Chapter 1
Natural Products Analysis: Instrumentation, Methods, and Applications

Vladimír Havlíček and Jaroslav Spížek

Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

This book aims at highlighting the newest trends in analytical chemistry that have recently been, or soon will be, employed in the analysis of natural products and their complex mixtures. All contributing authors were motivated to stress the innovative aspects in emerging natural product chemistries and were asked to formulate their own personal visions clearly indicating which milestones can be achieved in their fields of expertise in a five-year frame. The book is structured according to analytical instrumental approaches used either routinely or experimentally for structure characterization and/or determination of both low- and high-molecular-weight natural products.

1.1 BOOK MOTIVATION

This book enumerates the most recent and cutting-edge analytical approaches including those that have not yet been commercialized into the rejuvenated natural products field. For example, less-traditional applications of synchrotron irradiation to small molecules are reported when referring to standard X-ray diffraction. Likewise, examples of the newest hyphenation techniques with impact on screening and secondary metabolism studies are described in cases in which well-known multidimensional NMR spectroscopy is discussed.

The revitalization of the natural product field is documented by an increase in the number of peer-reviewed articles illustrated by a Web of Science search (Figure 1.1). The number of hits is seen to have increased threefold if the term “natural product activity” is evaluated. Antibacterial, antifungal, antineoplastic, anti-inflammatory, and other activities are also reported in patent literature. SciFinder returned constant data for the 2007–2013 period oscillating between 60 and 80 patent applications published annually. Diverse applications of natural products are also subjects of many review articles and book chapters. Interestingly, no monograph focused on instrumentation used for identification of natural products has been published in the past decade. This market gap was identified by Wiley senior editor Jonathan T. Rose: “In my opinion, given that plants and natural products are major sources for current and potential drugs, there is need for a book geared to researchers and professionals to facilitate natural product analysis, synthesis, and drug discovery. This type of book could explain the basics of natural products as pharmaceuticals, analytical tools and techniques, methods for isolation and elucidation, and applications for library design and in drug discovery. Such a book will find a welcome audience in organic and medicinal chemists, biochemists, analytical and medicinal chemists, microbiologists, and biomedical researchers.”

Figure 1.1 Report of published items accessed from the Web of Knowledge (Thomson Reuters) on December 31, 2013 illustrates the number of papers published annually in the field of “Natural Product Structure.”

In this book the instrumentation represents the common denominator. The contributors were motivated to make a very brief introduction to physicochemical principles of their methods and give an up-to-date overview of the most important applications relevant to natural products. In a limited number of chapters the tutorial part was extended, giving the reader the opportunity to get acquainted with both the fundamentals and future trends in one place. Personal views and mutual instrumental evaluations will help the newcomers to find a suitable technique. For instance, whereas nuclear magnetic resonance spectroscopy is nonselective and less sensitive (“always tells the truth”), mass spectrometry is selectively sensitive (“tells you what you want to hear”).

1.2 THE BROAD FIELD OF NATURAL PRODUCTS

Chapters 2–4 represent medically oriented introductory chapters. Chapter 2 focuses specifically on fungi and malaria and defines the current microbiology challenges in the field of natural product discovery. These two application areas were deliberately selected because they are rather underestimated in the review literature. The importance of tackling antimicrobial resistance and the application of standardized combination therapies is stressed. Drug degradation products arising from enzyme-specific reactions, drug target reprogramming, or ejecting the drug out of the bacterial or fungal cells belong to known mechanisms by which microbes fight against antimicrobial drugs. In the field of drug resistance, cultivation of microorganisms in drug-containing stable isotope-labeled media are particularly promising. Mass spectrometry (MS) is then used for the determination of natural isotope shift reflecting the viability of the microorganism and its ability to consume and metabolize the labeled nutrients. The potential and limitations of NextGen or NextNext sequencing methods are briefly described in the perspective section in Chapter 3. The importance of peptidogenomic methods for the determination of virulence mechanisms of pathogens is accentuated by means of imaging mass spectrometry in Chapter 10.

The introductory segment of this book is terminated by Chapter 4, in which the major fractionation and isolation procedures of natural products are briefly outlined. Major attention is dedicated to the respective biological activities of natural products. The chapter is subdivided according to plant and marine origin of most important metabolites that have found significant medical applications. The authors faced a difficult task to select the clinically most important active principles, of both marine and plant origin, and align their pharmacokinetic and biological properties with medical applications. Attention was paid to organic compounds in different phases of biological trials. Most important applications of natural compounds in cardiovascular, infectious, cancer and other areas are summarized.

1.3 DISCOVERY PHASES

Recent applications of metabolomics, proteomics, mutagenomics, and genomics in exploiting bacterial natural products are summarized in Chapter 5. In mass spectrometry-based metabolomics, the problem of silent or cryptic NP biosynthesis pathways (the “silent parvome”) is discussed in the context of the quest for novel chemistries. Mass spectral alignment strategies are outlined (XCMS, MZMine, commercial products) and supported by principal component analysis program packs (SIMCA, MATLAB, etc.), the importance of which is documented (for example) on strain prioritization. Two proteomic approaches in natural product discoveries are reviewed. The first is the Kelleher group proteomic investigation of secondary metabolism (PrISM) utilizing the phosphopantetheinyl ejection assay [1]. The second proteomic technique is represented by an Orthogonal Active Site Identification System (OASIS) [2]. In the (meta)genomic part, the amplicon sequencing, shotgun libraries/metagenomics, and single-cell genomics methods are outlined and supported by success stories. Genome annotation pipelines are provided (CloVR-microbe, AntiSMASH, NCBI, SMART). The importance of concerted application of density functional theory and 2D NMR spectroscopy for absolute structure determination in natural products is stressed in the final part of the chapter. The applications of residual dipolar couplings in nuclear magnetic resonance (NMR), circular dichroism, and classical chemistry are also emphasized and create the bridge to molecular tools.

Some of them are further structured in tutorial Chapter 6 referring to the applications of electronic and vibrational spectroscopies. Advances and challenges in optical molecular spectroscopy of biomolecules and natural products are supported by chiroptic methods and placed in the context with surface-enhanced techniques and surface plasmon resonance (SPR) sensing. NMR users and fans will appreciate Chapter 7, a substantial part of which is dedicated to sample preparation and handling. Attention is also paid to LC-NMR setup, with most common instrumental variants and practical recipes (on-flow, stop-flow, and the combination of solid-phase extraction and MS). Their properties in terms of sensitivity, sample concentration, and sample nature are discussed in detail. Similarly, both supervised and unsupervised methods of statistical data evaluation are reported. Differential analysis is addressed in specialized subchapters dedicated to statistical heterospectroscopy, statistical total correlation spectroscopy, and other methods. The reader can benefit from public databases of NMR spectra and web servers dedicated to NMR metabolomics. Covariance NMR data processing of TOCSY and NOESY spectra is described. Virtual NMR chromatography (including its 3D variant) is used for distinguishing signals coming from small or large molecules. Food adulteration, plant extract analysis, and tens of other NMR applications in metabolomics are presented.

1.4 ABSOLUTE STRUCTURE

Chapter 8 describes the general technique of X-ray diffraction including the single-crystal and powder methods, and it covers advances in the instrumentation currently in use. The central argument...