Design of high energy intermediate analogues to study sterol biosynthesis in higher plants.

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.

Non-specific biosynthesis of hopane triterpenes by a cell-free system from Acetobacter pasteurianum.

1. A cell-free system 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. 2. The same cell-free system transformed (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene into labelled 3 alpha-hydroxyhop-22(29)-ene, 3 beta-hydroxyhop-22(29)-ene, hopane-3 alpha,22-diol and hopane-3 beta,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. 3. The 3 alpha-hydroxy and 3 beta-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 3 alpha and 3 beta-hydroxyhopanoids. This excludes an isomerization via a 3-oxo compound which would give unlabelled 3-hydroxyhopanoids. 4. In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, squalene, by the corresponding epoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.

A new approach to the synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene from 3 beta, 21-diacetoxy-5-pregnen-20-one.

Synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene in six steps from 3 beta, 21-diacetoxy-5-pregnen-20-one has been achieved by a new approach. The method would allow to label the side chain of a bile acid at carbon-atom 21.