Residual Complexity of Bioactive Natural Products
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Background: The term residual complexity (RC) refers to the subtle but significant convolution of major and minor chemical species in materials that originate from reaction mixtures, such as natural products.
Because natural products are formed biosynthetically in living organisms, they are part of a complex mixture of often structurally related metabolites. Certain levels of these metabolites usually remain present even after a number of purification steps, and this RC is to some degree conserved even in highly purified materials.
In principle, RC affects all "pure" materials. RCs can be divided into two main groups: static RC describes the thermodynamically stable case, whereas dynamic RC refers to a situation where the impurity pattern changes over time due to reactivity or other chemical change that occurs during the timeframe and under the conditions of the observation (e.g., a bioassay).
Publication Series on Residual Complexity
This series encompasses peer-reviewed publications since 2008, starting with work on Purity Activity Relationships and Chemical Subtraction. It comprises articles that deal with all aspects of residual complexity (RC) of natural products, ranging from the analytical assessment of high-purity reference materials to the biological evaluation of dynamically changing extracts and mixtures. The following bibliography contains all publications of this series, brief synopses of each contribution, and their DOI links for easy access.
Series Overviews: The Supporting Information of the articles typically contains a table of all preceeding parts of this publication, as well as a brief synopsis of each.
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Part 32- Residual Complexity of Bioactive Natural Products
Part 31- Residual Complexity of Bioactive Natural Products
Part 30- Residual Complexity of Bioactive Natural Products
C Simmler, D Lankin, D Nikolic, R van Breemen, G Pauli
Isolation and structural characterization of dihydrobenzofuran congeners of licochalcone A
Fitoterapia 121, 6-15 (2017)
PubMedCentral: PMC in progress
Part 29- Residual Complexity of Bioactive Natural Products
J.W. Nam, R. Phansalkar, D.C. Lankin, J. McAlpine, A. Leme-Kraus, C. Vidal, A. Bedran-Russo, S.-N. Chen, G. Pauli
Absolute Stereochemistry of Native Oligomeric Proanthocyanidins with Dentin Biomodification Potency
Journal of Organic Chemistry 82, 1316-1329 (2017)
PubMedCentral: PMC in progress
Part 28- Residual Complexity of Bioactive Natural Products
Bisson J, McAlpine J, Friesen JB, Chen SN, Graham J, Pauli GF
Can Invalid Bioactives Undermine Natural Product-Based Drug Discovery?
Journal of Medicinal Chemistry 59: 1671-1690 (2016)
PubMedCentral: PMC4791574 [Open Access Article]
Part 27- Residual Complexity of Bioactive Natural Products
Nam JW, Phansalkar R, Lankin D, Bisson J, McAlpine J, Leme A, Vidal C, Ramirez B, Niemitz M, Bedran-Russo A, Chen SN, Pauli GF
Subtle Chemical Shifts Explain the NMR Fingerprints of Oligomeric Proanthocyanidins with High Dentin Biomodification Potency
Journal of Organic Chemistry 80: 7495-7507 (2015)
Brief Synposis Regarding Residual Complexity and Bioactivity: Detection and quantification of H/D-exchange effects of by dentin enhancing oligomeric proanthocyanidins (OPACs) QM-HiFSA identified C-8 and C-6 as (re-)active sites, explain preferences in biosynthetic linkage, and suggest their involvement in dentin cross-linking activity. Mapping of these molecular properties required high d precision at low- to sub-ppb in both 1H and 13C NMR. Newly characterized chemical shift differences in ppb are small but diagnostic measures of dynamic processes inherent to the OPAC pharmacophores and can help augment our understanding of nm-scale intermolecular interactions in biomodified dentin macromolecules.
Part 26- Residual Complexity of Bioactive Natural Products
Napolitano J, Simmler C, Napolitano J, McAlpine J, Lankin D, SN Chen, Pauli GF
Digital NMR Profiles as Building Blocks: Assembling 1H Fingerprints of Steviol Glycosides
Journal of Natural Products 78: 658-665 (2015)
Brief Synposis Regarding Residual Complexity and Bioactivity: A fragment-based approach 1D 1H NMR for the examination of congeneric organic compounds that utilizes characteristic HiFSA profiles of key structural motifs to understand spectra of increasingly complex molecules with near identical (residually complex) moieties (repeating sugar units). Illustrated using seven steviol glycosides and two related isosteviols, the method supports structural verification, chemical dereplication, and mixture analysis.
Part 25- Residual Complexity of Bioactive Natural Products
Pauli GF, Chen SN, Lankin D, Bisson J, Case R, Chadwick L, Goedecke T, Inui T, Krunic A, Jaki B, McAlpine J, Mo S, Napolitano J, Orjala J, Lehtivarjo J, Korhonen SP, Niemitz M
Essential Parameters for Spectral Analysis and Structural Dereplication by 1H NMR Spectroscopy
Journal of Natural Products 77: 1473-1487 (2014)
Brief Synposis Regarding Residual Complexity and Bioactivity: Demonstrates that 0.1-1 ppb and 10 mHz are adequate and required precisions for reporting the d and J parameters of frequency domain 1H NMR data. Shown for a variety of structural classes (terpenoids, phenolics, alkaloids) from different taxa (plants, cyanobacteria), spectral simulations paired with iteration are shown to be essential for spectral interpretation, adequate precision, and unambiguous HNMR-driven dereplication and metabolomic analysis. As HNMR spectra can be (residually) complex, decoding the wealth of structural information from the underlying spin parameters is a major challenge. Accordingly, the reproducibility of the spectral data is limited when translated into tables, hampering subsequent structure elucidation and dereplication. Handling and documenting 1H NMR data with adequate precision is critical for establishing unequivocal links between chemical structure, analytical data, metabolomes, and biological activity, collectively minimizing unwanted impact by residual complexity.
Part 24 - Residual Complexity of Bioactive Natural Products
Ramos Alvarenga R, Friesen JB, Nikolic D, Simmler C, Napolitano J, van Breemen R, Lankin D, McAlpine J, Pauli GF, SN Chen
K-Targeted Metabolomic Profiling Extends Chemical Subtraction to DESIGNER Extracts: Selective Depletion of Extracts of Hops (Humulus lupulus)
Journal of Natural Products 77: 2595-2604 (2014)
Brief Synposis Regarding Residual Complexity and Bioactivity: Introduces a flexible, compound targeted approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources (DESIGNER extracts). Uses chemical subtraction or augmentation of metabolites via K targeting using countercurrent separation methodology to remove single or multiple compounds from a chemically complex mixture. Demonstrates feasibility by single- or multi-targeted removal of four bioactive prenylated phenols (isoxanthohumol, 8-prenylnaringenin, 6-prenylnaringenin, and xanthohumol) from a standardized hops (Humulus lupulus L.). DESIGNER extracts innovate the characterization of chemically complex extracts through integration of residual complexity concepts with enabling technologies including countercurrent, K by bioactivity, qNMR, LC-MS, and HiFSA-based NMR fingerprinting.
Part 23 - Residual Complexity of Bioactive Natural Products
Riihinen K, Ou Z, Goedecke T, Lankin D, Pauli GF, Wu C
The Antibiofilm Activity of Lingonberry Flavonoids against Oral Pathogens is a Case Connected to Residual Complexity
Fitoterapia 97, 78-86 (2014)
Brief Synposis Regarding Residual Complexity and Bioactivity: The antimicrobial activity of isolated lingonberry flavonoid glycosides could not be confirmed with identical reference materials from other source. Representing a case of both static and dynamic residual complexity, even after HPLC-PDA, NMR, and MALDI-TOF analyses, the structural identity of the bioactive minor constituents in the cranberry-derived bioactive markers remained unknown, while color and analytical characteristics point to flavonoid oxidation products as underlying active principles.
Part 22- Residual Complexity of Bioactive Natural Products
Qiu F, McAlpine J, Lankin D, Burton I, Karakach T, Chen SN,
2D NMR Barcoding and Differential Analysis of Complex Mixtures for Chemical Identification: the Actaea Triterpenes
Analytical Chemistry 86: 3964-3972 (2014)
ADDITIONAL INFO: Topic Page for this paper
Brief Synposis Regarding Residual Complexity and Bioactivity: Similar to general barcoding technology, 2D-NMR barcoding utilizes clusters of fingerprint signals for the direct analysis of complex mixtures. It enables the identification of both known and new structures according to their structural subtypes. Demonstrated for HMBC data, 2D-NMR barcoding uses the structural information encoded in the 2-D chemical shift space in the form of relative cross peak patterns. The specificity of the patterns enables the distinction between congeneric metabolites in (residually) complex mixtures from extracts to purified compounds.
Part 21- Residual Complexity of Bioactive Natural Products
Dong SH, Cai G, Napolitano JG, Nikolic N, Lankin DC, McAlpine JB, van Breemen R, Soejarto DD, Pauli GF, Chen SN
Lipidated Steroid Saponins from Dioscorea villosa
Fitoterapia 84: 91: 113-124 (2013)
Brief Synposis Regarding Residual Complexity and Bioactivity: While known for its rich content in spirostanol glycosides, this study shows that wild yam (Dioscorea villosa) also performs lipidation of these otherwise polar molecules. Employing a fractionation protocol of metabolomic mining, seven new compounds were isolated and characterized to contain analogue long-chain fatty acids with different degrees of unsaturation, generating entire series of compounds which are difficult to purify and analyze. The discovery of this structural variation with largely different physicochemical properties may inspire biological studies which take into account their lipophilic character and and demonstrates that nature uses ubiquituous biosynthetic building blocks such as fatty acids to achieve a major shift int the polarity window of secondary metabolites, which can easily be overlooked.
Part 20 - Residual Complexity of Bioactive Natural Products
Napolitano JG, Lankin D, Graf T, Friesen JB, Chen SN, McAlpine JB, Oberlies, NH, Pauli GF
HiFSA Fingerprinting of Isomers with Near Identical NMR Spectra:
The Silybin/Isosilybin Case
Journal of Organic Chemistry 78: 2827-2839 (2013)
dx.doi.org/10.1021/jo302720h [Featured Article]
Brief Synposis Regarding Residual Complexity and Bioactivity: Demonstrates how the "hidden" complexity of regio- and diastereoisomers with near-identical NMR spectra can be distinguished and unambiguously assigned by quantum mechanical driven, 1H iterative Full Spin Analysis (HiFSA). The method is illustrated with four the four main flavonolignans von Silybum marianum ([iso]silybins A/B) which have posed an analytical challenge for nearly six decades. The highly reproducible HiFSA 1H NMR fingerprints allow distinction of the near-identical our isomers at 1H frequencies from 300 to 900 MHz, as well as their parallel quantification, even in difficult to characterize mixtures. The methodology opens new opportunities to explore hidden diversity in the chemical space of organic molecules.
Part 19 - Residual Complexity of Bioactive Natural Products
Gödecke T, Napolitano JG, Rodriguez Brasco MF, Chen SN, Jaki BU, Lankin D, Pauli GF
Validation of a Generic qHNMR Method for Natural Products Analysis
Phytochemical Analysis 24: 581-597 (2013)
Brief Synposis Regarding Residual Complexity and Bioactivity: Introduces a validated protocol for quantitative 1H NMR (qHNMR) analysis of complex samples with relatively high dynamic range. Comprehensive coverage of acquisition and processing parameters as well as software, and specific attention to the requirements of natural products makes the protocol suitable for a wide range of analytes and applications, from the quality control of highly complex crude botanical extracts to the assessment of the purity and residual complexity of reference compounds.
Part 18 - Residual Complexity of Bioactive Natural Products
Simmler C, Hajirahimkhan A; Lankin DC, Bolton J, Jones T,
Soejarto DD; Chen SN, Pauli GF
Dynamic Residual Complexity of the Isoliquiritigenin-Liquiritigenin Interconversion During
Journal of Agricultural and Food Chemistry 61: 2146–2157 (2013)
Brief Synposis Regarding Residual Complexity and Bioactivity: Demonstrates the relevance of the dynamic residual complexity of chalcones and flavanones under in vitro bioassay conditions by time-resolved investigation of the isomerization of the chalcone, isoliquiritigenin, and the flavanone, liquiritigenin in cell culture media, with and without cells. Isomerization occurs and leads to equilibrium mixtures with enantiomeric excess of the R-flavanone. The findings underscore the importance of choice and definition of in situ conditions in biological test systems as they affect chemical reactivity and, thus, residual complexity of the test agents.
Part 17 - Residual Complexity of Bioactive Natural Products
Qiu F, Cai G, Jaki BU, Lankin DC, Franzblau SG, Pauli GF
Quantitative Purity-Activity Relationships of Natural Products: The Case of Anti-Tuberculosis Active Triterpenes from Oplopanax horridus
Journal of Natural Products 76: 413-419 (2013)
Brief Synposis Regarding Residual Complexity and Bioactivity: Extends the previously developed concept of purity‒activity relationships (PARs) for the quantitative evaluation of the effects of multiple minor components on the bioactivity of residually complex natural products. The anti-tuberculosis active triterpenes were selected as a case for the development of the quantitative PAR (QPAR) concept. The residual complexity of the purified triterpenes from the Alaskan ethnobotanical, Oplopanax horridus, studied by 1D- and 2D-NMR and identified as a combination of structurally related and unrelated impurities. Using biochemometric methodology, the qHNMR purity and anti-TB activity of successive chromatographic fractions of O. horridus triterpenes were correlated by linear regression analysis to generate a mathematical QPAR model. The QPAR concept enables a quantitative assessment when residually complexity affects the biological activity of natural products.
Part 16 - Residual Complexity of Bioactive Natural Products
Dong S, Nikolic D, Simmler C, Qiu F, van Breemen R, Soejarto D,
Pauli GF, Chen SN
Diarylheptanoids from Dioscorea villosa (Wild Yam)
Journal of Natural Products 75: 2168-2177 (2012)
Brief Synposis Regarding Residual Complexity and Bioactivity: Establishes a link between the ubiquitous RC of crude metabolomes of Dioscorea villosa and the approach of qHNMR-guided metabolomic mining of the minor diarylheptanoid constituents. These findings not only extend the utility of qHNMR applications, but also complement previous conclusions about the relevance of RC. In addition, the establishment of HiFSA profiles of the isolates lays the groundwork for the quantifiable assessment of the RC of Dioscorea preparations.
Part 15 - Residual Complexity of Bioactive Natural Products
Inui T, Wang Y, Pro S, Franzblau SG, Pauli GF
Unbiased Evaluation of Bioactive Secondary Metabolites in Complex Matrices
Fitoterapia 83: 1218-1225 (2012)
Brief Synposis Regarding Residual Complexity and Bioactivity:
Establishes biochemometric methodology capable of identifying bioactive principles in crude metabolomic mixtures, as an alternative to bioassay-guided fractionation; establishes chemometric links between the bioassay and the preparative and analytical chemistry of (residually) complex natural products; exemplifies the concept for the anti-TB active principles of the ethnobotanical, Oplopanax horridus.
Part 14 - Residual Complexity of Bioactive Natural Products
Riihinen K, Gödecke T, Pauli GF
Journal of Chromatography A, 1244: 20-27 (2012)
Brief Synposis Regarding Residual Complexity and Bioactivity:
Establishes long-bed gel permeation chromatography (GPC) on Sephadex LH-20 as an efficient method for the purification of bioactive berry polyphenols; despite its capability to resolve closely related compounds, qHNMR analysis reveals an un expected degree of RC in the GPC fractions.
Part 13 - Residual Complexity of Bioactive Natural Products
Napolitano J, Lankin D, Chen SN, Pauli GF
Complete 1H NMR Spectral Analysis of Ten Chemical Markers of Ginkgo biloba
Magnetic Resonances in Chemistry 50: 569-575 (2012)
Brief Synposis Regarding Residual Complexity and Bioactivity: Establishes the methodology for the generation of unambiguous 1H NMR fingerprints of bioactive markers, exemplified for terpene lactones and flavonoids from Ginkgo biloba; the fingerprints are suitable for both structural dereplication and qHNMR quantitation at various levels of RC, can be scaled to all existing NMR field strength and are independent of instrumentation.
Part 12 - Residual Complexity of Bioactive Natural Products
Pauli GF, Chen SN, Friesen JB, McAlpine J, Jaki BU
Analysis and Purification of Bioactive Natural Products - The AnaPurNa Study
Journal of Natural Products, 75: 1243-1255 (2012)
dx.doi.org/10.1021/np300066q [Open Access Article - ACS Authors Choice]
Brief Synposis Regarding Residual Complexity and Bioactivity: Comprehensive meta-analysis of the literature (1999-2010) focusing on the role of analytical methodology in the purification and characterization of bioactive compounds from natural sources; addresses the role of RC in their purification and characterization and discusses the impact of RC on the biological evaluation and validation of lead compounds.
Part 11 - Residual Complexity of Bioactive Natural Products
Qiu F, Friesen JB, McAlpine JB, Pauli GF
Journal of Chromatography A 1242: 26-34 (2012)
Brief Synposis Regarding Residual Complexity and Bioactivity: Introduces the use of qHNMR for both the design and the analysis of countercurrent separation (CS) of bioactive botanical markers; demonstrates the measurement of partition coefficients of target markers in mixtures; performs the evaluation of chromatographic orthogonality in CS; establishes quantitative links between predicted and measured chromatographic CS performance and the RC of the purified markers.
Part 10 - Residual Complexity of Bioactive Natural Products
Napolitano J, Gödecke T, Rodriguez Brasco MF, Jaki BU, Chen SN, Lankin DC, Pauli GF
Journal of Natural Products 75: 238-248 (2012)
NEW LINK: Research Topic Page for this paper
Brief Synposis Regarding Residual Complexity and Bioactivity: Establishes 1H iterative full spin analysis (HiFSA)as the basis of a qHNMR approach for the parallel quantitation of eight bioactive markers in Ginkgo biloba; exemplifies how multi-target standardization can be achieved without the need for identical calibrants in (residually) complex samples including reference materials, fractions, and extracts; addresses the role of RC in reference materials of calibrants.
Part 9 - Residual Complexity of Bioactive Natural Products
Qiu F, Imai A, McAlpine J, Lankin D, Burton I, Karakach T, Farnsworth N, Chen SN, Pauli GF
Dereplication, Residual Complexity and Rational Naming - the Case of the Actaea Triterpenes
Journal of Natural Products 75: 432-443 (2012)
NEW LINK: Research Topic Page for this paper
Brief Synposis Regarding Residual Complexity and Bioactivity: Determines the RC of purified botanical reference materials of triterpenes from black cohosh; demonstrates the assessment of RC by computer-aided dereplication using classification binary trees (CBTs) to derive both structural information and quantitative measures for minor components contained in residually complex samples.
Part 8 - Residual Complexity of Bioactive Natural Products
Goedecke T, Napolitano J, Yao P, Nikolic D, Dietz B, Bolton J, van Breemen R, Chen SN, Lankin D, Farnsworth N, Pauli GF
Integrated standardization concept for Angelica botanicals using quantitative NMR
Fitoterapia 83: 18-32 (2012)
Brief Synposis Regarding Residual Complexity and Bioactivity: Establishes a qHNMR-based protocol for the simultaneous quantitation of multiple marker compounds in the bioactive fraction ([anti-]estrogenicity, cytotoxicity) of Angelica sinensis botanicals; demonstrates the advanced role qHNMR can have in botanical standardization and evaluation of RC of the plant extracts.
Part 7- Residual Complexity of Bioactive Natural Products
Molina-Salinas G, Rivas-Galindo V, Said-Fernández S, Lankin D, Muñoz M, Joseph-Nathan P, Pauli GF*, Waksman N* [*corresponding authors]
Stereochemical Analysis of Leubethanol, an Anti-TB Active Serrulatane, from Leucophyllum frutescens
Journal of Natural Products 74: 1842-1850 (2011)
Brief Synposis Regarding Residual Complexity and Bioactivity: Establishes the subtle but significant diastereomeric difference between elisabethanol, which had been isolated from a gorgonian organism, and leubethanol, the anti-TB active lead compound isolated from a plant; utilizes VCD for the determination of absolute stereochemistry and emphasizes 1H iterative full spin analysis (HiFSA) as a dereplication tool and for the analysis of RC of natural products.
Part 6 - Residual Complexity of Bioactive Natural Products
Pauli GF, Friesen B, Goedecke T, Farnsworth N, Glodny B
Occurrence of Progesterone and Related Animal Steroids in Two Higher Plants
Journal of Natural Products 73: 338-345 (2010)
[Featured in C&E News, Feb 2010: "Progesterone Proof"]
Brief Synposis Regarding Residual Complexity and Bioactivity: Unambiguously demonstrates the unexpected occurrence of the mammalian steroid, progesterone, in higher plants and shows that small amounts of this hormone as well as mammalian-like steroid metabolites (e.g., 3-O-sulfates) can form a small but integral part of the RC of plant metabolomes.
Part 5 - Residual Complexity of Bioactive Natural Products
Chen SN, Lankin D, Chadwick L, Jaki B, Pauli GF
Dynamic Residual Complexity of Natural Products by qHNMR: Solution Stability of Desmethylxanthohumol
Planta Medica 75: 757-762 (2009)
Brief Synposis Regarding Residual Complexity and Bioactivity: Exemplifies how the dynamic form of RC can lead to the generation of a highly potent phytoestrogen (8-PN) from the inactive precursor (DMX); institutes qHNMR methodology to assess RC in a time-resolved fashion, enabling correlation with bioassay outcome.
Part 4 - Residual Complexity of Bioactive Natural Products
Gödecke T, Chen SN, Lankin D, Nikolic D, van Breemen R, Pauli GF
Phytochemistry of Cimicifugic Acids and Associated Bases in Cimicifuga racemosa Root Extracts
Phytochemical Analysis 20: 120-131 (2009)
Brief Synposis Regarding Residual Complexity and Bioactivity: Establishes the new phytochemical methodology that leads to the LC-MS-driven discovery of trace amounts of N-Methyl-serotonin as serotonergic active principle in black cohosh; demonstrates the relevance of low-abundance constituents (RC) as potential bioactive markers for metabolomic mixtures such a botanical extracts.
Part 3 - Residual Complexity of Bioactive Natural Products
Schinkovitz A, Pro S, Main M, Chen SN, Jaki BU, Lankin DC, Pauli GF
The Dynamic Nature of the Ligustilide Complex
Journal of Natural Products 71: 1606-1611 (2008)
Brief Synposis Regarding Residual Complexity and Bioactivity: Shows how RC is generated and varies in purified samples of ligustilide, a designated bioactive marker present in Angelica and Ligusticum species; compares analytical methods suitable to assess dynamic RC.
Part 2 - Residual Complexity of Bioactive Natural Products
Chen S, Turner A, Jaki B, Nikolic D, van Breemen R, Friesen B, Pauli GF
An Experimental Implementation of Chemical Subtraction
Journal of Pharmaceutical and Biomedical Analysis 46: 692-698 (2008)
Brief Synposis Regarding Residual Complexity and Bioactivity: Performs selective removal of a single, interfering phytoconstituent from a bioactive (E. coli anti-adherence) fraction and demonstrates the presence of RC in the removed (“subtracted”) compound and its assessment by qNMR and MS methods.
Part 1 - Residual Complexity of Bioactive Natural Products
Jaki BU, Franzblau SG, Chadwick LR, Lankin DC, Zhang F, Wang Y, Pauli GF
Purity Bioactivity Relationships – The Case of Anti-TB Active Ursolic Acid
Journal of Natural Products 71: 1742-1748 (2008)
Brief Synposis Regarding Residual Complexity and Bioactivity: Demonstrates the relationship between purity, RC and anti-TB activity of different batches of a natural product; uses qHNMR methodology to establish quantitative relationships between purity/RC and activity.