THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND

Medicinal Natural Products. Paul M DewickCopyright 2002 John Wiley & Sons, Ltd

ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic)

4THE SHIKIMATE PATHWAY:

AROMATIC AMINO ACIDS ANDPHENYLPROPANOIDS

Shikimic acid and its role in the formation of aromatic amino acids, benzoic acids, and cinnamic acids isdescribed, 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 biosynthesisof styrylpyrones, flavonoids and stilbenes, flavonolignans, and isoflavonoids. Terpenoid quinones areformed by a combination of the shikimate pathway with the terpenoid pathway. Monograph topicsgiving more detailed information on medicinal agents include folic acid, chloramphenicol, podophyllum,volatile oils, dicoumarol and warfarin, psoralens, kava, Silybum marianum, phyto-oestrogens, derris andlonchocarpus, vitamin E, and vitamin K.

The shikimate pathway provides an alternativeroute to aromatic compounds, particularly the aro-matic amino acids L-phenylalanine, L-tyrosineand L-tryptophan. This pathway is employed bymicroorganisms and plants, but not by animals,and accordingly the aromatic amino acids fea-ture among those essential amino acids for manwhich have to be obtained in the diet. A cen-tral intermediate in the pathway is shikimic acid(Figure 4.1), a compound which had been iso-lated from plants of Illicium species (Japanese‘shikimi’) many years before its role in metabolismhad been discovered. Most of the intermediates inthe pathway were identified by a careful study ofa series of Escherichia coli mutants prepared byUV irradiation. Their nutritional requirements forgrowth, and any by-products formed, were thencharacterized. A mutant strain capable of growthusually differs from its parent in only a singlegene, and the usual effect is the impaired syn-thesis of a single enzyme. Typically, a mutantblocked in the transformation of compound A intocompound B will require B for growth whilstaccumulating A in its culture medium. In thisway, the pathway from phosphoenolpyruvate (from

glycolysis) and D-erythrose 4-phosphate (from thepentose phosphate cycle) to the aromatic aminoacids was broadly outlined. Phenylalanine andtyrosine form the basis of C6C3 phenylpropaneunits found in many natural products, e.g. cin-namic acids, coumarins, lignans, and flavonoids,and along with tryptophan are precursors of a widerange of alkaloid structures. In addition, it is foundthat many simple benzoic acid derivatives, e.g.gallic acid (Figure 4.1) and p-aminobenzoic acid(4-aminobenzoic acid) (Figure 4.4) are producedvia branchpoints in the shikimate pathway.

AROMATIC AMINO ACIDS ANDSIMPLE BENZOIC ACIDS

The shikimate pathway begins with a couplingof phosphoenolpyruvate (PEP) and D-erythrose4-phosphate to give the seven-carbon 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP)(Figure 4.1). This reaction, shown here as analdol-type condensation, is known to be mecha-nistically more complex in the enzyme-catalysedversion; several of the other transformations in thepathway have also been found to be surprisingly

122 THE SHIKIMATE PATHWAY

CO2H

OH

OH

HO

OH

NAD+

OH

OH

CO2HHO

HO

NADH

CO2H

OH

OH

HO

OPO

H

DAHP

O

OH

OH

CO2HHO

CO2H

OP

HO

PO

HO

OH

H

CO2H

O

OH

OH

HO

OH

CO2H

OH

NADPH

CO2H

HO

OH

OH

HO

OH

CO2H

D-erythrose 4-P

formally an elimination; it actually involves oxidation of the

hydroxyl adjacent to the proton lost and therefore requires NAD+

cofactor; the carbonyl is subsequently reduced back to an alcohol

aldol-type reaction

– HOP

aldol-type reaction

– H2O

3-dehydroquinic acid

3-dehydroshikimic acid

quinic acidshikimic acid

– H2O – 2H

protocatechuic acid

gallic acid

dehydration and enolization

oxidation and enolization

PEP

Figure 4.1

complex. Elimination of phosphoric acid fromDAHP followed by an intramolecular aldol reac-tion generates the first carbocyclic intermediate 3-dehydroquinic acid. However, this also representsan oversimplification. The elimination of phos-phoric acid actually follows an NAD+-dependentoxidation of the central hydroxyl, and this is thenre-formed in an NADH-dependent reduction reac-tion on the intermediate carbonyl compound priorto the aldol reaction occurring. All these changesoccur in the presence of a single enzyme. Reduc-tion of 3-dehydroquinic acid leads to quinic acid,a fairly common natural product found in thefree form, as esters, or in combination with alka-loids such as quinine (see page 362). Shikimicacid itself is formed from 3-dehydroquinic acidvia 3-dehydroshikimic acid by dehydration andreduction steps. The simple phenolic acids pro-tocatechuic acid (3,4-dihydroxybenzoic acid) and

gallic acid (3,4,5-trihydroxybenzoic acid) can beformed by branchpoint reactions from 3-dehy-droshikimic acid, which involve dehydration andenolization, or, in the case of gallic acid, dehy-drogenation and enolization. Gallic acid featuresas a component of many tannin materials (gal-lotannins), e.g. pentagalloylglucose (Figure 4.2),found in plants, materials which have been usedfor thousands of years in the tanning of animalhides to make leather, due to their ability to cross-link protein molecules. Tannins also contribute tothe astringency of foods and beverages, especiallytea, coffee and wines (see also condensed tannins,page 151).

A very important branchpoint compound in theshikimate pathway is chorismic acid (Figure 4.3),which has incorporated a further molecule ofPEP as an enol ether side-chain. PEP combineswith shikimic acid 3-phosphate produced in a

AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 123

OO

O

O

OO O

OO

OH

HO OH

OH

OH

OHOH

OH

OH

OH

OHHOOH

OH

HO

O

O

HN

P

O

CO2H

OHOH

CO2H

O

P

OHOHO

glyphosate PEP

pentagalloylglucose

Figure 4.2

simple ATP-dependent phosphorylation reaction.This combines with PEP via an addition–elimi-nation reaction giving 3-enolpyruvylshikimic acid3-phosphate (EPSP). This reaction is catalysedby the enzyme EPSP synthase. The syntheticN-(phosphonomethyl)glycine derivative glypho-sate (Figure 4.2) is a powerful inhibitor of this

enzyme, and is believed to bind to the PEP bind-ing site on the enzyme. Glyphosate finds con-siderable use as a broad spectrum herbicide, aplant’s subsequent inability to synthesize aromaticamino acids causing its death. The transformationof EPSP to chorismic acid (Figure 4.3) involves a1,4-elimination of phosphoric acid, though this isprobably not a concerted elimination.

4-hydroxybenzoic acid (Figure 4.4) is pro-duced in bacteria from chorismic acid byan elimination reaction, losing the recentlyintroduced enolpyruvic acid side-chain. However,in plants, this phenolic acid is formed by abranch much further on in the pathway viaside-chain degradation of cinnamic acids (seepage 141). The three phenolic acids so far encoun-tered, 4-hydroxybenzoic, protocatechuic, and gallicacids, demonstrate some of the hydroxylationpatterns characteristic of shikimic acid-derivedmetabolites, i.e. a single hydroxy para to the side-chain function, dihydroxy groups arranged ortho toeach other, typically 3,4- to the side-chain, and tri-hydroxy groups also ortho to each other and 3,4,5-to the side-chain. The single para-hydroxylationand the ortho-polyhydroxylation patterns contrastwith the typical meta-hydroxylation patterns char-acteristic of phenols derived via the acetate path-way (see page 62), and in most cases allow thebiosynthetic origin (acetate or shikimate) of an aro-matic ring to be deduced by inspection.

CO2H

HO

OH

OH

ATP

CO2H

PO

OH

OH

CO2HPO

H

CO2H

OH

O CO2H

CO2H

PO

OH

O CO2H

HH H

OP

CO2H

PO

OH

O CO2H

shikimic acid shikimic acid 3-P

nucleophilic attack on to protonated double bond of PEP

chorismic acid EPSP

– HOP1,4-elimination of phosphoric acid

– HOP

1,2-elimination of phosphoric acid

prephenic acid

L-Phe

L-Tyr

PEP

EPSP synthase

Figure 4.3


CO2H

OH

CO2H

O CO2H

OH

CO2H

OH

OH

NAD+

OH2

CO2H

OH

OH

CO2H

OH

O CO2H

CO2H

O CO2H

NH2

H

CO2H

OH

CO2H

NH2

CO2H

NH2

O CO2H

CO2H

NH2

chorismic acid4-hydroxybenzoicacid

p-aminobenzoic acid(PABA)

anthranilic acid

isochorismic acid salicylic acid

L-Trp

2,3-dihydroxybenzoicacid

L-Gln L-Gln

elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring isomerization via

SN2′ reaction

elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring

amination using ammonia (generated from glutamine) as nucleophile

elimination of pyruvic acid

hydrolysis of enol ether side-chain oxidation of 3-hydroxyl to

ketone, then enolization

4-amino-4-deoxy-chorismic acid

2-amino-2-deoxy-isochorismic acid

Figure 4.4

2,3-dihydroxybenzoic acid, and salicylic acid(2-hydroxybenzoic acid) (in microorganisms, butnot in plants, see page 141), are derived fromchorismic acid via its isomer isochorismicacid (Figure 4.4). The isomerization involvesan SN2′-type of reaction, an incoming waternucleophile attacking the diene system anddisplacing the hydroxyl. Salicyclic acid arises byan elimination reaction analogous to that producing4-hydroxybenzoic acid from chorismic acid. Inthe formation of 2,3-dihydroxybenzoic acid, theside-chain of isochorismic acid is first lost byhydrolysis, then dehydrogenation of the 3-hydroxyto a 3-keto allows enolization and formationof the aromatic ring. 2,3-Dihydroxybenzoicacid is a component of the powerful ironchelator (siderophore) enterobactin (Figure 4.5)found in Escherichia coli and many otherGram-negative bacteria. Such compounds play animportant role in bacterial growth by making

available sufficient concentrations of essentialiron. Enterobactin comprises three molecules of2,3-dihydroxybenzoic acid and three of the aminoacid L-serine, in cyclic triester form.

Simple amino analogues of the phenolicacids are produced from chorismic acid byrelated transformations in which ammonia, gen-erated from glutamine, acts as a nucleophile(Figure 4.4). Chorismic acid can be aminated atC-4 to give 4-amino-4-deoxychorismic acid andthen p-aminobenzoic (4-aminobenzoic) acid, or atC-2 to give the isochorismic acid analogue whichwill yield 2-aminobenzoic (anthranilic) acid. Ami-nation at C-4 has been found to occur with reten-tion of configuration, so perhaps a double inver-sion mechanism is involved. p-Aminobenzoic acid(PABA) forms part of the structure of folic acid(vitamin B9)∗ (Figure 4.6). The folic acid struc-ture is built up (Figure 4.6) from a dihydropterindiphosphate which reacts with p-aminobenzoic


CO2H

OH

OH

ATP

COAMP

OH

OH O

O

O

O

O

O

NHO

NH

O

HN

O

OH

OH

HO

OH

OH

HO

O O

OO

O

NH

O

HNHN

OH

OH HOHO

HO HO

O OO

Fe3+

enterobactin

2,3-dihydroxy-benzoic acid

activation to AMP derivative, compare peptide formation, Figure 7.15

enterobactin as iron chelator

L-Ser

Figure 4.5

H2N

SO2NH2

N

N

N

HN

HN

CO2H

H2N

OH

N

N

N

HN

HN

HN

CO2H

H2N

OH

O

CO2H

H2N

CO2HPABA

NADPH

HN

HN

CO2HO

CO2H

N

N

N

NH2N

OH

NADPH

N

N

N

HN

OPP

H2N

OH

ATP

N

N

NH

HN

HN

HN

CO2H

H2N

OH

O

CO2H

HN

N

N

NH2N

O

N

N

N

HN

OH

H2N

OH

the pteridine system is sometimesdrawn as the tautomeric amide form:

SN2 reaction

reduction

a pteridine L-Glu

hydroxymethyl-dihydropterin

p-amino-benzoic acid

(PABA)

dihydrofolic acid(FH2)

L-GluATP

dihydrofolatereductase(DHFR)

reduction

sulphanilamide(acts as antimetabolite of PABA

and is enzyme inhibitor)

dihydropteroic acid

folic acid

tetrahydrofolic acid(FH4)

hydroxymethyl-dihydropterin PP

dihydrofolatereductase(DHFR)

Figure 4.6


acid to give dihydropteroic acid, an enzymicstep for which the sulphonamide antibiotics areinhibitors. Dihydrofolic acid is produced from thedihydropteroic acid by incorporating glutamic acid,and reduction yields tetrahydrofolic acid. Thisreduction step is also necessary for the continual

regeneration of tetrahydrofolic acid, and forms animportant site of action for some antibacterial, anti-malarial, and anticancer drugs.

Anthranilic acid (Figure 4.4) is an intermediatein the biosynthetic pathway to the indole-containingaromatic 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 maydestroy up to 90% of the vitamin. Deficiency gives rise to anaemia, and supplementation isoften necessary during pregnancy. Otherwise, deficiency is not normally encountered unlessthere is malabsorption, or chronic disease. Folic acid used for supplementation is usuallysynthetic, and it becomes sequentially reduced in the body by the enzyme dihydrofolatereductase to give dihydrofolic acid and then tetrahydrofolic acid (Figure 4.6). Tetrahydrofolicacid 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 groupsare involved in amino acid and nucleotide metabolism. Thus a methyl group is transferredin the regeneration of methionine from homocysteine, purine biosynthesis involves methenyland formyl transfer, and pyrimidine biosynthesis utilizes methylene transfer. Tetrahydrofolatederivatives 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 theuse of sulphanilamide and other antibacterial sulpha drugs, compounds which competitivelyinhibit dihydropteroate synthase, the biosynthetic enzyme incorporating p-aminobenzoic acidinto the structure. These sulpha drugs thus act as antimetabolites of p-aminobenzoate. Spe-cific dihydrofolate reductase inhibitors have also become especially useful as antibacterials,

N

N

NH

HNH2N

OH HN

HCO2HATP

FH4

ADP

Gly

N

N

NH

HNH2N

OH NH

O

H2O

N

N

N

HNH2N

OH HNH O

N

N

N

HNH2N

OH HNMe

N

N

N

HNH2N

OH NNAD+ NADH

NADPHNADP+

ATP

ADP

N

N

N

HNH2N

OH N

10

5

N5-formyl-FH4

(folinic acid)

N5,N10-methylene-FH4

N10-formyl-FH4

N5,N10-methenyl-FH4N5-methyl-FH4

L-Ser

6

Figure 4.7

(Continues )


(Continued )

N

N

N

N

NHN

CO2H

H2N

NH2

O

CO2H

Me

methotrexate

N

NH2N

NH2Cl

pyrimethamine

N

NH2N

NH2

OMe

OMe

OMe

trimethoprim

Figure 4.8

HN

N

O

O

deoxyribose-P

N

N

N

HNH2N

OH N

N

N

N

HNH2N

OH HN

HN

N

O

O

deoxyribose-P

FH2

NADPH

Gly

FH4

N

N

NH

HNH2N

OH HN

N5,N10-methylene-FH4 dUMP

L-Ser

dTMP

Figure 4.9

e.g. trimethoprim (Figure 4.8), and antimalarial drugs, e.g. pyrimethamine, relying onthe differences in susceptibility between the enzymes in humans and in the infectiveorganism. Anticancer agents based on folic acid, e.g. methotrexate (Figure 4.8), primarilyblock pyrimidine biosynthesis, but are less selective than the antimicrobial agents, andrely on a stronger binding to the enzyme than the natural substrate has. Regenerationof tetrahydrofolate from dihydrofolate is vital for DNA synthesis in rapidly proliferatingcells. The methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) requiresN5,N10-methylenetetrahydrofolate as the methyl donor, which is thereby transformedinto 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 likemethotrexate. The natural 6S isomer is termed levofolinic acid (levoleucovorin); folinic acidin drug use is usually a mixture of the 6R and 6S isomers.

In a sequence of complex reactions, which willnot be considered in detail, the indole ring sys-tem is formed by incorporating two carbons fromphosphoribosyl diphosphate, with loss of the orig-inal anthranilate carboxyl. The remaining ribosylcarbons are then removed by a reverse aldol reac-tion, to be replaced on a bound form of indoleby those from L-serine, which then becomes the

side-chain of L-tryptophan. Although a precursor ofL-tryptophan, anthranilic acid may also be producedby metabolism of tryptophan. Both compounds fea-ture as building blocks for a variety of alkaloidstructures (see Chapter 6).

Returning to the main course of the shikimatepathway, a singular rearrangement process occurstransforming chorismic acid into prephenic acid


CO2H

NH2

NH

CO2H

NH2

HOCO2H

NH2

CH2OP

PPO OH OHO

NH

CO2H

NH CH2OPP

HO OHO

H

NH

OP

OH

HO

NH

HOOP

CO2H OH

OHH

NH

HOOP

CO2H OH

OH

NH

OOP

OO OH

OH

H

anthranilicacid

L-Trp

phosphoribosylanthranilic acid

indole-3-glycerol P

– CO2– H2O

phosphoribosyl PP

L-Ser

PLP

SN2 reaction

enol−keto tautomerism

indole(enzyme-bound)

reverse aldol reaction

imine−enamine tautomerism

Figure 4.10

OH

CO2H

HO2C

OCO2H

OH

O

HO2C

CO2H

OH

O

HO2C

Claisen rearrangement

prephenic acidchorismic acid(pseudoaxialconformer)

chorismic acid(pseudoequatorial

conformer)

Figure 4.11

(Figure 4.11). This reaction, a Claisen rearrange-ment, transfers the PEP-derived side-chain so thatit becomes directly bonded to the carbocycle, andso builds up the basic carbon skeleton of phenylala-nine and tyrosine. The reaction is catalysed in natureby the enzyme chorismate mutase, and, although itcan also occur thermally, the rate increases some106-fold in the presence of the enzyme. The enzymeachieves this by binding the pseudoaxial conformerof chorismic acid, allowing a transition state withchairlike geometry to develop.

Pathways to the aromatic amino acids L-pheny-lalanine and L-tyrosine via prephenic acid mayvary according to the organism, and often morethan one route may operate in a particular speciesaccording to the enzyme activities that are avail-able (Figure 4.12). In essence, only three reac-tions are involved, decarboxylative aromatization,

transamination, and in the case of tyrosinebiosynthesis an oxidation, but the order in whichthese reactions occur differentiates the routes.Decarboxylative aromatization of prephenic acidyields phenylpyruvic acid, and PLP-dependenttransamination leads to L-phenylalanine. In thepresence of an NAD+-dependent dehydrogenaseenzyme, decarboxylative aromatization occurswith retention of the hydroxyl function, thoughas yet there is no evidence that any inter-mediate carbonyl analogue of prephenic acidis involved. Transamination of the resultant4-hydroxyphenylpyruvic acid subsequently givesL-tyrosine. L-Arogenic acid is the result oftransamination of prephenic acid occurring priorto the decarboxylative aromatization, and canbe transformed into both L-phenylalanine andL-tyrosine depending on the absence or presence


of a suitable enzymic dehydrogenase activity.In some organisms, broad activity enzymesare known to be capable of accepting bothprephenic acid and arogenic acid as substrates.In microorganisms and plants, L-phenylalanineand L-tyrosine tend to be synthesized separatelyas in Figure 4.12, but in animals, which lackthe shikimate pathway, direct hydroxylation ofL-phenylalanine to L-tyrosine, and of L-tyrosineto L-DOPA (dihydroxyphenylalanine), may beachieved (Figure 4.13). These reactions are catal-ysed by tetrahydropterin-dependent hydroxylaseenzymes, the hydroxyl oxygen being derivedfrom molecular oxygen. L-DOPA is a precursorof the catecholamines, e.g. the neurotransmitternoradrenaline and the hormone adrenaline (seepage 316). Tyrosine and DOPA are also converted

by oxidation reactions into a heterogeneouspolymer melanin, the main pigment in mammalianskin, hair, and eyes. In this material, the indolesystem is not formed from tryptophan, but arisesfrom DOPA by cyclization of DOPAquinone, thenitrogen of the side-chain then attacking the ortho-quinone (Figure 4.13).

Some organisms are capable of synthesizing anunusual variant of L-phenylalanine, the aminatedderivative L-p-aminophenylalanine (L-PAPA) (Fig-ure 4.14). This is known to occur by a series ofreactions paralleling those in Figure 4.12, but uti-lizing the PABA precursor 4-amino-4-deoxychori-smic acid (Figure 4.4) instead of chorismic acid.Thus, amino derivatives of prephenic acid and py-ruvic acid are elaborated. One important metaboliteknown to be formed from L-PAPA is the antibiotic

CO2H

O

CO2H

O

OH

C

OH

O

OH

CO2H

O

CO2H

OH

O CO2H

NAD+

H

C

OH

O

OH

CO2H

NH2

CO2H

NH2

OH

CO2H

NH2

NAD+an additional oxidation step (of alcohol to ketone) means OH is retained on decarboxylationand aromatization; no discrete ketone intermediate is formed

chorismic acid prephenic acid

phenylpyruvic acid L-Phe

L-arogenic acid

4-hydroxyphenyl-pyruvic acid

L-Tyr

PLP

PLP

PLP

oxidation means OH isretained on decarboxylationand aromatization; no discreteketone intermediate is formed

decarboxylation,aromatization andloss of leaving group

transamination:keto acid amino acid

decarboxylation,aromatization andloss of leaving group

Figure 4.12


CO2H

NH2

CO2H

NH2HO

CO2H

NH2HO

HO

NH

CO2H

HO

HO

NCO2H

HO

OO

CO2H

NH2O

O

L-DOPAL-Tyr

DOPAchrome DOPAquinone

L-Phe

CATECHOLAMINESnoradrenaline,adrenaline

MELANINS

nucleophilic attack on to enone

O2tetrahydro-biopterin

O2tetrahydro-biopterin

Figure 4.13

chloramphenicol∗, produced by cultures of Strep-tomyces venezuelae. The late stages of the path-way (Figure 4.14) have been formulated to involvehydroxylation and N-acylation in the side-chain,the latter reaction probably requiring a coenzymeA ester of dichloroacetic acid. Following reductionof the carboxyl group, the final reaction is oxida-tion of the 4-amino group to a nitro, a fairly raresubstituent in natural product structures.

CINNAMIC ACIDS

L-Phenylalanine and L-tyrosine, as C6C3 build-ing blocks, are precursors for a wide range of

natural products. In plants, a frequent first step isthe elimination of ammonia from the side-chainto generate the appropriate trans (E ) cinnamicacid. In the case of phenylalanine, this wouldgive cinnamic acid, whilst tyrosine could yield4-coumaric acid (p-coumaric acid) (Figure 4.15).All plants appear to have the ability to deami-nate phenylalanine via the enzyme phenylalanineammonia lyase (PAL), but the corresponding trans-formation of tyrosine is more restricted, beingmainly limited to members of the grass family (theGraminae/Poaceae). Whether a separate enzymetyrosine ammonia lyase (TAL) exists, or whethergrasses merely have a broad specificity PAL also

Chloramphenicol

Chloramphenicol (chloromycetin) (Figure 4.14) was initially isolated from cultures ofStreptomyces venezuelae, but is now obtained for drug use by chemical synthesis. Itwas one of the first broad spectrum antibiotics to be developed, and exerts its antibacterialaction by inhibiting protein biosynthesis. It binds reversibly to the 50S subunit of thebacterial ribosome, and in so doing disrupts peptidyl transferase, the enzyme that catalysespeptide bond formation (see page 408). This reversible binding means that bacterial cells notdestroyed may resume protein biosynthesis when no longer exposed to the antibiotic. Somemicroorganisms have developed resistance to chloramphenicol by an inactivation processinvolving enzymic acetylation of the primary alcohol group in the antibiotic. The acetate bindsonly very weakly to the ribosomes, so has little antibiotic activity. The value of chloramphenicolas an antibacterial agent has been severely limited by some serious side-effects. It can causeblood disorders including irreversible aplastic anaemia in certain individuals, and these canlead to leukaemia and perhaps prove fatal. Nevertheless, it is still the drug of choice forsome life-threatening infections such as typhoid fever and bacterial meningitis. The bloodconstitution must be monitored regularly during treatment to detect any abnormalities oradverse changes. The drug is orally active, but may also be injected. Eye-drops are useful forthe treatment of bacterial conjunctivitis.

CINNAMIC ACIDS 131

CO2H

O

NH2

CO2H

NH2

NH2

CHCl2COSCoA

NH2

HO2C

CO2HO

CO2H

NH2

HONHCOCHCl2

O CO2H

NH2

CO2H

CH2OH

NO2

HONHCOCHCl2

4-amino-4-deoxy-chorismic acid PLP

chloramphenicol L-p-aminophenylalanineL-PAPA

Claisen rearrangement

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

transamination

hydroxylationN-acylation

reduction of CO2H to CH2OH; oxidation of NH2 to NO2

Figure 4.14

CO2H

NH2

CO2H

NH2

OH

CO2H

O2NADPH

CO2H

OH

O2NADPH

CO2H

OHHO

CH2OH

OH

SAM

CO2H

OHMeO

O2NADPH

CO2H

OHMeO OH

CH2OH

OHMeO

SAM

CO2H

OHMeO OMe

CH2OH

OHMeO OMe

L-Phe

L-Tyr

cinnamic acid

4-coumaric acid(p-coumaric acid)

caffeic acid ferulic acid sinapic acid

4-hydroxycinnamyl alcohol(p-coumaryl alcohol)

coniferyl alcohol sinapyl alcohol

PAL

LIGNIN

E2 elimination of ammonia

x n

LIGNANS

x 2POLYMERS

sequence of hydroxylation and methylation reactionshydroxylation

Figure 4.15


CO2H

OH

OHO

OH

OH

O

OH

OHOHO

OH

OH

O

O

Me3NO

O

OH

OMe

OMe

chlorogenic acid(5-O-caffeoylquinic acid)

1-O-cinnamoylglucose

sinapine

Figure 4.16

capable of deaminating tyrosine, is still debated.Those species that do not transform tyrosine syn-thesize 4-coumaric acid by direct hydroxylation ofcinnamic acid, in a cytochrome P-450-dependentreaction, and tyrosine is often channelled insteadinto other secondary metabolites, e.g. alkaloids.Other cinnamic acids are obtained by furtherhydroxylation and methylation reactions, sequen-tially building up substitution patterns typical ofshikimate pathway metabolites, i.e. an ortho oxy-genation pattern (see page 123). Some of the morecommon natural cinnamic acids are 4-coumaric,caffeic, ferulic, and sinapic acids (Figure 4.15).These can be found in plants in free form and ina range of esterified forms, e.g. with quinic acidas in chlorogenic acid (5-O-caffeoylquinic acid)(see coffee, page 395), with glucose as in 1-O-cinnamoylglucose, and with choline as in sinapine(Figure 4.16).

LIGNANS AND LIGNIN

The cinnamic acids also feature in the pathways toother metabolites based on C6C3 building blocks.

Pre-eminent amongst these, certainly as far asnature is concerned, is the plant polymer lignin,a strengthening material for the plant cell wallwhich acts as a matrix for cellulose microfibrils(see page 473). Lignin represents a vast reservoirof aromatic materials, mainly untapped becauseof the difficulties associated with release of thesemetabolites. The action of wood-rotting fungioffers the most effective way of making these use-ful products more accessible. Lignin is formedby phenolic oxidative coupling of hydroxycin-namyl alcohol monomers, brought about by perox-idase enzymes (see page 28). The most importantof these monomers are 4-hydroxycinnamyl alco-hol (p-coumaryl alcohol), coniferyl alcohol, andsinapyl alcohol (Figure 4.15), though the mono-mers used vary according to the plant type.Gymnosperms polymerize mainly coniferyl alco-hol, dicotyledonous plants coniferyl alcohol andsinapyl alcohol, whilst monocotyledons use allthree alcohols. The alcohols are derived by reduc-tion of cinnamic acids via coenzyme A esters andaldehydes (Figure 4.17), though the substitutionpatterns are not necessarily elaborated completelyat the cinnamic acid stage, and coenzyme A estersand aldehydes may also be substrates for aromatichydroxylation and methylation. Formation of thecoenzyme A ester facilitates the first reduction stepby introducing a better leaving group (CoAS−)for the NADPH-dependent reaction. The secondreduction step, aldehyde to alcohol, utilizes a fur-ther molecule of NADPH and is reversible. Theperoxidase enzyme then achieves one-electron oxi-dation of the phenol group. One-electron oxidationof a simple phenol allows delocalization of theunpaired electron, giving resonance forms inwhich the free electron resides at positions orthoand para to the oxygen function (see page 29).With cinnamic acid derivatives, conjugation allowsthe unpaired electron to be delocalized also intothe side-chain (Figure 4.18). Radical pairing ofresonance structures can then provide a range ofdimeric systems containing reactive quinoneme-thides, which are susceptible to nucleophilic attackfrom hydroxyl groups in the same system,or by external water molecules. Thus, coniferylalcohol monomers can couple, generating linkagesas exemplified by guaiacylglycerol β-coniferylether (β-arylether linkage), dehydrodiconi-feryl alcohol (phenylcoumaran linkage), and

LIGNANS AND LIGNIN 133

CO2H

OH

R1 R2

HSCoA

COSCoA

OH

R1 R2

NADPH

CHO

OH

R1 R2

NADPH

CH2OH

OH

R1 R2

Figure 4.17

OH

MeO

OH

O

MeO

OH

O

MeO

OH

O

MeO

OH

OH

OMe

O

HO

OMe

O

OH

OMe

O

HO

OMe

OH

HO

OH

OMe

O

HO

OMe

O

H

OH

OMe

OH

HO

OMe

O

OH

OMe

O

HO

OMe

HO

A B C

H2O

H H

H

O

MeO

OH

O

OMe

HO

O

O

OH

OMe

HO

MeO

O

MeO

OH

D

H

– e

– H+

A + D B + D D + D

pinoresinol(resinol linkage)

dehydrodiconiferyl alcoholguaiacylglycerolβ-coniferyl ether

coniferyl alcohol

one-electron oxidation

resonance forms of free radical

radical pairing

radical pairing

radical pairing

enolization

nucleophilic attack of water on to quinonemethide

nucleophilic attack of hydroxyls on to quinonemethides

nucleophilic attack of hydroxyl on to quinonemethide

(β-arylether linkage) (phenylcoumaran linkage)

Figure 4.18


OH

MeO

OH

O

O

O

OMe

MeO OMe

O

O

O

O

OMe

MeO OMe

O

O

O

OH

OMe

HO

MeOHH

O

O

MeO

HO

OH

MeO

O

O

O

OMe

MeO OMe

O

NADPH

O

HO

MeOHH

NAD+

H

O

O

O

OMe

MeO OMe

O

OH

OH

OH

MeO

HO

OH

MeO

OH

HO

OH

OMe

HO

MeOHH

OH

HO

MeOHH

H

matairesinol

desoxypodophyllotoxin

(–)-secoisolariciresinolyatein

(+)-pinoresinol

podophyllotoxin

phenolic oxidative coupling

oxidation of one CH2OH to CO2H, then lactone ring formation

coniferyl alcohol

modification of aromatic substitution patterns

nucleophilic attack on to quinonemethide system

hydroxylation

≡

(–)-secoisolariciresinol

this step probably involves ring opening to the quinonemethide followed by reduction

Figure 4.19

OGlc

OGlc

MeO

HO

OH

MeO

OH

OH

HO

HO

O

O

HO

HO

secoisolariciresinol diglucoside

intestinal bacteria

enterolactoneenterodiol

Figure 4.20

LIGNANS AND LIGNIN 135

pinoresinol (resinol linkage). These dimers canreact further by similar mechanisms to produce alignin polymer containing a heterogeneous seriesof inter-molecular bondings as seen in the var-ious dimers. In contrast to most other naturalpolymeric materials, lignin appears to be devoidof ordered repeating units, though some 50–70%of the linkages are of the β-arylether type. Thedimeric materials are also found in nature and arecalled lignans. Some authorities like to restrictthe term lignan specifically to molecules in whichthe two phenylpropane units are coupled at thecentral carbon of the side-chain, e.g. pinoresinol,whilst compounds containing other types of cou-pling, e.g. as in guaiacylglycerol β-coniferyl etherand dehydrodiconiferyl alcohol, are then referredto as neolignans. Lignan/neolignan formationand lignin biosynthesis are catalysed by differ-ent enzymes, and a consequence is that naturallignans/neolignans are normally enantiomericallypure because they arise from stereochemicallycontrolled coupling. The control mechanisms forlignin biosynthesis are less well defined, but theenzymes appear to generate products lacking opti-cal activity.

Further cyclization and other modifications cancreate a wide range of lignans of very differ-ent structural types. One of the most importantof the natural lignans having useful biologi-cal activity is the aryltetralin lactone podophyl-lotoxin (Figure 4.19), which is derived fromconiferyl alcohol via the dibenzylbutyrolactonesmatairesinol and yatein, cyclization probablyoccurring as shown in Figure 4.19. Matairesinol

is known to arise by reductive opening of thefuran rings of pinoresinol, followed by oxida-tion of a primary alcohol to the acid and thenlactonization. The substitution pattern in the twoaromatic rings is built up further during the path-way, i.e. matairesinol → yatein, and does notarise by initial coupling of two different cinnamylalcohol residues. The methylenedioxy ring sys-tem, as found in many shikimate-derived naturalproducts, is formed by an oxidative reaction onan ortho-hydroxymethoxy pattern (see page 27).Podophyllotoxin and related lignans are foundin the roots of Podophyllum∗ species (Berberi-daceae), and have clinically useful cytotoxic andanticancer activity. The lignans enterolactoneand enterodiol (Figure 4.20) were discovered inhuman urine, but were subsequently shown tobe derived from dietary plant lignans, especiallysecoisolariciresinol diglucoside, by the action ofintestinal microflora. Enterolactone and enterodiolhave oestrogenic activity and have been impli-cated as contributing to lower levels of breastcancer amongst vegetarians (see phyto-oestrogens,page 156).

PHENYLPROPENES

The reductive sequence from an appropriate cin-namic acid to the corresponding cinnamyl alcoholis not restricted to lignin and lignan biosynthesis,and is utilized for the production of variousphenylpropene derivatives. Thus cinnamaldehyde(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, andthe Himalayas and yields Indian podophyllum, whilst P. peltatum (May apple or Americanmandrake) comes from North America and is the source of American podophyllum. Plants arecollected from the wild. Both plants are large-leafed perennial herbs with edible fruits, thoughother 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 theactive principles is obtained by pouring an ethanolic extract of the root into water, and dryingthe precipitated podophyllum resin or ‘podophyllin’. Indian podophyllum yields about 6–12%of resin containing 50–60% lignans, and American podophyllum 2–8% of resin containing14–18% lignans.

(Continues )


(Continued )

O

O

O

OH

MeO OMe

O

OS

O

OH

OO

HO

O

O

O

OH

MeO OMe

O

OH

O

O

O

OR

MeO OMe

O

O

O

OH

OO

HO

4′

R = H, etoposideR = P, etopophos

teniposide4′-demethylepipodophyllotoxin

O

O

O

OR

MeO OMe

O

OH

O

O

O

OR

MeO OMe

O

OH

O

O

O

OMe

MeO OMe

OO

O

O

OMe

MeO OMe

O

O

4′

R = Me, β-peltatinR = H, α-peltatin

desoxypodophyllotoxin podophyllotoxoneR = Me, podophyllotoxinR = H, 4′-demethylpodophyllotoxin

Figure 4.21

The lignan constituents of the two roots are the same, but the proportions are markedlydifferent. The Indian root contains chiefly podophyllotoxin (Figure 4.21) (about 4%) and 4′-demethylpodophyllotoxin (about 0.45%). The main components in the American root arepodophyllotoxin (about 0.25%), β-peltatin (about 0.33%) and α-peltatin (about 0.25%).Desoxypodophyllotoxin and podophyllotoxone are also present in both plants, as arethe glucosides of podophyllotoxin, 4′-demethylpodophyllotoxin, and the peltatins, thoughpreparation 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 cytotoxicproperties of podophyllotoxin and related compounds has now made podophyllum acommercially important drug plant. Preparations of podophyllum resin (the Indian resinis preferred) are effective treatments for warts, and pure podophyllotoxin is available as apaint for venereal warts, a condition which can be sexually transmitted. The antimitotic effect ofpodophyllotoxin 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 ofthese 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 asanticancer agents due to toxic side-effects, but the semi-synthetic derivatives etoposide andteniposide (Figure 4.21), which are manufactured from natural podophyllotoxin, have provedexcellent antitumour agents. They were developed as modified forms (acetals) of the natural

(Continues )

PHENYLPROPENES 137

(Continued )

OMe

MeO

O

O

OMe

O

O

OH

HNaOAc

B

OMe

MeO

O

O

OMe

O

O

OH

HB

OMe

MeO

O

O

OMe

O

O

OH

H

podophyllotoxin picropodophyllin

base removes acidicproton α to carbonyl and generates enolate anion

reformation of keto form resultsin change of stereochemistry

Figure 4.22

4′-demethylpodophyllotoxin glucoside. Attempted synthesis of the glucoside inverted thestereochemistry at the sugar–aglycone linkage, and these agents are thus derivatives of 4′-demethylepipodophyllotoxin (Figure 4.21). Etoposide is a very effective anticancer agent, andis used in the treatment of small cell lung cancer, testicular cancer and lymphomas, usually incombination therapies with other anticancer drugs. It may be given orally or intravenously. Thewater-soluble pro-drug etopophos (etoposide 4′-phosphate) is also available. Teniposidehas similar anticancer properties, and, though not as widely used as etoposide, has value inpaediatric neuroblastoma.

Remarkably, the 4′-demethylepipodophyllotoxin series of lignans do not act via a tubulin-binding mechanism as does podophyllotoxin. Instead, these drugs inhibit the enzymetopoisomerase II, thus preventing DNA synthesis and replication. Topoisomerases areresponsible 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 toinhibit strand-rejoining ability by stabilizing the topoisomerase II–DNA complex in a cleavagestate, leading to double-strand breaks and cell death. Development of other topoisomeraseinhibitors based on podophyllotoxin-related lignans is an active area of research. Biologicalactivity 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 strainis markedly reduced in the corresponding cis-fused system, and the natural compoundsare easily and rapidly converted into these cis-fused lactones by treatment with very mildbases, via enol tautomers or enolate anions (Figure 4.22). Picropodophyllin is almost devoidof cytotoxic properties.

Podophyllotoxin is also found in significant amounts in the roots of other Podophyllumspecies, and in closely related genera such as Diphylleia (Berberidaceae).

oil from the bark of cinnamon (Cinnamomumzeylanicum; Lauraceae), widely used as a spiceand flavouring. Fresh bark is known to containhigh levels of cinnamyl acetate, and cinnamalde-hyde is released from this by fermentation pro-cesses which are part of commercial preparation ofthe bark, presumably by enzymic hydrolysis andparticipation of the reversible aldehyde–alcoholoxidoreductase. Cinnamon leaf, on the other hand,

contains large amounts of eugenol (Figure 4.23)and much smaller amounts of cinnamaldehyde.Eugenol is also the principal constituent in oilfrom cloves (Syzygium aromaticum; Myrtaceae),used for many years as a dental anaesthetic, aswell as for flavouring. The side-chain of eugenolis derived from that of the cinnamyl alcohols byreduction, but differs in the location of the doublebond. This change is accounted for by resonance


OMe

MeO OMe

elemicin

OOMeO

myristicin

OH

MeO

eugenol

OMe

estragole(methylchavicol)

OMe

anethole

OCOCH3

cinnamyl acetate

CHO

cinnamaldehyde

Figure 4.23

Ar OH

H

Ar

Ar

NADPH

H H

Ar

Ar

NADPHOMe OH

OMe

Ar =

cinnamyl alcohol

loss of hydroxyl as leaving group

resonance-stabilized allylic cation

propenylphenol allylphenol

etc

Figure 4.24

forms of the allylic cation (Figure 4.24), and addi-tion of hydride (from NADPH) can generate eitherallylphenols, e.g. eugenol, or propenylphenols, e.g.anethole (Figure 4.23). Loss of hydroxyl froma cinnamyl alcohol may be facilitated by proto-nation, or perhaps even phosphorylation, thoughthere is no evidence for the latter. Myristicin(Figure 4.23) from nutmeg (Myristica fragrans;Myristicaceae) is a further example of an allylphe-nol found in flavouring materials. Myristicin alsohas a history of being employed as a mild hallu-cinogen via ingestion of ground nutmeg. Myris-ticin is probably metabolized in the body viaan amination reaction to give an amfetamine-like derivative (see page 385). Anethole is themain component in oils from aniseed (Pimpinellaanisum; Umbelliferae/Apiaceae), star anise (Illi-cium verum; Illiciaceae), and fennel (Foeniculumvulgare; Umbelliferae/Apiaceae). The propenylcomponents of flavouring materials such as cin-namon, star anise, nutmeg, and sassafras (Sas-safras albidum; Lauraceae) have reduced theircommercial use somewhat since these constituents

have been shown to be weak carcinogens inlaboratory tests on animals. In the case of saf-role (Figure 4.25), the main component of sas-safras oil, this has been shown to arise fromhydroxylation in the side-chain followed by sul-phation, giving an agent which binds to cel-lular macromolecules. Further data on volatileoils containing aromatic constituents isolated fromthese and other plant materials are given inTable 4.1. Volatile oils in which the main compo-nents are terpenoid in nature are listed in Table 5.1,page 177.

OO

OO

HO

OO

HO3SO

safrole

Figure 4.25

Tab

le4.

1V

olat

ileoi

lsco

ntai

ning

prin

cipa

llyar

omat

icco

mpo

unds

Vol

atile

ores

sent

ial

oils

are

usua

llyob

tain

edfr

omth

eap

prop

riat

epl

ant

mat

eria

lby

stea

mdi

still

atio

n,th

ough

ifce

rtai

nco

mpo

nent

sar

eun

stab

leat

thes

ete

mpe

ratu

res,

othe

rle

ssha

rsh

tech

niqu

essu

chas

expr

essi

onor

solv

ent

extr

actio

nm

aybe

empl

oyed

.T

hese

oils

,w

hich

typi

cally

cont

ain

aco

mpl

exm

ixtu

reof

low

boili

ngco

mpo

nent

s,ar

ew

idel

yus

edin

flavo

urin

g,pe

rfum

ery,

and

arom

athe

rapy

.O

nly

asm

all

num

ber

ofoi

lsha

veus

eful

ther

apeu

ticpr

oper

ties,

e.g.

clov

ean

ddi

ll,th

ough

aw

ide

rang

eof

oils

isno

wex

ploi

ted

for

arom

athe

rapy

.M

ost

ofth

ose

empl

oyed

inm

edic

ines

are

sim

ply

adde

dfo

rfla

vour

ing

purp

oses

.So

me

ofth

em

ater

ials

are

com

mer

cial

lyim

port

ant

asso

urce

sof

chem

ical

sus

edin

dust

rial

ly,

e.g.

turp

entin

e.

For

conv

enie

nce,

the

maj

oroi

lslis

ted

are

divi

ded

into

two

grou

ps.

Tho

sew

hich

cont

ain

prin

cipa

llych

emic

als

whi

char

ear

omat

icin

natu

rean

dw

hich

are

deri

ved

byth

esh

ikim

ate

path

way

are

give

nin

Tabl

e4.

1be

low

.Tho

seoi

lsw

hich

are

com

pose

dpr

edom

inan

tlyof

terp

enoi

dco

mpo

unds

are

liste

din

Tabl

e5.

1on

page

177,

sinc

eth

eyar

ede

rive

dvi

ath

ede

oxyx

ylul

ose

phos

phat

epa

thw

ay.

Itm

ust

beap

prec

iate

dth

atm

any

oils

may

cont

ain

arom

atic

and

terp

enoi

dco

mpo

nent

s,bu

tus

ually

one

grou

ppr

edom

inat

es.

The

oil

yiel

ds,

and

the

exac

tco

mpo

sitio

nof

any

sam

ple

ofoi

lw

illbe

vari

able

,de

pend

ing

onth

epa

rtic

ular

plan

tm

ater

ial

used

inits

prep

arat

ion.

The

qual

ityof

anoi

lan

dits

com

mer

cial

valu

eis

depe

nden

ton

the

prop

ortio

nof

the

vari

ous

com

pone

nts.

Oil

Plan

tso

urce

Plan

tpa

rtO

ilM

ajor

cons

titue

nts

with

Use

s,no

tes

used

cont

ent

(%)

typi

cal

(%)

com

posi

tion

Ani

seed

Pim

pine

lla

anis

umri

pefr

uit

2–

3an

etho

le(8

0–

90)

flavo

ur,

carm

inat

ive,

(Ani

se)

(Um

belli

fera

e/es

trag

ole

(1–

6)ar

omat

hera

pyA

piac

eae)

Star

anis

eIl

lici

umve

rum

ripe

frui

t5

–8

anet

hole

(80

–90

)fla

vour

,ca

rmin

ativ

e(I

llici

acea

e)es

trag

ole

(1–

6)fr

uits

cont

ain

subs

tant

ial

amou

nts

ofsh

ikim

ican

dqu

inic

acid

s

Cas

sia

Cin

nam

omum

cass

iadr

ied

bark

,1

–2

cinn

amal

dehy

de(7

0–

90)

flavo

ur,

carm

inat

ive

(Lau

race

ae)

orle

aves

2-m

etho

xyci

nnam

al-

and

twig

sde

hyde

(12)

know

nas

cinn

amon

oil

inU

SA

Cin

nam

onba

rkC

inna

mom

umdr

ied

bark

1–

2ci

nnam

alde

hyde

(70

–80

)fla

vour

,ca

rmin

ativ

e,ze

ylan

icum

euge

nol

(1–

13)

arom

athe

rapy

(Lau

race

ae)

cinn

amyl

acet

ate

(3–

4)

Cin

nam

onle

afC

inna

mom

umle

af0.

5–

0.7

euge

nol

(70

–95

)fla

vour

zeyl

anic

um(L

aura

ceae

)

(Con

tinu

edov

erle

af)

Tab

le4.

1(C

onti

nued

)

Oil

Plan

tso

urce

Plan

tpa

rtO

ilM

ajor

cons

titue

nts

with

Use

s,no

tes

used

cont

ent

typi

cal

(%)

com

posi

tion

(%)

Clo

veSy

zygi

umar

omat

icum

drie

dflo

wer

15–

20eu

geno

l(7

5–

90)

flavo

ur,

arom

athe

rapy

,an

tisep

tic(E

ugen

iaca

ryop

hyll

us)

buds

euge

nyl

acet

ate

(10

–15

)(M

yrta

ceae

)β-c

aryo

phyl

lene

(3)

Fenn

elF

oeni

culu

mvu

lgar

eri

pefr

uit

2–

5an

etho

le(5

0–

70)

flavo

ur,

carm

inat

ive,

arom

athe

rapy

(Um

belli

fera

e/fe

ncho

ne(1

0–

20)

Api

acea

e)es

trag

ole

(3–

20)

Nut

meg

Myr

isti

cafr

agra

nsse

ed5

–16

sabi

nene

(17

–28

)fla

vour

,ca

rmin

ativ

e,ar

omat

hera

py(M

yris

ticac

eae)

α-p

inen

e(1

4–

22)

β-p

inen

e(9

–15

)al

thou

ghth

em

ain

cons

titu

ents

are

terp

inen

-4-o

l(6

–9)

terp

enoi

ds,

mos

tof

the

flavo

urm

yris

ticin

(4–

8)co

mes

from

the

min

orar

omat

icel

emic

in(2

)co

nstit

uent

s,m

yris

ticin

,el

emic

in,

etc

myr

isti

cin

isha

lluc

inog

enic

(see

page

385)

Win

terg

reen

Gau

lthe

ria

proc

umbe

nsle

aves

0.7

–1.

5m

ethy

lsa

licyl

ate

(98%

)fla

vour

,an

tisep

tic,

antir

heum

atic

(Eri

caca

e)or

Bet

ula

lent

aba

rk0.

2–

0.6

prio

rto

dist

illat

ion,

plan

tm

ater

ial

(Bet

ulac

eae)

ism

acer

ated

with

wat

erto

allo

wen

zym

ichy

drol

ysis

ofgl

ycos

ides

met

hyl

salic

ylat

eis

now

prod

uced

synt

hetic

ally

BENZOIC ACIDS FROM C6C3 COMPOUNDS 141

BENZOIC ACIDS FROM C6C3

COMPOUNDS

Some of the simple hydroxybenzoic acids (C6C1

compounds) such as 4-hydroxybenzoic acid andgallic acid can be formed directly from inter-mediates early in the shikimate pathway, e.g.3-dehydroshikimic acid or chorismic acid (seepage 121), but alternative routes exist in whichcinnamic acid derivatives (C6C3 compounds) arecleaved at the double bond and lose two car-bons from the side-chain. Thus, 4-coumaric acidmay act as a precursor of 4-hydroxybenzoicacid, and ferulic acid may give vanillic acid(4-hydroxy-3-methoxybenzoic acid) (Figure 4.26).A sequence analogous to that involved in theβ-oxidation of fatty acids (see page 18) is possi-ble, so that the double bond in the coenzyme Aester would be hydrated, the hydroxyl group oxi-dized to a ketone, and the β-ketoester would thenlose acetyl-CoA by a reverse Claisen reaction, giv-ing the coenzyme A ester of 4-hydroxybenzoicacid. Whilst this sequence has been generallyaccepted, newer evidence supports another side-chain cleavage mechanism, which is different fromthe fatty acid β-oxidation pathway (Figure 4.26).

Coenzyme A esters are not involved, and thougha similar hydration of the double bond occurs,chain shortening features a reverse aldol reac-tion, generating the appropriate aromatic alde-hyde. The corresponding acid is then formedvia an NAD+-dependent oxidation step. Thus,aromatic aldehydes such as vanillin, the mainflavour compound in vanilla (pods of the orchidVanilla planiflora; Orchidaceae) would be formedfrom the correspondingly substituted cinnamic acidwithout proceeding through intermediate benzoicacids or esters. Whilst the substitution patternin these C6C1 derivatives is generally built upat the C6C3 cinnamic acid stage, prior to chainshortening, there exists the possibility of furtherhydroxylations and/or methylations occurring atthe C6C1 level, and this is known in certainexamples. Salicylic acid (Figure 4.27) is synthe-sized in microorganisms directly from isochorismicacid (see page 124), but can arise in plants by twoother mechanisms. It can be produced by hydrox-ylation of benzoic acid, or by side-chain cleavageof 2-coumaric acid, which itself is formed by anortho-hydroxylation of cinnamic acid. Methyl sal-icylate is the principal component of oil of win-tergreen from Gaultheria procumbens (Ericaceae),used for many years for pain relief. It is derived by

HSCoAATP

CO2H

OH

R

COSCoA

OH

R

COSCoA

OH

HO

R

COSCoA

OH

O

R

H2O NAD+

HSCoAH2O

CH3COSCoA

CO2H

OH

HO

R

NAD+

CHO

OH

R

CO2H

OH

R

COSCoA

OH

R

R = H, 4-coumaroyl-CoAR = OMe, feruloyl-CoA

reversealdol

reverseClaisen

R = H, 4-coumaric acidR = OMe, ferulic acid

β-oxidation pathway, as in fatty acid metabolism (Figure 2.11)

R = H, 4-hydroxy- benzaldehydeR = OMe, vanillin

R = H, 4-hydroxybenzoic acidR = OMe, vanillic acid

Figure 4.26


CO2H

CO2H CO2H

OH

CO2H

OH SAM

CO2Me

OH

CHO

OH

CHO

OGlc

CO2H

OCOCH3

CH2OH

OGlc

salicylaldehyde

reduction

salicin2-coumaric acid

hydroxylation side-chain cleavage

glucosylation

salicylic acid methyl salicylate

hydroxylation

side-chain cleavage

benzoic acid

UDPGlc

aspirin(acetylsalicyclic acid)

methylation

Figure 4.27

SAM-dependent methylation of salicylic acid. Thesalicyl alcohol derivative salicin, found in manyspecies of willow (Salix species; Salicaceae), is notderived from salicylic acid, but probably via gluco-sylation of salicylaldehyde and then reduction ofthe carbonyl (Figure 4.27). Salicin is responsiblefor the analgesic and antipyretic effects of wil-low barks, widely used for centuries, and the tem-plate for synthesis of acetylsalicylic acid (aspirin)(Figure 4.27) as a more effective analogue.

COUMARINS

The hydroxylation of cinnamic acids ortho to theside-chain as seen in the biosynthesis of salicylicacid is a crucial step in the formation of a group ofcinnamic acid lactone derivatives, the coumarins.Whilst the direct hydroxylation of the aromaticring of the cinnamic acids is common, hydrox-ylation generally involves initially the 4-positionpara to the side-chain, and subsequent hydrox-ylations then proceed ortho to this substituent(see page 132). In contrast, for the coumarins,hydroxylation of cinnamic acid or 4-coumaric acidcan occur ortho to the side-chain (Figure 4.28).In the latter case, the 2,4-dihydroxycinnamic acidproduced confusingly seems to possess the metahydroxylation pattern characteristic of phenolsderived via the acetate pathway. Recognition ofthe C6C3 skeleton should help to avoid this confu-sion. The two 2-hydroxycinnamic acids then suffera change in configuration in the side-chain, from

the trans (E) to the less stable cis (Z) form. Whilsttrans–cis isomerization would be unfavourablein the case of a single isolated double bond, inthe cinnamic acids the fully conjugated systemallows this process to occur quite readily, andUV irradiation, e.g. daylight, is sufficient to pro-duce equilibrium mixtures which can be separated(Figure 4.29). The absorption of energy promotesan electron from the π-orbital to a higher energystate, the π∗-orbital, thus temporarily destroyingthe double bond character and allowing rotation.Loss of the absorbed energy then results in re-formation of the double bond, but in the cis-configuration. In conjugated systems, the π–π∗energy difference is considerably less than with anon-conjugated double bond. Chemical lactoniza-tion can occur on treatment with acid. Both thetrans–cis isomerization and the lactonization areenzyme-mediated in nature, and light is not nec-essary for coumarin biosynthesis. Thus, cinnamicacid and 4-coumaric acid give rise to the coumarinscoumarin and umbelliferone (Figure 4.28). Othercoumarins with additional oxygen substituents onthe aromatic ring, e.g. aesculetin and scopoletin,appear to be derived by modification of umbellif-erone, rather than by a general cinnamic acid tocoumarin pathway. This indicates that the hydrox-ylation meta to the existing hydroxyl, discussedabove, is a rather uncommon occurrence.

Coumarins are widely distributed in plants,and are commonly found in families such as theUmbelliferae/Apiaceae and Rutaceae, both in thefree form and as glycosides. Coumarin itself is

COUMARINS 143

CO2H

HO

CO2H

O OGlcO

MeO

CO2H

HO OH

CO2H

OH

O OHO

MeO

CO2HOHHO

CO2HOH

O OHO

O OHO

HO

O O

aesculetin

umbelliferone

coumarin

scopoletinscopolin

cinnamic acid 2-coumaric acid

4-coumaric acid 2,4-dihydroxy-cinnamic acid

trans-cis isomerization lactone formation

Figure 4.28

CO2H

OHCO2H

OH

H

OH O O

coumarin

Ehν

Z

Figure 4.29

CO2HO

Glc

CO2H

OGlc

O O

(E)-2-coumaric acidglucoside

enzymichydrolysis

(Z)-2-coumaric acidglucoside

coumarin

Figure 4.30

found in sweet clover (Melilotus species; Legu-minosae/Fabaceae) and contributes to the smellof new-mown hay, though there is evidencethat the plants actually contain the glucosides of(E)- and (Z)-2-coumaric acid (Figure 4.30), andcoumarin is only liberated as a result of enzymic

hydrolysis and lactonization through damage tothe plant tissues during harvesting and process-ing (Figure 4.30). If sweet clover is allowed toferment, 4-hydroxycoumarin is produced by theaction of microorganisms on 2-coumaric acid(Figure 4.31) and this can react with formalde-hyde, which is usually present due to micro-bial degradative reactions, combining to givedicoumarol. Dicoumarol∗ is a compound with pro-nounced blood anticoagulant properties, which cancause the deaths of livestock by internal bleeding,and is the forerunner of the warfarin∗ group ofmedicinal anticoagulants.

Many other natural coumarins have a morecomplex carbon framework and incorporateextra carbons derived from an isoprene unit(Figure 4.33). The aromatic ring in umbelliferoneis activated at positions ortho to the hydroxylgroup and can thus be alkylated by a suitablealkylating agent, in this case dimethylallyldiphosphate. The newly introduced dimethylallyl


H

O

H H

O

OH

O

OH

O

OH

O

COSCoA

OH

OCOSCoA

OH

HCHO

H

O

OH

OO

O

OO O

OH

O

OH

O

4-hydroxycoumarin

dicoumarol

– H2O

lactone formation and enolization

aldol reaction; it may help toconsider the diketo tautomer

dehydration follows

nucleophilic attack on to the enone system

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). Thisagent interferes with the effects of vitamin K in blood coagulation (see page 163), theblood 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 ofthrombosis, where the risk of blood clots becomes life threatening. It has been supersededby salts of warfarin and acenocoumarol (nicoumalone) (Figure 4.32), which are syntheticdevelopments from the natural product. An overdose of warfarin may be countered byinjection of vitamin K1.

Warfarin was initially developed as a rodenticide, and has been widely employedfor many years as the first choice agent, particularly for destruction of rats. After

O

OH

O

O

O

OH

O

NO2

O

O

OH

O

Cl

O

O

OH

O O

OH

O

R

RSRS

RS

RS RS

RS

warfarin

acenocoumarol(nicoumalone)

coumachlor

R = H, difenacoumR = Br, brodifenacoum

coumatetralyl

Figure 4.32

(Continues )

COUMARINS 145

(Continued )

consumption of warfarin-treated bait, rats die from internal haemorrhage. Other coumarinderivatives employed as rodenticides include coumachlor and coumatetralyl (Figure 4.32).In an increasing number of cases, rodents are becoming resistant to warfarin, an abilitywhich has been traced to elevated production of vitamin K by their intestinal microflora.Modified structures defenacoum and brodifenacoum have been found to be more potentthan warfarin, and are also effective against rodents that have become resistant to warfarin.

group in demethylsuberosin is then able tocyclize with the phenol group giving marmesin.This transformation is catalysed by a cytochromeP-450-dependent mono-oxygenase, and requirescofactors NADPH and molecular oxygen. Formany years, the cyclization had been postulatedto involve an intermediate epoxide, so thatnucleophilic attack of the phenol on to theepoxide group might lead to formation of eitherfive-membered furan or six-membered pyranheterocycles as commonly encountered in naturalproducts (Figure 4.34). Although the reactions ofFigure 4.34 offer a convenient rationalization forcyclization, epoxide intermediates have not beendemonstrated in any of the enzymic systems so farinvestigated, and therefore some direct oxidativecyclization mechanism must operate. A secondcytochrome P-450-dependent mono-oxygenase

enzyme then cleaves off the hydroxyisopropylfragment (as acetone) from marmesin givingthe furocoumarin psoralen (Figure 4.35). Thisdoes not involve any hydroxylated intermediate,and cleavage is believed to be initiated by aradical abstraction process. Psoralen can act as aprecursor for the further substituted furocoumarinsbergapten, xanthotoxin, and isopimpinellin(Figure 4.33), such modifications occurring latein the biosynthetic sequence rather than atthe cinnamic acid stage. Psoralen, bergapten,etc are termed ‘linear’ furocoumarins. ‘Angular’furocoumarins, e.g. angelicin (Figure 4.33), canarise by a similar sequence of reactions, butthese involve dimethylallylation at the alternativeposition ortho to the phenol. An isoprene-derivedfuran ring system has already been noted in theformation of khellin (see page 74), though the

O2NADPH

O OOHO

O OO

O2NADPH

O2NADPH

O2NADPH

O OO

O OHO

O OO

OH

O OO

OH

SAM

SAM

O OHO

O OO

OMe

O OO

OMe

OPP

DMAPP

O OO

OMe

OMe

umbelliferone demethylsuberosin marmesin

psoralen(linear furocoumarin)

bergaptolbergapten

xanthotoxin xanthotoxol angelicin(angular furocoumarin)

isopimpinellin

C-alkylation at activated position ortho to phenol

hydroxylationmethylation

Figure 4.33


HO

O

OHO

O

O

HO

OHO

O

nucleophilic attackon to epoxide

– H2O

– H2O

5-membered furan ring

6-membered pyran ring

H

H

Figure 4.34

Enz Fe

O

O OOHO

H HO2

NADPH

O OOHO

O

HO

Enz Fe

OH

HO OH

O OO

marmesin psoralen

oxidation leading to radical cleavage of side-chain carbons

side-chain carbons released as acetone

– H2O

Figure 4.35

aromatic ring to which it was fused was in thatcase a product of the acetate pathway. Linearfurocoumarins (psoralens)∗ can be troublesome tohumans since they can cause photosensitization

towards UV light, resulting in sunburn or seriousblistering. Used medicinally, this effect may bevaluable in promoting skin pigmentation andtreating psoriasis.

Psoralens

Psoralens are linear furocoumarins which are widely distributed in plants, but are particularlyabundant in the Umbelliferae/Apiaceae and Rutaceae. The most common examplesare psoralen, bergapten, xanthotoxin, and isopimpinellin (Figure 4.33). Plants containingpsoralens have been used internally and externally to promote skin pigmentation andsun-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 inexternal suntan preparations. The psoralen, because of its extended chromophore, absorbsin the near UV and allows this radiation to stimulate formation of melanin pigments (seepage 129).

Methoxsalen (xanthotoxin; 8-methoxypsoralen) (Figure 4.36), a constituent of the fruits ofAmmi majus (Umbelliferae/Apiaceae), is used medically to facilitate skin repigmentationwhere severe blemishes exist (vitiligo). An oral dose of methoxsalen is followed bylong wave UV irradiation, though such treatments must be very carefully regulated to

(Continues )

STYRYLPYRONES 147

(Continued )

HN

N

O

O

O

O

O

OMe

O

O

O

OMe

HN

N

O

O

O

O

O

OMe

HN

N

O

O

NN

O

OH

xanthotoxin(methoxsalen)

psoralen−DNA adductthymine in DNA

hν

psoralen−DNA di-adduct

hν

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 inthe treatment of psoriasis, a widespread condition characterized by proliferation of skincells. Similarly, methoxsalen is taken orally, prior to UV treatment. Reaction with psoralensinhibits 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 betweenpyrimidine bases (primarily thymine) in DNA and the furan ring of psoralens (Figure 4.36). Insome cases, di-adducts can form involving further cycloaddition via the pyrone ring, thuscross-linking the nucleic acid.

A troublesome extension of these effects can arise from the handling of plants thatcontain significant levels of furocoumarins. Celery (Apium graveolens; Umbelliferae/Apiaceae)is normally free of such compounds, but fungal infection with the natural parasite Sclerotiniasclerotiorum induces the synthesis of furocoumarins (xanthotoxin and others) as a responseto the infections. Some field workers handling these infected plants have become verysensitive to UV light and suffer from a form of sunburn termed photophytodermatitis. Infectedparsley (Petroselinum crispum) can give similar effects. Handling of rue (Ruta graveolens;Rutaceae) or giant hogweed (Heracleum mantegazzianum; Umbelliferae/Apiaceae), whichnaturally contain significant amounts of psoralen, bergapten, and xanthotoxin, can causesimilar unpleasant reactions, or more commonly rapid blistering by direct contact with thesap. The giant hogweed can be particularly dangerous. Individuals vary in their sensitivitytowards furocoumarins; some are unaffected whilst others tend to become sensitized by aninitial exposure and then develop the allergic response on subsequent exposures.

STYRYLPYRONES

Cinnamic acids, as their coenzyme A esters, mayalso function as starter units for chain exten-sion with malonyl-CoA units, thus combiningelements of the shikimate and acetate pathways(see page 80). Most commonly, three C2 unitsare added via malonate giving rise to flavonoidsand stilbenes, as described in the next section(page 149). However, there are several examplesof products formed from a cinnamoyl-CoA starter

plus one or two C2 units from malonyl-CoA. Theshort poly-β-keto chain frequently cyclizes to forma lactone derivative (compare triacetic acid lac-tone, page 62). Thus, Figure 4.37 shows the pro-posed derivation of yangonin via cyclization of thedi-enol tautomer of the polyketide formed from4-hydroxycinnamoyl-CoA and two malonyl-CoAextender units. Two methylation reactions com-plete the sequence. Yangonin and a series of relatedstructures form the active principles of kava root(Piper methysticum; Piperaceae), a herbal remedypopular for its anxiolytic activity.


SCoA

O

HO

O

MeO

O

OMe

O

HO

O

OSCoA

SAM

OH

HO

OH

OSCoA

O

HO

O

OH4-hydroxycinnamoyl-CoA

2 x malonyl-CoA

chain extension; acetate pathway witha cinnamoyl-CoA starter group

≡

lactone formation

yangonin

di-enol tautomer

Figure 4.37

Kava

Aqueous extracts from the root and rhizome of Piper methysticum (Piperaceae) have longbeen consumed as an intoxicating beverage by the peoples of Pacific islands comprisingPolynesia, 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 forthe treatment of anxiety, nervous tension, agitation and insomnia. The pharmacologicalactivity is associated with a group of styrylpyrone derivatives termed kavapyrones orkavalactones, good quality roots containing 5–8% kavapyrones. At least 18 kavapyroneshave been characterized, the six major ones being the enolides kawain, methysticin, andtheir dihydro derivatives reduced in the cinnamoyl side-chain, and the dienolides yangoninand demethoxyyangonin (Figure 4.38). Compared with the dienolides, the enolides have areduced pyrone ring and a chiral centre. Clinical trials have indicated kava extracts to beeffective as an anxiolytic, the kavapyrones also displaying anticonvulsive, analgesic, andcentral muscle relaxing action. Several of these compounds have been shown to have aneffect on neurotransmitter systems including those involving glutamate, GABA, dopamine,and serotonin.

O O

OMe

H

O

MeO

O

OMe

O O

OMe

HO O

OMe

HO

O

O O

OMe

HO

O

O O

OMe

yangonin demethoxyyangonin

kawain methysticindihydrokawain dihydromethysticin

Figure 4.38

FLAVONOIDS AND STILBENES 149

FLAVONOIDS AND STILBENES

Flavonoids and stilbenes are products from acinnamoyl-CoA starter unit, with chain exten-sion using three molecules of malonyl-CoA.This initially gives a polyketide (Figure 4.39),which, according to the nature of the enzymeresponsible, can be folded in two differentways. These allow aldol or Claisen-like reac-tions to occur, generating aromatic rings asalready seen in Chapter 3 (see page 80). Enzymesstilbene synthase and chalcone synthase cou-ple a cinnamoyl-CoA unit with three malonyl-CoA units giving stilbenes, e.g. resveratrol orchalcones, e.g. naringenin-chalcone respectively.

Both structures nicely illustrate the different char-acteristic oxygenation patterns in aromatic ringsderived from the acetate or shikimate pathways.With the stilbenes, it is noted that the terminalester function is no longer present, and there-fore hydrolysis and decarboxylation have alsotaken place during this transformation. No inter-mediates, e.g. carboxylated stilbenes, have beendetected, and the transformation from cinnamoyl-CoA/malonyl-CoA to stilbene is catalysed by thesingle enzyme. Resveratrol has assumed greaterrelevance in recent years as a constituent ofgrapes and wine, as well as other food products,with antioxidant, anti-inflammatory, anti-platelet,and cancer preventative properties. Coupled with

CoAS

O

OH

NADPH

OH

O

O

O

SCoA

O

OH

O

O

OH

SCoA

O

OH

O

O

SCoA

O

O

CO2

OH

OOH

HO

OH

OH

O

HO

OH

OH

HO

OH

O

OOH

HO

OH

O

O

HO

OH

(reductase)

resveratrol(a stilbene)

naringenin-chalcone(a chalcone)

isoliquiritigenin(a chalcone)

4-hydroxycinnamoyl-CoA

aldol(stilbene synthase)

3 x malonyl-CoA

≡

liquiritigenin(a flavanone)

naringenin(a flavanone)

chain extension; acetate pathway with a cinnamoyl-CoA starter group

Michael-type nucleophilic attack of OH on to α,β-unsaturated ketone

ClaisenClaisen

(chalcone synthase)

Figure 4.39


the cardiovascular benefits of moderate amountsof alcohol, and the beneficial antioxidant effectsof flavonoids (see page 151), red wine has nowemerged as an unlikely but most acceptable medic-inal agent.

Chalcones act as precursors for a vast rangeof flavonoid derivatives found throughout theplant kingdom. Most contain a six-memberedheterocyclic ring, formed by Michael-type nucle-ophilic attack of a phenol group on to the unsatu-rated ketone giving a flavanone, e.g. naringenin(Figure 4.39). This isomerization can occur chem-ically, acid conditions favouring the flavanoneand basic conditions the chalcone, but in naturethe reaction is enzyme catalysed and stereospe-cific, resulting in formation of a single fla-vanone enantiomer. Many flavonoid structures,e.g. liquiritigenin, have lost one of the hydroxylgroups, so that the acetate-derived aromatic ring

has a resorcinol oxygenation pattern rather thanthe phloroglucinol system. This modification hasbeen tracked down to the action of a reductaseenzyme concomitant with the chalcone synthase,and thus isoliquiritigenin is produced rather thannaringenin-chalcone. Flavanones can then giverise to many variants on this basic skeleton, e.g.flavones, flavonols, anthocyanidins, and cate-chins (Figure 4.40). Modifications to the hydroxy-lation patterns in the two aromatic rings may occur,generally at the flavanone or dihydroflavonol stage,and methylation, glycosylation, and dimethylal-lylation are also possible, increasing the rangeof compounds enormously. A high proportion offlavonoids occur naturally as water-soluble gly-cosides. Considerable quantities of flavonoids areconsumed daily in our vegetable diet, so adversebiological effects on man are not particularlyintense. Indeed, there is growing belief that some

O

OOH

HO

OH

R

O

OOH

HO

OH

R

O

OOH

HO

OH

R

OH

O

OHOH

HO

OH

R

OH

O

OH

HO

OH

R

OH

NADPH

NADPH

O

O

OH

HO

OH

R

OH

O

OOH

HO

OH

R

OH

O

OHOH

HO

OH

R

OH

HO

R = H, apigeninR = OH, luteolin

(flavones)

R = H, afzalechinR = OH, (+)-catechin

(catechins)

R = H, naringeninR = OH, eriodictyol

(flavanones)

R = H, dihydrokaempferolR = OH, dihydroquercetin

(dihydroflavonols)

R = H, kaempferolR = OH, quercetin

(flavonols)

R = H, pelargonidinR = OH, cyanidin(anthocyanidins)

O22-oxoglutarate

O22-oxoglutarate

O22-oxoglutarate

R = H, leucopelargonidinR = OH, leucocyanidin

(flavandiols; leucoanthocyanidins) – 2 H2O

Figure 4.40

FLAVONOIDS AND STILBENES 151

O

OH

HO

OH

OH

OH

O

OH

HO

OH

OH

OH

O

OH

HO

OH

OH

OH

O

OH

HO

OH O

OHHO

HO

OH

OH

OHO

epicatechin trimer

theaflavin

Figure 4.41

flavonoids are particularly beneficial, acting asantioxidants and giving protection against cardio-vascular disease, certain forms of cancer, and,it is claimed, age-related degeneration of cellcomponents. Their polyphenolic nature enablesthem to scavenge injurious free radicals such assuperoxide and hydroxyl radicals. Quercetin inparticular is almost always present in substan-tial amounts in plant tissues, and is a power-ful antioxidant, chelating metals, scavenging freeradicals, and preventing oxidation of low densitylipoprotein. Flavonoids in red wine (quercetin,kaempferol, and anthocyanidins) and in tea (cate-chins and catechin gallate esters) are also demon-strated to be effective antioxidants. Flavonoidscontribute to plant colours, yellows from chal-cones and flavonols, and reds, blues, and violetsfrom anthocyanidins. Even the colourless mate-rials, e.g. flavones, absorb strongly in the UVand are detectable by insects, probably aidingflower pollination. Catechins form small poly-mers (oligomers), the condensed tannins, e.g. theepicatechin trimer (Figure 4.41) which contributeastringency to our foods and drinks, as do the sim-pler gallotannins (see page 122), and are commer-cially important for tanning leather. Theaflavins,antioxidants found in fermented tea (see page 395),are dimeric catechin structures in which oxida-tive processes have led to formation of a seven-membered tropolone ring.

The flavonol glycoside rutin (Figure 4.42)from buckwheat (Fagopyrum esculentum; Polygo-naceae) and rue (Ruta graveolens; Rutaceae), andthe flavanone glycoside hesperidin from Citrus

peels have been included in dietary supplements asvitamin P, and claimed to be of benefit in treatingconditions characterized by capillary bleeding, buttheir therapeutic efficacy is far from conclusive.Neohesperidin (Figure 4.42) from bitter orange(Citrus aurantium; Rutaceae) and naringin fromgrapefruit peel (Citrus paradisi ) are intenselybitter flavanone glycosides. It has been foundthat conversion of these compounds into dihy-drochalcones by hydrogenation in alkaline solu-tion (Figure 4.43) produces a remarkable changeto their taste, and the products are now intenselysweet, being some 300–1000 times as sweet assucrose. These and other dihydrochalcones havebeen investigated as non-sugar sweetening agents.

FLAVONOLIGNANS

An interesting combination of flavonoid and lig-nan structures is found in a group of compoundscalled flavonolignans. They arise by oxidativecoupling processes between a flavonoid and aphenylpropanoid, usually coniferyl alcohol. Thus,the dihydroflavonol taxifolin through one-electronoxidation may provide a free radical, which maycombine with the free radical generated fromconiferyl alcohol (Figure 4.44). This would leadto an adduct, which could cyclize by attack ofthe phenol nucleophile on to the quinone methidesystem provided by coniferyl alcohol. The prod-uct would be silybin, found in Silybum marianum(Compositae/Asteraceae) as a mixture of two transdiastereoisomers, reflecting a lack of stereospeci-ficity for the original radical coupling. In addition,


O

OOH

HO

OH

OH

O-Glc-Rha

O

OOH

O

OMe

OH

O

HOOH

HO

O

OHO

OHHO

O

OOH

O

OMe

OH

O

HOHO

OH

O

O

OHHO

HO

O

OOH

Rha-Glc-O

OH

hesperetin

neohesperidin

rutin

L-Rha

hesperidin

quercetin

D-Glc

rutinose= rhamnosyl(α1→6)glucose

D-Glc

L-Rha

naringenin

hesperetin rutinose

naringin

neohesperidoseneohesperidose= rhamnosyl(α1→2)glucose

Figure 4.42

O

OOH

Rha-Glc-O

R1

OH

OOH

Rha-Glc-O

R1

OH

OOH

Rha-Glc-O

R1

R2 R2 R2

H

R1 = OH, R2 = H,

naringin dihydrochalcone

R1 = OMe, R2 = OH,

neohesperidin dihydrochalcone

R1 = OH, R2 = H, naringin

R1 = OMe, R2 = OH, neohesperidin

H2 / catalyst

OH

OH

Figure 4.43

the regioisomer isosilybin (Figure 4.45), again amixture of trans diastereoisomers, is also found inSilybum. Silychristin (Figure 4.45) demonstratesa further structural variant which can be seen tooriginate from a mesomer of the taxifolin-derivedfree radical, in which the unpaired electron islocalized on the carbon ortho to the original 4-hydroxyl function. The more complex structurein silydianin is accounted for by the mechanismshown in Figure 4.46, in which the initial couplingproduct cyclizes further by intramolecular attack

of an enolate nucleophile on to the quinoneme-thide. Hemiketal formation finishes the process.The flavonolignans from Silybum∗ (milk thistle)have valuable antihepatotoxic properties, and canprovide protection against liver-damaging agents.Coumarinolignans, which are products arising by asimilar oxidative coupling mechanism which com-bines a coumarin with a cinnamyl alcohol, maybe found in other plants. The benzodioxane ringas seen in silybin and isosilybin is a characteristicfeature of many such compounds.

FLAVONOLIGNANS 153

O

OOH

HO

OH

OH

OH

O

OOH

HO

O

OH

OH

O

OOH

HO

O

O

OH

OH

OH

OMe

OH

O

OMe

O

OOH

HO

O

O

OH

OH

OH

OMe

OH

OH

OMe

O

OOH

HO

O

OH

OH

OH

O

OMe

H

taxifolin

+

silybin(diastereoisomeric pair)

coniferyl alcohol

– H

– e


radical coupling

– H

nucleophilic attack of OH on to quinonemethide (two stereochemistries possible)

– e


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 flavonolignanscollectively termed silymarin. This mixture contains mainly silybin (Figure 4.44), together withsilychristin (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. Silybummarianum is widely used in traditional European medicine, the fruits being used to treata variety of hepatic and other disorders. Silymarin has been shown to protect animallivers against the damaging effects of carbon tetrachloride, thioacetamide, drugs such asparacetamol, and the toxins α-amanitin and phalloin found in the death cap fungus (Amanitaphalloides) (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 benefitin the treatment of poisoning by the death cap fungus. These agents appear to havetwo main modes of action. They act on the cellular membrane of hepatocytes inhibitingabsorption of toxins, and secondly, because of their phenolic nature, they can act asantioxidants and scavengers for free radicals which cause liver damage originating from liverdetoxification of foreign chemicals. Derivatives of silybin with improved water-solubility and/orbioavailability have been developed, e.g. the bis-hemisuccinate and a phosphatidylcholinecomplex.


O

HOOMe

O

OOH

HO

O

OH

OH

O

OOH

HO

O

O

OHOH

OH

OMe

O

OOH

HO

O

O

OHOH

OH

OMe

O

OOH

HO

OH

OOH

OMe

OH

OH

isosilybin(diastereoisomeric pair)

+

silychristin

Figure 4.45

O

OOH

HO

O

OH

OH

OH

O

MeO

H

O

OOH

HO

O

OH

OH

OH

O

MeO

H

O

OOH

HO

OH

HO

MeO O

OH

O

H

silydianin

(ii)

(i)

(i) nucleophilic attack of enolate on to quinonemethide (ii) hemiketal formation

radical coupling

Figure 4.46

ISOFLAVONOIDS

The isoflavonoids form a quite distinct subclassof flavonoid compound, being structural vari-ants in which the shikimate-derived aromaticring has migrated to the adjacent carbon ofthe heterocycle. This rearrangement process isbrought about by a cytochrome P-450-dependentenzyme requiring NADPH and O2 cofactors,which transforms the flavanones liquiritigeninor naringenin into the isoflavones daidzein orgenistein respectively via intermediate hydrox-yisoflavanones (Figure 4.47). A radical mecha-nism has been proposed. This rearrangement isquite rare in nature, and isoflavonoids are almostentirely restricted to the plant family the Legu-minosae/Fabaceae. Nevertheless, many hundreds

of different isoflavonoids have been identified,and structural complexity is brought about byhydroxylation and alkylation reactions, varying theoxidation level of the heterocyclic ring, or formingadditional heterocyclic rings. Some of the manyvariants are shown in Figure 4.48. Pterocarpans,e.g. medicarpin from lucerne (Medicago sativa),and pisatin from pea (Pisum sativum), have anti-fungal activity and form part of these plants’natural defence mechanism against fungal attack.Simple isoflavones such as daidzein and coumes-tans such as coumestrol from lucerne and clovers(Trifolium species), have sufficient oestrogenicactivity to seriously affect the reproduction of graz-ing animals, and are termed phyto-oestrogens∗.These planar molecules undoubtedly mimic theshape and polarity of the steroid hormone estradiol

ISOFLAVONOIDS 155

O

O

HO

OH

R

H

H

O2NADPH

O

Fe Enz

O

O

HO

OH

R

O

O

HO

ROH

O

O

HO

ROH

O

O

HO

ROH

OH

OH

Fe Enz

R = H, liquiritigeninR = OH, naringenin

(flavonoid)

oxidation to free radical

– H2O

R = H, daidzeinR = OH, genistein

(isoflavonoid)

1,2-aryl migration

7

4′

Figure 4.47

O

O

HO

OH

O

O

HO

OMe

OHO

OOH

O

O

OOMe

O

OMe

H

H

O

H

OHO

OOMe

H

H

OMeO

O O

OOH

H

daidzein(an isoflavone)

formononetin(an isoflavone)

medicarpin(a pterocarpan)

coumestrol(a coumestan)

pisatin(a pterocarpan)rotenone

(a rotenoid)

Figure 4.48

(see page 276). The consumption of legume fod-der crops by animals must therefore be restricted,or low isoflavonoid producing strains have to beselected. Isoflavonoids in the human diet, e.g.from soya (Glycine max ) products, are believed togive some protection against oestrogen-dependentcancers such as breast cancer, by restricting theavailability of the natural hormone. In addition,they can feature as dietary oestrogen supplementsin the reduction of menopausal symptoms, ina similar way to hormone replacement therapy

(see page 279). The rotenoids take their namefrom the first known example rotenone, and areformed by ring cyclization of a methoxyisoflavone(Figure 4.49). Rotenone itself contains a C5 iso-prene unit (as do virtually all the natural rotenoids)introduced via dimethylallylation of demethyl-munduserone. The isopropenylfurano system ofrotenone, and the dimethylpyrano of deguelin,are formed via rotenonic acid (Figure 4.49) with-out any detectable epoxide or hydroxy intermedi-ates (compare furocoumarins, page 145). Rotenone


O

OOMe

O

OMe

HHOO

O

HO

OMe

OMe

OMeO

O

HO

OMe

OMe

O

H

O

OOMe

O

OMe

H

H

O

H

OPP

DMAPPO

OOMe

O

OMe

H

H

HO O

OOMe

O

OMe

H

H

HO

O

OOMe

O

OMe

H

H

O

rotenone

deguelin

demethylmunduseronerotenonic acid

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

C-alkylation at activated position ortho to phenol

cyclization to 5-membered ring

cyclization to 6-membered ring

addition of hydride(reduction)

Figure 4.49

and other rotenoids are powerful insecticidal andpiscicidal (fish poison) agents, interfering withoxidative phosphorylation. They are relativelyharmless to mammals unless they enter the bloodstream, being metabolized rapidly upon ingestion.

Rotenone thus provides an excellent biodegradableinsecticide, and is used as such either in pure orpowdered plant form. Roots of Derris elliptica∗or Lonchocarpus∗ species are rich sources ofrotenone.

Phyto-oestrogens

Phyto-oestrogen (phytoestrogen) is a term applied to non-steroidal plant materials displayingoestrogenic properties. Pre-eminent amongst these are isoflavonoids. These planar moleculesmimic the shape and polarity of the steroid hormone estradiol (see page 279), and are ableto bind to an oestrogen receptor, though their activity is less than that of estradiol. In sometissues, they stimulate an oestrogenic response, whilst in others they can antagonize theeffect of oestrogens. Such materials taken as part of the diet therefore influence overalloestrogenic activity in the body by adding their effects to normal levels of steroidaloestrogens (see page 282). Foods rich in isoflavonoids are valuable in countering someof the side-effects of the menopause in women, such as hot flushes, tiredness, andmood swings. In addition, there is mounting evidence that phyto-oestrogens also provide arange of other beneficial effects, helping to prevent heart attacks and other cardiovasculardiseases, protecting against osteoporosis, lessening the risk of breast and uterine cancer,and in addition displaying significant antioxidant activity which may reduce the risk ofAlzheimer’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 isoflavonesdaidzein, and genistein (Figure 4.47), in free form and as their 7-O-glucosides. Totalisoflavone levels fall in the range 0.1–0.4%, according to variety. Soya products suchas soya milk, soya flour, tofu, and soya-based textured vegetable protein may allbe used in the diet for their isoflavonoid content. Breads in which wheat flour isreplaced by soya flour are also popular. Extracts from red clover (Trifolium pratense;Leguminosae/Fabaceae) are also used as a dietary supplement. Red clover isoflavonesare 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 onlignans such as secoisolariciresinol or matairesinol ingested in the diet. A particularlyimportant 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 originswere traced back to dietary fibre-rich foods. Levels in the urine were much higherin vegetarians, and have been related to a lower incidence of breast cancer invegetarians.

Derris and Lonchocarpus

Species of Derris (e.g. D. elliptica, D. malaccensis) and Lonchocarpus (e.g. L. utilis, L.urucu) (Leguminosae/Fabaceae) have provided useful insecticides for many years. Roots ofthese plants have been employed as a dusting powder, or extracts have been formulatedfor sprays. Derris plants are small shrubs cultivated in Malaysia and Indonesia, whilstLonchocarpus includes shrubs and trees, with commercial material coming from Peru andBrazil. The insecticidal principles are usually supplied as a black, resinous extract. Both Derrisand Lonchocarpus roots contain 3–10% of rotenone (Figure 4.49) and smaller amounts ofother rotenoids, e.g. deguelin (Figure 4.49). The resin may contain rotenone (about 45%) anddeguelin (about 20%).

Rotenone and other rotenoids interfere with oxidative phosphorylation, blocking transfer ofelectrons to ubiquinone (see page 159) by complexing with NADH:ubiquinone oxidoreductaseof the respiratory electron transport chain. However, they are relatively innocuous tomammals 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 hasbeen exploited for centuries in a number of tropical countries, allowing lazy fishing bythe 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 undesirablefish species prior to restocking with other species. As insecticides, the rotenoidsstill 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 tocompete effectively with other insecticides such as the modern pyrethrin derivatives (seepage 188).


TERPENOID QUINONES

Quinones are potentially derivable by oxi-dation of suitable phenolic compounds, cat-echols (1,2-dihydroxybenzenes) giving rise to

ortho-quinones and quinols (1,4-dihydroxyben-zenes) yielding para-quinones (see page 25).Accordingly, quinones can be formed from phe-nolic systems generated by either the acetate orshikimate pathways, provided a catechol or quinol

O

O

MeO

MeO H

O

OH

OH

OH

R2R1

H

O

OH

O

O

n

n = 1−13

plastoquinone-n 3

R1 = R2 = Me, α-tocopherol

R1 = H, R2 = Me, β-tocopherol

R1 = Me, R2 = H, γ-tocopherol

R1 = R2 = H, δ -tocopherol

3

phylloquinone(vitamin K1; phytomenadione) menaquinone-n

(vitamin K2)

n

n

n = 1−12

(vitamin E)

ubiquinone-n(coenzyme Qn)

n = 3−10

Figure 4.50

CO2H

OH

O CO2H

OH

CO2H

CH3COCO2H

O

O

MeO

MeO H

CO2H

OH

OPPH

SAM O2 SAM

CO2H

OHH

OHHMeO

O2

SAM

O

O

MeO H

O2

C-alkylation with a polyisoprenyl PP

plants / animals

bacteria

n

n

n

n

1. hydroxylation2. O-methylation3. decarboxylationor: 3, 1, 2

n

1. C-methylation2. hydroxylation3. O-methylation

ubiquinone-n

chorismic acid

4-coumaric acid

4-hydroxybenzoicacid

oxidation to quinone

Figure 4.51

TERPENOID QUINONES 159

system has been elaborated, and many examplesare found in nature. A range of quinone deriva-tives and related structures containing a terpenoidfragment as well as a shikimate-derived portionare also widely distributed. Many of these haveimportant biochemical functions in electron trans-port systems for respiration or photosynthesis, andsome examples are shown in Figure 4.50.

Ubiquinones (coenzyme Q) (Figure 4.50) arefound in almost all organisms and function as elec-tron carriers for the electron transport chain inmitochondria. The length of the terpenoid chainis variable (n = 1−12), and dependent on species,but most organisms synthesize a range of com-pounds, of which those where n = 7−10 usuallypredominate. The human redox carrier is coenzymeQ10. They are derived from 4-hydroxybenzoicacid (Figure 4.51), though the origin of thiscompound varies according to organism (seepages 123, 141). Thus, bacteria are known totransform chorismic acid by enzymic eliminationof pyruvic acid, whereas plants and animals uti-lize a route from phenylalanine or tyrosine via4-hydroxycinnamic acid (Figure 4.51). 4-Hydro-xybenzoic acid is the substrate for C-alkylationortho to the phenol group with a polyisoprenyldiphosphate of appropriate chain length (seepage 231). The product then undergoes furtherelaboration, the exact sequence of modifications,i.e. hydroxylation, O-methylation, and decarboxy-lation, varying in eukaryotes and prokaryotes.Quinone formation follows in an O2-dependentcombined hydroxylation–oxidation process, andubiquinone production then involves further hydro-xylation, and O- and C-methylation reactions.

Plastoquinones (Figure 4.50) bear considerablestructural similarity to ubiquinones, but are notderived from 4-hydroxybenzoic acid. Instead, theyare produced from homogentisic acid, a pheny-lacetic acid derivative formed from 4-hydroxyphe-nylpyruvic acid by a complex reaction involvingdecarboxylation, O2-dependent hydroxylation, andsubsequent migration of the −CH2CO2H side-chain to the adjacent position on the aromatic ring(Figure 4.52). C-Alkylation of homogentisic acidortho to a phenol group follows, and involvesa polyisoprenyl diphosphate with n = 3−10, butmost commonly with n = 9, i.e. solanesyl diphos-phate. However, during the alkylation reaction,the −CH2CO2H side-chain of homogentisic acid

suffers decarboxylation, and the product is thus analkyl methyl p-quinol derivative. Further aromaticmethylation (via S-adenosylmethionine) and oxida-tion of the p-quinol to a quinone follow to yield theplastoquinone. Thus, only one of the two methylgroups on the quinone ring of the plastoquinone isderived from SAM. Plastoquinones are involved inthe photosynthetic electron transport chain in plants.

Tocopherols are also frequently found in thechloroplasts and constitute members of the vita-min E∗ group. Their biosynthesis shares many ofthe features of plastoquinone biosynthesis, withan additional cyclization reaction involving thep-quinol and the terpenoid side-chain to give achroman ring (Figure 4.52). Thus, the tocopherols,e.g. α-tocopherol and γ-tocopherol, are not infact quinones, but are indeed structurally related toplastoquinones. The isoprenoid side-chain added,from phytyl diphosphate, contains only four iso-prene units, and three of the expected double bondshave suffered reduction. Again, decarboxylationof homogentisic acid cooccurs with the alkylationreaction. C-Methylation steps using SAM, and thecyclization of the p-quinol to γ-tocopherol, havebeen established as in Figure 4.52. Note once againthat one of the nuclear methyls is homogentisate-derived, whilst the others are supplied by SAM.

The phylloquinones (vitamin K1) and mena-quinones (vitamin K2) are shikimate-derived na-phthoquinone derivatives found in plants andalgae (vitamin K1

∗) or bacteria and fungi (vita-min K2). The most common phylloquinone struc-ture (Figure 4.50) has a diterpenoid side-chain,whereas the range of menaquinone structures tendsto be rather wider with 1–13 isoprene units.These quinones are derived from chorismic acidvia its isomer isochorismic acid (Figure 4.55).Additional carbons for the naphthoquinone skele-ton are provided by 2-oxoglutaric acid, which isincorporated by a mechanism involving the coen-zyme thiamine diphosphate (TPP). 2-Oxoglutaricacid is decarboxylated in the presence of TPPto give the TPP anion of succinic semialdehyde,which attacks isochorismic acid in a Michael-typereaction. Loss of the thiamine cofactor, elimina-tion of pyruvic acid, and then dehydration yieldthe intermediate o-succinylbenzoic acid (OSB).This is activated by formation of a coenzyme Aester, and a Dieckmann-like condensation allowsring formation. The dihydroxynaphthoic acid is the


CO2HO

HO OO2

HO

OH

HO2C

PPOH

HPPO

HO

HOH

HO

HOH

HO

HO

CO2

CO2

H

SAM

SAM

CO2

HO

OH

H

HO

OH

H

O

O

H

O

HO

HO

SAM

C-methylation ortho to phenol

n

C-alkylation ortho to phenol; also decarboxylation

n

n = 9, solanesyl PP

n

3

phytyl PP

homogentisic acid

3

3


plastoquinone-n

γ-tocopherol α-tocopherol

3

oxidation of quinol to quinone

3

n


cyclization to 6-membered ring via protonation of double bond

4-hydroxyphenyl-pyruvic acid

a complex sequence involving hydroxylation, migration of side-chain, and decarboxylation

Figure 4.52

Vitamin E

Vitamin E refers to a group of fat-soluble vitamins, the tocopherols, e.g. α-, β-, γ-, andδ-tocopherols (Figure 4.53), which are widely distributed in plants, with high levels in cerealseeds such as wheat, barley, and rye. Wheat germ oil is a particularly good source. Theproportions of the individual tocopherols vary widely in different seed oils, e.g. principallyβ- in wheat oil, γ- in corn oil, α- in safflower oil, and γ- and δ- in soybean oil. Vitamin Edeficiency is virtually unknown, with most of the dietary intake coming from food oils andmargarine, though much can be lost during processing and cooking. Rats deprived of thevitamin display reproductive abnormalities. α-Tocopherol has the highest activity (100%), withthe 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 )


(Continued )

HO

O

HO

O

HO

O

HO

O

β-tocopherol

α-tocopherol

δ-tocopherolγ-tocopherol

Figure 4.53

HO

O

ROOO

O

O

O

OH

O

O

O

OOH

ROO

H2O

O

OOOR

O

O

loss of peroxide leavinggroup

resonance-stabilized free radical

α-tocopherol

hydrolysis ofhemiketal

initiation of free radical reaction by peroxy radical quenching of second

peroxy radical

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 unsaturatedfatty acids in biological membranes. It is used commercially to retard rancidity in fattymaterials in food manufacturing, and there are also claims that it can reduce the effects ofageing and help to prevent heart disease. Its antioxidant effect is likely to arise by reactingwith peroxyl radicals, generating by one-electron phenolic oxidation a resonance-stabilizedfree radical that does not propagate the free radical reaction, but instead mops up furtherperoxyl radicals (Figure 4.54). In due course, the tocopheryl peroxide is hydrolysed to thetocopherolquinone.

more favoured aromatic tautomer from the hydrol-ysis of the coenzyme A ester. This compound isnow the substrate for alkylation and methylationas seen with ubiquinones and plastoquinones.However, the terpenoid fragment is found toreplace the carboxyl group, and the decarboxylatedanalogue is not involved. The transformation of

1,4-dihydroxynaphthoic acid to the isoprenylatednaphthoquinone appears to be catalysed by a singleenzyme, and can be rationalized by the mech-anism in Figure 4.56. This involves alkylation(shown in Figure 4.56 using the diketo tautomer),decarboxylation of the resultant β-keto acid, andfinally an oxidation to the p-quinone.


H

CO2HO

HO2C

HO

H2O OH

CO

HO2C

OH

O

HO2C CO2H

O

HO CO2H

TPP

CO2H CO2H

O

C COSCoA

O

OOH

O

COSCoA

O

OH

OH

CO2H

O

OH

HPPO

CO2

TPP

HSCoA ATP

CO2

HSCoA

SAM

CH3COCO2H

H

H

CO2HOH

O

HO2C

CO2H

O TPPH

CO2HOH

O

HO2C

CO2H

O

H

CO2HOH

CO2H

O

H

O

OH

C-methylation

isochorismicacid

chorismic acid

o-succinylbenzoic acid

(OSB)

2-oxoglutaric acid

n

menaquinone-n(vitamin K2)

Michael-type addition

succinic semi-aldehyde-TPP anion

1,4-eliminationof pyruvic acid

dehydration to form aromatic ring

Claisen-like condensation (Dieckmann reaction)

TPP-dependent decarboxylation of α-keto acid to aldehyde; nucleophilic addition of TPP anion on to aldehyde then allows removal of aldehydicproton which has become acidic

hydrolysis of thioester; enolization to morestable tautomer

1,4-dihydroxy-naphthoic acid

C-alkylation with concomitant decarboxylation

n n

Figure 4.55

PPOR

O

O

R

OH

O

O

CO2H

O

R

CO2

O

O

O

R

O

O

R

decarboxylationof β-keto acid

Figure 4.56


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 greenvegetable, 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 malabsorptionof the vitamin, which is lipid soluble. Vitamin K1 (phytomenadione) or the water-solublemenadiol phosphate (Figure 4.57) may be employed as supplements. Menadiol is oxidizedin the body to the quinone, which is then alkylated, e.g. with geranylgeranyl diphosphate, toyield a metabolically active product.

Vitamin K is involved in normal blood clotting processes, and a deficiency would lead tohaemorrhage. Blood clotting requires the carboxylation of glutamate residues in the proteinprothrombin, generating bidentate ligands that allow the protein to bind to other factors. Thiscarboxylation requires carbon dioxide, molecular oxygen, and the reduced quinol form ofvitamin K (Figure 4.57). During the carboxylation, the reduced vitamin K suffers epoxidation,and vitamin K is subsequently regenerated by reduction. Anticoagulants such as dicoumaroland warfarin (see page 144) inhibit this last reduction step. However, the polysaccharideanticoagulant heparin (see page 477) does not interfere with vitamin K metabolism, but actsby complexing with blood clotting enzymes.

HN

NH

CO2H

O

OH

OH

O

O

O

HN

NH

CO2H

O

CO2H

OP

OP

O

O

prothrombin

vitamin K epoxide

vitamin K (quinol)vitamin KO2, CO2

reductase

menadiol phosphate

Figure 4.57

OSB, and 1,4-dihydroxynaphthoic acid, or itsdiketo tautomer, have been implicated in thebiosynthesis of a wide range of plant naphtho-quinones and anthraquinones. There are parallelswith the later stages of the menaquinone sequenceshown in Figure 4.55, or differences according tothe plant species concerned. Some of these path-ways are illustrated in Figure 4.58. Replacementof the carboxyl function by an isoprenyl sub-stituent is found to proceed via a disubstitutedintermediate in Catalpa (Bignoniaceae) and

Streptocarpus (Gesneriaceae), e.g. catalponone(compare Figure 4.56), and this can be transformedto deoxylapachol and then menaquinone-1 (Fig-ure 4.58). Lawsone is formed by an oxidativesequence in which hydroxyl replaces the carboxyl.A further interesting elaboration is the synthesisof an anthraquinone skeleton by effectively cycliz-ing a dimethylallyl substituent on to the naph-thaquinone system. Rather little is known abouthow this process is achieved but many examplesare known from the results of labelling studies.


O

O

O

O

OH

OH

OH

deoxylapachol

lucidin

O

CO2H

O

O

OCO2H

O

O

O

OH

O

O

O

OHO

O

OH

OH

CO2H

OH

OH

CO2H

O

O OH

OH

1,4-dihydroxy-naphthoic acid

alizarin

menaquinone-1catalponone

lawsone

Figure 4.58

Some of these structures retain the methyl fromthe isoprenyl substituent, whilst in others this hasbeen removed, e.g. alizarin from madder (Rubiatinctorum; Rubiaceae), presumably via an oxi-dation–decarboxylation sequence. Hydroxylation,particularly in the terpenoid-derived ring, is also afrequent feature.

Some other quinone derivatives, althoughformed from the same pathway, are producedby dimethylallylation of 1,4-dihydroxynaphthoic

O

O OHOH

HO

O

O OHOH

O

O

OH

O

O

OH

OH

OH

OH

OH

emodin aloe-emodin

alizarin lucidin

acetate / malonate

Shikimate / 2-oxoglutarate / isoprenoid

Figure 4.59

acid at the non-carboxylated carbon. Obviously,this is also a nucleophilic site and alkylationhere is mechanistically sound. Again, cyclizationof the dimethylallyl to produce an anthraquinonecan occur, and the potently mutagenic lucidinfrom Galium species (Rubiaceae) is a typicalexample. The hydroxylation patterns seen in theanthraquinones in Figure 4.58 should be com-pared with those noted earlier in acetate/malonate-derived structures (see page 63). Remnants ofthe alternate oxygenation pattern are usuallyvery evident in acetate-derived anthraquinones(Figure 4.59), whereas such a pattern cannoteasily be incorporated into typical shikimate/2-oxoglutarate/isoprenoid structures. Oxygen sub-stituents are not usually present in positions fittingthe polyketide hypothesis.

FURTHER READING

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Chloramphenicol

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Volatile Oils

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Lignin

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THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND