General Biosynthetic Pathways of Secondary Metabolites

Chapter 13

General Biosynthetic Pathways of Secondary Metabolites

INTRODUCTION

• All organisms need to transform and interconvert a vast number of organic compounds to enable them to live, grow, and reproduce. They need to provide themselves with energy in the form of ATP, and a supply of building blocks to construct their own tissues. An integrated network of enzyme-mediated and carefully regulated chemical reations is used for this purpose, collectively referred to as ‘intermediary metabolism’, and the pathways involved are termed ‘metabolic pathways’. Some of the crucially important molecules of life are carbohydrates, proteins, fats, and nucleic acids.

• Despite the extremely varied characteristics of living organisms, the pathways for generally modifying and synthesizing carbohydrates, proteins, fats, and nucleic acids are found to be essentially the same in all organisms, apart from minor variations. These processes demonstrate the fundamental unity of all living matter, and are collectively described as ‘primary metabolism’, with the compounds involved in the pathways being termed ‘primary metabolites’. Thus degradation of carbohydrates and sugars generally proceeds via the well-characterized pathways known as glycolysis and the Krebs/citric acid/tricarboxylic acid cycle, which release energy from the organic compounds by oxidative reactions. Oxidation of fatty acids from fats by the sequence called β-oxidation also provides energy.

• In contrast to these primary metabolic pathways, which synthesize, degrade, and generally interconvert compounds commonly encountered in all organisms, there also exists an area of metabolism concerned with compounds which have a much more limited distribution in nature. Such compounds, called ‘secondary metabolites’, are found in only specific organisms, or groups of organisms, and are an expression of the individuality of species. Secondary metabolites are not necessarily produced under all conditions, and in the vast majority of cases the function of these compounds and their benefit to the organism is not yet known. Some are undoubtedly produced for easily appreciated reasons, for example, as toxic materials providing defence against predators, as volatile attractants towards the same or other species, or as colouring agents to attract or warn other species, but it is logical to assume that all do play some vital role for the well-being of the producer. It is this area of ‘secondary metabolism’ that provides most of the pharmacologically active natural products. It is thus fairly obvious that the human diet could be both unpalatable and remarkably dangerous if all plants, animals, and fungi produced the same range of compounds.

THE BUILDING BLOCKS

• The building blocks for secondary metabolites are derived from primary metabolism as indicated in Figure 13.1. This scheme outlines how metabolites from the fundamental processes of photosynthesis, glycolysis, and the Krebs cycle are tapped off from energy-generating processes to provide biosynthetic intermediates. The number of building blocks needed is surprisingly few, and as with any child’s construction set a vast array of objects can be built up from a limited number of basic building blocks. By far the most important building blocks employed in the biosynthesis of secondary metabolites are derived from the intermediates acetyl coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid, and 1-deoxyxylulose 5-phosphate. These are utilized respectively in the acetate, shikimate, mevalonate, and deoxyxylulose phosphate pathways.

• In addition to acetyl-CoA, shikimic acid, mevalonic acid, and deoxyxylulose phosphate, other building blocks based on amino acids are frequently employed in natural product synthesis.

• Peptides, proteins, alkaloids, and many antibiotics are derived from amino acids, and the origins of the most important amino acid components of these are briefly indicated in Figure 13.1. Intermediates from the glycolytic pathway and the Krebs cycle are used in constructing many

• of them, but the aromatic amino acids phenylalanine, tyrosine, and tryptophan are themselves products from the shikimate pathway. Ornithine, a nonprotein amino acid, and its homologue lysine, are important alkaloid precursors having their origins in Krebs cycle intermediates.

• Relatively few building blocks are routinely employed, and the following list, though not comprehensive, includes those most frequently encountered in producing the carbon and nitrogen skeleton of a natural product.

• C1 : The simplest of the building blocks is composed of a single carbon atom, usually in the form of a methylgroup, and most frequently it is attached to oxygen or nitrogen, but occasionally to carbon. It is derived from the S-methyl of L-methionine. The methylenedioxy group (OCH2 O) is also an example of a C1 unit.

• C2 : A two-carbon unit may be supplied by acetyl-CoA. This could be a simple acetyl group, as in an ester, but more frequently it forms part of a long alkyl chain (as in a fatty acid) or may be part of an aromatic system (e.g. phenols). Of particular relevance is that in the latter examples, acetyl-CoA is first converted into the more reactive malonyl-CoA before its incorporation.

• C5 : The branched-chain C5 ‘isoprene’ unit is a feature of compounds formed from mevalonate or deoxyxylulose

•  phosphate. Mevalonate itself is the product from three acetyl-CoA molecules, but only five of mevalonate’s six carbons are used, the carboxyl group being lost. The alternative precursor deoxyxylulose phosphate, a straightchain sugar derivative, undergoes a skeletal rearrangement to form the branched chain isoprene unit.

• C6 C3 : This refers to a phenylpropyl unit and is obtained from the carbon skeleton of either L-phenylalanine or L-tyrosine, two of the shikimate-derived aromatic amino acids. This, of course, requires loss of the amino group. The C3 side chain may be saturated or unsaturated, and may be oxygenated. Sometimes the side chain is cleaved, removing one or two carbons. Thus, C6 C2 and C6 C1 units represent modified shortened forms of the C6 C3 system.

• C6 C2 N: Again, this building block is formed from either L-phenylalanine or L-tyrosine, L-tyrosine being by far the more common. In the elaboration of this unit, the carboxyl carbon of the amino acid is removed.

• Indole.C2 N: The third of the aromatic amino acids is L-tryptophan. This indole-containing system can undergo decarboxylation in a similar way to L-phenylalanine and L-tyrosine so providing the remainder of the skeleton as an indole. C2 N unit.

• C4 N: The C4 N unit is usually found as a heterocyclic pyrrolidine system and is produced from the nonprotein amino acid L-ornithine. In marked contrast to the C6 C2 N and indole.C2 N units described above, ornithine supplies not its α-amino nitrogen, but the δ-amino nitrogen. The carboxylic acid function and the α-amino nitrogen are both lost.

• C5 N: This is produced in exactly the same way as the C4 N unit, but using L-lysine as precursor. The ε-amino nitrogen is retained, and the unit tends to be found as a piperidine ring system.

• These eight building blocks form the basis of many of the natural product structures discussed in this chapter. Simple examples of how compounds can be visualized as a combination of building blocks are shown in Figure 13.3.

 

• Although primary and secondary metabolism are interrelated to the extent that an absolute distinction is meaningless, for the purpose of this chapter some division has had to be made, and this has been based on biosynthetic pathways. Excluding the primary processes of sugar and protein biosynthesis, there are three main routes to the wealth of chemical compounds found in plants, that is, shikimic acid pathways, acetate-malonate, and acetatemevalonate pathways, which are interrelated as shown in figure 13.4.

Shikimic Acid Pathway

• The shikimate pathway provides an alternative route to aromatic compounds, particularly the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan. This pathway is employed by microorganisms and plants, but not by animals, and accordingly the aromatic amino acids feature among those essential amino acids for men whom have to be obtained in the diet. A central intermediate in the pathway is shikimic acid, a compound which had been isolated from plants of Illicium species (Japanese ‘shikimi’) many years before its role in metabolism had been discovered. Most of the intermediates in the pathway were identified by a careful study of a series of Escherichia coli mutants.

• prepared by UV irradiation. Their nutritional requirements for growth, and any by-products formed, were then characterized. A mutant strain capable of growth usually differs from its parent in only a single gene, and the usual effect is the impaired synthesis of a single enzyme. Typically, a mutant blocked in the transformation of compound A into compound B will require B for growth whilst accumulating A in its culture medium. In this way, the pathway from.

• phosphoenolpyruvate (from glycolysis) and D-rythrose 4-phosphate (from the pentose phosphate cycle) to the aromatic amino acids was broadly outlined. Phenylalanine and tyrosine form the basis of C6 C3 phenylpropane units found in many natural products, for example, cinnamic acids, coumarins, lignans, and flavonoids, and along with tryptophan are precursors of a wide range of alkaloid structures. In addition, it is found that many simple benzoic acid derivatives, for example, gallic acid and p-aminobenzoic acid (4-aminobenzoic acid) are produced via branchpoints in the shikimate pathway (Figure 13.5).

• The shikimic acid pathway contains several branch points, the first of these, dehydroquinic acid, can be converted either to 3-dehydroshikimic acid, which continues the pathway, or to quinic acid. The enzymes catalysing the dehydration of dehydroquinic acid are of two kinds. Form

• 1, associated with shikimate dehydrogenase, is independent of shikimate concentration, while form 2 is specifically activated by shikimate. 

• The formation of chorismic acid is an important branch point in the shikimic acid pathway as this compound can undergo three different types of conversion. The name ‘chorismic’ is derived from a Greek word for separate, indicating the multiple role of this compound. In the presence of glutamine, chorismic acid is converted to anthranilic acid, whereas chorismate mutase catalyses the formation of prephenic acid. chorismic acid is also converted into p-aminobenzoic acid.

• Then after anthranilic acid is converted first to phosphoribosylanthranilic acid and then to carboxyphenylaminodeoxyribulose-5-phosphate, these reactions being catalysed.

• The biosynthesis of phenylalanine involves first the aromatization of prephanic acid to phenylpyruvic acid, a reaction catalysed by prephenate dehydratase, and then transamination catalysed by phenylalanine aminotransferase, which gives phenylalanine.

Acetate-Mevalonate Pathway

• Since a long time biochemists were aware of the involvement of acetic acid in the synthesis of cholesterol, squalene and rubber-like compounds. The discovery of acetyl coenzyme A called as ‘active acetate’ in 1950, further supported the role of acetic acid in biogenetic pathways. Later, mevalonic acid was found to be associated with the acetate. Mevalonic acid further produced isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). These two main intermediates IPP and DMAPP set the ‘active isoprene’ unit as a basic building block of isoprenoid compounds. Both of these units yield geranyl pyrophosphate (C10-monoterpenes) which further association with IPP produces farnesyl pyrophosphate (C15-sesquiterpenes).

• Farnesyl pyrophosphate with one more unit of IPP develops into geranyl—eranyl pyrophosphate (C20-diterpenes). The farnesyl pyrophosphate multiplies with its own unit to produce squalene, and its subsequent cyclization gives rise to cyclopentanoperhydrophenantherene skeleton containing steroidal compounds like cholesterols and other groups like triterpenoids. The acetate mevalonate pathway thus works through IPP and DMAPP via squalene to produce two different skeleton containing compounds, that is, steroids and triterpenoids. It also produces vast array of monoterpenoids, sesquiterpenoids, diterpenoids, carotenoids, polyprenols, and also the compounds like glycosides and alkaloids in association with other pathways (Figure 13.6).

Acetate-Malonate Pathway

• Acetate pathway operates functionally with the involvement of acyl carrier protein (ACP) to yield fatty acyl thioesters of ACP. These acyl thioesters forms the important intermediates in fatty acid synthesis. These C2 acetyl CoA units at the later stage produces even number of fatty acids from n-tetranoic (butyric) to n-ecosanoic (arachidic acid). The synthesis of fatty acids is thus explained by the reactions given in Figure 13.7. Unsaturated fatty acids are produced by subsequent direct dehydrogenation of saturated fatty acids. Enzymes play important role in governing the position of newly introduced double bonds in the fatty acids. 

 BIOSYNTHESIS OF CARBOHYDRATES

• Carbohydrates are the products of photosynthesis, a biological process that converts light energy into chemical energy. The general process of photosynthesis can be described by:

• CO2 + H2 O Sugars + O2

• All green plants and certain algae and bacteria have the capacity to synthesize adenosine triphosphate (ATP) and nicotine adenine dinucleotide phosphate (NADPH). These compounds mediate most of the biosynthetic reactions in plants. There are basically two primary lights:

• 1. Absorption of light by chlorophyll or energy transfer to chlorophyll by other light absorbing pigments leading to production of ATP and NADPH. 

• 2. Photolysis of water to produce oxygen and electrons which are transferred via carrier species and produces ATP and NADPH, two reactive molecules which work as activating and reducing agents.

• Blackmann Reaction: In the subsequent ‘dark reaction’, carbon dioxide is reduced to produce four, five, six, and seven carbon sugars. The reactions were firstly given by Blackmann and hence called as Blackmann reaction. It is estimated that about 4000 × 109 tons of CO2 is fixed annually through the photosynthetic process. The path of carbon in photosynthesis was first given by Calvin is termed as Calvin cycle.

CO2 + 2 NADPH2 + 2 ATP (CH2 O)n + H2 O + 2 ADP + 2 NADPH (Carbohydrate)

BIOSYNTHESIS OF GLYCOSIDES

• The glycosides are the condensation products of sugar and the acceptor unit called as aglycone. The reaction occurs in two parts as given below. Firstly sugar phosphates bind with uridine triphosphate (UTP) to produce sugar—uridine diphosphate sugar complex. This sugar nucleotide complex reacts with acceptor units in the second reaction which leads to glycoside production.

• UTP + Sugar 1-P UDP - Sugar + Ppi (1) Uridyl transferase UDP - Sugar + Acceptor Acceptor - Sugar + UDP (2) Glycosyl transferase.

• Once such glycosides are formed, other specific enzymes may transfer another sugar unit in the later reactions in which the glycoside formed in the previous reaction work as an acceptor to provide di-, tri-, or tetraglycosides and so on by subsequent reactions.

BIOSYNTHESIS OF ALKALOIDS

• The biosynthesis of different groups of alkaloids has now been investigated to some extent using precursors labelled with radioactive atoms. Very little work has, however, been published in the area of enzymology of alkaloid biosynthesis, some exceptions being in studies of ergot and Amaryllidaceae alkaloids. The biosynthetic pathways of pharmacognostically important alkaloids are given below.

Ornithine Derivatives

• L-Ornithine is a nonprotein amino acid forming part of the urea cycle in animals, where it is produced from L-arginine in a reaction catalysed by the enzyme arginase. In plants it is formed mainly from L-glutamate. Ornithine contains both δ- and α-amino groups, and it is the nitrogen from the former group which is incorporated into alkaloid structures along with the carbon chain, except for the carboxyl group. Thus ornithine supplies a C4 N building block to the alkaloid, principally as a pyrrolidine ring system, but also as part of the tropane alkaloids (Figure 13.8.).

• Ornithine is a precursor of the cyclic pyrrolidines that occur in the alkaloids of tobacco (nicotine, nornicotine) and in solanaceae family. Most of the tobacco alkaoids have nicotine as the starting compound. Few of the intermediates produced during the biosynthesis of tropane are also the starting compounds for hyoscamine and cocaine.

Biosynthesis of tropane

• The starting compound of this synthesis is ornithine and methylornithine is the first intermediate.

Lysine Derivatives

• L-Lysine is the homologue of L-ornithine, and it too functions as an alkaloid precursor, using pathways analogous to those noted for ornithine. The extra methylene group in lysine means this amino acid participates in forming six-membered piperidine rings, just as ornithine provided five-membered pyrrolidine rings. As with ornithine, the carboxyl group is lost, the ε-amino nitrogen rather than the α-amino nitrogen is retained, and lysine thus supplies a C5 N building block (Figure 13.11).

• Lysine is a precursor for piperidine. Piperidine forms the basic skeleton for numerous alkaloids. Lysine and its derivatives are responsible for the biogenesis of some of the bitter principles of the lupine, lupinine, lupanine, anabasine, pelletierine, and some other alkaloidal compounds. Lycopodium, a substance obtained from Lycopodium spp., also belongs to this group.

Phenylalanine Derivatives

• Whilst the aromatic amino acid L-tyrosine is a common and extremely important precursor of alkaloids, L-phenylalanine is less frequently utilized, and usually it contributes only carbon atoms, for example, C6 C3 , C6 C2 , or C6 C1 units, without providing a nitrogen atom from its amino group. Ephedrine (Figure 13.13.), the main alkaloid in species of Ephedra (Ephedraceae) and a valuable nasal decongestant and bronchial dilator, is a prime example.

   

Tyrosine Derivatives

• The first essential intermediate is dopamine; dopamine is the precursor in the biosynthesis of papaverine, berberine, and morphine. Tyrosine is considered to be a precursor for the huge family containing alkaloids.

Biosynthesis of morphine

• A range of similar compounds like the opium alkaloids, thebaine, codeine, etc. are derived during the formation of morphine.

Tryptophan Derivatives

• About 1,200 dissimilar compounds, the entire of which are tryptophan derivatives have been isolated till today. The tryptophan derivatives correspond to 25% of all known alkaloids and many of them are medicinally valuable. Tryptophan and its decarboxylated product (tryptamine) are precursors for the biosynthesis of broad range of indole alkaloids of which the vinca and rauwolfia alkaloids are examples; and also in the alkaloids belonging to families like Apocynaceae, Loganiaceae, and Rubiaceae. D-Tubocurarine, the active components of curare, is also a tryptophan derivative. Tryptamine on condensation with secologanin produces vincoside a nitrogenous glucoside. Some of the indole alkaloids in vinca are formed from vincoside.

Biosynthesis of quinoline alkaloids 

• Some of the most remarkable examples of terpenoid indole alkaloid modifications are to be found in the genus Cinchona (Rubiaceae), in the alkaloids quinine, quinidine, cinchonidine, and cinchonine (Figure 13.17), long prized for their antimalarial properties. These structures are remarkable in 

• that the indole nucleus is no longer present, having been rearranged into a quinoline system.

Biosynthesis of Lysergic Acid

• The building blocks for lysergic acid are tryptophan (less the carboxyl group) and an isoprene unit (Figure 13.18). Alkylation of tryptophan with dimethylallyl diphosphate gives 4-dimethylallyl-L-tryptophan, which then undergoes N-methylation. Formation of the tetracyclic ring system of lysergic acid is known to proceed through chanoclavine-I and agroclavine, though the mechanistic details are far from clear. Labelling studies have established that the double bond in the dimethylallyl substituent must become a single bond on two separate occasions, allowing rotation to occur as new rings are established. This gives the appearance of cis–trans isomerizations as 4-dimethylallyl-L-tryptophan is transformed into chanoclavine-I, and as chanoclavine-I aldehyde cyclizes to agroclavine. A suggested sequence to account for the first of these is shown. In the later stages, agroclavine is hydroxylated to elymoclavine, further oxidation of the primary alcohol occurs giving paspalic acid, and lysergic acid then results from a spontaneous allylic isomerization.

BIOSYNTHESIS OF PHENOLIC COMPOUNDS

• Most of the phenolic compounds belong to the category of flavonoids, with acidic nature due to the presence of –OH group in it. The flavonoids have their basic structure from C15 body of flavone. Flavones occur both as coloured and in colourless nature, for example, Anthocyanins are normally red or yellow. They also form chelate complexes with metals and get easily oxidized to form a polymer. Some of the common phenolic compounds are coumarine, flavone, flavonol, anthrocyanidines, etc.