L.J. Goad

Phytosterol side chain biosynthesis

The typical plant sterols contain a substituent at C-24 of the side chain. This can be a methylene, ethylidene, methyl, or ethyl group; with the last three groups, all possible isomers have been reported in nature. The C-24 alkyl groups are derived by a transmethylation reaction from methionine. The details of several distinct alkylation mechanisms, which are now recognized in a range of lower and higher plants, have been reviewed. The operation of these different routes may have some phylogenetic significance.

Sterols of coffee

Abstract The sterols of the bean of the coffee tree ( Coffea arabica L.) have been identified as cycloartenol, 24-methylenecyloartanol, cycloeucalenol, obtusifoliol, citrostadienol, 24-methylenelophenol, stigmasterol, sitosterol, campesterol, stigmastanol, and campestanol. No trace of the previously reported lanosterol, dihydrolanostero, or coffeasterol could be detected in either the beans or in the commercial coffee oil. Trace amounts of cholesterol and cholestanol were also indicated. Combined gas chromatography-mass spectrometry has tentatively identified 4α,24 R -dimethyl-5α-cholest-8-en-3β-ol, 4α-24 R -dimethyl-5α-cholest-7-en-3β-ol, and 4α-methyl-5α-stigmast-7-en-3β-ol.

Observations on the biosynthesis of 24-methylcholesterol and 24-ethylcholesterol by Zea mays

Abstract Examination of the sterols of Zea mays shoots has established that the 24-ethylcholesterol is predominately the 24α-epimer, sitosterol, but the 24-methylcholesterol is a mixture of the 24α- and 24β-epimers. After incubation of Z. mays shoots with [2- 14 C, (4 R )4- 3 H 1 ]mevalonic acid the sitosterol had a 3 H: 14 C atomic ratio of 2.09:5 which is consistent with previous results indicating that a Δ 24(25) -sterol is implicated in its biosynthesis. By contrast, the 24α- and 24β-methylcholesterol mixture had a higher 3 H: 14 C atomic ratio of 2.82:5. This can be explained by the operation of two routes for the elaboration of the 24-methylcholesterol side chain. One may proceed via Δ 24(25) - and Δ 24(25) -sterols to produce the 24α-methylcholesterol with a 3 H: 14 C atomic ratio of 2:5. The other route may involve reduction of either a Δ 24(28) -, a Δ 23 - or a Δ 25 -sterol intermediate to give the 24β1-methylcholesterol with a 3 H: 14 C atomic ratio of 3:5. The proportion of these two labelled compounds in the mixture then determines the observed 3 H: 14 C atomic ratio (2.82:5). Some evidence for the formation of a Δ 25 -compound, cyclolaudenol, by Z. mays shoots was provided by incorporation studies employing either [2- 14 C]mevalonic acid or [Me- 14 C]methionine as the sterol precursor.

The conversion of 24-ethylidene sterols into poriferasterol by the alga Ochromonas malhamensis

Abstract A convenient method is described for the preparation of fucosterol-[7- 3 H 2 ] and 28-isofucosterol-[7- 3 H 2 ]. Both of these 24-ethylidene sterols, as well as 5α-stigmasta-7, Z -24(28)-diene-3β-ol-[2,4- 3 H 4 ], were converted into the 24β-ethyl sterol, poriferasterol, by cultures of the chrysophyte alga Ochromonas malhamensis . However, fucosterol-[7- 3 H 2 ] was not so efficiently incorporated as the other two compounds thus indicating that the configuration of the 24-ethylidene group is of some importance. It is suggested that a 24-ethylidene sterol of the Z -configuration is produced in de novo poriferasterol synthesis and that a Δ 22,24(28) -diene may be an important subsequent intermediate.

Isolation of 14α-methyl-9β,19-cyclo-5α-ergost-24(28)-en-3β-ol from Musa sapientum

Abstract 14α-Methyl-9β,19-cyclo-5α-ergost-24(28)-en-3β-ol has been isolated from Musa sapientum and is found primarily in the esterified sterol fraction. It is also released by saponification of tissue from which all solvent-extractable lipid has been previously removed. The relevance of these results is discussed in relation to the metabolism of sterols in this tissue.

A 37-kDa peroxidase secreted from liverworts in response to chemical stress

A peroxidase was purified from the culture medium of a suspension culture of Marchantia polymorpha (liverwort) after treatment with bornyl acetate, which acts as a chemical stress agent to the cells. The peroxidase was characterised as a glycoprotein of molecular mass 37-kDa having a pl of about 10 and an optimal pH of 6.5. The peroxidase was thermally stable at 50 degrees C for up to 60 min. The partial amino acid sequence of the peroxidase was determined and found to be dissimilar to the amino acid sequences of other higher plant peroxidases. The oxidative polymerization of lunularin by this peroxidase was examined and the formation of a dimer, a trimer and a tetramer was demonstrated by negative ion Fast Atom Bombardment (FAB)-mass spectroscopy of the reaction products.

Isolation of a new sterol from potato leaves

Abstract A new sterol of probable structure 4α,14α-dimethylcholesta-8,24-dien-3°-ol has been identified in potato leaves. A possible intermediate role for this compound in cholesterol biosynthesis in plants is discussed.

Four Δ5,7-sterols from terbinafine treated celery cell suspension cultures

Abstract Incubation of celery cell suspension cultures with the squalene epoxidase inhibitor, terbinafine, caused an accumulation of squalene, a depletion of steryl esters and the appearance of appreciable amounts of di- and tri-unsaturated sterols in the free sterol fraction. The four major unsaturated sterols were isolated by preparative reverse phase HPLC and identified by UV, 1 H NMR and mass spectrometry as campesta-5,7-dien-3β-ol, stigmasta-5,7-dien-3β-ol, [24(24 1 )Z]-stigmasta-5,7,24(24 1 )-trien-3β-ol and (22 E )-stigmasta-5,7,22-trien-3β-ol. Small amounts of Δ 8 - and Δ 8, 14 -sterols were also observed to accumulate in the terbinafine treated cultures. These results apparently reveal other sites of terbinafine inhibition of the sterol biosynthetic pathway in plants at the Δ 7 -reductase and Δ 14 -reductase steps, in addition to its primary inhibitory effect on squalene epoxidase.

Synthesis of deuterium labeled cholesterol and steroids and their use for metabolic studies.

A simple method is described for the preparation of [6,7,7−2H3] sterols and steroids. The synthesis starts with a Δ5-sterol or steroid and involves preparation of the 6-oxo-3α,5α-cyclosteroid, base exchange in the presence of deuterium oxide to introduce two deuteriums at the C-7 position and sodium borodeuteride reduction of the 6-oxo group to introduce the third deuterium atom at C-6. Rearrangement of the [6,7,7−2H3]6α-hydroxy-3α,5α-cyclosteroid then gives the desired [6,7,7-2H3]-Δ5 sterol or steroid. [6,7,7−2H3]Cholesterol, [6,7,7−2H3]pregnenolone and [6,7,7−2H3]3β-hydroxyandrost-5-en-17-one were synthesized in this fashion and [6,7,7−2H3]progesterone was prepared from the [6,7,7−2H3]pregnenolone. Three examples of the use of these deuchromatography-mass spectrometry. The chrysophyte alga,Ochromonas malhamensis, was shown to be capable of introducing an extra methyl or ethyl group at C-24 of the side chain of [6,7,7−2H3]cholesterol to yield brassicasterol and poriferasterol, respectively. The ovary of the echinoderm,Asterias rubens, was demonstrated to metabolize [6,7,7−2H3]progesterone to yield mainly the 5α-isomers of pregnane-3,20-dione and 3β-hydroxypregnan-20-one. However, the 5β-isomers of these compounds were also detected as minor products for the first time as progesterone metabolites in this animal. Isolated oocytes of the frog,Xenopus laevis, produced a number of metabolites of [6,7,7−2H3]progesterone. In this report, two of them were shown to be 17α-hydroxy-pregn-4-en-3,20-dione and 20α-hydroxypregn-4-en-3-one.

The stereochemistry of polyporenic acid a methyl ester

Abstract The absolute configuration of the terminal asymmetric carbon centre in the side-chain of the methyl ester of polyporenic acid A, from Piptoporus betulinus Fries, is shown to be 25(S)(2).