5/C-5/C-6/H-6’ dihedral angles. In the corresponding Drciding model of 2 the primary

329

C. L. M. HERVEDU PENHOATet al.

330

HO H

1

R’ =

3

H, R’ = OH

2 R’ = OH, R2 = H

Table 1. ‘H chemical shifts [multipliciti~ and 3JX,x+t or 2JX,x‘ coupling constants (Hz)] of compounds 1, 2 and 3 (400 MHz, DMSO-d,, 6t, 2.72) 3 H

Glc 1

Glc 2

4.881

4.R8t

5.148

5.117

4.663

1

2

5.694 V. 4.5) 5.00 (d. 4.5) 5.021 (d, 7.5) 5.120 (d, 7.5) 5.617 5.095 (d, 12) 4.184

4.852 t&r s) 5.158 (hr s) 4.509 (d, 7) 5.113 0, 7) 5649 5.059 (d, 11.5) 4.202

4.188

4.541 (d, 6.8) s.105 (d, 6.3) 5.456 4.330 (d, 12.7) 2.807

(d, 12)

fd, 11.5)

(d, 10.7)

(d, 11.7)

6.733

4.723 (s) 6.734 (s) 6.581 (s)

6.619

GIG 1

2 3 4 5 6 6 Aromatics H’“’1) &pV”’

HV”’

(S) 6.84 w 6.587 b)

5.191 0, 7.3) 5.505 4.663

fs) 7.296 (s) 6.557, (s)

7.325 (s) 6.736 (.r) 7.5337 (s)

*Numbering of polyphenols according to formula I. +3Jn_s,H_6 3Hz and 3Jn_s,H-6s, 2 Hz from a phase-sensitive double quantum-filtered COSY spectrum in acetone-d,. TTentative assignment from spectrum acquired with a 30 set recycle time. Extracted from the x + 1 multiplet.

group is situated below IV with H-6’ pointing towards the centre of this ring. Flexibility of the HHDP moiety is accompanied by variation in the methylene-IV distance. Thus, it appeared that the high field shifts might be related to a small change in the virtual conformation of the flexible HHDP group. The polyphenolic groups of hydrolysable tannins can often be recognized in ‘H spectra, far example, one 2H singlet between 86.95 and 7.25 for a galloyl group, two (one) 1H singlets between fi6.3 and 6.8 for a HHDP [nonahydroxytriphenoyl (NHTP)] group, three 1H singlets, two near 86.4 and one near 67.2 for a valoneoyl group, etc. [ 12, 131. Although six aromatic proton signals were expected in the spectrum of 3 (three per vescalagin subunit) only five were observed. Moreover, two of these latter protons presented signals which were shifted to low field (+OS ppm) (ride supraf. However, when the iH spectrum of 3 was acquired with a 30 set recycle delay the integral of a proton at 67.533 increased from O.tH to 0.25H. It was possible that 3 contains an aromatic proton which relaxes much more slowly than the others. Significant differences in relaxation rates, which may be correlated with interproton distances, have been described for carbohydrate molecules [ZO]. Broad-band and J-modulated ‘“C spectra were recorded for tannins 1 -3 and 2D heteronuclear chemical shift correlation data were obtained for 1 and 2. In the case of 3 this data could not be obtained, due either to insufficient material or short T,s or both. This information is collected in Table 2. The “C spectrum of 3 contained 80-85 peaks, ostensibly twice the corresponding number in the spectra of monomeric compounds 1 and 2. The chemical shifts of the C-2 signals appear at lowest field among the glucose signals of C-glucosidic tannins and are dependent on the configuration at hydroxyl

Ellagitannin from Quercus robw Table 2. i3C chemical shifts of compounds 1, 2 and 3 (100 MHz, DMSO-d,, 6c 39.5 ppm).

331 3JC,H

3 C

1

2

65.97 12.13 65.12 67.90 69.83 64.63

64.08 76.31 67.23 68.28 69.71 64.50

Glc 1

Glc 2

Glc 1 2 3 4 5 6

76.24. 76.84’ Fig. 1. Glucose-polyphenol 63.82, 64.67’

Aromatics 1 2 3

121.14 115.29 142.41

123.84 116.45 143.0

2 3 4 6

106.77 144.48 135.54 114.28

106.49 144.43 135.45 114.24

144.60 135.38 114.06

143.93 135.06 114.52

2 IV”’ 3 4 6

106.86 144.53 135.72 115.20

106.62 144.43 135.87 115.39

136.26 116.01

144.60 135.72 114.90

2 3 4 6

105.54 144.56 135.02 114.50

105.29 144.49 134.86 114.36

144.60’ 134.54’ 114.29”

162.91 165.37 165.83 165.83 168.17

163.30 165.21 165.84 165.79 168.18

162.65 163.25 164.85 165.23 166.52 165.12 165.72 166.34 166.74, 168.35’

I

VW

Carbonylst G2 to I”’ G3 to II”’ G4 to IV”’ G5 to III”’ G6 to V”’

linkage network.

The dimeric nature of tannin 3 was confirmed by its FAB mass spectrum which indicated that its M, ([M -H] - m/z 1849) is twice that of 1 or 2 ([M -HIm/z 933) less one water molecule. Acidic treatment of 3 under conditions which hydrolyse only the HHDP moiety of 2 afforded 1 mol ellagic acid per mol substrate. A structure such as 3 with an ether linkage between C”‘-3 of Glcl and Cm’-3 of Glc2 would be compatible with both the FAB mass spectrum and the acidic hydrolysis results. This linkage would explain the low field shifts observed for aromatic protons HIV-2 (Glcl) and Hut’-2 (Glc2) due to the proximity to the III’ and IV rings, respectively. However, this proposal is only tentative as the unambiguous assignment of the V and V’ aromatic protons are lacking. In conclusion, the majority of the ‘H and 13CNMR parameters for castalagin (1) and vescalagin (2) have been determined by selective long-range heteronuclear correlation experiments. Comparison of these NMR data with those of the polar tannin of Q. robur, roburin A, suggests that the latter compound has a dimeric structure possibly with a Cn” 3-o-C” 3 linkage. To the best of our knowledge, this is the first dimer of a C-glucosidic tannin to be reported. EXPERIMENTAL.

*Numbering of polyphenols according to the given formulae. Multiplicity from J-modulated spectrum. t Ester carbonyl between glucose carbon, Gx, and polyphenol. aAssignmentmay be reversed.

C-l [16]. Thus, the resonances at 676.24 and 76.84 could be attributed to the C-2s of the glucose units and are analogous to the value observed for 2. Both the number and chemical shift range of the various groups of quaternary aromatic carbons are those expected for two vescalagin units (see Experimental). In order to assign the aromatic protons, the carbonyls and certain aromatic carbons, selective long-range heteronuclear chemical shift correlation (INAPT [21]) experiments were performed. This technique provides the glucose-polyphenol linkage network through 3J,. c coupling to the carbonyls. (Fig. 1). These assignments are collected in Table 2. The low-field aromatic protons were identified as Hrv-2 (Glcl) and Hi*“-2 (Glc2). Very similar aromatic and carbonyl carbon chemical shifts are observed for tannins l-3 corroborating the presence of analogously linked HHDP and NHTP groups in tannin 3. This approach could not be applied to the slowlyrelaxing proton at 6 7.533 as 5 000-10 000 transients were required in order to obtain a reasonable S/N ratio.

NMR. Solvents (DMSO-I, dist. over charcoal), int. standards and spectrometer frequencies for NMR spectra are indicated in Tables 1 and 2. The digital resolution of the ‘H spectra was 0.5 Hz pt-i and the acquisition time was 2.01 sec. i3C spectra were recorded with complete proton decoupling, an acquisition time of 1.11 set, digital resolution of 0.9 Hzpt-’ and a recycle time of 4.1 sec. INAPT [21] spectra were acquired under similar conditions using a 5 Hz filter for polarization transfer. FABMS. Spectra were acquired on a double focussing sector instrument in the negative mode with a resolution of 3.000 and an acceleration voltage of 10 kV using a glycerol matrix. Extraction and purification of tannins. Castalagin (I), vesca!agin (2) and roburin A (3) were extracted by aq. MeOH from Q. robur L. heartwood and purified by Sephadex LH-20 chromatography and reverse-phase HPLC as previously described [7]. Yields were respectively, 1.1, 3.1 and 0.4 g kg-i wood. UV spectra were obtained with a diode array detector coupled to an analytical HPLC. Chromatographic conditions were the following Merck Lichrospher RP-18e (5 pm) column (25 cm x 4 mm i.d.); linear gradient O-10% Solvent B from 0 to 40 mm, Solvent A:H,O-H,PO, (99O:lk Solvent B: MeOH-HsPO, (990:lk flow rate 1 ml min-i. R,s were 28.9, 25.1 and 17.0 min. for 1, 2 and 3, respectively, 3 being the fastest-sluted component among the eight ellagitannins identified. The three compounds had identical UV spectra with no maximum between 240 and 400 nm

C. L. M. HERVEDU PENHOATd a!.

332

but a shoulder at 280 nm. i3C NMR (DMSO-d,): Unassigned glucosyl carbons-63.97 (IQ, 67.34 (iC), 68.04 (HZ), 69.34 (IC) 69.74 (2 or 3C), 70.59 (lC), (Gl, G3-GS); methine aromatic carbons-104.76, 106.38, 108.16 [all 1 or 2C: C2 (III”‘-V”))]; quaternary aromatic carbons---li1.57-116.85 [lSC (14 expected): C2 (I(‘), II(‘)), C6 (I(‘)-V(‘)}], 122.24-125.95 [IOC: Cl (I”‘--V”‘)], 132.89-136.80 [IOU: C4 (I”‘-V”‘)J, f43.06147.24 [2OC: C3 and C5 (Ir’)-V(“)], 162.65-168.36 [lOC C=O (pi-V(‘))]. Three methine carbones at 226.48 (OX), 126.60 (IC) and 128.11 (lC), probably due to impurities, were also observed. Acid hydrolysis o~~unains. Purified tannins (5 mg) were solubilized in MeOH- M HCI (9: 1) (5 ml) contaimng I-naphthol (0.5 mg) as int. standard. The soins (0.5 ml), in tubes with teflonlined screw caps, were kept at 120” for 160 min. The resulting ellagic acid was estimated by HPLC. The chromatograph~c conditions were the same as descrtbed above apart from the linear gradient: (r90% Solvent B from 0 to 30 min. Acknow~edge~n~s-We

thank the Pierre and Marie Curie University (Paris VI) and the CNRS (URA DllOl) for financial support.

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