Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ(8)-Δ(7)-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10(-8) M, Ki/Km=10(-3)) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.

1959 (s), 1811 (s), 1753 (m) aSpectra in cm 1 recorded on a Perkin-Elmer 267 spectrometer and calibrated with polystyrene [CpCo(CO)]3 form an isosceles triangle with the two distinct cobalt-cobalt bond lengths of 2.521 Á (Col-Co3) and 2.449 Á (average of Col-Co2 and Co2-Co3). The molecule has approximate Cs-m symmetry (though no crystallographic symmetry is imposed) with the minor plane passing through Co2, C3, 03, and bisecting the Col-Co3 bond. Though the cobalt-cobalt bond lengths of [CpCo(CO)]3 vary from 2.440 to 2.521 Á, this variation is well within the range found for other reported cobalt-cobalt single bonds: 2.365 A, Cp3Co3(M3-CO)(Ai3-0);6 2.463 A, (/-Bu2-C2)Co2(CO)6;9 and 2.457-2.527 A, Co4(CO)12.10The solid-state infrared spectrum of [CpCo(CO)]3, 1833, 1775, and 1673 cm-1, (cf. 1835, 1775, and 1675 cm-1 reported by Vollhardt et al.7) is readily interpreted in terms of the structure reported here. The 1673-cm-1 band is assigned to the C-O stretch of the triply bridging CO while the 1833-and 1775-cm-1 bands can be assigned to symmetric and antisymmetric stretching modes of the semibridging CO's. The solid-state infrared spectrum is somewhat dependent upon what solvent was used for crystallization in that peaks corresponding to King's "second isomer" may or may not be present. However, we feel that in light of the apparent ease with which the positions of the CO ligands can be changed, vide infra, this "second isomer" is basically the same as the structure reported here but with only slight changes in the three CO positions.

Squalene, squalene-2,3-oxide, triterpenes and 4a-methylsterols of Euglena gracilis Z . have been characterised by thin-layer chromatography, gas chromatography and mass spectrometry. Their distribution in dark-grown and light-grown Euglena cells is quantitatively and qualitatively different, reflecting differences in the sterol compositions. However the sterol precursor is found to be in each case cycloartenol, whereas lanosterol is not detected. A new 4wmethylsterol is isolated and the structure of 4a-24-dimethy1-5a-cholesta-8(9)-en-3~-01 proposed. 24-Methylene lanostenol is formed in light-grown Euglena. None of the compounds described have been found in water-soluble fractions nor in the form of fatty-acid esters. The following compounds have also been isolated : 24-methylene cycloartanol, 24-methylene agnostenol ( T ), @-amyrin, obtusifoliol, 24-methylene lophenol and 4a-methylzymosterol.In a preceding paper [l] the free and bound sterols of the unicellular algae Euglena gracilia Z. were described. In summary it was shown that, when grown in the light, Euglena contains mainly free A7-sterols with a methyl or ethyl substituent a t (2-24. By contrast, dark-grown Euglena elaborates a small amount of free sterols and mainly sterol esters with ad6-double bond either not substituted a t C-24 or with an ethyl substituent.The finding that free A7-sterols occur almost only in green cells suggested that they might be associated with the chloroplast, probably as constituents of the chloroplast membranes, the other cell membranes being poor in sterols. It seems therefore that the sterol metabolism is greatly influenced by the chloroplast system. On the other hand it is established that the sterol precursor is cycloartenol for chlorophyllcontaining organisms and lanosterol for organisms containing no chlorophyll (animals and fungi).Euglena gracilis, with its ability to exist with or without chlorophyll, seems to be especially favourable for comparing biochemical patterns. As part of our study, we examined the sterol precursors in green and etiolated Euglena and found that both contain cycloartenol, while lanosterol could not be detected. However the squalene, triterpene and 4a-methylsterol fractions are qualitatively and quantitatively Trivial Names. Lanosterol, 8,24-lanoetadien-3D-o1; 24-methylene lanostenol, 38-hydroxy-24-methyIene-S-lanostene ; cycloartenol, 9,19-cyclolanost-24-en-3~-ol; 24-methylene cycloartanol, 3~-hydroxy-24-methylene-9,19-cyclolanostane ; 24-methylene agnosterol, 3p-hydroxy-24-methylene-7,9( 11)-Ianostadiene ; cycloeucslenol, 4a, 14a-dimethyl-24-methylene-9,19-cyclocholestan-3~-ol; obtusifoliol, 4~,14a-dimethyl-24-methylene-8(9)-cholestene-3~-01.Merent, reflecting differences observed in the sterol fractions. In an earlier report we described a watersoluble form of sterols making up to 40°/, of the total sterols of Euglena [2]. However sterol precursors were not found in the present study in the watersoluble fraction nor in the form of fatty-acid esters. MATERIALS AND METHODS Cultures of E. gracil...

A cell‐free syqtem from the bacterium Acetobacter pasteurianum was incubated with [12‐3H]‐squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin‐layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum. The same cell‐free system transformed (RS)‐2,3‐epoxy‐2,3‐dihydro‐[12,13‐3H]squalene into labelled 3α‐hydroxyhop‐22(29)‐ene, 3β‐hydroxyhop‐22(29)‐ene, hopane‐3α,22‐diol and hopane3β,22‐diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3‐epoxy‐squalene, into 3‐hydroxyhopanoids normally absent in the bacterium. The 3α‐hydroxy and 3β‐hydroxyhopanoids could have been enzymatically interconverted via the 3‐oxo compound. Synthetic racemic (RS)‐2,3‐epoxy‐2,3‐dihydro‐[3‐3H]squalene was incubated and gave rise to 3‐3H‐labelled 3α and 3β‐hydroxyhopanoids. This excludes an isomerization via a 3‐oxo compound which would give unlabelled 3‐hydroxyhoparoids. In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, saualene, by the corresponding cpoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.

An analysis of the total sterol content has been made of a yeast strain during aerobic adaptation. Transmethylation occurs principally a t the 4-demethylsterol level, but conditions were obtained wherein a small fraction of the transmethylations were with 4a-methyl precursors. A'-Zymostenol has been tentatively identified as a product previously unreported in yeast. Esterification was observed in all sterol intermediates and products. Cell-free cyclization of 2,3-epoxy-22-methylene squalene to 24-methylene lanosterol occurs with yeast.'In the synthesis of ergosterol in yeast the methylation event a t C-24 donates to the nascent sterol skeleton the only carbon atom not immediately derived from acetate [i]. S-Adenosyl-methionine provides the methyl group, and although the mechanism of the reaction has been elucidated [2 -41, the recipient substrate for the methyl group has not been precisely determined. Towards this end two general lines of approach have been made on the problem, often with conflicting findings. The first involves a detailed study of the sterolic components of yeast followed by feeding suspected intermediates and observing their conversion to ergosterol. A second approach has been to isolate the transmethylating enzyme and determine possible substrates and products in vitro.Because zymosterol had been reported not to be a precursor of ergosterol [5], and was the only 4-demethyl-sterol in yeast not methylated a t C-24 [S], it was assumed that transmethylation must occur sometime prior to the oxidative removal of all the extra methyl groups a t 401, 4j3, and 14a positions, and the rearrangement of the unsaturation in ring B.Significant amounts of 4,4-dimethyl and 4a-methyl sterols have been demonstrated in yeast preparations [7].Using the first experimental approach described above, evidence has been obtained allowing for the methylation a t the 4,4-dimethyl and 4a-methyl sterol levels [S -101. Although preliminary experiments were interpreted to the contrary [ill, the in vitro approach seems unequivocal in its interpretation that transmethylation occurs a t the level of zymosterol [12,13]. However, our own early experiments [I41 and those of Katsuki and Bloch [15] showed the methylation of endogenous sterols to be unstimulated and, in fact, inhibited by zymosterol.I n order to assess the importance of transmethylation a t the level of the 4,4-dimethyl and 4a-methyl sterols, experiments were needed wherein transmethylation would occur under physiological eonditions and unperturbed by exogenous substrates.Under anaerobic conditions, yeast is unable to synthesize sterols but accumulates large amounts of squalene [16]. This hydrocarbon is rapidly converted to sterols, when molecular oxygen becomes available to the culture [17,18]. The sterol transmethylase is present (although presumably non-functional [ 191) under anaerobic conditions, but a further synthesis of the enzyme is also stimulated by aeration [ZO]. For the experiments we report here, yeast was grown under anaerobic condition ; the cultures...