Natural Products (Secondary Metabolites)

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CHAPTER 24 Natural Products (Secondary Metabolites)

Rodney Croteau Toni M. Kutchan Norman G. Lewis

CHAPTER OUTLINE Introduction

Introduction Natural products have primary ecological functions. 24.1 Terpenoids 24.2 Synthesis of IPP Plants produce a vast and diverse assortment of organic compounds, 24.3 Prenyltransferase and terpene the great majority of which do not appear to participate directly in synthase reactions growth and development. These substances, traditionally referred to 24.4 Modification of terpenoid as secondary metabolites, often are differentially distributed among skeletons limited taxonomic groups within the plant kingdom. Their functions, 24.5 Toward transgenic terpenoid many of which remain unknown, are being elucidated with increas- production ing frequency. The primary metabolites, in contrast, such as phyto- 24.6 Alkaloids sterols, acyl lipids, nucleotides, amino acids, and organic acids, are 24.7 Alkaloid biosynthesis found in all plants and perform metabolic roles that are essential 24.8 Biotechnological application and usually evident. of alkaloid biosynthesis Although noted for the complexity of their chemical structures research and biosynthetic pathways, natural products have been widely per- 24.9 Phenylpropanoid and ceived as biologically insignificant and have historically received lit- phenylpropanoid-acetate tle attention from most plant biologists. Organic chemists, however, pathway metabolites have long been interested in these novel phytochemicals and have 24.10 Phenylpropanoid and investigated their chemical properties extensively since the 1850s. phenylpropanoid-acetate Studies of natural products stimulated development of the separa- biosynthesis tion techniques, spectroscopic approaches to structure elucidation, and synthetic methodologies that now constitute the foundation of 24.11 Biosynthesis of lignans, lignins, contemporary organic chemistry. Interest in natural products was and suberization not purely academic but rather was prompted by their great utility 24.12 Flavonoids as dyes, polymers, fibers, glues, oils, waxes, flavoring agents, per- 24.13 Coumarins, stilbenes, fumes, and drugs. Recognition of the biological properties of myriad styrylpyrones, and arylpyrones natural products has fueled the current focus of this field, namely, 24.14 Metabolic engineering of the search for new drugs, antibiotics, insecticides, and herbicides. phenylpropanoid production: Importantly, this growing appreciation of the highly diverse biologi- a possible source of enhanced cal effects produced by natural products has prompted a reevalua- fibers, pigments, pharmaceuti- tion of the possible roles these compounds play in plants, especially cals, and flavoring agents in the context of ecological interactions. As illustrated in this chapter, many of these compounds now have been shown to have important

1250 Chapter 24 Natural Products (Secondary Metabolites) adaptive significance in protection against thus a natural product (Fig. 24.1). Even herbivory and microbial infection, as attrac- lignin, the essential structural polymer of tants for pollinators and seed-dispersing ani- wood and second only to cellulose as the mals, and as allelopathic agents (allelochem- most abundant organic substance in plants, icals that influence competition among plant is considered a natural product rather than species). These ecological functions affect a primary metabolite. plant survival profoundly, and we think it In the absence of a valid distinction reasonable to adopt the less pejorative term based on either structure or biochemistry, we “plant natural products” to describe sec- return to a functional definition, with prima- ondary plant metabolites that act primarily ry products participating in nutrition and es- on other species. sential metabolic processes inside the plant, and natural (secondary) products influenc- ing ecological interactions between the plant The boundary between primary and and its environment. In this chapter, we secondary metabolism is blurred. provide an overview of the biosynthesis of the major classes of plant natural products, Based on their biosynthetic origins, plant emphasizing the origins of their structural natural products can be divided into three diversity, as well as their physiological func- major groups: the terpenoids, the alkaloids, tions, human uses, and potential biotechno- and the phenylpropanoids and allied pheno- logical applications. lic compounds. All terpenoids, including both primary metabolites and more than 25,000 secondary compounds, are derived 24.1 Terpenoids from the five-carbon precursor isopentenyl diphosphate (IPP). The 12,000 or so known Terpenoids perhaps are the most structurally alkaloids, which contain one or more nitro- varied class of plant natural products. The gen atoms, are biosynthesized principally name terpenoid, or terpene, derives from the from amino acids. The 8000 or so phenolic fact that the first members of the class were compounds are formed by way of either the shikimic acid pathway or the malonate/ acetate pathway. Primary metabolite Secondary metabolite Primary and secondary metabolites cannot readily be distinguished on the basis of precursor molecules, chemical structures, or biosynthetic origins. For example, both primary and secondary metabolites are found among the diterpenes (C ) and triterpenes 20 COOH COOH (C30). In the diterpene series, both kaurenoic Kaurenoic acid Abietic acid acid and abietic acid are formed by a very similar sequence of related enzymatic reactions (Fig. 24.1); the former is an essential intermediate in the synthesis of gibberellins, N COOH N COOH i.e., growth hormones found in all plants H H (see Chapter 17), whereas the latter is a resin Proline Pipecolic acid component largely restricted to members of the Fabaceae and Pinaceae. Similarly, the es- Figure 24.1 sential amino acid proline is classified as a Kaurenoic acid and proline are primary metabolites, whereas the closely related compounds abietic primary metabolite, whereas the C6 analog acid and pipecolic acid are considered secondary pipecolic acid is considered an alkaloid and metabolites.

24.1–Terpenoids 1251 isolated from turpentine (“terpentin” in Ger- known hemiterpene is isoprene itself, a vol- man). All terpenoids are derived by repeti- atile product released from photosynthe- tive fusion of branched five-carbon units tically active tissues. The enzyme isoprene based on isopentane skeleton. These mono- synthase is present in the leaf plastids of nu-

mers generally are referred to as isoprene merous C3 plant species, but the metabolic units because thermal decomposition of rationale for the light-dependent production many terpenoid substances yields the alkene of isoprene is unknown (acclimation to high gas isoprene as a product (Fig. 24.2, upper temperatures has been suggested). Estimated panel) and because suitable chemical condi- annual foliar emissions of isoprene are quite tions can induce isoprene to polymerize in substantial (5 × 108 metric tons of carbon), multiples of five carbons, generating numer- and the gas is a principal reactant in the ous terpenoid skeletons. For these reasons, NOx radical–induced formation of tropo- the terpenoids are often called isoprenoids, spheric ozone (see Chapter 22, Fig. 22.37).

although researchers have known for well C10 terpenoids, although they consist of over 100 years that isoprene itself is not two isoprene units, are called monoterpenes; the biological precursor of this family of as the first terpenoids isolated from turpen- metabolites. tine in the 1850s, they were considered to be the base unit from which the subsequent nomenclature is derived. The monoterpenes 24.1.1 Terpenoids are classified by the are best known as components of the vol- number of five-carbon units they contain. atile essences of flowers and of the essential oils of herbs and spices, in which they make The five-carbon (isoprene) units that make up as much as 5% of plant dry weight. Mono- up the terpenoids are often joined in a “head- terpenes are isolated by either distillation or to-tail” fashion, but head-to-head fusions are extraction and find considerable industrial also common, and some products are formed use in flavors and perfumes. by head-to-middle fusions (Fig. 24.2, lower The terpenoids that derive from three panel). Accordingly, and because extensive isoprene units contain 15 carbon atoms and structural modifications with carbon–carbon are known as sesquiterpenes (i.e., one and bond rearrangements can occur, tracing the one-half terpenes). Like monoterpenes, original pattern of isoprene units is some- many sesquiterpenes are found in essential times difficult. oils. In addition, numerous sesquiterpenoids The smallest terpenes contain a single act as phytoalexins, antibiotic compounds isoprene unit; as a group, they are named produced by plants in response to microbial hemiterpenes (half-terpenes). The best challenge, and as antifeedants that discourage opportunistic herbivory. Although the plant hormone abscisic acid is structurally a

sesquiterpene, its C15 precursor, xanthoxin, is not synthesized directly from three isoprene Isopentane Isoprene units but rather is produced by asymmetric cleavage of a C40 carotenoid (see Chapter 17). The diterpenes, which contain 20 car-

bons (four C5 units), include phytol (the hydrophobic side chain of chlorophyll), the gibberellin hormones, the resin acids of conifer and legume species, phytoalexins, Head-to-tail Head-to-head Head-to-middle and a host of pharmacologically important metabolites, including taxol, an anticancer Figure 24.2 agent found at very low concentrations Terpenes are synthesized from C5 units. The upper panel shows the structures of the isopentane skeleton and isoprene gas. The lower panel shows how different (0.01% dry weight) in yew bark, and for- patterns of isoprene unit assembly yield a variety of different structures. For ex- skolin, a compound used to treat glaucoma. ample, the triterpene squalene is formed by head-to-head fusion of two molecules Some gibberellins have only 19 carbon atoms of farnesyl diphosphate (FPP), which itself is the product of the head-to-tail fu- and are considered norditerpenoids since sion of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) (see Fig. 24.7). The monoterpene pyrethrin I (see Fig. 24.10) results from a head-to-middle they have lost 1 carbon through a metabolic fusion of two C5 units. cleavage reaction (see Chapter 17).

1252 Chapter 24 Natural Products (Secondary Metabolites) The triterpenes, which contain 30 car- rule,” emphasizing mechanistic considera- bon atoms, are generated by the head-to- tions of terpenoid synthesis in terms of

head joining of two C15 chains, each of electrophilic elongations, cyclizations, and which constitutes three isoprene units joined rearrangements. This hypothesis ignores the head-to-tail. This large class of molecules in- precise character of the biological precursors cludes the brassinosteroids (see Chapter 17), and assumes only that they are “isoprenoid” the phytosterol membrane components (see in structure. As a working model for ter- Chapter 1), certain phytoalexins, various penoid biosynthesis, the biogenetic isoprene toxins and feeding deterrents, and compo- rule has proved essentially correct. nents of surface waxes, such as oleanolic Despite great diversity in form and acid of grapes. function, the terpenoids are unified in their The most prevalent tetraterpenes (40 common biosynthetic origin. The biosynthe- carbons, eight isoprene units) are the carote- sis of all terpenoids from simple, primary noid accessory pigments which perform metabolites can be divided into four overall essential functions in photosynthesis (see steps: (a) synthesis of the fundamental pre- Chapter 12). The polyterpenes, those con- cursor IPP; (b) repetitive additions of IPP to taining more than eight isoprene units, in- form a series of prenyl diphosphate ho- clude the prenylated quinone electron carri- mologs, which serve as the immediate pre- ers (plastoquinone and ubiquinone; see cursors of the different classes of terpenoids; Chapters 12 and 14), long-chain polyprenols (c) elaboration of these allylic prenyl diphos- involved in sugar transfer reactions (e.g., phates by specific terpenoid synthases to dolichol; see Chapters 1 and 4), and enor- yield terpenoid skeletons; and (d) secondary mously long polymers such as rubber (aver- enzymatic modifications to the skeletons age molecular mass greater than 106 Da), (largely redox reactions) to give rise to the often found in latex. functional properties and great chemical di- Natural products of mixed biosynthetic versity of this family of natural products. origins that are partially derived from terpenoids are often called meroterpenes. For example, both cytokinins (see Chapter 17) 24.2 Synthesis of IPP and numerous phenylpropanoid compounds contain C5 isoprenoid side chains. Certain al- 24.2.1 Biosynthesis of terpenoids is kaloids, including the anticancer drugs vin- compartmentalized, as is production of the cristine and vinblastine, contain terpenoid terpenoid precursor IPP. fragments in their structures (see Fig. 24.34). Additionally, some modified proteins in- Although terpenoid biosynthesis in plants, clude a 15- or 20-carbon terpenoid side chain animals, and microorganisms involves simi- that anchors the protein in a membrane (see lar classes of enzymes, important differences Chapter 1). exist among these processes. In particular, plants produce a much wider variety of terpenoids than do either animals or microbes, 24.1.2 A diverse array of terpenoid a difference reflected in the complex organi- compounds is synthesized by various zation of plant terpenoid biosynthesis at the conserved reaction mechanisms. tissue, cellular, subcellular, and genetic levels. The production of large quantities of ter- At the turn of the 20th century, structural in- penoid natural products as well as their sub- vestigations of many terpenoids led Otto sequent accumulation, emission, or secretion Wallach to formulate the “isoprene rule,” is almost always associated with the pres- which postulated that most terpenoids could ence of anatomically highly specialized be constructed hypothetically by repetitively structures. The glandular trichomes (Fig. joining isoprene units. This principle provid- 24.3A, B) and secretory cavities of leaves ed the first conceptual framework for a com- (Fig. 24.3C) and the glandular epiderms of mon structural relationship among terpenoid flower petals generate and store or emit ter- natural products (Box 24.1). Wallach’s idea penoid essential oils that are important be- was refined in the 1930s, when Leopold cause they encourage pollination by insects. Ruzicka formulated the “biogenetic isoprene The resin ducts and blisters of conifer species

24.2–Synthesis of IPP 1253 Early investigators formulated rules for identifying and naming Box 24.1 isoprenoid structures.

In the late 1800s, chemists struggled concept, known as the isoprene rule, ophile to yield the observed terpenoid to define the structures of the monoter- earned Wallach the Nobel Prize in Chem- products. This proposal, which Ruzicka penes. The mixed results achieved by istry in 1910. called the biogenetic isoprene rule, can these efforts are illustrated by the numer- By the 1930s, faced with a bewilder- be stated simply: A compound is “iso- ous structures proposed for camphor ing array of terpenoid substances, prenoid” if it is derived biologically from

(C10H16O; see structures at left of figure, Leopold Ruzicka and his contemporaries an “isoprenoid” precursor, with or with- which include the names of the proposers sought to develop a unifying principle out rearrangements. Ruzicka’s concept and the dates proposed). Chromatograph- that could rationalize the natural occur- differs from Wallach’s in its emphasis on ic purification techniques and spectro- rence of all of the known terpenoids, biochemical origin rather than structure. scopic methods for structure elucidation even those that did not strictly fit Wal- The great strength of the biogenetic iso- were not available to these early lach’s isoprene rule. Ruzicka’s ingenious prene rule lay in its use of mechanistic chemists, who relied on the preparation solution to the problem was to focus on considerations to classify the bulk of of crystalline derivatives to assess purity reaction mechanisms and ignore the pre- known terpenoids, including structures and on chemical degradation studies to cise character of the biological precursor, that did not strictly follow Wallach’s iso- determine structures. Systematic study of assuming only that it had a terpenoid prene rule. Application of the biogenetic the monoterpenes led the German structure during reaction. He hypothe- isoprene rule to the origin of several of chemist Otto Wallach to recognize that sized the involvement of electrophilic re- the common monoterpene skeletons is il- many terpenoid compounds might be actions that generated carbocationic inter- lustrated in the right panel of the figure constructed by joining isoprene units, mediates, which underwent subsequent (note the bornane skeleton from which

generally in a repetitive head-to-tail C5 addition, cyclization, and in some cas- camphor is derived). Ruzicka was award- fashion, as in Bredt’s correct proposed es skeletal rearrangement before elimina- ed the Nobel Prize in Chemistry in 1939. structure for camphor (see figure). This tion of a proton or capture by a nucle-

+ O

(Meyer, 1870) (Hlasiwetz, 1870) +

+ O O + Pinane skeleton α-Terpinyl cation Terpinen-4-yl cation

(Bredt, 1893) (Tiemann, 1895)

+ O + +

(Bouveault, 1897) (Perkin, 1898) Fenchane skeleton Bornane skeleton Thujane skeleton

(Fig. 24.3D) produce and accumulate a defen- polyterpenes such as rubber. These special- sive resin consisting of turpentine (monoter- ized structures sequester natural products pene olefins) and rosin (diterpenoid resin away from sensitive metabolic processes and acids). Triterpenoid surface waxes are formed thereby prevent autotoxicity. Most structures and excreted from specialized epidermis, of this type are nonphotosynthetic and must and laticifers produce certain triterpenes and therefore rely on adjacent cells to supply

1254 Chapter 24 Natural Products (Secondary Metabolites) the carbon and energy needed to drive ter- IPP to the cytosol for use in biosynthesis, penoid biosynthesis. and vice versa. Mitochondria, a third com- A more fundamental, and perhaps uni- partment, may generate the ubiquinone versal, feature of the organization of ter- prenyl group by the acetate/mevalonate penoid metabolism exists at the subcellular pathway, although little is known about the

level. The sesquiterpenes (C15), triterpenes capability of these organelles for terpenoid (C30), and polyterpenes appear to be pro- biosynthesis. duced in the cytosolic and endoplasmic reticulum (ER) compartments, whereas iso- 24.2.2 Hydroxymethylglutaryl-CoA

prene, the monoterpenes (C10), diterpenes reductase, an enzyme in the (C20), tetraterpenes (C40), and certain preny- acetate/mevalonate pathway, lated quinones originate largely, if not exclu- is highly regulated. sively, in the plastids. The evidence now indicates that the biosynthetic pathways for The basic enzymology of IPP biosynthesis by the formation of the fundamental precursor way of the acetate/mevalonate pathway is IPP differ markedly in these compartments, widely accepted (Fig. 24.4). This cytosolic with the classical acetate/mevalonate path- IPP pathway involves the two-step conden- way being active in the cytosol and ER and sation of three molecules of acetyl-CoA the glyceraldehyde phosphate/pyruvate catalyzed by thiolase and hydroxymethyl- pathway operating in the plastids. Regula- glutaryl-CoA synthase. The resulting tion of these dual pathways may be difficult product, 3-hydroxy-3-methylglutaryl-CoA to assess, given that plastids may supply (HMG-CoA), is subsequently reduced by

(A) (B)

St B E 100 µ

X Sh

P S L L S Sh

Figure 24.3 (A) Scanning electron micrograph of the leaf surface of thyme. The round structures are peltate glandular trichomes, in which monoterpenes and sesquiterpenes are synthesized. (B) Light micrograph of a glandular tri- chome from spearmint, shown in longitudinal section. C, subcuticular space; S, secretory cells; St, stalk; B, basal cell; E, epidermal cell. (C) Light micrograph of a secretory cavity in a lemon leaf, shown in cross-section. L, lumen; Sh, sheath cells; P, parenchyma cell. (D) Light micrograph of a resin duct in wood of Jeffrey pine, shown in cross-section. X, secondary xylem.

24.2–Synthesis of IPP 1255 HMG-CoA reductase in two coupled reac- HMG-CoA reductase is one of the most tions that form mevalonic acid. Two sequen- highly regulated enzymes in animals, being tial ATP-dependent phosphorylations of largely responsible for the control of choles- mevalonic acid and a subsequent phospho- terol biosynthesis. Accumulated evidence rylation/elimination-assisted decarboxylation indicates that the plant enzyme, which is yield IPP. located in the ER membrane, is also highly regulated. In many cases, small gene families, each containing multiple members, en- O Acetyl-CoA code this reductase. These gene families are CH3 C S CoA expressed in complex patterns, with individ- O ual genes exhibiting constitutive, tissue- or Acetyl-CoA development-specific, or hormone-inducible CH3 C S CoA expression. Specific HMG-CoA reductase Thiolase genes can be induced by wounding or path- CoASH ogen infection. The activity of HMG-CoA re- O O ductase may be subject to posttranslational Acetoacetyl- CH3 C CH2 C S CoA CoA regulation, for example, by a protein kinase O cascade that phosphorylates and thereby in- Acetyl-CoA activates the enzyme. Allosteric modulation HMG-CoA CH3 C S CoA probably also plays a regulatory role. Prote- synthase olytic degradation of HMG-CoA reductase CoASH protein and the rate of turnover of the corre- HO O 3-Hydroxy- sponding mRNA transcripts may also influ- 3-methyl- ence enzyme activity. Researchers have not CH3 C CH2 C S CoA glutaryl-CoA (HMG-CoA) arrived at a unified scheme that explains CH2 COOH how the various mechanisms that regulate 2 NADPH HMG-CoA reductase facilitate the produc- HMG-CoA + tion of different terpenoid families. The 2 NADP reductase precise biochemical controls that influence CoASH activity have been difficult to assess in vitro HO because the enzyme is associated with the Mevalonic acid ER membrane. A model proposed to ration- CH3 C CH2 CH2OH (MVA) alize the selective participation of HMG-CoA

CH2 COOH reductase in the biosynthesis of different ATP mevalonate-derived terpenoids is shown MVA kinase in Figure 24.5. ADP HO 24.2.3 In plastids, IPP is synthesized from Mevalonic acid pyruvate and glyceraldehyde 3-phosphate. CH3 C CH2 CH2O P 5-phosphate (MVAP)

CH2 COOH The plastid-localized route to IPP involves ATP a different pathway, demonstrated in green MVAP kinase algae and many eubacteria as well as plants. ADP In this pathway, pyruvate reacts with thia- HO mine pyrophosphate (TPP) to yield a Mevalonic acid two-carbon fragment, hydroxyethyl-TPP, CH3 C CH2 CH2O P P 5-diphosphate (MVAPP) which condenses with glyceraldehyde 3-

CH2 COOH phosphate (see Chapter 12, Fig. 12.41, for Figure 24.4 similar TPP-mediated C transfers catalyzed The acetate/mevalonate ATP 2 pathway for the forma- MVAPP by transketolase). TPP is released to form a tion of IPP, the basic decarboxylase five-carbon intermediate, 1-deoxy-D-xylulose ADP + P i five-carbon unit of ter- 5-phosphate, which is rearranged and re- penoid biosynthesis. CO2 + H2O duced to form 2-C-methyl-D-erythritol 4- Synthesis of each IPP CH3 Isopentenyl phosphate and subsequently transformed unit requires three C CH2 CH2O P P diphosphate molecules of acetyl-CoA. CH2 (IPP) to yield IPP (Fig. 24.6, upper pathway). The

1256 Chapter 24 Natural Products (Secondary Metabolites) HMG-CoA MVA Phytoalexins Sterols MVA HMG-CoA

Cytoplasm Glycosylation Lumen site Lumen

Transmembrane domains

N C N-terminal Catalytic domain sequence Linker Linker

Figure 24.5 Model for the membrane topology of HMG-CoA reductase sis of sesquiterpenoid phytoalexins contain an N-linked glycosyla- (HMGR). The protein includes a highly variable hydrophilic N-ter- tion site exposed to the ER lumen. Differences in N-terminal se- minal sequence (blue), a conserved membrane anchor (orange), a quences and extent of glycosylation may affect targeting of HMGR highly variable linker sequence (green and purple), and a highly to various ER domains and to other organelles of the endomem- conserved, cytosol-exposed, C-terminal catalytic domain (yellow). brane system (see Chapters 1 and 4). ER, endoplasmic reticulum; Isoforms of HMGR that are associated with elicitor-induced synthe- MVA, mevalonic acid.

E CH2O P CH2O P HO TPP CH3 OH COO– C H C OH TPP E H C OH NADPH C CH2

C O CH3 HO C H H O C H H2C C O P

CO CH O P H OH CH3 2 2 HO C TPP E O C + OH NADP Pyruvate + H C OH H TPP E CH3 CH3

C 1-Deoxy-D-xylulose- 2-C-Methyl-D-erythritol- H O 5-phosphate 4-phosphate

GAP

2 Acetyl-CoA OS–CoA 5

C CH2OH 1 O S–CoA 3 C 4 O P P CH2 CH2 2 IPP COOH CH3 HO C CH HO C CH 3 2 NADPH 3 CH3 5 C O CH2 CH2 3 1 C 4 O P P CH3 O S–CoA COOH COOH 2 2 CoASH + IPP Pyruvate Acetyl-CoA HMG-CoA 2 NADP Mevalonic + acid CoASH

Figure 24.6 Feeding studies distinguish two pathways of isoprenoid biosynthe- from labeled pyruvate and GAP by the plastid-localized pathway sis. When glucose isotopically labeled at C-1 is transformed by gly- will be labeled at C-1 and C-5 (upper panel), whereas IPP formed colytic enzymes and pyruvate dehydrogenase, the label subse- from labeled acetyl-CoA by way of the cytosolic acetate/meval- quently appears in the methyl groups of pyruvate and acetyl-CoA onate pathway will be labeled at C-2, C-4, and C-5 (lower panel). and in C-3 of glyceraldehyde 3-phosphate (GAP). IPP synthezised

1257 discovery of this new pathway for IPP for- can be used to determine the 13C-labeling mation in plastids suggests that these organ- pattern of each isoprene unit in a terpenoid elles, presumed to have originated as pro- compound, allowing researchers to infer the karyotic endosymbionts, have retained the labeling pattern of the corresponding IPP bacterial machinery for the production of this units (Fig. 24.6). key intermediate of terpenoid biosynthesis. The details of the glyceraldehyde 3- phosphate/pyruvate pathway and the en- 24.3 Prenyltransferase and terpene zymes responsible have not yet been fully synthase reactions defined. However, products of the two IPP biosynthesis pathways can be easily dis- Prenyltransferase enzymes generate the al- tinguished in experiments that utilize lylic diphosphate esters geranyl diphosphate [1-13C]glucose as a precursor for terpenoid (GPP), farnesyl diphosphate (FPP), and ger- biosynthesis. Nuclear magnetic resonance anylgeranyl diphosphate (GGPP). Reactions (NMR) spectroscopy (see Chapter 2, Box 2.2) that these compounds undergo (often cyclizations), which are catalyzed by terpene synthases, yield a wide variety of terpenoid compounds. Both prenyltransferases and ter- C5 CH2OCHP P 2O P P pene synthases utilize electrophilic reaction Isopentenyl Dimethylallyl Hemiterpenes mechanisms involving carbocationic inter- diphosphate diphosphate IPP isomerase mediates, a feature of terpenoid biochemistry. Enzymes in both groups share similar P P i properties and contain conserved sequence elements, such as an aspartate-rich DDxxD C10 CH2O P P motif involved in substrate binding, which Geranyl Monoterpenes may participate in initiating divalent metal diphosphate ion–dependent ionizations. CH2O P P (IPP)

P P i 24.3.1 Repetitive addition of C units is C CH O P P 5 15 2 carried out by prenyltransferases. Farnesyl Sesquiterpenes 2 diphosphate IPP is utilized in a sequence of elongation CH2O P P (IPP) reactions to produce a series of prenyl P P i diphosphate homologs, which serve as the

C20 CH2O P P immediate precursors of the different families of terpenoids (Fig. 24.7). Isomerization of 2 Geranylgeranyl Diterpenes IPP by IPP isomerase produces the allylic diphosphate isomer dimethylallyl diphosphate (DMAPP), 2 P P i

C30

Squalene Triterpenes

2 P P i

C40

Phytoene Tetraterpenes

Figure 24.7 The major subclasses of terpenoids are biosynthesized from the derived from the corresponding intermediates by sequential head- basic five-carbon unit, IPP, and from the initial prenyl (allylic) to-tail addition of C5 units. Triterpenes (C30) are formed from two diphosphate, dimethylallyl diphosphate, which is formed by iso- C15 (farnesyl) units joined head-to-head, and tetraterpenes (C40) are merization of IPP. In reactions catalyzed by prenyltransferases, formed from two C20 (geranylgeranyl) units joined head-to-head. monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20) are

1258 Chapter 24 Natural Products (Secondary Metabolites) which is considered the first prenyl diphos- wide range of nonterpenoid compounds, phate. Because DMAPP and related prenyl including proteins. diphosphates contain an allylic double bond, The most extensively studied prenyl- these compounds can be ionized to generate transferase, farnesyl diphosphate synthase, resonance-stabilized carbocations. Once plays an important role in cholesterol bio- formed, a carbocation intermediate of n car- synthesis in humans. Farnesyl diphosphate bons can react with IPP to yield a prenyl synthases from microbes, plants, and ani- diphosphate homolog containing n + 5 car- mals exhibit high sequence conservation. The bons. Thus, the reactive primer DMAPP un- first enzyme of the terpenoid pathway to be dergoes condensation with IPP to yield the structurally defined is recombinant avian

C10 intermediate GPP. Repetition of the reac- farnesyl diphosphate synthase, the crystal tion cycle by addition of one or two structure of which has been determined. molecules of IPP provides FPP (C15) or GGPP (C20), respectively. Each prenyl homolog in the series arises as an allylic diphos- 24.3.2 The enzyme limonene synthase is a phate ester that can ionize to form a reso- model for monoterpene synthase action. nance-stabilized carbocation and condense with IPP in another round of elongation The families of enzymes responsible for the (Fig. 24.8). formation of terpenoids from GPP, FPP, and The electrophilic elongation reactions GGPP are known as monoterpene, sesquiter-

that yield C10, C15, and C20 prenyl diphos- pene, and diterpene synthases, respectively. phates are catalyzed by enzymes known col- These synthases use the corresponding lectively as prenyltransferases. GPP, FPP, and prenyl diphosphates as substrates to form GGPP are each formed by specific prenyl- the enormous diversity of carbon skeletons transferases named for their products (e.g., characteristic of terpenoids. Most terpenoids farnesyl diphosphate synthase). The new al- are cyclic, and many contain multiple ring lylic double bond introduced in the course systems, the basic structures of which are of the prenyltransferase reaction is common- determined by the highly specific synthases. ly in the trans geometry, although this is not Terpenoid synthases that produce cyclic always the case: The transferase responsible products are also referred to as “cyclases,” for rubber biosynthesis introduces cis-double although examples of synthases producing bonds, which are responsible for the elastici- acyclic products are also known. ty of that polymer. Prenylation reactions are A diverse array of monoterpene syn- not limited to elongations involving IPP; the thases has been isolated from essential oil- same basic carbocationic mechanism permits producing angiosperm species and resin- the attachment of prenyl side chains to atoms producing gymnosperms. These enzymes of carbon, oxygen, nitrogen, or sulfur in a use a common mechanism in which ionization

IPP + O P P OPP O P P + H H 2+ H M + R R 1 2 3 R O P P R P P i P P Allylic diphosphate ester (n carbons)i Allylic diphosphate ester (n + 5 carbons) 1 A divalent metal cation promotes 2 The cation is added to IPP, generating 3 Deprotonation of the enzyme-bound the ionization of an allylic diphosphate a tertiary carbocation that corresponds intermediate yields an allylic (prenyl) substrate, yielding a charge-delocalized to the next C5 isoprenolog. diphosphate five carbons longer than cation that probably remains paired with the starting substrate. the pyrophosphate anion.

Figure 24.8 The prenyltransferase reaction.

24.3–Prenyltransferase and Terpene Synthase Reactions 1259 O P P of GPP leads initially to the tertiary allylic pines, spruces, and firs. The compounds are isomer linalyl diphosphate (LPP; Fig. 24.9). toxic to bark beetles and their pathogenic This isomerization step is required because fungal symbionts, which cause serious dam- GPP cannot cyclize directly, given the pres- age to conifer species worldwide. Many con- ence of the trans-double bond. Ionization ifers respond to bark beetle infestation by of the enzyme-bound LPP intermediate pro- up-regulating synthesis of monoterpenes, Geranyl diphosphate motes cyclization to a six-membered ring a process analogous to the production of 2+ M carbocation (the α-terpinyl cation), which antimicrobial phytoalexins, when under P P i may undergo additional electrophilic cycli- pathogen attack (Fig. 24.11). Other monoter- + zations, hydride shifts, or other rearrange- penes have quite different functions. Thus, ments before the reaction is terminated by linalool (see Fig. 24.10) and 1,8-cineole emit- deprotonation of the carbocation or capture ted by flowers serve as attractants for polli- by a nucleophile (e.g., water). Variations on nators, including bees, moths, and bats. this simple mechanistic scheme, involving 1,8-Cineole and camphor act as foliar feed- subsequent reactions of the α-terpinyl carbo- ing deterrents to large herbivores such as O P P cation, are responsible for the enzymatic for- hares and deer and also may provide a mation of most monoterpene skeletons (see competitive advantage to several angio- Box 24.1). sperm species as allelopathic agents that The simplest monoterpene synthase re- inhibit germination of the seeds of other action is catalyzed by limonene synthase, a species. (3S)-Linalyl diphosphate useful model for all terpenoid cyclizations Exceptions to the general pattern of (transoid rotamer) (Fig. 24.9). The electrophilic mechanism of head-to-tail joining of isoprene units seen action used by limonene synthase can be in limonene, the pinenes, and most other O P P viewed as an intramolecular equivalent of monoterpenes derived from GPP are the the prenyltransferase reaction (see Fig. 24.8). “irregular” monoterpenes. An example of Synthases that produce acyclic olefin prod- this type is the family of insecticidal mono- ucts (e.g., myrcene) and bicyclic products (α- terpene esters called pyrethrins, found in and β-pinene) from GPP are also known, as Chrysanthemum and Tanacetum species. are enzymes that transform GPP to oxy- These monoterpenoids, which exhibit a (3S)-Linalyl diphosphate (cisoid rotamer) genated derivatives such as 1,8-cineole and head-to-middle joining of C5 units, have bornyl diphosphate (Fig. 24.10), the precur- gained wide use as commercial insecticides 2+ M sor of camphor (see Box 24.1). because of their negligible toxicity to mam- An interesting feature of the monoter- mals and their limited persistence in the pene synthases is the ability of these en- environment (see Fig. 24.10). P P i + zymes to produce more than one product; for example, pinene synthase from several plant sources produces both α- and β-pinene. 24.3.3 Sesquiterpene synthases generate The pinenes are among the most common several compounds that function in monoterpenes produced by plants and are plant defense. principal components of turpentine of the The electrophilic mechanisms for the forma-

tion of the C15 sesquiterpenes from FPP Figure 24.9 closely resemble those used by monoterpene (–)-Limonene synthase catalyzes the simplest of all synthases, although the increased flexibility P P i terpenoid cyclizations and serves as a model for this of the 15-carbon farnesyl chain eliminates + reaction type. Ionization of GPP, assisted by divalent α-Terpinyl cation metal ions, provides the delocalized carbocation– the need for the preliminary isomerization diphosphate anion pair, which collapses to form the step except in forming cyclohexanoid-type P P i enzyme-bound tertiary allylic intermediate linalyl compounds. The additional C5 unit and dou- diphosphate. This required isomerization step, fol- ble bond of FPP also permit formation of a lowed by rotation about the C-2–C-3 single bond, overcomes the original stereochemical impediment to greater number of skeletal structures than direct cyclization of the geranyl precursor. A subse- in the monoterpene series. The best known quent assisted ionization of the linalyl diphosphate sesquiterpene synthase of plant origin is epi- α ester promotes an anti-endo-cyclization to the -ter- aristolochene synthase from tobacco, the pinyl cation, which undergoes deprotonation to form limonene, a compound now thought to be an impor- crystal structure of which has been deter- (–)-Limonene tant cancer preventive in humans. mined (Fig. 24.12). This enzyme cyclizes FPP

1260 Chapter 24 Natural Products (Secondary Metabolites) and catalyzes a methyl migration to yield the olefin precursor of the phytoalexin capsidiol, which is elicited by pathogen attack. Vetispiradiene synthase from potato provides the olefin precursor of the phytoalexin lubimin in this species, whereas α Myrcene -Pinene δ-cadinene synthase from cotton yields the OH olefin precursor of the important defense compound gossypol, the latter being currently studied as a possible male contraceptive (Fig. 24.13). Some sesquiterpene synthases involved in the production of conifer resin are capable of individually producing more Limonene Linalool than 25 different olefins.

O 24.3.4 Diterpene synthases catalyze two distinct types of cyclization reactions.

Two fundamentally different types of enzy- β-Pinene 1,8-Cineole matic cyclization reactions occur in the COOR transformation of GGPP to diterpenes (Fig. O P P 24.14). The first resembles the reactions catalyzed by monoterpene and sesquiterpene synthases, in which the cyclization involves ionization of the diphosphate ester and at- Bornyl diphosphate Pyrethrin I tack of the resulting carbocation on an interi- Figure 24.10 or double bond of the geranylgeranyl sub- Structures of monoterpenes, including insecticidal strate. An example of this type is casbene α β compounds ( - and -pinene, pyrethrin), pollinator synthase, which is responsible for produc- attractants (linalool and 1,8-cineole), and antiher- bivory agents (1,8-cineole). tion of the phytoalexin casbene in castor bean. Taxadiene synthase from yew species uses a mechanistically similar, but more complex, cyclization to produce the tricyclic olefin precursor of taxol. Abietadiene synthase from grand fir exemplifies the second type of cyclization, in which protonation of the terminal double bond to generate a carbonium ion initiates the first cyclization to a bicyclic intermediate (labdadienyl diphosphate, also known as copalyl diphosphate). Ionization of the diphosphate ester promotes the second cyclization step to give the tricyclic olefin product, abietadiene; a single enzyme catalyzes both

Figure 24.11 Mass attack by mountain pine beetles on a lodgepole pine (Pinus contorta) bole. Each white spot on the trunk represents a beetle entry point at which resin has been secreted. This tree has survived the attack because turpentine production was sufficient to kill all of the bark beetles, which have been “pitched out” by resin outflow. On evaporation of the turpentine and exposure to air, the diterpenoid resin acids form a solid plug that seals the wound.

24.3–Prenyltransferase and Terpene Synthase Reactions 1261 cyclization steps. Oxidation of a methyl 24.3.5 Triterpene synthesis proceeds group yields abietic acid (see Fig. 24.1), one from squalene, tetraterpene synthesis of the most common diterpenoid resin acids from phytoene. of conifers and important for wound sealing in these species. Fossilization of this resin Before cyclization can occur in the triterpene

produces amber. (C30) series, two molecules of FPP (C15) are first joined in a head-to-head condensation to produce squalene (see Fig. 24.7). The cata- lyst, squalene synthase, is a prenyltransferase that catalyzes a complex series of cationic C terminus (548) rearrangements to accomplish the chemically difficult chore of joining the C-1 carbons of 2 3 1 two farnesyl residues. Squalene is usually oxidized to form the 2,3-epoxide, oxidosqua- 4 8 lene, and then cyclized in a protonation- 7 5 6 initiated reaction to produce, for example, 36 534 A K J 521 I D J/K loop OH C H1 W273 G2 FHP R266 H2 E HO

R264 H3 α -1 Capsidiol 255 D1 F Mg2+ G1 D2 A/C loop N terminus (17)

Figure 24.12 epi-Aristolochene Schematic view of epi-aristolochene synthase com- plexed with the substrate analog farnesyl hydroxy- epi-Aristolochene phosphonate (FHP). Blue rods represent α-helices in synthase the N-terminal domain; red rods represent α-helices in the C-terminal domain. Loop regions shown in purple are disordered in the native enzyme. Three Mg2+ ions and arginines 264 and 266 are involved in the initial steps of the reaction and are labeled near δ-Cadinene Vetispiradiene the entrance to the active site. Tryptophan 273, which synthase O P P synthase serves as the general base in the final deprotonation FPP step, is shown within the hydrophobic active-site pocket. The substrate analog FHP is shown in ball- and-stick representation, highlighted with yellow carbon–carbon bonds. Naming of helices in the C-terminal domain corresponds to the convention used for FPP synthase.

δ-Cadinene Vetispiradiene

CHO OH OH CHO CHO HO OH

HO HO OH Figure 24.13 Structures of sesquiterpenes biosynthetical- ly derived from FPP. The end products Gossypol Lubimin function in plant defense. (Sesquiterpene dimer)

1262 Chapter 24 Natural Products (Secondary Metabolites) Casbene synthase O P P

Taxadiene Geranylgeranyl synthase Casbene diphosphate

Abietadiene synthase

Taxadiene

O P P

Abietadiene synthase

Labdadienyl Abietadiene (copalyl) diphosphate

Figure 24.14 Cyclization of GGPP to form the diterpenes casbene, taxadiene, and abietadiene. Cyclization can proceed by one of two distinct mechanims, only one of which yields the intermediate labdadienyl (copalyl) diphosphate.

the common sterol cycloartenol (Fig. 24.15), thase. A series of desaturation steps precedes a precursor of many other phytosterols and cyclization in the tetraterpene (carotenoid) brassinosteroids (see Chapter 17). Several al- series, usually involving formation of six- ternative modes of cyclization in the triter- membered (ionone) rings at the chain termi- pene series are also known, such as that ni to produce, for example, β-carotene from leading to the pentacyclic compound lycopene (see Chapter 12, Fig. 12.7). β-amyrin, the precursor of oleanolic acid found in the surface wax of several fruits (Fig. 24.15). Preliminary evidence suggests 24.4 Modification of terpenoid skeletons that sesquiterpene biosynthesis and triterpene biosynthesis (both of which utilize cy- Subsequent modifications of the basic parent tosolic FPP as a precursor) are reciprocally skeletons produced by the terpenoid syn- regulated during the induced defense re- thases are responsible for generating the

sponses, such that production of C15 defen- myriad different terpenoids produced by sive compounds is enhanced and C30 synthe- plants. These secondary transformations sis is repressed. most commonly involve oxidation, reduc-

The tetraterpenes (C40) are produced by tion, isomerization, and conjugation reac- joining two molecules of GGPP in head-to- tions, which impart functional properties to head fashion to produce phytoene, in a man- the terpenoid molecules. Several oxygenated ner analogous to the formation of squalene derivatives of parent terpenoids have al- (see Fig. 24.7). The reaction is catalyzed by ready been described in this chapter, includ- phytoene synthase, which deploys a mecha- ing capsidiol, lubimin, gossypol, abietic acid, nism very similar to that of squalene syn- and oleanolic acid.

24.4–Modification of Terpenoid Skeletons 1263 HO

OH HO

HO HO

O Cycloartenol O Brassinolide

COOH O

Oxidosqualene

HO HO Figure 24.15 β Structures of triterpenes. -Amyrin Oleanolic acid This class of squalene- derived products includes brassinosteroid regulators of plant Many of the hydroxylations or epoxida- reductase to produce (+)-cis-isopulegone. An growth and surface wax tions involved in introducing oxygen atoms isomerase next moves the remaining double components. into the terpenoid skeletons are performed bond into conjugation with the carbonyl by cytochrome P450 mixed-function oxidas- group, yielding (+)-pulegone. One regiospe- es. Because these reactions are not unique to cific, NADPH-dependent, stereoselective terpenoid biosynthesis, this section will not reductase converts (–)-pulegone to either focus on specific enzyme types but rather on (+)-isomenthone or the predominant species, the general role of secondary transforma- (–)-menthone. Similar reductases produce tions as the wellspring of diversity in ter- the menthol isomers from these ketones. penoid structure and function. (–)-Menthol greatly predominates among the menthol isomers (constituting as much as 40% of the essential oil) and is the compo- 24.4.1 The conversion of (–)-limonene to nent primarily responsible for the character- (–)-menthol in peppermint and carvone in istic flavor and cooling sensation of pepper- spearmint illustrates the biochemistry of mint. The menthol isomers are often found terpenoid modification. as acetate esters, formed by the action of an acetyl CoA-dependent acetyltransferase. The The principal and characteristic essential oil menthol and menthyl acetate content of pep- components of peppermint (Mentha piperita) permint oil glands increases with leaf matu- and spearmint (M. spicata) are produced by rity. Environmental factors greatly influence secondary enzymatic transformations of oil composition. Water stress and warm (–)-limonene (Fig. 24.16). In peppermint, night growth conditions both promote the a microsomal cytochrome P450 limonene accumulation of the more-oxidized pathway 3-hydroxylase introduces an oxygen atom intermediates such as (+)-pulegone. at an allylic position to produce (–)-trans- The pathway in spearmint is much isopiperitenol. A soluble NADP+-dependent shorter. In this instance, a cytochrome P450 dehydrogenase oxidizes the alcohol to a ke- limonene 6-hydroxylase specifically intro- tone, (–)-isopiperitenone, thereby activating duces oxygen at the alternative allylic posi- the adjacent double bond for reduction by a tion to produce (–)-trans-carveol, which soluble, NADPH-dependent, regiospecific is oxidized to (–)-carvone by the soluble

1264 Chapter 24 Natural Products (Secondary Metabolites) O P P 1 6 2

5 3 O 4 OH

Geranyl (–)-Limonene (–)-trans- (–)-Isopiperitenone diphosphate Isopiperitenol

(–)-trans-Carveol (+)-cis-Isopulegone

(–)-Carvone (+)-Pulegone

O O

(–)-Menthone (+)-Isomenthone

Figure 24.16 Essential oil synthesis in peppermint and spearmint. In peppermint, (–)-limonene is converted to (–)-isopiperitenone, OOCCH OH OH OH OH which is modified to form 3 (–)-menthol and related compounds. In spearmint, (–)-limonene is converted (–)-Menthyl (–)-Menthol (+)-Neomenthol (+)-Isomenthol (+)-Neoisomenthol to (–)-carvone by a acetate two-step pathway.

1265 NADP+-dependent dehydrogenase. Al- mals. Monoterpene lactones include nepeta- though most of the enzymatic machinery lactone (the active principle of catnip as well present in peppermint oil glands is also as an aphid pheromone), a member of the present in spearmint, the specificity of these iridoid family of monoterpenes, which are enzymes is such that (–)-carvone is a very formed by a cyclization reaction quite different poor substrate. Consequently, carvone, the from that of other monoterpenes (Fig. 24.17). characteristic component of spearmint flavor, The limonoids are a family of oxygenat- accumulates as the major essential oil com- ed nortriterpene antiherbivore compounds. ponent (about 70%). Similar reaction se- Like the sesquiterpene lactones, these sub- quences initiated by allylic hydroxylations stances taste very bitter to humans and prob- and subsequent redox metabolism and con- ably to other mammals as well. A powerful jugations are very common in the monoter- insect antifeedant compound is azadirachtin pene, sesquiterpene, and diterpene classes. A, a highly modified limonoid from the neem tree (Azadirachta indica). Other oxygenated triterpenoid natural products with unusual 24.4.2 Some terpenoid skeletons are biological properties include the phytoecdy- extensively decorated. sones, a family of plant steroids that act as hormones and stimulate insect molting; the Reactions similar to those responsible for saponins, so named because of their soap- essential oil production in mints generate like, detergent properties; and the cardeno- myriad terpenoid compounds of biological lides, which, like the saponins, are glyco- or pharmaceutical interest. Such reactions sides, in that they bear one or more attached convert sesquiterpene olefin precursors to sugar residues. Ingestion of α-ecdysone by phytoalexins (see Fig. 24.13), allelopathic insects disrupts the molting cycle, usually agents, and pollinator attractants. Additional with fatal consequences. The saponins and sesquiterpenes generated by modifying cardenolides are toxic to many vertebrate olefin precursors include juvabione (Fig. herbivores; this family of compounds in- 24.17), a compound from fir species that cludes well-known fish poisons and snail exhibits insect juvenile hormone activity; poisons of significance in the control of sirenin, a sperm attractant of the water mold schistosomiasis. Many of these products are allomyces; and artemisinin, a potent anti- also cardioactive and anticholesterolemic malarial drug from annual wormwood agents of pharmacological significance. Digi- (Artemisia annua, also known as Qinghaosu, toxin, the glycone (glycosylated form) of a plant used in traditional Chinese medicine digitoxigenin (Fig. 24.17) extracted from fox- since about 200 B.C.). A related enzymatic glove (Digitalis), is used widely in carefully reaction sequence converts the parent diter- prescribed doses for treatment of congestive pene olefin taxadiene to the anticancer drug heart disease. taxol in yew species, in which the basic ter- The broad range of insect and higher an- penoid nucleus is modified extensively by a imal toxins and deterrents among the modi- complex pattern of hydroxylations and acy- fied triterpenes leaves little doubt as to their lations. Esters of phorbol (another highly role in plant defense. Interestingly, some her- oxygenated diterpene) produced by species bivores have developed the means to cir- of the Euphorbiaceae are powerful irritants cumvent the toxic effects of these terpenoids and cocarcinogens. After introduction of a and adapt them to their own defense pur- hydroxyl group, subsequent oxidation can poses. The classical example of this phe- generate a carboxyl function such as that nomenon is the monarch butterfly, a special- found in abietic acid (see Fig. 24.1) and ist feeder on milkweeds (Asclepias) which oleanolic acid, and also provide the struc- contain cardenolides that are toxic to most tural elements for lactone ring formation. herbivores and are even associated with live- Sesquiterpenes bearing such lactone rings, stock poisoning. Monarch caterpillars, how- e.g., costunolide, are produced and accumu- ever, feed on milkweeds and accumulate the lated in the glandular hairs on the leaf sur- cardenolides without apparent ill effects. As faces of members of the Asteraceae, where a result, both caterpillars and the adult but- some of these compounds serve as feeding terflies contain enough cardenolides to be repellents to herbivorous insects and mam- toxic to their own predators such as birds.

1266 Chapter 24 Natural Products (Secondary Metabolites) O O O OH O COOCH3 O HO O Juvabione Sirenin Artemisinin (insect juvenile (sperm attractant in (antimalarial drug) hormone analog) water molds)

OH OOCCH3 O OH OH NH O O HO

O O OH OH H OOCCH3 O O O HO OH O Taxol O Phorbol Costunolide (anticancer drug) (irritant and cocarcinogen) (insect repellent, mammal antifeedant)

OH COOCH O 3 HO O OH OH C O O O O

HO O H3CCOO OH OH H3COOC O O HO O Nepetalactone Azadirachtin A α-Ecdysone (active principle of catnip) (insect antifeedant) (disrupts insect molting cycle)

O O

O O Figure 24.17 Terpenoids formed by OH secondary transforma- HO tions of parent cyclic HO H compounds. The yellow highlighting delineates Hecogenin, Digitoxigenin, the terpenoid portion of the aglycone of a saponin the aglycone of digitoxin, a cardenolide the molecule taxol. (a detergent) (treatment of congestive heart disease)

1267 24.5 Toward transgenic an even greater challenge, given that little terpenoid production metabolic context exists in these cases. In such species, issues of subcellular sites of With recent success in the cloning of genes synthesis, requirements for sufficiency of that encode enzymes of terpenoid synthesis, precursor flux, and the fate of the desired the transgenic manipulation of plant ter- product might present additional difficulties. penoid metabolism may present a suitable Clearly, targeting a terpenoid synthase to the avenue for achieving a number of goals. Sev- cellular compartment containing the appro-

eral agriculturally important crop species priate C10, C15, C20, or C30 precursor will be have been bred selectively to produce rela- an important consideration. Sufficient flux of tively low amounts of unpalatable terpenoid IPP at the production site to drive the path- defense compounds; in the process, these way also will be essential. Because con- cultivars have lost not only defense capabili- straints in precursor flow ultimately will ties but also, in some cases, quality attributes limit the effectiveness of transgenes for sub- such as flavor and color. The selective rein- sequent pathway steps, information about troduction of terpenoid-based defense the flux controls on IPP biosynthesis in both chemistry is certainly conceivable, as is the cytosol and plastid, and about the interac- engineering of pathways into fruits and veg- tions of these controls, is sorely needed. etables to impart desirable flavor properties. Very few published examples of the ge- The aroma profiles of ornamental plant netic engineering of terpenoid metabolism species might be modified by similar ap- are currently available, although two notable proaches. Likewise, transgene expression successes have been achieved in the area of might accelerate the rate of slow biosynthetic terpenoid vitamins. The ratio of beneficial steps and thereby increase the yields of tocopherol (vitamin E) isomers in oilseeds essential oils used in flavors and perfumes, has been altered by this means, and an in- phytopharmaceuticals (e.g., artemisinin creased concentration of β-carotene (a vita- and taxol), insecticides (e.g., pyrethrins and min A precursor) in both rice kernels and azadirachtin), and a wide range of industrial rapeseed has been obtained by manipulating intermediates that are economically inacces- the carotenoid pathway. In another, caution- sible by traditional chemical synthesis. ary example, however, overexpression in a The genetic engineering of terpenoid- transgenic tomato of the enzyme that diverts based insect defenses is particularly appeal- GGPP to carotenoids resulted in a dwarf ing, given the array of available monoterpene, phenotype, an unintended consequence of sesquiterpene, diterpene, and triterpene depleting the precursor of the gibberellin compounds that are toxic to insects not plant hormones. adapted to them. Attracting predators and parasitoids of the target insect or modifying host attractants, oviposition stimulators, and 24.6 Alkaloids pheromone precursors offers even more so- phisticated strategies for pest control. For ef- 24.6.1 Alkaloids have a 3000-year history fective transgenic manipulation of such ter- of human use. penoid biosynthetic pathways, promoters for tissue-specific, developmentally controlled, For much of human history, plant extracts and inducible expression are required, as are have been used as ingredients in potions promoters for targeting production to secre- and poisons. In the eastern Mediterranean, tory structures of essential oil plants and use of the latex of the opium poppy (Papaver conifers. The latter are the most likely somniferum; Fig. 24.18) can be traced back at species for initial manipulation because they least to 1400 to 1200 B.C. The Sarpagandha already are adapted for terpenoid accumula- root (Rauwolfia serpentina) has been used in tion, and the antecedent and subsequent India since approximately 1000 B.C. Ancient metabolic steps are largely known. people used medicinal plant extracts as The engineering of terpenoid biosyn- purgatives, antitussives, sedatives, and treat- thetic pathways into plant species that do ments for a wide range of ailments, includ- not ordinarily accumulate these natural ing snakebite, fever, and insanity. As the use products presents a greater opportunity but of medicinal plants spread westward across

1268 Chapter 24 Natural Products (Secondary Metabolites) Arabia and Europe, new infusions and de- coctions played a role in famous events. Dur- ing his execution in 399 B.C., the philoso- (A) (B) pher Socrates drank an extract of coniine- containing hemlock (Conium maculatum; Fig. 24.19). In the last century B.C., Queen Cleopatra used extracts of henbane (Hyoscya- mus), which contains atropine (Fig. 24.20), to dilate her pupils and appear more alluring to her male political rivals. Over the centuries, the king of all medic- inals has been opium, which was widely consumed in the form of Theriak, a concoc- tion consisting mainly of opium, dried snake meat, and wine (Box 24.2). Analysis of the individual components of opium led to the identification of morphine (Fig. 24.21A), named for Morpheus, the god of dreams in Figure 24.18 Greek mythology. The isolation of morphine (A) Maturing capsule of the opium poppy Pa- in 1806 by German pharmacist Friedrich paver somniferum. When the capsule is wound- Sertürner gave rise to the study of alkaloids. ed, a white, milky latex is exuded. Poppy latex The term alkaloid, coined in 1819 in contains morphine and related alkaloids such as codeine. When the exuded latex is allowed Halle, Germany, by another pharmacist, Carl to dry, a hard, brown substance called opium Meissner, finds its origin in the Arabic name is formed. (B) Statuette from Gazi of a goddess al-qali, the plant from which soda was first of sleep crowned with capsules of the opium isolated. Alkaloids were originally defined poppy (1250–1200 B.C.). as pharmacologically active, nitrogen- containing basic compounds of plant origin.

(B)

H H N Conium maculatum

Coniine

Figure 24.19 (A). The piperidine alkaloid coniine, the first alkaloid to be synthe- ing an extract of coniine-containing poisonous hemlock. This depic- sized, is extremely toxic, causing paralysis of motor nerve endings. tion of the event, “The Death of Socrates,” was painted by Jacques- (B) In 399 B.C., the philosopher Socrates was executed by consum- Louis David in 1787.

24.6–Alkaloids 1269 sis de novo occurs in each organism. Many CH3 of the alkaloids that have been discovered N are not pharmacologically active in mammals and some are neutral rather than basic in character, despite the presence of a nitrogen atom in the molecule. CH2OH Alkaloid-containing plants were OOCCH mankind’s original “materia medica.” Many are still in use today as prescription drugs Figure 24.20 (Table 24.1). One of the best-known prescrip- Stucture of the anti- tion alkaloids is the antitussive and anal- cholinergic tropane alkaloid atropine from gesic codeine from the opium poppy (Fig. Hyoscyamus niger. Hyoscyamus niger Atropine 24.21A). Plant alkaloids have also served as models for modern synthetic drugs, such as the tropane alkaloid atropine for tropicamide used to dilate the pupil during eye examina- After 190 years of alkaloid research, this def- tions and the indole-derived antimalarial al- inition as such is no longer comprehensive kaloid quinine for chloroquine (Fig. 24.22). enough to encompass the alkaloid field, but In addition to having a major impact on in many cases it is still appropriate. Alka- modern medicine, alkaloids have also influ- loids are not unique to plants. They have enced world geopolitics. Notorious examples also been isolated from numerous animal include the Opium Wars between China and sources (Fig. 24.21B and Box 24.3), although Britain (1839–1859) and the efforts currently still to be determined is whether biosynthe- underway in various countries to eradicate

Theriak, an ancient antipoisoning nostrum containing opium, Box 24.2 wine, and snake meat, is still used today in rare instances.

One of the oldest and most long-lived (A) (B) medications in the history of mankind is Theriak. Originating in Greco-Roman culture, Theriak consists of mainly opium and wine with a variety of plant, animal, and mineral constituents. Panel A of the figure shows a recipe for Theriak from the French Pharmacopée Royale in 1676. Theriak was developed as an antidote against poisoning, snake bites, spider bites, and scorpion stings. History has it that the Roman Emperor Nero contracted the Greek physician Andromachus to dis- cover a medicine that was effective against all diseases and poisons. Andro- machus improved the then-existing recipe to include, in addition to opium, five other plant poisons and 64 plant drugs. An- other crucial component was dried snake meat, believed to act against snake bites by neutralizing the venom. Today, Theriak is still prescribed in rare cases in Europe for pain and other ailments. Panel B shows a valuable Theriak-holding vessel made of Nym- phenburg porcelain (in about 1820), which is on display in the Residenz Phar- macy in Munich, Germany.

1270 Chapter 24 Natural Products (Secondary Metabolites) (A) (B)

H3CO HO

O O

N CH3 N CH3 H H

HO HO Papaver somniferum Codeine Morphine

Figure 24.21 (A) Structures of the alkaloids codeine and mor- illicit production of heroin, a semisynthetic phine from the opium poppy Papaver somnifer- compound derived by acetylation of mor- um. Asymmetric (chiral) carbons are highlighted phine (Fig. 24.23), and cocaine, a naturally with red dots. (B) The frog Bufo marinus accu- occurring alkaloid of the coca plant (Fig. mulates a considerable amount of morphine in its skin. 24.24). Because of their various pharmacological activities, alkaloids have influenced

Some butterflies and moths use alkaloids for sexual signaling or for Box 24.3 protection against predators.

Alkaloid-bearing species have been courtship success of these male butterflies (A) found in nearly all classes of organisms, therefore depends on their ingesting alka- including frogs, ants, butterflies, bacteria, loids from higher plants. sponges, fungi, spiders, beetles, and The larvae of a second insect group, mammals. Alkaloids of various structures the Ithomiine butterflies, feed on solana- have been isolated from a variety of ma- ceous plants and sequester the plant tox- rine creatures. Some animals, such as am- ins, including tropane alkaloids and phibians, produce an array of either toxic steroidal glycoalkaloids. However, the or noxious alkaloids in the skin or the se- adult Ithomiinae do not contain these cretory glands. Others, such as the insects Solanaceae alkaloids but prefer to ingest described below, use plant alkaloids as a plants that produce pyrrolizidine alka- source of attractants, pheromones, and loids, sequestering these bitter substances defense substances. as N-oxides and monoesters. The pyr- Some butterflies gather alkaloidal pre- rolizidine alkaloid derivatives protect cursors from plants that are not their food Ithomiinae butterflies from an abundant sources and convert these compounds predator, the giant tropical orb spider. The into pheromones and defense com- spider will release a field-caught butterfly pounds. Larvae of the cinnabar moth, Tyr- from its web but will readily eat a freshly (B) ia jacobaea, continuously graze their emerged adult that has not yet had an plant host Senecio jacobaea until the opportunity to feed on the preferred host plant is completely defoliated (see panel plant. When palatable butterflies were A). The alkaloids thus obtained by the lar- painted externally with a solution of vae are retained throughout metamorpho- pyrrolizidine alkaloids, the spider re- sis. Male Asian and American arctiid leased them from its web. In contrast, moths incorporate pyrrolizidine alkaloids palatable butterflies treated the same way into their reproductive biology by seques- with Solanaceae alkaloids were devoured. tering these alkaloids in abdominal scent In general, mostly male butterflies are organs called coremata, which are evert- found feeding on the pyrrolizidine alka- ed in the final stages of their courtship to loid-accumulating plants; however, as release the pheromones necessary to gain much as 50% of the pyrrolizidine alka- acceptance by a female. The coremata of loids present in these males is sequestered a male Asian arctiid moth (Creatonotos in the spermatophores and transferred to transiens) are directly proportional to the females at mating. In some butterfly pyrrolizidine alkaloid content of its diet species, the protective alkaloids are then during the larval stage (see panel B). The transferred to the eggs.

24.6–Alkaloids 1271 Table 24.1—Physiologically active alkaloids used in modern medicine Alkaloid Plant source Use Ajmaline Rauwolfia serpentina Antiarrythmic that functions by inhibiting glucose uptake by heart tissue mitochondria Atropine, Hyoscyamus niger Anticholinergic, antidote to nerve gas poisoning -(±)-hyoscyamine Caffeine Coffea arabica Widely used central nervous system stimulant Camptothecin Camptotheca acuminata Potent anticancer agent Cocaine Erythroxylon coca Topical anaesthetic, potent central nervous system stimulant, and adrenergic blocking agent; drug of abuse Codeine Papaver somniferum Relatively nonaddictive analgesic and antitussive Coniine Conium maculatum First alkaloid to be synthesized; extremely toxic, causes paralysis of motor nerve endings, used in homeopathy in small doses Emetine Uragoga ipecacuanha Orally active emetic, amoebicide Morphine P. somniferum Powerful narcotic analgesic, addictive drug of abuse Nicotine Nicotiana tabacum Highly toxic, causes respiratory paralysis, horticultural insecticide; drug of abuse Pilocarpine Pilocarpus jaborandi Peripheral stimulant of the parasympathetic system, used to treat glaucoma Quinine Cinchona officinalis Traditional antimalarial, important in treating Plasmodium falciparum strains that are resistant to other antimalarials Sanguinarine Eschscholzia californica Antibacterial showing antiplaque activity, used in toothpastes and oral rinses Scopolamine H. niger Powerful narcotic, used as a sedative for motion sickness Strychnine Strychnos nux-vomica Violent tetanic poison, rat poison, used in homeopathy (+)-Tubocurarine Chondrodendron tomentosm Nondepolarizing muscle relaxant producing paralysis, used as an adjuvant to anaesthesia Vinblastine Catharanthus roseus Antineoplastic used to treat Hodgkin’s disease and other lymphomas.

24.6.2 Physiologically active alkaloids participate in plant chemical defenses.

More than 12,000 alkaloids have been isolated since the discovery of morphine. About 20% of the species of flowering plants pro- CH2 CH duce alkaloids, and each of these species ac- H cumulates the alkaloids in a unique, defined pattern. Some plants, such as the periwinkle (Catharanthus roseus) contain more than 100 H N different monoterpenoid indole alkaloids. Cinchona officinalis HO Why should a plant invest so much nitrogen H into synthesizing such a large number of al- CH3O kaloids of such diverse structure? The role of Quinine alkaloids in plants has been a longstanding question, but a picture has begun to emerge N that supports an ecochemical function for Figure 24.22 these compounds. Structure of the monoterpenoid indole alkaloid- The role of chemical defense for alka- derived quinine from Cinchona officinalis. An anti- loids in plants is supported by their wide malarial quinine-containing tonic prepared from range of physiological effects on animals and the bark of C. officinalis greatly facilitated European exploration and inhabitation of the tropics during by the antibiotic activities many alkaloids the past two centuries. possess. Various alkaloids also are toxic to insects or function as feeding deterrents. For example, nicotine, found in tobacco, was human history profoundly, both for good and one of the first insecticides used by humans ill. Of interest to plant biologists, however, is and remains one of the most effective (Fig. the evolutionary selection process in plants 24.25). Herbivory has been found to stimu- that has caused alkaloids to evolve into such late nicotine biosynthesis in wild tobacco a large number of complex structures and to plants. Another effective insect toxin is caf- remain effective over the millennia. feine, found in seeds and leaves of cocoa,

1272 Chapter 24 Natural Products (Secondary Metabolites) O

H3C C O

N CH3 H

H3C C O H O Heroin N

Figure 24.23 CH3 Structure of diacetyl morphine, commonly N known as heroin. Nicotiana tabacum Nicotine Figure 24.25 Structure of nicotine from Nicotiana tabacum. The coffee, cola, maté, and tea (Fig. 24.26). At a asymmetric chiralcarbon is highlghted with a red dot. dietary concentration well below that found in fresh coffee beans or tea leaves, caffeine kills nearly all larvae of the tobacco horn- S. vernalis, 60% to 80% of the pyrrolizidine worm (Manduca sexta) within 24 hours— alkaloids is found to accumulate in the inflo- primarily by inhibiting the phosphodieste- rescences. Members of the Senecio genus are rase that hydrolyzes cAMP. The steroid alka- responsible for livestock poisonings and also α loid -solanine, a cholinesterase inhibitor represent a potential health hazard for hu- found in potato tuber (Fig. 24.27), is the trace mans. Naturally occurring pyrrolizidine al- toxic constituent thought to be responsible kaloids are harmless but become highly toxic for the teratogenicity of sprouting potatoes. when transformed by cytochrome P450 Two groups of alkaloids that have been monooxygenases in the liver. On the other well studied with respect to ecochemical hand, several insect species have adapted to function are the pyrrolizidine and quino- the pyrrolizidine alkaloids that accumulate lizidine alkaloids. The pyrrolizidine alka- in plants and have evolved mechanisms for loids, frequently found in members of the using these alkaloids to their own benefit. tribe Senecioneae (Asteraceae) and in the Some insects can feed on pyrrolizidine alka- Boraginaceae, render most of these plants loid-producing plants and effectively and toxic to mammals. In Senecio species (Fig. 24.28), senecionine N-oxide is synthesized in the roots and translocated throughout the plant. In species such as Senecio vulgaris and

CH3 COOCH3 N

OOC O CH3 H CH3 N Cocaine N

N Figure 24.24 O N Structure of the tropane alka- Figure 24.26 loid cocaine, a central nervous Structure of the purine CH3 system stimulant derived from alkaloid caffeine from Erythroxylon coca Erythroxylon coca. Coffea arabica Caffeine Coffea arabica.

24.6–Alkaloids 1273 H H

N H OH OH OH H H H HO O O O HO O OH O Solanidine O HO HO HO

Solanum tuberosum α-Solanine

Figure 24.27 seed-bearing stage of the plant life cycle— α Structure of the steroid alkaloid glycoside -solanine from Solanum tuberosum the seeds being the plant parts that accumu- (potato). The aglycone solanidine is derived from cholesterol. late the greatest quantities of these alkaloids. Because of their bitter taste, lupine alkaloids efficiently eliminate the alkaloids after enzy- can also function as feeding deterrents. Given matic modification, such as formation of N- a mixed population of sweet and bitter lu- oxide derivatives. Other insects not only pines, rabbits and hares will readily eat the feed on these plants, but also store the pyr- alkaloid-free sweet variety and avoid the rolizidine alkaloids for their own defense or lupine alkaloid-accumulating bitter variety, convert the ingested pyrrolizidine alkaloids indicating that lupine alkaloids in plants to pheromones that attract prospective mates serve to reduce herbivory by functioning (Box 24.3). both as bitter-tasting deterrents and toxins. The quinolizidine alkaloids occur pri- Given this collection of examples, alkaloids marily in the genus Lupinus and are fre- can be viewed as a part of the chemical de- quently referred to as lupine alkaloids (Fig. fense system of the plant that evolved under 24.29); they are toxic to grazing animals, par- the selection pressure of predation. ticularly to sheep. The highest incidence of livestock losses attributable to lupine alkaloid poisoning occurs in autumn during the

HO O H N

O O O N H H

N Lupinus polyphyllus Lupanine Senecio jacobaea Senecionine Figure 24.29 Structure of the quinolizidine alkaloid lupanine Figure 24.28 from the bitter lupine Lupinus polyphyllus. Lupa- Structure of the pyrrolizidine alkaloid senecionine nine is a bitter compound that functions as a feed- from ragwort (Senecio jacobaea). ing deterrent.

1274 Chapter 24 Natural Products (Secondary Metabolites) 24.6.3 Alkaloid biosynthesis research has been greatly aided by the development of techniques for culturing plant cells.

Many alkaloids have complex chemical structures and contain multiple asymmetric centers, complicating structure elucidation Figure 24.30 and making study of the biosynthesis of al- Callus cultures estab- kaloids quite difficult until relatively recent- lished from plants can be optimized to produce ly. For example, although nicotine (one high concentrations of a asymmetric center; see Fig. 24.25) was dis- wide variety of natural covered in 1828, its structure was not known products. In some of until it was synthesized in 1904, and the the examples shown, metabolite pigments structure of morphine (five asymmetric cen- give the calli distinctive ters; see Fig. 24.21) was not unequivocally colors. elucidated until 1952, almost 150 years after its isolation. Almost all of the enzymes in- tages over whole-plant studies, including the volved in the biosynthesis of these two alka- year-round availability of plant material; the loids have been identified, but 190 years af- undifferentiated, relatively uniform state of ter morphine was first isolated, its development of the cells; the absence of in- biosynthetic pathway remains incomplete. terfering microorganisms; and most impor- Why has it been so difficult to elucidate tantly, the compressed vegetative cycle. Plant alkaloid biosynthetic pathways? Plants syn- cell cultures can synthesize large amounts of thesize natural products at a relatively slug- secondary products within a two-week culti- gish rate, so steady-state concentrations of vation period. This is very favorable in com- the alkaloid biosynthetic enzymes are low. In parison with in planta production, for which addition, the large amounts of tannins and the time frame for alkaloid accumulation other phenolics that accumulate in plants in- may vary from one season for annual plants terfere with the extraction of active enzymes. to several years for some perennial species. Even when plants are treated with radiola- In plant cell culture, the rate of alkaloid beled precursors and the resulting radioactive biosynthesis can be increased, greatly facili- alkaloids are chemically degraded to identify tating its study (Table 24.2). Moreover, the the position of the label, the low rate of nat- greater metabolic rates associated with cell ural product metabolism can prevent the cultures promote the incorporation of la- high rates of incorporation that yield clearly beled precursors during alkaloid biosynthe- interpretable results. The use of polyvinyl- sis. Hormones regulate the accumulation of pyrrolidone and Dowex-1 in preparing pro- alkaloids in culture, and in many cases, al- tein extracts from plant tissues has helped kaloid biosynthesis can be induced by the overcome the enzyme inactivation by pheno- addition of abiotic and biotic elicitor sub- lic compounds, but isolation of the enzymes stances to the culture. These advances have involved in natural product synthesis has provided a powerful system with which to had only limited success because of their analyze the regulation of alkaloid biosynthe- very low concentrations in the plant. sis. Since the advent of alkaloid production Not until the 1970s were suspension cul- in culture, more than 80 new enzymes that tures of plant cells established that were ca- catalyze steps in the biosynthesis of indole, pable of producing high concentrations of isoquinoline, tropane, pyrrolizidine, acri- alkaloids (Fig. 24.30). As an experimental done, and purine classes of alkaloids have system, cell culture provides several advan- been discovered and partially characterized.

Table 24.2—Production of selected alkaloids in plant cell culture Secondary metabolite Species Yield (g/l) % Dry weight

Berberine Coptis japonica 7.0 12 Jatrorrhizine Berberis wilsoniae 3.0 12 Raucaffricine Rauwolfia serpentina 1.6 3

24.6–Alkaloids 1275 HO

C2H5

N N H H

H3CO2C

C2H5

CH O N Figure 24.31 3 OCOCH3 Structure of the HO CO2CH3 monoterpenoid indole CH3 alkaloid vinblastine from Catharanthus roseus. Catharanthus roseus Vinblastine

At one time, plant cell suspension cul- 24.6.4 Although typically considered tures were considered an alternative source constitutive defense compounds, some of industrially significant secondary metabo- alkaloids are synthesized in response to lites, particularly alkaloids of pharmaceutical plant tissue damage. importance. However, many important compounds such as vincristine, vinblastine (Fig. Alkaloids are thought to be part of the con- 24.31), pilocarpine (Fig. 24.32), morphine, stitutive chemical defense system of many and codeine, among many others, are not plants. The ultimate test of this hypothesis synthesized to any appreciable extent in cell may be future research into molecular genet- culture. The reason for this is thought to be ic suppression of alkaloid biosynthesis. The tissue-specific expression of alkaloid biosyn- phenotypes of mutants lacking specific gene thesis genes, because in some cases plants products in an alkaloid biosynthesis path- regenerated from nonproducing callus cells way may provide a direct demonstration of contained the same alkaloid profile as the the role of noninducible alkaloids produced parent plant. Although not currently used constitutively in plants. Near-isogenic for commercial alkaloid production, plant species of alkaloid-producing and nonpro- cell culture continues to provide biochemists ducing plants might then be subjected to ex- with a rich source of certain alkaloid biosyn- perimental conditions to test their relative thesis enzymes and a convenient system resistance. with which to study enzyme regulation. In a few cases, such as that of nicotine in tobacco, convincing evidence has been pre- sented that an alkaloid is involved in induced chemical defense. Wild species of tobacco have been found to be highly toxic to the hornworm, a tobacco-adapted species that is insensitive to nicotine but susceptible to N-acyl nicotines found in the tobacco leaf. The N-acyl derivatives are not found in unwounded Nicotiana repanda but their formation is induced by methyl jasmonate treat- O N O ment. In response to leaf wounding, tobacco plants increase the alkaloid content of leaves C2H5 CH2 N that have not been subjected to wounding. CH3 N-Acetylnicotine accumulates very rapidly H H (within 10 hours). The alkaloid content in- Pilocarpus jaborandi Pilocarpine creases and then returns to basal concentra- Figure 24.32 tions over a 14-day period. Recent isotope The imidazole alkaloid pilocarpine from Pilocarpus jaborandi. labeling experiments indicate that this

1276 Chapter 24 Natural Products (Secondary Metabolites) derivative is formed from a preexisting pool sidered feasible within the realm of organic of nicotine. De novo nicotine biosynthesis chemistry. In the 1950s, however, alkaloid occurs in roots, followed by transport to biosynthesis became an experimental sci- leaves, but only after 36 hours. The increase ence, as radioactively labeled organic in nicotine biosynthesis results in a 10-fold molecules became available for testing hy- increase of the alkaloid in the xylem fluid. potheses. These early precursor-feeding ex- Freshly hatched hornworm larvae fed periments clearly established that alkaloids wounded leaves achieve only half the are in most cases formed from L-amino acids weight gain obtained by counterparts fed (e.g., tryptophan, tyrosine, phenylalanine, ly- leaf material from unwounded plants. Re- sine, and arginine), either alone or in combi- cent studies demonstrate that, given the nation with a steroidal, secoiridoid (e.g., sec- choice, hornworms will abandon a wounded ologanin), or other terpenoid-type moiety. plant. Hornworms not permitted to leave a One or two transformations can convert wounded plant exhibit much higher mortali- these ubiquitous amino acids from primary ty rates and much lower growth rates than metabolites to substrates for highly species- those fed on unwounded plants. specific alkaloid metabolism. Although we Inducible synthesis of nicotine and other do not thoroughly understand how most of alkaloids appears to involve methyl jas- the 12,000 known alkaloids are made by monate, a volatile plant growth regulator plants, several well-investigated systems (see Chapter 17). Endogenous jasmonate can serve as examples of types of building pools increase rapidly when plant cells are blocks and enzymatic transformations that treated with an elicitor prepared from yeast have evolved in alkaloid biosynthesis. cell walls. In turn, jasmonates are known to The L-tryptophan–derived monoter- induce accumulation of secondary metabo- penoid indole alkaloid ajmalicine was the lites in cell culture. More than 140 different first alkaloid for which biosynthesis was cultured plant species respond to the addi- clarified at the enzyme level (Fig. 24.33); in tion of methyl jasmonate by increasing their that study plant cell suspension cultures of production of natural products. Although the Madagascar periwinkle C. roseus (see Fig. studies of this type with intact plants are not 24.31) were used. In plants, the biosynthesis as extensive as with cell suspension cultures, of ajmalicine and more than 1800 other clear examples have been demonstrated with monoterpenoid indole alkaloids begins with tobacco plants, in which leaf wounding pro- the decarboxylation of the amino acid L- duces an increase in endogenous jasmonic tryptophan by tryptophan decarboxylase to acid pools in shoots and roots. Moreover, the form tryptamine. Then tryptamine, by action application of methyl jasmonate to tobacco of strictosidine synthase, is stereospecifically leaves increases both endogenous jasmonic condensed with the secoiridoid secologanin acid in roots and de novo nicotine biosyn- (derived in multiple enzymatic steps from thesis. These results imply that jasmonate geraniol) to form 3α-strictosidine. Strictosi- may play a role in regulating the defense re- dine can then be enzymatically permutated sponses of alkaloid-producing plants. in a species-specific manner to form a multi- tude of diverse structures (Fig. 24.34). The elucidation of the enzymatic formation 24.7 Alkaloid biosynthesis of ajmalicine by using plant cell cultures laid the groundwork for analysis of more- 24.7.1 Plants biosynthesize alkaloids complex biosynthetic pathways, such as from simple precursors, using many those leading to two other L-tryptophan– unique enzymes. derived monoterpenoid indole alkaloids, ajmaline (Fig. 24.35) and vindoline. Until the mid-20th century, our view of how alkaloids are synthesized in plants was 24.7.2 The berberine synthesis pathway has based on biogenic hypotheses. Pathways been defined completely. suggested by illustrious natural product chemists such as Sir Robert Robinson, The first alkaloid for which each biosynthet- Clemens Schöpf, Ernst Winterstein, and ic enzyme has been identified, isolated, and Georg Trier were based on projections con- characterized from the primary metabolite

24.7–Alkaloid Biosynthesis 1277 CHO H O-Glucosyl

+ H O NH2 N CH3O2C H Tryptamine Secologanin

Strictosidine synthase

Glucosidases 3 NH I and II NH N N H H H H H H O-Glucosyl OH Glucose H H O O CH3O2C CH3O2C

Strictosidine Aglycone

Spontaneous

4 + NH N N CHO N 21 H Spontaneous H H H H

H H

CH3O2C CH3O2C

OH OH Dialdehyde 4,21-Dehydrogeissoschizine

+ NADPH NADP

N N N Reductase N H H H H H 20 CH3 CH3 19 H H H O O CH3O2C CH3O2C

OH 19-H 20-H β β Cathenamine Ajmalicine 19-epi-Ajmalicine α β Tetrahydroalstonine α α Figure 24.33 Biosynthesis of the monoterpenoid indole alkaloid ajmalicine and related compounds in Catharanthus roseus. Tryptamine is derived from L-tryptophan by decarboxylation through the action of tryptophan decarboxylase, and the secoiridoid secologanin is derived in multiple steps from the monoterpene geraniol.

1278 HO

C2H5

N N H H

H3CO2C

C2H5 H CO N 3 OCOCH3 N HO CO2CH3 N H R H H H N R = CH3 R = CHO N Vinblastine Vincristine H Catharanthus roseus H3CO2C H3CO2C OH CH2CH3 OH Vincamine Yohimbine Vinca minor Corynanthe johimbe

H2C CH H

NH N H H H N H N OGlc N HO H H H H CH3 H O H CO H 3 H3CO2C H O 3α(S)-Strictosidine H3CO2C N Ajmalicine Quinine Rauwolfia serpentina Cinchona officinalis

N OH

H H H N OH H N Figure 24.34 N H Strictosidine, the prod- CH3 uct of tryptamine and O O secologanin, is the precursor for many H species-specific Strychnine Ajmaline alkaloids. Strychnos nux-vomica Rauwolfia serpentina

precursor through to the end product alka- substrate-specific enzymes and of compart- loid is the antimicrobial tetrahydrobenzyl- mentalization in alkaloid biosynthesis. isoquinoline alkaloid, berberine, in Berberis The biosynthesis of tetrahydrobenzyliso- (barberry) cell suspension cultures (Fig. quinoline alkaloids in plants begins in the 24.36). This pathway will be described in de- cytosol with a matrix of reactions that gener- tail because it exemplifies the role of highly ates the first tetrahydrobenzylisoquinoline

24.7–Alkaloid Biosynthesis 1279 condensed to form (S)-norcoclaurine. A series of methylation and oxidation reactions yield the branchpoint intermediate of benzylisoquinoline alkaloid biosynthesis, OH (S)-reticuline (Fig. 24.38). In Berberis, the N-methyl group of H (S)-reticuline is oxidized to the berberine H N OH bridge carbon C-8 of (S)-scoulerine (see N H Fig. 24.37). The specific pathway from (S)-scoulerine that leads to berberine pro- CH3 ceeds with O-methylation to (S)-tetrahydrocolumbamine. The 3-O-methyl moiety of Rauwolfia serpentina Ajmaline tetrahydrocolumbamine is converted to the Figure 24.35 methylenedioxy bridge of canadine by cana- Structure of the monoterpenoid indole alkaloid ajmaline from Rauwolfia serpentina. dine synthase, a microsomal cytochrome P450–dependent oxidase. The final step in the biosynthesis of berberine is catalyzed by alkaloid, (S)-norcoclaurine (Fig. 24.37). The (S)-tetrahydroprotoberberine oxidase, an en- pathway proceeds from two molecules of zyme shown to contain a covalently bound L-tyrosine. One is decarboxylated to form flavin. The end product alkaloid berberine tyramine or is acted on by a phenol oxidase accumulates in the central vacuole of the to form L-dopa. Dopamine can then be Berberis cell. formed by decarboxylation of L-dopa or by The berberine bridge enzyme and the action of a phenol oxidase on tyramine. (S)-tetrahydroprotoberberine oxidase are Determining which of these two pathways is compartmentalized together in vesicles ap- predominant in a given plant is difficult be- parently derived from the smooth endoplas- cause all of the enzyme activities are present mic reticulum. Each of these enzymes con- in protein extracts. The benzyl moiety of sumes 1 mol of O2 and produces 1 mol of (S)-norcoclaurine is formed by transamina- H2O2 per mole of berberine formed. Overall, tion of the second L-tyrosine molecule to the course of reactions from 2 mol of L-tyro- form p-hydroxyphenylpyruvate, which is sine to 1 mol of berberine consumes 4 mol next decarboxylated to p-hydroxyphenyl- of S-adenosylmethionine and 2 mol of acetaldehyde. Dopamine and p-hydroxy- NADPH. phenylacetaldehyde are then stereoselectively

24.7.3 Elucidation of other alkaloid biosynthetic pathways is progressing.

The enzymes that catalyze the biosynthesis of the benzophenanthridine alkaloid macarpine in the California poppy Eschscholzia californica have also been identified, isolated, and characterized, as have nearly all of the enzymes of morphine biosynthesis in the opium poppy (Fig. 24.39). Good progress has been made toward understanding the enzymatic formation of the tropane alkaloid scopolamine in Hyoscyamus niger and of the acridone alkaloid furofoline-I in Ruta graveolens. Figure 24.36 Studies have revealed that the chemical The Berberis wilsoniae plant (left) and cell suspension culture (right). The cell sus- transformations required for alkaloid biosyn- pension culture derives its color from optimized production of the highly oxi- thesis are catalyzed by highly stereo-, regio-, dized benzylisoquinoline alkaloid berberine. Plant cell cultures (like this one) that produce large quantities of alkaloids have led to the complete elucidation of sev- and substrate-specific enzymes that are pres- eral alkaloid biosynthetic pathways. ent only in specific species. These enzymes

1280 Chapter 24 Natural Products (Secondary Metabolites) Phenol oxidase

2+ NH2 O ′ Cu HO 2 Tyramine HO

HO 6 Tyrosine CO2 Dopa decarboxylase NH2 decarboxylase HO Dopamine Phenol NH HO COOH CO2 HO COOH oxidase H (S)-Norcoclaurine 2+ NH O ′ Cu 2 synthase NH2 2 HO HO L-Tyrosine L-Dopa O HO (S)-Norcoclaurine H HO p-Hydroxyphenyl- SAM Norcoclaurine COOH acetaldehyde CO2 6-O-methyl- O transferase Transaminase HO SAH Decarboxylase p-Hydroxyphenyl- H3CO pyruvate H3CO H3CO (S)-N-methyl- NCH3 HO coclaurine Coclaurine NCH3 NH H 3’-hydroxylase HO N-methyltransferase HO HO 3’ H H 3’ HO 4’ SAH SAM (S)-3’-Hydroxy-N- H2O O2 HO 4’ HO methylcoclaurine + (S)-Coclaurine NADP NADPH (S)-N-Methylcoclaurine

SAM 3’-Hydroxy-N- methylcoclaurine 4’-O-methyltrans- SAH ferase

Berberine Scoulerine H CO H3CO bridge H3CO 9-O-methyl- 3 3 enzyme transferase N N N 2 HO CH3 HO 8 HO H H H OH 9 OH OCH3 O H O 2 2 2 SAM SAH OCH OCH3 OCH3 3 (S)-Reticuline (S)-Scoulerine (S)-Tetrahydrocolumbamine

Canadine NADPH synthase O2 O (S)-Tetrahydro- O protoberberine oxidase + N N O O H H Figure 24.37 OCH3 OCH3 Biosynthesis of berberine from H2O2 O2 two molecules of L-tyrosine. OCH OCH SAM, S-adenosylmethionine; 3 3 SAH, S-adenosylhomocysteine. Berberine (S)-Canadine

1281 CH O HO 3 O

CH3 N O O O H H O O N CH3 N CH3

HO HO O Codeine Morphine Protopine Papaver somniferum Papaver somniferum Fumaria officinalis

H3CO O

N CH3 O O N CH H 3 H HO CH 3 + H3CO H N O OH O O OCH3 H3CO OCH3 (S)-Reticuline O Noscapine Sanguinarine Papaver somniferum Sanguinaria canadensis

O H3CO H3CO + N N N O H3CO H3CO H OCH3 OCH3 OCH3 H3C

OCH OCH 3 OCH Papaverine 3 Berberine Corydaline 3 Papaver somniferum Berberis vulgaris Corydalis cava

Figure 24.38 (S)-Reticuline has been called the chemical chameleon. Depending on how the molecule is twisted and turned before undergoing enzymatic oxidation, plant enzymes are highly substrate-specific a vast array of tetrahydrobenzylisoquinoline-derived alkaloids of remark- and catalyze reactions previously unknown ably different structures can be formed. until discovered in the plant kingdom.

24.8 Biotechnological application of do not appear to participate in primary alkaloid biosynthesis research metabolism. For example, the cytochrome P450–dependent monooxygenases and 24.8.1 Available techniques for biochemical oxidases of alkaloid biosynthesis differ from and molecular genetic analysis facilitate the hepatic cytochrome P450–dependent identification, purification, and production monooxygenases and oxidases of mammals. of useful alkaloids. Unlike the individual mammalian enzymes, which share a common catalytic mechanism The current status of the alkaloid branch and modify a broad range of substrates, the of the field of natural products reflects the

1282 Chapter 24 Natural Products (Secondary Metabolites) H3CO H3CO H3CO 2 + Dehydro- N CH3 N CH3 N CH3 HO (S)-Reticuline HO reticulinium ion HO 1 1 1 H oxidase reductase H HO HO HO

+ H3CO H3CO NADPH NADP H3CO (S)-Reticuline 1,2-Dehydroreticulinium ion (R)-Reticuline

H3CO H3CO H3CO

HO HO HO Salutaridine 12 Salutaridine reductase 13 synthase N CH3 N CH3 N CH3 H H NADPH , O2 H 7 H3CO + H3CO H3CO NADP NADPH HO H O OH Salutaridinol Salutaridine (R)-Reticuline

Acetyl-CoA Salutaridinol O-acetyltransferase CoASH

H3CO H3CO H3CO 3 4 HO Spontaneous O Demethylation O

N CH3 N CH3 N CH3 H 5 H H

7 H3CO Acetate H3CO O

H3C C O H Thebaine Neopinone O Salutaridinol-7-O-acetate

HO H3CO H3CO 3

Codeinone O O O Demethylation reductase

N CH3 N CH3 N CH3 H H H

6 HO HO + O NADP NADPH Morphine Codeine Codeinone

Figure 24.39 Isolation and characterization of all the enzymes of morphine biosynthesis in opium poppy are nearly complete 190 years after discovery of that alkaloid. Many of the equivalent morphine biosynthetic enzymes have been discovered in mammalian liver. Demonstration that the mammalian liver biosynthesizes morphine de novo would have tremendous implications concerning evolutionary development of the opiate receptor in humans.

1283 many new advances in analytical chemistry, on plantations in Australia, Indonesia, and enzymology, and pharmacology. Only mini- Brazil. Certain other tropane alkaloid–pro- mal quantities of a pure alkaloid are now ducing species accumulate hyoscyamine in- necessary for a complete structure to be stead of scopolamine as the major alkaloid. elucidated by mass and NMR spectroscopic The question arises whether expression of a analyses. Absolute stereochemistry can be transgene in a medicinal plant would alter unambiguously assigned by determining the the alkaloid-producing pattern such that crystal structure. The pharmacological activi- more of the pharmaceutically useful alka- ties of crude plant extracts or pure substances loid, scopolamine, is obtained. To this end, a are determined by fully automated systems, such that millions of data points are collected each year in industrial screening programs. The factor that limits the number of biological activities for which we can test is the number of available target enzymes and re- Product 1 ceptors. As more of the underlying biochemical bases for diseases continue to be discovered, the number of test systems will increase. Precursor Product 2 What happens when a small quantity of an alkaloid of complex chemical structure from a rare plant is found to be physiologically active? The alkaloid must first pass ani- (A) mal and clinical trials; if these are successful, eventually enough material will be needed to satisfy market demand. Researchers can (B) develop biomimetic syntheses, which dupli- Introduce cate at least part of the biosynthetic pathways transgene of plants to yield synthetic compounds; alternatively, they can alter the metabolism of Product 3 the plant to change the alkaloid profile (Fig. 24.40). The regulation of alkaloid biosynthe- (C) sis in cell culture can also be influenced to produce a desired alkaloid. The following Introduce successful studies demonstrate the viability transgene of these approaches. Product 4

24.8.2 Metabolic engineering of medicinal plants may be the pharmaceutical Figure 24.40 biotechnology of the future. Using antisense/cosuppression technologies (see Chapter 7) or overexpression, medicinal plants can be tailored to produce pharmaceutically important The tropane class of alkaloids, found mainly alkaloids by eliminating interfering metabolic steps in the Solanaceae, contains the anticholiner- or by introducing desired metabolic steps. Express- gic drugs hyoscyamine and scopolamine. ing an entire alkaloid biosynthesis pathway of 20 to Solanaceous plants have been used tradition- 30 enzymes in a single microorganism is currently beyond our technical capability. However, altering ally for their medicinal, hallucinogenic, and the pathway in a plant and producing the desired al- poisonous properties, which derive, in part, kaloid either in culture or in the field may now be from tropane alkaloids. For obtaining im- possible. For example, to accumulate more of the end proved sources of pharmaceuticals, metabol- product alkaloid, a side pathway that also uses the same precursor may have to be blocked (A). To accu- ic engineering of the plants that serve as mulate an alkaloid not normally produced in a par- commercial sources of scopolamine could ticular plant species, a transgene (from another plant augment classical breeding in the effort to or a microorganism) may be introduced (B). If the develop plants with an optimal alkaloid end product alkaloid would be more useful as a particular derivative, for example, as a more soluble gly- pattern. The current commercial source of coside, a gene that encodes a glycosyl transferase scopolamine is Duboisia, which is cultivated could be introduced (C).

1284 Chapter 24 Natural Products (Secondary Metabolites) Hyoscyamine CH3 6β-hydroxylase CH3 CH3 N N N α-Ketoglutarate Succinate HO Hyoscyamine O 6β-hydroxylase OH Fe2+ OH OH

O O O Ascorbate

O O2 CO2 O O Hyoscyamine 6β-Hydroxyhyoscyamine Scopolamine

Figure 24.41 The reaction catalyzed by hyoscyamine 6β-hydroxylase along Datura. This one enzyme catalyzes two consecutive steps, hy- the biosynthetic pathway leading to the tropane alkaloid scopo- droxylation of hyoscyamine followed by epoxide formation to lamine in species of the genera Hyoscyamus, Duboisia, and produce scopolamine.

cDNA encoding hyoscyamine 6β-hydroxylase from H. niger (black henbane) has been introduced into Atropa belladonna (deadly nightshade) by using Agrobacterium tumefa- A B C D E Gene 1 Gene 2 Gene 3 Gene 4 ciens– and A. rhizogenes–mediated transformation (Fig. 24.41). The resulting transgenic plants and hairy roots each contained greater concentrations of scopolamine than did the Gene 3 wild-type plants. These transgenic Atropa Single gene expression plants provided the first example of how for biomimetic medicinal plants could be successfully al- synthesis tered by using molecular genetic techniques to produce increased quantities of a medici- nally important alkaloid. CD Designing meaningful transformation experiments requires a thorough knowledge of alkaloid biosynthetic pathways. Such stud- Gene 2 Gene 3 Expression ies are also limited by our ability to trans- Gene 1 Gene 4 of short form and regenerate medicinal plants. To pathway date, expertise in this important area lags well behind that for tobacco, petunia, and cereal crops. For example, in the area of tropane alkaloids, transformation and regenera- A E tion of Duboisia, a plant for which plantation, harvesting, and purification techniques have Figure 24.42 already been established commercially, will One alternative approach to the use of metabolically have to be developed before any potential engineered plants is to use microbes to produce alka- commercialization can be considered. Genet- loids. Short biosynthetic pathways can be expressed in either yeast or bacteria and used as a source of al- ic manipulation of plant cell cultures may kaloid. Plant alkaloid genes can be functionally ex- increase the concentrations of rate-limiting pressed in microorganisms to produce either single enzymes or may result in expression of gene biotransformation steps or short biosynthetic path- products not normally induced in cultured ways. For example, a known plant biosynthetic pathway contains an enzyme encoded by gene 3, which cells. If so, alkaloid production in plant cell catalyzes a transformation step that is difficult to or tissue culture may become a viable indus- achieve by chemical synthesis. After heterologous ex- trial approach (Fig. 24.42). pression of gene 3 in a microorganism, the protein Another successful example of how me- product can be used in a biomimetic synthesis of alkaloid D. Likewise, expression of the biosynthetic tabolic engineering can alter natural prod- genes 1 through 4 in a microorganism might produce ucts synthesis has been provided by the alkaloid E directly from precursor A.

24.8–Biotechnological Application of Alkaloid Biosynthesis Research 1285 transformation of Brassica napus (canola) the development of alternative systems of with the cDNA encoding the C. roseus tryp- production, such as plant cell or microbial tophan decarboxylase used in biosynthesis cultures, and in the development of plants of monoterpenoid indole alkaloids. Useful- with an improved spectrum of alkaloids for ness of seed from this oil-producing crop a more efficient production of the pharma- as animal feed has been limited in part by ceuticals currently isolated from field-grown the presence of indole glucosinolates (see plants. The design of these alternative sys- Chapters 8 and 16), sulfur-containing com- tems and optimized plants requires molecu- pounds that make the protein meal less lar manipulation, which in turn requires palatable. The tryptophan decarboxylase knowledge of alkaloid biosynthetic path- transgene in canola redirects tryptophan ways at the enzyme level. Much progress pools away from indole glucosinolate has been made with select alkaloids, but biosynthesis and into tryptamine (Fig. 24.43). much remains to be discovered about the The mature seed of the transgenic canola enzymatic synthesis of pharmaceutically plants contains less of the indole glucosi- important alkaloids such as camptothecin, nates and does not accumulate tryptamine, quinine, and emetine, to name only a few making it more suitable for use as animal examples. cDNAs have now been isolated feed and achieving a potentially economical- for approximately 20 enzymes of alkaloid ly useful product. biosynthesis, and the rate at which new To date, the elucidation of enzymatic clones are identified is certain to increase in syntheses of at least eight alkaloids is either the coming years. As genes are isolated, we complete or nearly complete: ajmaline, vin- can anticipate that heterologous expression doline, berberine, corydaline, macarpine, systems will be developed in bacterial, yeast, morphine, berbamunine, and scopolamine. and insect cell culture systems to allow pro- Of these alkaloids, those in current industrial duction of single enzymes, and perhaps use, such as morphine and scoploamine, are even short pathways, for biomimetic synthe- still being isolated from the plants that pro- ses of alkaloids. Our understanding of how duce them rather than synthesized. The fu- the expression of alkaloid biosynthesis genes ture for research on these alkaloids lies in is regulated by elicitors or in specific tissues will also improve as the promoters of alkaloid biosynthetic genes are analyzed. The future will almost certainly bring genetically Indole glucosinolate engineered microorganisms and eukaryotic S Glc cell cultures that produce alkaloids, metabolically engineered medicinal plants with tai- – N O SO3 lored alkaloid spectra, pharmaceutically im- WT N portant alkaloids in plant cell culture, and canola H even enzymatic synthesis of as yet unknown alkaloids through combinatorial biochemistry. COOH

CO2 NH2 24.9 Phenylpropanoid and N H phenylpropanoid-acetate Tryptophan pathway metabolites L-Tryptophan decarboxylase 24.9.1 Plants contain a remarkably diverse Transgenic NH2 canola N array of phenolic compounds. H Tryptamine Plants originated in an aquatic environment. Figure 24.43 Their successful evolutionary adaptation to Metabolic engineering to improve the quality of canola oil. A canola cultivar land was achieved largely by massive forma- is transformed with a gene from Catharanthus roseus that encodes tryptophan tion of “plant phenolic” compounds. Al- decarboxylase, an enzyme involved in biosynthesis of monoterpenoid indole though the bulk of these substances as- alkaloids. The transgene effectively directs the L-tryptophan pool away from use in biosynthesis of the bitter indole glucosinolate and into the production sumed cell wall structural roles, a vast of tryptamine. WT, wild type. array of nonstructural constituents was

1286 Chapter 24 Natural Products (Secondary Metabolites) also formed, having such various roles as only in specific plant families. Placing undue defending plants, determining certain distin- emphasis on any single plant species can ob- Phenyl guishing features of different woods and scure the extremely broad variation in bio- ring barks (e.g., durability), establishing flower synthetic capabilities that has yielded this color, and contributing substantially to cer- spectrum of different plant types. OH “Acidic” tain flavors (tastes and odors). These func- hydroxyl tions and others performed by plant pheno- or 24.9.2 Most, but not all, plant phenolic lics are essential for the continued survival phenolic compounds are products of group of all types of vascular plants. Accounting phenylpropanoid metabolism. for about 40% of organic carbon circulating Phenol in the biosphere, these phenolic compounds Most plant phenolics are derived from the Figure 24.44 are primarily derived from phenylpropanoid, phenylpropanoid and phenylpropanoid- Structure of phenol. phenylpropanoid-acetate, and related bio- acetate pathways (Fig. 24.45) and fulfill a chemical pathways such as those leading to very broad range of physiological roles “hydrolyzable” tannins. Furthermore, it is in planta. In ferns, fern allies, and seed their reassimilation back to carbon dioxide plants, polymeric lignins reinforce special- during biodegradation (mineralization) that ized cell walls, enabling them to support presents the rate-limiting step in recycling their massive weights on land and to trans- biological carbon. port water and minerals from roots to Plant phenolics are generally character- leaves. Closely related to lignins, the lignans ized as aromatic metabolites that possess, or can vary from dimers to higher oligomers. formerly possessed, one or more “acidic” Widespread throughout the plant kingdom, hydroxyl groups attached to the aromatic lignans can, for example, either help defend arene (phenyl) ring (Fig. 24.44). These compounds plagued plant scientists for years by interfering with experimental methods. For example, when exposed to air, plant pheno- C lics readily oxidize and turn brown, generat- C ing products that form complexes with pro- C3 teins and inhibit enzyme activity. Many C protocols now used to isolate plant proteins and nucleic acids include special precautions C designed to minimize interference by pheno- 6 lic compounds. Cultured plant tissues can 3 C2 C C also release phenolics that inhibit growth of 6 3 callus and regeneration of plantlets. At the Phenylpropanoid Phenylpropanoid–acetate skeleton (C6C3–C6), same time, phenolic compounds are increas- skeleton (C6C3) with phenylpropanoid-derived (C6C3) and ingly being recognized for their profound acetate-derived (3 C2) rings impact on plant growth, development, re- OH production, and defense; indeed, scientists have come to appreciate their significance HO more fully, particularly over the past few decades. HO O OH The discussion of plant phenolic substances is a discussion of plant diversity itself. Characteristics unique to each of the OH H CO roughly 250,000 species of vascular plants 3 HO O arise, at least in part, through differential OH deposition of highly specialized phenyl- Coniferyl alcohol, Quercetin, a flavonoid propanoid and phenylpropanoid-acetate a component of (C6C3–C6) derivatives. No single species can be used to lignins and many lignans illustrate the extraordinary diversity of “secondary” metabolites that exists within the Figure 24.45 Phenylpropanoid skeleton Phenylpropanoid and phenylpropanoid- Acetate-derived rings plant kingdom, because many branches of acetate skeletons and representative plant the pathways are found or are amplified compounds based on those structures.

24.9–Phenylpropanoid and Phenylpropanoid-Acetate Pathway Metabolites 1287 against various pathogens or act as antioxi- potato skin), suberized tissues function, for dants in flowers, seeds, seed coats, stems, example, by providing a protective barrier, nuts, bark, leaves, and roots. Suberized tis- thereby limiting the effects of desiccation sues contain alternating layers of hydropho- from the atmosphere and pathogen attack. bic (aliphatic) and hydrophilic (phenolic) The flavonoids comprise an astonishingly structural substances. Present in cork, bark, diverse group of more than 4500 com- roots, and certain periderm tissues (e.g., pounds. Among their subclasses are the anthocyanins (pigments), proanthocyanidins or condensed tannins (feeding deterrents and wood protectants), and isoflavonoids (defen- COOH COOH sive products and signaling molecules). The coumarins, furanocoumarins, and stilbenes NH2 NH2 protect against bacterial and fungal pathogens, discourage herbivory, and inhibit seed Figure 24.46 The aromatic amino germination. Numerous miscellaneous pheno- acids phenylalanine lics also play defensive roles or impart char- and tyrosine are deriva- acteristic tastes and odors to plant material. tives of the shikimic– Although most plant phenolics are prod- chorismic acid pathway OH (see Chapter 8). Phenylalanine Tyrosine ucts of phenylpropanoid metabolism, with the phenylpropanoids, in turn, being derived from phenylalanine and tyrosine (Fig. 24.46), some phenolic compounds are generated through alternative pathways. For example, hydrolyzable tannins, a group of (A) (B) (D) mostly polymeric substances that appear to C COOH act in plant defense, are typically copoly- mers of carbohydrates and the shikimate- derived gallic and ellagic acids (Fig. 24.47). Found in the leaves, fruits, pods, and galls HO OH of some woody dicots, hydrolyzable tannins Shikimate-derived OH have not yet been identified in monocots. aromatic core constituent of “Condensed tannins,” on the other hand, are hydrolyzable tannins Gallic acid widespread and occur in practically all trees (C6C1) (C6C1) Chestnut and shrubs. Known as proanthocyanidins, these compounds are synthesized by the (C) HO OH phenylpropanoid-acetate pathway. Some HO others, such as the phenolic psychoactive HO OH compounds of cannabis, the tetrahydro- Ellagic cannabinoids, are polyketide (acetate) or acid OH terpenoid derivatives (Fig. 24.48). O O O O 24.10 Phenylpropanoid and OH phenylpropanoid-acetate biosynthesis

O O O O OH 24.10.1 Phenylalanine (tyrosine) HO O ammonia-lyase is a central enzyme in O phenylpropanoid metabolism. OH HO HO One enzyme directs carbon from aromatic OH amino acids to the synthesis of phenyl- HO OH Figure 24.47 propanoid metabolites. This enzyme con- The shikimate-derived skeleton (A) verts phenylalanine (PAL) to cinnamic acid OH forms the core of gallic acid (B), a com- Castalagin ponent of hydrolyzable tannins, includ- and tyrosine (TAL) to p-coumaric acid (Fig. ing castalagin (C) from chestnut (D). 24.49, reactions 1 and 2). Interestingly, in

1288 Chapter 24 Natural Products (Secondary Metabolites) (A) (B) NH2

H3CO OCH3 O OCH3 Mescaline Peyote cactus ∆1-3,4-cis-Tetrahydrocannabinol Cannabis/Hemp

Figure 24.48 Not all plant phenolic compounds are derived from phenyl- compound ∆1-3,4-cis-tetrahydrocannabinol (B), a psychoactive com- propanoid substrates. Whereas mescaline, the psychoactive compo- ponent of cannabis, is a product of polyketide synthesis, the repeat- nent of peyote, is a phenylpropanoid derivative (A), the phenolic ed condensation of acetyl-CoA units derived from malonyl-CoA.

most vascular plants, Phe is the highly pre- cinnamic acid to the monolignols (see Fig. ferred substrate, but the monocot enzyme 24.49). This pathway essentially comprises can utilize both Phe and Tyr. PAL has been four types of enzymatic reactions: aromatic detected in a few aquatic plants, where it hydroxylations, O-methylations, CoA liga- probably functions in formation of simple tions, and NADPH-dependent reductions. flavonoids, such as the C-glucosyl–linked More recently, the precise enzymology in- lucenin and vicenin of Nitella species (Fig. volved in earlier parts of the pathway has 24.50). Lignins, however, are not present in come under renewed attention, focusing par- aquatic plants. Thus, this PAL (TAL) enzy- ticularly on aromatic hydroxylations and on matic step and the products of the various whether the O-methylation steps utilize the phenylpropanoid and phenylpropanoid-ac- free acids or CoA esters. etate pathways appear to have been key to Aromatic ring hydroxylation involves the plant colonization of land. three distinct hydroxylation conversions, all PAL is the most extensively studied en- of which are believed to be microsomal. The zyme in the phenylpropanoid pathway, per- best studied of these enzymes, cinnamate- haps in all secondary metabolism. In some 4-hydroxylase, is an oxygen-requiring, plants, PAL appears to be encoded by a sin- NADPH-dependent, cytochrome P450 en- gle gene, whereas in others it is the product zyme that catalyzes the regiospecific hydrox- of a multigene family. The enzyme requires ylation at the para-position of cinnamic acid no cofactor for activity. The ammonium ion to give p-coumaric acid (see Fig. 24.49, reac- liberated by the PAL reaction is recycled by tion 3). The other two hydroxylases original- way of glutamine synthetase and glutamate ly were thought to introduce hydroxyl synthetase (GS-GOGAT; see Chapter 8). groups into the free acids p-coumarate or Once assimilated into glutamate, the amino ferulate (or their CoA ester forms), yielding group can be donated to prephenate, form- the diphenol (catechol) products caffeic acid ing arogenate, a precursor of both phenylala- or 5-hydroxyferulic acid (or their CoA nine and tyrosine (Fig. 24.51). This nitrogen- derivatives), respectively (see Fig. 24.49, recycling process ensures a steady supply of action 4). At this time, however, substantial the aromatic amino acids from which plant confusion remains as to how the caffeoyl phenolics are derived. moiety of caffeic acid or caffeoyl-CoA is formed. Whether this biosynthesis involves a 24.10.2 Biochemical pathways to nonspecific phenolase-catalyzed conversion distinct phenolic classes share many or whether some other enzymatic step (e.g., common features. one involving an NADPH-dependent cytochrome P450) is used is still not known. During the 1960s and early 1970s, impressive Additionally, although ferulate-5-hydroxy- progress was made in defining many of the lase has been established as an NADPH- salient features of the pathway that converts dependent cytochrome P450 enzyme, there

24.10–Phenylpropanoid and Phenylpropanoid-Acetate Biosynthesis 1289 OH OH COOH HO OH HO O NH 2 10 Flavonoids

(HO) O (HO) O 4,2’,4’,6’-Tetrahydroxychalcone Naringenin OH (Isoliquiritigenin) (Liquiritigenin) 2 Tyrosine 9 3 Malonyl-CoA OH COOH COOH COOH COAMP COSCoA CHO

NH2

1 3 5 5 7 8

OH OH OH OH OH Phenylalanine Cinnamic acid p-Coumaric acid p-Coumaroyl-CoA p-Coumaraldehyde p-Coumaryl alcohol

5 4 4 4 CoASOC COOH COOH COAMP COSCoA ? Stilbenes

3 11 Aromatic domain of Malonyl- OH 5 5 suberized tissue CoA

OH OH OH OH OH OH OH ? 2 Malonyl-CoA 12 Caffeic acid Caffeoyl-CoA 6 6 OH COOH COAMP COSCoA CHO Styrylpyrones Coumarins

5 5 7 8 Lignins and Figure 24.49 OCH3 OCH3 OCH3 OCH3 OCH3 lignans Phenylpropanoid metab- OH OH OH OH OH olism leading to produc- Ferulic acid Feruloyl-CoA Coniferaldehyde Coniferyl alcohol tion of the monolignols, p-coumaryl, coniferyl, and 4 4 4 sinapyl alcohols, as well COOH COAMP COSCoA CHO as to other (sub)classes of plant phenolics. Conver- sions from p-coumaric 5 5 acid to sinapic acid and corresponding CoA esters HO OCH3 HO OCH3 HO OCH3 HO OCH3 are illustrated as a grid, OH OH OH OH because dual pathways 5-Hydroxyferulic acid 5-Hydroxyferuloyl-CoA 5-Hydroxyconiferaldehyde may be in effect. Produc- tion of the aromatic do- 6 6 6 main of suberized tissue OH COOH COSCoA CHO (yellow) may mainly COAMP involve hydroxycinnamates, including p-coumaroyl and feruloyl 5 5 7 8 tyramines (see Section 24.11.5), as well as small H3CO OCH3 H3CO OCH3 H3CO OCH3 H3CO OCH3 H3CO OCH3 amounts of monolignols. OH OH OH OH OH The tyramine derivatives Sinapic acid Sinapoyl-CoA Sinapaldehyde Sinapyl alcohol are, in turn, derived from p-coumaroyl-CoA and feruloyl-CoA. Enzymes (and their cofactors) are as follows: 1. PAL; 2. PAL (or TAL), found mainly in grasses; 3. cinnamate-4-hydroxylase (O2, cytochrome P450, NADPH); 4. hydroxylases (O2, cyt. P450, NADPH); 5. CoA ligases that participate in ligation of AMP and CoA (CoASH, ATP); 6. O-methyltransferases (SAM); 7. cinnamoyl-CoA:NADPH oxidoreductases (NADPH); 8. cinnamoyl alcohol dehydrogenases (NADPH); 9. chalcone synthase; 10. chalcone isomerase; 11. stilbene synthase; 12. styrylpyrone synthase. Products in parentheses refer to less common pathways. [Note: Sequence of intermediates in the pathways leading to sinapyl alcohol awaits experimental confirmation at the time of writing. The reader is encouraged to read the pertinent literature on developments in this area.]

1290 R steps precede CoA-ligation, or whether both routes are possible (see Fig. 24.49, reaction 6). OH C-Glycosyl In any case, O-methyltransferases, whether acting on free acids or CoA esters, introduce O HO methyl groups in a highly regiospecific manner, methylating the meta-hydroxyl group Glycosyl-C but not the group at the para-position. The enzyme catalyzing this transformation uses HO O S-adenosylmethionine (SAM) as a cofactor, R = OH Lucenin whereas CoA ligation requires ATP and R = H Vicenin CoASH. This two-step ligation first generates the AMP derivative, then converts it Figure 24.50 C-Glycosyl flavonoid types reported to be present in into the corresponding CoA ester. a green alga, Nitella hookeri (Charophyceae). After the CoA ester is formed, two sequential NADPH-dependent reductions produce the monolignols, completing the gener- is still some uncertainty as to whether it is al phenylpropanoid pathway (see Fig. 24.49, ferulic acid, feruloyl-CoA, coniferaldehyde, reactions 7 and 8). The first of these enzymes, or coniferyl alcohol that serves as the physio- cinnamoyl-CoA reductase, catalyzes forma- logical substrate. tion of p-coumaraldehyde (p-hydroxycin- Researchers have not yet determined namaldehyde), coniferaldehyde, and possi- whether in some instances the O-methylation bly sinapaldehyde. This type B reductase

1 – 1 – – COO 1 COO COO COO– H H 2 2 HR 2 HR + + 3 NH3 + 3 NH3 H 3 NH3 H S –OOC S Arogenate Arogenate dehydrogenase dehydratase PAL

OH OH Tyrosine Arogenate Phenylalanine Cinnamic acid

+ ADP + P (from –NH3 and i pro-S hydrogen – of phenylalanine) COO Fdxred α -KG L-Gln NH + O 4 –OOC Prephenate aminotransferase Glutamine GOGAT synthetase

OH L-Gln L-Glu Prephenate

Fdxox ATP

Figure 24.51 During active phenylpropanoid metabolism, nitrogen from pheny- glutarate aminotransferase; L-Gln, glutamine; L-Glu, glutamate; α α lalanine is recycled. Although TAL activity has been reported in -KG, -ketoglutarate; Fdxred, reduced ferredoxin; Fdxox, oxidized certain plant species, no report has yet established a comparable ferredoxin. nitrogen-recycling system for tyrosine. GOGAT, glutamine: α-keto-

24.10–Phenylpropanoid and Phenylpropanoid-Acetate Biosynthesis 1291 abstracts the pro-S hydride (Fig. 24.52) from thetic pathway is directed to the production behind the nicotinamide plane of NADPH of the lignins, which are structural compo- during reduction. The second enzyme, cin- nents of cell walls. Free radicals participate namyl alcohol dehydrogenase, is a type A in the reactions that produce both dimeric/ reductase that abstracts the pro-R hydride oligomeric lignans and lignins as well as from in front of the nicotinamide plane to related complex plant polymers such as yield the monolignols p-coumaryl, coniferyl, those in suberized tissue. and sinapyl alcohols (Fig. 24.52). The above description is a brief account of the overall biochemical steps that culmi- nate in monolignol formation. However, the 24.11.1 Dimeric and oligomeric lignans are pathway shown in Figure 24.49 is deceptive. formed primarily from coniferyl alcohol. Not all cells, tissues, or species of plants utilize the entire pathway. In many instances, The term lignan was initially coined by plants utilize only a small pathway segment Robert Downs Haworth in 1936 to describe a

that directs substrates to one or more of the class of dimeric phenylpropanoid (C6C3) main metabolic branchpoints; moreover, metabolites linked by way of their 8–8’ they may express that truncated pathway bonds. More recently, another term, neolig- only in specific tissues. Researchers do not nan, was used to define all of the other yet fully understand metabolic flux and types of linkages (e.g., 8–1’-linked dimers), compartmentalization in the phenyl- but has since been modified to encompass propanoid pathway. Elucidation of these substances derived from allylphenol com- processes will be a necessary step toward pounds, such as isoeugenol (Fig. 24.53). In defining or identifying the control points this chapter, however, we have chosen to in the pathway. use the more convenient name lignan to de-

scribe all possible phenylpropanoid (C6C3) coupling products, so long as the coupling 24.11 Biosynthesis of lignans, lignins, mode (e.g., 8–8’, 8–5’) is specified (Fig. and suberization 24.54). Interestingly, although several thou- sand lignans are now known in nature, The monolignols are primarily converted relatively few coupling modes have been into two distinct classes of plant metabolites: encountered. the lignans and the lignins. Most metabolic Lignan dimers are found in ferns, gym- flux through the phenylpropanoid biosyn- nosperms, and angiosperms, but higher

NADPH NADP+ NADPH NADP+

O O O O HA HB HB HA HB HA C C C C NH2 NH2 NH2 NH2 OH + + N N N N HB HA C COSCoA CHBO R R R R

OCH3 OCH3 CCR OCH3 CAD OH OH Type B reductase OH Type A reductase Feruloyl-CoA ester Coniferyl aldehyde Coniferyl alcohol

Figure 24.52 The stereospecificity of a type B reductase, NADPH-dependent plane of the page); HB, pro-S (the hydrogen projecting upward cinnamoyl-CoA reductase (CCR), and a type A reductase, cinnamyl from the B-face of the nicotinamide ring, i.e., behind the plane alcohol dehydrogenase (CAD). HA, pro-R (the hydrogen projecting of the page). R, adenine nucleotide diphosphate. upward from the A-face of the nicotinamide ring, i.e., out from the

1292 Chapter 24 Natural Products (Secondary Metabolites) phenolic coupling was the in vitro synthesis of (+)-pinoresinol (Fig. 24.55). This overall reaction, discovered in Forsythia species, is as follows: A laccase or laccase-like enzyme catalyzes a one-electron oxidation that forms the corresponding free radicals, and a dirigent protein (Latin: dirigere, to guide or H3CO Figure 24.53 align) orients the putative free radical sub- OH Isoeugenol, an allylphenol. strates in such a way that random coupling cannot occur; only formation of the 8–8’-coupled intermediate, (+)-pinoresinol, is permit- oligomeric forms also occur. Lignan forma- ted. The particular antipode (optical form) tion utilizes coniferyl alcohol predominantly, of pinoresinol formed also varies with the along with other monolignols, allylphenols, plant species in question; for example, flax and phenylpropanoid monomers to a lesser seeds accumulate (–)-pinoresinol. Once extent. Most lignans are optically active, formed, pinoresinol can then undergo a vari- although the particular antipode (enantio- ety of conversions, depending on the plant mer) can vary with the plant source. species. The biochemistry of lignan formation has The gene encoding the Forsythia dirigent only very recently begun to be delineated. protein has been cloned and the functional To date, work has focused mainly on genera- recombinant protein expressed. It is not ho- tion of the most common 8–8’-linked lignans. mologous to any other protein. Given the This class of natural products is formed existence of lignans linked by way of other by a strict stereoselective coupling of two distinct bonding modes and the increasing coniferyl alcohol molecules. The first demon- number of homologous genes and expressed strated example of stereoselective control of sequence tags found in this and other species,

(A) O (B) (C)

H3CO 8 85’ O 8’ OCH HO H CO 3 3 O 8 O OCH3 4’ HO H3CO OH OCH3

OH H3CO α-Conidendrin (8–8’) Licarin A (8–5’) Virolin (8–O–4’)

C 9 C C 9′ C C C ′ 5 C 4′ 8 C C 8′ 8 C 8 C O C C C 7 C C 7′ C C 1 1′ HO 6 2 6′ 2′ OCH3 5 3 5′ 3′ 4 4′ (8–8’) (8–5’)(8–O–4’)

Figure 24.54 Examples of lignans derived by distinct coupling modes, e.g., 8–8’, 8–5’, and 8–O–4’.

24.11–Biosynthesis of Lignans, Lignins, and Suberization 1293 Forsythia intermedia OH OH OH OH

Dirigent 8 • protein • 8’ + Oxidase + or oxidant

OCH3 OCH3 OCH3 OCH3 OH OH O O E-Coniferyl alcohol

+ Figure 24.55 we can easily assume that the dirigent pro- H3O Proposed biochemical tein represents a new class of proteins. Addi- mechanism accounting O tionally, the mode of action of this protein for stereoselective con- OCH trol (regio- and stereo- is of particular interest and may provide 3 chemistry) of E-coniferyl new and definitive insight into the macro- H alcohol coupling in molecular assembly processes that lead to Forsythia species. The O: particular enantiomer of lignins and suberins (see Sections 24.11.3 pinoresinol formed can and 24.11.5). 8 vary with plant species. Pinoresinol can be enantiospecifically 8’ converted into lariciresinol and secoisolariciresinol, followed by dehydrogenation to :O H give matairesinol (Fig. 24.56 and Box 24.4). This last is the presumed precursor of plicatic acid (Figs. 24.56 and 24.57A) and its H3CO analogs in western red cedar (Thuja plicata), O as well as of podophyllotoxin (Figs. 24.56 + and Fig. 24.57B) in the Indian plant H3O

(Podophyllum hexandrum) and may apple OCH3 (P. peltatum). Podophyllotoxin is used to treat OH venereal warts, whereas its semisynthetic derivative, teniposide, is widely used in can- O cer treatment. Interestingly, pinoresinol/ lariciresinol reductase, which converts 8 H H pinoresinol into lariciresinol and secoisolari- 8’ ciresinol, shows considerable homology to the phytoalexin-forming isoflavonoid reduc- O tases, indicative perhaps of a common evo- HO lutionary thread in plant defense for both the lignans and isoflavonoids. Pinoresinol is OCH3 also the precursor of the antioxidant sesamin (+)-Pinoresinol (Fig. 24.57C) in the seeds of sesame (Sesa- mum indicum). the noncellulosic encrusting substance present in woody tissue. After cellulose, lignins 24.11.2 Lignin biosynthesis has been are the most abundant organic natural prod- described as a largely nonenzymatic ucts known, accounting for as much as 20% process, but differences between synthetic to 30% of all vascular plant tissue. Deposi- and biologically derived lignins cast doubt tion of lignins in plants results in the forma- on this premise. tion of woody secondary xylem tissues in trees, as well as reinforcement of vascular Derived from the Latin lignum (wood), the tissues in herbaceous plants and grasses. term lignin initially was coined to describe There are still no methods available for

1294 Chapter 24 Natural Products (Secondary Metabolites) OCH3 OCH3 OH OH

O O

H3CO 8’ OH 8 8 + H H NADPH + H H NADPH NADP 8’ NADP 8’ OH HO 8 O OH Pinoresinol/ Pinoresinol/ HO lariciresinol HO lariciresinol reductase reductase OCH3 OCH3 OCH3 (+)-Pinoresinol (+)-Lariciresinol OH (–)-Secoisolariciresinol + isolating lignins in their native state that do NADP not markedly alter the original structure of Secoisolariciresinol the biopolymers during dissolution. In con- dehydrogenase trast to many of the lignans, lignins are NADPH thought to be racemic (optically inactive). Gymnosperm lignins are primarily derived H3CO 8’ from coniferyl alcohol, and to a lesser extent, O p-coumaryl alcohol, whereas angiosperms Figure 24.56 HO 8 contain coniferyl and sinapyl alcohols in Proposed biochemical pathway for interconversions in the various O roughly equal proportions (see Fig. 24.49). 8–8’-linked lignan classes in Forsythia, For decades, the perceived formation of western red cedar (Thuja plicata), and lignins in vivo has been biochemically incon- Podophyllum species. The pathway from pinoresinal to matairesinol is gruous. Investigators originally proposed common to all three plants. OCH3 that monolignols were transported into the OH cell walls and that the only subsequent en- (–)-Matairesinol zymatic requirement for biopolymer formation was the one-electron oxidation of the monolignols to give the corresponding free radical intermediates, as shown with coniferyl Transformations Transformations alcohol (Fig. 24.58). Even today, there is no in Thuja plicata in Podophyllum full agreement on the oxidative enzymes re- species sponsible for free radical generation (monolignol oxidation) in lignin biosynthesis. Five or six candidate proteins are still under consideration, although peroxidase remains the (–)-Plicatic acid (–)-Podophyllotoxin most favored. The free radical intermediates formed by oxidation were initially believed to couple natural product for which its formation is together in a manner requiring no further not under enzymatic control. enzymatic control or input. These nonenzy- However, although it is rarely recog- matic free radical coupling reactions were nized, natural and synthetic lignins differ in thought to generate dimeric lignan struc- terms of bonding frequency, bonding type, tures that underwent further reoxidation and and macromolecular size. For example, for coupling to yield the lignin biopolymer (Fig. lignins in vivo, the 8–O–4’ interunit linkage 24.58). In other words, the random reactions predominates (more than 50%), with the 8–5’ of monolignol-derived free radical interme- substructure found in much lower amounts diates in a test tube were considered to give (about 9–12%) (Fig. 24.59). In contrast, in preparations identical to the lignins formed synthetic in vitro preparations, the 8–O–4’ in vivo. According to this model, nature’s substructure is present to only a very small second most abundant substance is the only extent, and the 8–5’ and 8–8’ linkages

24.11–Biosynthesis of Lignans, Lignins, and Suberization 1295 Box 24.4 Dietary lignans have health-protecting functions.

Secoisolariciresinol and matairesinol ure). These “mammalian” lignans under- rate of incidence of prostate and breast are common constituents of various go enterohepatic circulation, in which cancers. The protection accrues to indi- plants, including Forsythia intermedia, they are conjugated in the liver, excreted viduals on diets rich in grains, vegeta- flax, and certain vegetables and grains in the bile, deconjugated in the intestine bles, and berries that contain high con- (e.g., green beans and rye). These lignans by bacterial enzymes, absorbed across centrations of secoisolariciresinol and have important nutritional functions in the intestinal mucosa, and returned to the matairesinol. In contrast, typical Western health protection. During digestion, in- liver in the portal circulation (see figure). diets tend to be poor in these foods and testinal bacteria convert secoisolari- Enterodiol and enterolactone are be- do not afford comparable protection. ciresinol and matairesinol to enterodiol lieved to be responsible for preventing the and enterolactone, respectively (see fig- onset of and substantially reducing the

H3CO OH OH H3CO HO O

HO O

Renal clearance Enterodiol Enterolactone OCH3 (urine)

OH Glucuronides Secoisolariciresinol, OCH3 Lignan, (–)-Secoisolariciresinol matairesinol glucuronides OH

(–)-Matairesinol Facultative aerobes

Absorption Enterodiol, enterolactone HO HO OH O OH

O Fecal loss, unconjugated lignans

OH OH Enterodiol Enterolactone

predominate. This disparity suggests that ly, more details will emerge as this impor- within woody tissues some mechanism in tant process is investigated systematically. the lignifying cell wall regulates or man- dates the interunit linkage pattern within the native biopolymer. 24.11.3 Lignin biosynthesis is controlled As is becoming increasingly clear, the spatially and temporally and may involve a lignification process in situ is under very proteinaceous template. tight biochemical control as part of a cell- specific programmed process. In the follow- Before lignin biosynthesis is initiated, the ing section, we describe known elements cells destined to form secondary xylem that control lignification in vivo. Undoubted- (i.e., wood; Fig. 24.60) undergo specific

1296 Chapter 24 Natural Products (Secondary Metabolites) (A) (B) OH

HO O H3CO O OH O HO COOH O OH

Thuja plicata H3CO OCH3

H3CO OH OCH3 OH Podophyllum peltatum (–)-Plicatic acid (–)-Podophyllotoxin

H O O O O O OH O (C) S OH O O O H H O O O

O H3CO OCH3 O OH Sesamum indicum (+)-Sesamin Teniposide (semisynthetic)

Figure 24.57 Examples of 8–8’-linked lignans. (A) Plicatic acid (and its (B) Podophyllotoxin, from the may apple. The etoposide or tenipo- oligomeric congeners, not shown) are deposited en masse during side derivatives of this compound are also used in treatment of heartwood formation in western red cedar. The congeners con- certain cancers. (C) Sesamin, from the sesame seed, has in vitro an- tribute substantially to the color, quality, and durability of this tioxidant properties that stabilize sesame oil against turning rancid heartwood and are major components of the biochemical protec- during commercial storage. tion that enables such species to survive for more than 3000 years.

irreversible changes that ultimately lead to synthesis is initiated at defined sites in the cell death and the formation of conducting cell corners and middle lamella, i.e., at the elements (e.g., tracheids, vessels) and struc- locations farthest from the cytosol and plas- tural supporting tissues, such as fibers. ma membrane. These loci in the cell walls These cells experience a programmed expan- then form distinctive domains that extend sion of their primary walls, followed by so- inward through the various cell wall layers, called secondary thickenings, which involve toward the plasma membrane. The domains ordered deposition of cellulose, hemicellu- ultimately coalesce. lose, pectin, and structural proteins. Thus, UV-microscopy and radiochemical la- the overall architecture of the plant cell wall beling indicate that individual monolignols is established largely before lignification are deposited differentially. For example, in takes place. conifers, p-coumaryl alcohol is primarily laid At the start of lignin biosynthesis, down at the early stages of lignin biosynthesis monolignols are transported from the cyto- in the cell corners and middle lamella, where- sol into the cell wall during a specific stage as coniferyl alcohol is deposited predomi- of wall development. Electron microscopy nantly in the secondary wall (Fig. 24.60A). investigations have shown that lignin bio- This controlled deposition of specific

24.11–Biosynthesis of Lignans, Lignins, and Suberization 1297 OH OH OH OH OH 9

8 • 7

1 • 6 2 (Per)Oxidase

3 5 • 4 OCH3 OCH3 OCH3 OCH3 OCH3 OH O• O O O Coniferyl alcohol

Figure 24.58 The random coupling hypothesis for “lignin” Coupling with neighboring free radical by way of a nonenzymatic process, followed by either formation in vitro. Free intramolecular cyclization or reaction with H O radical intermediates are 2 putatively generated by peroxidase or laccase. HO OH The free radicals then H3CO OH couple nonenzymatically H3CO to generate (±)-racemic dimers. Repetition of this process, involving further enzymatic oxida- H3CO 1’ O HO OH tion of the dimeric phe- 8 nols, was originally con- OH 5’ HO 8 H O O 8’ sidered to continue until O H 8 8 “lignin” was formed. H3CO 4’ OH O H3CO

H3CO O

OCH OH 3 HO OCH3

(±)-8–O–4’ (±)-8–5’ (±)-8–1’ (±)-8–8’ HO

Repetition of process (enzymatic oxidation followed by nonenzymatic coupling)

“Lignin” polymer formation in vitro

monolignols creates domains with distinct cation is initiated. Thus, lignin biopolymer structural configurations. assembly may be under the control of a pro- Perhaps most interesting of all, immuno- teinaceous template. chemical studies demonstrate that initiation Taken together, this evidence suggests of lignin biosynthesis is both temporally and that lignin assembly in vivo is subject to bio- spatially associated with the secretion of dis- chemical regulation, whereby the appropri- tinct proteins from the Golgi apparatus and ate monomers are linked in a specific man- their deposition into the cell wall, including ner to yield a limited number of coupling some that are proline-rich. These or related modes in characteristic proportions. This polypeptides, including some proline-rich model assumes that elongation of the primary proteins, may participate in lignification and lignin chain occurs by end-wise polymeriza- may be related to the dirigents identified in tion and is guided by an array of proteina- lignan biosynthesis. Indeed, dirigent sites ceous sites that stipulate or control linkage have been detected in regions where lignifi- type and configuration. Moreover, in this

1298 Chapter 24 Natural Products (Secondary Metabolites) way, the cytosol predetermines the outcome of phenoxy radical coupling. Lignin chain Estimated replication is thus envisaged to involve frequencies: primitive self-replicating polymerization OCH3 templates, and even the presumed lack of O optical activity in lignins might result from, for example, the self-replication process in- O volving generation of mirror-image polymer- 8 H H ic assemblies. How lignification is ultimately Traces 8’ achieved and what is the precise nature and O mechanism of the putative proteinaceous templates now await full clarification at the O biochemical level. As we establish the salient OCH details of how lignin biopolymer assembly 3 (±)-8–8’ Pinoresinol is controlled, plants are beginning to yield some of the long-hidden secrets involved in cell wall formation. OH 8′ 5′ OH 24.11.4 Variations on lignin deposition can be observed in the formation of O reaction wood and in lignification in 9–12% OCH nonwoody plants. O 3

OCH3 A programmed plasticity of sorts is built (±)-8–5’ into the overall macromolecular assembly of Dehydrodiconiferyl alcohol lignified cell walls. Perhaps the best example of this is seen in the formation of so-called reaction wood. When the woody stem be- OH OH comes misaligned from its vertical axis, reac- HO 8 O tion wood forms to buttress the growing 4′ stem and gradually realign the photosyn- OCH thetic canopy (Fig. 24.61). In this region, >50% 3 some of the cells originally fated to form ordinary xylem (see Fig. 24.60B) are repro- O grammed to generate reaction wood instead. OCH3 These cells then undergo massive changes in (±)-8–O–4’ the macromolecular assembly of their cell (erythro/threo) Guaiacylglycerol 8–O–4’-coniferyl alcohol ethers walls (Fig. 24.61). In conifers, the cell walls of reaction wood, called compression wood, OH become thicker and rounder, the cellulose content is reduced relative to normal wood, OH and the cellulose microfibril angle is in- 8 ′ creased; the quantity of lignin also increas- 8 es, primarily through an increase in the p- coumaryl alcohol content. In contrast, the 5 5′ reaction wood formed in angiosperms is 18–20% H CO 4 4′ known as tension wood, because the affect- 3 OCH3 O O ed tissue is placed under tension rather than 7′′ 8′′ compression. Tension wood forms on the OH O

OCH3 Figure 24.59 Prevalence of selected interunit linkages in native (±)-5–5’–O–4’ lignin biopolymers from the gymnosperm Norway Dibenzodioxocin spruce (Picea abies).

24.11–Biosynthesis of Lignans, Lignins, and Suberization 1299 (A) OH (B) Normal secondary xylem

Tertiary wall

Secondary wall 2 OCH3

OH Secondary wall 1

Coniferyl Primary wall alcohol 24.11.5 Suberization protects tissues from water loss and pathogen invasion. OH Suberized tissues are found in various un- derground organs (e.g., roots, stolons, tubers) as well as in periderm layers (e.g., Middle lamella cork, bark). They are also formed as part of the wound- and pathogen-induced defenses Figure 24.60 Telescopic portrayal of a conifer tracheid. of specific organs and cell types, perhaps the (A) p-Coumaryl alcohol is preferentially deposited most familiar example being the browning in the compound middle lamella and cell corners, and subsequent encrustation of sliced potato coniferyl alcohol in the secondary wall. The parallel OH tubers. Suberized tissues are formed as mul- and hatched lines shown in such telescopic diagrams p-Coumaryl alcohol indicate the orientation of cellulose microfibrils. tilamellar domains consisting of alternating (B) Light micrograph cross-section of normal second- polyaliphatic and polyaromatic layers (Fig. ary xylem of Douglas fir (Pseudotsuga menziesii). 24.62), as shown in the wound-healing layers in potato. These layers contribute to cell wall strength and provide a means to limit upperside of the stem, compression wood on uncontrolled water loss by the intact organ- the underside. Characteristics of tension ism by forming impenetrable barriers. From wood include increased cellulose content an evolutionary perspective, suberization and the presence of a carbohydrate-derived was of utmost importance in plant adapta- gelatinous layer. The amount of lignin pres- tion to living on land and may even have ent may decrease or remain the same, de- preceded lignification. pending on the species. The underlying bio- As with lignin, no methods are yet chemical mechanisms that engender available to obtain either of the two domains formation of both compression and tension of suberin in a native or unaltered condition. wood are not known. The aliphatic component is located between Lignification in nonwoody herbaceous the primary wall and the plasmalemma. plants and grasses differs to some extent Suberin aliphatics are generally long-chain from lignin biosynthesis during wood for- (more than 20 carbons) lipid substances; mation. Nonwoody plants contain lignins they also include α,ω-fatty dioic acids, such α,ω that appear to be formed from mixtures of as C16- or C18-alkan- -dioic acids, which monolignols and hydroxycinnamic acids. are considered diagnostic of suberized tissue The lignin interunit linkages seem to follow (Fig. 24.63). Interestingly, the polyaromatic those generally described for woody tissue domain located in the cell wall is apparently lignin, except that hydroxycinnamic acids formed before the aliphatics, primarily from are also involved. To date, no extensive bio- distinctive monomeric building blocks that chemical studies have focused on how the contain hydroxycinnamate-derived sub- macromolecular assembly of lignin in non- stances (Fig. 24.64). Thus, the formation of woody plants actually occurs, although the suberized tissue is very distinct from the lig- involvement of proline-rich polymers has nification of secondary xylem (where deposi- been implicated here as well through the tion is the last biochemical act of the xylem- immunochemical studies discussed above. forming cells before cell death).

1300 Chapter 24 Natural Products (Secondary Metabolites) (A) (B) Sequoia sempervirens (C) Compression wood xylem stem

Pith

Compression wood

(D) Compression (reaction) wood tracheid

Secondary wall 2 (S ) 2 Intercellular Compression wood space Figure 24.61 Compression wood (reaction wood). (A) Gym- nosperm showing region of compression wood tis- Secondary wall 1 (S1) sue. (B) Cross-section of Sequoia sempervirens Primary wall showing pith and compression wood. (C) Light Intercellular material micrograph cross-section of compression wood xylem of Douglas fir (Pseudotsuga menziesii). Intercellular material (D) Telescopic portrayal of a tracheid in compression wood.

A further complication to the study of the aromatic domain in suberization is the presence of related phenolic substances. For example, in wound-healing suberizing pota- to periderm tissues, chlorogenic acid and miscellaneous other phenolics are also present (Fig. 24.65). These compounds do not appear to function in suberization per se but rather may provide a means for topical dis- infection of the exposed cell surfaces, thereby preventing or limiting infection/contami- nation. Some evidence also suggests the presence of low amounts of monolignols in suberized tissues, but these may be from small amounts of lignin. Although the polymeric suberin phenolic constituents are predominantly derived from hydroxycinnamate, how this aromatic 0.5 µm domain is assembled is unknown. Recent studies demonstrated that potato tuber wound-healing suberizing tissues contain Figure 24.62 two hydroxycinnamoyl-CoA transferases, Suberized tissue consists of layered polyaliphatic and which catalyze formation of various alkyl polyaromatic domains, as shown for wound-healing potato tuber slices exposed to air. A suberized layer ferulates and (p-coumaroyl) feruloyl tyra- (see arrows) forms five days after exposure of potato mine derivatives, respectively. How, or if, tuber slices to air.

24.11–Biosynthesis of Lignans, Lignins, and Suberization 1301 Fatty acid COOH

H3C(CH2)nCOOH n = 14, 16, 18, 20, 22

α,ω-Fatty dioic acids

HOOC(CH2)nCOOH n = 14, 16, 18, 20, 22 R

α,ω-Hydroxy fatty acids OH R = H p-Coumaric acid HO(CH2)nCOOH R = OCH3 Ferulic acid n = 15, 17, 19, 21, 23, 25

Figure 24.63 O OCH2(CH2)nCH3 Aliphatic components of suberized tissue. Found in combination, these compounds are considered diagnostic for suberin. An α,ω-dioic acid has carboxyl groups on both of the end carbons. An α,ω-hydroxy acid has a hydroxyl on one of the end carbons. n = 14, 16, 17, 18, 19, 20, 22, 24, 26

these are integrated into the aromatic do- OCH3 main of suberin of potato remains to be es- OH tablished, although an anionic peroxidase Alkyl ferulates has been implicated in the polymerization process. Additionally, the finding that the H appearance of proline-rich proteins seems to O N correlate temporally and spatially with deposition of the aromatic domain of suberized tissue may be important. OH Much remains to be understood about formation of both the polyaromatic and the polyaliphatic domains of suberized tissue. In particular, we do not know yet which fea- R tures are common to all plants and which OH are species-specific. For example, the suberized tissues seen in various root, periderm, R = H p-Coumaroyl tyramine and woody bark tissues are not identical to R = OCH3 Feruloyl tyramine one another. This underscores the need to identify the basic biochemical requirements OH for suberization and to determine how these differ with regard to tissue-specific addition of particular phenolic substances.

24.12 Flavonoids R

With more than 4500 different representa- OH tives known thus far, the flavonoids constitute an enormous class of phenolic natural R = H p-Coumaryl alcohol R = OCH Coniferyl alcohol products. Present in most plant tissues, 3 often in vacuoles, flavonoids can occur as Figure 24.64 monomers, dimers, and higher oligomers. Aromatic components of suberized tissue, derived They are also found as mixtures of colored primarily from hydroxycinnamates, including alkyl ferulates and p-coumaroyl and feruloyl tyramines. oligomeric/polymeric components in vari- Suberized tissue may also contain small amounts of ous heartwoods and barks. monolignols.

1302 Chapter 24 Natural Products (Secondary Metabolites) O O (purple, mauve, and blue). Related flavo- COOH noids, such as flavonols, flavones, chalcones, and aurones, also contribute to color defini- HO OH tion. Manipulating flower color by targeting OH various enzymatic steps and genes in flavonoid biosynthesis has been quite successful, OH particularly in petunia. OH Specific flavonoids can also function to Suberized tissue Chlorogenic acid protect plants against UV-B irradiation, a role sometimes ascribed to kaempferol (Fig. Figure 24.65 24.67). Others can act as insect feeding at- Suberin deposition has been studied in wounded tractants, such as isoquercetin in mulberry, a potato tubers. In these tissues, suberin formation is accompanied by the production of an unrelated factor involved in silkworm recognition of phenolic compound, chlorogenic acid. its host species. In contrast, condensed tannins such as the proanthocyanidins add a distinct bitterness or astringency to the taste of certain plant tissues and function as anti- 24.12.1 Flavonoids comprise a diverse feedants (Fig. 24.68). The flavonoids api- set of compounds and perform a wide genin and luteolin serve as signal molecules range of functions. in legume–rhizobium bacteria interactions, facilitating nitrogen fixation (Fig. 24.69). In a Many plant–animal interactions are influ- related function, isoflavonoids are involved enced by flavonoids. The colors of flowers in inducible defense against fungal attack in and fruits, which often function to attract alfalfa (e.g., medicarpin; Fig. 24.69) and oth- pollinators and seed dispersers, result pri- er plant species. Perhaps the most poorly marily from vacuolar anthocyanins (Fig. studied and least understood classes of the 24.66) such as the pelargonidins (orange, flavonoids are the oligomeric and polymeric salmon, pink, and red), the cyanidins (ma- substances associated with formation of genta and crimson), and the delphinidins certain heartwood and bark tissues. These

HO OH

OH OH OH + + + HO O HO O HO O OH

OH OH OH

OH OH OH Pelargonidin Cyanidin Delphinidin

Pelargonium Rosa Delphinium (Geranium) (Rose) (Larkspur)

Figure 24.66 Selected anthocyanin pigments: pelargonidin, cyanidin, and delphinidin from geranium, rose, and larkspur, respectively.

24.12–Flavonoids 1303 Soybean Various flavonoids have also been studied extensively from the perspectives of health protection and pharmacological utility, for which mammalian enzyme systems have been used to assess flavonoid activity. Flavonoids have been analyzed as modula- tors of immune and inflammatory responses, for their impact on smooth muscle function, and as anticancer, antiviral, antitoxic, and hepatoprotective agents. There is considerable current interest in the use of OH isoflavonoids in cancer prevention. Dietary consumption of the isoflavonoids daidzein HO O and genistein (Fig. 24.70), which are present in soybeans, is thought to reduce substantially the incidence of breast and prostate Figure 24.67 OH cancers in humans. Kaempferol, a UV-B protectant, is OH O present in many plants such as soybean (Glycine max). Kaempferol 24.12.2 The flavonoid biosynthesis pathway has several important branchpoints. compounds include proanthocyanidins and their congeners in woody gymnosperms and The flavonoids consist of various groups of isoflavonoids in woody legumes from the plant metabolites, which include chalcones, tropics. In both cases, their massive deposi- aurones, flavonones, isoflavonoids, flavones, tion during heartwood formation contributes flavonols, leucoanthocyanidins (flavan-3,4- significantly and characteristically to the diols), catechins, and anthocyanins (Figs. overall color, quality, and rot resistance of 24.70 and 24.71). wood. These metabolites can be misidenti- The first committed step of the flavo- fied as lignins because some constituents are noid pathway is catalyzed by chalcone syn- not readily solubilized and are frequently thase (CHS; see Fig. 24.70). Three molecules dissolved only under the same conditions of acetate-derived malonyl-CoA and one that effect lignin dissolution. molecule of p-coumaryl-CoA are condensed to generate a tetrahydroxychalcone (see Fig. 24.49, reaction 9). CHS, a dimeric polyketide OH synthase with each subunit at about 42 kDa, OH has no cofactor requirements. In certain

HO O species, the coordinated action of CHS and an NADPH-dependent reductase generates OH OH a 6-deoxychalcone (isoliquiritigenin). Both OH OH n chalcones can then be converted into au- HO O rones, a subclass of flavonoids found in certain plant species. Beyond CHS, the next OH OH step shared by most of the flavonoid biosyn- OH OH thesis pathways is catalyzed by chalcone HO O isomerase (CHI), which catalyzes a stereospecific ring closure isomerization step OH to form the 2S-flavanones, naringenin, and OH (less commonly) liquiritigenin (see Fig. Red sorghum Proanthocyanidin (n = 1–30) 24.70). The flavanones may represent the most important branching point in Figure 24.68 flavonoid metabolism, because isomerization Red sorghum produces proanthocyanidin antifeedant compounds—condensed of these compounds yields the phytoalexin tannins, which deter birds from feeding on the seed. White sorghum, which is deficient in these compounds, is rapidly consumed by birds. Similar compounds isoflavonoids (Fig. 24.70), introduction of a are present in the heartwood of Douglas fir (not shown). C-2–C-3 double bond affords flavones and

1304 Chapter 24 Natural Products (Secondary Metabolites) Alfalfa HO O H

H O OCH3 (–)-Medicarpin

HO O R

OH O Nodule R = H Apigenin R = OH Luteolin

Figure 24.69 Flavonoids perform diverse functions in alfalfa (Medicago sativa). The flavonoids apigenin and luteolin function as signaling molecules that induce Nod gene expression in compatible Rhizobium bacteria, facilitating the development of nitrogen-fixing root nodules. The phytoalexin isoflavonoid medicarpin participates in inducible plant defense.

flavonols (Fig. 24.71), and hydroxylation of pinoresinol/lariciresinol reductase (see Fig. the 3-position generates dihydroflavonols 24.56 and Section 24.11.1), suggesting a phy- (Fig. 24.71). logenetic link between both lignan and Entry into the isoflavonoid branchpoint isoflavonoid pathways for plant defense. occurs by way of two enzymes (see Fig. 24.70). The second branching point in general The first, isoflavone synthase (IFS), catalyzes flavonoid metabolism involves that of dehy- an unusual C-2 to C-3 aryl migration and dration of naringenin at the C-2/C-3 posi- hydroxylation to give the 2-hydroxyisofla- tions to give such abundant flavones as vanones and has recently been shown to apigenin (Fig. 24.71). This conversion is cat- be an NADPH-dependent cytochrome P450 alyzed by flavone synthase (FNS), which var- enzyme. Dehydration of the 2-hydroxyiso- ies in enzymatic type depending on the plant flavanones, catalyzed by 2-hydroxyisoflava- species. For example, in parsley cell cultures, none dehydratase (IFD), forms the isofla- flavone formation is catalyzed by an α-ketog- vonoids genistein and daidzein. The lutarate–dependent dioxygenase (FNS I in isoflavonoids can be further metabolized, Fig. 24.71), whereas an NADPH-dependent primarily in the Fabaceae, to yield phy- microsomal preparation engenders this toalexins (e.g., medicarpin in alfalfa; see Fig. reaction in Antirrhinum flowers (FNS II in 24.70) or to generate isoflavonoid-derived Fig. 24.71). substances known as rotenoids in tropical The third major branchpoint in flavonoid legumes (e.g., 9-demethylmunduserone metabolism is stereospecific 3-hydroxylation from Amorpha fruticosa; see Fig. 24.70). The of naringenin (or its 3’-hydroxylated analog) rotenoids, which are isolated mainly from to give dihydroflavonols (Fig. 24.71) such as Derris elliptica and related species, are used dihydrokaempferol (or dihydroquercetin). extensively as insecticidal agents but have The enzyme involved, flavanone 3-hydroxy- other applications as well. For example, lase, is an Fe2+-requiring, α-ketoglutarate– rotenone is used as a rat poison and an in- dependent dioxygenase. Specific hydroxyla- hibitor of NADH dehydrogenase. Interest- tion involving an NADPH-dependent cyto- ingly, the NADPH-dependent isoflavone re- chrome P450 monooxygenase of naringenin ductase (IFR) step involved in isoflavonoid can also directly give dihydroquercetin, which formation shows considerable homology to can be converted to quercetin (a flavanol) by

24.12–Flavonoids 1305 p-Coumaryl-CoA + 3 malonyl-CoA

Chalcone synthase Chalcone synthase NADPH reductase 5 OH 6 4 OH OH OH

OH OH OH OH 4’ 2’ OH 3 OH O 1 O 2

6’ O O OH O OH O Hispidol Isoliquiritigenin 4,2’,4’,6’-Tetrahydroxychalcone 4,4’,6’-Trihydroxyaurone (an aurone) (a chalcone) (a chalcone) (an aurone) CHI CHI

5’ 5’ OH O OH 6’ 4’ OH 6’ 4’ OH B B OH OH 8 8 O OH 7 O 2 3’ OH 7 O 2 1’ 1’ 3’ ACC 2’ A 2’ IFS 3 IFS OH O 6 6 3 OH 5 4 54 O O OH O OH Liquiritigenin Naringenin 2-Hydroxyisoflavanone 2-Hydroxyisoflavanone (a flavanone) (a flavanone) IFD IFD

8 OH 7 O 2 OH O AC 6’ 6 3 Figure 24.70 4 5’ 5 B Biosynthetic pathways for production of specific flavonoid 4’ O 2’ subclasses, including the chalcones, aurones, flavanones, OH OH O 3’ and isoflavones (isoflavonoids). The enzymes involved OH Daidzein Genistein (and their cofactors) are as follows: CHI, chalcone iso- (an isoflavonoid) (an isoflavonoid) merase; IFS, 2-hydroxyisoflavanone synthase (O2, cytochrome P450, NADPH); IFD, 2-hydroxyisoflavanone dehy- IOMT IOMT dratase; IOMT, isoflavanone O-methyltransferase (SAM); I-2’H, isoflavone 2’-hydroxylase (O , cyt. P450, NADPH); OH O 2 IFR, isoflavone reductase (NADPH); vestitone reductase OH O (NADPH); DMI reductase, 7,2’-dihydroxy-4’-methoxyisoflavanol dehydratase.

O OH OCH3 O OCH3 Formononetin Biochanin A I-2’-H H OH O OH O OH O O OCH3

H O O O HO OCH3 OCH3 OCH3 2’-Hydroxyformononetin OCH3 OCH3 2’,4’,5’-Trimethoxyformononetin 9-Demethylmunduserone IFR (a rotenoid) OH O OH O OH O H H H Vestitone reductase DMI dehydratase H H OH O O HO OCH3 HO OCH3 OCH3 (–)-Vestitone 7,2’-Dihydroxy-4’-methoxyisoflavanol (–)-Medicarpin (DMI)

1306 5’ OH OH 6’ 4’ B 8 OH O 2 3’ OH O 7 1’ 2’ A C 63FNS 54 OH O OH O Naringenin Apigenin (a flavanone) (a flavone)

FHT

OH OH OH

OH O OH O OH O OH Ring B hydroxylation FLS OH OH OH F3H OH O OH O OH O

Dihydroquercetin Dihydrokaempferol Kaempferol (dihydroflavonol) (dihydroflavonol) (flavonol)

FLS

DFR

OH DFR OH

OH O OH O OH

OH Flavan- OH Flavan- 3,4-diols 3,4-diols OH O OH HO

Quercetin Leucopelargonidin (flavonol) (leucoanthocyanidins)

ANS

OH + Catechins Oligomeric/polymeric Oligomeric/polymeric Catechins OH O proanthocyanidins anthocyanidins

OH OH Phlobaphenes, Pelargonidin etc. (anthocyanin)

FGT Figure 24.71 Selected major enzymatic reactions in the flavonoids. OH The enzymes involved (and their cofactors) are as fol- + lows: FNS, flavone synthase (FNS I: 2-oxoglutarate, OH O O2; FNS II: O2, cytochrome P450, NADPH; apigenin is formed by the action of FNS I); FHT, flavanone α OGlc 3-hydroxylase ( -ketoglutarate, O2); F3H, flavonoid 3’-hydroxylase (cytochrome P450, NADPH); FLS, OH α flavonol synthase ( -ketoglutarate, O2); DFR, dihydroflavonol 4-reductase (NADPH); ANS, anthocyani- Pelargonidin 3-glucoside din synthase; FGT, UDP–glucose:flavonoid 3-O- (anthocyanin) glucosyltransferase (UDP–glucose).

1307 flavonol synthase (FLS)–catalyzed C-2–C-3 of Heracleum mantegazzianum (giant hog- double bond formation; FLS is an α-ketoglu- weed), can cause photophytodermititis on tarate–dependent dioxygenase. Alternatively, skin contact and subsequent exposure to dihydroquercetin can be reduced by an UV-A radiation (Fig. 24.73). A comparable NADPH-dependent dihydroflavonol form of coumarin-induced dermatitis can reductase (DFR) to give the corresponding also occur during celery handling. Psoralen flavan-3,4-diols (Fig. 24.71). (Fig. 24.74), however, is now successfully Subsequent species- and tissue-specific used to treat various skin disorders (eczema, enzymatic conversions can create vast arrays psoriasis) by means of a combination of oral of structurally diverse groups of flavonoids. ingestion and UV-A treatment. For example, in flower petals, the leucoan- The structure of a representative simple thocyanidins (e.g., leucopelargonidin) can be coumarin, 7-hydroxycoumarin, is shown in converted to the colored anthocyanins (e.g., Figure 24.75. Additional families of plant pelargonidin) through the action of a dehy- coumarins (see Fig. 24.74) include linear dratase, anthocyanidin synthase (ANS), furanocoumarins (e.g., psoralen), angular which is thought to be an α-ketoglutarate– furanocoumarins (e.g., angelicin), pyra- dependent dioxygenase (Fig. 24.71). Leu- nocoumarins (e.g., seselin), and pyrone- coanthocyanidins can also serve as precur- substituted coumarins (e.g., 4-hydroxy- sors of the (epi-)catechins and condensed coumarin). tannins. The enzymology associated with those coupling processes, chain extension mechanisms, and oxidative modifications, 24.13.2 Coumarin biosynthesis pathways however, is not yet established. have not yet been fully elucidated.

The biosynthetic pathways to the coumarins 24.13 Coumarins, stilbenes, are only partially determined at this point; styrylpyrones, and arylpyrones they mainly involve aromatic hydroxylations and additional reactions catalyzed by 24.13.1 Some coumarins, a class of plant trans/cis-hydroxycinnamic acid isomerases, defense compounds, can cause internal dimethylallyltransferases, various P450/

bleeding or dermatitis. NADPH/O2–dependent synthases and O- methyltransferases (Fig. 24.75). The simplest Coumarins (e.g., coumarin; Fig. 24.72A) belong to a widespread family of plant metabolites called the benzopyranones, with (A) more than 1500 representatives in more than 800 species. In plants, these compounds can occur in seed coats, fruits, flowers, roots, leaves, and stems, although in general the O O greatest concentrations are found in fruits Coumarin and flowers. Their roles in plants appear to be mainly defense-related, given their an- (B) timicrobial, antifeedant, UV-screening, and germination inhibitor properties. O OH The best known properties of coumarins indirectly highlight their roles in plant defense. Ingesting coumarins from plants such as clover can cause massive internal bleeding in mammals. This discovery ultimately O O led to the development of the rodenticide Warfarin Warfarin (Fig. 24.72B) and to the use of relat- (synthetic coumarin) ed compounds to treat and prevent stroke. Figure 24.72 Likewise, the photosensitizing compound Structures of (A) coumarin (from clover), and (B) a 8-methoxypsoralen, present in leaf tissue synthetic coumarin, the rodenticide Warfarin.

1308 Chapter 24 Natural Products (Secondary Metabolites) O O O

OCH3

Heracleum 8-Methoxypsoralen (a furanocoumarin)

Figure 24.73 A linear furanocoumarin, 8-methoxypsoralen, sensitizes human skin to UV-A light. This compound, present in external tissues of Heracleum species, causes severe blistering on skin contact followed by exposure to UV-irradiation.

examples, coumarin and 7-hydroxycoumarin systems. Beyond the initial synthases, how- (umbelliferone), are believed to be formed ever, little has yet been described about sub- by O-hydroxylation of cinnamic and p- sequent transformations. coumaric acids, respectively, followed by Stilbenes are present in bryophytes, trans/cis-isomerization and ring closure. pteridophytes, gymnosperms, and angio- However, neither the enzymes nor their en- sperms, with more than 300 different stil- coding genes have yet been obtained. benoids known today. The stilbenes play On the other hand, much more is known important roles in plants, particularly in about the biosynthesis of both linear and an- heartwood protection, and also have signifi- gular furanocoumarins. These involve re- cance in pharmacology and human health. giospecific prenylation through the action In plants, they can function as both constitu- of the corresponding tranferases to yield tive and inducible defense mechanisms. Stil- demethylsuberosin and osthenol, respective- benes display weak antibacterial properties ly. The subsequent transformations are believed to involve various NADPH-dependent, cytochrome P450 oxidase–catalyzed conversions and O-methylations (Fig. 24.75). Various fungi and yeasts also biosynthesize coumarins, e.g., the toxic aflatoxins. O O O O O However, these metabolites are polyketide O derivatives and hence are biochemically distinct from their plant analogs. Psoralen Angelicin (a linear furanocoumarin) (an angular furanocoumarin)

24.13.3 Stilbenes, styrylpyrones, and OH arylpyrones constitute another class of chemical defense compounds.

In addition to the products of the flavonoid O O O O O pathway, cinnamoyl-CoA and malonyl-CoA (acetate-derived) pathways can in certain plant species also undergo condensation re- Seselin 4-Hydroxycoumarin actions to yield the corresponding stilbenes, (a pyranocoumarin) (a pyrone-substituted styrylpyrones, and arylpyrones (Fig. 24.76). coumarin) Comparison of gene sequences for each entry-point enzyme (CHS and stilbene syn- Figure 24.74 Structures of the linear furanocoumarin psoralen, the angular fura- thase) reveals significant homology, as nocoumarin angelicin, the pyranocoumarin seselin, and the pyrone- would be expected for similar enzymatic substituted coumarin 4-hydroxycoumarin.

24.13–Coumarins, Stilbenes, Styrylpyrones, and Arylpyrones 1309 H3CO Figure 24.75 Selected aspects of coumarin and furanocoumarin biosynthesis. The enzymes involved (and their cofactors) are as follows: HO O O 1. DMAPP:umbelliferone dimethylallyl transferase, modifying Scopoletin the 6 position; 2. marmesin synthase (O2, cytochrome P450, NADPH); 3. psoralen synthase (O2, cyt. P450, NADPH); 4. bergaptol synthase (O2, cyt. P450, NADPH); 5. xanthotoxol synthase (O2, cyt. P450, NADPH); 6. bergaptol O-methyltransferase (SAM); 7. DMAPP:umbelliferone dimethylallyl transferase, modifying the 8 position; 8. columbianetin synthase H3CO COOH (O2, cyt. P450, NADPH); and 9, angelicin synthase (O2, cyt. P450, NADPH). HO Ferulic acid

H C 5 4 3 COOH COOH 6 6 3 H3C Phenylalanine HO HO 7 2 O 1 HO O 8 O O 1 Demethylsuberosin Cinnamic acid p-Coumaric acid 7-Hydroxycoumarin (umbelliferone) 2 7

HO O O H3C 8 O O O 8 H3C (+)-Marmesin

O O O Osthenol

9 H3C (–)-Columbianetin HO O O O CH 3 Psoralen 5 O O O 4 OH Angelicin

O O O OH O O O Xanthotoxol Bergaptol

6 OCH but their antifungal effects are more potent, 3 inhibiting fungal spore germination and hyphal growth; stilbenes also function in dormancy and growth inhibition of plants. O O O Certain stilbenoids, besides being toxic to in- Bergapten sects and other organisms, have mammalian antifeedant and nematicidal properties. Stil- benoid formation can be induced by insect From a pharmacological perspective, the attack, as illustrated by the colored deposits stilbene combretastatin has important anti- formed in radiata pine sapwood when at- neoplastic activities, and resveratrol, present tacked by the Sarix wasp (see photographs in red wine, helps suppress tumor formation in Box 24.5). (Fig. 24.77).

1310 Chapter 24 Natural Products (Secondary Metabolites) R1

COOH + H2C COSCoA

Malonyl-CoA

CoAS O

R1 R2 =H Cinnamoyl-CoA R1 = OH R2 = H p-Coumaroyl-CoA R1 R2 = OH Caffeoyl-CoA R2

R1 R2

R1 1 1 O O H C 3 Benzalacetone Arylpyrone synthase synthase O Benzalacetones Arylpyrones e.g., p-Hydroxyphenylbut-3-ene-2-one e.g., Psilotinin R2 [R1 = OH, R2 = H] [R1 = OH, R2 = H] R1

2 O O Styrylpyrone synthase

R2 OH Styrylpyrones R1 R e.g., Hispidin [R1, R2 = OH] 2

R1 Figure 24.76 OH OH Cinnamoyl-CoA, 3 3 p-coumaroyl-CoA, and OH caffeoyl-CoA, precursors Chalcone Stilbene for the biosynthesis of synthase synthase arylpyrones, styrylpy- OH O rones, and stilbenes. The designation 1×, 2×, and Chalcones OH 3× refers to the number Stilbenes of molar equivalents of e.g., Naringenin [R1 = OH, R2 = H] e.g., Pinosylvin [R1, R2 = H] malonyl-CoA required. Eriodictyol [R1, R2 = OH] Resveratrol [R1 = OH, R2 = H]

24.14 Metabolic engineering of economic implications, affording new oppor- phenylpropanoid production: a possible tunities for systematic modification of com- source of enhanced fibers, pigments, mercially important plants to engineer or pharmaceuticals, and flavoring agents specify particular traits that can benefit hu- manity. The biochemical, chemical, and molecular Many biotechnological possibilities characterization of how plants produce vari- await our manipulation of plant phenolic ous metabolic substances is essential to un- metabolism: plants with increased resistance derstanding the very basis of the biodiversi- to pathogens; improvements in the quality ty and life of plants. This pursuit also has of wood and fiber products; new or improved

24.13–Coumarins, Stilbenes, Styrylpyrones, and Arylpyrones 1311 Postlignification metabolism and heartwood formation require Box 24.5 nonstructural plant phenolic compounds.

Heartwood represents more than 95% content of this wood (about 28%) indicate only now becoming known, heartwood of the merchantable bole of harvested that lignin biopolymers themselves are metabolites are first deposited in the cen- wood. The heartwood of commercially nearly colorless. tral (pith) region of the lignified woody important woody plants accounts for Heartwood production is a postsec- stem tissues, which primarily consist of more than 60% of the revenues generated ondary xylem-forming process, whereby dead, lignified cells. Over years of subse- from harvesting plant materials before fur- nonstructural highly colored phenolics quent growth, heartwood formation grad- ther factory processing. Heartwood serves (primarily lignan, stilbene, and flavonoid- ually extends radially, until almost all of as the main source of raw material for derived compounds) and other character- the woody xylem tissue is encompassed. lumber, solid wood products, fine furni- istic substances (e.g., terpenes or alka- A transition zone sometimes visible be- ture, paper, and many miscellaneous ap- loids) are infused into wood that has tween the heartwood and sapwood is pre- plications. Despite its economic signifi- already been lignified. Substances similar sumed to be involved in the final stages of cance, however, the general mechanism (if not identical, in some cases) to those in biosynthesis of heartwood metabolites responsible for its formation is one of the heartwood can also be formed in regions preceding cell death. The composition of most poorly studied and poorly under- where insects or pathogens have attacked heartwood metabolites varies extensively stood areas of plant science today. sapwood, but these are manifested as a among species. For example, Douglas fir Heartwood is formed by the species- more-localized containment response. For accumulates flavonoids and lignans in its specific deposition of distinct and varied example, panel B of the figure shows sap- heartwood, whereas yellow poplar de- metabolites that frequently alter the color, wood of radiata pine into which a Sirex posits lignans, terpenoids, and alkaloids. durability, texture, and odor of particular noctilio wasp has bored, forming two tun- Given that wood is composed largely woods relative to that of sapwood. Heart- nels, one for the wasp’s eggs and one for of dead cells, how are heartwood woods contain strikingly distinctive col- a fungus, Amylostereum noctilio, that metabolites deposited? Investigators rec- ored metabolites that can readily be ob- serves as a foodstuff for the larvae. The at- ognized 50 years ago that ray parenchy- served by inspecting cross-sections of tacked plant responds by increasing the ma cells remain living in lignified sap- woody stems of plants, such as tamarack deposition of various phenolic sub- wood. As their last function before death, (see panel A of figure), western red stances, in this case stilbenes, which are these cells accumulate or biosynthesize cedar (reddish- or pinkish-brown to dark primarily localized in the affected regions, substances (often in complex species-spe- brown), ebony (jet black), and southern making them appear lighter-colored than cific mixtures) that are then infused into pine (yellow-orange). In contrast, spruce the background in the stained wood sec- lignified woody secondary xylem by way wood, highly valued for pulp and paper tion shown in panel C of the figure. of pit apertures (see figure, panel D). This manufacture, contains less of the highly Constitutive heartwood formation, on infusion process may explain why many colored heartwood metabolites and hence the other hand, follows several years or of the heartwood substances also occur has a pale whitish-yellow color. Indeed, decades of sapwood growth and develop- at much lower concentrations in sap- the pale color and the very high lignin ment. According to biochemical details wood, where the ray parenchyma cells

(A) (B)

HO OH

OCH3 OH

H3CO OCH3 HO

OCH3

Combretastatin Resveratrol (a stilbene)

Figure 24.77 (A) The stilbene, combretastatin, has antineoplastic activity. (B) Another stilbene, resveratrol, present in red grapes and red wine, has potent antitumor properties.

1312 Chapter 24 Natural Products (Secondary Metabolites) (A) (B) are located. At some point during heartwood formation, the infused phenolic substances apparently undergo oxidation to form nonstructural oligomeric and polymeric components, some of which can be removed only under the conditions typically used for lignin extraction. With the further sealing of bordered pits, heartwood ceases to function in conducting nutrients and water and essentially be- comes a protective structural tissue, highly durable and resistant to rot.

Bark Sapwood Heartwood

Ray parenchyma

Substances secreted through pit apertures

Neighboring cell lumen

sources of pharmaceuticals, nutriceuticals, sociated metabolic functions. This approach pigments, flavors, and fragrances; and selec- could involve modification of lignin, either tive adjustments to the taste and odor of se- to render it more susceptible to removal, or lected plant species. Indeed, a biotechnological to increase its content, thereby increasing the revolution is now being witnessed in the strength and rigidity of certain fragile crops. plant sciences. The combined use of molecu- Modifying heartwood metabolite formation lar genetic techniques and conventional may allow researchers to tailor traits such as plant breeding approaches is expected to rot resistance, texture, color, and durability produce a new generation of plants that are in various commercially important woody even further optimized for human use. plant heartwoods. These goals require fur- By far the largest and economically most ther study of the fundamental mechanisms significant deployment of plant materials is controlling both macromolecular assembly as a fiber source, whether for pulp/paper, patterns involved in the biosynthesis of lumber for housing and shelter, wood for plant cell wall polymers and exploration of furniture, or other applications. Accordingly, how and where the heartwood-forming many biotechnological strategies are directed metabolites are generated. toward improving fiber and wood properties To this point, most of the biotechnological by manipulating the biochemical processes emphasis placed on attempting to engineer responsible for cell wall biosynthesis and as- lignin content and composition has involved

24.14–Metabolic Engineering of Phenylpropanoid Production 1313 Box 24.6 Phenolics flavor our world.

Phenylpropanoid-derived plant phe- establish the characteristic tastes and is thus ingested daily by millions. Green nolics contribute significantly to impart- odors of oil of cloves, widely used in and black tea leaves contain other plant ing specific fragrances/odors, flavors, and toothache treatment, and of the spices phenolics, such as (epi-)catechins and tastes to various plants widely utilized in nutmeg and mace. Vanillin, from the various other tannins that impart charac- the food and beverage industries today vanilla bean, is used extensively in both teristic tastes to these popular beverages. (see figure). Although the biochemistry of baking and confectionery. In most in- Most drinks consumed today would be their formation is scarcely addressed in stances, precise biochemical pathways to watery indeed if not for various pheno- this brief chapter, their importance cannot these compounds are not yet established lics, such as vanillin, ferulic acid, certain be discounted. at the levels of either the enzymes or the flavonoids, tannins, and others. An impor- The capsaicinoids, such as capsaicin, genes. tant endeavor of the flavor and fragrance are responsible for the pungent properties Plant phenolics are important compo- industry is to define or identify the mix- of the red peppers, whereas the piperi- nents of the characteristic aromas, flavors, tures of various phenolic substances that noids flavor black pepper. The delightful and colors of many beverages, whether create pleasing flavors ranging from tastes of cinnamon and ginger are impart- for alcoholic or nonalcoholic consump- maple syrup to whisky. ed by various cinnamate and gingerol tion. Chlorogenic acid, for example, con- derivatives, respectively; and allylphenols stitutes about 4% of the coffee bean and

using antisense and sense strategies to target pursue various interesting questions, includ- the genes that encode various enzymatic ing how heartwood is formed. steps in the pathway from phenylalanine to Advances in lignan and (iso)flavonoid the monolignols (see Fig. 24.49). This work biochemistry and molecular biology offer has focused primarily on cinnamyl alcohol the opportunity to modify concentrations dehydrogenase and cinnamoyl-CoA reduc- of health protectants and pharmacologically tase and has targeted such plants as tobacco, active species in particular plants of choice. poplar, and eucalyptus. Although the effects Eventually, we should be able to engineer on lignin formation per se have often been the formation of secoisolariciresinol, matai- quite small, the transgenic plant tissues pro- resinol, daidzein, genistein, and similar com- duced were highly colored, unlike the origi- pounds in staple crops that do not ordinarily nal wild-type plants. Whether these trans- produce them in significant quantities. The genic plants will have any beneficial corresponding transgenic plants thus would properties, for example, greater ease of provide long-term health benefits as sources lignin removal for pulp/paper applications, of cancer preventives. A similar target for is unclear. The pigmentation effects observed enhanced production might be podophyllo- were not anticipated by the researchers in- toxin, one of a handful of plant anticancer volved and point to the fact that attempts to compounds already in use today. alter lignin-forming processes must also take The potential being unleashed is perhaps into account the related biochemical path- most vividly demonstrated by the impres- ways that utilize the same substrates. sive advances in plant metabolic engineering The finding that lignin formation proper seen in the manipulation of flower color by is somehow temporally and spatially associ- application of sense/antisense technologies. ated with various presumedly proline-rich Several laboratories in Europe and New proteins and dirigent sites holds much Zealand have successfully transformed vari- promise. Full details of the influence of these ous plants such as petunia to alter petal color. proteins on lignin structure may result in Lastly, knowledge of these pathways the design of new strategies for modifying will eventually lead to the systematic modi- both lignin deposition and structure. The fication and improvement of plant flavors discovery of dirigent proteins, pinoresinol/ and fragrances, the properties of which de- lariciresinol reductases, and their correspond- fine the very essence of many of our food- ing genes also affords the opportunity to stuffs, such as pepper, ginger, and vanilla.

1314 Chapter 24 Natural Products (Secondary Metabolites) Coffee beans O O COOH Cloves

HO OH OH

R OH OH OH R = H Chavicol Chlorogenic acid R = OCH3 Eugenol

Cinnamon bark Nutmeg CHO O

R R = H Safrole R = OCH3 Myristicin Cinnamaldehyde

(CH ) CH Ginger rhizome O 2 n 3 Orchid

CH2CH2OH

n=4,6,8

OCH3 OH Phenylethyl alcohol Gingerols

Red and black peppers O

H3CO N R

H Vanilla HO

R = CHO Nordihydrocapsaicin

R =

Capsaicin OCH3 O OH O N Vanillin

O Piperine

R Green tea OH

HO O OH

OH R = H (–)-Epicatechin R = OH (–)-Epigallocatechin

24.14–Metabolic Engineering of Phenylpropanoid Production 1315 These modifications will ultimately impact the capacity to produce and safely store such the quality of many of our alcoholic and ecologically useful metabolites has become nonalcoholic beverages, which in turn are widely established in the plant kingdom. often largely determined by their aromatic Pressures from herbivores and pathogens, as phenolic constituents (Box 24.6). well as constant competition, continue to select for new natural products. In cultivated species, however, such chemical defenses Summary have often been artificially selected against. Study of the biochemistry of plant natu- Plants produce a great variety of organic ral products has many practical applications. compounds that are not directly involved in Biotechnological approaches can selectively primary metabolic processes of growth and increase the amounts of defense compounds development. The roles these natural prod- in crop plants, thereby reducing the need for ucts or secondary metabolites play in plants costly and potentially toxic pesticides. Simi- have only recently come to be appreciated in larly, genetic engineering can be utilized to an analytical context. Natural products ap- increase the yields of pharmaceuticals, flavor pear to function primarily in defense against and perfumery materials, insecticides, fungi- predators and pathogens and in providing cides, and other natural products of com- reproductive advantage as attractants of pol- mercial value. Although many natural prod- linators and seed dispersers. They may also ucts and their functions have been described act to create competitive advantage as poi- in this chapter, the metabolism of natural sons of rival species. products in most plant species remains to be Most natural products can be classified elucidated. A great deal of fascinating bio- into three major groups: terpenoids, alka- chemistry remains to be discovered. loids, and phenolic compounds (mostly phenylpropanoids). Terpenoids are composed of five-carbon units synthesized by Further Reading way of the acetate/mevalonate pathway or the glyceraldehyde 3-phosphate/pyruvate Terpenoids pathway. Many plant terpenoids are toxins and feeding deterrents to herbivores or are Cane, D. E., ed. (1999) Comprehensive Natural attractants of various sorts. Alkaloids are Products Chemistry, Vol. 2, Isoprenoids In- synthesized principally from amino acids. cluding Carotenoids and Steroids. Perga- These nitrogen-containing compounds pro- mon/Elsevier, Amsterdam. tect plants from a variety of herbivorous ani- Chappell, J. (1995) Biochemistry and molecu- mals, and many possess pharmacologically lar biology of the isoprenoid biosynthetic important activity. Phenolic compounds, pathway in plants. Annu. Rev. Plant Physi- which are synthesized primarily from ol. Plant Mol. Biol. 46: 521–547. products of the shikimic acid pathway, have Eisenreich, W., Schwarz, M., Cartayrade, A., several important roles in plants. Tannins, Arigoin, D., Zenk, M. H., Bacher, A. (1998) lignans, flavonoids, and some simple pheno- The deoxylulose phosphate pathway of lic compounds serve as defenses against her- terpenoid biosynthesis in plants and mi- bivores and pathogens. In addition, lignins croorganisms. Chem. Biol. 5: R221–R233. strengthen cell walls mechanically, and Gershenzon, J., Croteau, R. (1993) Terpenoid many flavonoid pigments are important at- biosynthesis: the basic pathway and for- tractants for pollinators and seed dispersers. mation of monoterpenes, sesquiterpenes Some phenolic compounds have allelopathic and diterpenes. In Lipid Metabolism in activity and may adversely influence the Plants, T. S. Moore, Jr., ed. CRC Press, Boca growth of neighboring plants. Raton, FL, pp. 339–388. Throughout the course of evolution, Harborne, J. B., and Tomas-Barberan, F. A., plants have developed defenses against her- eds. (1991) Ecological Chemistry and Bio- bivory and microbial attack and produced chemistry of Plant Terpenoids. Clarendon other natural products to aid competitive- Press, Oxford, UK. ness. The better-defended, more-competitive Langenheim, J. H. (1994) Higher plant plants have generated more progeny, and so terpenoids: a phytocentric overview of

1316 Chapter 24 Natural Products (Secondary Metabolites) their ecological roles. J. Chem. Ecol. 20: Sarkanen, S., Lewis, N. G. (1998) Biosynthe- 1223–1280. sis of lignins and lignans. ACS Symp. Ser. Lichtenthaler, H. K. (1999) The 1-deoxy- 697: 1–421. D-xylulose-5-phosphate pathway of iso- Timell, T. E. (1986) Compression Wood in prenoid biosynthesis in plants. Annu. Rev. Gymnosperms, Vols. 1–3. Springer- Plant Physiol. Plant Mol. Biol. 50: 47–65. Verlag, Berlin. McGarvey, D., Croteau, R. (1995) Terpenoid biosynthesis. Plant Cell 7: 1015–1026. Flavonoids Dixon, R. A. (1999) Isoflavonoids: biochem- Alkaloids istry, molecular biology and biological Cordell, G., ed. (1997) The Alkaloids, Vol. 50. functions. In Comprehensive Natural Prod- Academic Press, San Diego. ucts Chemistry, Vol. 1, Polyketides and Other Rosenthal, G. A., and Berenbaum, M. R., eds. Secondary Metabolites Including Fatty Acids (1991) Herbivores: Their Interactions with Sec- and Their Derivatives, D.H.R. Barton, K. ondary Plant Metabolites, Vol. 1: The Chemi- Nakanishi, and O. Meth-Cohn, eds.-in- cal Participants, 2nd ed. Academic Press, chief. Elsevier, Amsterdam, pp. 773–824. San Diego. Forkmann, G. (1991) Flavonoids as flower Southon, I. W., and Buckingham, J., eds. pigments: the formation of the natural (1989) Dictionary of Alkaloids. Chapman spectrum and its extension by genetic en- and Hall, London. gineering. Plant Breed. 106: 1–26. Forkmann, G., Heller, W. (1999) Biosynthesis of flavonoids. In Comprehensive Natural Suberin Products Chemistry, Vol. 1, Polyketides and Other Secondary Metabolites Including Fatty Bernards, M. A., Lewis, N. G. (1998) The Acids and Their Derivatives, D.H.R. Barton, macromolecular aromatic domain in suber- K. Nakanishi, and O. Meth-Cohn, eds.-in- ized tissues: a changing paradigm. Phyto- chief. Elsevier, Amsterdam, pp. 713–748. chemistry 47: 583–591. Harborne, J. B., ed. (1994) The Flavonoids: Ad- vances in Research Since 1986. Chapman and Hall, London. Lignins and lignans Gang, D. R., Costa, M. A., Fujita, M., Coumarins and furanocoumarins Dinkova-Kostova, A. T., Wang, H.-B., Burlat, V., Martin, W., Sarkanen, S., Davin, Berenbaum, M. R., and Zangerl, A. R. (1996) L. B., Lewis, N. G. (1999) Regiochemical Phytochemical diversity: adaptation or ran- control of monolignol radical coupling: a dom variation. Rec. Adv. Phytochem. 30: 1–24. new paradigm for lignin and lignan Keating, G. J., O’Kennedy, R. (1997) The chem- biosynthesis. Chem. Biol. 6: 143–151. istry and occurrence of coumarins. In Cou- Lewis, N. G., Davin, L. B. (1999) Lignans: marins: Biology, Application and Mode of Ac- biosynthesis and function. In Comprehensive tion, R. O’Kennedy and R. D. Thornes, eds. Natural Products Chemistry, Vol. 1, Polyke- John Wiley & Sons, Chichester, pp. 23–66. tides and Other Secondary Metabolites Includ- Matern, U., Lüer, P., Kreusch, D. (1999) ing Fatty Acids and Their Derivatives, D.H.R. Biosynthesis of coumarins. In Comprehen- Barton, K. Nakanishi, and O. Meth-Cohn, sive Natural Products Chemistry, Vol. 1, eds.-in-chief. Elsevier, Amsterdam, pp. Polyketides and Other Secondary Metabolites 639–712. Including Fatty Acids and Their Derivatives, Lewis, N. G., Davin, L. B., Sarkanen, S. D.H.R. Barton, K. Nakanishi, and O. Meth- (1999) The nature and functions of lignins. Cohn, eds.-in-chief. Elsevier, Amsterdam, In Comprehensive Natural Products Chemistry, pp. 623–638. Vol. 3, Carbohydrates and Their Derivatives In- Matern, U., Strasser, H., Wendorff, H., cluding Tannins, Cellulose and Related Lignins, Hamerski, D. (1988) Coumarins and fura- D.H.R. Barton, K. Nakanishi, and O. Meth- nocoumarins. In Cell Culture and Somatic Cohn, eds.-in-chief. Elsevier, Amsterdam, Cell Genetics of Plants, Vol. 5, I. K. Vasil, ed. pp. 617–745. Academic Press, Orlando, FL, pp. 3–21.

Further Reading 1317 Zobel, A. M. (1997) Coumarins in fruits and Biochemistry of the Stilbenoids, Vol. 1, Bio- vegetables. Proc. Phytochem. Soc. Eur. 41: chemistry of Natural Products Series, J. B. 173–203. Harborne, ed. Chapman and Hall, London. Schröder, J. (1999) The chalcone/stilbene synthase-type family of condensing Stilbenes, styrylpyrones, and arylpyrones enzymes. In Comprehensive Natural Products Berkert, C., Horn, C., Schnitzler, J.-P., Lehning, Chemistry, Vol. 1, Polyketides and Other Sec- A., Heller, W., Veit, M. (1997) Styrylpyrone ondary Metabolites Including Fatty Acids and biosynthesis in Equisetum arvense. Phyto- Their Derivatives, D.H.R. Barton, K. Naka- chemistry 44: 275–283. nishi, and O. Meth-Cohn, eds.-in-chief. El- Gorham, J., Tori, M., Asakawa, Y. (1995) The sevier, Amsterdam, pp. 749–772.

1318 Chapter 24 Natural Products (Secondary Metabolites)