History of Natural Products and Phytochemistry

Natural Products and Phytochemistry

What is natural product chemistry and why should we be interested in studying it? The broadest definition of a natural product is anything that is produced by life, and includes biotic materials (e.g. wood, silk), bio-based materials (e.g. bioplastics, cornstarch), bodily fluids (e.g. milk, plant exudates), and other natural materials that were once found in living organisms (e.g. soil, coal). A more restrictive definition of a natural product is any organic compound that is synthesized by a living organism.  The science of phytochemistry, in fact, has its origins in the study of natural products, and has given rise to the fields of synthetic organic chemistry where scientists create organic molecules in the laboratory, and semi-synthetic organic chemistry where scientists modify existing natural products to improve or alter their activities.

Natural products have high structural diversity and unique pharmacological or biological activities due to the natural selection and evolutionary processes that have shaped their utility over hundreds of thousands of years.  In fact, the structural diversity of natural products far exceeds the capabilities of synthetic organic chemists within the laboratory. Thus, natural products have been utilized in both traditional and modern medicine for treating diseases. Currently, natural products are often used as starting points for drug discovery followed by synthetic modifications to help reduce side effects and increase bioavailabilty. In addition to medicine, natural products and their derivatives are commonly used as food additives in the form of spices and herbs, antibacterial agents, and antioxidants to protect food freshness and longevity. In fact, natural organic products find their way into almost every facet of our lives, from the clothes on our backs, to plastics and rubber products, health and beauty products, and even the energy we use to power our automobiles.

Natural products may be classified according to their biological function, biosynthetic pathway, or their source.

2. Natural Product Function

Natural products are often divided into two major classes: primary and secondary metabolites. Primary metabolites are organic molecules that have an intrinsic function that is essential to the survival of the organism that produces them (i.e. the organism would die without these metabolites). Examples of primary metabolites include the core building block molecules (nucleic acids, amino acids, sugars, and fatty acids) required to make the major macromolecules (DNA, RNA, proteins, carbohydrates, and lipids) responsible for sustaining life. Secondary metabolites in contrast are organic molecules that typically have an extrinsic function that mainly affects other organisms outside of the producer. Secondary metabolites are not essential to survival but do increase the competitiveness of the organism within its environment.

Natural products, especially within the field of organic chemistry, are often defined as primary and secondary metabolites. A more restrictive definition limiting natural products to secondary metabolites is commonly used within the fields of medicinal chemistry and pharmacognosy, the study and use of natural products in medicine.

3. Primary Metabolites

Primary metabolites are components of basic metabolic pathways that are required for life. They are associated with essential cellular functions such as nutrient assimilation, energy production, and growth/development. They have wide species distributions that span many phyla and frequently more than one kingdom. Primary metabolites include the building blocks required to make the four major macromolecules within the body: carbohydrates, lipids, proteins, and nucleic acids (DNA and RNA).

These are large polymers of the body that are built up from repeating smaller monomer units (Fig. 1.1). The monomer units for building the nucleic acids, DNA and RNA, are the nucleotide bases, whereas the monomers for proteins are amino acids, for carbohydrates are sugar residues, and for lipids are fatty acids or acetyl groups.

Figure 1.1:  The Molecular building blocks of life are made from organic compounds.

Primary metabolites that are involved with energy production include numerous enzymes that breakdown food molecules, such as carbohydrates and lipids, and capture the energy released in molecules of adenosine triphosphate (ATP). Enzymes are biological catalysts that speed up the rate of chemical reactions. Typically they are proteins, which are composed of amino acid building blocks.

The basic structures of cells and of organisms are also composed of primary metabolites. These include cell membranes (e.g. phospholipids), cell walls (e.g. peptidoglycan, chitin), and cytoskeletons (proteins). DNA and RNA which store and transmit genetic information are composed of nucleic acid primary metabolites. Primary metabolites also include molecules involved in cellular signaling, communication and transport.

4. Secondary Metabolites

Secondary metabolites, in contrast to primary metabolites are dispensable and not absolutely required for survival. Furthermore, secondary metabolites typically have a narrow species distribution. For example, the deadly nightshade, Atropa belladonna, produces toxic hallucinogenic compounds, like scopolamine, but other plant species do not have this capacity. To date hundreds of thousands of secondary metabolites have been discovered!

Secondary metabolites have a broad range of functions. These include pheromones that act as social signaling molecules with other individuals of the same species, other communication molecules that attract and activate symbiotic organisms, agents that solubilize and transport nutrients, known as siderophores, and competitive weapons (repellants, venoms, toxins etc.) that are used against competitors, prey, and predators. 

Secondary metabolites have a diversity of structures and include examples such as alkaloids, phenylpropanoids, polyketides and terpenoids, as shown in Fig. 1.2  Alkaloids are secondary metabolites that contain nitrogen as a component of their organic structure and can be divided into many subclasses of compounds. Nicotine, the addictive substance in tobacco is provided as an example alkaloid (Fig. 1.2). The Phenylpropanoids are a diverse family of organic compounds that are synthesized from the amino acids phenylalanine and tyrosine (phenylalanine is shown in Fig. 1.2). Cinnamic acid one of the volatile flavor molecules found in cinnamon is a phenylpropanoid. Polyketides are assembled from the building blocks of acetate and malonate to form large, complex structures.  Alflatoxin B1, shown below, is a polyketide structure produced by fungi from the Aspergillus genus. These types of molds commonly grow of stored food crops, such as corn and peanuts and contaminate them with aflatoxins. Aflatoxins damage DNA molecules and act as a carcinogen, or cancer causing agent. Food crops contaminated with aflatoxins have been linked with cases of liver cancer. Terpenoids are another large class of natural products that are constructed from 5-carbon monomer units called isoprene (Fig. 1.2). Natural rubber is a good example of a terpenoid-based structure.  It is assembled from multiple reapeating isoprene units (Fig. 1.2). As we explore organic structures in more detail in the next few chapters we will continue to evaluate examples from these diverse classes of metabolites and how they impact our lives.

Figure 1.2: Representative examples of each of the major classes of secondary metabolites

5. Where Do We Find Natural Products?

Natural products may be extracted from the cells, tissues, and secretions of microorganisms, plants and animals. A crude (unfractionated) extract from any one of these sources will contain a range of structurally diverse and often novel chemical compounds. Chemical diversity in nature is based on biological diversity, so researchers travel around the world obtaining samples to analyze and evaluate in drug discovery screens or bioassays. This effort to search for natural products is known as bioprospecting. Examples of biological sources used to find new natural products are described below.

5.1 Prokaryotic Organisms

A prokaryote is a unicellular organism that lacks a membrane-bound nucleus (karyon), mitochondria, or any other membrane-bound organelle. The word prokaryote comes from the Greek (pro) “before” and (karyon) “nut” or “kernel”. Prokaryotes can be divided into two domains, Archaea and Bacteria. In contrast, species with nuclei and organelles (Animals, Plants, Fungi and Protists) are placed in the domain Eukaryota.

Figure 1.3: Phylogenetic tree of life based on genetic sequencing of ribosomal RNA.

In the prokaryotes, all the intracellular water-soluble components (proteins, DNA and metabolites) are located together in the cytoplasm enclosed by the cell membrane, rather than in separate cellular compartments. Prokaryotes are also much smaller than eukaryotic cells.

5.1.1 Bacteria

Typically a few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep portions of  Earth’s crust. Bacteria also live in symbiotic and parasitic relationships with plants and animals. Most bacteria have not been characterised, and only about half of the bacterial phyla have species that can be grown in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology.  There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water. Bacteria are a prominent source of natural products.  Figure 1.4 shows a few examples of bacterial natural products that have had an impact on our society, including several antibiotics.

The serendipitous discovery and subsequent clinical success of penicillin prompted a large-scale search for other environmental microorganisms that might produce anti-infective natural products. Soil and water samples were collected from all over the world, leading to the discovery of streptomycin (derived from the bacterium, Streptomyces griseus), and the realization that bacteria, not just fungi, represent an important source of antibacterial natural products.  This, in turn, led to the development of an impressive arsenal of antibacterial and antifungal agents including amphotericin B, chloramphenicol, erythromycin, neomycin B, daptomycin and tetracycline (all from Streptomyces spp.), the polymyxins (from Paenibacillus polymyxa), and the rifamycins (from Amycolatopsis rifamycinica).

Figure 1.4: Bacteria isolated from soil are prolific producers of antibacterial compounds.

Although most of the drugs derived from bacteria are employed as anti-infectives, some have found use in other fields of medicine. Botulinum toxin (from Clostridium botulinum) and bleomycin (from Streptomyces verticillus) are two examples. Botulinum toxin is the neurotoxin responsible for botulism food poisoning (Fig. 1.5).  It is caused by the bacterium, Clostridium botulinum, which can grow in improperly sterilized canned meats and other preserved foods. The poisoning can be fatal depending on how much of the toxin is ingested.  It causes muscle weakness and paralysis.  This toxin is now used cosmetically to help reduce facial wrinkles. It is injected in small doses into areas such as the forehead to cause paralysis to the muscles that create wrinkles.  Also, the glycopeptide bleomycin is used for the treatment of several cancers including Hodgkin’s lymphoma, head and neck cancer, and testicular cancer. Newer trends in the field include the metabolic profiling and isolation of natural products from novel bacterial species present in underexplored environments. Examples include secondary metabolite discovery from symbionts or endophytes. Symbionts are organisms that live in close association with another, often larger, organism known as a host. Endophytes are non-harmful symbionts that are associated with plants for at least part of their life cycle. In addition, discovery of organisms from tropical environments, subterranean bacteria found deep underground via mining/drilling, and marine bacteria continue to add to the complexity of secondary metabolites discovered.

Figure 1.5: Botulinum toxin. (A) Diagram of botulinum toxin A. Consuming food products tainted with the neurotoxin produced by (B) the bacterium Clostridium botulinum, can cause paralysis and death.  Interestingly, the neurotoxin has been adapted for medicinal use to reduce epileptic seizures and for cosmetic use to reduce wrinkles and frown lines by paralyzing muscle tissue in the forehead. 

5.1.2 Archaea

The discovery of organisms now classified as Archaea is fairly recent in our history, dating back to 1977 by the researchers. Genetic sequencing was used to show that a separate branch of ancient prokaryotic organisms diverged at an early stage in the history of life on Earth (Fig. 1.3). Thus, Woese suggested dividing the prokaryotic organisms into two major categories, Bacteria and Archaea, based on these genetic differences.  It is noteworthy that many Archaea have adapted to life in extreme environments such as the polar regions, hot springs, acidic springs, alkaline springs, salt lakes, and the high pressure of deep ocean water. These Archaea species are known as extremophiles. 

For example, Pyrococcus furiosus is an extremophilic species of Archaea (Fig. 1.6). It can be classified as a hyperthermophile because it thrives best under extremely high temperatures—higher than those preferred of a thermophile. It is notable for having an optimum growth temperature of boiling water – 100°C (a temperature that would destroy most living organisms). Recently, an isolation of a thermostable enzyme from this species that can breakdown lactose, a disaccharide sugar found in milk (Fig. 1.6).  Early explorations of this enzyme show that it has optimal activity at 100^oC and that it is thermostable even at 110^oC (Fig. 1.6).

Figure 1.6: The Extremophile Pyrococcus furiosus. (A) Shows a computer recreation of P. furiosus.  (B) Shows the effects of temperature on the stability of the lactase enzyme, β-glucosidase.

5.2 Eukaryotic Organisms

Eukaryotic organisms include four major kingdoms: Protista, Fungi, Plantae, and Animalia (Fig. 1.7).  Fungi are heterotrophic, eukaryotic organisms, either single-celled or multicellular, that are primarily decomposers within the environment. Heterotrophs are organisms that cannot produce their own food. Plants are multicellular eukaryotic organisms that are autotrophic, or capable of producing their own food. Plants are also characterized by having true roots, stems and leaves. Animals are multicellular, eukaryotic organisms that are heterotrophic, and are characterized by being mobile at some point in their lifetime. The term Protista (or sometimes Protoctista) is still often used to describe all other eurkaryotic organisms that do not fit in the Fungi, Plantae, or Animalia kingdoms.  However, it is not an ideal grouping, as there are protists that are animal-like, plant-like and fungi-like grouped under one umbrella term. Many scientists prefer to reclassify the protist kingdom into sub-groupings of related organisms based on phylogenetic data, rather than use the older protist classification. In fact, the phylogenetic classification proposed by Carl Woese breaks Kingdom Protista into three major groups;  the ciliates, the flagellates, and the microsporidia (Fig 1.3). 

Figure 1.7: The major domains and kingdoms of life. 

5.2.1 Fungi

As mentioned above, Fungi are heterotrophic, eukaryotic organisms that are primarily decomposers within the environment.  They include single-celled organisms such as yeast and molds, and multicellular organisms that have fruiting bodies, such as mushrooms. Fungi produce a myriad of secondary natural products.  Some are very toxic and have spurred common names such as death cap, destroying angel, and fool’s mushroom.  Others have found great utility in medicine. For example, several anti-infective medications have been derived from fungi including the penicillins and the cephalosporins (antibacterial drugs from Penicilium chrysogenum and Cephalosporium acremonium respectively), and Griseofulvin (an antifungal drug from Penicilium griseofulvum) (Fig. 1.8, parts A-C).  Another medicinally useful fungal metabolite is Lovastatin (from Aspergillus terreus), which became a lead for the statins, a series of drugs commonly used to lower cholesterol levels (Fig. 1.8, part D). 

Figure 1.8:  Examples of fungal secondary metabolites.

Cyclosporin is another amazing example of a fungal metabolite with important medical implications. Cyclosporin is an alkaloid structure that is assembled from amino acid building blocks that forms a cyclic peptide structure (Fig. 1.9).  Its major biological activity is to suppress the immune response.  Thus, it is widely prescribed to patients following an organ transplant, to help reduce the chance of organ rejection. Cyclosporin was isolated in 1971 from the fungus Tolypocladium inflatum (Fig.1.9). After 12 years of laboratory investigations and clinical testing, it was approved by the FDA for use in 1983. It is on the WHO’s list of essential medicines, as one of the most effective and safe medicines needed in a health system. Of note, T. inflatum is the asexual, single-celled form of a fungus that can also take on a sexually-reproducing multicellular life-stage, where it is known as the fungi, Cordyceps subsessilis (Fig. 1.9).

Figure 1.9: Fungal production of cyclosporin.  (A) Multicellular life-stage of the fungus, known as Cordyceps subsessilis, (B) unicellular life-stage of the fungus, known as Tolypocladium inflatum. (C) Structure of cyclosporine.

5.2.2 Plants

Life forms that are classified in the plant kingdom are multicellular eukaryotic organisms that are autotrophic, or capable of producing their own food. They produce their own food through the process of photosynthesis, where they utilize light energy from the sun to convert carbon dioxide and water into simple sugars.  Oxygen is a by-product of this reaction.  Thus, plants are a major source of oxygen on the planet. It is estimated that there are approximately 250,000 to 300,000 different species of plants on the planet. In addition to producing oxygen and being utilized as a food source, plants are also a major source of complex and highly structurally diverse secondary metabolites. Clinically useful examples include the anticancer agents paclitaxel and vinblastine (from Taxux brevifolia and Catharanthus roseus respectively),        

The antimalarial agent artemisinin (from Artemisia annua), the opioid analgesic drug morphine (from Papaver somniferum), and gagalantamine (from Galanthus), used to treat Alzheimer’s disease (Fig. 1.10).

Figure 1.10: Examples of biologically active metabolites from plants.

5.2 3 Animals

Animals are multicellular, eukaryotic organisms of the kingdom Animalia. As described earlier, animals are heterotrophic organisms and are characterized by being mobile at some point in their lifetime.  Animals can be divided broadly into vertebrates and invertebrates. Animals also represent a source of bioactive natural products. In particular, venomous animals such as snakes, spiders, scorpions, caterpillars, bees, wasps, centipedes, ants, toads, and frogs have attracted much attention. This is because venom constituents (peptides, enzymes, nucleotides, lipids, biogenic amines etc.) often have very specific interactions with a macromolecular target in the body. 

For example, Chlorotoxin is a 36-amino acid peptide found in the venom of the deathstalker scorpion (Leiurus quinquestriatus) which blocks small-conductance chloride channels (Fig. 1.11). It uses this toxin to immobilize it’s prey.

Figure 1.11: Chlorotoxin from the deathstalker scorpion (Leiurus quinquestriatus).  A ribbon diagram of the chlorotoxin protein is shown on the right. 

Other novel drugs that have arisen from animal venoms include, teprotide, a peptide isolated from the venom of the Brazilian pit viper Bothrops jararaca (Fig. 1.12). Teprotide was found to have activity as an antihypertensive agent and provided an initial lead compound for the development of blood pressure lowering medications. It was not a good drug candidate on its own, due to the expense in isolating it and the lack of oral availability.  However the structure was used as a lead compound and many derivative structures were made to try and find smaller, more soluble, orally active compounds that had the same biological activity. This has resulted in the development of the currently prescribed antihypertensive agents, cilazapril and captopril (Fig. 1.12).

Figure 1.12: Teprotide and its synthetic derivatives cilazapril and captoprial.  Teprotide (B) is a toxin produced by the pit viper, Bothrops jararaca (A)., Antihypertensive drugs, Cilazapril (C) and Captopril (D).

In addition to the terrestrial animals described above, many marine animals have been examined for pharmacologically active natural products, with corals, sponges, tunicates, sea snails, and bryozoans yielding chemicals with interesting analgesic, antiviral, and anticancer activities. Two examples developed for clinical use include ɷ-conotoxin (from the marine snail Conus magus) and ecteinascidin 743 (from the tunicate Ecteinascidia turbinata) (Fig. 1.13). The former, ω-conotoxin, is used to relieve severe and chronic pain, while the latter, ecteinascidin 743 is used to treat cancer.

Figure 1.13: Medicines from the sea.  In the upper panel the marine snail, Conus magnus and its active metabolite, ω-conotoxin, are shown.  Note that ω-conotoxin is a protein.  Thus, its structure is much too large to show all the organic bonds.  Proteins are often depicted in ribbon diagrams to give you a sense of the 3-dimensional folding patterns. In the lower panel the tunicate Ecteinascidia turbinate and its metabolite, ecteinascidin 743 (ET-743) are shown.