J. Brent Friesen

G.U.E.S.S : A generally useful estimate of solvent systems in CCC

Abstract The choice of an appropriate solvent system for Countercurrent Chromatography (CCC) is a critical step in the purification of natural products. Targeted towards their high sample diversity, G.U.E.S.S. is a practical approach for the prediction of CCC distribution constants, K values, by standard thin layer chromatography (TLC). G.U.E.S.S. allows a major reduction in workload by direct use of routine TLC information. The separation capability of CCC focuses on an optimal “window of opportunity” that can be described as the “sweet spot” of CCC separation. The sweet spot of optimal CCC performance may be described as an area where compound K values are between 0.4 and 2.5. Two useful CCC solvent systems: hexane/ethyl acetate/methanol/water and chloroform/methanol/water are organized and recommended as the HEMWat and ChMWat methods of solvent system selection. The relationship of (i) P values, measured by the ratio of UV‐vis absorption, (ii) TLC R f values and (iii) CCC retention volumes for over 20 diverse commercially available natural products are described. The HEMWat method characterizes a versatile solvent selection technique. TLC R f values will often give practical predictions, even with simple single‐phase mixtures. Additional information can be acquired from equivalent solvent systems and by calibration with the G.U.E.S.S. standard compounds. The latter will also aid in the important selection of which phase will function as the mobile phase. The choice of normal vs. reverse phase will depend on the polarity of compounds that are desired to be gathered into the sweet spot. In addition, G.U.E.S.S. has been shown to be readily applicable to natural product purification necessary for drug discovery, bioassay guided fractionation, and metabolome analysis.

Countercurrent separation of natural products.

An assessment of the technology and method development in countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC), collectively referred to as countercurrent separation (CS), is provided. More than six decades of CS theory and applications are critically reviewed and developed into a practical guide to CS for natural products research. The necessary theoretical foundation is given for better use of CS in the separation of biological molecules of any size, small to large, and from any matrix, simple to complex. The three operational fundamentals of CS--instrumentation, biphasic solvent systems, and theory--are covered in a prismatic fashion. The goal of this review is to provide the necessary background and references for an up-to-date perspective of CS and to point out its potential for the natural products scientist for applications in natural products chemistry, metabolome, and proteome research involving organisms from terrestrial and marine sources.

Countercurrent Separation of Natural Products: An Update

This work assesses the current instrumentation, method development, and applications in countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC), collectively referred to as countercurrent separation (CCS). The article provides a critical review of the CCS literature from 2007 since our last review (J. Nat. Prod.2008, 71, 1489–1508), with a special emphasis on the applications of CCS in natural products research. The current state of CCS is reviewed in regard to three continuing topics (instrumentation, solvent system development, theory) and three new topics (optimization of parameters, workflow, bioactivity applications). The goals of this review are to deliver the necessary background with references for an up-to-date perspective of CCS, to point out its potential for the natural product scientist, and thereby to induce new applications in natural product chemistry, metabolome, and drug discovery research involving organisms from terrestrial and marine sources.

Importance of Purity Evaluation and the Potential of Quantitative 1H NMR as a Purity Assay

In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical 1H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material.

HiFSA Fingerprinting Applied to Isomers with Near-Identical NMR Spectra: The Silybin/Isosilybin Case

This study demonstrates how regio- and diastereo-isomers with near-identical NMR spectra can be distinguished and unambiguously assigned using quantum mechanical driven (1)H iterative Full Spin Analysis (HiFSA). The method is illustrated with four natural products, the flavonolignans silybin A, silybin B, isosilybin A, and isosilybin B, which exhibit extremely similar coupling patterns and chemical shift differences well below the commonly reported level of accuracy of 0.01 ppm. The HiFSA approach generated highly reproducible (1)H NMR fingerprints that enable distinction of all four isomers at (1)H frequencies from 300 to 900 MHz. Furthermore, it is demonstrated that the underlying numeric (1)H NMR profiles, combined with iterative computational analysis, allow parallel quantification of all four isomers, even in difficult to characterize reference materials and mixtures. The results shed new light on the historical challenges to the qualitative and quantitative analysis of these therapeutically relevant flavonolignans and open new opportunities to explore hidden diversity in the chemical space of organic molecules.

Can Invalid Bioactives Undermine Natural Product-Based Drug Discovery?

High-throughput biology has contributed a wealth of data on chemicals, including natural products (NPs). Recently, attention was drawn to certain, predominantly synthetic, compounds that are responsible for disproportionate percentages of hits but are false actives. Spurious bioassay interference led to their designation as pan-assay interference compounds (PAINS). NPs lack comparable scrutiny, which this study aims to rectify. Systematic mining of 80+ years of the phytochemistry and biology literature, using the NAPRALERT database, revealed that only 39 compounds represent the NPs most reported by occurrence, activity, and distinct activity. Over 50% are not explained by phenomena known for synthetic libraries, and all had manifold ascribed bioactivities, designating them as invalid metabolic panaceas (IMPs). Cumulative distributions of ∼200,000 NPs uncovered that NP research follows power-law characteristics typical for behavioral phenomena. Projection into occurrence–bioactivity–effort space produces the hyperbolic black hole of NPs, where IMPs populate the high-effort base.

Phytoconstituents from Vitex agnus-castus fruits.

A new labdane-diterpene, viteagnusin I (1), together with 23 known phytoconstituents were isolated from the fruits of Vitex agnus-castus L, and their structures characterized by spectroscopic methods (NMR and MS). The known compounds include ten flavonoids, five terpenoids, three neolignans, and four phenolic compounds, as well as one glyceride. Biological evaluation identified apigenin, 3-methylkaempferol, luteolin, and casticin as weak ligands of delta and mu opioid receptors, exhibiting dose-dependent receptor binding.

Occurrence of Progesterone and Related Animal Steroids in Two Higher Plants

Previously, the presence of a wide variety of chemically diverse steroids has been identified in both flora and fauna. Despite the relatively small differences in chemical structures and large differences in physiological function of steroids, new discoveries indicate that plants and animals are more closely related than previously thought. In this regard, the present study gathers supporting evidence for shared phylogenetic roots of structurally similar steroids produced by these two eukaryotic taxa. Definitive proof for the presence of progesterone (3) in a vascular plant, Juglans regia, is provided. Additional evidence is gleaned from the characterization of five new plant steroids from Adonis aleppica: three 3-O-sulfated pregnenolones (6a/ b, 7), a sulfated H-5beta cardenolide, strophanthidin-3-O-sulfate (8), and spirophanthigenin (10), a novel C-18 oxygenated spirocyclic derivative of strophanthidin. The ab initio isolation and structure elucidation (NMR, MS) of these genuine minor plant steroids offers information on preparative metabolomic profiling at the ppm level and provides striking evidence for the conserved structural space of pregnanes and its congeners across the phylogenetic tree.

Analysis and Purification of Bioactive Natural Products: The AnaPurNa Study

Based on a meta-analysis of data mined from almost 2000 publications on bioactive natural products (NPs) from >80 000 pages of 13 different journals published in 1998–1999, 2004–2005, and 2009–2010, the aim of this systematic review is to provide both a survey of the status quo and a perspective for analytical methodology used for isolation and purity assessment of bioactive NPs. The study provides numerical measures of the common means of sourcing NPs, the chromatographic methodology employed for NP purification, and the role of spectroscopy and purity assessment in NP characterization. A link is proposed between the observed use of various analytical methodologies, the challenges posed by the complexity of metabolomes, and the inescapable residual complexity of purified NPs and their biological assessment. The data provide inspiration for the development of innovative methods for NP analysis as a means of advancing the role of naturally occurring compounds as a viable source of biologically active agents with relevance for human health and global benefit.

Qualitative and quantitative evaluation of solvent systems for countercurrent separation

Rational solvent system selection for countercurrent chromatography and centrifugal partition chromatography technology (collectively known as countercurrent separation) studies continues to be a scientific challenge as the fundamental questions of comparing polarity range and selectivity within a solvent system family and between putative orthogonal solvent systems remain unanswered. The current emphasis on metabolomic investigations and analysis of complex mixtures necessitates the use of successive orthogonal countercurrent separation (CS) steps as part of complex fractionation protocols. Addressing the broad range of metabolite polarities demands development of new CS solvent systems with appropriate composition, polarity (π), selectivity (σ), and suitability. In this study, a mixture of twenty commercially available natural products, called the GUESSmix, was utilized to evaluate both solvent system polarity and selectively characteristics. Comparisons of GUESSmix analyte partition coefficient (K) values give rise to a measure of solvent system polarity range called the GUESSmix polarity index (GUPI). Solvatochromic dye and electrical permittivity measurements were also evaluated in quantitatively assessing solvent system polarity. The relative selectivity of solvent systems were evaluated with the GUESSmix by calculating the pairwise resolution (αip), the number of analytes found in the sweet spot (Nsw), and the pairwise resolution of those sweet spot analytes (αsw). The combination of these parameters allowed for both intra- and inter-family comparison of solvent system selectivity. Finally, 2-dimensional reciprocal shifted symmetry plots (ReSS(2)) were created to visually compare both the polarities and selectivities of solvent system pairs. This study helps to pave the way to the development of new solvent systems that are amenable to successive orthogonal CS protocols employed in metabolomic studies.