Forskolin

In a screening programme of Indian medicinal plants, extracts from the roots of Coleus forskohlii (Labiatae/Lamiaceae) were discovered to lower blood pressure and have cardioactive properties. This led to the isolation of the diterpene forskolin (= coleonol) (Figure 5.51) as the active principle in yields of about 0.1%. Forskolin has been shown to exert its effects by direct stimulation of adenylate cyclase, and has become a valuable pharmacological tool in the study of this enzyme and its functions. It has shown promising potential for the treatment of glaucoma, congestive heart failure, hypertension, and bronchial asthma, though drug use is limited by poor water-solubility, and derivatives or analogues will need to be developed.

concerted cyclizations initiated by allylic cation c

geranylfarnesyl PP (GFPP)

concerted cyclizations initiated by allylic cation

Geranylfarnesyl Diphosphate

W-M 1,5-hydride shift generates allylic cation; cyclization on to this cation follows, and the cation is eventually quenched by water

W-M 1,5-hydride shift generates allylic cation; cyclization on to this cation follows, and the cation is eventually quenched by water

Sesquiterpene Ophiobolins

ophiobolin A

Figure 5.52

Sesquiterpene Ophiobolins

ophiobolene (ophiobolin F)

ophiobolin A

ophiobolene (ophiobolin F)

Figure 5.52

GFPP

sclarin

GFPP

sclarin

Figure 5.53

as a precursor of triterpenes and steroids; several seed oils are now recognized as quite rich sources of squalene, e.g. Amaranthus cruentus (Amaran-thaceae). During the coupling process, which on paper merely requires removal of the two diphos-phate groups, a proton from a C-1 position of one molecule of FPP is lost, and a proton from NADPH is inserted. Difficulties with formulating a plausible mechanism for this unlikely reaction were resolved when an intermediate in the process, presqualene diphosphate, was isolated from rat liver. Its characterization as a cyclopropane derivative immediately ruled out all the hypotheses current at the time.

The formation of presqualene PP is represented in Figure 5.54 as attack of the 2,3-double bond of FPP on to the farnesyl cation, analogous to the chain extension using IPP (see also the proposal for the origins of irregular monoterpenes, page 186). The resultant tertiary cation is discharged by loss of a proton and formation of the cyclopropane ring, giving presqualene PP. Obviously, to form squa-lene, carbons-1 of the two FPP units must eventually be coupled, whilst presqualene PP formation has actually joined C-1 of one molecule to C-2 of the other. To account for the subsequent change in bonding of the two FPP units, a further cyclopropane cationic intermediate is proposed. Loss of

squalene synthase

squalene synthase

Squalene Synthase Mechanism

allylic cation electrophilic addition giving tertiary cation

,OPP

allylic cation electrophilic addition giving tertiary cation

,OPP

Presqualene Diphosphate Mechanism

loss of proton with formation of cyclopropane ring loss of diphosphate gives primary cation

presqualene PP

presqualene PP

(NADPH)

cation quenched by attack of hydride

(NADPH)

loss of proton with formation of cyclopropane ring loss of diphosphate gives primary cation le gives _

W-M 1,3-alkyl shift

1,3-alkyl shift generates new cyclopropane ring and more favourable tertiary cation

bond cleavage produces alkene and favourable allylic cation cation quenched by attack of hydride squalene

Figure 5.54

squalene

Figure 5.54

diphosphate from presqualene PP would give the unfavourable primary cation, which via Wagner -Meerwein rearrangement can generate a tertiary carbocation and achieve the required C-1 -C-1' bond. Breaking the original but now redundant C-1 -C-2' bond can give an allylic cation, and the generation of squalene is completed by supply of hydride from NADPH.

Cyclization of squalene is via the intermediate squalene-2,3-oxide (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 cycliza-tions, 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 step-wise sequence (Figure 5.56). Thus, protonation of the epoxide group will allow opening of this ring and generation of the preferred tertiary carbocation, suitably placed to allow electrophilic addition to a double bond, formation of a six-membered ring and production of a new tertiary carbocation. This process continues twice more, generating the preferred tertiary carbocation (Markovnikov addition) after each ring formation, though the third ring formed is consequently a five-membered one. This is expanded to a six-membered ring via a Wagner - Meerwein 1,2-alkyl shift, resulting in some relief of ring strain, though sacrificing a tertiary carbocation for a secondary one. A further elec-trophilic addition generates the tertiary protosteryl

Geranylfarnesyl Diphosphate

sequence of W-M 1,2-hydride and 1,2-methyl shifts

Figure 5.55

sequence of W-M 1,2-hydride and 1,2-methyl shifts lanosterol

Figure 5.55

H-hH

loss of proton leads to plants cyclopropane

cycloartenol

protonation of epoxide allows ring opening to tertiary cation electrophilic addition gives tertiary cation + 6-membered ring

HO^CP

squalene oxide squalene oxide

Protosteryl Cation

electrophilic addition H gives tertiary cation protosteryl cation electrophilic addition H gives tertiary cation

electrophilic addition gives tertiary cation + 6-membered ring

HO^CP

electrophilic addition gives tertiary cation + 5-membered ring

W-M rearrangement; ring expansion at expense of tertiary^ secondary cation

W-M rearrangement; ring expansion at expense of tertiary^ secondary cation

protosteryl cation

cation (Figure 5.56). The stereochemistries in this cation are controlled by the type of folding achieved on the enzyme surface, and this probably also limits the extent of the cyclization process. Thus, if the folded squalene oxide approximates to a chair - boat-chair - boat conformation (Figure 5.57), the transient protosteryl cation will

Cholesterol Chair Conformation
squalene oxide
cyclopropane ring formation and loss of proton from C-10 methyl

be produced with these conformational characteristics. This cation then undergoes a series of Wagner - Meerwein 1,2-shifts, firstly migrating a hydride and generating a new cation, migrating the next hydride, then a methyl and so on until a proton is lost forming a double bond and thus creating lanosterol (Figure 5.57). The stereochemistry

Chair Euphol
III
HO

protosteryl cation OH

protosteryl cation OH

various oxidative OAc modifications cucurbitacin E

cucurbitacin E

first sequence of W-M 1,2-hydride and 1,2-methyl shifts

various oxidative OAc modifications

Figure 5.58

first sequence of W-M 1,2-hydride and 1,2-methyl shifts

further sequence of W-M 1,2-methyl and 1,2-hydride shifts, terminated by loss of proton

of the protosteryl cation in Figure 5.57 shows how favourable this sequence will be, and emphasizes that in the ring system, the migrating groups are positioned anti to each other, one group entering whilst the other leaves from the opposite side of the stereocentre. This, of course, inverts configurations at each appropriate centre. No anti group is available to migrate to C-9 (steroid numbering), and the reaction terminates by loss of proton H-9. Lanosterol is a typical animal triterpenoid, and the precursor for cholesterol and other sterols in animals (see page 233) and fungi (see page 254). In plants, its intermediate role is taken by cycloartenol (Figure 5.57), which contains a cyclopropane ring, generated by inclusion of carbon from the methyl at C-10. For cycloartenol, H-9 is not lost, but migrates to C-8, and the carbocation so formed is quenched by cyclopropane formation and loss of one of the methyl protons. For many plant steroids, this cyclopropane ring has then to be reopened (see page 235). Most natural triterpenoids and steroids contain a 3-hydroxyl group, the original epoxide oxygen from squalene oxide.

An additional feature of the protosteryl cation is that the C-10 methyl and H-5 also share an anti-axial relationship, and are also susceptible to Wagner-Meerwein rearrangements, so that the C-9 cation formed in the cycloartenol sequence may then initiate further migrations. This can be terminated by formation of a 5,6-double bond (Figure 5.58), as in the pathway to the cucurbitacins, a group of highly oxygenated triter-penes encountered in the cucumber/melon/marrow family, the Cucurbitaceae. These compounds are characteristically bitter tasting, purgative, and extremely cytotoxic.

Should squalene oxide be folded on to another type of cyclase enzyme, this time in a roughly chair-chair-chair-boat conformation (Figure 5.59), then an identical carbocation mechanism ensues, and the transient dammarenyl cation formed now has different stereochemical features to the protosteryl cation. Whilst a series of Wagner - Meerwein migrations can occur, there is relatively little to be gained on purely chemical grounds, since these would invert stereochemistry and destroy the already favourable conformation. Instead, the dammarenyl cation typically undergoes further carbocation promoted cyclizations, without any major changes to the ring system already formed. There are occasions in which the migrations do occur, however, and euphol from Euphorbia species (Euphorbiaceae) is a stereoisomer of lanosterol (Figure 5.55).

Should the Wagner - Meerwein rearrangements not occur, the dammarenyl cation could be quenched with water, giving the epimeric dammarene-diols, as found in Dammar resin from Bal-anocarpus heimii (Dipterocarpaceae) and ginseng* (Panax ginseng; Araliaceae) (Figure 5.60). Alternatively, the migration shown to give the

chair - chair - chair - boat squalene oxide chair - chair - chair - boat squalene oxide

sequence of W-M 1,2-hydride and 1,2-methyl shifts terminated by loss of proton

sequence of W-M 1,2-hydride and 1,2-methyl shifts terminated by loss of proton

Figure 5.59

Labdadienyl
euphol

Figure 5.59

baccharenyl cation relieves some ring strain by creating a six-membered ring, despite sacrificing a tertiary carbocation for a secondary one. A pentacyclic ring system can now be formed by cyclization on to the double bond, giving a new five-membered ring and the tertiary lupenyl cation. Although this appears to contradict the reasoning used above for the dammarenyl ^ baccharenyl transformation, the contribution of the enzyme involved must also be considered in each case. A five-membered ring is not highly strained as evidenced by all the natural examples encountered. Loss of a proton from the lupenyl cation gives lupeol, found in lupin (Lupinus luteus; Legumi-nosae/Fabaceae). Ring expansion in the lupenyl cation by bond migration gives the oleanyl system, and labelling studies have demonstrated this ion is discharged by hydride migrations and loss of a proton, giving the widely distributed P-amyrin. Formation of the isomeric a-amyrin involves first the migration of a methyl in the oleanyl cation, then discharge of the new taraxasteryl cation by three hydride migrations and loss of a proton. Loss of a proton from the non-migrated methyl in the taraxasteryl cation is an alternative way of achieving a neutral molecule, and yields taraxasterol found in dandelion (Taraxacum officinale; Compositae/Asteraceae). Comparison with a-amyrin shows the subtly different stereochemistry present because the inversions of configuration caused by hydride migrations have not occurred. Where evidence is available, these extensive series of cyclizations and Wagner - Meerwein rearrangements appear to be catalysed by a single enzyme, which converts squalene oxide into the final product, e.g. lanosterol, cycloartenol, a-amyrin, or P-amyrin.

Bacterial membranes frequently contain hopa-noids (Figure 5.61), triterpenoid compounds that appear to take the place of the sterols that are typically found in the membranes of higher organisms, helping to maintain the structural integrity and to control permeability. Hopanoids arise from squa-lene by a similar carbocation cyclization mechanism, but do not involve the initial epoxidation to squalene oxide. Instead, the carbocation is produced by protonation (compare the cyclization of GGPP to labdadienyl PP, page 207), and the resultant compounds tend to lack the characteristic 3-hydroxyl group, e.g. hopan-22-ol from Alicy-clobacillus acidocaldarius (Figure 5.61). On the other hand, tetrahymanol from the protozoan ring expansion at expense of tertiary — secondary cation

ring expansion at expense of tertiary — secondary cation

5-membered ring formation gives tertiary cation baccharenyl cation cation quenched by H2O; attack may occur from either side of cation OH

5-membered ring formation gives tertiary cation cation quenched by H2O; attack may occur from either side of cation OH

dammarenediols baccharenyl cation two 1,2-hydride shifts then loss of proton; note inversion of stereochemistry H

Ha^Hj

two 1,2-hydride shifts then loss of proton; note inversion of stereochemistry H

Ha^Hj

P-amyrin

dammarenediols ring expansion at expense of tertiary - secondary cation

lupenyl cation

lupeol lupenyl cation

W-M 1,2-alkyl shift

W-M 1,2-alkyl shift

1,2-methyl shift oleanyl cation

1,2-methyl shift allows secondary - tertiary cation

1,2-methyl shift lupeol three 1,2-hydride shifts, then loss of proton; note inversions of stereochemistry

oleanyl cation

Hydride Shift

taraxasterol

Figure 5.60

three 1,2-hydride shifts, then loss of proton; note inversions of stereochemistry

a-amyrin

P-amyrin taraxasterol a-amyrin

Figure 5.60

Tetrahymena pyriformis, because of its symmetry, might appear to have a 3-hydroxyl group, but this is derived from water, and not molecular oxygen as would be the case if squalene oxide were involved. As in formation of the proto-steryl cation (page 214), Wagner - Meerwein ring expansions occur during the cyclization mechanisms shown in Figure 5.61 so that the first-formed tertiary carbocation/five-membered ring

(Markovnikov addition) becomes a secondary carbocation/six-membered ring.

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  • Ermes Pinto
    How to form carbocation in chair chair chair boat conformation of squalene?
    8 years ago

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