Living plants are solar-powered biochemical and biosynthetic laboratory which manufactures both primary and secondary metabolites from air, water, minerals and sunlight. The primary metabolites like sugars, amino acids and fatty acids that are needed for general growth and physiological development of plant are widely distributed in nature and are also utilized as food by man. The secondary metabolites such as alkaloids, glycosides, flavonoids, volatile oils etc are biosynthetically derived from primary metabolites. They represent chemical adaptations to environmental stresses, or serve as defensive, protective or offensive chemicals against microorganisms, insects and higher herbivorous predators. They are some times considered as waste or secretory products of plant metabolism and are of pharmaceutical importance. A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes, but usually has important ecological function.
MEVALONIC ACID PATHWAY Biosynthesis of Mevalonic Acid A molecule of acetyl-CoA undergoes a Claisen condensation with a molecule of malonyl-CoA (as in fatty acid biosynthesis; see Biochemistry of Yeast Fermentation – Fatty Acids) to form acetoacetyl-CoA which reacts with a third molecule of manonyl-CoA in an Aldol Condensation to give 3-hydroxy-3methylglutaryl-CoA (HMG-CoA). Reduction with HMG-CoA reductase yields mevaldic acid which is reduced again to mevalonic acid.
Conversion of Mevalonic acid to Isopentenyl pyrophosphate (3-methylbut-3enylpyrophosphate) Mevalonate is converted into 3-phospho-5-pyrophosphomevalonate by three consecutive phosphorylations. This labile intermediate loses CO2 and phosphate and yields 3-isopentenyl pyrophosphate (IPP).
Isomerization Of Isopentenyl Pyrophosphate And Prenyl Transferase Reactions IPP is isomerized to dimethylallyl pyrophosphate (DMAPP) with IPP isomerase. The isomerization involves addition of a proton to the sp2 carbon of isopentenyl
pyrophosphate and generation of the more stable tertiary carbocation. Elimination of a proton yields the most stable alkene (trisubstituted, Zaitev’s Rule).
Next IPP reacts with DMAPP to give geranyl pyrophosphate (GPP). In the first step of the reaction, IPP acts as a nucleophile and displaces a pyrophosphate group from DMAPP. Pyrophosphate is an excellent leaving group. Its four OH groups have pKa values of 0.9, 2.0, 6.6, and 9.4. Therefore three of the four groups will be primarily in their basic forms at physiological pH (pH 7.3). A proton is removed in the next step, again yielding the most stable alkene. GPP rearranges to give the monoterpenes. GPP reacts with a second molecule of IPP to produce farnesyl pyrophosphate (FPP). FPP is the precursor to the sesquiterpenoids, and the steroids. FPP reacts with a third molecule of IPP to produce geranylgeranyl pyrophosphate (GGPP). GGPP is the precursor of the carotenoids.
Significance of Mevalonic acid pathway: Isoprenoid Compounds: The isoprenoids are built up of C5 isoprene units and the nomenclature of the main classes reflects the number of isoprene units present (Table 1).
Monoterpenes: Acyclic Monoterpenes: In monoterpene diphosphate, geranyl PP, the double bond is trans (E). Linalyl PP and neryl PP are isomers of geranyl PP, and are likely to be formed from geranyl PP by ionization to the allylic cation, which can thus allow a change in attachment
of the diphosphate group (to the tertiary carbon in linalyl PP) or a change in stereochemistry at the double bond (to Z in neryl PP) (Figure 5.9). These three compounds can give rise to a range of linear monoterpenes found as components of volatile oils used in flavouring and perfumery (Figure 5.10). E.g.: Nerol, Geraniol, citronellol, linalool, hotrienol
MonoCyclic Monoterpenes: Cyclic monoterpenes can not be expected to occur with the precursor geranyl diphosphate, because the E stereochemistry of the double bond being unfavourable for ring formation (Figure 5.9). Neryl PP or linalyl PP, however, do have favourable stereochemistry, and either or both of these would seem more immediate precursors of the monocyclic menthane system, formation of which could be represented as shown in Figure 5.12, generating a carbocation (termed menthyl or α-terpinyl) having the menthane skeleton. It has been found that monoterpene cyclase enzymes are able to accept all three diphosphates, with linalyl PP being the best substrate, and it appears they have the ability to isomerize the substrates initially as well as to cyclize them. It is convenient therefore to consider the species involved in the cyclization as the delocalized allylic cation tightly bound to the diphosphate anion, and bond formation follows due to the proximity of the π-electrons of the double bond (Figure 5.12).
The menthyl cation could be quenched by attack of water, in which case the alcohol α-terpineol would be formed, or it could lose a proton to give limonene (Figure 5.13). The menthyl cation, although it is a tertiary, may be converted by a 1,3-hydride shift into a favourable resonance stabilized allylic cation (Figure 5.13). This allows the formation of α- and β-phellandrenes by loss of a proton from the phellandryl carbocation. E.g. of monocyclic terpenoids are p-cymene, thymol, carvacrol, menthol, isomenthol, neomenthol, menthone, isomenthone, neoisomenthone,
pulegone,
isopulegone,
piperitenone, isopiperitenone, isopiperitenol,
carveol,
carvone,
piperitone,
Dicyclic Monoterpenes: Alternatively, folding the cationic side-chain towards the double bond (via the surface characteristics of the enzyme) would allow a repeat of the cyclization mechanism, and produce bicyclic bornyl and pinyl cations, according to which end of the double bond was involved in forming the new bonds (Figure 5.14). Borneol would result from quenching of the bornyl cation with water, and then oxidation of the secondary alcohol could generate the ketone camphor. As an alternative to discharging the positive charge by adding a nucleophile, loss of a proton would generate an alkene. Thus α-pinene and β-pinene arise by loss of different protons from the pinyl cation, producing the double bonds as cyclic or exocyclic respectively. A less common termination step involving loss of a proton is the formation of a cyclopropane ring as exemplified by 3-carene and generation of the carane skeleton. The bicyclic pinyl cation, with a strained four membered ring, rearranges to the less strained five-membered fenchyl cation (Figure 5.14), a change which presumably more than makes up for the unfavourable tertiary to secondary carbocation transformation. This produces the fenchane skeleton, exemplified by
fenchol and fenchone. The isocamphyl tertiary carbocation is formed from the bornyl secondary carbocation by a Wagner–Meerwein rearrangement, and so leads to camphene. A hydride shift converting the menthyl cation into the terpinen-4-yl cation only changes one tertiary carbocation system for another, but allows formation of α-terpinene, γ-terpinene, and the α-terpineol isomer, terpinen-4-ol. A further cyclization reaction on the terpinen-4-yl cation generates the thujane skeleton, e.g. sabinene and thujone.
Sesquiterpenes: FPP can then give rise to linear and cyclic sesquiterpenes. Because of the increased chain length and additional double bond, the number of possible cyclization modes is also increased, and a huge range of mono-, bi-, and tri-cyclic structures can result. The stereochemistry of the double bond nearest the diphosphate can adopt an E configuration (as in FPP), or a Z configuration via
ionization, as found with geranyl/neryl PP (Figure 5.26). In some systems, the tertiary diphosphate nerolidyl PP (compare linalyl PP, page 172) has been implicated as a more immediate precursor than farnesyl PP (Figure 5.26). This allows different possibilities for folding the carbon chain, dictated of course by the enzyme involved, and cyclization by electrophilic attack on to an appropriate double bond.
E,E-Farnasyl cation, E,Z-Farnasyl cation and nerolidyl cation give rise to matricin, α -santonin, humulene, thapsigargin, bisabolene, α -bisabolol,
caryophyllene,
zingiberene,
sesqui-phellandrene,
chamazulene.
acid,
α -cardinene,
thapsigargin,
parthenolide,
artemisinic
Diterpenoids: The diterpenes arise from geranylgeranyl diphosphate (GGPP). One of the simplest and most important of the diterpenes is phytol, a reduced form of
geranylgeraniol, which forms the lipophilic side-chain of the chlorophylls, e.g. chlorophyll a. E.g. of diterpenoids are
Triterpenoids: Two molecules of farnesyl PP are joined tail to tail to yield the hydrocarbon squalene which act as precursor of triterpenes and steroids. Cyclization of squalene is via the intermediate squalene-2,3oxide (Figure 5.55), produced in a reaction catalysed by a flavoprotein requiring O2 and NADPH cofactors. If squalene oxide is suitably positioned and folded on the enzyme surface, the polycyclic triterpene structures formed can be rationalized in terms of a series of cyclizations, followed by a sequence of concerted Wagner–Meerwein migrations of methyls and hydrides (Figure 5.55). The cyclizations are carbocation mediated and proceed in a stepwise sequence (Figure 5.56).
Examples of triterpenoids prepared from squalene are euphol, lupeol, dammarenediol, taraxasterol, α -amyrin, β -amyrin. E.g. of triterpenoid saponin are hopan-22-ol, tetrahymanol, quillaic acid, barringtogenol, glycyrrhizic acid,
liquiritin, isoliquiritin, carbenoxolone sodium, panaxadiol, panaxatriol, oleanolic acid, syringin. Tetraterpenes: Formation of the tetraterpene skeleton, e.g. phytoene, involves tail-to-tail
coupling
of
two
molecules
of
geranylgeranyl
diphosphate (GGPP) in a sequence essentially analogous to that seen for squalene and triterpenes (Figure 5.67). For phytoene biosynthesis, a proton is lost, generating a double bond in the centre of the molecule, and thus a short conjugated chain is developed. This triene system prevents the type of cyclization seen with squalene. Conjugation is extended then by a sequence of desaturation reactions, removing pairs of hydrogens alternately from each side of the triene system, giving eventually lycopene (Figure 5.67). The tetraterpenes are represented by only
one
group
Lycopene,
of
compounds,
α -carotene,
the
carotenoids.
β -carotene,
E.g.
γ -carotene,
zeathanthin, lutein, violaxanthin, capsanthin, fucoxanthin, astaxanthin.
Steroids: The steroids are modified triterpenoids containing the tetracyclic ring system of lanosterol but lacking the three methyl groups at C-4 and C-14.
