Elsevier

Phytochemistry

Volume 68, Issue 14, July 2007, Pages 1872-1881
Phytochemistry

Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway

https://doi.org/10.1016/j.phytochem.2007.04.034Get rights and content

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.

Graphical abstract

Biosynthetic analysis of diterpene salvinorin A proved its formation via the DOXP pathway. Salvia divinorum was administered [1-13C]-d-glucose in sterile culture of microshoots. Results were analyzed by LCMS and NMR spectroscopic techniques.

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Introduction

Salvia divinorum, commonly referred to as Maria Pastora sage, has been known for its hallucinogenic properties for generations by Mazatec Indians of Oaxaca, Mexico (Valdés et al., 1983). The active component of S. divinorum, salvinorin A (1) (Fig. 1), was discovered by Ortega and colleagues in 1982 (Ortega et al., 1982), and its psychoactive activity in mice tests were reported a few years later (Valdés et al., 1987). Subsequent work established a threshold dose of 200 μg of 1 for humans (Siebert, 1994). Salvinorin A (1) is a first non-nitrogenous, potent and selective kappa-opioid receptor agonist, and is being intensively studied as a lead compound for the treatment of mental disorders (Roth et al., 2002, Vortherms and Roth, 2006).

Terpenoids are among the most abundant plant secondary metabolites (reviewed by Gershenzon and Croteau, 1991). All terpenoids result from the assembly of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) building blocks (reviewed by Eisenreich et al., 2004). It was long thought that these precursors originate exclusively from the mevalonic acid (MVA) pathway, which is ubiquitous in plants and animals (Porter and Spurgeon, 1981). However, this paradigm was challenged by Rohmer in labeling studies of bacterial hopanoids (Rohmer et al., 1993). Broers and Schwarz showed that the new pathway involves the monophosphate of 1-deoxy-d-xylulose (DOX), and that higher plants utilize both the MVA and DOX pathways (Broers, 1994, Schwarz, 1994). This biosynthetic route is now called the DOXP or MEP pathway, in reference to the early pentose intermediates, 1-deoxy-d-xylulose 5-phosphate and 2-C-methyl-d-erythritol 4-phosphate (Eisenreich et al., 1998, Eisenreich et al., 2001, Eisenreich et al., 2004, Rohmer, 1999).

The biosynthetic pathways of many different metabolites have been studied extensively using stable isotopes, primarily 13C and 2H (Simpson, 1998). The biosynthetic pathway of the hallucinogenic diterpenoid 1 has not yet been elucidated. Recent studies have shown that 1 is compartmentalized within the glandular trichomes located on abaxial side of the leaves of S. divinorum (Siebert, 2004). Since monoterpenes produced in glandular trichomes are normally derived from the DOXP pathway (Samanani and Facchini, 2006), we hypothesized that this psychoactive diterpenoid was also formed by this alternative route (Fig. 2). This hypothesis was tested by incorporation experiments with [1-13C]-glucose and [1-13C; 3,4-2H2]-1-deoxy-d-xylulose in Salvia divinorum. The source of the methyl ester of 1 was also investigated in a feeding experiment with [Me-13C]-methionine.

Section snippets

Method of incorporation

Salvinorin A (1) is being extensively studied as a lead compound for the treatment of mental disorders (Roth et al., 2002, Vortherms and Roth, 2006), however, its biosynthesis has not been investigated previously. Preliminary experiments with [2-13C]-glucose were designed to determine whether isotopically labeled substrates were taken up by S. divinorum cuttings and to estimate the duration of the administration period required for sufficient incorporation. Our initial attempts to label 1 using

Concluding remarks

Retrobiosynthetic NMR spectroscopic analysis of the biogenic origin of salvinorin A (1) yielded an incorporation pattern consistent with the DOXP-dependent pathway. Labeling with [1-13C]-glucose and [1-13C; 3,4-2H2]-1-deoxy-d-xylulose were in agreement with each other. Additionally, enrichment of the C-23 methoxy group in samples grown in the presence of [Me-13C]-methionine strongly suggested the participation of a SAM-dependent type III O-methyltransferase. The microshoot tissue culture

General experimental procedures

All chemicals were purchased from Fisher Scientific unless specified otherwise. [2-13C]-Glucose (99% 13C enrichment) was purchased from Sigma–Aldrich (St. Louis, MO), [1-13C]-glucose (99% 13C enrichment) and [Me-13C]-methionine (99% 13C enrichment) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). 1-Deoxy-d-xylulose (DOX), [1-13C]-1-deoxy-d-xylulose ([1-13C]-DOX) and [1-13C, 3,4-2H2]-1-deoxy-d-xylulose ([13C,2H2]-DOX) were synthesized as previously described (Giner, 1998).

Acknowledgments

The authors acknowledge Dr. Jeremy Stewart for scientific inspiration on research of Salvia divinorum, Dr. Ruslan Bikbulatov for valuable discussions during the preparation of this manuscript and Dr. Abbas Shilabin for the mass spectrometric analysis. We also thank David J. Kiemle (Analytical and Technical Services, SUNY-ESF, Syracuse, NY) for assistance with the spectrometry involving the 600 MHz Bruker NMR instrument. This work was supported by NIH Grant P20 RR 021929-01 (Center of Research

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