Biosynthesis of Carbohydrates and Amino Acids

We have already seen that some of the basic building blocks used in the biosynthesis of natural products are amino acids such as phenylalanine, tyrosine, and others. These and other crucial construction materials such as the acyl group in acetyl-CoA are all ultimately derived from carbohydrates. In this chapter, we will present an abbreviated overview of the components of carbohydrate structure and metabolism sufficient for our purposes going forward, with a schematic flowchart showing how carbohydrates and amino acids are modified, combined, and branched off in various ways to yield the distinct set of biosynthetic pathways that will form the core of the remainder of the text. We will finish the chapter with a brief, general review of amino acid nomenclature and structure with emphasis on the key amino acids that will be used throughout the remainder of the text. We know that plants make glucose (C6H12O6) by photosynthesis using light, water (H2O), and carbon dioxide (CO2). Another way of looking at the formula for glucose is C6(H2O)6, that is, six carbon atoms and six water molecules. Thus, glucose was originally referred to as a hydrated form of carbon—a carbohydrate. But this is a very general term since there are many different types of carbohydrate compounds. One way to broadly classify carbohydrates is to identify them as either mono- (one), di- (two), oligo- (a few) or poly- (many) saccharides. For example, glucose (C6H12O6) cannot be broken down into simpler carbohydrates by simple hydrolysis, so it is classified as a monosaccharide, that is, a single, discrete carbohydrate compound. On the other hand, the carbohydrate sucrose (C12H22O11) is classified as a disaccharide since when it is subjected to aqueous hydrolysis, it yields two different monosaccharide carbohydrates, namely glucose (C6H12O6) and fructose (C6H12O6). Noting that glucose and fructose are different compounds but with the same molecular formula, they must be related to one another either as stereoisomers or as constitutional isomers, so further refinement of classification is needed. Structurally speaking, most monosaccharide carbohydrates are simply polyhydroxyaldehydes (aldoses) or polyhydroxyketones (ketoses) which can be further classified using a combination of aldo- or keto- prefixes along with suffixes such as triose, tetrose, pentose, or hexose to designate the number of carbon atoms.

Brief Organic Review

In addition to simple hydrocarbon structures (alkanes, alkenes, alkynes, and aromatic systems) and alkyl groups (methyl, ethyl, propyl, isopropyl, etc.), this text assumes a familiarity with the most common functional groups associated with organic chemical structures and their basic reactivity patterns. Table 1.1 summarizes the names and structures of some of the more important functional groups we will be dealing with throughout the remainder of the book. It is important to remember that functional groups containing O or N with nonbonding electrons have an affinity for both protic and Lewis acids and are important participators in H-bonding. Groups containing a carbonyl (C=O) function are especially important, as these bonds are strongly polarized (δ+C=Oδ–), the C atom being electron deficient and the O atom electron excessive; this strong polarization is mainly responsible for the familiar reactivity patterns associated with carbonyl compounds. Figure 1.1 depicts the standard classification of isomers in organic chemical structures. Recall that constitutional isomers are compounds with the same molecular formula but different atom connectivity, such as 1-butanol versus 2-butanol. Stereoisomers, on the other hand, are compounds with the same formula and the same atom connectivity, differing from one another only in the three-dimensional orientation of their atoms in space. These are divided into two groups: enantiomers and diastereomers. Enantiomers are nonsuperimposable mirror image molecules whose asymmetry is usually the result of a tetrahedral carbon atom with four different atoms or groups attached to it, as in the 2-butanol enantiomers. Such chiral molecules rotate the plane of polarized light either (+) or (−) and so are said to be optically active. Achiral molecules, such as 1-butanol, do not rotate the plane of polarized light and so are optically inactive. A standard formalism for representation of a chiral center is to use bond line drawings with two of the four atoms or groups lying in the plane of the paper, a third projecting outward (wedge bond), and the fourth projecting inward (dashed bond).

The Terpenoid Pathway: Products from Mevalonic Acid and Deoxyxylulose Phosphate

It was Otto Wallach (1847– 1931) who first coined the term “terpene” and made the observation that many plant-derived essential oils had chemical structures whose composition was based on multiples of a basic five-carbon unit. His work with turpentine and the organic products derived from it was consistent with earlier studies of natural rubber which had shown that its thermal decomposition released “isoprene” (2-methyl-1,3-butadiene) as the principal product. This led eventually to the formulation of the so-called biogenetic isoprene rule of Leopold Ruzicka (1887–1976) in 1953 which stated that “the carbon skeleton of the terpenes is composed of isoprene units linked in regular or irregular arrangement.” As it turns out, biosynthetic pathways to terpenes are found in nearly all organisms, producing a remarkable variety of different structural types, as we will soon see. In fact, something in excess of over 25,000 different terpenes with a wide variety of biological functions have been isolated from the plant kingdom over the years. Interestingly, while many terpenes are simple achiral compounds, others are chiral as can be seen in the case of α-pinene in Fig. 4.1. But unlike the naturally occurring L-amino acids and D-carbohydrates, different organisms may produce the same terpene product but in different enantiomeric forms. For example, limonene is formed by more than 300 plants, with the (+)-(R) enantiomer being the most widespread form as the major constituent of citrus peel essential oils (orange oil). As the most abundant of all terpenes, its pleasant citrus fragrance and flavor have led to its worldwide use in the food and fragrance industries and also as a botanical insecticide. A number of plants produce both enantiomers of limonene, while others produce only the (−)-(S)-enantiomer which possesses a strong pine smell reminiscent of turpentine. This obviously speaks to the chirality and enantioselectivity of our own olfactory receptor sites which can readily distinguish between the two enantiomers, thus signaling a different odor response in each case.

Organic Synthesis in the Laboratory

The German chemist Friedrich Wöhler is generally credited with the first laboratory synthesis of a known organic compound (urea) from inorganic materials. He accomplished this by the simple heating of an inorganic salt, ammonium cyanate (NH4OCN). “I must tell you,” he wrote to his mentor Jöns Jakob Berzelius in 1828, “that I can prepare urea without requiring a kidney of an animal, either man or dog.” While this report may seem relatively minor given the structural simplicity of urea, its impact was revolutionary. For the first time, the preparation and isolation of an organic compound had been achieved in the absence of the elemental “vital force” of living systems previously believed to be required for the construction of all such compounds. This milestone of 19th century organic chemistry was later followed by many others, including Kolbe’s synthesis of acetic acid in 1847 and Fischer’s synthesis of glucose in 1890. With the support of evolving methods for compound separation, purification, and spectroscopic analysis, rapid advances in the sophistication of organic synthesis followed throughout the 20th century, developing in tandem with an ever-deepening understanding of the underlying organic processes associated with living systems. While it is certainly true that syntheses of many structurally complex unnatural compounds of theoretical interest are also among the most remarkable achievements in synthetic strategy, tactical execution, and perseverance, the realm of natural products remains the dominant source for the most challenging and potentially beneficial targets available for such synthetic efforts. Figure 8.1 shows a small selection of some natural (and unnatural) products which have been produced via synthesis over the years, from Wöhler’s time to the present. Note the increasing levels of structural sophistication and stereochemical complexity that have eventually been mastered by practitioners of organic synthesis. In our own time, the traditional boundaries between organic and biological chemistry are disappearing in ways that are likely to transform the design and synthesis of organic molecules, from the construction of synthetic biologicals designed to act as biomarkers, biosensors, or drug delivery agents, to the development of molecular motors, self-replicating macromolecular systems, and even synthetic life forms.

The Acetate Pathway: Biosynthesis of Polyketides and Related Compounds

We saw in the previous chapter how Otto Wallach’s early proposal regarding the structural origin of terpenoid natural products was later refined by the insightful work of Leopold Rudzicka, leading to the biogenetic isoprene rule and all that it implies. In a nearly parallel fashion, we find in our present chapter a second, unrelated class of naturally occurring compounds whose characteristic structural features prompted an initial innovative hypothesis by J. N. Collie near the turn of the 20th century. Collie proposed that certain natural compounds might arise from precursors containing repeated “ketide” (–CH2CO–) units which could then undergo subsequent condensations and other reactions typical of carbonyl compounds to produce some of the observed structures. Unfortunately, Collie’s work was more or less ignored and largely forgotten for nearly a half century, only to be reimagined and expanded in the middle of the century by A. J. Birch, another pioneer whose proposals met with considerable initial resistance. But unlike his predecessor, Birch ultimately prevailed by providing experimental results that supported a comprehensive theory of the biochemical origin of the group of compounds now universally known as “polyketide” natural products. This structurally diverse family includes some of the most useful medicinal agents now known to us, with many members possessing powerful antibacterial, antifungal, anticancer, immunosuppressant, and even cholesterol-lowering biological properties. As we see in Fig. 5.1, such structures range from the relatively simple to the exceedingly complex and may include large macrocyclic lactone rings (macrolides) such as erythromycin, polycyclic ethers such as monensin A, polycyclic structures which may be partly or mostly aromatic as in tetracycline, griseofulvin, or daunorubicin, or nonaromatic polycyclics such as tacrolimus and lovastatin. Some also contain noncyclic linear components that may be saturated, oxygenated, or unsaturated, as seen in different regions of amphotericin B which, like erythromycin, daunorubicin, and many other polyketides, also possesses an aglycone core which has been glycosylated with a carbohydrate component at a specific position. But in spite of this range of structures, many polyketide compounds share some common features that ultimately become more evident upon closer inspection; six-membered rings (either aromatic or nonaromatic) and multiple oxygens which tend to appear in a repeating 1,3-relationship to one another on both acyclic, cyclic, and aromatic structures.

Biosynthesis of Alkaloids and Related Compounds

Though definitions may vary from source to source, the term alkaloid generally refers to members of a large set of naturally occurring, slightly basic (i.e., alkaline) nitrogen-containing organic compounds. Generally excluded from this group are amino acids, peptides, proteins, N-containing carbohydrates, and nitrogenous bases used in the construction of nucleotides. Though a small number are produced by animals or microorganisms, the vast majority of alkaloids are plant-produced compounds possessing a remarkably diverse range of structural features, from simple cycloaliphatic amines to highly complex polycyclic N-heterocycles. Some representative alkaloids are shown in Fig. 7.1. Alkaloid-containing plants and their extracts have been used by humans for thousands of years, mainly on the basis of their stimulant, therapeutic, or poisonous properties. References to plants containing compounds such as morphine (from opium poppies), strychnine (from seeds of the Strychnos nux-vomica tree), ephedrine (from the plant Ephedra chinensis), and coniine (from the poison hemlock plant) may be found in some of our earliest known writings. Today, it has been estimated that the health care of over 5 billion people worldwide benefits from the use of plant-based medicinal agents, many of which are alkaloids. With that in mind, it is worth noting concerns that deforestation, environmental damage, large-scale development, and unregulated harvesting programs may ultimately lead to the extinction of hundreds of known medicinal plants and perhaps even more whose medicinal properties have yet to be discovered, thereby endangering the prospects for future discoveries of new curative agents for the benefit of all humankind. As a scientific field, alkaloid chemistry itself dates back to the early 1800s with the first isolation of pure crystalline morphine from opium. This milestone achievement allowed the delivery of accurate, therapeutic doses of a drug that was immensely valuable for the relief of pain but which could also lead to fatal overdoses when administered from simple extracts of variable composition and strength. The subsequent rapid development of increasingly sophisticated techniques for the isolation and purification of the active components (often alkaloids) from many other medicinal plants essentially spawned the field of organic chemistry.

The Shikimate Pathway: Biosynthesis of Phenolic Products from Shikimic Acid

Like other amino acids, the aromatic amino acids phenylalanine, tyrosine, and tryptophan are vitally important for protein synthesis in all organisms. However, while animals can synthesize tyrosine via oxidation of phenylalanine, they can synthesize neither phenylalanine itself nor tryptophan and so these essential amino acids must be obtained in the diet, usually from plant material. Though many other investigators made significant contributions in this area over the years, it was Bernhard Davis in the early 1950s whose use of mutant stains of Escherichia coli led to a full understanding of the so-called shikimic acid pathway that is used by plants and also by some microorganisms for the biosynthesis of these essential amino acids. The pathway is almost completely devoted to their synthesis for protein production in bacteria, while in plants the pathway extends their use to the construction of a wide array of secondary metabolites, many of which are valuable medicinal agents. These secondary metabolites range from simple and familiar compounds such as vanillin (vanilla flavor and fragrance) and eugenol (oil of clove, a useful dental anesthetic) to more complex structures such as pinoresinol, a common plant biochemical, and podophyllotoxin, a powerful cancer chemotherapy agent. Earlier in Chapter 3, we encountered two important intermediates, erythrose-4-phosphate and phosphoenolpyruvate (PEP), each of which was derived from a different pathway utilized in carbohydrate metabolism. Erythrose-4-P was an intermediate in one of the steps of the pentose phosphate pathway while hydrolysis of PEP to pyruvic acid was the final step in glycolysis. These two simple intermediates provide the seven carbon atoms required for construction of shikimic acid itself. The two are linked to one another via a sequence of enzyme-mediated aldol-type reactions, the first being a bimolecular reaction and the second an intramolecular variant that ultimately leads to a cyclic precursor of shikimic acid known as 3-dehydroquinic acid as shown in Fig. 6.3. Subsequent dehydration of 3-dehydroquinic acid leads to 3-dehydroshikimic acid which then leads directly to shikimic acid via NADPH reduction.

Bioorganic Reactions

It is not essential to have a background in enzymology or biochemistry to gain at least an introductory-level understanding of many biosynthetic processes, so this book does not deal with enzymology or enzyme structure or function in any significant way, even though much of the chemistry we will be examining depends almost entirely on enzyme catalysis. Nevertheless, we will refer to enzyme catalysis and the names of specific enzymes throughout the text as we examine biosynthetic processes and reactions in significant detail. So what exactly are enzymes? Simply put, enzymes are naturally occurring proteins that catalyze various biochemical reactions in living systems. As we will see, many of the reactions they catalyze are familiar organic reactions, but have specific purposes and target structures. Generally speaking, enzymes catalyze organic reactions by lowering transition state energies or raising ground state energies of reactants in much the same way as nonenzymatic catalysts in laboratory chemical reactions, though in the case of enzyme catalysis, rate enhancements of as much as 1023 have been reported, far exceeding rate enhancements currently achievable by conventional chemical means. Understanding the interaction of enzymes and substrates (reactants) to form an enzyme–substrate complex (E–S complex) is fundamental to having some appreciation for how enzymes carry out their work. While overly simplistic, the “lock-and-key” model of enzyme–substrate interaction provides an intuitive context for understanding the remarkable substrate specificity of enzyme-mediated reactions. Thus, so-called enzyme active sites or binding sites (the “lock”) will only accept certain specific substrate structures (the “key”), with shape, conformation, intermolecular forces, and other factors determining the lock-and-key fit. Enzymes not only catalyze specific kinds of reactions, they can act specifically on certain compounds or classes of compounds or functional groups, often showing remarkable selectivity and stereospecificity, especially in the recognition and/or introduction of chirality centers in organic molecules. In terms of nomenclature, enzyme names always end with an ase suffix and are typically named in accordance with the substrate they act upon and/or the kind of reaction process they catalyze.