J Biol Chem, Vol. 274, Issue 51, 36312-36320, December 17, 1999

Biosynthesis of a Neo-epi-verrucosane Diterpene in the Liverwort Fossombronia alaskana
A RETROBIOSYNTHETIC NMR STUDY^*

Wolfgang Eisenreich^§, Christoph Rieder, Carola Grammes^¶, Gerhard Heßler, Klaus-Peter Adam^¶, Hans Becker^¶, Duilio Arigoni, and Adelbert Bacher

From the  Institut für Organische Chemie und Biochemie, Technische Universität München, D-85747 Garching, Germany, ^¶ Pharmakognosie und Analytische Phytochemie der Universität des Saarlandes, D-66041 Saarbrücken, Germany, and  Laboratorium für Organische Chemie, Eidgenössische Technische Hochschule Zürich, CH-8092 Zürich, Switzerland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The biosynthesis of the diterpene 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane in the arctic liverwort Fossombronia alaskana was studied by incorporation experiments using [1-¹³C]- and [U-¹³C₆]glucose as precursors. The ¹³C-labeling patterns of acetyl-CoA, pyruvate, and phosphoenolpyruvate in intermediary metabolism were reconstructed from the ¹³C NMR data of biosynthetic amino acids (leucine, alanine, phenylalanine) and were used to predict hypothetical labeling patterns for isopentenyl pyrophosphate formed via the mevalonate pathway and the deoxyxylulose pathway. The labeling patterns observed for the neoverrucosane diterpene were consistent with the intermediate formation of geranyllinaloyl pyrophosphate assembled from dimethylallyl pyrophosphate and three molecules of isopentenyl pyrophosphate generated predominantly or entirely via 1-deoxyxylulose 5-phosphate. The experimental data can be integrated into a detailed biosynthetic scheme involving a 1,5-hydride shift. The postulated involvement of the 1,5-hydride shift was confirmed by an incorporation experiment with [6,6-²H₂]glucose.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Verrucosanes are a family of tetracyclic diterpenes initially observed in the liverworts Mylia anomala (1) and Mylia verrucosa (2-6). More recently, verrucosane type terpenes have also been found in other liverworts (7-14), in marine sponges (15-17) and in the eubacterium Chloroflexus aurantiacus (18). The structures of 5-hydroxy-13-epi-neoverrucosane from the liverwort Plagiochila stephensoniana (9) and of 5-oxo-13-epi-neoverrucosane from Fossombronia alaskana (7) have been determined by x-ray structure analysis. The major diterpene from axenic cultures of F. alaskana was found to be 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1, Fig. 1) (7). Hypothetical biosynthetic schemes for verrucosane-type diterpenes have been proposed without experimental substantiation (8, 11).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structures of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1), 2-hydroxyverrucosane (2).

In the eubacterium C. aurantiacus, the biosynthesis of 2-hydroxyverrucosane (2) has been analyzed by in vivo incorporation of ¹³C-labeled acetate samples (19). On the basis of the observed ¹³C-labeling patterns, the isoprene dissection of the diterpene has been deduced, and a mechanism yielding the tetracyclic ring system from geranyllinaloyl pyrophosphate (3, Fig. 2) has been proposed. Briefly, the solvolysis of the pyrophosphate moiety from compound 3 triggers a reaction sequence via the monocyclic intermediate 4 involving a Wagner-Meerwein rearrangement (from bicyclic ionic intermediate 5 to intermediate 6), a 1,5-hydride shift (from tricyclic ionic intermediate 7 to intermediate 8), and a cyclopropyl carbinyl to cyclopropyl carbinyl rearrangement (from intermediate 9 to intermediate 10, Fig. 2). The terpenoid building blocks, isopentenyl pyrophosphate (IPP,¹ 13, Fig. 3) and dimethylallyl pyrophosphate (DMAPP, 14), from which compound 3 had been assembled were derived from acetate (11) via mevalonate (12).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Mechanism for the formation of 2-hydroxyverrucosane (2) in C. aurantiacus as proposed by Rieder et al. (19). Carbon atoms contributed by individual C5 monomers are boxed in compound 2.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Diversion of carbon atoms from acetyl-CoA (11), pyruvate (15), and glyceraldehyde 3-phosphate (16) to the isoprenoid monomers IPP (13) and DMAPP (14) by the mevalonate pathway and the deoxyxylulose pathway. The deoxyxylulose pathway involves a rearrangement that interrupts the contiguity of the carbon atoms derived from triose phosphate. TPP, thiamin pyrophosphate.

For several decades the mevalonate pathway had been assumed to be the universal source for the building blocks of all terpenes (for review, see Ref. 20). The existence of a second pathway was established relatively recently on the basis of independent studies by Rohmer, Arigoni, Sahm, and their co-workers (for review see Refs. 21 and 22). Specifically, it was shown that the nonmevalonate pathway involved a transketolase type condensation of glyceraldehyde 3-phosphate (16) with "activated acetaldehyde" obtained by decarboxylation of pyruvate (15) (Fig. 3). The first committed intermediate of the novel pathway was shown to be 1-deoxy-D-xylulose or its 5-phosphate (17) (23-25). An enzyme catalyzing the formation of 1-deoxy-D-xylulose 5-phosphate from glyceraldehyde 3-phosphate and pyruvate has been found recently in Escherichia coli (26, 27) and Mentha piperita (28). A NADPH-dependent reductoisomerase catalyzing the formation of 2-C-methylerythritol 4-phosphate (18) from 1-deoxyxylulose 5-phosphate (17) has been isolated from E. coli (29). The reactions leading from the tetrol phosphate (18) to IPP (13) and DMAPP (14) are still unknown.

Whereas vertebrates and archaebacteria appear to use exclusively the mevalonate pathway for the biosynthesis of isoprenoids, different eubacteria have been found to use either the mevalonate or deoxyxylulose pathway (19, 30, 31). Several higher plants were found to use predominantly the mevalonate pathway for the biosynthesis of steroids and the deoxyxylulose pathway for the biosynthesis of monoterpenoids, carotenoids, phytol and other diterpenes (for review, see Ref. 21). The liverworts Conocephalum conicum and Ricciocarpos natans have been shown to synthesize the monoterpenes borneol and bornyl acetate as well as phytol via the deoxyxylulose pathway, whereas the sesquiterpenes cubebanol and ricciocarpin A, as well as stigmasterol, were derived via the mevalonate pathway (32).

The mevalonate pathway appears to be operative in the cytoplasmic compartment of plant cells, whereas the deoxyxylulose pathway seems to be limited to the plastid compartment. Segregation of the two pathways, however, is not necessarily complete, and exchange of metabolites between the two pools has been observed in plants and plant cell cultures (24, 25, 33-35). In retrospect, it is clear that this cross-talk was responsible for the wide belief in a universal mevalonate origin for all terpenoids. Due to partial metabolite exchange between the two compartments, at least a fraction of the mevalonate-derived building blocks was incorporated, if only in strongly reduced yields, into those terpenoids that are now known to originate predominantly from the deoxyxylulose pathway.

This study was initiated to elucidate the biosynthesis of the diterpene 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1, Fig. 1) in the arctic liverwort F. alaskana.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- [U-¹³C₆]Glucose and [1-¹³C]glucose were purchased from Omicron (South Bend, IN). [6,6-²H₂]Glucose was purchased from Promochem (Wesel, Germany).

Organism-- F. alaskana STEERE & INOUE was kindly provided by Professor Basile, New York Botanical Garden.

Culture Conditions-- Plants were cultivated in 200-ml Erlenmeyer flasks under aseptic conditions on Gamborg B5 medium (36) supplemented with 0.9% agar and 1% sucrose. They were kept under continuous light at 20 °C.

Isotope Incorporation Studies-- Before experiments with isotope-labeled glucose, plants were cultivated for at least one culture period on the basic medium with substitution of sucrose by glucose. The cultures were harvested after 12 weeks.

Isolation of 8-Acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1)-- The plant material was air-dried and pulverized (25 g, dry weight). Percolation with diethyl ether afforded a crude extract that was fractionated by gradient vacuum liquid chromatography on silica gel 60 (3 × 8 cm, 15 µm, 100% hexane to 100% ethyl acetate). The fractions were checked by thin layer chromatography on silica gel using hexane/ethyl acetate (4:1; v/v) as mobile phase. Compound 1 (R_f, 0.15) was eluted at 55% ethyl acetate. Fractions containing compound 1 were subjected to gradient vacuum liquid chromatography on Lichroprep Diol (2 × 7 cm, 40-63 µm, 100% hexane to 60% ethyl acetate). Compound 1 was eluted with 35% ethyl acetate. Typically, 0.6 mg of compound 1 was isolated/g of dry plant material.

Isolation of Amino Acids-- The hydrolysis of biomass and the isolation of amino acids has been described earlier (37, 38).

NMR Spectroscopy-- Parameters for one- and two-dimensional NMR experiments and quantitative analysis of ¹³C enrichment as well as ¹³C¹³C coupling have been described earlier (19). Simulation of NMR signals was performed using the NMRSIM software package (Bruker, Germany).

Molecular Modeling-- Molecular modeling studies were performed using the program package DISCOVER from MSI (Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NMR Signal Assignments and Conformational Study-- To interpret biosynthetic ¹³C- and ²H-labeling data, it was mandatory to assign unequivocally all ¹H and ¹³C NMR signals of the target molecule. ¹H and ¹³C NMR chemical shifts of compound 1 in CDCl₃ have been reported earlier (7). A more detailed NMR analysis was performed using HMQC, HMBC, and NOE spectroscopy experiments (Table I). Additional information was obtained by INADEQUATE spectroscopy of a biosynthetic sample obtained from a feeding experiment with [U-¹³C₆]glucose. Interproton distances were estimated from NOE build-up rates (Table II). The assignment of diastereotopic protons was based on the calculated interproton distances in conjunction with molecular modeling studies yielding the conformation shown in Fig. 4. The assignment of the diastereotopic methyl groups 16 (Re) and 17 (Si) was also confirmed from the vicinal long range carbon carbon coupling between C-12 and C-16 of 2.4 Hz, indicating a dihedral angle of approximately 60° between these centers (39-41).

View this table: [in this window] [in a new window]   Table I 1H NMR and 13C NMR assignments of 8-acetoxy-13-hydroxy-5-oxo-epi-neoverrucosane (1) in CDCl3 HMBC, heteronuclear multiple bond correlation; DQF-COSY, double quantum-filtered COSY.

View this table: [in this window] [in a new window]   Table II Interproton distances (in Å), calculated from NOE initial rates and from energy minimization by molecular dynamic methods of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Conformation of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane. This conformation was gleaned from the analysis of NOE effect data and molecular dynamics calculations.

Incorporation Experiments-- F. alaskana was grown for 12 weeks on a mineral medium solidified with agar supplemented with isotope-labeled precursors (see below). Starting from approximately 2 g of F. alaskana cells (fresh weight), approximately 20 g of cells (dry weight) were harvested after the incorporation period of 12 weeks. Compound 1 was extracted from the cell mass and was purified as described under "Experimental Procedures." Hydrolysis of biomass afforded amino acids that were isolated chromatographically. ¹³C abundance and ¹³C¹³C couplings of the diterpene and amino acids were determined by ¹H and ¹³C NMR spectroscopy as described earlier (19).

In the feeding experiment with [U-¹³C₆]glucose, the labeled precursor was proffered together with a 10-fold excess of unlabeled glucose (culture volume, 2 liters; unlabeled glucose, 18 g; [U-¹³C₆]glucose, 2 g). This feeding strategy leads to the formation of downstream metabolites by the random combination of metabolic intermediates derived from multiple ¹³C-labeled or unlabeled glucose molecules. The resulting diterpene was therefore a complex mixture of various multiple ¹³C-labeled isotopomers.

The average ¹³C abundance of the diterpene in the experiment with [U-¹³C₆]glucose was 4.5%. The ratio of ¹³C-labeled and unlabeled glucose in the culture medium would have predicted an averaged enrichment of about 11%. Since the cell mass of the liverwort increased by a factor larger than 10 during the incorporation experiment, it can be excluded that the diterpene was significantly diluted by unlabeled material that was already present at the beginning of the feeding experiment. Apparently, the ¹³C label has been diluted by photosynthetic CO₂ fixation. Analysis of ¹³C¹³C couplings in the one-dimensional ¹³C NMR spectrum and in two-dimensional ¹³C INADEQUATE experiments (Table I) indicated that seven pairs of carbon atoms were incorporated jointly from the glucose precursor into the ring system of the diterpene (Fig. 5A). Moreover, the acetyl moiety at carbon atom eight showed the presence of a double-labeled acetate isotopomer.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   13C-Labeling patterns of 8-acetoxy-13-hydroxy-5- oxo-13-epi-neoverrucosane (1). A, from the experiment with [U-13C6]glucose; pairs of adjacent 13C atoms are shown by bold lines, and multiple 13C couplings are shown by arrows; B, from the experiment with [1-13C]glucose; the numbers indicate 13C abundance in %; C, dissection of isoprenoid monomers; carbon atoms contributed by individual C5 monomers are boxed.

In addition to one-bond ¹³C¹³C couplings, several ¹³C¹³C couplings via two or three bonds (indicated by arrows in Fig. 5A) were observed in the ¹³C NMR spectrum and in a two-dimensional INADEQUATE experiment optimized for magnetization transfer via small coupling constants.

In the experiment with [1-¹³C]glucose (culture volume, 1 liter; unlabeled glucose, 5 g; [1-¹³C]glucose, 5 g), nine ¹³C signals of compound 1 (carbon atoms 2, 6, 9, 12, 17, 18, 19, 20, and 22) displayed increased intensities (Fig. 5B). By comparison with the spectra of the unlabeled diterpene, an average ¹³C content of 9.3 ± 0.2% was determined for these centers. A ¹³C content of 4.0 ± 0.3% was found for the other carbon atoms of the diterpene, reflecting significant reshuffling of [1-¹³C]glucose during the growth period of 12 weeks.

The C₅ building blocks contributed by the isoprenoid precursors IPP and DMAPP were assigned on the basis of the observed labeling patterns (Fig. 5C). It is immediately obvious that one C₅ moiety (comprising C-9, C-10, C-11, C-14, and C-20 of the diterpene) must have undergone reshuffling of the original isoprenoid precursor via at least one rearrangement reaction. On the other hand, three isoprenoid units have retained the connectivity present in the precursor.

The arrangement of the C₅ building blocks in the neoverrucosane system (Fig. 5C) was similar to that in 2-hydroxyverrucosane (Fig. 2) formed by the eubacterium C. aurantiacus and a mechanism for the formation of the neoverrucosane skeleton from geranyllinaloyl pyrophosphate can be adapted from the one proposed earlier for the 2-hydroxyverrucosane cyclase (19) (see "Discussion").

Biogenetic Origin of the C₅ Building Blocks-- The formation of isoprenoid C₅ units, IPP and DMAPP, starts from acetyl-CoA in case of the mevalonate pathway and from pyruvate and glyceraldehyde 3-phosphate in case of the deoxyxylulose pathway (Fig. 3). The labeling patterns of acetyl CoA, pyruvate, and glyceraldehyde 3-phosphate are difficult to determine directly because these central intermediates represent only trace amounts of total biomass. However, their labeling patterns can be reconstructed tentatively by retrobiosynthetic analysis from the labeling patterns of amino acids (42). For this purpose, we hydrolyzed the residual cell mass after verrucosane isolation and isolated amino acids from the hydrolysate by ion exchange chromatography and high performance liquid chromatography. Specifically, the carboxylic atom and the  carbon atom of leucine are derived from acetyl CoA via condensation with -ketoisovalerate. The labeling pattern of C-1 and C-2 of leucine, therefore, can be taken as a reference for the labeling pattern of acetyl-CoA. Indeed, the validity of this method is vindicated by comparison with the observed labeling pattern of the acetyl moiety of the diterpene. The labeling pattern of acetyl CoA (11, as reconstructed from leucine, Figs. 6C, 7C) and the labeling pattern of the acetyl moiety of the verrucosane diterpene (Figs. 6A, 7A) were in close correspondence. Alanine is biosynthetically obtained by reductive amination of pyruvate. Hence, the labeling pattern of pyruvate can be reconstructed from that of alanine. The labeling pattern of glyceraldehyde 3-phosphate cannot be easily determined directly; however, it can be expected to be similar to that of phosphoenolpyruvate, which is obtained from labeling patterns of the side chains of phenylalanine and tyrosine. The reconstructed labeling patterns of acetyl CoA (11), pyruvate (15) and glyceraldehyde 3-phosphate/phosphoenolpyruvate (16) from the incorporation experiments with [U-¹³C₆]glucose and [1-¹³C]glucose are summarized in Figs. 6 and 7, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Observed and predicted labeling patterns of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1) and 13C NMR signals of C-12 (indicated by an arrow) after feeding of [U-13C6]glucose diluted with unlabeled glucose (1:9; w/w) to a culture of F. alaskana. The arabic numbers indicate 13C abundance of individual carbon atoms; the bold lines indicate isotopomers with adjacent 13C atoms; the arrows indicate isotopomers involving long range 13C13C couplings; the italic numbers indicate fractions of 13C13C-coupled satellite signals in the global 13C NMR signal intensities. A, observed isotopomer pattern in the diterpene; the isotopomer pattern of IPP (13) was reconstructed from the isoprene dissection of the diterpene (see Fig. 5C). B, prediction via the deoxyxylulose pathway on the basis of the labeling patterns of pyruvate (15) and glyceraldehyde 3-phosphate (16) reconstructed from the observed labeling patterns of alanine and tyrosine, respectively; C, prediction via the mevalonate pathway on the basis of the labeling pattern of acetyl CoA (11) reconstructed from the observed labeling pattern of leucine.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Observed and predicted labeling patterns of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane after feeding of [1-13C]glucose. The numbers indicate 13C abundance in %. The dotted atoms indicate high 13C enrichment from [1-13C]glucose; for details see also legend to Fig. 6. A, observed; the isotopomer pattern of IPP (13) was reconstructed from the observed labeling pattern in the diterpene. B, prediction via the deoxyxylulose pathway; C, prediction via the mevalonate pathway.

As in previous studies (19, 42), hypothetical labeling patterns for IPP (13) were predicted on the basis of reconstructed labeling patterns of the building blocks, acetyl-CoA in the mevalonate route (Figs. 6C and 7C) and pyruvate/glyceraldehyde 3-phosphate in the deoxyxylulose route (Figs. 6B and 7B), respectively. Figs. 6A and 7A show the observed labeling pattern of the diterpene and the reconstruction of the IPP precursor based on the isoprenoid dissection (see Fig. 5C) by averaging of the enrichment data over the three isoprenoid units, which have conserved their connectivities in the cyclization process. The comparison of the experimentally determined labeling pattern with the hypothetical predictions shows unequivocally that the DMAPP and the three IPP moieties used as building blocks of the diterpene were generated predominantly, if not entirely, via the deoxyxylulose pathway. This conclusion was confirmed by simulation of ¹³C NMR signals based on the predicted isotopomer compositions in the diterpene. As shown for the ¹³C NMR signal of C-12, the signal simulated from the deoxyxylulose prediction was in close correspondence to the observed ¹³C NMR signal, whereas the predicted signal from the mevalonate hypothesis was clearly at odds (Fig. 6).

Evidence for a 1,5-Hydride Shift in Verrucosane Formation-- The mechanism proposed for the formation of the verrucosane system in C. aurantiacus (19) postulates a 1,5-hydride shift from position 2 to position 15 of intermediate 7 (Fig. 2). To check the validity of this hypothesis in the biosynthesis of compound 1, we decided to use a strategy leading to in vivo deuteration of C-2 in intermediate 7a (Fig. 8). According to the isoprene dissection shown in Fig. 5C and the 1-deoxyxylulose 5-phosphate origin of the C₅ building blocks, C-2 in the ionic intermediate, 7a is derived from C-1 of IPP and ultimately from C-3 of glyceraldehyde 3-phosphate. A feeding experiment was performed with [6,6-²H₂]glucose (culture volume, 1 liter; [6,6-²H₂]glucose, 10 g), which is expected to afford [3,3-²H₂] glyceraldehyde 3-phosphate next to [3,3-²H₂]pyruvate by glycolysis (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   2H isotopomer composition of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1) after feeding of [6,6-2H2]glucose. Carbon atoms contributed by individual C5 monomers are boxed (see also Fig. 5C). The numbers in italics indicate 2H enrichments in % calculated from - and -shifted satellite signals in the 13C NMR spectrum. The 2H distribution in the precursors were adjusted to accommodate the observed labeling pattern in compound 1.

The ²H NMR spectrum of the resulting compound 1 was complex (Fig. 9B), and the deuterium substitution pattern in compound 1 was therefore determined in the ¹H,²H-decoupled ¹³C spectrum analyzing satellite lines caused by ²H isotope shifts. As an example, the ¹³C signatures of carbon atoms 16 and 17 are shown in Fig. 10B. C-16 and C-17 displayed satellite signals characterized by up-field shifts of 106 and 111 ppb, respectively. Both satellite lines could be attributed unequivocally to compound 1 via a two-dimensional ²H-decoupled ¹³C¹H-COSY experiment (Fig. 10A) showing that the unshifted ¹³C signals as well as the up-field-shifted satellites display correlations to the same ¹H signals of the diterpene. As expected, the cross-peaks of the up-field ¹³C satellites showed a slight up-field shift of the protons, sensing the presence of an adjacent ²H atom. The size of the up-field shift in the ¹³C NMR signals for C-16 and C-17 was typical for a ²H isotope effect via two bonds, thus indicating that the adjacent methine group 15 carried a deuterium atom. The complete breakdown of ²H distribution in compound 1 obtained from the detailed quantitative analysis of  and  shifts in the ¹³C NMR spectrum (Table III) is summarized in Fig. 8. In addition, the data confirm the presence of deuterium at positions originating from C-1 of IPP (21.3 ± 2.3% ²H) and from C-5 of IPP (5.3 ± 1.8% ²H). The low ²H enrichment values in the methyl groups derived from C-5 of IPP are explained by deuterium loss via keto-enol tautomerization of pyruvate. A similar ²H distribution pattern has been observed earlier in deoxyxylulose-derived IPP in an incorporation experiment with Ginkgo biloba using [6,6-²H₂]glucose as a precursor (24).

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Parts of 1H and 2H NMR spectra of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1) after feeding of [6,6-2H2]glucose. A, simulated 2H NMR spectrum on the basis of the 2H isotopomer composition deduced from the 13C(1H,2H) NMR experiment; B, observed 2H NMR spectrum; C, observed 1H NMR spectrum.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   NMR signals of 8-acetoxy-13-hydroxy-5-oxo-epi-neoverrucosane after feeding of [6,6-2H2]glucose. A, signals from a two-dimensional 13C1H-COSY experiment; B, 1H,2H-decoupled 13C NMR signals.

View this table: [in this window] [in a new window]   Table III 2H isotope effects on 13C NMR frequencies in 8-acetoxy-13-hydroxy-5-oxo-epi-neoverrucosane (1) after feeding with [6,6-2H2]glucose The position of deuteration in compound 1 conducive to a shifted satellite of the respective index 13C NMR signal is indicated in parenthesis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

On the basis of incorporation studies using radioactively labeled mevalonate or acetate and despite low incorporation yields, the biosynthesis of numerous plant terpenoids has been taken to rest entirely on the operation of the mevalonate pathway. Even in those cases where the expected location of the expected label was subsequently verified by painstaking degradation procedures, it is nowadays clear that the observed results do not constitute proof for the exclusive operation of the expected pathway. More recent studies have shown that metabolite exchange between mevalonate- and deoxyxylulose-derived terpenoid building blocks (IPP or others) is occurring in plants.

Thus, in seedlings of G. biloba, deoxyxylulose-derived precursors were found to contribute 0.5% of steroids supposed to be biosynthesized via the mevalonate pathway in the cytosolic compartment of the cell (24). Involvement of both IPP biosynthetic pathways has also been reported in cell cultures of Catharanthus roseus (33) and in Matricaria recutitas (34), as well as for the formation of terpenoids in some Actinomycetes (43, 44).

Recently, a variety of plant terpenoids have been reported to be formed via the deoxyxylulose route on the basis of incorporation experiments with 1-deoxy-D-xylulose (33, 35, 45-48). Clearly, these experiments must be interpreted with the same qualifications as the earlier experiments with mevalonate. Unless the incorporation rates of 1-deoxy-D-xylulose are very high, it remains open whether the compound is diverted to a specific terpenoid via the major route or via cross-talk of this major route with the other pathway. This question can be solved only by consideration of the quantitative aspects of metabolite flux within the organism under investigation. Flux parameters can be investigated by quantitative NMR analysis using general ¹³C-labeled precursors (glucose, acetate) in conjunction with the retrobiosynthesis concept (for review, see Ref. 42). The comparative analysis of isotopomer patterns in terpenoids and amino acids after incorporation of various ¹³C-labeled glucoses was used in this study to analyze the origin of a neoverrucosane-type diterpene in the arctic liverwort, F. alaskana.

The data show conclusively that the C₅ building blocks IPP and DMAPP of the neoverrucosane are biosynthesized predominantly (>95%) via the deoxyxylulose pathway. In contrast, earlier studies with the eubacterium C. aurantiacus have shown that the isoprenoid building blocks of the structurally related 2-hydroxyverrucosane are derived from the mevalonate pathway (19).

In addition, the labeling patterns observed in compound 1 cast light on the mechanisms responsible for the formation of the 13-epi-neoverrucosane ring system. More specifically, the labeling data were consistent with the formation of the neoverrucosane system from geranyllinaloyl pyrophosphate (3) via the proposed intermediate 6 of bacterial verrucosane biosynthesis (Fig. 2). With the appropriate conformation of the side chain (see below), attack of the cationic center in intermediate 6 on the isopropylidene double bond leads to the tricyclic ion 7a with "epi-configuration" at carbon atom 13 (Fig. 11). As proposed earlier for the biosynthesis of 2-hydroxyverrucosane, a 1,5-hydride transfer from the C-2 methylene group generates the homoallylic intermediate 8a, which collapses to the cyclopropylcarbinyl ion 9a. Saturation of the positive charge in intermediate 9a by the addition of a hydroxyl group from the solvent then yields 5-hydroxy-13-epi-neoverrucosane (19), which is known as a natural product from cultures of F. alaskana (7). The formation of compound 1 is then completed by hydroxylations of compound 19 at C-13 and C-8, acetylation of the hydroxyl group at position 8, and oxidation of the hydroxyl group at position 5. 

View larger version (15K): [in this window] [in a new window]   Fig. 11.   Late steps of 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1) formation in F. alaskana.

In the case of the verrucosane system, a boat conformation for the chain segment of precursor 6 involved in the formation of the five-membered ring was implied by the trans-arrangement of the hydrogen atoms at C-14 and C-13 and shown to be responsible for the observed stereospecific labeling of the diastereotopic methyl group C-17 in the isopropyl chain of compound 2 from the methyl group of mevalonate (the Re-methyl group of compound 2 corresponding to the Z-methyl group of the aliphatic precursor) (19). In contrast to this result, the experimental data disclose that in the case of compound 1 it is the Si-methyl group (responsible for the signal at 19.92 ppm in the ¹³C NMR spectrum) that maintains its bond-labeling to the adjacent C-15 in the experiment with [U-¹³C₆]glucose and that is specifically enriched from [1-¹³C]glucose and therefore corresponds to the Z-methyl group of the aliphatic precursor. This "inverse" label distribution is compatible only with a prechair conformation of the cyclizing chain segment in intermediate 6a, resulting in the formation of a tetracyclic intermediate 7a with a cis arrangement of the hydrogen atoms of C-14 and C-13 (Fig. 12). It follows that the subsequent hydroxylation at C-13 is indeed occurring as expected with retention of configuration.

View larger version (31K): [in this window] [in a new window]   Fig. 12.   Stereochemical course of the five-membered ring formation for 8-acetoxy-13-hydroxy-5-oxo-13-epi-neoverrucosane (1) in F. alaskana (left) and for 2-hydroxyverrucosane (2) in C. aurantiacus (right). The dotted methyl groups indicate biosynthetic origin from the methyl group of 1-deoxyxylulose (Fossombronia) and the methyl group of mevalonate (Chloroflexus).

Evidence for an unusual 1,5-hydride shift before formation of the cyclopropyl moiety in compound 1 was obtained by an incorporation experiment with [6,6-²H₂]glucose, confirming the earlier proposal for 2-hydroxyverrucosane cyclases (19). The content of ²H at C-15 (12.3%) was perfectly in line with its origin by transfer from a center derived from one of the hydrogen atoms of C-1 of the IPP precursor (10.7 ± 1.2% ²H). To double-check the ²H substitution pattern deduced from the ¹³C spectrum, the ²H NMR spectrum was simulated (Fig. 9A). For that purpose, ²H signals were modeled as Gaussian line shapes with a line width of 5 Hz, and intensities of individual lines were taken from Fig. 8. The simulated spectrum is similar to the experimental ²H NMR spectrum (Fig. 9B). The peak at 2.30 ppm in the experimental ²H spectrum thus confirms the presence of deuterium at C-15. In summary, the data provide firm evidence that a deuteride ion was transferred from C-2 to the positively charged center located at C-15 in intermediate 7a. This experimental finding rules out the validity of a previous proposal (8) in which generation of intermediate 8a from intermediate 7a was suggested to imply four successive 1,2-hydride shifts.

The data presented in this study demonstrate again that the quantitative analysis of ¹³C- and ²H-labeling patterns from universal precursors (i.e. [¹³C]glucose, [²H]glucose) in conjunction with the retrobiosynthesis concept (42) is a powerful tool for metabolite flux analysis. Even in the complex biological system used in this study (i.e. liverwort growing for 3 months on agar supplemented with the isotope-labeled glucoses), the building blocks of a specific biosynthetic pathway are clearly discernable.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 369 and a grant from the Fonds der Chemischen Industrie.

^§ To whom correspondence should be addressed: Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany. Tel.: 49-89-289-13043; Fax: 49-89-289-13363; E-mail: wolfgang.eisenreich@ch.tum.de.

	ABBREVIATIONS

The abbreviations used are: IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; NOE, nuclear Overhauser effect.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES