Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway
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.
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
References (40)
- et al.
Incorporation of 1-[1-13C]deoxy-d-xylulose in chamomile sesquiterpenes
Arch. Biochem. Biophys.
(1999) - et al.
Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.)
Sci. Hort.
(2004) - et al.
Glandular hairs and essential oils in micropropagated plants of Salvia officinalis L
Plant Sci.
(2005) - et al.
Elucidation of the biosynthetic pathway of the allelochemical sorgoleone using retrobiosynthetic NMR analysis
J. Biol. Chem.
(2003) - et al.
Deoxyxylulose phosphate pathway to terpenoids
Trends Plant Sci.
(2001) - et al.
The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms
Chem. Biol.
(1998) New and efficient synthetic routes to 1-deoxy-d-xylulose
Tetrahedron Lett.
(1998)- et al.
Structural, functional, and evolutionary basis for methylation of plant small molecules
Rec. Adv. Phytochem.
(2003) - et al.
The effects of exogenous amino acid on acetylene reduction activity of Vicia faba L. cv. Fiord
Ann. Bot.
(1993) - et al.
Genetics and biochemistry of secondary metabolites in plants: an evolutionary perspective
Trends Plant Sci.
(2000)
S-Adenosyl-l-methionine: beyond the universal methyl group donor
Phytochemistry
Compartmentalization of plant secondary metabolism
Incorporation of 1-deoxy-d-xylulose into isoprene and phytol by higher plants and algae
FEBS Lett.
Biosynthesis of Camptotheca acuminate alkaloids
Phytochemistry
Salvia divinorum and salvinorin A: new pharmacologic findings
J. Ethnopharmacol.
Biosynthesis of the sesquiterpene germacrene D in Solidago canadensis: 13C and 2H labeling studies
Phytochemistry
Incorporation of [1-13C]1-deoxy-d-xylulose into isoprenoids of the liverwort Conocephalum conicum
Phytochemistry
Ethnopharmacology of Ska Marı´a Pastora (Salvia divinorum, Epling and Játiva-M.)
J. Ethnopharmacol.
Biosynthesis of plaunotol in Croton stellatopilosus proceeds via the deoxyxylulose phosphate pathway
Tetrahedron Lett.
Divinatorins A–C, new neoclerodane diterpenoids from the controlled sage Salvia divinorum
J. Nat. Prod.
Cited by (41)
Progress in the study of natural neo-clerodane diterpenoids
2024, Studies in Natural Products ChemistryThe Plant Salvia divinorum (Lamiaceae)-Chemistry and Pharmacology
2016, Neuropathology of Drug Addictions and Substance MisuseThe Plant Salvia divinorum (Lamiaceae)-Chemistry and Pharmacology
2016, Neuropathology of Drug Addictions and Substance Misuse Volume 2: Stimulants, Club and Dissociative Drugs, Hallucinogens, Steroids, Inhalants and International AspectsA Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms
2016, Methods in EnzymologyCitation Excerpt :For plants, labeled tracers can be added to the growth media, which often show uptake through the roots (Weng, Li, Mo, & Chapple, 2012). However, in other cases, injection of biosynthetic precursors into specific organs of the target organism is necessary (Kutrzeba et al., 2007; Sheriha & Rapoport, 1976). Once optimal labeling condition is found, in which the end target metabolite gets labeled, computational analysis can be performed to search for other features that have also become labeled (eg, light ions accompanied by a corresponding heavy ion of expected mass).
Salvinorin A content in legal high products of Salvia divinorum sold in Mexico
2015, Forensic Science InternationalCitation Excerpt :S. divinorum is also used for medicinal purposes as anti-parasitic, anti-diarrhoea and a cure for an abdominal inflammation condition called “panzón de borrego” [8,9]. Previous phytochemical studies of S. divinorum have led to the isolation of several diterpenes including salvinorins A–J, saldividins A–D, divinatorins D and F, and salvinicins A and B [7,10,11]. Salvinorin A (Fig. 1), the main active diterpene of this plant has shown diverse biological effects such as anti-inflammatory [12], antidepressant [13], gastrointestinal [14] and hallucinogenic [15].