Research Article Received: 30 December 2013, Revised: 29 January 2014, Accepted: 2 February 2014 Published online in Wiley Online Library: 9 May 2014 (wileyonlinelibrary.com) DOI 10.1002/pca.2511 Semi-automated Separation of the Epimeric Dehydropyrrolizidine Alkaloids Lycopsamine and Intermedine: Preparation of their N-oxides and NMR Comparison with Diastereoisomeric Rinderine and Echinatine Steven M. Colegate,a* Dale R. Gardner,a Joseph M. Betzb and Kip E. Pantera ABSTRACT: Introduction – The diversity of structure and, particularly, stereochemical variation of the dehydropyrrolizidine alkaloids can present challenges for analysis and the isolation of pure compounds for the preparation of analytical standards and for toxicology studies. Objective – To investigate methods for the separation of gram-scale quantities of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine and to compare their NMR spectroscopic data with those of their heliotridine-based analogues echinatine and rinderine. Methods – Lycopsamine and intermedine were extracted, predominantly as their N-oxides and along with their acetylated derivatives, from commercial samples of comfrey (Symphytum officinale) root. Alkaloid enrichment involved liquid–liquid partitioning of the crude methanol extract between dilute aqueous acid and n-butanol, reduction of N-oxides and subsequent continuous liquid–liquid extraction of free base alkaloids into CHCl3. The alkaloid-rich fraction was further subjected to semi- automated flash chromatography using boronated soda glass beads or boronated quartz sand. Results – Boronated soda glass beads (or quartz sand) chromatography adapted to a Biotage Isolera Flash Chromatography System enabled large-scale separation (at least up to 1–2 g quantities) of lycopsamine and intermedine. The structures were confirmed using one- and two-dimensional 1H- and 13C-NMR spectroscopy. Examination of the NMR data for lycopsamine, intermedine and their heliotridine-based analogues echinatine and rinderine allowed for some amendments of literature data and provided useful comparisons for determining relative configurations in monoester dehydropyrrolizidine alkaloids. A similar NMR comparison of lycopsamine and intermedine with their N-oxides showed the effects of N-oxidation on some key chemical shifts. A levorotatory shift in specificrotationfrom+3.29°toÀ1.5° was observed for lycopsamine when dissolved in ethanol or methanol respectively. Conclusion – A semi-automated flash chromatographic process using boronated soda glass beads was standardised and confirmed as a useful, larger scale preparative approach for separating the epimers lycopsamine and intermedine. The useful NMR correlations to stereochemical arrangements within this specific class of dehydropyrrolizidine alkaloid cannot be confidently extrapolated to other similar dehydropyrrolizidine alkaloids. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Supporting information can be found in the online version of this article. Keywords: Boronated soda glass chromatography; HPLC–ESI/MS; NMR; optical rotation; echinatine; dehydropyrrolizidine alkaloids; intermedine; lycopsamine; rinderine; comfrey; Symphytum officinale Introduction The C9 monoester DHPAs lycopsamine (1) and intermedine (2) are epimeric about C13 (Fig. 1) (Mackay et al., 1983) and are In vivo oxidative metabolites of the plant-derived esters of components in many DHPA-producing plants (Bull et al., 1968). dehydropyrrolizidine alkaloids (DHPAs) and their N-oxides are potentially acutely and chronically toxic to livestock and humans (Edgar et al., 2011; Molyneux et al., 2011). Consequently, to enable monitoring of animal feed, the human food supply, dietary * Correspondence to: Steven M. Colegate, Poisonous Plant Research Laboratory, Agriculture Research Service, US Department of Agriculture, 1150 East 1400 supplements and herbal medicines for the presence of the DHPAs, North, Logan, Utah 84341, USA. and to define the actual risk to health presented by individual E-mail: [email protected] DHPAs, pure alkaloids are required for analytical standards and, a on a larger scale, for toxicological studies. However, the sometimes Poisonous Plant Research Laboratory, Agriculture Research Service, US subtle diversities of structure and stereochemistry (relative and Department of Agriculture, 1150 East 1400 North, Logan, Utah, 84341, USA absolute) of the several hundreds of DHPAs known can present a b Office of Dietary Supplements, National Institutes of Health, 6100 Executive challenge for analysis and for the isolation of pure alkaloids. Blvd., Room 3B01, Bethesda, Maryland, 20892, USA 429 Phytochem. Anal. 2014, 25, 429–438 Published 2014. This article is a U.S. Government work and is in the public domain in the USA. S. M. Colegate et al. Lycopsamine (1) and intermedine (2) in extracts of Amsinckia intermedia have been previously separated from each other by taking advantage of their different affinities for the boronate anion (Frahn et al., 1980). Two methods were described: (i) an affinity-type column where 1 and 2 were selectively partitioned between aqueous borax-coated powdered soda glass and the CHCl3 elution solvent; and (ii) an ion exchange process in which the anionic nature of the respective boronate complexes differentially hindered retention on a cation exchange resin. Only one of these approaches, that is, the cation exchange, seems to subsequently have been utilised successfully, albeit only once and with mannitol as the lycopsamine/intermedine displacement ligand instead of glucose (Roitman, 1983). In the present study, both methods were re-examined for their ability to separate lycopsamine, intermedine and related alkaloids derived from Symphytum officinale roots with the intent of standardising an approach with modern availability of reagents, substrates and equipment. Experimental Chemicals and reagents Dry, powdered comfrey (S. officinale) root (Frontier Natural Products Co-op) was purchased from Take Herb (Alhambra, CA, USA). The Figure 1. Structures of dehydropyrrolizidine alkaloids. following were reagent ACS/USP/NF grade (Pharmaco Products, Brookfield, CT, USA): methanol, for extractions; petroleum ether (b.p. – fl Each can be biosynthesised relatively exclusively of the other, 35 60°C) for column packing; and CHCl3,for ashcolumnchromatog- such as the lycopsamine-N-oxide chemotype of Amsinckia raphy. All other reagents, that is, zinc powder, n-butanol, ammonia solution and concentrated sulphuric acid were of analytical grade intermedia (Colegate et al., 2013) and intermedine-N-oxide from and generally sourced. Acetonitrile was HPLC-certified solvent Cerinthe glabra (Luber et al., 2012), but more often they co-occur (Honeywell Burdick and Jackson, Muskegon, MI, USA) and water was at various relative levels as the free bases and/or their N-oxides Milli-Q-purified (18.2 MΩ/cm) (Millipore, Bedford, MA, USA). Formic (Kelley and Seiber 1992; Williams et al., 2011). acid was ‘For Analysis’ grade (> 99%; Acros Organics/Thermo Fisher In some cases a fortuitous combination of solubility properties Scientific, Bridgewater, NJ, USA). Ammoniated methanol was prepared and a lack of other DHPA co-metabolites has allowed for a by bubbling dry ammonia gas through cooled (ice bath) methanol. The relatively simple extraction, purification and precipitation resultant saturated ammoniated methanol solution was diluted 1:9 process to result in large quantities of relatively pure DHPA. Such with methanol for use in strong cation exchange, solid phase was the case, for example, in the isolation of the macrocyclic extraction (SCX SPE). The HPLC columns, with their guard columns, μ diester DHPAs riddelliine (Adams et al., 1942) and senecionine bulk SCX packing material (Sepra SCX, 50 m, 65A) and SCX SPE cartridges (Strata SCX 55 μm, 70 A; 5 g/20 mL Giga tube), were sourced (Culvenor, 1962). However, when the DHPAs are less tractable, from Phenomenex (Torrence, CA, USA). The indigo-carmine-based methods of separation include thin-layer chromatography redox resin was prepared and used as previously described (Colegate (TLC), column chromatography, centrifugal planar chromatogra- et al., 2005; Williams et al., 2011). Echinatine (3), rinderine (4), heliotrine phy, semi-preparative and preparative scale high-pressure liquid (5) (Fig. 1) and lasiocarpine (all > 99% pure based on HPLC–ESI(+)/MS chromatography (HPLC), rotation locular counter current and NMR analysis) were sourced from the stocks of extracted and chromatography (RLCCC), droplet counter current chromatogra- purified pyrrolizidine alkaloids kept by the USDA/ARS Poisonous phy (DCCC) and high-speed counter current chromatography Plant Research Laboratory. One- and two-dimensional (COSY, HSQC) 1 13 (HSCCC). Because of the time, effort and higher levels of losses H- (300 MHz) and C- (75 MHz) NMR data were acquired using a JEOL through degradation or adsorption-related effects usually Eclipse NMR spectrometer using solutions in deuterochloroform involved with necessary repetitive application of the solid (Sigma-Aldrich, St Louis, MO, USA) and the residual proton in the CHCl3 as the chemical shift reference. Optical activity was measured using an stationary phase approaches, they have mostly been used to iso- Autopol IV Automatic Polarimeter (Rudolph Research Analytical, late relatively small quantities of pure alkaloid for NMR-based Hackettstown, NJ, USA). structural studies (e.g. Roeder et al., 1991; Colegate et al., 2012). High-speed