2-3-oxidosqualene and diploptene

To provide insight into the polycyclization mechanism of squalene by squalene-hopene cyclase (SHC) from Alicyclobacilus acidocaldarius, some analogs of nor- and bisnorsqualenes were synthesized including the deuterium-labeled squalenes and incubated with the wild-type SHC, leading to the following inferences. (1) The deprotonation reaction for the introduction of the double bond of the hopene skeleton occurs exclusively from the Z-methyl group on the terminal double bond of squalene. (2) 3R-Oxidosqualene was folded in a boat conformation for the A-ring construction, while the 3S-form was in a chair structure. (3) The terminal two methyl groups are indispensable both for the formation of the 5-membered E-ring of the hopene skeleton and for the initiation of the polycyclization cascade, but the terminal Z-methyl group has a more crucial role for the construction of the 5-membered E-ring than the E-methyl group. (4) Some of the novel terpene skeletons, 36, 37, 39 and 40, were created from the analogs employed in this investigation.

Topics: Alkenes; Binding Sites; Bridged-Ring Compounds; Chromatography, Gas; Cyclization; Deuterium; Gram-Positive Bacteria; Intramolecular Transferases; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Structure; Polycyclic Compounds; Recombinant Proteins; Squalene; Stereoisomerism; Structure-Activity Relationship; Triterpenes; Water

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.

Topics: Acetobacter; Cyclization; Lyases; Squalene; Stereoisomerism; Substrate Specificity; Triterpenes