Squalene-Hopene Cyclase: Mechanistic Insights into the Polycyclization Cascades of Squalene Analogs Bearing Ethyl and Hydroxymethyl Groups at the C-2 and C-23 Positions.

Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene 1 into 6,6,6,6,5-fused pentacyclic hopene 2 and hopanol 3. To elucidate the binding sites for the terminal positions of 1, four analogs, having the larger ethyl (Et) and the hydrophilic CH2 OH groups at the 23E or 23Z positions of 1, were incubated with SHC. The analog with the Et group at the 23E position (23E-Et-1) yielded two tetra- and three pentacyclic products; however, the analog possessing the Et group at the 23Z position (23Z-Et-1) gave two hopene homologs and the neohopane skeleton, but no hopanol homologs. Hopene homolog (C31 ) was generated from 23E-Et-1 by deprotonation from 23Z-Me (normal cyclization cascade). Intriguingly, the same homolog was also generated from the geometrical isomer 23Z-Et-1, indicating C-C bond rotation about the C-21-C-22 axis of the hopanyl cation and the more compact nature of the binding domain at 23Z compared with 23E. On the other hand, analogs with the CH2 OH group gave novel hopane skeletons having 1-formylethyl and 1-hydroxyprop-2-en-2-yl residues at C-21. Products bearing an aldehyde group were generated in higher yield from 23Z-CH2 OH-1 (89 %), than from 23E-CH2 OH-1 (26 %). The significant yield (26 %) of the aldehyde products from 23E-CH2 OH-1 indicated that C-C bond rotation had occurred owing to the absence of hydrophobic interactions between the hydrophilic 23E-CH2 OH and its binding site. The polycyclization mechanisms of the four different analogs are discussed.

ß-Amyrin Biosynthesis: Promiscuity for Steric Bulk at Position 23 in the Oxidosqualene Substrate and the Significance of Hydrophobic Interaction between the Methyl Group at Position 30 and the Binding Site.

To examine how the sterics at the 23 position of (3S)-2,3-oxidosqualene 1 influence the polycyclization cascade in ß-amyrin biosynthesis, substrate analogues substituted with an ethyl group (10, 11), a hydrogen atom (12, 13), or a propyl residue (14) at the 23 position were incubated with ß-amyrin synthase. The bulkier ethyl group was accepted as a substrate, leading to formation of the ß-amyrin skeleton (42, 43) without truncation of the multiple cyclization reactions. Analogue 13, possessing a hydrogen atom and an ethyl group at the 23E and 23Z positions, respectively, was also converted into the ß-amyrin skeleton 45. However, the analogue lacking an ethyl group at the 23Z position (12) underwent almost no conversion, strongly indicating that an alkyl group must exist at the Z position. The cyclization of the analogue with a propyl substituent at the Z position (14) was poor. Analogue 15 possessing CH2OH at the 23E position afforded a new compound 47 in a high yield as a result of trapping of the final oleanyl cation. Conversely, 16 with 23Z-CH2OH afforded novel compounds 48-50 in low yields, which resulted from the intermediary dammarenyl and baccharenyl cations. Therefore, the hydrophobic interaction between the 23Z-alkyl group and its binding site (possibly via CH/π interaction) is critical for adopting the correct chair-chair-chair-boat-boat conformation and for the full cyclization cascade.

ß-Amyrin Biosynthesis: The Methyl-30 Group of (3S)-2,3-Oxidosqualene Is More Critical to Its Correct Folding To Generate the Pentacyclic Scaffold than the Methyl-24 Group.

Oxidosqualene cyclases catalyze the transformation of oxidosqualene (1) into numerous cyclic triterpenes. Enzymatic reactions of 24-noroxidosqualene (8) and 30-noroxidosqualene (9) with Euphorbia tirucalli ß-amyrin synthase were conducted to examine the role of the branched methyl groups of compound 1 in the ß-amyrin biosynthesis. Substrate 8 almost exclusively afforded 30-nor-ß-amyrin (>95.5 %), which was produced through a normal cyclization pathway, along with minor products (<4.5 %). However, a lack of the Me-30 group (analogue 9) resulted in significantly high production of premature cyclization products, including 6/6/6/5-fused tetracyclic and 6/6/6/6/5-fused pentacyclic skeletons (64.6 %). In addition, the fully cyclized product (35.4 %) having the 6/6/6/6/6-fused pentacycle was produced; however, the normally cyclized product, 29-nor-ß-amyrin was present in only 18.6 % of these products. The conversion yield of substrate 8 possessing a Z-Me group at the terminus was approximately twofold greater than that of compound 9 with an E-Me group. Thus, the Me-30 group is essential for the correct folding of a chair-chair-chair-boat-boat conformation of compound 1 for the production of the ß-amyrin scaffold, whereas the Me-24 group exerts little influence on the normal polycyclization cascade. Here, we show that the Me-30 group plays critical roles in constructing the ordered architecture of a chair-chair-chair-boat-boat structure, in facilitating the ring-expansion reactions, and in performing the final deprotonation reaction at the correct position.