Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway

[Paradoxic]

Title: Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway (Summary)
Authors: Lukasz Kutrzeba, Franck E. Dayan, J’Lynn Howell, Ju Feng, José-Luis Giner, and Jordan K. Zjawiony
Journal: Phytochemistry
Date: 26 February 2007

Abstract
Salvinorin A, a neoclerodane diterpenoid, isolated from the Mexican hallucinogenic plant Salvia divinorum, is a potent kappa-opioid receptor agonist. Its biosynthetic route was studied by NMR and HR-ESI-MS analysis of the products of the incorporation of [1-¹³C]-glucose, [Me-¹³C]-methionine, and [1-¹³C; 3,4-²H₂]-1-deoxy-d-xylulose into its structure. While the use of cuttings and direct-stem injection were unsuccessful, incorporation of ¹³C into salvinorin A was achieved using in vitro sterile culture of microshoots. NMR spectroscopic analysis of salvinorin A (2.7 mg) isolated from 200 microshoots grown in the presence of [1-¹³C]-glucose established that this pharmacologically important diterpene is biosynthesized via the 1-deoxy-d-xylulose-5-phosphate pathway, instead of the classic mevalonic acid pathway. This was confirmed further in plants grown in the presence of [1-¹³C; 3,4-²H₂]-1-deoxy-d-xylulose. In addition, analysis of salvinorin A produced by plants grown in the presence of [Me-¹³C]-methionine indicates that methylation of the C-4 carboxyl group is catalyzed by a type III S-adenosyl-l-methionine-dependent O-methyltransferase.

Introduction
Salvinorin A is the first non-nitrogenous, potent and selective kappa-opioid receptor agonist, and therefore is being intensively studied as a lead compound for the treatment of various mental disorders.

Fig. 1. [VIEW] Structures of salvinorin A (1) with atoms numbered according to Ortega et al. (2), salvinorins B (2), F (3), and divinatorins A (4) and B (5) isolated from Salvia divinorum.

Salvinorin A is a diterpenoid. Terpenoids are common secondary metabolites in plants, and all of them are assembled from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) building blocks. It was previously thought that these building blocks all originated from the mevalonic acid (MVA) pathway. However, it was found that some plants use both the MVA and deoxy-d-xylulose (DOXP) pathways.

Studies have shown that salvinorin A is contained in the glandular trichomes located on abaxial side (underside) of the leaves of S. divinorum.

Fig. 2. [VIEW] Simplified biosynthetic scheme showing the predicted incorporation pattern of [1-¹³C]-glucose isotopically labeled IPP in salvinorin A derived from either the MVA route (blue labels) or DOXP pathway (green labels).

Research Questions
Is salvinorin A biosynthesized using the “alternative” DOXP pathway?
What is the source of the methyl ester of salvinorin A?

Methods
General procedures: Deoxy-d-xylulose was synthesized using a method described by Giner, 1998. Metabolites from crude Salvia divinorum plant extracts were purified using a Waters XTerra column connected to an HPLC equipped with a dual wavelength detector. Then metabolites were separated using MeCN:H2O (40:60) isocratic mobile phase and a UV detector then evaluated by LC-MS (Liquid chromatography-mass spectrometer). They were analyzed using a LC-MS with a C8 column. A Bruker Advance was used for NMR (Nuclear magnetic resonance) spectroscopic analyses.

Plant growth: Hofmann & Wasson strain used; purchased from Theatrum Botanicum and grown in a greenhouse under natural light with some fertilizer.

Direct-stem injection: Syringe was used to inject [2-¹³C]-glucose solution into the stem just below the terminal node. New leaves developing above the point of injection were harvested 7 days later and extracted (extraction was analyzed by LC-MS for incorporation of [2-¹³C]-glucose into salvinorin A).

Plant cuttings:  Grown in Hoagland’s medium and then transferred to solutions containing either 1% total glucose (0.5% actual [2-¹³C]-glucose) or 0.1% [1-¹³C]-1-deoxy-d-xylulose. The culture media were filtered through sterile 0.2 mm filters every other day to limit the microbial contamination.

Tissue culture: Stem sections from plant were washed with 7:3 EtOH:Water for 40 sec and a sterile 60 sec wash in water. Then placed in sterile glass culture tubes containing Murashige and Skoog medium supplemented with 2% glucose, Gamborg’s Vitamin solution, 0.2% Phytagel, and 5 μM 6-benzylaminopurine and put into a grow chamber.

Small scale incorporation with [2-¹³C]-glucose: Tissue cultures started as described above, but an additional 1% [2-¹³C]-glucose (2% total glucose concentration) was used. This special [¹³C]-glucose will be incorporated into the plants and is used to label compounds of interest (ex: salvinorin A)

Large scale experiment with [1-¹³C]-glucose: Same procedure as stated above except [1-¹³C]-glucose was used for labeling (instead of [2-¹³C]-glucose) for ¹³C NMR spectroscopic analysis.

[1-¹³C,3,4-²H₂]-1-deoxy-d-xylulose experiment: Labeling with the specific precursor of the DOXP pathway was done using the methods described above but with incorporation of [1-¹³C,3,4-²H₂]-DOX (0.1% of medium)

[Me-¹³C]-methionine experiment: Same procedure use above but with [Me-¹³C]-methionine (0.1% of medium).

Extraction and purification (HPLC) of salvinorin A: Salvinorin A and its derivatives were extracted from tissue culture microshoots by sonicating the tissue in CHCl3 for 5min. Solvent evaporated and residue was redissolved in MeCN and filtered through 0.2 μm HPLC filter.

Results and Discussion
Incorporation Method
Although successful with other plants, direct stem injection used to incorporate ¹³C-glucose and ¹³C-DOX into salvinorin A could not be detected by HR-MS or ¹³C NMR spectroscopic analyses. Other experiments (with other plants) have shown that incorporation of [1-14C]-tryptophan into camptothecin was increased 25–100 fold by using direct-stem injection compared with feeding through roots, however stem injection can cause severe tissue damage and necrosis.

The tissue culture method was used to overcome these problems and led to successful incorporation of labeled substrates. Incorporation of [¹³C]-glucose in salvinorin A was achieved in microshoots grown in 1% labeled/1% unlabeled glucose (1:1 mix used to enhance uptake).

Spectroscopic analysis of salvinorins labeled with [2-¹³C]-glucose
Enrichment from [M+2]⁺ to [M+4]⁺, was in the range between 2.1% to 7.8% for salvinorin A, and 2.6% to 13.1% for salvinorin B.

Fig. 3. [VIEW] Mass spectra showing incorporation of ¹³C from the experiment with [2-¹³C]-glucose. (a) standard of salvinorin A, (b) m/z 433.1159 [M+H]⁺ refers to salvinorin A parent peak. Subsequent peaks form an isotope cluster reflecting the incorporation of ¹³C into the molecule of salvinorin A. (c) standard of salvinorin B, (d) m/z 391.1810 [M+H]⁺ corresponds to salvinorin B. Isotope cluster is spread to [M+5]⁺.

Spectroscopic analysis of salvinorins labeled with [1-¹³C]-glucose
For salvinorin A [M+2]⁺ was 3.6% enriched, while salvinorin B was 4.4% incorporated.

Fig. 4. [VIEW] Mass spectra of: (a) standard of salvinorin A, (b) salvinorin A isolated from experiment with [1-¹³C]-glucose, (c) standard of salvinorin B, and (d) salvinorin B isolated from experiment with [1-¹³C]-glucose. Isotope clusters are spread up to [M+5]⁺ peak in case of isotopically labeled salvinorin A and to [M+5]⁺ in salvinorin B.

The pattern of incorporation of ¹³C was consistent with predictions for the DOXP pathway. The carbonyl C-21 and methyl C-22 from the acetate functional group in salvinorin A were also found to be labeled with the [1-¹³C]-glucose, due to the non-specificity of glucose as a substrate. The methyl ester in salvinorin A at position C-23 was also observed, and most likely results from the enzymatic action of a type III S-adenosyl-l-methionine(SAM)-dependent O-methyltransferase.
It has been experimentally shown that salvinorin A is stored in glands, but no genomic or biochemical confirmation was found to suggest that biosynthesis of this compound occurs in the glands. The data shows that salvinorin A is derived from the DOXP biosynthetic pathway, which would suggest that it is biosynthesized in the glandular trichomes. However, many secondary metabolites produced in plant secretory glands (ex: cannabinoids and other terpenoids) are also produced via the DOXP pathway ([Gang et al., 2001] and [Mahlberg and Kim, 2004]).

Fig. 5. [VIEW] (a) Reference carbon NMR spectrum of salvinorin A; (b) spectrum of salvinorin A labeled with [1-¹³C]-glucose; (c) spectrum of salvinorin A labeled with [Me-¹³C]-methionine. Spectra were recorded in CDCl3 with a Bruker NMR with BBO 5 mm carbon probe at 151 MHz (standard, and labeled with [1-¹³C]-glucose salvinorin A), and Bruker NMR with 3 mm carbon direct probe at 100 MHz (salvinorin A labeled with [Me-¹³C]-methionine). Carbons with enhanced peak heights relative to the reference spectrum are labeled accordingly.

1-¹³C,3,4-²H₂]-1-deoxy-d-xylulose incorporation
Labeling with [1-¹³C,3,4-²H₂]-1-deoxy-d-xylulose (DOX) was done to confirm the results found using [1-¹³C]-glucose. Due to the phytotoxicity of DOX, concentration could not exceed 0.1%. Low, but  significant, ¹³C enrichments were found (at about 0.3–0.6%) to be consistent with [1-¹³C]-glucose findings. Low enrichment of DOX could possibly be due to: poor phosphorylation rate of the exogenous DOX (Adam et al., 1999), partial degradation of DOX into acetate (reducing available DOX) (Thiel and Adam, 2002), limitations of DOX associated with active transport to plastids and with membrane permeability (Fluegge and Gao, 2005), or the kinetic isotope effects associated with deuterium labeling.

[Me-¹³C]-methionine incorporation
The experiment done with 0.2% labeled methionine was unsuccessful because it caused phytotoxic damage. However, reducing the concentration to 0.1% of ¹³C-labeled methionine resulted in a 2.9% incorporation. The methionine incorporation was a highly successful, comparable to the level of incorporation for labeled glucose at a 10-fold higher concentration.

Retrobiosynthetic NMR analysis of salvinorin A showed the the methoxy ester at C-23 likely originated from SAM. As mentioned above, these reactions are catalyzed by type III SAM-dependent O-methyltransferases (Noel et al., 2003). In plants methyltransferases play important roles in the biosynthesis of primary and secondary metabolites (Roje, 2006), but some are highly specific of substrates while others are rather indiscriminate ([Dayan et al., 2003] and [Pichersky and Gang, 2000]).

Conclusion
The pattern of incorporation found using retrobiosynthetic NMR spectroscopic analysis was consistent with the DOXP-dependent pathway. The labeling with [1-¹³C]-glucose and [1-¹³C; 3,4-²H₂]-1-deoxy-d-xylulose are both consistent with this finding. The observed enrichment of the C-23 methoxy group in samples incorporated with [Me-¹³C]-methionine strongly supports the hypothesis that that SAM-dependent type III O-methyltransferase participates in these reactions. The microshoot tissue technique used in this study proved successful and is a valuable tool for the characterization of the steps involved in the synthesis of salvinorin A.

Further Research Questions
Does the biosynthesis of salvinorin A occur in the glands at all or is it only in the glandular trichomes?

[Paradoxic]

This is a paper I read and is probably the most relevant to the research I will be conducting. Many of the methods described I will be doing for my research. This the summary I did, which a bit less dense than than the actual paper. Its missing some of the more insignificant data recordings, but all the major findings and important procedures are there.

If anyone is interested in this kind of stuff (its biochemistry) they should definitely give this a read. This is the first (surely more to come) paper that gets into the inner workings of the plant, apart from past papers that simply identified the chemicals present in the plant.