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United States Department of Agriculture

Agricultural Research Service

Research Project: Understanding and Mitigating the Adverse Effects of Poisonous Plants on Livestock Production Systems

Location: Poisonous Plant Research

Title: Semi-automated separation of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine: Preparation of their N-oxides and NMR comparison with diastereoisomeric rinderine and echinatine

item Colegate, Steven
item Gardner, Dale
item Betz, Joseph -
item Panter, Kip

Submitted to: Phytochemical Analysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 2, 2014
Publication Date: N/A

Interpretive Summary: Dehydropyrrolizidine alkaloids are natural poisons present in many plants and have been shown to poison livestock and humans. Humans can be poisoned by contamination of their food or via medicinal herb preparations. The approximately 500 dehydropyrrolizidine alkaloids that are known, most also occurring as their N-oxides, can have subtle variations in their structure which makes them difficult to isolate in large amounts for analytical standards and toxicology studies. A method was developed to separate gram-scale quantities of very closely related alkaloids that have featured in livestock and human poisoning cases. A concurrent, detailed examination of the nuclear magnetic resonance spectroscopy data for these alkaloids and some closely related analogues allowed for some amendments to literature data and provided useful comparisons for determining structures in similar dehydropyrrolizidine alkaloids.

Technical 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 semiautomated 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 specific rotation from +3.29° to 1.5° 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.

Last Modified: 2/28/2015
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