Medicinal Natural Products. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 4 THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND PHENYLPROPANOIDS

Shikimic acid and its role in the formation of aromatic amino acids, benzoic acids, and cinnamic acids is described, along with further modifications leading to lignans and lignin, phenylpropenes, and coumarins. Combinations of the shikimate pathway and the acetate pathway are responsible for the biosynthesis of styrylpyrones, flavonoids and stilbenes, flavonolignans, and isoflavonoids. Terpenoid quinones are formed by a combination of the shikimate pathway with the terpenoid pathway. Monograph topics giving more detailed information on medicinal agents include folic acid, chloramphenicol, podophyllum, volatile oils, dicoumarol and warfarin, psoralens, kava, Silybum marianum, phyto-oestrogens, derris and lonchocarpus, vitamin E, and vitamin K.

The shikimate pathway provides an alternative glycolysis) and D-erythrose 4-phosphate (from the route to aromatic compounds, particularly the aro- pentose phosphate cycle) to the aromatic amino matic amino acids L-phenylalanine,L-tyrosine acids was broadly outlined. Phenylalanine and and L-tryptophan. This pathway is employed by tyrosine form the basis of C6C3 phenylpropane microorganisms and plants, but not by animals, units found in many natural products, e.g. cin- and accordingly the aromatic amino acids fea- namic acids, coumarins, lignans, and flavonoids, ture among those essential amino acids for man and along with tryptophan are precursors of a wide which have to be obtained in the diet. A cen- range of alkaloid structures. In addition, it is found tral intermediate in the pathway is shikimic acid that many simple benzoic acid derivatives, e.g. (Figure 4.1), a compound which had been iso- gallic acid (Figure 4.1) and p-aminobenzoic acid lated from plants of Illicium species (Japanese (4-aminobenzoic acid) (Figure 4.4) are produced ‘shikimi’) many years before its role in metabolism via branchpoints in the shikimate pathway. had been discovered. Most of the intermediates in the pathway were identified by a careful study of AROMATIC AMINO ACIDS AND aseriesofEscherichia coli mutants prepared by SIMPLE BENZOIC ACIDS UV irradiation. Their nutritional requirements for growth, and any by-products formed, were then The shikimate pathway begins with a coupling characterized. A mutant strain capable of growth of phosphoenolpyruvate (PEP) and D-erythrose usually differs from its parent in only a single 4-phosphate to give the seven-carbon 3-deoxy- gene, and the usual effect is the impaired syn- D-arabino-heptulosonic acid 7-phosphate (DAHP) thesis of a single enzyme. Typically, a mutant (Figure 4.1). This reaction, shown here as an blocked in the transformation of compound A into aldol-type condensation, is known to be mecha- compound B will require B for growth whilst nistically more complex in the enzyme-catalysed accumulating A in its culture medium. In this version; several of the other transformations in the way, the pathway from phosphoenolpyruvate (from pathway have also been found to be surprisingly 122 THE SHIKIMATE PATHWAY

formally an elimination; it actually involves oxidation of the hydroxyl adjacent to the proton lost and therefore requires NAD+ PEP CO H 2 aldol-type cofactor; the carbonyl is subsequently reduced back to an alcohol reaction CO H P O 2 CO2H H PO PO O H – HOP O H NAD+ HO O H HO OH HO OH OH OH OH D-erythrose 4-P DAHP aldol-type reaction

CO2H CO2H HO CO2H HO CO2H

NADPH – H2O NADH

HO OH O OH O OH HO OH OH OH OH OH shikimic acid 3-dehydroshikimic 3-dehydroquinic quinic acid acid acid

– 2H – H O oxidation and dehydration and 2 enolization enolization CO2H CO2H

HO HO OH OH OH protocatechuic gallic acid acid

Figure 4.1 complex. Elimination of phosphoric acid from gallic acid (3,4,5-trihydroxybenzoic acid) can be DAHP followed by an intramolecular aldol reac- formed by branchpoint reactions from 3-dehy- tion generates the first carbocyclic intermediate 3- droshikimic acid, which involve dehydration and dehydroquinic acid. However, this also represents enolization, or, in the case of gallic acid, dehy- an oversimplification. The elimination of phos- drogenation and enolization. Gallic acid features phoric acid actually follows an NAD+-dependent as a component of many tannin materials (gal- oxidation of the central hydroxyl, and this is then lotannins), e.g. pentagalloylglucose (Figure 4.2), re-formed in an NADH-dependent reduction reac- found in plants, materials which have been used tion on the intermediate carbonyl compound prior for thousands of years in the tanning of animal to the aldol reaction occurring. All these changes hides to make leather, due to their ability to cross- occur in the presence of a single enzyme. Reduc- link protein molecules. Tannins also contribute to tion of 3-dehydroquinic acid leads to quinic acid, the astringency of foods and beverages, especially a fairly common natural product found in the tea, coffee and wines (see also condensed tannins, free form, as esters, or in combination with alka- page 151). loids such as quinine (see page 362). Shikimic A very important branchpoint compound in the acid itself is formed from 3-dehydroquinic acid shikimate pathway is chorismic acid (Figure 4.3), via 3-dehydroshikimic acid by dehydration and which has incorporated a further molecule of reduction steps. The simple phenolic acids pro- PEP as an enol ether side-chain. PEP combines tocatechuic acid (3,4-dihydroxybenzoic acid) and with shikimic acid 3-phosphate produced in a AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 123

OH enzyme, and is believed to bind to the PEP bind- OH OH ing site on the enzyme. Glyphosate finds con- HO OH siderable use as a broad spectrum herbicide, a O OH plant’s subsequent inability to synthesize aromatic OH O amino acids causing its death. The transformation OH of EPSP to chorismic acid (Figure 4.3) involves a O O O O O O 1,4-elimination of phosphoric acid, though this is OO OH probably not a concerted elimination. O 4-hydroxybenzoic acid (Figure 4.4) is pro- duced in bacteria from chorismic acid by HO OH HO OH an elimination reaction, losing the recently OH introduced enolpyruvic acid side-chain. However, OH pentagalloylglucose in plants, this phenolic acid is formed by a branch much further on in the pathway via side-chain degradation of cinnamic acids (see CO H CO H 2 2 page 141). The three phenolic acids so far encoun- HN O O tered, 4-hydroxybenzoic, protocatechuic, and gallic O P P OH OH acids, demonstrate some of the hydroxylation OH OH patterns characteristic of shikimic acid-derived glyphosate PEP metabolites, i.e. a single hydroxy para to the side- chain function, dihydroxy groups arranged ortho to Figure 4.2 each other, typically 3,4- to the side-chain, and tri- hydroxy groups also ortho to each other and 3,4,5- simple ATP-dependent phosphorylation reaction. to the side-chain. The single para-hydroxylation This combines with PEP via an addition–elimi- and the ortho-polyhydroxylation patterns contrast nation reaction giving 3-enolpyruvylshikimic acid with the typical meta-hydroxylation patterns char- 3-phosphate (EPSP). This reaction is catalysed acteristic of phenols derived via the acetate path- by the enzyme EPSP synthase. The synthetic way (see page 62), and in most cases allow the N-(phosphonomethyl)glycine derivative glypho- biosynthetic origin (acetate or shikimate) of an aro- sate (Figure 4.2) is a powerful inhibitor of this matic ring to be deduced by inspection.

nucleophilic attack on to protonated double bond of PEP H PEP CO H CO2H CO2H 2 H PO CO2H HH ATP

HO OH PO OH EPSP synthase PO OCO2H OH OH OH OP shikimic acid shikimic acid 3-P 1,2-elimination of – HOP phosphoric acid 1,4-elimination of phosphoric acid CO2H CO2H

L-Phe – HOP prephenic acid L-Tyr OCO2H PO OCO2H OH OH chorismic acid EPSP

Figure 4.3 124 THE SHIKIMATE PATHWAY

elimination of pyruvic acid elimination of pyruvic acid (formally as enolpyruvic acid) (formally as enolpyruvic acid) generates aromatic ring isomerization via generates aromatic ring CO H S 2′ reaction 2 CO2H N CO2H CO2H OH2 OH OH

OCO2H OCO2H OH OH isochorismic acid salicylic acid 4-hydroxybenzoic H chorismic acid acid hydrolysis of L-Gln L-Gln enol ether amination using side-chain oxidation of 3-hydroxyl to ammonia (generated ketone, then enolization from glutamine) as CO2H CO H CO2H nucleophile 2 CO2H NH2 OH NAD+ OH

OCOH 2 OCO2H OH OH NH 2 2,3-dihydroxybenzoic 2-amino-2-deoxy- 4-amino-4-deoxy- acid chorismic acid isochorismic acid

elimination of pyruvic acid

CO2H CO2H

NH2 L-Trp

NH2 anthranilic acid p-aminobenzoic acid (PABA)

Figure 4.4

2,3-dihydroxybenzoic acid,andsalicylic acid available sufficient concentrations of essential (2-hydroxybenzoic acid) (in microorganisms, but iron. Enterobactin comprises three molecules of not in plants, see page 141), are derived from 2,3-dihydroxybenzoic acid and three of the amino chorismic acid via its isomer isochorismic acid L-serine, in cyclic triester form. acid (Figure 4.4). The isomerization involves Simple amino analogues of the phenolic  an SN2 -type of reaction, an incoming water acids are produced from chorismic acid by nucleophile attacking the diene system and related transformations in which ammonia, gen- displacing the hydroxyl. Salicyclic acid arises by erated from glutamine, acts as a nucleophile an elimination reaction analogous to that producing (Figure 4.4). Chorismic acid can be aminated at 4-hydroxybenzoic acid from chorismic acid. In C-4 to give 4-amino-4-deoxychorismic acid and the formation of 2,3-dihydroxybenzoic acid, the then p-aminobenzoic (4-aminobenzoic) acid, or at side-chain of isochorismic acid is first lost by C-2 to give the isochorismic acid analogue which hydrolysis, then dehydrogenation of the 3-hydroxy will yield 2-aminobenzoic (anthranilic) acid. Ami- to a 3-keto allows enolization and formation nation at C-4 has been found to occur with reten- of the aromatic ring. 2,3-Dihydroxybenzoic tion of configuration, so perhaps a double inver- acid is a component of the powerful iron sion mechanism is involved. p-Aminobenzoic acid chelator (siderophore) enterobactin (Figure 4.5) (PABA) forms part of the structure of folic acid ∗ found in Escherichia coli and many other (vitamin B9) (Figure 4.6). The folic acid struc- Gram-negative bacteria. Such compounds play an ture is built up (Figure 4.6) from a dihydropterin important role in bacterial growth by making diphosphate which reacts with p-aminobenzoic AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 125

OH OO O activation to AMP derivative, compare peptide formation, O Figure 7.15 OH O O CO H COAMP 2 L-Ser O NH O NH O HN HN O OH OH ATP O OH HO HO 3+ OH OH O O O Fe O HO 2,3-dihydroxy- OH HO benzoic acid O OH HN N enterobactin as iron chelator H HO O O HO OH enterobactin

Figure 4.5

H2N

SO2NH2 H2N sulphanilamide (acts as antimetabolite of PABA and is enzyme inhibitor) CO2H PABA H H H H2N N N H2N N N H2N N N ATP H N OH N OPP N N N N N SN2 reaction OH OH OH CO2H hydroxymethyl- hydroxymethyl- dihydropteroic acid dihydropterin dihydropterin PP L-Glu ATP H H reduction H2N N N H2N N N H NADPH H N N N N N H N H H dihydrofolate OH N CO2H reductase OH N CO2H (DHFR) O CO H O CO H tetrahydrofolic acid 2 dihydrofolic acid 2 (FH4) (FH2) dihydrofolate NADPH reductase reduction (DHFR) the pteridine system is sometimes H2N N N drawn as the tautomeric amide form: H N N H N N N 2 N H OH N CO H HN 2 N O CO2H O p-amino- benzoic acid a pteridine (PABA) L-Glu

folic acid

Figure 4.6 126 THE SHIKIMATE PATHWAY acid to give dihydropteroic acid, an enzymic regeneration of tetrahydrofolic acid, and forms an step for which the sulphonamide antibiotics are important site of action for some antibacterial, anti- inhibitors. Dihydrofolic acid is produced from the malarial, and anticancer drugs. dihydropteroic acid by incorporating glutamic acid, Anthranilic acid (Figure 4.4) is an intermediate and reduction yields tetrahydrofolic acid.This in the biosynthetic pathway to the indole-containing reduction step is also necessary for the continual aromatic amino acid L-tryptophan (Figure 4.10).

Folic Acid (Vitamin B9)

Folic acid (vitamin B9) (Figure 4.6) is a conjugate of a pteridine unit, p-aminobenzoic acid, and glutamic acid. It is found in yeast, liver, and green vegetables, though cooking may destroy up to 90% of the vitamin. Deficiency gives rise to anaemia, and supplementation is often necessary during pregnancy. Otherwise, deficiency is not normally encountered unless there is malabsorption, or chronic disease. Folic acid used for supplementation is usually synthetic, and it becomes sequentially reduced in the body by the enzyme dihydrofolate reductase to give dihydrofolic acid and then tetrahydrofolic acid (Figure 4.6). Tetrahydrofolic acid then functions as a carrier of one-carbon groups, which may be in the form of methyl, methylene, methenyl, or formyl groups, by the reactions outlined in Figure 4.7. These groups are involved in amino acid and nucleotide metabolism. Thus a methyl group is transferred in the regeneration of methionine from homocysteine, purine biosynthesis involves methenyl and formyl transfer, and pyrimidine biosynthesis utilizes methylene transfer. Tetrahydrofolate derivatives also serve as acceptors of one-carbon units in degradative pathways. Mammals must obtain their tetrahydrofolate requirements from their diet, but microorgan- isms are able to synthesize this material. This offers scope for selective action and led to the use of sulphanilamide and other antibacterial sulpha drugs, compounds which competitively inhibit dihydropteroate synthase, the biosynthetic enzyme incorporating p-aminobenzoic acid into the structure. These sulpha drugs thus act as antimetabolites of p-aminobenzoate. Spe- cific dihydrofolate reductase inhibitors have also become especially useful as antibacterials,

H HCO2H H H ATP ADP H2N N N H2N N N H2N N N

N 5 N N 6 N N N H 10 H OH HN OH N OH HN H HO 10 O 5 FH4 N -formyl-FH4 N -formyl-FH4 L-Ser ATP (folinic acid) Gly ADP

H2O H H H + H2N N N H2N N N NADP NADPH H2N N N

N N N N N N + NADH OHMe HN NAD OH N OH N

5 5 10 5 10 N -methyl-FH4 N ,N -methylene-FH4 N ,N -methenyl-FH4

Figure 4.7

(Continues) AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 127

(Continued)

OMe H2N N N H2N N Me H2N N OMe N N N N H N OMe NH2 N CO2H NH2 Cl NH2 O CO2H trimethoprim pyrimethamine methotrexate

Figure 4.8

H L-Ser Gly H O H2N N N H2N N N HN N N N N H O N OH HN OH N deoxyribose-P 5 10 FH4 N ,N -methylene-FH4 dUMP

NADPH H O H2N N N HN N N O N OH HN deoxyribose-P

FH2 dTMP

Figure 4.9

e.g. trimethoprim (Figure 4.8), and antimalarial drugs, e.g. pyrimethamine, relying on the differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate (Figure 4.8), primarily block pyrimidine biosynthesis, but are less selective than the antimicrobial agents, and rely on a stronger binding to the enzyme than the natural substrate has. Regeneration of tetrahydrofolate from dihydrofolate is vital for DNA synthesis in rapidly proliferating cells. The methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) requires N5,N10-methylenetetrahydrofolate as the methyl donor, which is thereby transformed into dihydrofolate (Figure 4.9). N5-Formyl-tetrahydrofolic acid (folinic acid, leucovorin) (Figure 4.7) is used to counteract the folate-antagonist action of anticancer agents like methotrexate. The natural 6S isomer is termed levofolinic acid (levoleucovorin); folinic acid in drug use is usually a mixture of the 6R and 6S isomers.

In a sequence of complex reactions, which will side-chain of L-tryptophan. Although a precursor of not be considered in detail, the indole ring sys- L-tryptophan, anthranilic acid may also be produced tem is formed by incorporating two carbons from by metabolism of tryptophan. Both compounds fea- phosphoribosyl diphosphate, with loss of the orig- ture as building blocks for a variety of alkaloid inal anthranilate carboxyl. The remaining ribosyl structures (see Chapter 6). carbons are then removed by a reverse aldol reac- Returning to the main course of the shikimate tion, to be replaced on a bound form of indole pathway, a singular rearrangement process occurs by those from L-serine, which then becomes the transforming chorismic acid into prephenic acid 128 THE SHIKIMATE PATHWAY

OH CO2H CO2H CO2H SN2 reaction HO OP H NH2 NH CH OPP OH CH2OP 2 N H imine−enamine anthranilic O O acid OH OH HO OH tautomerism PPO H phosphoribosyl PP phosphoribosyl anthranilic acid OH CO2H HO OP OH N H enol−keto

CO2H tautomerism HO H CO2H NH2 reverse aldol HO OP L-Ser OOHO reaction – CO2 NH OH – H2O O 2 OP PLP OH N N N N H H H H L-Trp indole indole-3-glycerol P (enzyme-bound)

Figure 4.10

HO2C HO C Claisen 2 CO2H O rearrangement O CO2H O CO2H OH HO2C OH OH chorismic acid chorismic acid prephenic acid (pseudoequatorial (pseudoaxial conformer) conformer)

Figure 4.11

(Figure 4.11). This reaction, a Claisen rearrange- transamination, and in the case of tyrosine ment, transfers the PEP-derived side-chain so that biosynthesis an oxidation, but the order in which it becomes directly bonded to the carbocycle, and these reactions occur differentiates the routes. so builds up the basic carbon skeleton of phenylala- Decarboxylative aromatization of prephenic acid nine and tyrosine. The reaction is catalysed in nature yields phenylpyruvic acid, and PLP-dependent by the enzyme chorismate mutase, and, although it transamination leads to L-phenylalanine. In the can also occur thermally, the rate increases some presence of an NAD+-dependent dehydrogenase 106-fold in the presence of the enzyme. The enzyme enzyme, decarboxylative aromatization occurs achieves this by binding the pseudoaxial conformer with retention of the hydroxyl function, though of chorismic acid, allowing a transition state with as yet there is no evidence that any inter- chairlike geometry to develop. mediate carbonyl analogue of prephenic acid Pathways to the aromatic amino acids L-pheny- is involved. Transamination of the resultant lalanine and L-tyrosine via prephenic acid may 4-hydroxyphenylpyruvic acid subsequently gives vary according to the organism, and often more L-tyrosine. L-Arogenic acid is the result of than one route may operate in a particular species transamination of prephenic acid occurring prior according to the enzyme activities that are avail- to the decarboxylative aromatization, and can able (Figure 4.12). In essence, only three reac- be transformed into both L-phenylalanine and tions are involved, decarboxylative aromatization, L-tyrosine depending on the absence or presence AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 129 of a suitable enzymic dehydrogenase activity. by oxidation reactions into a heterogeneous In some organisms, broad activity enzymes polymer melanin, the main pigment in mammalian are known to be capable of accepting both skin, hair, and eyes. In this material, the indole prephenic acid and arogenic acid as substrates. system is not formed from tryptophan, but arises In microorganisms and plants, L-phenylalanine from DOPA by cyclization of DOPAquinone, the and L-tyrosine tend to be synthesized separately nitrogen of the side-chain then attacking the ortho- as in Figure 4.12, but in animals, which lack quinone (Figure 4.13). the shikimate pathway, direct hydroxylation of Some organisms are capable of synthesizing an L-phenylalanine to L-tyrosine, and of L-tyrosine unusual variant of L-phenylalanine, the aminated to L-DOPA (dihydroxyphenylalanine), may be derivative L-p-aminophenylalanine (L-PAPA) (Fig- achieved (Figure 4.13). These reactions are catal- ure 4.14). This is known to occur by a series of ysed by tetrahydropterin-dependent hydroxylase reactions paralleling those in Figure 4.12, but uti- enzymes, the hydroxyl oxygen being derived lizing the PABA precursor 4-amino-4-deoxychori- from molecular oxygen. L-DOPA is a precursor smic acid (Figure 4.4) instead of chorismic acid. of the catecholamines, e.g. the neurotransmitter Thus, amino derivatives of prephenic acid and py- noradrenaline and the hormone adrenaline (see ruvic acid are elaborated. One important metabolite page 316). Tyrosine and DOPA are also converted known to be formed from L-PAPA is the antibiotic

transamination: keto acid amino acid CO2H CO2H

O NH2

PLP

phenylpyruvic acid L-Phe decarboxylation, decarboxylation, aromatization and aromatization and loss of leaving group loss of leaving group

O CO2H O CO2H CO2H H C H C O O O PLP NH2

OCO2H OH H OH OH chorismic acid prephenic acid L-arogenic acid

+ oxidation means OH is an additional oxidation step (of NAD + NAD retained on decarboxylation alcohol to ketone) means OH is and aromatization; no discrete retained on decarboxylation ketone intermediate is formed and aromatization; no discrete CO2H CO2H ketone intermediate is formed O NH2

PLP

OH OH 4-hydroxyphenyl- L-Tyr pyruvic acid

Figure 4.12 130 THE SHIKIMATE PATHWAY

O2 O2 tetrahydro- tetrahydro- CO H CO H HO CO2H 2 biopterin 2 biopterin CATECHOLAMINES noradrenaline, NH NH2 NH2 2 HO HO adrenaline L-Phe L-Tyr L-DOPA

nucleophilic attack

O HO on to enone O CO2H O MELANINS CO2H CO2H NH2 HO N HO N O H DOPAchrome DOPAquinone

Figure 4.13

chloramphenicol∗, produced by cultures of Strep- natural products. In plants, a frequent first step is tomyces venezuelae. The late stages of the path- the elimination of ammonia from the side-chain way (Figure 4.14) have been formulated to involve to generate the appropriate trans (E) cinnamic hydroxylation and N-acylation in the side-chain, acid. In the case of phenylalanine, this would the latter reaction probably requiring a coenzyme give cinnamic acid, whilst tyrosine could yield A ester of dichloroacetic acid. Following reduction 4-coumaric acid (p-coumaric acid) (Figure 4.15). of the carboxyl group, the final reaction is oxida- All plants appear to have the ability to deami- tion of the 4-amino group to a nitro, a fairly rare nate phenylalanine via the enzyme phenylalanine substituent in natural product structures. ammonia lyase (PAL), but the corresponding trans- formation of tyrosine is more restricted, being CINNAMIC ACIDS mainly limited to members of the grass family (the Graminae/Poaceae). Whether a separate enzyme L-Phenylalanine and L-tyrosine, as C6C3 build- tyrosine ammonia lyase (TAL) exists, or whether ing blocks, are precursors for a wide range of grasses merely have a broad specificity PAL also

Chloramphenicol

Chloramphenicol (chloromycetin) (Figure 4.14) was initially isolated from cultures of Streptomyces venezuelae, but is now obtained for drug use by chemical synthesis. It was one of the first broad spectrum antibiotics to be developed, and exerts its antibacterial action by inhibiting protein biosynthesis. It binds reversibly to the 50S subunit of the bacterial ribosome, and in so doing disrupts peptidyl transferase, the enzyme that catalyses peptide bond formation (see page 408). This reversible binding means that bacterial cells not destroyed may resume protein biosynthesis when no longer exposed to the antibiotic. Some microorganisms have developed resistance to chloramphenicol by an inactivation process involving enzymic acetylation of the primary alcohol group in the antibiotic. The acetate binds only very weakly to the ribosomes, so has little antibiotic activity. The value of chloramphenicol as an antibacterial agent has been severely limited by some serious side-effects. It can cause blood disorders including irreversible aplastic anaemia in certain individuals, and these can lead to leukaemia and perhaps prove fatal. Nevertheless, it is still the drug of choice for some life-threatening infections such as typhoid fever and bacterial meningitis. The blood constitution must be monitored regularly during treatment to detect any abnormalities or adverse changes. The drug is orally active, but may also be injected. Eye-drops are useful for the treatment of bacterial conjunctivitis. CINNAMIC ACIDS 131

decarboxylation and aromatization; amino group is retained via an additional oxidation step (amine → imine)

O CO2H CO2H

CO2H HO C O Claisen 2 rearrangement

OCO2H NH NH2 2 NH2 4-amino-4-deoxy- transamination chorismic acid PLP hydroxylation CH2OH CO2H N-acylation CO2H HO HO NHCOCHCl2 NHCOCHCl2 CHCl2COSCoA NH2

reduction of CO2H to CH2OH; oxidation of NH NO2 NH2 to NO2 2 NH2 chloramphenicol L-p-aminophenylalanine L-PAPA

Figure 4.14

E2 elimination of ammonia CO2H CO2H

NH2

PAL

L-Phe cinnamic acid O 2 hydroxylation NADPH sequence of hydroxylation and methylation reactions

CO2H CO2H CO2H CO2H CO2H CO2H

NH2 O O2 2 SAM NADPH SAM NADPH

HO MeO MeO OH MeO OMe OH OH OH OH OH OH L-Tyr 4-coumaric acid caffeic acid ferulic acid sinapic acid (p-coumaric acid)

CH2OH CH2OH CH2OH

MeO MeO OMe OH OH OH 4-hydroxycinnamyl alcohol coniferyl alcohol sinapyl alcohol (p-coumaryl alcohol)

POLYMERS x 2 x n

LIGNANS LIGNIN

Figure 4.15 132 THE SHIKIMATE PATHWAY

HO Pre-eminent amongst these, certainly as far as O O CO2H nature is concerned, is the plant polymer lignin, OH OH a strengthening material for the plant cell wall which acts as a matrix for cellulose microfibrils (see page 473). Lignin represents a vast reservoir of aromatic materials, mainly untapped because OH of the difficulties associated with release of these OH metabolites. The action of wood-rotting fungi chlorogenic acid offers the most effective way of making these use- (5-O-caffeoylquinic acid) ful products more accessible. Lignin is formed OH by phenolic oxidative coupling of hydroxycin- namyl alcohol monomers, brought about by perox- HO O HO O idase enzymes (see page 28). The most important OH of these monomers are 4-hydroxycinnamyl alco- O hol (p-coumaryl alcohol), coniferyl alcohol,and 1-O-cinnamoylglucose sinapyl alcohol (Figure 4.15), though the mono- mers used vary according to the plant type. OMe Gymnosperms polymerize mainly coniferyl alco- OH hol, dicotyledonous plants coniferyl alcohol and O sinapyl alcohol, whilst monocotyledons use all Me3N OMe three alcohols. The alcohols are derived by reduc- O tion of cinnamic acids via coenzyme A esters and sinapine aldehydes (Figure 4.17), though the substitution patterns are not necessarily elaborated completely Figure 4.16 at the cinnamic acid stage, and coenzyme A esters and aldehydes may also be substrates for aromatic capable of deaminating tyrosine, is still debated. hydroxylation and methylation. Formation of the Those species that do not transform tyrosine syn- coenzyme A ester facilitates the first reduction step thesize 4-coumaric acid by direct hydroxylation of − by introducing a better leaving group (CoAS ) cinnamic acid, in a cytochrome P-450-dependent for the NADPH-dependent reaction. The second reaction, and tyrosine is often channelled instead reduction step, aldehyde to alcohol, utilizes a fur- into other secondary metabolites, e.g. alkaloids. ther molecule of NADPH and is reversible. The Other cinnamic acids are obtained by further peroxidase enzyme then achieves one-electron oxi- hydroxylation and methylation reactions, sequen- dation of the phenol group. One-electron oxidation tially building up substitution patterns typical of of a simple phenol allows delocalization of the shikimate pathway metabolites, i.e. an ortho oxy- unpaired electron, giving resonance forms in genation pattern (see page 123). Some of the more which the free electron resides at positions ortho common natural cinnamic acids are 4-coumaric, para caffeic, ferulic,andsinapic acids (Figure 4.15). and to the oxygen function (see page 29). These can be found in plants in free form and in With cinnamic acid derivatives, conjugation allows a range of esterified forms, e.g. with quinic acid the unpaired electron to be delocalized also into as in chlorogenic acid (5-O-caffeoylquinic acid) the side-chain (Figure 4.18). Radical pairing of (see coffee, page 395), with glucose as in 1-O- resonance structures can then provide a range of cinnamoylglucose, and with choline as in sinapine dimeric systems containing reactive quinoneme- (Figure 4.16). thides, which are susceptible to nucleophilic attack from hydroxyl groups in the same system, or by external water molecules. Thus, coniferyl LIGNANS AND LIGNIN alcohol monomers can couple, generating linkages as exemplified by guaiacylglycerol β-coniferyl The cinnamic acids also feature in the pathways to ether (β-arylether linkage), dehydrodiconi- other metabolites based on C6C3 building blocks. feryl alcohol (phenylcoumaran linkage), and LIGNANS AND LIGNIN 133

CO2H COSCoA CHO CH2OH

HSCoA NADPH NADPH

R1 R2 R1 R2 R1 R2 R1 R2 OH OH OH OH

Figure 4.17

OH OH OH OH OH one-electron oxidation

– H+ – e MeO MeO MeO MeO MeO OH O O O O coniferyl alcohol A BCD resonance forms of free radical

A + DB + DD + D radical radical radical pairing pairing pairing OH OH O H OMe

OH HO H HO OMe OMe O O HO enolization H2O O MeO nucleophilic attack of hydroxyls on to OMe OH O O H H quinonemethides OMe nucleophilic attack OH of water on to HO quinonemethide O OH OMe OMe OH MeO O H O OMe HO OH pinoresinol HO OMe nucleophilic attack (resinol linkage) O of hydroxyl on to quinonemethide HO HO

OH OMe OMe O guaiacylglycerol dehydrodiconiferyl alcohol β-coniferyl ether HO β (phenylcoumaran linkage) ( -arylether linkage) OMe

Figure 4.18 134 THE SHIKIMATE PATHWAY

H H H H MeO MeO OH O H H HO HO

OH OH this step probably involves ring OH phenolic oxidative opening to the quinonemethide coupling O followed by reduction OH OMe OMe H H NADPH H H MeO MeO O HO MeO OH HO HO coniferyl alcohol (+)-pinoresinol (–)-secoisolariciresinol

modification of oxidation of one CH2OH ≡ aromatic substitution to CO2H, then lactone ring patterns formation MeO MeO O OH O O OH O HO NAD+ HO O O

MeO OMe MeO MeO OMe OH OH yatein matairesinol (–)-secoisolariciresinol

OH hydroxylation O O O O O O O O O nucleophilic O O O attack on to quinonemethide system MeO OMe MeO OMe MeO OMe OMe OMe OMe

desoxypodophyllotoxin podophyllotoxin

Figure 4.19

MeO HO HO OGlc OH O OGlc intestinal OH HO bacteria O

MeO HO HO OH enterodiol enterolactone secoisolariciresinol diglucoside

Figure 4.20 LIGNANS AND LIGNIN 135 pinoresinol (resinol linkage). These dimers can is known to arise by reductive opening of the react further by similar mechanisms to produce a furan rings of pinoresinol, followed by oxida- lignin polymer containing a heterogeneous series tion of a primary alcohol to the acid and then of inter-molecular bondings as seen in the var- lactonization. The substitution pattern in the two ious dimers. In contrast to most other natural aromatic rings is built up further during the path- polymeric materials, lignin appears to be devoid way, i.e. matairesinol → yatein, and does not of ordered repeating units, though some 50–70% arise by initial coupling of two different cinnamyl of the linkages are of the β-arylether type. The alcohol residues. The methylenedioxy ring sys- dimeric materials are also found in nature and are tem, as found in many shikimate-derived natural called lignans. Some authorities like to restrict products, is formed by an oxidative reaction on the term lignan specifically to molecules in which an ortho-hydroxymethoxy pattern (see page 27). the two phenylpropane units are coupled at the Podophyllotoxin and related lignans are found central carbon of the side-chain, e.g. pinoresinol, in the roots of Podophyllum∗ species (Berberi- whilst compounds containing other types of cou- daceae), and have clinically useful cytotoxic and pling, e.g. as in guaiacylglycerol β-coniferyl ether anticancer activity. The lignans enterolactone and dehydrodiconiferyl alcohol, are then referred and enterodiol (Figure 4.20) were discovered in to as neolignans. Lignan/neolignan formation human urine, but were subsequently shown to and lignin biosynthesis are catalysed by differ- be derived from dietary plant lignans, especially ent enzymes, and a consequence is that natural secoisolariciresinol diglucoside, by the action of lignans/neolignans are normally enantiomerically intestinal microflora. Enterolactone and enterodiol pure because they arise from stereochemically have oestrogenic activity and have been impli- controlled coupling. The control mechanisms for cated as contributing to lower levels of breast lignin biosynthesis are less well defined, but the cancer amongst vegetarians (see phyto-oestrogens, enzymes appear to generate products lacking opti- page 156). cal activity. Further cyclization and other modifications can create a wide range of lignans of very differ- PHENYLPROPENES ent structural types. One of the most important of the natural lignans having useful biologi- The reductive sequence from an appropriate cin- cal activity is the aryltetralin lactone podophyl- namic acid to the corresponding cinnamyl alcohol lotoxin (Figure 4.19), which is derived from is not restricted to lignin and lignan biosynthesis, coniferyl alcohol via the dibenzylbutyrolactones and is utilized for the production of various matairesinol and yatein, cyclization probably phenylpropene derivatives. Thus cinnamaldehyde occurring as shown in Figure 4.19. Matairesinol (Figure 4.23) is the principal component in the

Podophyllum

Podophyllum consists of the dried rhizome and roots of Podophyllum hexandrum (P. emodi) or P. peltatum (Berberidaceae). Podophyllum hexandrum is found in India, China, and the Himalayas and yields Indian podophyllum, whilst P. peltatum (May apple or American mandrake) comes from North America and is the source of American podophyllum. Plants are collected from the wild. Both plants are large-leafed perennial herbs with edible fruits, though other parts of the plant are toxic. The roots contain cytotoxic lignans and their glucosides, P. hexandrum containing about 5%, and P. peltatum about 1%. A concentrated form of the active principles is obtained by pouring an ethanolic extract of the root into water, and drying the precipitated podophyllum resin or ‘podophyllin’. Indian podophyllum yields about 6–12% of resin containing 50–60% lignans, and American podophyllum 2–8% of resin containing 14–18% lignans.

(Continues) 136 THE SHIKIMATE PATHWAY

(Continued)

OH OH O O O O O O O O O O O O O O O O O

MeO4′ OMe MeO OMe MeO OMe MeO OMe OR OR OMe OMe R = Me, podophyllotoxin R = Me, β-peltatin desoxypodophyllotoxin podophyllotoxone R = H, 4′-demethylpodophyllotoxin R = H, α-peltatin

O O O O O S O HO O HO O OH OH OH O O O O O O O O O O O O

MeO OMe MeO4′ OMe MeO OMe OH OR OH 4′-demethylepipodophyllotoxin R = H, etoposide teniposide R = P, etopophos

Figure 4.21

The lignan constituents of the two roots are the same, but the proportions are markedly different. The Indian root contains chiefly podophyllotoxin (Figure 4.21) (about 4%) and 4- demethylpodophyllotoxin (about 0.45%). The main components in the American root are podophyllotoxin (about 0.25%), β-peltatin (about 0.33%) and α-peltatin (about 0.25%). Desoxypodophyllotoxin and podophyllotoxone are also present in both plants, as are the glucosides of podophyllotoxin, 4-demethylpodophyllotoxin, and the peltatins, though preparation of the resin results in considerable losses of the water-soluble glucosides. Podophyllum resin has long been used as a purgative, but the discovery of the cytotoxic properties of podophyllotoxin and related compounds has now made podophyllum a commercially important drug plant. Preparations of podophyllum resin (the Indian resin is preferred) are effective treatments for warts, and pure podophyllotoxin is available as a paint for venereal warts, a condition which can be sexually transmitted. The antimitotic effect of podophyllotoxin and the other lignans is by binding to the protein tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules (compare vincristine, page 356, and colchicine, page 343). During mitosis, the chromosomes separate with the assistance of these microtubules, and after cell division the microtubules are transformed back to tubulin. Podophyllotoxin and other Podophyllum lignans were found to be unsuitable for clinical use as anticancer agents due to toxic side-effects, but the semi-synthetic derivatives etoposide and teniposide (Figure 4.21), which are manufactured from natural podophyllotoxin, have proved excellent antitumour agents. They were developed as modified forms (acetals) of the natural

(Continues) PHENYLPROPENES 137

(Continued)

base removes acidic reformation of keto form results proton α to carbonyl and OH generates enolate anion OH in change of stereochemistry OH O O O O O O O NaOAc O O H H O HB O O B MeO OMe MeO OMe MeO OMe OMe OMe OMe podophyllotoxin picropodophyllin

Figure 4.22

4-demethylpodophyllotoxin glucoside. Attempted synthesis of the glucoside inverted the stereochemistry at the sugar–aglycone linkage, and these agents are thus derivatives of 4- demethylepipodophyllotoxin (Figure 4.21). Etoposide is a very effective anticancer agent, and is used in the treatment of small cell lung cancer, testicular cancer and lymphomas, usually in combination therapies with other anticancer drugs. It may be given orally or intravenously. The water-soluble pro-drug etopophos (etoposide 4-phosphate) is also available. Teniposide has similar anticancer properties, and, though not as widely used as etoposide, has value in paediatric neuroblastoma. Remarkably, the 4-demethylepipodophyllotoxin series of lignans do not act via a tubulin- binding mechanism as does podophyllotoxin. Instead, these drugs inhibit the enzyme topoisomerase II, thus preventing DNA synthesis and replication. Topoisomerases are responsible for cleavage and resealing of the DNA strands during the replication process, and are classified as type I or II according to their ability to cleave one or both strands. Camptothecin (see page 365) is an inhibitor of topoisomerase I. Etoposide is believed to inhibit strand-rejoining ability by stabilizing the topoisomerase II–DNA complex in a cleavage state, leading to double-strand breaks and cell death. Development of other topoisomerase inhibitors based on podophyllotoxin-related lignans is an active area of research. Biological activity in this series of compounds is very dependent on the presence of the trans-fused five- membered lactone ring, this type of fusion producing a highly-strained system. Ring strain is markedly reduced in the corresponding cis-fused system, and the natural compounds are easily and rapidly converted into these cis-fused lactones by treatment with very mild bases, via enol tautomers or enolate anions (Figure 4.22). Picropodophyllin is almost devoid of cytotoxic properties. Podophyllotoxin is also found in significant amounts in the roots of other Podophyllum species, and in closely related genera such as Diphylleia (Berberidaceae).

oil from the bark of cinnamon (Cinnamomum contains large amounts of eugenol (Figure 4.23) zeylanicum; Lauraceae), widely used as a spice and much smaller amounts of cinnamaldehyde. and flavouring. Fresh bark is known to contain Eugenol is also the principal constituent in oil high levels of cinnamyl acetate, and cinnamalde- from cloves (Syzygium aromaticum; Myrtaceae), hyde is released from this by fermentation pro- used for many years as a dental anaesthetic, as cesses which are part of commercial preparation of well as for flavouring. The side-chain of eugenol the bark, presumably by enzymic hydrolysis and is derived from that of the cinnamyl alcohols by participation of the reversible aldehyde–alcohol reduction, but differs in the location of the double oxidoreductase. Cinnamon leaf, on the other hand, bond. This change is accounted for by resonance 138 THE SHIKIMATE PATHWAY

OCOCH3 CHO

MeO MeO O MeO OMe cinnamaldehyde cinnamyl acetate OMe OMe OH O OMe anethole estragole eugenol myristicin elemicin (methylchavicol)

Figure 4.23

loss of hydroxyl as resonance-stabilized leaving group allylic cation H = Ar OH Ar Ar Ar etc cinnamyl alcohol H H OMe OMe OH NADPH NADPH

Ar Ar propenylphenol allylphenol

Figure 4.24 forms of the allylic cation (Figure 4.24), and addi- have been shown to be weak carcinogens in tion of hydride (from NADPH) can generate either laboratory tests on animals. In the case of saf- allylphenols, e.g. eugenol, or propenylphenols, e.g. role (Figure 4.25), the main component of sas- anethole (Figure 4.23). Loss of hydroxyl from safras oil, this has been shown to arise from a cinnamyl alcohol may be facilitated by proto- hydroxylation in the side-chain followed by sul- nation, or perhaps even phosphorylation, though phation, giving an agent which binds to cel- there is no evidence for the latter. Myristicin lular macromolecules. Further data on volatile (Figure 4.23) from nutmeg (Myristica fragrans; oils containing aromatic constituents isolated from Myristicaceae) is a further example of an allylphe- these and other plant materials are given in nol found in flavouring materials. Myristicin also Table 4.1. Volatile oils in which the main compo- has a history of being employed as a mild hallu- nents are terpenoid in nature are listed in Table 5.1, cinogen via ingestion of ground nutmeg. Myris- page 177. ticin is probably metabolized in the body via an amination reaction to give an amfetamine- like derivative (see page 385). Anethole is the main component in oils from aniseed (Pimpinella HO HO3SO anisum; Umbelliferae/Apiaceae), star anise (Illi- cium verum; Illiciaceae), and fennel (Foeniculum vulgare; Umbelliferae/Apiaceae). The propenyl O O O components of flavouring materials such as cin- O O O namon, star anise, nutmeg, and sassafras (Sas- safrole safras albidum; Lauraceae) have reduced their commercial use somewhat since these constituents Figure 4.25 Table 4.1 Volatile oils containing principally aromatic compounds Volatile or essential oils are usually obtained from the appropriate plant material by steam distillation, though if certain components are unstableatthese temperatures, other less harsh techniques such as expression or solvent extraction may be employed. These oils, which typically contain a complex mixture of low boiling components, are widely used in flavouring, perfumery, and aromatherapy. Only a small number of oils have useful therapeutic properties, e.g. clove and dill, though a wide range of oils is now exploited for aromatherapy. Most of those employed in medicines are simply added for flavouring purposes. Some of the materials are commercially important as sources of chemicals used industrially, e.g. turpentine. For convenience, the major oils listed are divided into two groups. Those which contain principally chemicals which are aromatic in nature and which are derived by the shikimate pathway are given in Table 4.1 below. Those oils which are composed predominantly of terpenoid compounds are listed in Table 5.1 on page 177, since they are derived via the deoxyxylulose phosphate pathway. It must be appreciated that many oils may contain aromatic and terpenoid components, but usually one group predominates. The oil yields, and the exact composition of any sample of oil will be variable, depending on the particular plant material used in its preparation. The quality of an oil and its commercial value is dependent on the proportion of the various components. Oil Plant source Plant part Oil Major constituents with Uses, notes used content (%) typical (%) composition Aniseed Pimpinella anisum ripe fruit 2–3 anethole (80–90) flavour, carminative, (Anise) (Umbelliferae/ estragole (1–6) aromatherapy Apiaceae) Star anise Illicium verum ripe fruit 5–8 anethole (80–90) flavour, carminative (Illiciaceae) estragole (1–6) fruits contain substantial amounts of shikimic and quinic acids Cassia Cinnamomum cassia dried bark, 1–2 cinnamaldehyde (70–90) flavour, carminative (Lauraceae) or leaves 2-methoxycinnamal- and twigs dehyde (12) known as cinnamon oil in USA Cinnamon bark Cinnamomum dried bark 1–2 cinnamaldehyde (70–80) flavour, carminative, zeylanicum eugenol (1–13) aromatherapy (Lauraceae) cinnamyl acetate (3–4) Cinnamon leaf Cinnamomum leaf 0.5–0.7 eugenol (70–95) flavour zeylanicum (Lauraceae) (Continued overleaf ) Table 4.1 (Continued) Oil Plant source Plant part Oil Major constituents with Uses, notes used content typical (%) composition (%) Clove Syzygium aromaticum dried flower 15–20 eugenol (75–90) flavour, aromatherapy, antiseptic (Eugenia caryophyllus) buds eugenyl acetate (10–15) (Myrtaceae) β-caryophyllene (3) Fennel Foeniculum vulgare ripe fruit 2–5 anethole (50–70) flavour, carminative, aromatherapy (Umbelliferae/ fenchone (10–20) Apiaceae) estragole (3–20) Nutmeg Myristica fragrans seed 5–16 sabinene (17–28) flavour, carminative, aromatherapy (Myristicaceae) α-pinene (14–22) β-pinene (9–15) although the main constituents are terpinen-4-ol (6–9) terpenoids, most of the flavour myristicin (4–8) comes from the minor aromatic elemicin (2) constituents, myristicin, elemicin, etc myristicin is hallucinogenic (see page 385) Wintergreen Gaultheria procumbens leaves 0.7–1.5 methyl salicylate (98%) flavour, antiseptic, antirheumatic (Ericacae) or Betula lenta bark 0.2–0.6 prior to distillation, plant material (Betulaceae) is macerated with water to allow enzymic hydrolysis of glycosides methyl salicylate is now produced synthetically BENZOIC ACIDS FROM C6C3 COMPOUNDS 141

BENZOIC ACIDS FROM C6C3 Coenzyme A esters are not involved, and though COMPOUNDS a similar hydration of the double bond occurs, chain shortening features a reverse aldol reac- tion, generating the appropriate aromatic alde- Some of the simple hydroxybenzoic acids (C C 6 1 hyde. The corresponding acid is then formed compounds) such as 4-hydroxybenzoic acid and via an NAD+-dependent oxidation step. Thus, gallic acid can be formed directly from inter- aromatic aldehydes such as vanillin,themain mediates early in the shikimate pathway, e.g. flavour compound in vanilla (pods of the orchid 3-dehydroshikimic acid or chorismic acid (see Vanilla planiflora; Orchidaceae) would be formed page 121), but alternative routes exist in which from the correspondingly substituted cinnamic acid cinnamic acid derivatives (C6C3 compounds) are without proceeding through intermediate benzoic cleaved at the double bond and lose two car- acids or esters. Whilst the substitution pattern bons from the side-chain. Thus, 4-coumaric acid in these C6C1 derivatives is generally built up may act as a precursor of 4-hydroxybenzoic at the C6C3 cinnamic acid stage, prior to chain acid, and ferulic acid may give vanillic acid shortening, there exists the possibility of further (4-hydroxy-3-methoxybenzoic acid) (Figure 4.26). hydroxylations and/or methylations occurring at A sequence analogous to that involved in the the C6C1 level, and this is known in certain β-oxidation of fatty acids (see page 18) is possi- examples. Salicylic acid (Figure 4.27) is synthe- ble, so that the double bond in the coenzyme A sized in microorganisms directly from isochorismic ester would be hydrated, the hydroxyl group oxi- acid (see page 124), but can arise in plants by two dized to a ketone, and the β-ketoester would then other mechanisms. It can be produced by hydrox- lose acetyl-CoA by a reverse Claisen reaction, giv- ylation of benzoic acid, or by side-chain cleavage ing the coenzyme A ester of 4-hydroxybenzoic of 2-coumaric acid, which itself is formed by an acid. Whilst this sequence has been generally ortho-hydroxylation of cinnamic acid. Methyl sal- accepted, newer evidence supports another side- icylate is the principal component of oil of win- chain cleavage mechanism, which is different from tergreen from Gaultheria procumbens (Ericaceae), the fatty acid β-oxidation pathway (Figure 4.26). used for many years for pain relief. It is derived by

CO2H COSCoA COSCoA COSCoA HO O

HSCoA ATP H2O NAD+ β R R R R -oxidation pathway, as in fatty acid metabolism OH OH OH OH (Figure 2.11) R = H, 4-coumaric acid R = H, 4-coumaroyl-CoA R = OMe, ferulic acid R = OMe, feruloyl-CoA HSCoA reverse H2O Claisen CH3COSCoA

CO2H HO CHO CO2H COSCoA reverse aldol NAD+

R R R R OH OH OH OH R = H, 4-hydroxy- R = H, 4-hydroxy- benzaldehyde benzoic acid R = OMe, vanillin R = OMe, vanillic acid

Figure 4.26 142 THE SHIKIMATE PATHWAY

hydroxylation methylation CO2H CO2H CO2Me CO2H

OH SAM OH OCOCH3

benzoic acid salicylic acid methyl salicylate aspirin (acetylsalicyclic acid) side-chain cleavage

CO2HCO2H

hydroxylation side-chain CHO glucosylation CHO reduction CH OH cleavage 2 OH OH UDPGlc OGlc OGlc

2-coumaric acid salicylaldehyde salicin

Figure 4.27

SAM-dependent methylation of salicylic acid. The the trans (E) to the less stable cis (Z) form. Whilst salicyl alcohol derivative salicin, found in many trans–cis isomerization would be unfavourable species of willow (Salix species; Salicaceae), is not in the case of a single isolated double bond, in derived from salicylic acid, but probably via gluco- the cinnamic acids the fully conjugated system sylation of salicylaldehyde and then reduction of allows this process to occur quite readily, and the carbonyl (Figure 4.27). Salicin is responsible UV irradiation, e.g. daylight, is sufficient to pro- for the analgesic and antipyretic effects of wil- duce equilibrium mixtures which can be separated low barks, widely used for centuries, and the tem- (Figure 4.29). The absorption of energy promotes plate for synthesis of acetylsalicylic acid (aspirin) an electron from the π-orbital to a higher energy (Figure 4.27) as a more effective analogue. state, the π∗-orbital, thus temporarily destroying the double bond character and allowing rotation. Loss of the absorbed energy then results in re- COUMARINS formation of the double bond, but in the cis- configuration. In conjugated systems, the π–π∗ The hydroxylation of cinnamic acids ortho to the energy difference is considerably less than with a side-chain as seen in the biosynthesis of salicylic non-conjugated double bond. Chemical lactoniza- acid is a crucial step in the formation of a group of tion can occur on treatment with acid. Both the cinnamic acid lactone derivatives, the coumarins. trans–cis isomerization and the lactonization are Whilst the direct hydroxylation of the aromatic enzyme-mediated in nature, and light is not nec- ring of the cinnamic acids is common, hydrox- essary for coumarin biosynthesis. Thus, cinnamic ylation generally involves initially the 4-position acid and 4-coumaric acid give rise to the coumarins para to the side-chain, and subsequent hydrox- coumarin and umbelliferone (Figure 4.28). Other ylations then proceed ortho to this substituent coumarins with additional oxygen substituents on (see page 132). In contrast, for the coumarins, the aromatic ring, e.g. aesculetin and scopoletin, hydroxylation of cinnamic acid or 4-coumaric acid appear to be derived by modification of umbellif- can occur ortho to the side-chain (Figure 4.28). erone, rather than by a general cinnamic acid to In the latter case, the 2,4-dihydroxycinnamic acid coumarin pathway. This indicates that the hydrox- produced confusingly seems to possess the meta ylation meta to the existing hydroxyl, discussed hydroxylation pattern characteristic of phenols above, is a rather uncommon occurrence. derived via the acetate pathway. Recognition of Coumarins are widely distributed in plants, the C6C3 skeleton should help to avoid this confu- and are commonly found in families such as the sion. The two 2-hydroxycinnamic acids then suffer Umbelliferae/Apiaceae and Rutaceae, both in the a change in configuration in the side-chain, from free form and as glycosides. Coumarin itself is COUMARINS 143

trans-cis isomerization lactone formation CO H 2 CO2H

CO2H OH OH OO cinnamic acid 2-coumaric acid coumarin

CO2H CO2H

CO2H HO HO OH HO OH HO OO 4-coumaric acid 2,4-dihydroxy- umbelliferone cinnamic acid

MeO MeO HO

GlcO OO HO OO HO OO scopolin scopoletin aesculetin

Figure 4.28

E Z CO2H hν H

CO2H OH OH OH OO coumarin

Figure 4.29

CO2H hydrolysis and lactonization through damage to the plant tissues during harvesting and process- OGlc ing (Figure 4.30). If sweet clover is allowed to (E)-2-coumaric acid ferment, 4-hydroxycoumarin is produced by the glucoside action of microorganisms on 2-coumaric acid (Figure 4.31) and this can react with formalde- enzymic hydrolysis hyde, which is usually present due to micro- bial degradative reactions, combining to give CO2H ∗ O OO dicoumarol. Dicoumarol is a compound with pro- Glc coumarin nounced blood anticoagulant properties, which can (Z)-2-coumaric acid cause the deaths of livestock by internal bleeding, glucoside and is the forerunner of the warfarin∗ group of medicinal anticoagulants. Figure 4.30 Many other natural coumarins have a more complex carbon framework and incorporate found in sweet clover (Melilotus species; Legu- extra carbons derived from an isoprene unit minosae/Fabaceae) and contributes to the smell (Figure 4.33). The aromatic ring in umbelliferone of new-mown hay, though there is evidence is activated at positions ortho to the hydroxyl that the plants actually contain the glucosides of group and can thus be alkylated by a suitable (E)- and (Z)-2-coumaric acid (Figure 4.30), and alkylating agent, in this case dimethylallyl coumarin is only liberated as a result of enzymic diphosphate. The newly introduced dimethylallyl 144 THE SHIKIMATE PATHWAY

aldol reaction; it may help to lactone formation consider the diketo tautomer and enolization H O OH O OH OH COSCoA COSCoA HH dehydration follows OH OH O O O O

4-hydroxycoumarin nucleophilic – H2O attack on to the HCHO enone system

H OH OH O OH

OOO O O O O O dicoumarol

Figure 4.31

Dicoumarol and Warfarin The cause of fatal haemorrhages in animals fed spoiled sweet clover (Melilotus officinalis; Leguminosae/Fabaceae) was traced to dicoumarol (bishydroxycoumarin) (Figure 4.31). This agent interferes with the effects of vitamin K in blood coagulation (see page 163), the blood loses its ability to clot, and thus minor injuries can lead to severe internal bleeding. Synthetic dicoumarol has been used as an oral blood anticoagulant in the treatment of thrombosis, where the risk of blood clots becomes life threatening. It has been superseded by salts of warfarin and acenocoumarol (nicoumalone) (Figure 4.32), which are synthetic developments from the natural product. An overdose of warfarin may be countered by injection of vitamin K1. Warfarin was initially developed as a rodenticide, and has been widely employed for many years as the first choice agent, particularly for destruction of rats. After

OH O OH OH RS RS RS RS NO Cl O O 2 O O O O warfarin coumatetralyl R = H, difenacoum R = Br, brodifenacoum R OH O OH O RS RS

O O O O acenocoumarol coumachlor (nicoumalone)

Figure 4.32

(Continues) COUMARINS 145

(Continued)

consumption of warfarin-treated bait, rats die from internal haemorrhage. Other coumarin derivatives employed as rodenticides include coumachlor and coumatetralyl (Figure 4.32). In an increasing number of cases, rodents are becoming resistant to warfarin, an ability which has been traced to elevated production of vitamin K by their intestinal microflora. Modified structures defenacoum and brodifenacoum have been found to be more potent than warfarin, and are also effective against rodents that have become resistant to warfarin. group in demethylsuberosin is then able to enzyme then cleaves off the hydroxyisopropyl cyclize with the phenol group giving marmesin. fragment (as acetone) from marmesin giving This transformation is catalysed by a cytochrome the furocoumarin psoralen (Figure 4.35). This P-450-dependent mono-oxygenase, and requires does not involve any hydroxylated intermediate, cofactors NADPH and molecular oxygen. For and cleavage is believed to be initiated by a many years, the cyclization had been postulated radical abstraction process. Psoralen can act as a to involve an intermediate epoxide, so that precursor for the further substituted furocoumarins nucleophilic attack of the phenol on to the bergapten, xanthotoxin,andisopimpinellin epoxide group might lead to formation of either (Figure 4.33), such modifications occurring late five-membered furan or six-membered pyran in the biosynthetic sequence rather than at heterocycles as commonly encountered in natural the cinnamic acid stage. Psoralen, bergapten, products (Figure 4.34). Although the reactions of etc are termed ‘linear’ furocoumarins. ‘Angular’ Figure 4.34 offer a convenient rationalization for furocoumarins, e.g. angelicin (Figure 4.33), can cyclization, epoxide intermediates have not been arise by a similar sequence of reactions, but demonstrated in any of the enzymic systems so far these involve dimethylallylation at the alternative investigated, and therefore some direct oxidative position ortho to the phenol. An isoprene-derived cyclization mechanism must operate. A second furan ring system has already been noted in the cytochrome P-450-dependent mono-oxygenase formation of khellin (see page 74), though the

C-alkylation at activated position OPP ortho to phenol O2 NADPH DMAPP HO HO OO HO OO O OO umbelliferone demethylsuberosin marmesin

O2 NADPH OMe OH O2 SAM NADPH

O OO O OO O OO bergapten bergaptol psoralen O2 (linear furocoumarin) OMe hydroxylation NADPH methylation SAM

O O O OO OO OO O OO OMe OMe OH isopimpinellin xanthotoxin xanthotoxol angelicin (angular furocoumarin)

Figure 4.33 146 THE SHIKIMATE PATHWAY

H O – H2O HO HO O O

nucleophilic attack 5-membered furan ring H on to epoxide

O HO – H2O

HO O O 6-membered pyran ring

Figure 4.34

oxidation leading to radical cleavage of Enz Fe Enz Fe O side-chain carbons H H OH O2 NADPH HO HO HO O OO O OO O OO marmesin psoralen – H O side-chain carbons 2 O HO OH released as acetone

Figure 4.35 aromatic ring to which it was fused was in that towards UV light, resulting in sunburn or serious case a product of the acetate pathway. Linear blistering. Used medicinally, this effect may be furocoumarins (psoralens)∗ can be troublesome to valuable in promoting skin pigmentation and humans since they can cause photosensitization treating psoriasis.

Psoralens Psoralens are linear furocoumarins which are widely distributed in plants, but are particularly abundant in the Umbelliferae/Apiaceae and Rutaceae. The most common examples are psoralen, bergapten, xanthotoxin, and isopimpinellin (Figure 4.33). Plants containing psoralens have been used internally and externally to promote skin pigmentation and sun-tanning. Bergamot oil obtained from the peel of Citrus aurantium ssp. bergamia (Rutaceae) (see page 179) can contain up to 5% bergapten, and is frequently used in external suntan preparations. The psoralen, because of its extended chromophore, absorbs in the near UV and allows this radiation to stimulate formation of melanin pigments (see page 129). Methoxsalen (xanthotoxin; 8-methoxypsoralen) (Figure 4.36), a constituent of the fruits of Ammi majus (Umbelliferae/Apiaceae), is used medically to facilitate skin repigmentation where severe blemishes exist (vitiligo). An oral dose of methoxsalen is followed by long wave UV irradiation, though such treatments must be very carefully regulated to

(Continues) STYRYLPYRONES 147

(Continued)

H O N O N

O O O O O O O hν O hν O HN HN HN

O N O OMe O N O OMe O N O OMe xanthotoxin (methoxsalen) thymine in DNA psoralen−DNA adduct psoralen−DNA di-adduct

Figure 4.36

minimize the risk of burning, cataract formation, and the possibility of causing skin cancer. The treatment is often referred to as PUVA (psoralen + UV-A). PUVA is also of value in the treatment of psoriasis, a widespread condition characterized by proliferation of skin cells. Similarly, methoxsalen is taken orally, prior to UV treatment. Reaction with psoralens inhibits DNA replication and reduces the rate of cell division. Because of their planar nature, psoralens intercalate into DNA, and this enables a UV-initiated cycloaddition reaction between pyrimidine bases (primarily thymine) in DNA and the furan ring of psoralens (Figure 4.36). In some cases, di-adducts can form involving further cycloaddition via the pyrone ring, thus cross-linking the nucleic acid. A troublesome extension of these effects can arise from the handling of plants that contain significant levels of furocoumarins. Celery (Apium graveolens; Umbelliferae/Apiaceae) is normally free of such compounds, but fungal infection with the natural parasite Sclerotinia sclerotiorum induces the synthesis of furocoumarins (xanthotoxin and others) as a response to the infections. Some field workers handling these infected plants have become very sensitive to UV light and suffer from a form of sunburn termed photophytodermatitis. Infected parsley (Petroselinum crispum) can give similar effects. Handling of rue (Ruta graveolens; Rutaceae) or giant hogweed (Heracleum mantegazzianum; Umbelliferae/Apiaceae), which naturally contain significant amounts of psoralen, bergapten, and xanthotoxin, can cause similar unpleasant reactions, or more commonly rapid blistering by direct contact with the sap. The giant hogweed can be particularly dangerous. Individuals vary in their sensitivity towards furocoumarins; some are unaffected whilst others tend to become sensitized by an initial exposure and then develop the allergic response on subsequent exposures.

STYRYLPYRONES plus one or two C2 units from malonyl-CoA. The short poly-β-keto chain frequently cyclizes to form a lactone derivative (compare triacetic acid lac- Cinnamic acids, as their coenzyme A esters, may tone, page 62). Thus, Figure 4.37 shows the pro- also function as starter units for chain exten- posed derivation of yangonin via cyclization of the sion with malonyl-CoA units, thus combining di-enol tautomer of the polyketide formed from elements of the shikimate and acetate pathways 4-hydroxycinnamoyl-CoA and two malonyl-CoA (see page 80). Most commonly, three C2 units extender units. Two methylation reactions com- are added via malonate giving rise to flavonoids plete the sequence. Yangonin and a series of related and stilbenes, as described in the next section structures form the active principles of kava root (page 149). However, there are several examples (Piper methysticum; Piperaceae), a herbal remedy of products formed from a cinnamoyl-CoA starter popular for its anxiolytic activity. 148 THE SHIKIMATE PATHWAY

chain extension; acetate pathway with O a cinnamoyl-CoA starter group OH di-enol tautomer O 2 x malonyl-CoA

SCoA O O ≡ OH O SCoA SCoA HO HO HO 4-hydroxycinnamoyl-CoA OMe lactone OH formation

SAM O O O O

MeO HO yangonin

Figure 4.37

Kava Aqueous extracts from the root and rhizome of Piper methysticum (Piperaceae) have long been consumed as an intoxicating beverage by the peoples of Pacific islands comprising Polynesia, Melanesia and Micronesia, and the name kava or kava-kava referred to this drink. In herbal medicine, the dried root and rhizome is now described as kava, and it is used for the treatment of anxiety, nervous tension, agitation and insomnia. The pharmacological activity is associated with a group of styrylpyrone derivatives termed kavapyrones or kavalactones, good quality roots containing 5–8% kavapyrones. At least 18 kavapyrones have been characterized, the six major ones being the enolides kawain, methysticin, and their dihydro derivatives reduced in the cinnamoyl side-chain, and the dienolides yangonin and demethoxyyangonin (Figure 4.38). Compared with the dienolides, the enolides have a reduced pyrone ring and a chiral centre. Clinical trials have indicated kava extracts to be effective as an anxiolytic, the kavapyrones also displaying anticonvulsive, analgesic, and central muscle relaxing action. Several of these compounds have been shown to have an effect on neurotransmitter systems including those involving glutamate, GABA, dopamine, and serotonin.

OMe OMe OMe OMe

O O OO OO OO OO H H H H O O kawaindihydrokawain methysticin dihydromethysticin OMe OMe

O O OO

MeO yangonin demethoxyyangonin

Figure 4.38 FLAVONOIDS AND STILBENES 149

FLAVONOIDS AND STILBENES Both structures nicely illustrate the different char- acteristic oxygenation patterns in aromatic rings Flavonoids and stilbenes are products from a derived from the acetate or shikimate pathways. cinnamoyl-CoA starter unit, with chain exten- With the stilbenes, it is noted that the terminal sion using three molecules of malonyl-CoA. ester function is no longer present, and there- This initially gives a polyketide (Figure 4.39), fore hydrolysis and decarboxylation have also which, according to the nature of the enzyme taken place during this transformation. No inter- responsible, can be folded in two different mediates, e.g. carboxylated stilbenes, have been ways. These allow aldol or Claisen-like reac- detected, and the transformation from cinnamoyl- tions to occur, generating aromatic rings as CoA/malonyl-CoA to stilbene is catalysed by the already seen in Chapter 3 (see page 80). Enzymes single enzyme. Resveratrol has assumed greater stilbene synthase and chalcone synthase cou- relevance in recent years as a constituent of ple a cinnamoyl-CoA unit with three malonyl- grapes and wine, as well as other food products, CoA units giving stilbenes, e.g. resveratrol or with antioxidant, anti-inflammatory, anti-platelet, chalcones, e.g. naringenin-chalcone respectively. and cancer preventative properties. Coupled with

OH

CoAS

O 4-hydroxycinnamoyl-CoA chain extension; acetate pathway with 3 x malonyl-CoA a cinnamoyl-CoA starter group OH OH OH O SCoA O SCoA O NADPH ≡ O O O SCoA (reductase) O O OH O O O Claisen aldol Claisen (stilbene synthase) (chalcone synthase) CO2 OH OH OH

HO HO OH HO OH

OH OH O O resveratrol naringenin-chalcone isoliquiritigenin (a stilbene) (a chalcone) Michael-type nucleophilic (a chalcone) attack of OH on to α,β-unsaturated ketone

OH OH

HO O HO O

OH O O naringenin liquiritigenin (a flavanone) (a flavanone)

Figure 4.39 150 THE SHIKIMATE PATHWAY the cardiovascular benefits of moderate amounts has a resorcinol oxygenation pattern rather than of alcohol, and the beneficial antioxidant effects the phloroglucinol system. This modification has of flavonoids (see page 151), red wine has now been tracked down to the action of a reductase emerged as an unlikely but most acceptable medic- enzyme concomitant with the chalcone synthase, inal agent. and thus isoliquiritigenin is produced rather than Chalcones act as precursors for a vast range naringenin-chalcone. Flavanones can then give of flavonoid derivatives found throughout the rise to many variants on this basic skeleton, e.g. plant kingdom. Most contain a six-membered flavones, flavonols, anthocyanidins,andcate- heterocyclic ring, formed by Michael-type nucle- chins (Figure 4.40). Modifications to the hydroxy- ophilic attack of a phenol group on to the unsatu- lation patterns in the two aromatic rings may occur, rated ketone giving a flavanone,e.g.naringenin generally at the flavanone or dihydroflavonol stage, (Figure 4.39). This isomerization can occur chem- and methylation, glycosylation, and dimethylal- ically, acid conditions favouring the flavanone lylation are also possible, increasing the range and basic conditions the chalcone, but in nature of compounds enormously. A high proportion of the reaction is enzyme catalysed and stereospe- flavonoids occur naturally as water-soluble gly- cific, resulting in formation of a single fla- cosides. Considerable quantities of flavonoids are vanone enantiomer. Many flavonoid structures, consumed daily in our vegetable diet, so adverse e.g. liquiritigenin, have lost one of the hydroxyl biological effects on man are not particularly groups, so that the acetate-derived aromatic ring intense. Indeed, there is growing belief that some

OH OH OH

HO O HO O HO O R O2 R O2 R 2-oxoglutarate 2-oxoglutarate OH OH OH O OH O OH O R = H, naringenin R = H, dihydrokaempferol R = H, kaempferol R = OH, eriodictyol R = OH, dihydroquercetin R = OH, quercetin (flavanones) (dihydroflavonols) (flavonols) O2 2-oxoglutarate NADPH OH OH OH HO O HO O HO R HO O R O R

OH OH OH O OH OH OH OH R = H, apigenin R = H, leucopelargonidin R = OH, luteolin R = OH, leucocyanidin (flavones) (flavandiols; leucoanthocyanidins) – 2 H2O

NADPH OH OH

HO O HO O R R

OH OH OH OH R = H, afzalechin R = H, pelargonidin R = OH, (+)-catechin R = OH, cyanidin (catechins) (anthocyanidins)

Figure 4.40 FLAVONOIDS AND STILBENES 151

OH

O HO O HO HO OH OH OH OH OH HO O HO O OH OH O OH OH OH OH OH HO O OH HO OH theaflavin OH OH epicatechin trimer

Figure 4.41

flavonoids are particularly beneficial, acting as peels have been included in dietary supplements as antioxidants and giving protection against cardio- vitamin P, and claimed to be of benefit in treating vascular disease, certain forms of cancer, and, conditions characterized by capillary bleeding, but it is claimed, age-related degeneration of cell their therapeutic efficacy is far from conclusive. components. Their polyphenolic nature enables Neohesperidin (Figure 4.42) from bitter orange them to scavenge injurious free radicals such as (Citrus aurantium; Rutaceae) and naringin from superoxide and hydroxyl radicals. Quercetin in grapefruit peel (Citrus paradisi) are intensely particular is almost always present in substan- bitter flavanone glycosides. It has been found tial amounts in plant tissues, and is a power- that conversion of these compounds into dihy- ful antioxidant, chelating metals, scavenging free drochalcones by hydrogenation in alkaline solu- radicals, and preventing oxidation of low density tion (Figure 4.43) produces a remarkable change lipoprotein. Flavonoids in red wine (quercetin, to their taste, and the products are now intensely kaempferol, and anthocyanidins) and in tea (cate- sweet, being some 300–1000 times as sweet as chins and catechin gallate esters) are also demon- sucrose. These and other dihydrochalcones have strated to be effective antioxidants. Flavonoids been investigated as non-sugar sweetening agents. contribute to plant colours, yellows from chal- cones and flavonols, and reds, blues, and violets FLAVONOLIGNANS from anthocyanidins. Even the colourless mate- rials, e.g. flavones, absorb strongly in the UV An interesting combination of flavonoid and lig- and are detectable by insects, probably aiding nan structures is found in a group of compounds flower pollination. Catechins form small poly- called flavonolignans. They arise by oxidative mers (oligomers), the condensed tannins,e.g.the coupling processes between a flavonoid and a epicatechin trimer (Figure 4.41) which contribute phenylpropanoid, usually coniferyl alcohol. Thus, astringency to our foods and drinks, as do the sim- the dihydroflavonol taxifolin through one-electron pler gallotannins (see page 122), and are commer- oxidation may provide a free radical, which may cially important for tanning leather. Theaflavins, combine with the free radical generated from antioxidants found in fermented tea (see page 395), coniferyl alcohol (Figure 4.44). This would lead are dimeric catechin structures in which oxida- to an adduct, which could cyclize by attack of tive processes have led to formation of a seven- the phenol nucleophile on to the quinone methide membered tropolone ring. system provided by coniferyl alcohol. The prod- The flavonol glycoside rutin (Figure 4.42) uct would be silybin, found in Silybum marianum from buckwheat (Fagopyrum esculentum; Polygo- (Compositae/Asteraceae) as a mixture of two trans naceae) and rue (Ruta graveolens; Rutaceae), and diastereoisomers, reflecting a lack of stereospeci- the flavanone glycoside hesperidin from Citrus ficity for the original radical coupling. In addition, 152 THE SHIKIMATE PATHWAY

OH HO HO O L-Rha O OMe OH HO O O O HO OH HO O OH OH D-Glc O-Glc-Rha OH O rutinose OH O = rhamnosyl(α1→6)glucose hesperetinquercetin rutinose

hesperidin rutin

OH OMe HO O D-Glc O O HO OH OH O Rha-Glc-O O O HO OH O HO L-Rha OH OH O neohesperidose = rhamnosyl(α1→2)glucose hesperetin neohesperidose naringenin

neohesperidin naringin

Figure 4.42

R1 R1 R1

Rha-Glc-O O Rha-Glc-O OH Rha-Glc-O OH R2 R2 R2

OH H2 / catalyst H OH O OH O OH O OH R1 = OH, R2 = H, R1 = OH, R2 = H, naringin naringin dihydrochalcone R1 = OMe, R2 = OH, neohesperidin R1 = OMe, R2 = OH, neohesperidin dihydrochalcone

Figure 4.43 the regioisomer isosilybin (Figure 4.45), again a of an enolate nucleophile on to the quinoneme- mixture of trans diastereoisomers, is also found in thide. Hemiketal formation finishes the process. Silybum. Silychristin (Figure 4.45) demonstrates The flavonolignans from Silybum∗ (milk thistle) a further structural variant which can be seen to have valuable antihepatotoxic properties, and can originate from a mesomer of the taxifolin-derived provide protection against liver-damaging agents. free radical, in which the unpaired electron is Coumarinolignans, which are products arising by a localized on the carbon ortho to the original 4- similar oxidative coupling mechanism which com- hydroxyl function. The more complex structure bines a coumarin with a cinnamyl alcohol, may in silydianin is accounted for by the mechanism be found in other plants. The benzodioxane ring shown in Figure 4.46, in which the initial coupling as seen in silybin and isosilybin is a characteristic product cyclizes further by intramolecular attack feature of many such compounds. FLAVONOLIGNANS 153

OH OH one-electron one-electron oxidation OH oxidation O

HO O HO O OH – H OH – H – e – e OH OH OMe OMe OH O OH O O OH taxifolin coniferyl alcohol OH radical coupling O OH HO O OMe O O

OH OH HO O OMe OH OH O + OH OH O H O OH O nucleophilic attack of OH HO O OMe O on to quinonemethide (two stereochemistries possible) OH OH OH O silybin (diastereoisomeric pair)

Figure 4.44

Silybum marianum

Silybum marianum (Compositae/Asteraceae) is a biennial thistle-like plant (milk thistle) common in the Mediterranean area of Europe. The seeds yield 1.5–3% of flavonolignans collectively termed silymarin. This mixture contains mainly silybin (Figure 4.44), together with silychristin (Figure 4.45), silydianin (Figure 4.46), and small amounts of isosilybin (Figure 4.45). Both silybin and isosilybin are equimolar mixtures of two trans diastereoisomers. Silybum marianum is widely used in traditional European medicine, the fruits being used to treat a variety of hepatic and other disorders. Silymarin has been shown to protect animal livers against the damaging effects of carbon tetrachloride, thioacetamide, drugs such as paracetamol, and the toxins α-amanitin and phalloin found in the death cap fungus (Amanita phalloides) (see page 433). Silymarin may be used in many cases of liver disease and injury, though it still remains peripheral to mainstream medicine. It can offer particular benefit in the treatment of poisoning by the death cap fungus. These agents appear to have two main modes of action. They act on the cellular membrane of hepatocytes inhibiting absorption of toxins, and secondly, because of their phenolic nature, they can act as antioxidants and scavengers for free radicals which cause liver damage originating from liver detoxification of foreign chemicals. Derivatives of silybin with improved water-solubility and/or bioavailability have been developed, e.g. the bis-hemisuccinate and a phosphatidylcholine complex. 154 THE SHIKIMATE PATHWAY

OH OH O O OMe O HO O HO O O OMe OH OH HO OH OH O OH O + OH OH O OMe O HO O OH O HO O OH OMe OH OH OH OH O isosilybin OH O (diastereoisomeric pair) silychristin

Figure 4.45

(i) nucleophilic attack of enolate on to quinonemethide MeO (ii) hemiketal formation OH HO MeO radical O (ii) OH coupling O H O H MeO O O H HO O HO O OH OH OH HO O (i) O OH OH OH OH O OH O OH O silydianin

Figure 4.46

ISOFLAVONOIDS of different isoflavonoids have been identified, and structural complexity is brought about by The isoflavonoids form a quite distinct subclass hydroxylation and alkylation reactions, varying the of flavonoid compound, being structural vari- oxidation level of the heterocyclic ring, or forming ants in which the shikimate-derived aromatic additional heterocyclic rings. Some of the many ring has migrated to the adjacent carbon of variants are shown in Figure 4.48. Pterocarpans, the heterocycle. This rearrangement process is e.g. medicarpin from lucerne (Medicago sativa), brought about by a cytochrome P-450-dependent and pisatin from pea (Pisum sativum), have anti- enzyme requiring NADPH and O2 cofactors, fungal activity and form part of these plants’ which transforms the flavanones liquiritigenin natural defence mechanism against fungal attack. or naringenin into the isoflavones daidzein or Simple isoflavones such as daidzein and coumes- genistein respectively via intermediate hydrox- tans such as coumestrol from lucerne and clovers yisoflavanones (Figure 4.47). A radical mecha- (Trifolium species), have sufficient oestrogenic nism has been proposed. This rearrangement is activity to seriously affect the reproduction of graz- quite rare in nature, and isoflavonoids are almost ing animals, and are termed phyto-oestrogens∗. entirely restricted to the plant family the Legu- These planar molecules undoubtedly mimic the minosae/Fabaceae. Nevertheless, many hundreds shape and polarity of the steroid hormone estradiol ISOFLAVONOIDS 155

oxidation to 1,2-aryl OH free radical OH migration O OH HO O 2 HO O HO O NADPH Fe Enz H H O R O R O R O Fe Enz OH R = H, liquiritigenin R = OH, naringenin

(flavonoid) HO 7 O HO O OH – H2O

R O 4′ R O OH OH R = H, daidzein R = OH, genistein (isoflavonoid)

Figure 4.47

HO O HO O HO O H

H O O O OH OMe OMe daidzein formononetin medicarpin (an isoflavone) (an isoflavone) (a pterocarpan)

H HO O O MeO O H O O OH O O H O O OH H O O coumestrol OMe pisatin (a coumestan) rotenone OMe (a pterocarpan) (a rotenoid)

Figure 4.48

(see page 276). The consumption of legume fod- (see page 279). The rotenoids take their name der crops by animals must therefore be restricted, from the first known example rotenone,andare or low isoflavonoid producing strains have to be formed by ring cyclization of a methoxyisoflavone selected. Isoflavonoids in the human diet, e.g. (Figure 4.49). Rotenone itself contains a C5 iso- from soya (Glycine max) products, are believed to prene unit (as do virtually all the natural rotenoids) give some protection against oestrogen-dependent introduced via dimethylallylation of demethyl- cancers such as breast cancer, by restricting the munduserone. The isopropenylfurano system of availability of the natural hormone. In addition, rotenone, and the dimethylpyrano of deguelin, they can feature as dietary oestrogen supplements are formed via rotenonic acid (Figure 4.49) with- in the reduction of menopausal symptoms, in out any detectable epoxide or hydroxy intermedi- a similar way to hormone replacement therapy ates (compare furocoumarins, page 145). Rotenone 156 THE SHIKIMATE PATHWAY

oxidation of OMe group (hydroxylation and loss of hydroxide, compare H HO O Figure 2.21) HO O HO O OMe O O

O O O H OMe OMe OMe OMe OMe OMe H

H C-alkylation at addition of O O activated position hydride O cyclization to 5-membered ring ortho to phenol (reduction)

H O OPP OMe H H HO O DMAPP HO O rotenone OMe O O

H H O O H O O O OMe cyclization to OMe 6-membered ring OMe OMe H rotenonic acid demethylmunduserone O OMe deguelin OMe

Figure 4.49 and other rotenoids are powerful insecticidal and Rotenone thus provides an excellent biodegradable piscicidal (fish poison) agents, interfering with insecticide, and is used as such either in pure or ∗ oxidative phosphorylation. They are relatively powdered plant form. Roots of Derris elliptica ∗ harmless to mammals unless they enter the blood or Lonchocarpus species are rich sources of stream, being metabolized rapidly upon ingestion. rotenone.

Phyto-oestrogens

Phyto-oestrogen (phytoestrogen) is a term applied to non-steroidal plant materials displaying oestrogenic properties. Pre-eminent amongst these are isoflavonoids. These planar molecules mimic the shape and polarity of the steroid hormone estradiol (see page 279), and are able to bind to an oestrogen receptor, though their activity is less than that of estradiol. In some tissues, they stimulate an oestrogenic response, whilst in others they can antagonize the effect of oestrogens. Such materials taken as part of the diet therefore influence overall oestrogenic activity in the body by adding their effects to normal levels of steroidal oestrogens (see page 282). Foods rich in isoflavonoids are valuable in countering some of the side-effects of the menopause in women, such as hot flushes, tiredness, and mood swings. In addition, there is mounting evidence that phyto-oestrogens also provide a range of other beneficial effects, helping to prevent heart attacks and other cardiovascular diseases, protecting against osteoporosis, lessening the risk of breast and uterine cancer, and in addition displaying significant antioxidant activity which may reduce the risk of Alzheimer’s disease. Whilst some of these benefits may be obtained by the use of steroidal

(Continues) ISOFLAVONOIDS 157

(Continued) oestrogens, particularly via hormone replacement therapy (HRT; see page 279), phyto- oestrogens offer a dietary alternative. The main food source of isoflavonoids is the soya bean (Glycine max; Legumi- nosae/Fabaceae) (see also page 256), which contains significant levels of the isoflavones daidzein, and genistein (Figure 4.47), in free form and as their 7-O-glucosides. Total isoflavone levels fall in the range 0.1–0.4%, according to variety. Soya products such as soya milk, soya flour, tofu, and soya-based textured vegetable protein may all be used in the diet for their isoflavonoid content. Breads in which wheat flour is replaced by soya flour are also popular. Extracts from red clover (Trifolium pratense; Leguminosae/Fabaceae) are also used as a dietary supplement. Red clover isoflavones are predominantly formononetin (Figure 4.48) and daidzein, together with their 7-O- glucosides. The lignans enterodiol and enterolactone (see page 135) are also regarded as phyto- oestrogens. These compounds are produced by the action of intestinal microflora on lignans such as secoisolariciresinol or matairesinol ingested in the diet. A particularly important precursor is secoisolariciresinol diglucoside from flaxseed (Linum usitatissimum; Linaceae), and flaxseed may be incorporated into foodstuffs along with soya products. Enterolactone and enterodiol were first detected in human urine, and their origins were traced back to dietary fibre-rich foods. Levels in the urine were much higher in vegetarians, and have been related to a lower incidence of breast cancer in vegetarians.

Derris and Lonchocarpus

Species of Derris (e.g. D. elliptica, D. malaccensis)andLonchocarpus (e.g. L. utilis, L. urucu) (Leguminosae/Fabaceae) have provided useful insecticides for many years. Roots of these plants have been employed as a dusting powder, or extracts have been formulated for sprays. Derris plants are small shrubs cultivated in Malaysia and Indonesia, whilst Lonchocarpus includes shrubs and trees, with commercial material coming from Peru and Brazil. The insecticidal principles are usually supplied as a black, resinous extract. Both Derris and Lonchocarpus roots contain 3–10% of rotenone (Figure 4.49) and smaller amounts of other rotenoids, e.g. deguelin (Figure 4.49). The resin may contain rotenone (about 45%) and deguelin (about 20%). Rotenone and other rotenoids interfere with oxidative phosphorylation, blocking transfer of electrons to ubiquinone (see page 159) by complexing with NADH:ubiquinone oxidoreductase of the respiratory electron transport chain. However, they are relatively innocuous to mammals unless they enter the blood stream, being metabolized rapidly upon ingestion. Insects and also fish seem to lack this rapid detoxification. The fish poison effect has been exploited for centuries in a number of tropical countries, allowing lazy fishing by the scattering of powdered plant material on the water. The dead fish were collected, and when subsequently eaten produced no ill effects on the consumers. More recently, rotenoids have been used in fish management programmes to eradicate undesirable fish species prior to restocking with other species. As insecticides, the rotenoids still find modest use, and are valuable for their selectivity and rapid biodegradability. However, they are perhaps inactivated too rapidly in the presence of light and air to compete effectively with other insecticides such as the modern pyrethrin derivatives (see page 188). 158 THE SHIKIMATE PATHWAY

TERPENOID QUINONES ortho-quinones and quinols (1,4-dihydroxyben- zenes) yielding para-quinones (see page 25). Quinones are potentially derivable by oxi- Accordingly, quinones can be formed from phe- dation of suitable phenolic compounds, cat- nolic systems generated by either the acetate or echols (1,2-dihydroxybenzenes) giving rise to shikimate pathways, provided a catechol or quinol

O O OH = − MeO n 1 12 n = 3−10 R1 R2

MeO n H n H O O O H ubiquinone-n plastoquinone-n 3 (coenzyme Qn) R1 = R2 = Me, α-tocopherol O O R1 = H, R2 = Me, β-tocopherol = − n 1 13 R1 = Me, R2 = H, γ-tocopherol R1 = R2 = H, δ -tocopherol H 3 nH (vitamin E) O O phylloquinone menaquinone-n (vitamin K1; phytomenadione) (vitamin K2)

Figure 4.50

CO2H

plants / H animals nOPP CO2H CO2H

OH 4-coumaric acid C-alkylation with a n H bacteria polyisoprenyl PP OH OH CO2H 4-hydroxybenzoic 1. hydroxylation CH3COCO2H acid 2. O-methylation O2 3. decarboxylation SAM OCO2H or: 3, 1, 2 OH chorismic acid MeO n H OH

1. C-methylation O2 2. hydroxylation oxidation O 3. O-methylation O to quinone MeO SAM O2 SAM

MeO H MeO n n H O O ubiquinone-n

Figure 4.51 TERPENOID QUINONES 159 system has been elaborated, and many examples suffers decarboxylation, and the product is thus an are found in nature. A range of quinone deriva- alkyl methyl p-quinol derivative. Further aromatic tives and related structures containing a terpenoid methylation (via S-adenosylmethionine) and oxida- fragment as well as a shikimate-derived portion tion of the p-quinol to a quinone follow to yield the are also widely distributed. Many of these have plastoquinone. Thus, only one of the two methyl important biochemical functions in electron trans- groups on the quinone ring of the plastoquinone is port systems for respiration or photosynthesis, and derived from SAM. Plastoquinones are involved in some examples are shown in Figure 4.50. the photosynthetic electron transport chain in plants. Ubiquinones (coenzyme Q) (Figure 4.50) are Tocopherols are also frequently found in the found in almost all organisms and function as elec- chloroplasts and constitute members of the vita- tron carriers for the electron transport chain in min E∗ group. Their biosynthesis shares many of mitochondria. The length of the terpenoid chain the features of plastoquinone biosynthesis, with is variable (n = 1−12), and dependent on species, an additional cyclization reaction involving the but most organisms synthesize a range of com- p-quinol and the terpenoid side-chain to give a pounds, of which those where n = 7−10 usually chroman ring (Figure 4.52). Thus, the tocopherols, predominate. The human redox carrier is coenzyme e.g. α-tocopherol and γ-tocopherol, are not in Q10. They are derived from 4-hydroxybenzoic fact quinones, but are indeed structurally related to acid (Figure 4.51), though the origin of this plastoquinones. The isoprenoid side-chain added, compound varies according to organism (see from phytyl diphosphate, contains only four iso- pages 123, 141). Thus, bacteria are known to prene units, and three of the expected double bonds transform chorismic acid by enzymic elimination have suffered reduction. Again, decarboxylation of pyruvic acid, whereas plants and animals uti- of homogentisic acid cooccurs with the alkylation lize a route from phenylalanine or tyrosine via reaction. C-Methylation steps using SAM, and the 4-hydroxycinnamic acid (Figure 4.51). 4-Hydro- cyclization of the p-quinol to γ-tocopherol, have xybenzoic acid is the substrate for C-alkylation been established as in Figure 4.52. Note once again ortho to the phenol group with a polyisoprenyl that one of the nuclear methyls is homogentisate- diphosphate of appropriate chain length (see derived, whilst the others are supplied by SAM. page 231). The product then undergoes further The phylloquinones (vitamin K1)andmena- elaboration, the exact sequence of modifications, quinones (vitamin K2) are shikimate-derived na- i.e. hydroxylation, O-methylation, and decarboxy- phthoquinone derivatives found in plants and ∗ lation, varying in eukaryotes and prokaryotes. algae (vitamin K1 ) or bacteria and fungi (vita- Quinone formation follows in an O2-dependent min K2). The most common phylloquinone struc- combined hydroxylation–oxidation process, and ture (Figure 4.50) has a diterpenoid side-chain, ubiquinone production then involves further hydro- whereas the range of menaquinone structures tends xylation, and O-andC-methylation reactions. to be rather wider with 1–13 isoprene units. Plastoquinones (Figure 4.50) bear considerable These quinones are derived from chorismic acid structural similarity to ubiquinones, but are not via its isomer isochorismic acid (Figure 4.55). derived from 4-hydroxybenzoic acid. Instead, they Additional carbons for the naphthoquinone skele- are produced from homogentisic acid, a pheny- ton are provided by 2-oxoglutaric acid, which is lacetic acid derivative formed from 4-hydroxyphe- incorporated by a mechanism involving the coen- nylpyruvic acid by a complex reaction involving zyme thiamine diphosphate (TPP). 2-Oxoglutaric decarboxylation, O2-dependent hydroxylation, and acid is decarboxylated in the presence of TPP subsequent migration of the −CH2CO2Hside- to give the TPP anion of succinic semialdehyde, chain to the adjacent position on the aromatic ring which attacks isochorismic acid in a Michael-type (Figure 4.52). C-Alkylation of homogentisic acid reaction. Loss of the thiamine cofactor, elimina- ortho to a phenol group follows, and involves tion of pyruvic acid, and then dehydration yield a polyisoprenyl diphosphate with n = 3−10, but the intermediate o-succinylbenzoic acid (OSB). most commonly with n = 9, i.e. solanesyl diphos- This is activated by formation of a coenzyme A phate. However, during the alkylation reaction, ester, and a Dieckmann-like condensation allows the −CH2CO2H side-chain of homogentisic acid ring formation. The dihydroxynaphthoic acid is the 160 THE SHIKIMATE PATHWAY

a complex sequence involving C-alkylation ortho to phenol; hydroxylation, migration of H side-chain, and decarboxylation also decarboxylation PPO n n = 9, solanesyl PP HO O HO O2 HO H n OH OH CO2 CO O CO2H HO2C 2 4-hydroxyphenyl- homogentisic acid SAM pyruvic acid C-methylation H ortho to phenol PPO 3 HO H CO2 phytyl PP n

OH HO

H oxidation of O O quinol to quinone H 3

C-methylation SAM ortho to phenol O H n

HO H O

H O H 3 plastoquinone-n cyclization to 6-membered ring via protonation of double bond

HO HO SAM H H O O 3 C-methylation 3 ortho to phenol γ-tocopherol α-tocopherol

Figure 4.52

Vitamin E Vitamin E refers to a group of fat-soluble vitamins, the tocopherols, e.g. α-, β-, γ-, and δ-tocopherols (Figure 4.53), which are widely distributed in plants, with high levels in cereal seeds such as wheat, barley, and rye. Wheat germ oil is a particularly good source. The proportions of the individual tocopherols vary widely in different seed oils, e.g. principally β- in wheat oil, γ- in corn oil, α- in safflower oil, and γ-andδ-insoybeanoil.VitaminE deficiency is virtually unknown, with most of the dietary intake coming from food oils and margarine, though much can be lost during processing and cooking. Rats deprived of the vitamin display reproductive abnormalities. α-Tocopherol has the highest activity (100%), with the relative activities of β-, γ-, and δ-tocopherols being 50%, 10%, and 3% respectively. α-Tocopheryl acetate is the main commercial form used for food supplementation and

(Continues) TERPENOID QUINONES 161

(Continued)

HO

O α-tocopherol

HO HO HO

O O O

β-tocopherol γ-tocopherol δ-tocopherol

Figure 4.53

initiation of free radical reaction by peroxy radical resonance-stabilized quenching of second free radical peroxy radical HO O O O ROO ROO

O O O O OOR α loss of -tocopherol peroxide leaving group OH hydrolysis of O hemiketal O O H2O

O O O OH α-tocopherolquinone

Figure 4.54

for medicinal purposes. The vitamin is known to provide valuable antioxidant properties, probably preventing the destruction by free radical reactions of vitamin A and unsaturated fatty acids in biological membranes. It is used commercially to retard rancidity in fatty materials in food manufacturing, and there are also claims that it can reduce the effects of ageing and help to prevent heart disease. Its antioxidant effect is likely to arise by reacting with peroxyl radicals, generating by one-electron phenolic oxidation a resonance-stabilized free radical that does not propagate the free radical reaction, but instead mops up further peroxyl radicals (Figure 4.54). In due course, the tocopheryl peroxide is hydrolysed to the tocopherolquinone. more favoured aromatic tautomer from the hydrol- 1,4-dihydroxynaphthoic acid to the isoprenylated ysis of the coenzyme A ester. This compound is naphthoquinone appears to be catalysed by a single now the substrate for alkylation and methylation enzyme, and can be rationalized by the mech- as seen with ubiquinones and plastoquinones. anism in Figure 4.56. This involves alkylation However, the terpenoid fragment is found to (shown in Figure 4.56 using the diketo tautomer), replace the carboxyl group, and the decarboxylated decarboxylation of the resultant β-keto acid, and analogue is not involved. The transformation of finally an oxidation to the p-quinone. 162 THE SHIKIMATE PATHWAY

Michael-type HO C HO2C HO C addition 2 H2O 2 OH O OH CO H O 2 CO H O CO2H O C 2 OH H HO HO CO2H H OTPP chorismic acid isochorismic acid TPP succinic semi-aldehyde -TPP anion TPP-dependent decarboxylation of CO2 α-keto acid to aldehyde; nucleophilic H TPP addition of TPP anion on to aldehyde HO C then allows removal of aldehydic 2 OH HO2CCO2H proton which has become acidic CO2H O CO2H O H 2-oxoglutaric acid O 1,4-elimination of pyruvic acid CH COCO H Claisen-like condensation 3 2 (Dieckmann reaction) O O HSCoA H OH OH H C ATP CO2H CO2H COSCoA COSCoA CO2H CO2H

dehydration to O O O O hydrolysis of thioester; o-succinyl form aromatic ring enolization to more benzoic acid stable tautomer (OSB) HSCoA H OH PP O n O SAM O CO2H H H n n

C-methylation CO2 OH C-alkylation with O O 1,4-dihydroxy- concomitant menaquinone-n naphthoic acid decarboxylation (vitamin K2)

Figure 4.55

R PPO H decarboxylation O O O of β-keto acid CO H 2 O R R O O

CO2 O O R R O

O O

Figure 4.56 TERPENOID QUINONES 163

Vitamin K

Vitamin K comprises a number of fat-soluble naphthoquinone derivatives, with vitamin K1 (phylloquinone) (Figure 4.50) being of plant origin whilst the vitamins K2 (menaquinones) are produced by microorganisms. Dietary vitamin K1 is obtained from almost any green vegetable, whilst a significant amount of vitamin K2 is produced by the intestinal microflora. As a result, vitamin K deficiency is rare. Deficiencies are usually the result of malabsorption of the vitamin, which is lipid soluble. Vitamin K1 (phytomenadione) or the water-soluble menadiol phosphate (Figure 4.57) may be employed as supplements. Menadiol is oxidized in the body to the quinone, which is then alkylated, e.g. with geranylgeranyl diphosphate, to yield a metabolically active product. Vitamin K is involved in normal blood clotting processes, and a deficiency would lead to haemorrhage. Blood clotting requires the carboxylation of glutamate residues in the protein prothrombin, generating bidentate ligands that allow the protein to bind to other factors. This carboxylation requires carbon dioxide, molecular oxygen, and the reduced quinol form of vitamin K (Figure 4.57). During the carboxylation, the reduced vitamin K suffers epoxidation, and vitamin K is subsequently regenerated by reduction. Anticoagulants such as dicoumarol and warfarin (see page 144) inhibit this last reduction step. However, the polysaccharide anticoagulant heparin (see page 477) does not interfere with vitamin K metabolism, but acts by complexing with blood clotting enzymes.

O OH O H N N H

O OH CO2H vitamin K vitamin K (quinol) prothrombin O2, CO2 OP O O reductase H N N O H CO2H

OP O CO2H menadiol phosphate vitamin K epoxide

Figure 4.57

OSB, and 1,4-dihydroxynaphthoic acid, or its Streptocarpus (Gesneriaceae), e.g. catalponone diketo tautomer, have been implicated in the (compare Figure 4.56), and this can be transformed biosynthesis of a wide range of plant naphtho- to deoxylapachol and then menaquinone-1 (Fig- quinones and anthraquinones. There are parallels ure 4.58). Lawsone is formed by an oxidative with the later stages of the menaquinone sequence sequence in which hydroxyl replaces the carboxyl. shown in Figure 4.55, or differences according to A further interesting elaboration is the synthesis the plant species concerned. Some of these path- of an anthraquinone skeleton by effectively cycliz- ways are illustrated in Figure 4.58. Replacement ing a dimethylallyl substituent on to the naph- of the carboxyl function by an isoprenyl sub- thaquinone system. Rather little is known about stituent is found to proceed via a disubstituted how this process is achieved but many examples intermediate in Catalpa (Bignoniaceae) and are known from the results of labelling studies. 164 THE SHIKIMATE PATHWAY

O O O O

CO2H CO2H

O O O O catalponone deoxylapachol menaquinone-1

O O O OH

O O O OH lawsone

OH OH O OH O OH CO H CO2H OH 2 OH

OH OH OH O O 1,4-dihydroxy- lucidin alizarin naphthoic acid

Figure 4.58

Some of these structures retain the methyl from acid at the non-carboxylated carbon. Obviously, the isoprenyl substituent, whilst in others this has this is also a nucleophilic site and alkylation been removed, e.g. alizarin from madder (Rubia here is mechanistically sound. Again, cyclization tinctorum; Rubiaceae), presumably via an oxi- of the dimethylallyl to produce an anthraquinone dation–decarboxylation sequence. Hydroxylation, can occur, and the potently mutagenic lucidin particularly in the terpenoid-derived ring, is also a from Galium species (Rubiaceae) is a typical frequent feature. example. The hydroxylation patterns seen in the Some other quinone derivatives, although anthraquinones in Figure 4.58 should be com- formed from the same pathway, are produced pared with those noted earlier in acetate/malonate- by dimethylallylation of 1,4-dihydroxynaphthoic derived structures (see page 63). Remnants of the alternate oxygenation pattern are usually acetate / malonate very evident in acetate-derived anthraquinones O O (Figure 4.59), whereas such a pattern cannot HO OH easily be incorporated into typical shikimate/2- oxoglutarate/isoprenoid structures. Oxygen sub- stituents are not usually present in positions fitting OH OOH OH OOH the polyketide hypothesis. emodin aloe-emodin

Shikimate / 2-oxoglutarate / isoprenoid O OH O OH FURTHER READING OH OH Shikimate Pathway OH O O Abell C (1999) Enzymology and molecular biology alizarin lucidin of the shikimate pathway. Comprehensive Natural Products Chemistry, Vol 1. Elsevier, Amsterdam, Figure 4.59 pp 573–607. FURTHER READING 165

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