Biosynthesis of the dimeric ellagitannin, cornusiin E, in Tellima grandiflora

Article abstract of DOI:10.1016/S0031-9422(03)00280-2

First evidence for the in vitro synthesis of a dimeric ellagitannin has been obtained with cell-free extracts from the weed Tellima gratidiflora (fringe cups, Saxifragaceae). Partially purified enzyme preparations from leaves of this plant catalyzed the oxidation of 1,2,3,4,6-pentagalloyl-β -D-glucose to the monomeric ellagitannin, tellimagrandin II, followed by oxidative coupling of two units of this intermediate to yield a dimeric derivative. Chemical degradation, MALDI-TOF mass spectrometry, ¹H and ¹³C nuclear magnetic resonance, and CD spectroscopy were employed to identify this enzyme reaction product as cornusiin E which is characterized by a (S)-valoneoyl bridge between glucose-positions 2, 4′ and 6′. This result was supported by comparison with data obtained for cornusiin E that had been isolated from leaves of intact T. grandiflora plants. No indication for the earlier proposed existence of rugosin D (an isomer with a 1,4′6′-bound valoneoyl unit) in T. grandiflora has been obtained in this investigation.

Full text of DOI:10.1016/S0031-9422(03)00280-2

Phytochemistry 64 (2003) 109–114
www.elsevier.com/locate/phytochem
Biosynthesis of the dimeric ellagitannin, cornusiin E, in
Tellima grandiflora
Ruth Niemetza, Gerhard Schillingb, Georg G. Grossa,*
aMolekulare Botanik, Universitat Ulm, D-89069 Ulm, Germany
¨
bUniversitat Heidelberg, Organisch-Chemisches Institut, Im Neuenheimer Feld 270, D-69120 Heidelberg, Germany
¨
Received 21 January 2003; received in revised form 24 March 2003
Dedicated to the memory of Professor Jeffrey B. Harborne
Abstract
First evidence for the in vitro synthesis of a dimeric ellagitannin has been obtained with cell-free extracts from the weed Tellima
grandiflora (fringe cups, Saxifragaceae). Partially purified enzyme preparations from leaves of this plant catalyzed the oxidation of
1,2,3,4,6-pentagalloyl-b-d-glucose to the monomeric ellagitannin, tellimagrandin II, followed by oxidative coupling of two units of
1
this intermediate to yield a dimeric derivative. Chemical degradation, MALDI-TOF mass spectrometry, H and 13C nuclear mag-
netic resonance, and CD spectroscopy were employed to identify this enzyme reaction product as cornusiin E which is characterized
by a (S)-valoneoyl bridge between glucose-positions 2, 40 and 60. This result was supported by comparison with data obtained for
cornusiin E that had been isolated from leaves of intact T. grandiflora plants. No indication for the earlier proposed existence of
rugosin D (an isomer with a 1,40,60-bound valoneoyl unit) in T. grandiflora has been obtained in this investigation.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Tellima grandiflora; Saxifragaceae; Fringe cups; Dimeric ellagitannin biosynthesis; Pentagalloyl-b-d-glucose; Tellimagrandin II;
Cornusiin E
1. Introduction
their infancy, in spite of the fact that this subclass
clearly predominates the rather limited number of gal-
lotannins in Nature, spanning over 500 structurally
characterized members (Feldman and Sahasrabudhe,
1999). Already decades ago, it has been postulated that
suitably orientated galloyl residues of the penta-
galloylglucose core should be connected by oxidative
processes that form the 3,4,5,30,40,50-hexahydroxy-
diphenol (HHDP) moieties characteristic of ellagi-
tannins (Schmidt and Mayer, 1956), a view that was
refined later by Haslam and coworkers (reviewed in
detail, e.g., by Haslam 1989, 1998). Eventual subsequent
oxidation steps were thought to produce a host of oli-
gomeric derivatives by formation of intermolecular C–O
and/or C–C linkages; so far, more than 150 ellagitannin
dimers, trimers and tetramers have been isolated
(Okuda et al., 1993).
According to a traditional definition (Freudenberg,
1920), plant tannins are classified as flavonoid-derived
proanthocyanins (or condensed tannins) and hydrolyz-
able tannins. The latter group comprises a wide variety
of polygalloyl ester derivatives of a central polyol car-
bohydrate moiety, typically b-d-glucopyranose, and is
usually divided into the gallotannin and ellagitannin
subclasses. It is now generally accepted that 1,2,3,4,6-
penta-O-galloyl-b-d-glucopyranose (compound
1 in
Fig. 4) represents the common and immediate precursor
of both groups. For the biosynthesis of gallotannins,
this pivotal intermediate is substituted with additional
meta-depsidically linked galloyl units. On the basis of
recent enzyme studies, a metabolic scheme summarizing
the major routes to hexa- and heptagalloylglucoses has
been presented (Frohlich et al., 2002). Our insights into
the biosynthesis of ellagitannins, in contrast, are still in
First experimental proof of the correctness of the
merely theoretical considerations on the biosynthesis of
ellagitannins has been presented in a recent paper (Nie-
metz et al., 2001) reporting the in vitro oxidation of
pentagalloylglucose (1) to the monomeric ellagitannin,
tellimagrandin II (2), by enzyme extracts from leaves of
* Corresponding author. Tel.: +49-731-50-22624; fax +49-731-50-
22809.
E-mail address: georg.gross@biologie.uni-ulm.de (G.G. Gross).
0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00280-2

110
R. Niemetz et al. / Phytochemistry 64 (2003) 109–114
the weed Tellima grandiflora (fringe cups, Saxi-
fragaceae). Closer investigations led to the isolation of
an O2-dependent laccase-type phenol oxidase that spe-
cifically formed tellimagrandin II (Niemetz and Gross,
2003). Analysis of side-products encountered in these
studies, obtained with crude enzyme preparations,
revealed the existence of a higher molecular weight
compound that was identified as cornusiin E (3), i.e. a
dimeric elagitannin that apparently resulted from the
oxidative condensation of two molecules of tell-
imagrandin II (2). The in vitro formation, structure
identification and isolation of this compound from
native T. grandiflora plants is reported below.
The above postulated intermediary role of tell-
imagrandin II (2) was corroborated in a time-course
experiment with [U-14C]pentagalloylglucose (1). As
depicted in Fig. 2, the substrate 1 was rapidly oxidized
to tellimagrandin II (2). This product, however, could
not accumulate because it was almost simultaneously
transformed to cornusiin E (3). After ca. 3 min, net
synthesis was observed only for this end product, at the
expense of tellimagrandin II (2). The experiment sug-
2. Results and discussion
2.1. Enzyme studies
Cell-free extracts were obtained by homogenizing
leaves of T. grandiflora in liquid N2 and extraction of
the powder with a mixture of strong Tris–HCl buffer
supplemented with borate that complexes inhibitory
endogenous plant phenolics. The resulting crude
extracts were depleted of residual contaminants by
stirring with Amberlite XAD ion-exchange resin, fol-
lowed by ammonium sulfate fractionation, hydro-
phobic-interaction chromatography (HIC) on butyl
sepharose and a final column chromatography step on
hydroxyapatite (for details, see Experimental). As
depicted in Fig. 1A, incubation for 30 min under stan-
ꢀ
dard assay conditions (45 C, pH 5.0) of this partially
purified enzyme with 1,2,3,4,6-pentagalloylglucose (1)
as substrate afforded only one new product that was
identified as the dimeric ellagitannin, cornusiin E (3)
(cf. Section 2.2). Theoretically, such a transformation
should require a monomeric ellagitannin as inter-
mediate, most likely tellimagrandin II (2). This latter
compound was actually detected after short-time incu-
bation (3 min) of reaction mixtures (Fig. 1B), indicating
its extremely efficient conversion to the dimeric end-
product. The supposed role of tellimagrandin II (2) as
intermediate metabolite was finally proven in a third
experiment in which this compound was found to be
directly converted to cornusiin E (3) (Fig. 1C). When
[U-14C]-labelled pentagalloylglucose was used as sub-
strate, both reaction products were found to be radio-
active. This finding was important as it provided
unequivocal proof that the isolated ellagitannins had
actually been synthesized in vitro. It should be empha-
sized that, owing to the ‘tanning’ properties of these
compounds, i.e. their strong tendency to form com-
plexes with proteins and carbohydrates (cf., e.g.,
Haslam, 1989, 1998), such investigations always bear
the inherent risk that enzyme assays are contaminated
with in vivo formed polyphenols.
Fig. 1. HPLC analysis of enzyme reactions forming the dimeric ella-
gitannin, cornusiin E (3), with partially purified extracts from Tellima
grandiflora leaves. (A) Oxidation of pentagalloylglucose (1) to cornu-
siin E (3) after incubation for 30 min at 45 ꢀC. (B) The same reaction
as in A, but analyzed in a short time experiment (3 min incubation),
indicating the intermediacy of the monomeric ellagitannin, tell-
imagrandin II (2), in the biosynthesis of cornusiin E (3). (C) Direct
dimerization (30 min, 45 ꢀC) of tellimagrandin II (2) to cornusiin E
(3). (ꢁ), Enzyme assays; (...), blank with heat-denatured enzyme. For
HPLC conditions, see Experimental.

R. Niemetz et al. / Phytochemistry 64 (2003) 109–114
111
(Fig. 3). (S)-Configuration of both the HHDP and valo-
neoyl moiety was shown in the circular dichroism (CD)
spectrum by a strong positive Cotton effect at 225 nm
(+95.3).
1
Definitive structural proof was achieved by H and
1
13C NMR spectroscopy. H NMR spectra revealed five
galloyl groups, one valoneoyl group and one HHDP
group. The d values of the two glucose units, deter-
mined by COSY and TOCSY experiments, showed
complete acylation of all OH groups and b-configur-
ation of glucoses I and II (for numbering, refer to 3 in
Fig. 4). The linkage between the two glucose units was
determined by HMQC and HMBC experiments via the
ester CO signals, showing that ring C of the valoneoyl
group was connected to O-2 of glucose I, while rings A
and B were bonded to O-60 and O-40 of glucose II,
respectively. The data provided clear evidence that this
compound was identical with cornusiin E (3) that had
been isolated from fruits of Cornus officinalis by Hatano
et al. (1989).
Fig. 2. Time course of the in vitro oxidation of [U-14C]penta-
galloylglucose to tellimagrandin II (2) (...&...) and subsequent dimer-
ization of this intermediate to cornusiin E (3) (–*–).
2.3. Concluding remarks
Our results present first evidence of the enzymatic
formation of a dimeric ellagitannin, cornusiin E (3). As
depicted in Fig. 4, the reaction involves oxidation of
pentagalloylglucose (1), the committed precursor of
both gallotannins and ellagitannins, to a monomeric
ellagitannin, tellimagrandin II (2), which in turn under-
goes dimerization to the end product. This metabolic
gested a pronounced affinity of the enzyme towards
tellimagrandin II (2). This assumption was supported by
analyzing the substrate dependence of this compound
which revealed an initial lag phase from 0 to ca. 4 mM
tellimagrandin II (2), followed by normal Michaelis–
Menten kinetics up to ca. 15 mM; pronounced substrate
inhibition was observed at higher concentrations.
Replotting the data according to Lineweaver and Burk
(1934) revealed a Km of 8 mM. For comparison, the
enzyme catalyzing the first step in this sequence, i.e.
oxidation of pentagalloylglucose (1) to tellimagrandin II
(2), displayed a relatively high Km of 80 mM (Niemetz
and Gross, 2003).
2.2. Reaction product identification
For the unambiguous identification of the dimeric
enzyme reaction product, 100 mg of pentagalloylglucose
(1) were incubated in a scaled-up assay from which 3 mg
of product with a purity of >95% could be isolated (for
details, see Experimental). To facilitate NMR spectro-
scopy studies, greater amounts of this compound were
purified from dried leaves of T. grandiflora (cf. Experi-
mental); both preparations were found to be identical.
MALDI-TOF analysis displayed a molecular weight
of 1874.3 (M+) which is consistent with the expected
formula C82H58O52 for a dimeric derivative of tell-
imagrandin II (2). RP-HPLC of a total acid hydrolysate
of the reaction product revealed gallic, ellagic and valo-
neic acid as phenolic constituents, while sanguisorbic
acid (an isomer of valoneic acid that functions as ana-
logous linking unit in various ellagitannins) was absent
Fig. 3. HPLC analysis of phenolic components after total acid
hydrolysis of the enzyme reaction product, cornusiin E (3). S, Solvent
peak; GA, gallic acid; VA, (S)-valoneic acid; EA, ellagic acid. The
retention time of (S)-sanguisorbic acid (SA), a naturally occurring
isomer of valoneic acid with the galloyl residue attached in ortho-
position to the HHDP moiety, is indicated by the arrow.

112
R. Niemetz et al. / Phytochemistry 64 (2003) 109–114
Fig. 4. Pathway from 1,2,3,4,6-pentagalloylglucose (1) to the dimeric ellagitannin, cornusiin E (2), in Tellima grandiflora. In reaction , penta-
galloylglucose (1) is oxidized to the monomeric ellagitannin, tellimagrandin II (2) (Niemetz et al., 2001; Niemetz and Gross, 2003). This intermediate
undergoes oxidative dimerization to yield cornusiin E (3) (reaction ). Labelling A–C, I, II in (3) designates groups relevant to NMR analyses (cf.
Section 3.5).
sequence is particularly fascinating because of its strict
product specificity—no formation of alternative lin-
kages as realized in other plants has been observed in
this investigation. Step 1 of the pathway, i.e. the for-
mation of tellimagrandin II (2), has been recently found
to be specifically catalyzed by a laccase-type phenol
oxidase (Niemetz and Gross, 2003). However, it is still
unknown to date whether the transitions reported here
depend on a single, rather unspecific enzyme, or whether
two individual enzymes are involved. Current activities in
our laboratory are devoted to this important question.
In an earlier broad survey on the taxonomic distribu-
tion of hydrolyzable tannins, based on paper chroma-
tography as principal analytical tool, two then
unidentified dimeric tannins (T2A, T2B) had been detec-
ted as prominent constituents of T. grandiflora leaves
(Haddock et al., 1982). Their structures were later
interpreted as rugosins D (T2B) and E (T2A), respectively
(Haslam, 1989). These two compounds have been iso-
lated by Hatano et al. (1990) from flowers of Rosa
rugosa and were identified as dimers originating from
two tellimagrandin II units (rugosin D) or from a com-
bination of tellimagrandin I and II (rugosin E), both
being connected by a 1,40,60-valoneoyl bridge. The
results of our studies, based on enzyme studies and
phytochemical analyses, do not lend any support to the
existence of rugosin-type ellagitannin dimers in T.
grandiflora. Instead, it must be concluded that meta-

R. Niemetz et al. / Phytochemistry 64 (2003) 109–114
113
bolic activities in this plant are specifically directed
towards the biogenesis of cornusiin-type (i.e. 2,40,60-
linked) ellagitannin dimers.
3.4. Enzyme preparation and assay
Enzyme from leaves of T. grandiflora was extracted
and partially purified according to Niemetz and Gross
(2003).The enzyme fraction eluting with 0.6 M ammo-
nium sulfate from the butyl-sepharose column was sub-
jected to a final purification step on a hydroxyapatite
column from which it was recovered with 0.3 M Na
phosphate buffer. Standard enzyme activity assays,
containing 12.5 mg (42 Bq) pentagalloylglucose (1) and
enzyme in 50 ml acetate buffer (50 mM, pH 5.0), were
incubated at 45 ꢀC for 30 min, stopped by heating
(100 ꢀC, 5 min), and analyzed by isocratic RP-18 HPLC
on Reprosil NE (cf. Section 3.2). Acid-denatured
enzyme was used as a control. Radioactivity was deter-
mined by fractionation of eluates and subsequent
liquid-scintillation counting.
3. Experimental
3.1. Plant material
Young leaves (2–4 months old) from greenhouse
grown T. grandiflora (Pursh) Lindley (fringe cups, Saxi-
fragacea) plants were washed with dist. H2O and either
immediately used as enzyme source, or frozen in liquid
N2 and stored at ꢁ20 ꢀC in evacuated plastic bags
where they could be kept for more than 6 months with-
out apparent loss of enzyme activity.
3.2. Chemicals
3.5. In vitro synthesis and analysis of cornusiin E (3)
[U-14C]Pentagalloylglucose (1) was prepared by pho-
toassimilation of 14CO2 with leaves of Rhus typhina
(staghorn sumac) and isolated in >99% purity by
Sephadex LH-20 chromatography and preparative
reversed-phase HPLC (Rausch and Gross, 1996); unla-
belled pentagalloylglucose (1) was isolated from dried
leaves of R. typhina by the same techniques. Tell-
imagrandin II (2) was prepared enzymatically (Niemetz
and Gross, 2003). Rugosin D (Hatano et al., 1990) and
Sanguiin H-6 (Okuda et al., 1992) were gifts of Prof. T.
Yoshida (Okayama). Valoneic and sanguisorbic acid,
respectively, were obtained from these compounds by
hydrolyzing ca. 0.5 mg of each compound in 200 ml 6 N
HCl at 100 ꢀC for 4 h. After adjusting the pH to 5.0, 50
ml aliquots were analyzed by isocratic RP-HPLC on
Reprosil NE (5 mm; 250ꢂ4 mm i.d.; solvent 0.01% aq.
TFA/EtOH/EtOAc=10:2:1, by vol.; flow rate 0.5 ml
minꢁ1). Cornusiin E (3) was hydrolyzed identically.
For the large-scale preparation of cornusiin E, T.
grandiflora leaves (150 g) were homogenized, extracted
and treated with Amberlite XAD ion-exchange resin
according to Niemetz and Gross (2003). The pellet (800
mg protein) of the subsequent 30–90% ammonium sul-
fate precipitation step was dissolved in acetate buffer (50
mM, pH 5.0), desalted on Sephadex G-25 (Pharmacia;
30ꢂ1.6 cm i.d.) and diluted to 350 ml with buffer for use
in a scaled-up enzyme assay mixture containing 100 mg
pentagalloylglucoꢀse (1) as substrate. After incubation
for 60 min at 45 C, the reaction was stopped by heat-
denaturing the enzyme. The supernatant of the cen-
trifuged mixture was brought to pH 4.0 with 1 N HOAc
and extracted 10 times with EtOAc. After removing the
organic solvent by rotary evaporation, the residue was
dissolved in 10 ml H2O and subjected in two portions to
semi-preparative gradient RP-18 HPLC on Kromasil (5
mm, 250ꢂ20 mm i.d.; gradient: solvent A=0.01% aq.
TFA, B=acetonitrile; 0–1 min 5% B, 1–2 min 5–18%
B, then isocratic at 18% B; flow rate 22 ml/min). Rele-
vant fractions were immediately depleted of organic
solvent and TFA in vacuo, lyophilized and subjected to
further isocratic RP-18 HPLC on the same column
(solvent 0.01% aq. TFA/EtOH/EtOAc=10:2:1; by vol.;
flow rate 12 ml minꢁ1). The product-containing frac-
tions were neutralized, depleted of solvent and lyophy-
lised, affording 3 mg material of >95% purity as
determined by analytical HPLC (for the isolation of
cornusiin E (3) from native plant material, see Section
3.3).
3.3. Cornusiin E (3) from native plant material
Cornusiin E (3) was isolated from T. grandiflora
ꢀ
leaves that had been dried overnight at 30 C, followed
by powdering in an ultracentrifugal mill (Retsch KG,
Haan, Germany) in liquid N2 and extracting with 70%
aq. acetone on a gyrotory shaker at room temp. for 3
days. After removing the acetone by rotary evaporation,
the aqueous phase was washed 5-times with petroleum
ꢀ
benzene (40–60 C) and lyophilized. A 200 mg aliquot
of the solid residue was dissolved in 5 ml H2O and
subjected to semi-preparative isocratic RP-HPLC
(modified from Yoshida et al., 1991) on Kromasil (5
mm, 250ꢂ20 mm i.d.; solvent: 0.01% aq. TFA/EtOH/
EtOAc=10:2:1; flow rate 12 ml minꢁ1; detection UV
280 nm). Relevant fractions were depleted of acid and
organic solvent by rotary evaporation and lyophilized
to yield 50 mg of >95% pure product.
3.5.1. Analysis of (3)
MALDI-TOF MS: the compound was dissolved in
MeOH, and a 40% solution of isopropanol, imidazole
and piperidine (1:1:1) in MeOH was added for ESI
spectrometry (Finnigan MAT TSQ 700); m/z 936.5

114
R. Niemetz et al. / Phytochemistry 64 (2003) 109–114
(M2+), 1874.3 (M+). CD (MeOH): 225 nm (+95.2),
Freudenberg, K., 1920. Die Chemie der naturlichen Gerbstoffe.
Springer Verlag, Berlin.
1
237 (sh), 260 (ꢁ13.3), 281 (+26.5), 315 (ꢁ3.6). H and
Frohlich, B., Niemetz, R., Gross, G.G., 2002. Gallotannin biosynth-
esis: two new galloyltransferases from Rhus typhina leaves pre-
ferentially acylating hexa- and heptagalloylglucoses. Planta 216,
168–172.
13C NMR spectra were recorded in acetone-d6 on a
Bruker DEX 500 MHz spectrometer; d ppm (TMS):
7.17, 7.14 (s, 2H, 1,3-galloyl glcI); 7.13, 6.99, 6.86 (s, 2H,
10,20,30-galloyl glcII); 6.72, 6.31, 6.85 (s, 1H, valoneoyl
H-A,B,C); 6.59, 6.53 (s, 1H, HHDP-A,B); 5.69 (glcI H-
1, J1H,2H=8.1 Hz), 5.55 (glcI H-2, J2H,3H=9.5 Hz), 5.53
(glcI H-3, J3H,4H=9.8 Hz), 5.11 (glcI H-4, J4H,5H=9.8
Hz), 4.46 (glcI H-5, J5H,6H=7.9 Hz), 5.16 (glcI H-6,
Haddock, E.A., Gupta, R.K., Al-Shafi, S.M.K., Layden, K., Haslam,
E., Magnolato, D., 1982. The metabolism of gallic and hexahy-
droxydiphenic acid in plants: biogenetic and molecular taxonomic
considerations. Phytochemistry 21, 1049–1062.
Haslam, E., 1989. Plant Polyphenols. Vegetable Tannins Revisited.
Cambridge University Press, Cambridge.
J5H,6 H=<1Hz), 3.76 (glcI H-60, J6H,6 H=13.3Hz); 6.22
(glcII H-1, J1H,2H=8.4 Hz), 5.58 (glcII H-2, J2H,3H=9.5
Hz), 5.68 (glcII H-3, J3H,4H=9.8 Hz), 5.15 (glcII H-1,
J4H,5H=10.1 Hz), 4.81 (glcII H-5, J5H,6H=10.1 Hz),
Haslam, E., 1998. Practical Polyphenols. From Structure to Molecular
Recognition and Physiological Action. Cambridge University Press,
Cambridge.
0
0
Hatano, T., Yasuhara, T., Okuda, T., 1989. Tannins of cornaceous
plants. II. Cornusiins D, E, and F, new dimeric and trimeric
hydrolyzable tannins from Cornus officinalis. Chemical and Phar-
maceutical Bulletin (Tokyo) 37, 2665–2669.
5.60 (glcII H-6, J5H,6 H=<1 Hz), 3.96 (glcII H-60,
0
0
J6H,6 H=13.1 Hz). These data were fully consistent with
those of Hatano et al. (1989). In addition, 13C shifts of
CO carbonyls were determined: 167.54 (valA), 166.92
(valB), 164.92 (valC); 167.21 (HHDPA), 166.83
(HHDPB); 164.40 (gal glcI O-1), 165.58 (gal glcI O-3),
164.98 (gal glcII O-1), 164.62 (gal glcII O-2), 164.92 (gal
glcII O-3).
Hatano, T., Ogawa, N., Shingu, T., Okuda, T., 1990. Tannins of
rosaceous plants. IX. Rugosins D, E, F and G, dimeric and trimeric
hydrolyzable tannins with valoneoyl group(s), from flower petals of
Rosa rugosa Thunb. Chemical and Pharmaceutical Bulletin (Tokyo)
38, 3341–3346.
Lineweaver, H., Burk, D., 1934. The determination of enzyme dis-
sociation constants. Journal of the American Chemical Society 56,
659–666.
Niemetz, R., Schilling, G., Gross, G.G., 2001. Ellagitannin biosynth-
esis: oxidation of pentagalloylglucose to tellimagrandin II by an
enzyme from Tellima grandiflora leaves. Chemical Communications
35–36.
Acknowledgements
Niemetz, R., Gross, G.G., 2003. Oxidation of pentagalloylglucose to
the ellagitannin, tellimagrandin II, by a phenol oxidase from Tellima
grandiflora leaves. Phytochemistry 62, 301–306.
We are indebted to Mrs. A. Muller for excellent tech-
nical assistance, Prof. G. Bringmann and Dr. M. Dreyer
(Wurzburg) for recording and interpretation of CD
spectra, and Prof. T. Yoshida (Okayama) for providing
ellagitannin references and original NMR spectra.
Generous financial support by the Deutsche For-
schungsgemeinschaft (Bonn) and the Fonds der Che-
mischen Industrie (Frankfurt/M.) is gratefully
acknowledged.
Okuda, T., Yoshida, T., Hatano, T., Iwasaki, M., Kubo, M., Orime,
T., Yoshizaki, M., Naruhashi, N., 1992. Hydrolysable tannins as
chemotaxonomic markers in the Rosaceae. Phytochemistry 31,
3091–3096.
Okuda, T., Yoshida, T., Hatano, T., 1993. Classification of oligomeric
hydrolysable tannins and specificity of their occurrence in plants.
Phytochemistry 32, 507–521.
Rausch, H., Gross, G.G., 1996. Preparation of [14C]-labelled 1,2,3,4,6-
penta-O-galloyl-b-d-glucose and related gallotannins. Zeitschrift fur
Naturforschung 51c, 473–476.
References
Schmidt, O.Th., Mayer, W., 1956. Naturliche Gerbstoffe. Angewandte
Chemie 68, 103–115.
Feldman, K.S., Sahasrabudhe, K., 1999. Ellagitannin chemistry.
Syntheses of tellimagrandin II and a dehydrodigalloyl ether-con-
taining dimeric gallotannin analogue of coriariin A. Journal of
Organic Chemistry 64, 209–216.
Yoshida, T., Jin, Z.-X., Okuda, T., 1991. Heterophylliins A, B, C, D
and E, ellagitannin monomers and dimers from Corylus hetero-
phylla Fisch. Chemical and Pharmaceutical Bulletin (Tokyo) 39,
49–54.