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

Article abstract of DOI:10.1002/chem.201801668

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 CH₂OH 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 CH₂OH 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-CH₂OH-1 (89 %), than from 23E-CH₂OH-1 (26 %). The significant yield (26 %) of the aldehyde products from 23E-CH₂OH-1 indicated that C?C bond rotation had occurred owing to the absence of hydrophobic interactions between the hydrophilic 23E-CH₂OH and its binding site. The polycyclization mechanisms of the four different analogs are discussed.

Full text of DOI:10.1002/chem.201801668

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Accepted Article
Title: 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
Authors: Tsutomu Hoshino, Ikki Kaneko, and Yuri Terasawa
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10.1002/chem.201801668
Chemistry - A European Journal
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
Ikki Kaneko,+ Yuri Terasawa,+ and Tsutomu Hoshino*
Graduate School of Science and Technology and Department of Applied Biological
Chemistry, Faculty of Agriculture, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata
950-2181, Japan
* Corresponding author, Prof. Dr.
E-mail: hoshitsu@agr.niigata-u.ac.jp
[+] the authors contributed equally to this work.
1
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Abstract
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 CH2OH
groups at 23E or 23Z-positions of 1, were incubated with SHC. The analog having 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 as
compared to 23E. On the other hand, analogs having the CH2OH 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-CH2OH-1 (89%),
than from 23E-CH2OH-1 (26%). The significant yield (26%) of the aldehyde products
from 23E-CH2OH-1 indicated that C-C bond rotation had occurred due to the absence of
hydrophobic interaction between the hydrophilic 23E-CH2OH and its binding site. The
polycyclization mechanisms of the four different analogs are discussed.
.
Introduction
Terpenoid cyclases have attracted much interest because the structural diversity of
terpenes originates primarily from the different cyclization cascades of the linear
isoprenoid molecules. [1] Naturally occurring cyclic triterpenes consist of more than 120
different carbon skeletons, which are generated by the enzymatic reactions of (3S)-2,3-
oxidosqualene or squalene with triterpene cyclases.[2] Squalene-hopene cyclase (SHC)
converts acyclic squalene (1; C30) into the 6,6,6,6,5-fused pentacyclic triterpenes,
hopene (2) and hopanol (3) in a 5:1 ratio (Scheme 1).[2a,b,h] As shown in Scheme 1, 1 is
folded into a fully pre-organized chair conformation. Proton attack on the terminal
double bond from the acidic DXDD motif,[3] gives tetracyclic 17-epi-dammarenyl cation
4 via the mono-, bi- and tricyclic cations.[2b] Intermediate 4 undergoes a ring expansion
2
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to afford prohopanyl cation 5. [2b] Further cyclization provides pentacyclic hopanyl
cation 6 as the final intermediate. Proton elimination from Me-29 of 6, which
corresponds to Me-30 (Z-Me at C-23) of 1 and is oriented toward the lower face of the
molecule (as indicated by the blue box), affords 2 (path a). Nucleophilic attack of a
water molecule onto cation 6 provides 3 (path b). The site-directed mutagenesis
experiments have revealed the active sites crucial for the catalysis.[2b] Recently, Hauer
and coworkers have reported the successful creation of the mutated SHCs that enable
the productions of novel and useful truncated cyclic terpenes and non-terpenoid
compounds.[2i, 5] The enzymatic products obtained from substrate analogs also have
provided important information on the cyclization mechanisms by SHC.[4, 6-17] We have
reported the enzymatic reactions of many squalene analogs by Alicyclobacillus
acidocaldarius SHC (AaSHC).[4, 7-17] Notably, our experiments on squalene analogs
lacking methyl groups at the two termini have demonstrated that the methyl groups
present at both the initiation site (C-2 position) and the terminal site (C-23 position) are
required for the normal polycyclization pathway, which leads to the formation of
hopene scaffold 2 (Fig. 1A).
1. Both Me-1 and Me-25 of 1 at the C-2 position are required for the initiation of the
polycyclization reaction, that is, if either group is missing, the cyclization reaction
will not proceed.[7,10]
2. 30-Norsqualene 7, which lacks the Z-Me at C-23, exclusively afforded product 8
with a 6,6,6,6,6-fused pentacyclic tetrahymanol scaffold, suggesting that the SHC
enzyme binds tightly to the Z-Me (Me-30 of 1), leading to the formation of the five-
membered E-ring of 2.[ 7,10]
3. 24-Norsqualene 9, which lacks the E-Me (Me-24) at C-23 but still has the Z-Me
(Me-30) at C-23, furnished the 6,6,6,6,5-fused neohopane skeleton 10 and
tetrahymanol skeleton 11 (10:11=33:37). This finding also suggests that Me-30 of 1
(marked with a blue box) binds more tightly than Me-24 (indicated by a pink box) to
the E-ring formation site of 2.[ 7,10]
These findings revealed that the 23Z-Me (Me-30 of 1, indicated by a blue box) is
required for the formation of the five-membered E-ring of the hopane skeleton. In fact,
24,30-bisnorsqulaene afforded the 6,6,6,6,6-fused pentacyclic tetrahymanol skeleton 13
as the main product.[10] The products generated from these norsqualenes indicate that
the two terminal Me groups are strongly bound by their respective Me binding or
catalytic sites.
3
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