Quantitative ¹H NMR. Development and Potential of an Analytical Method: An Update

Covering the literature from mid-2004 until the end of 2011, this review continues a previous literature overview on quantitative ¹H NMR (qHNMR) methodology and its applications in the analysis of natural products. Among the foremost advantages of qHNMR is its accurate function with external calibration, the lack of any requirement for identical reference materials, a high precision and accuracy when properly validated, and an ability to quantitate multiple analytes simultaneously. As a result of the inclusion of over 170 new references, this updated review summarizes a wealth of detailed experiential evidence and newly developed methodology that supports qHNMR as a valuable and unbiased analytical tool for natural product and other areas of research

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

Quantitative Purity–Activity Relationships of Natural Products: The Case of Anti-Tuberculosis Active Triterpenes from <i>Oplopanax horridus</i>

The present study provides an extension of the previously developed concept of purity–activity relationships (PARs) and enables the quantitative evaluation of the effects of multiple minor components on the bioactivity of residually complex natural products. The anti-tuberculosis active triterpenes from the Alaskan ethnobotanical <i>Oplopanax horridus</i> were selected as a case for the development of the quantitative PAR (QPAR) concept. The residual complexity of the purified triterpenes was initially evaluated by 1D- and 2D-NMR and identified as a combination of structurally related and unrelated impurities. Using a biochemometric approach, the qHNMR purity and anti-TB activity of successive chromatographic fractions of <i>O. horridus</i> triterpenes were correlated by linear regression analysis to generate a mathematical QPAR model. The results demonstrate that impurities, such as widely occurring monoglycerides, can have a profound impact on the observed antimycobacterial activity of triterpene-enriched fractions. The QPAR concept is shown to be capable of providing a quantitative assessment in situations where residually complex constitution contributes toward the biological activity of natural products

The Tandem of Full Spin Analysis and qHNMR for the Quality Control of Botanicals Exemplified with <i>Ginkgo biloba</i>

Botanical dietary supplements and herbal remedies are widely used for health promotion and disease prevention. Due to the high chemical complexity of these natural products, it is essential to develop new analytical strategies to guarantee their quality and consistency. In particular, the precise characterization of <i>multiple</i> botanical markers remains a challenge. This study demonstrates how a combination of computer-aided spectral analysis and 1D quantitative ¹H NMR spectroscopy (qHNMR) generates the analytical foundation for innovative means of simultaneously identifying and quantifying botanical markers in complex mixtures. First, comprehensive ¹H NMR profiles (fingerprints) of selected botanical markers were generated via ¹H iterative full spin analysis (HiFSA) with PERCH. Next, the ¹H fingerprints were used to assign specific ¹H resonances in the NMR spectra of reference materials, enriched fractions, and crude extracts of <i>Ginkgo biloba</i> leaves. These ¹H fingerprints were then used to verify the assignments by 2D NMR. Subsequently, a complete purity and composition assessment by means of 1D qHNMR was conducted. As its major strengths, this tandem approach enables the simultaneous quantification of multiple constituents without the need for identical reference materials, the semiquantitative determination of particular subclasses of components, and the detection of impurities and adulterants

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

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

Correction to Importance of Purity Evaluation and the Potential of Quantitative ¹H NMR as a Purity Assa

Residual Complexity Does Impact Organic Chemistry and Drug Discovery: The Case of Rufomyazine and Rufomycin

Residual complexity (RC) involves the impact of subtle but critical structural and biological features on drug lead validation, including unexplained effects related to unidentified impurities. RC commonly plagues drug discovery efforts due to the inherent imperfections of chromatographic separation methods. The new diketopiperazine, rufomyazine (<b>6</b>), and the previously known antibiotic, rufomycin (<b>7</b>), represent a prototypical case of RC that (almost) resulted in the misassignment of biological activity. The case exemplifies that impurities well below the natural abundance of ¹³C (1.1%) can be highly relevant and calls for advanced analytical characterization of drug leads with extended molar dynamic ranges of >1:1,000 using qNMR and LC-MS. Isolated from an actinomycete strain, <b>6</b> was originally found to be active against <i>Mycobacterium tuberculosis</i> with a minimum inhibitory concentration (MIC) of 2 μg/mL and high selectivity. As a part of lead validation, the dipeptide was synthesized and surprisingly found to be inactive. The initially observed activity was eventually attributed to a very minor contamination (0.24% [m/m]) with a highly active cyclic peptide (MIC ∼ 0.02 μM), subsequently identified as an analogue of <b>7</b>. This study illustrates the serious implications RC can exert on organic chemistry and drug discovery, and what efforts are vital to improve lead validation and efficiency, especially in NP-related drug discovery programs

Chlorinated Coumarins from the Polypore Mushroom <i>Fomitopsis officinalis</i> and Their Activity against <i>Mycobacterium tuberculosis</i>

An EtOH extract of the polypore mushroom <i>Fomitopsis officinalis</i> afforded two new naturally occurring chlorinated coumarins, which were identified as the previously synthesized compounds 6-chloro-4-phenyl-2<i>H</i>-chromen-2-one (<b>1</b>) and ethyl 6-chloro-2-oxo-4-phenyl-2<i>H</i>-chromen-3-carboxylate (<b>2</b>). The structures of the two isolates were deduced by <i>ab initio</i> spectroscopic methods and confirmed by chemical synthesis. In addition, an analogue of each was synthesized as 7-chloro-4-phenyl-2<i>H</i>-chromen-2-one (<b>3</b>) and ethyl 7-chloro-2-oxo-4-phenyl-2<i>H</i>-chromen-3-carboxylate (<b>4</b>). All four compounds were characterized physicochemically, and their antimicrobial activity profiles revealed a narrow spectrum of activity with lowest MICs against the <i>Mycobacterium tuberculosis</i> complex

Discovery and Characterization of the Tuberculosis Drug Lead Ecumicin

The new tuberculosis (TB) lead ecumicin (<b>1</b>), a cyclic tridecapeptide, was isolated from <i>Nonomuraea sp.</i> MJM5123, following a high-throughput campaign for anti-TB activity. The large molecular weight of 1599 amu detected by LC-HR-MS precluded the initial inference of its molecular formula. The individual building blocks were identified by extensive NMR experiments. The resulting two possible planar structures were distinguished by LC-MS². Determination of absolute configuration and unambiguous structural confirmation were carried out by X-ray crystallography and Marfey’s analysis