Ruth
Niemetz
a,
Gerhard
Schilling
b and
Georg G.
Gross
*a
aMolekulare Botanik, Universität Ulm, 89069, Ulm, Germany.. E-mail: ruth.niemetz@biologie.uni-ulm.de;
georg.gross@biologie.uni-ulm.de
bUniversität Heidelberg, Organisch-Chemisches Institut, Im Neuenheimer Feld 270, D-69120, Heidelberg, Germany.. E-mail: gerhard.schilling@urz.uni-heidelberg.de
First published on 11th December 2000
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).
We concluded that inadequate analytical techniques represented the decisive obstacle in such investigations, which suffered from minimal enzyme reaction rates yielding numerous structurally closely related reaction products and unspecific by-products, and particularly from the inherent risk of contamination 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 produced [U-14C]pentagalloylglucose by photoassimilation of 14CO2 in leaves of the gallotannin synthesizing plant Rhus typhina (staghorn sumac) in >99% purity.10 This compound was used as standard substrate in an extended screening program for enzymes that formed reaction products liberating [14C]ellagic acid (6) upon hydrolysis, thus providing a general probe for the in vitro synthesis of ellagitannins of widely differing structures.
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 ellagitannins. The partially purified protein11 was found to catalyze the conversion of [U-14C]pentagalloylglucose 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 14C-labeled degradation products as determined by HPLC and liquid-scintillation counting of the eluates, thus indicating the in vitro synthesis of a true ellagitannin.
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Fig. 1 RP-18 HPLC analysis of in vitro formed oxidation products of [14C]pentagalloylglucose with an enzyme from Tellima grandiflora leaves. Assay mixtures, containing 12.5 μg (2,500 dpm) 1 and enzyme (4 pkat) in 50 μl HEPES buffer (50 mM, pH 5.0), were incubated at 30 °C for 60 min, stopped by heat-denaturing of enzyme, and analyzed by RP-18 HPLC. (—), Enzyme assay; (⋯), blank with acid-denatured enzyme. (1), Pentagalloylglucose; (2) tellimagrandin II. HPLC conditions: Reprosil NE, 5 μm, 250 × 4 mm i.d.; solvent A = 0.05% aq. H3PO4, B =0.05% H3PO4, 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 min−1. Radioactivity was determined by fractionation of eluates and subsequent liquid-scintillation counting. |
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–Menten kinetics were observed for the substrate, pentagalloylglucose (1), up to a maximal concentration of 320 μM, while increasing substrate inhibition occurred above this value. Replots of this data according to Lineweaver-Burk revealed a Km value of 110 μM (Vmax = 33 pkat [0.12 μmol h−1]).
For the unequivocal determination of the structure of the reaction product, 100 mg of unlabeled pentagalloylglucose (1), chemically synthesized from triacetylgalloylchloride and β-D-glucose12) 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 revealed prominent peaks for the deprotonated molecular ion [M − H]− at m/z 937 (tellimagrandin II 2, Mr 938) and m/z 635 (trigalloylglucose, Mr 636).14 Proton NMR spectroscopy (500 MHz) displayed signals that corresponded to those of an authentic sample of tellimagrandin II (2). In particular, three characteristic singlets at δ ppm (TMS) 6.91 (2H), 6.94 (2H) and 7.05 (2H) were detected in d4-MeOH that corresponded to the aryl-2,6 hydrogens at C-1, C-2 and C-3 of 2, respectively, while the singlets at δ 6.47 (1H) and 6.60 (1H) were due to the 2,2′-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.
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 concluded by comparison with 1H 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 (2) and isomeric 1,4,6-tri-O-galloyl-2,3-O-hexahydroxydiphenoyl-β- 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 pterocarinin C, characterized by 2,3-R- and S-HHDP residues, respectively.15,16 (An earlier report proposing this structure for a compound named cercidinin A from Cercidiphyllum japonicum17 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 considerations 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 ellagitannins. Financial support from the Deutsche Forschungsgemeinschaft (Bonn), the Fonds der Chemischen Industrie (Frankfurt/M) and from research grants of the University of Ulm is gratefully acknowledged.
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