Phytosterols

The major sterol found in mammals is the C27 compound cholesterol, which acts as a precursor for other steroid structures such as sex hormones and corticosteroids. The main sterols in plants, fungi, and algae are characterized by extra one-carbon or two-carbon substituents on the side-chain, attached at C-24. These substituent carbons are numbered 241 and 242 (Figure 5.77); some older publications

Campesterol

ergosterol

campesterol Figure 5.100

ergosterol

campesterol Figure 5.100

may use 28 and 29. The widespread plant sterols campesterol and sitosterol (Figure 5.100) are respectively 24-methyl and 24-ethyl analogues of cholesterol. Stigmasterol contains additional unsaturation in the side-chain, a trans-A22 double bond, a feature seen in many plant sterols, but never in mammalian ones. The introduction of methyl and ethyl groups at C-24 generates a new chiral centre, and the 24-alkyl groups in campesterol, sitosterol, and stigmasterol are designated a. The predominant sterol found in fungi is ergosterol (Figure 5.100), which has a P-oriented 24-methyl, as well as a trans-A22 double bond and additional A7 unsaturation. The descriptors a and P unfortunately do not relate to similar terms for the steroid ring system, but are derived from consideration of Fischer projections for the side-chain, substituents to the left being designated a and those to the right as p. Systematic RS nomenclature is preferred, but note that this defines sitosterol as 24R whilst stigmasterol, because of its extra double bond, is 24S. The majority of plant sterols have a 24a-methyl or 24a-ethyl substituent, whilst algal sterols tend to have 24P-ethyls, and fungi 24^-methyls. The most abundant sterol in brown algae (Fucus spp.; Fucaceae) is fucosterol (Figure 5.100), which demonstrates a further variant, a 24-ethylidene substituent. Such groups can have E-configurations as in fucosterol, or the alternative Z-configuration. Sterols are found predominantly in free alcohol form, but also as esters with long chain fatty acids (e.g. palmitic, oleic, linoleic, and a-linolenic acids), as glycosides, and as fatty acylated glycosides. These sterols, termed phytosterols, are structural components of membranes in plants, algae, and fungi, and affect the permeability of these membranes. They also appear to play a role in cell proliferation.

The source of the extra methyl or ethyl side-chain carbons in both cases is S-adenosylmethio-nine (SAM), and to achieve alkylation the side-chain must have a A24 double bond, i.e. the side-chains seen in lanosterol and cycloartenol. The precise mechanisms involved have been found to vary according to organism, but some of the demonstrated sequences are given in Figure 5.101. Methylation of the A24 double bond at C-24 via SAM yields a carbocation which undergoes a hydride shift and loss of a proton from C-241 to generate the 24-methylene side-chain. This can be reduced to a 24-methyl either directly, or after allylic isomerization. Alternatively, the 24-methylene derivative acts as substrate for a second methylation step with SAM, producing a carbocation. Discharge of this cation by proton loss produces a 24-ethylidene side-chain, and reduction or isomerization/reduction gives a 24-ethyl group. The trans-A22 double bond is introduced only after alkylation at C-24 is completed. No stereochemistry is intended in

Ad electrophilic addition; C^. C-methylation using SAM

W-M 1,2-hydride shift followed by loss of proton

second electrophilic addition involving SAM

W-M 1,2-hydride shift followed by loss of proton

second electrophilic addition involving SAM

Double Bond

dehydrogenation allylic isomerization

NADPH

NADPH

NADPH

dehydrogenation dehydrogenation dehydrogenation

Figure 5.101

Figure 5.101. It is apparent that stereochemistries in the 24-methyl, 24-ethyl, and 24-ethylidene derivatives could be controlled by the reduction processes or by proton loss as appropriate. It is more plausible for different stereochemistries in the 24-methyl and 24-ethyl side-chains to arise from reduction of different double bonds, rather than reduction of the same double bond in two different ways. In practice, other mechanisms involving a 25(26)-double bond are also found to operate.

The substrates for alkylation are found to be cycloartenol in plants and algae, and lanos-terol in fungi. The second methylation step in plants and algae usually involves gramisterol (24-methylenelophenol) (Figure 5.102). This indicates that the processes of side-chain alkylation and the steroid skeleton modifications, i.e. loss of methyls, opening of the cyclopropane ring, and migration of the double bond, tend to run concurrently rather than sequentially. Accordingly, the range of plant and algal sterol derivatives includes products containing side-chain alkylation, retention of one or more skeletal methyls, and possession of a cyclopropane ring, as well as those more abundant examples such as sitosterol and stigmasterol based on a cholesterol-type skeleton. Most fungal sterols originate from lanosterol, so less variety is encountered. The most common pathway from lanosterol to ergosterol in fungi involves initial side-chain alkylation to eburicol (24-methylenedihydrolanosterol), which is the substrate for 14-demethylation (Figure 5.103). Loss of the 4-methyls then gives fecosterol, from which ergosterol arises by further side-chain and ring B modifications. Although the transformations are similar to those occurring in the mammalian pathway for lanosterol ^ cholesterol, the initial side-chain alkylation means the intermediates formed are different. Some useful anti-fungal agents, e.g. ketoconazole and miconazole, are specific inhibitors of the 14-demethylation reaction in fungi, but do not affect cholesterol biosynthesis in humans. Inability to synthesize the essential sterol components of their membranes proves fatal for the fungi. Similarly, 14-demethylation in plants proceeds via obtusifoliol (Figure 5.102) and plants are unaffected by azole derivatives developed as agricultural fungicides. The antifungal effect of polyene antibiotics such as amphotericin and nys-tatin depends on their ability to bind strongly to ergosterol in fungal membranes and not to cholesterol in mammalian cells (see page 102).

Sitosterol and stigmasterol (Figure 5.100) are produced commercially from soya beans* (Glycine max; Leguminosae/Fabaceae) as raw materials

Fecosterol Ergosterol
(24-ethylidenelophenol) (24-methylenelophenol)
Figure 5.102

lanosterol eburicol

(24-methylenedihydrolanosterol)

4,4-dimethylfecosterol lanosterol eburicol

(24-methylenedihydrolanosterol)

4,4-dimethylfecosterol demethylations at C-4

Chiral Centers Lanosterol

side-chain and ring B modifications ergosterol side-chain and ring B modifications

demethylations at C-4

Fecosterol Ergosterol

fecosterol

ergosterol fecosterol

Soya Bean Sterols

Soya beans or soybeans (Glycine max; Leguminosae/Fabaceae) are grown extensively in the United States, China, Japan, and Malaysia as a food plant. They are used as a vegetable, and provide a high protein flour, an important edible oil (Table 3.2), and an acceptable non-dairy soybean milk. The flour is increasingly used as a meat substitute. Soy sauce is obtained from fermented soybeans and is an indispensable ingredient in Chinese cookery. The seeds also contain substantial amounts (about 0.2%) of sterols. These include stigmasterol (about 20%), sitosterol (about 50%) and campesterol (about 20%) (Figure 5.100), the first two of which are used for the semi-synthesis of medicinal steroids. In the seed, about 40% of the sterol content is in the free form, the remainder being combined in the form of glycosides, or as esters with fatty acids. The oil is usually solvent extracted from the dried flaked seed using hexane. The sterols can be isolated from the oil after basic hydrolysis as a by-product of soap manufacture, and form part of the unsaponifiable matter.

The efficacy of dietary plant sterols in reducing cholesterol levels in laboratory animals has been known for many years. This has more recently led to the introduction of plant sterol esters as food additives, particularly in margarines, as an aid to reducing blood levels of low density lipoprotein (LDL) cholesterol, known to be a contributory factor in atherosclerosis and the incidence of heart attacks (see page 236). Plant sterol esters are usually obtained by esterifying sitosterol from soya beans with fatty acids to produce a fat-soluble product. Regular consumption of this material (recommended 1.3 g per day) is shown to reduce blood LDL cholesterol levels by 10-15%. The plant sterols are more hydrophobic than cholesterol and have a higher affinity for micelles involved in fat digestion, effectively decreasing intestinal cholesterol absorption. The plant sterols themselves are not absorbed from the GI tract. Of course, the average diet will normally include small amounts of plant sterol esters. Related materials used in a similar way are plant stanol esters. Stanols are obtained by hydrogenation of plant sterols, and will consist mainly of sitostanol (from sitosterol and stigmasterol) and campestanol (from campesterol) (Figure 5.104); these are then esterified with fatty acids. Regular consumption of plant stanol esters (recommended 3.4 g per day) is shown to reduce blood LDL cholesterol levels by an average of 14%. Much of the material used in preparation of plant stanol esters originates from tall oil, a by-product of the wood pulping industry. This contains campesterol, sitosterol, and also sitostanol. The stanols are usually transesterified with rapeseed oil, which is rich in unsaturated fatty acids (see page 43).

Sitostanol Campestanol

sitostanol campestanol

sitostanol campestanol

protosteryl cation

protosteryl cation

CO2H

CO2H

Sitostanol Conformations

CO2H

HO H

fusidic acid

CO2H

HO H

fusidic acid i n^o

Figure 5.105

for the semi-synthesis of medicinal steroids (see pages 266, 279). For many years, only stigmas-terol was utilized, since the A22 double bond allowed chemical degradation of the side-chain to be effected with ease. The utilization of sitosterol was not realistic until microbiological processes for removal of the saturated side-chain became available.

Fusidic acid* (Figure 5.105), an antibacterial agent from Acremonium fusidioides, has no additional side-chain alkylation, but has lost one C-4 methyl and undergone hydroxylation and oxidation of a side-chain methyl. Its relationship to the protosteryl cation is shown in Figure 5.105. The stereochemistry in fusidic acid is not typical of most steroids, and ring B adopts a boat conformation; the molecular shape is comparable to the protosteryl cation (Figure 5.57, page 216).

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