Ellagitannin biosynthesis: Oxidation of pentagalloylglucose to tellimagrandin II by an enzyme from Tellima grandiflora leaves

Article abstract of DOI:10.1039/b006270g

First evidence of the in vitro oxidation of 1,2,3,4,6-pentagalloylglucose to the ellagitannins, tellimagrandin II and 1,4,6-tri-O-galloyl-2,3-O-hexahydroxydiphenoyl-β-D-glucose, has been obtained with a partially purified enzyme from leaves of Tellima grandiflora (fringe cups, Saxifragaceae).

Full text of DOI:10.1039/b006270g

Ellagitannin biosynthesis: oxidation of pentagalloylglucose to tellimagrandin II
by an enzyme from Tellima grandiflora leaves
a
b
a
Ruth Niemetz, Gerhard Schilling and Georg G. Gross*
a
Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany. E-mail: ruth.niemetz@biologie.uni-ulm.de;
georg.gross@biologie.uni-ulm.de
Universität Heidelberg, Organisch-Chemisches Institut, Im Neuenheimer Feld 270, D-69120 Heidelberg, Germany.
E-mail: gerhard.schilling@urz.uni-heidelberg.de
b
Received (in Liverpool, UK) 28th July 2000, Accepted 15th November 2000
First published as an Advance Article on the web 11th December 2000
First evidence of the in vitro oxidation of 1,2,3,4,6-penta-
suffered from minimal enzyme reaction rates yielding numer-
galloylglucose to the ellagitannins, tellimagrandin II and
ous structurally closely related reaction products and unspecific
by-products, and particularly from the inherent risk of con-
tamination with in vivo formed ellagitannins that had been
transferred into the enzyme assays by complexation with
proteins. Such problems are eliminated by using radioactively
labeled compounds, a technique that dramatically increases
both the sensitivity and specificity of test systems. We therefore
1
,4,6-tri-O-galloyl-2,3-O-hexahydroxydiphenoyl-b- -glu-
D
cose, has been obtained with a partially purified enzyme
from leaves of Tellima grandiflora (fringe cups, Saxi-
fragaceae).
It was already recognized in the mid 1950’s that ellagitannins,
a widespread subclass of hydrolyzable plant tannins, were
characterized by 3,4,5,3A,4A,5A-hexahydroxydiphenic (HHDP)
acid residues (5) that occur in Nature in the form of various
glucose esters and which, after hydrolytic release, sponta-
neously rearranged to the dilactone, ellagic acid (6). It was
discussed very early on that the biogenesis of such ellagitannins
14
produced [U- C]pentagalloylglucose by photoassimilation of
1
4
2
CO in leaves of the gallotannin synthesizing plant Rhus
10
typhina (staghorn sumac) in > 99% purity. This compound
was used as standard substrate in an extended screening
program for enzymes that formed reaction products liberating
1
4
[ C]ellagic acid (6) upon hydrolysis, thus providing a general
probe for the in vitro synthesis of ellagitannins of widely
differing structures.
in plants should originate from 1,2,3,4,6-penta-O-galloyl-b- -
D
glucose (1) as the principal precursor,1 a view that was
corroborated and refined later by Haslam and coworkers. It was
By this strategy we were able to discover a novel soluble
enzyme in leaves of Tellima grandiflora (Pursh) Lindley (fringe
cups, Saxifragaceae), a weed that is known as a rich source of
4
proposed by these authors that the energetically preferred C
1
conformer of 1 was sequentially oxidized to tellimagrandin II
(
2) and casuarictin (3).2 The recently recognized role of
,3
ellagitannins. The partially purified protein was found to
11
4,5
14
ellagitannins as promising chemotherapeutic agents
has
catalyze the conversion of [U- C]pentagalloylglucose to
stimulated remarkable success in the challenge of chemically
synthesizing such compounds.6 The biosynthesis of ellagi-
tannins, in contrast, still remained completely obscure. The
present situation was illustrated in a recent review article with
the statement that ‘the in vitro biaryl coupling of 1 has yet to be
achieved by an isolated enzyme.’ In several attempts to
several radioactively labeled products, while no reaction
occurred in the presence of denatured enzyme (Fig. 1). The most
prominent peak among these compounds coincided with
authentic tellimagrandin II (2) in two different HPLC systems
with acetonitrile and MeOH gradients, respectively. This
fraction was isolated and hydrolyzed (4 M HCl, 100 °C, 4 h) to
afford glucose, gallic acid and ellagic acid (6) as sole C-
labeled degradation products as determined by HPLC and
liquid-scintillation counting of the eluates, thus indicating the
in vitro synthesis of a true ellagitannin.
,7
8
1
4
elucidate the mechanism of such a transition with the enzyme
9
systems laccase/O
2
or peroxidase/H
2
O
2
only free ellagic acid
(
6) had been detected, while the formation of true ellagitannins,
characterized by glucose-bound HHDP residues, has never been
achieved.
Under the conditions given in Fig. 1, the enzyme reaction
proceeded linearly for 30 min and had pH and temperature
optima at pH 5 and 45 °C, respectively. Normal Michaelis–
We concluded that inadequate analytical techniques repre-
sented the decisive obstacle in such investigations, which
DOI: 10.1039/b006270g
Chem. Commun., 2001, 35–36
This journal is © The Royal Society of Chemistry 2001
35

siderations on the eventual cooperation of product-specificity
guiding enzymes, analogous to the recently reported ‘dirigent’
protein involved in lignan biosynthesis.18
In summary, first evidence has been provided by our results
that the long sought clue to unravel the old enigma of
ellagitannin biosynthesis is now available.
We thank Angelika Müller (Ulm) for excellent technical
assistence, Dr G. Schmidtberg (Ulm) for MS analyses, and Dr
A. Scalbert (Clermont-Ferrand, France) and Professor T.
Yoshida (Okayama, Japan) for reference samples of ellagi-
tannins. Financial support from the Deutsche Forschungsge-
meinschaft (Bonn), the Fonds der Chemischen Industrie
(
Frankfurt/M) and from research grants of the University of
Ulm is gratefully acknowledged.
Fig. 1 RP-18 HPLC analysis of in vitro formed oxidation products of
14
[
C]pentagalloylglucose with an enzyme from Tellima grandiflora leaves.
Notes and references
Assay mixtures, containing 12.5 mg (2,500 dpm) 1 and enzyme (4 pkat) in
50 ml HEPES buffer (50 mM, pH 5.0), were incubated at 30 °C for 60 min,
1
2
O. Th. Schmidt and W. Mayer, Angew. Chem., 1956, 68, 103.
R. K. Gupta, S. M. K. Al-Shafi, K. Layden and E. Haslam, J. Chem.
Soc., Perkin Trans. 1, 1982, 2525.
stopped by heat-denaturing of enzyme, and analyzed by RP-18 HPLC. (—),
Enzyme assay; (…), blank with acid-denatured enzyme. (1), Pentagalloyl-
glucose; (2) tellimagrandin II. HPLC conditions: Reprosil NE, 5 mm, 250 3
3
4
E. A. Haddock, R. K. Gupta, S. M. K Al-Shafi, K. Layden, E. Haslam
and D. Magnolato, Phytochemistry, 1982, 21, 1049.
K. S. Feldman, K. Sahasrabudhe, R. S. Smith and W. J. Scheuchen-
zuber, Bioorg. Med. Chem. Lett., 1999, 9, 985.
4
3 3 4
mm i.d.; solvent A = 0.05% aq. H PO4, B = 0.05% H PO , in MeOH;
gradient 0–1 min 10% B, 1–3 min 10–30% B, 3–20 min 30–40% B, 20–40
min 40% B; flow rate 0.7 ml min2 . Radioactivity was determined by
fractionation of eluates and subsequent liquid-scintillation counting.
1
5
6
M. N. Clifford and A. Scalbert, J. Sci. Food Agric., 2000, 80, 1118.
K. S. Feldman, K. Sahasrabudhe, S. Quideau, K. L. Hunter and M. D.
Lawlor, in Plant Polyphenols 2. Chemistry, Biology, Pharmacology,
Ecology, G. G. Gross, R. W. Hemingway and T. Higuchi, eds., Kluwer
Academic/Plenum Publishers, New York, 1999, p. 101.
Menten kinetics were observed for the substrate, pentagalloyl-
glucose (1), up to a maximal concentration of 320 mM, while
increasing substrate inhibition occurred above this value.
Replots of this data according to Lineweaver-Burk revealed a
7
8
K. S. Feldman and K. Sahasrabudhe, J. Org. Chem., 1999, 64, 209.
R. F. Helm, L. Zhentian, T. Ranatunga, J. Jervis and T. Elder, in Plant
Polyphenols 2. Chemistry, Biology, Pharmacology, Ecology, G. G.
Gross, R. W. Hemingway and T. Higuchi, eds., Kluwer Academic/
Plenum Publishers, New York, 1999, p. 83.
2
1
K
m
value of 110 mM (Vmax = 33 pkat [0.12 mmol h ]).
For the unequivocal determination of the structure of the
reaction product, 100 mg of unlabeled pentagalloylglucose (1),
9
G. G. Gross, in Comprehensive Natural Products Chemistry. Vol. 3.
Carbohydrates and Their Derivatives Including Tannins, Cellulose, and
Related Lignins, B. M. Pinto, ed., Elsevier, Amsterdam, 1999, p. 799.
chemically synthesized from triacetylgalloylchloride and b- -
D
12
glucose ) was incubated in a scaled-up enzyme assay,
affording 1.7 mg pure reaction product of > 93% purity after
semi-preparative HPLC.13 Negative FAB-MS of this substance
1
0 H. Rausch and G. G. Gross, Z. Naturforsch. C: Biosci., 1996, 51,
73.
1 Fresh leaves (80 g) of Tellima grandiflora were homogenized in liquid
, extracted with 250 ml Tris-HCl (1.5 M, pH 8.0)–Na borate (0.2 M,
4
revealed prominent peaks for the deprotonated molecular ion
1
2
[
(
M 2 H] at m/z 937 (tellimagrandin II 2, M
r
938) and m/z 635
N
2
trigalloylglucose, M
r
636).14 Proton NMR spectroscopy (500
pH 7.5) (1+1, by vol.) and centrifuged (30000 3 g, 30 min). The
supernatant was depleted of phenolics by stirring with Amberlite XAD
(20 min), filtered and fractionated with (NH ) SO . The 30–80% pellet
4 2 4
was resuspended in HEPES buffer (50 mM, pH 6.0), desalted,
concentrated by ultrafiltration and chromatographed on a Sephacryl S-
MHz) displayed signals that corresponded to those of an
authentic sample of tellimagrandin II (2). In particular, three
characteristic singlets at d ppm (TMS) 6.91 (2H), 6.94 (2H) and
7
4
.05 (2H) were detected in d -MeOH that corresponded to the
3
00 (Pharmacia Biotech) column (40 3 2.4 cm i.d.) in HEPES
aryl-2,6 hydrogens at C-1, C-2 and C-3 of 2, respectively, while
the singlets at d 6.47 (1H) and 6.60 (1H) were due to the 2,2A-
hydrogens of diphenic acid (5) bound at C-4,6. Also the
chemical shifts for the glucose moiety were in full agreement
with those of authentic 2.
buffer.
1
1
2 G. G. Gross, Z. Naturforsch. C: Biosci., 1983, 38, 519
3 The reaction mixture (400 ml vol., pH 5, containing 100 mg 1 and 850
mg protein) was incubated for 60 min at 30 °C. After stopping the
reaction by heat-denaturing the enzyme, the mixture was extracted with
EtOAc. The contents of the dried organic phase were subjected to semi-
preparative RP-18 HPLC on Kromasil (5m, 250 3 20 mm i.d.; gradient:
The reaction product displayed, however, strong additional
singlets at 6.61 (1H) and 6.63 (1H) ppm that apparently were
due to a different compound with a C-2,3 linked HHDP unit, as
3 4
solvent A = 0.05% aq. H PO , B = acetonitrile; 0–1 min 5% B, 1–2
21
1
min 5–18% B, then isocratic at 18% B; flow rate 22 ml min ). Relevant
fractions were immediately neutralized, depleted of organic solvent in
vacuo, extracted with EtOAc and rechromatographed twice by RP-18
HPLC on Reprosil NE (5 mm, 250 3 8 mm i.d.; solvents and gradient as
in Fig. 1; flow rate 2.4 ml min ). The purified product was neutralized,
depleted of MeOH and applied to a column of Sephadex LH-20 (60 3
12 mm i.d., in water). After rinsing with water to remove H PO , the
product was eluted with MeOH and lyophylized, affording 1.7 mg
material of > 93% purity as determined by analytical HPLC under the
conditions given in Fig. 1.
concluded by comparison with H NMR data from authentic
samples of 2,3-O-hexahydroxydiphenoylglucose, casuarictin
(
3) and pedunculagin (structure as 3, but with a free anomeric
OH-group at C-1). Evidently, the enzyme preparation had
catalyzed the simultaneous synthesis of both tellimagrandin II
21
(
2) and isomeric 1,4,6-tri-O-galloyl-2,3-O-hexahydroxydiphe-
3
4
noyl-b-
D
-glucose (4) that had not been separated by RP-HPLC.
This latter, unusual ellagitannin is not known as a natural
product; it has been recently obtained, however, by total
synthesis in two enantiomerically pure forms, mahtabin A and
1
4 R. Isobe, T. Tanaka, G. Nonaka and I. Nishioka, Chem. Pharm. Bull.
(Tokyo), 1989, 37, 1748.
pterocarinin C, characterized by 2,3-R- and S-HHDP residues,
15 K. Khanbabaee and K. Lötzerich, Liebigs Ann., 1997, 1571.
16 K. Khanbabaee and K. Lötzerich, J. Org. Chem., 1998, 73, 8723.
17 G. Nonaka, M. Ishimatsu, M. Ageta and I. Nishioka, Chem. Pharm.
Bull. (Tokyo),1989, 37, 50.
respectively.1
5,16
(An earlier report proposing this structure for
a compound named cercidinin A from Cercidiphyllum jap-
onicum17 has been questioned by these authors.) Detection of
such a compound raises questions about the specificity of the
enzyme(s) catalyzing such transformations that prompt con-
18 L. B. Davin, H. B. Wang, A. L. Crowell, D. L. Bedgar, D. M. Martin,
S. Sarkanen and N. G. Lewis, Science, 1997, 275, 362.
36
Chem. Commun., 2001, 35–36