Chapter 1: Natural Product Chemistry: To See the World in a Single Leaf

Chemistry, like most disciplines, has both a particular way of thinking and a language and syntax that can exclude the non-specialist. While technical language can be very useful as a shorthand way of explaining ideas and concepts, it can also be part of a strategy designed to avoid close scrutiny of ideas and concepts. A casual glance at organic chemistry textbooks reveals a whole set of phrases that are only illuminating for those who understand them. After all, how many people actually know what an Arbuzov reaction is or, as will be mentioned later at various stages in this book, the Lossen rearrangement? As chemists, we cheerfully talk about the Pictet–Spengler reaction as a type of Mannich reaction and of Grignard reagents and Maillard reactions. In many cases, rather than illuminating ideas, the language of chemistry can be as arcane as that of an ancient ritual. The scientist and author C P Snow, in the Rede lecture he gave in 1959, gave voice to the opinion that “the intellectual life of the whole of western society is increasingly being split into two polar groups”. Snow labelled these groups as ‘The Two Cultures’ and complained that the chasm that had opened up between scientists and non-scientists was to the detriment of both. He took non-scientists to task for their assumption that culture was exclusive to their genre. Indeed Snow observed that “A good many times I have been present at gatherings of people who, by the standards of the traditional culture, are thought highly educated and who have with considerable gusto been expressing their incredulity at the illiteracy of scientists. Once or twice I have been provoked and have asked the company how many of them could describe the second law of thermodynamics. The response was cold: it was also negative. Yet I was asking something which is about the scientific equivalent of Have you read a work of Shakespeare’s?” While Snow’s overall point is valid, the use of very specialised language by scientists does not help them communicate either with the general population or with their intellectual peer group in other disciplines. There is no doubt that the second law of thermodynamics is profound, and most definitions require also an understanding of what is meant by entropy and the nature of energy in open and closed systems. Scientists tend to be somewhat dismissive of attempts to describe their fundamental rules in descriptive terms and this can be a barrier to understanding for non-specialists.

The Two Cultures described by Snow have continued to diverge. Indeed, the divisions caused by specialisation even within science have grown increasingly profound. Many scientists are obsessed with the ‘new’. They read the latest research results in increasingly narrowing fields and these results are increasingly only comprehendible to ever narrower groups of specialists. This type of behaviour is inherent in the philosophy of chemistry which has deep roots in empiricism and in reductionism and the consequences of this are clearly visible in most branches of chemistry.

In the case of natural product chemistry, a strong driver is the need to isolate, characterise and understand the chemical compounds produced by nature. Once this part is complete, the chemist then assigns a name (based on internationally established rules) to the substance which is incomprehensible to anyone not trained in chemistry. The development of complex and elegant chromatographic techniques provides a fundamental underpinning for this endeavour but these are also obscured by language and specialisation. The increasing sophistication of spectroscopic methods (particularly nuclear magnetic resonance spectroscopy) has revealed the structures of many (if not most) natural molecules but again only to those who know how to ‘read’ the spectra. There are still some intractable problems (for example, distinguishing individual components of the mixtures of truxillic and truxinic acids which are the subject of a chapter in this book), but natural products are far less of a structural and isolative challenge than they were during the 19th and early part of the 20th centuries – even if the knowledge about them is only available to a small number of people. Following from the identification of individual natural products was the realisation that they could be grouped into families by considering structural similarity or biogenesis, or both. The way compounds are formed in nature, the reactions that are catalysed by enzymes and the interrelationships between the pathways employed (and their manipulation) were the basis of incredible advances in biosynthetic chemistry.

The starting point for natural product chemistry is the elucidation of natural products themselves and how (and why) they are formed by their host organisms. A second aspect (of even greater complexity) is how natural products function in vivo. What is their purpose and why are they formed? Is the function of natural products different to the world at large compared to the species that creates them? Their primary function is often to prevent the plant or other organisms from being consumed. This creates a survival advantage for the species that creates them. They can also provide a useful store of compounds that can be metabolised to provide essential nutrients. Understanding how natural products interact with non-host species is hugely challenging. In many cases, they operate as active agents at more than one target within a species and, while our use for them may be highly specialised (as is the case for pharmaceuticals), the complexity in the biological profile of natural products (singly or in groups) continues to be a challenge to understanding structure and function relationships.

This book is not designed to be either an exhaustive text of natural products or even a fresh insight into techniques used to study natural products themselves and their uses. Instead, the book is a reflection of many years of investigation into natural products from different origins and which were studied for different reasons. The subject range is broad and it covers, inter alia, a range of techniques and challenges. The key themes are the study of natural products themselves and how they interact with other living systems. Chemistry is used as a starting point to discuss the interactions of chemistry and chemists with the world at large. Each chapter begins with a reference to a published paper. This provides the intellectual and scientific anchoring for the chapter. The chapter continues to describe the way the published work illustrates particular aspects of chemistry – but not in deep detail. For an analysis of the detailed chemistry, the reader can consult the papers that are referenced. The book begins with a reductionist starting point (a natural product) and, in successive chapters, this is investigated through its interactions with increasingly complex living systems. During this progression, a purveyor of a reductionist approach (a natural product chemist) is studied in their interactions with and relevance to the world at large. The philosophical basis for natural product chemistry is examined since this underpins the approaches that chemists adopt. It also helps to place chemists and chemistry in context.

Just as William Blake sought to see the world in a grain of sand, so the natural product chemist sees the natural world in a grain of pollen or in a leaf from a plant. The chemist seeks to examine the world and to illuminate all that is around and how we interact with it through this microcosm.

The progression is illustrated by a brief consideration of each of the chapters of the book. Chapter 2 begins by placing chemistry in the context of other scientific disciplines and this leads into a discussion of the importance of scale when considering how to investigate systems. The importance of equilibrium states and the failure of homeostasis to accurately represent living systems progresses to a consideration of reductionism as a tool for chemists. The isolation of glucosinolates is used as an example of an approach to understanding the role of a specific natural product and the discussion is then widened to consider other natural products including alkaloids and penicillins. Activity-led isolation is introduced as is the debate between synthesis and isolation of natural products. Finally, the use of analogy, empiricism and extrapolation to deduce structures of classes of natural products is discussed using ring-substituted indole glucosinolates as a specific example.

Having examined the reductionist isolation of natural products, a second approach is outlined in Chapter 3. The chapter opens with a discussion of biological activity since this has traditionally been the driving force for many studies of natural products. The use of animal and other model systems is briefly considered in the context of both biological activity and bioavailability. The use of an animal model (rodent) to assess one aspect of the biological activity of glucosinolates is considered. This leads to the concept of selective toxicity and the relevance of homeostasis and the ability to resist perturbation as an important aspect of health. Finally, the development of receptor-driven technologies in the isolation and study of natural products is discussed and the challenges of determining chronic rather than acute toxicity are reviewed.

One of the key enablers of natural product chemistry is the presence and use of enzymes. Enzyme reactions are important because they work under physiological conditions. The reductionist approach to measuring enzyme activity involves isolation and then assay under what is presumed to be physiological conditions – particularly at a pH of about 7.4, at room temperature and with optimal amounts of substrate present. Enzymes in a cell do not function under such conditions. Chapter 4 considers the use of enzymes under non-traditional conditions which may, nonetheless, have some degree of biological relevance. The examples used are the hydrolysis of DNA with restriction endonucleases and the hydrolysis of glucosinolates by the thioglucosidase enzyme myrosinase. Both reactions are investigated in ‘low water’ environments. While reverse hydrolytic reactions have been demonstrated to take place using, for example, proteases, there is no general rule that can be used to predict the outcome of such manipulations. Part of the rationale is bound up with the concept of entropy and its effect on enzyme reactions and this is discussed. One of the major uses of enzymes in living systems is to catalyse reactions leading to the biosynthesis of natural products. This is discussed in the context of reversibility but also, in a wider sense, expectation and free will. Finally, some aspects of the future study of natural products are discussed. This is examined in the context of knowledge and experience but also the difference between correlation and causality. A deepening knowledge of not just what natural products exist but why they occur and how they are linked to living systems where they exist enables the knowledge base to move increasingly beyond correlation and towards causality.

One of the challenges of natural product chemistry is the complexity of the biological matrix in which the compounds exist and discerning the role of a specific compound (or a set of compounds) in vivo. In some cases, it is not possible to study the compounds in situ and alternative approaches must be adopted. One example of this is the study of plant cell walls and, in particular, the phenolic components of the cell wall. The process of lignification is the oxidation (by peroxidases) followed by polymerisation of plant phenolic compounds. The traditional way of studying these complex natural products is to hydrolyse them and then extract some fragments that can be analysed, identified and characterised. The role of the various fragments and how they combine in vivo can then be hypothesised. Once the fragments have been characterised and the relationship between them has been hypothesised, their biogenesis can be considered. Such speculation often includes extrapolation from known systems and this is the subject of much of Chapter 5. In this chapter, putative starting materials for a series of cyclobutanes (truxillic and truxinic acids) that occur in plant cell walls are suggested and the conditions under which they might be formed are hypothesised and then tested using model compounds. In this way, it is possible to probe the most likely mechanism of formation. Such studies lead to a consideration of evolution and morphology since the mechanism of formation suggests a particular function for lignin in the plant cell wall. Natural product chemistry, like many other areas of science, has become increasingly specialised; however, the occurrence and formation of lignin and other phenolic compounds in plant cell walls cannot easily be explained in terms of traditional paradigms in which natural products are produced as a result of a specific, enzyme-catalysed biosynthetic pathway. Maintaining intellectual (and mechanistic) flexibility is an important aspect of trying to understand natural products and their function in complex biological matrices.

The importance of natural products has traditionally been closely linked to their biological activity and, hence, their interaction with living systems. Some natural products are inherently biologically active, while others require to be activated in order to exhibit their full biological potency. The activation of natural products by mammals occurs upon ingestion and most often by the phase 1 enzyme systems that are responsible for the insertion of a moiety that will enable the natural product to be later conjugated and excreted. The most common enzyme systems used are cytochrome P450s (commonly abbreviated to CYPs). This activation adds a further level of complexity to the interaction of natural products with living systems. Not all CYP systems are the same (and not all are ‘switched on’ all the time – some are inducible). One of the most extensively studied groups of compounds are the alkaloids. The impetus for the substantial interest in them was a combination of the relative ease of isolation (and their abundance) and their biological properties. Often those properties only became apparent after the molecules were activated by the CYP system. One group of the class, pyrrolizidine alkaloids, is the subject of Chapter 6. Pyrrolizidine alkaloids have limited therapeutic potential and the major reason for the interest in them is due to their toxicity. Members of the class are widely distributed and are responsible for poisoning of livestock. They are also suspected carcinogens and mutagens. In order to fulfil their toxic potential, pyrrolizidine alkaloids have to be activated by a specific member of the CYP class of enzymes. The activated compound is electrophilic and will readily bind to DNA inducing a mutation. One of the simplest ways of determining mutagenicity is the Ames test which relies upon a mutation which enables a bacterium to synthesise histidine (an essential amino acid). If a compound causes that mutation, then the bacterium will grow in the absence of added histidine since it can synthesise it. This is, therefore, a rapid way of screening for mutagens. Unfortunately, the test fails to pick up some known mutagens and can also produce false positive results so it is not 100% predictive. Finally, bacteria do not contain the CYP enzymes so these must be introduced into the test system in some way. There was considerable interest in the biological relevance of genes that limit the growth of tumours. One of the most celebrated of these is the tumour suppressor gene called p53. The presence of some specific mutations in the p53 gene appears to be linked to tumour growth and, in Chapter 6, this property of the gene is used as an assay for the mutagenicity (and potential carcinogenicity) of pyrrolizidine alkaloids. In the system developed, the presence of a biologically relevant and specific mutation in the p53 gene is used as a basis for determining if the alkaloid has the potential to initiate one step in the tumorigenesis process. Mutational analysis and the carcinogenic process are very complex and there is a tendency for large amounts of data to be generated. Not all of these data are hypothesis driven and the establishment of increasingly sophisticated data handling techniques has facilitated pattern recognition as a scientific approach rather than a deeper understanding of how complex living systems actually work. The development of ideas about living systems was in part impeded by the concept of vitalism and agency in nature. The former has been almost completely discredited and the latter seems to apply only in some very limited circumstances. A discussion of the philosophical basis for agency in nature (and by implication in natural product chemistry and how these species interact with living systems) provides the conclusion for Chapter 6.

The chemistry of natural products is driven, to varying degrees, by the nature of the interaction of the chemicals with living systems. Interaction with receptors gives one way of studying the impact of natural products with whole organisms. In many species, the interaction with bioactive compounds is not a simple linear relationship. Rather than, for example, a compound binding as an agonist (or an antagonist) to a receptor and that inducing a fixed physiological response, there are also instances where an interaction leads to a cascade of events subsequent to the initial interaction. One of the most significant of these is the allergic immune response. A second, non-linear challenge is the result of exposure to chronically rather than acutely bioactive natural products or exposure to compounds for extended periods of time at a level that is below the ‘No Observable Adverse Effect’ level. Both of these situations are examined in Chapter 7 in the context of developing valid and meaningful test systems that are able to give biological relevance to exposures. In the case of allergy, while there are animal models that are able to respond to human allergens – even to the extent of being able to respond to allergens through Th2 immune responses, IgE production and mast cell activation/expansion, these tests are expensive, require animal facilities and cannot reproduce complex human physiology. They are, therefore, neither clinically nor analytically relevant. At the other end of the scale, the measurement of binding to a specific IgE antibody has been used as a detection method for allergic epitopes. In this case, the problem is that antibody binding is insufficient to demonstrate clinical relevance since it does not predict absolutely the consequences (mast cell degranulation) and the production of the cascade of allergic response enablers. An alternative, which is discussed in Chapter 7, is to sensitise human tissue and then measure the release of allergic mediators upon subsequent exposure. This has the advantage that it is more clinically relevant than IgE binding alone and the test system is human rather than animal in origin. The major drawback to such systems is the availability of suitable human tissue samples. In general, human cells in secondary culture do not display a true allergic response so their use is severely limited and they are not a viable alternative to biopsy material; however, the use of co-cultures of epithelial cells and immune cells is becoming more common. The second challenge, which is relevant to a range of sub-acutely toxic natural products, is the measurement of the consequences of long-term, chronic exposure. Long-term animal studies are both expensive and of limited relevance to humans. Primary cell lines do not remain viable for long enough to be useful and while secondary cell lines are essentially immortal, they have to be subcultured (passaged) into fresh medium every 7–10 days. In essence, they are batch rather than continuous culture systems. One alternative to this – a flow cell bioreactor approach – is described in Chapter 7. The results, advantages and drawbacks of this approach are discussed. The chapter concludes with a discussion of complexity and extrapolation. An immune response is very complex and the development of a suitable, sensitive and clinically relevant model can be useful. Chronically bioactive natural products are temporally complex – what happens in the short term at a biologically measurable dose level may not be relevant to longer term effects. This progression from simple models to complex systems is a reflection of the nature of living systems. We cannot measure systems either in exquisite detail or in all of their complexity and therefore we strive to see the whole world (large and small) in Blake’s ‘grain of sand’.

The question of how to define natural products – or more accurately, the anomalies that arise – is part of the focus of Chapter 8. The question of derivation of natural products has been a source of debate. The dismissal of vitalism was key. If natural products, derived from living systems, did not contain any ‘essence’ that made them unique, then their definition had to rely upon other signifiers. The simplest approach is perhaps to define natural products as compounds that are both created by and isolable from living systems. Any subsequent alteration will remove the compounds from the ‘natural products’ group. The other part of the usual definition is that natural products should not be part of primary metabolism – they should not be essential for life. This means that essential vitamins and amino acids are not considered to be natural products. There is a problem associated with these definitions and an example will illustrate this. Mustard oils, such as those found in Brassicas, are not produced by living systems but arise from the enzymatic hydrolysis of parent glucosinolates, followed by a rearrangement reaction. The same applies to compounds such as allicin and the plethora of sulfur compounds produced by plants of the genus Allium. The fact that both the precursors and the enzymes that act upon them are true natural products suggests that the definition of natural products needs to be expanded to include compounds that are wholly derived from other natural products. The fact that a compound can be synthesised as well as obtained from living systems does not change its definition as a natural product. The focus is, therefore, upon the compounds themselves rather than on any process that is applied to them. By this definition, mustard oils are natural products but compounds such as amoxycillin which arise from chemical modification of the naturally produced core molecule 6-amino penicillanic acid (6-APA) are not. By this amended definition, acrylamide is a natural product since it arises from the reaction of one naturally produced molecule (asparagine) with another (a reducing sugar). Attempts to mitigate the production of acrylamide in foods can target the chemical precursors or the process by which the reaction occurs. These considerations are explored in Chapter 8 together with a discussion of the results of the processing of foods through techniques such as 3D printing and ultraprocessed foods (UPFs). At what stage (if any) does a process convert a natural product into something that is no longer a natural product? Is the idea that the definition of a natural product is something that is, directly or indirectly, wholly derived from substances produced by living entities actually valid?

The complexity of living systems and how natural products interact with these systems is a significant challenge. There is, however, a further level of complexity that is not too difficult to envision. Some complex organisms such as mammals have a number of components which have a specific role. Natural products may act upon specific parts of an organism (the liver or kidneys, for example) or may impact upon an aspect of primary metabolism that is common to all cells (the Krebs cycle, for example). In the case of unicellular organisms such as an individual species of bacteria, the impact of a given natural product will be the same on all members of that particular group. Bacteria and other microorganisms exist generally in communities of other species in a generally symbiotic relationship which is called a microbiome. One of the most important aspects of a microbiome is that it is a structured system. Any one species does not predominate and therefore grow to be pathogenic. Some members of a microbiome will produce extracellular polysaccharides which form an envelope (biofilm) for the community and protect it from external perturbations. This in turn will affect the flow of nutrients to all members of the microbiome so that even those that do not produce the polysaccharides will still have to adapt to the changing conditions. The nature of such a complex community in one particular example (the oral cavity) and the effect of natural products on it are the subject of Chapter 9. One of the ways that microbes control growth in a microbiome is through the use of quorum sensing molecules. These are small molecules whose production can stimulate the growth of certain bacteria. One class of these (called homoserine lactones) is effective for all Gram negative bacteria. Some other species exert control over Gram negative bacteria by producing an enzyme called lactonase that breaks down the homoserine lactones. Production of the enzyme can be stimulated by some natural products present in plants (phenolic compounds). In this way, natural products exert control over bacterial growth and help to maintain a healthy and balanced microbiome. Another level of control is exerted by a group of ultrasmall bacteria called Patescibacteria. This group is found to be closely associated with other bacteria because they lack the genes that enable synthesis of essential components of primary metabolism. They are able to stimulate the production of quorum sensing molecules and thus have a positive effect on the growth of the bacteria they use as hosts. Many of the functions of ultrasmall bacteria are unknown and, by analogy with the unknown parts of the universe, they have been described as ‘biological dark matter’. Another way in which natural products may facilitate the maintenance of a healthy microbiome is through the control of biofilm formation by Gram positive bacteria. A group of molecules with a particular structural characteristic (an exocyclic double bond) are able to control the attachment of polysaccharides to a surface (the first step in biofilm formation) by irreversibly inhibiting an enzyme (sortase A) which catalyses this reaction. Overall, Chapter 9 describes the interaction of natural products with microbial communities and extends the relevance of natural products to a further level of complexity of living systems.

The interest in natural products resides both in the molecules themselves (and how they are formed) and in their interactions with living systems. Unlike many very specialised areas of chemistry (and science more generally), natural product chemistry is a strongly poly-disciplinary activity. Not only are there a large number of different structures, they are formed by diverse (and divergent) routes and have a range of biological activities. A reductionist approach to chemistry produces highly specialised individuals with a high level of very specific expertise. In cellular terms, such individuals correspond to highly differentiated cells which have a defined function. Natural product chemistry calls for, at the very least, a pluripotent (if not totipotent) skill base and the ability to view systems in their entirety. Chapter 10 uses this as a starting point and builds upon the concept that natural products can be defined by their environment as well as by their properties. The example used is glucosinolates and their enzymatically-produced products, isothiocyanates. Understanding the chemistry of the formation and reactions of isothiocyanates suggests that they may have different properties under different sets of circumstances. They can be used for the elimination of halitosis, as anti-microbial agents via a novel mechanism and in the prevention of Alzheimer’s disease. All of these properties are linked by the types of reactions that the compounds themselves are able to carry out, but each is a consequence of a different aspect of the class of compounds and the environment in which they operate. The chapter concludes with a consideration of why natural products exist and the difference between primary and secondary metabolism in living systems.

The relevance and importance of natural products in terms of their formation and reactions, their interactions with living systems and how they change according to the environment are examined over eleven chapters. In addition, the techniques described provide a progression in chemistry over decades. The context of natural products and the development of scientific thought are also subjected to analysis. Finally, a number of areas of study are suggested and these help to demonstrate how natural product chemistry is aligned with wider society and with living systems – both individually and collectively.