Mutated variants of squalene-hopene cyclase: Enzymatic syntheses of triterpenes bearing oxygen-bridged monocycles and a new 6,6,6,6,6-fusded pentacyclic scaffold, named neogammacerane, from 2,3-oxidosqualene

Article abstract of DOI:10.1039/c8ob02009d

Squalene-hopene cyclase (SHC) catalyzes the conversion of acyclic squalene molecule into a 6,6,6,6,5-fused pentacyclic hopene and hopanol. SHC is also able to convert (3S)-2,3-oxidosqualene into 3β-hydroxyhopene and 3β-hydroxyhopanol and can generate 3α-hydroxyhopene and 3α-hydroxyhopanol from (3R)-2,3-oxidosqualene. Functional analyses of active site residues toward the squalene cyclization reaction have been extensively reported, but investigations of the cyclization reactions of (3R,S)-oxidosqualene by SHC have rarely been reported. The cyclization reactions of oxidosqualene with W169X, G600F/F601G and F601G/P602F were examined. The variants of the W169L generated new triterpene skeletons possessing a 7-oxabicyclo[2.2.1]heptane moiety (oxygen-bridged monocycle) with (1S,2S,4R)- and (1R,2S,4S)-stereochemistry, which were produced from (3R)- and (3S)-oxidosqualenes, respectively. The F601G/P602F double mutant also furnished a novel triterpene, named neogammacer-21(22)-en-3β-ol, consisting of a 6,6,6,6,6-fused pentacyclic system, in which Me-29 at C-22 of the gammacerane skeleton migrated to C-21. We propose to name this novel scaffold neogammacerane. The formation mechanisms of the enzymatic products from 2,3-oxidosqualene are discussed.

Full text of DOI:10.1039/c8ob02009d

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Fukuda, T.
Watanabe and T. Hoshino, Org. Biomol. Chem., 2018, DOI: 10.1039/C8OB02009D.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Page 1 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
Mutated variants of squalene-hopene cyclase: Enzymatic syntheses of triterpenes
bearing oxygen-bridged monocycles and a new 6,6,6,6,6-fusded pentacyclic
scaffold, named neogammacerane, from 2,3-oxidosqualene
Yoriyuki Fukuda, Takashi Watanabe, 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
Tsutomu Hoshino: Prof. Dr.
*Corresponding author
E-mail: hoshitsu@agr.niigata-u.ac.jp
Electronic Supporting Information (ESI) available: Primers used for the mutagenesis, GC
profiles of the enzymatic reactions, EIMS and NMR spectra of the enzymatic products,
and the NMR analyses for proposing structures.
1

Organic & Biomolecular Chemistry
Page 2 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Abstract
Squalene-hopene cyclase (SHC) catalyzes the conversion of acyclic squalene molecule
into a 6,6,6,6,5-fused pentacyclic hopene and hopanol. SHC is also able to convert (3S)-
2,3-oxidosqualene into 3-hydroxyhopene and 3-hydroxyhopanol and can generate
3-hydroxyhopene and 3-hydroxyhopanol from (3R)-2,3-oxidosqualene. Functional
analyses of active site residues toward the squalene cyclization reaction have
extensively been reported, but investigations of the cyclization reactions of (3R,S)-
oxidosqualene by SHC have rarely been reported. The cyclization reactions of
oxidosqualene with W169X, G600F/F601G and F601G/P602F were examined. The
variants of the W169L generated new triterpene skeletons possessing a 7-
oxabicyclo[2.2.1]heptane moiety (oxygen-bridged monocycle) with (1S,2S,4R)- and
(1R,2S,4S)-stereochemistry, which were produced from (3R)- and (3S)-oxidosqualenes,
respectively. The F601G/P602F double mutant also furnished a novel triterpene, named
neogammacer-21(22)-en-3-ol, consisting of a 6,6,6,6,6-fused pentacyclic system, in
which Me-29 at C-22 of the gammacerane skeleton migrated to C-21. We propose to
name this novel scaffold neogammacerane. The formation mechanisms of the enzymatic
products from 2,3-oxidosqualene are discussed.
Introduction
The polycyclization cascades of squalene 1 and (3S)-2,3-oxidosqualene 4 have been
attractive to organic chemists and biochemists for more than 70 years since Ruzicka and
coworkers proposed the “biogenetic isoprene rule”. 1 The reactions proceed with
complete regiospecificity and stereospecificity to yield sterols and triterpenes2 with
remarkable structural diversity; more than 100 different carbon frameworks that exhibit
important biological activities can be produced.3 Squalene-hopene cyclase (SHC)
converts squalene 1 into a 6,6,6,6,5-fused pentacyclic hopene 2 and hopanol 3 at a ratio
of approximately 5:1. Substrate 1 folds into an all-chair conformation inside the enzyme
cavity, as shown in Scheme 1A. SHC can also tolerate both (3S)-2,3-oxidosqualene 4
and (3R)-2,3-oxidosqualene 7, yielding 3-hydroxyhopene 5 and 3-hydroxyhopanol 6
from 4 and 3–hydroxyhopene 8 and 3–hydroxyhopanol 9 from 7 (Scheme 1B), in
which 4 folds into an all-chair structure, while 7 folds into a boat-chair-chair-chair-chair
conformation in the reaction cavity.4 These polycyclization cascades are attained by a
single enzyme to yield five C-C bonds and nine stereocenters. Recent studies on the
SHC from the bacterium sp. Alicyclobacillus acidocaldarius (AaSHC) have revealed
that AaSHC has high potential for developing new compounds, including terpene and
non-terpenoid skeletons, which result from SHC-mediated enzymatic reactions.4d,5, 6 We
2

Page 3 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
have reported functional analyses of active site residues for the cyclization reactions of
squalene molecule 1 by AaSHC.2b, 2j Intriguingly, we recently discovered that the site-
specific mutant A306V of AaSHC generated an epoxydammarane skeleton from (3R)-
oxidosqualene 7.7 This result indicated that the proton from the DXDD sequence8
attacked the terminal double bond, but not on the epoxide ring, despite the cyclization
reaction usually occurring from the epoxide ring, as seen in Scheme 1B. This unusual
cyclization outcome prompted us to reinvestigate the cyclization reactions of 4 and 7
with mutated SHCs.
We previously reported the cyclization reactions of 1 with W169X variants
(X=Val, Phe and Tyr) and that -electrons are necessary at this position for complete
polycyclization,9 but we have not reported on the reactions of 4 and 7. Prokaryotic
SHCs accept 1, 4 and 7 as substrates to yield 2, 3, 5, 6, 8 and 9, as shown in Schemes
1A and 1B, but eukaryotic oxidosqualene cyclases (OSCs) only accept (3S)-4 to
generate cyclic triterpenes possessing a 3-hydroxyl group.2 Comparison of the amino
acid alignment of prokaryotic SHCs to eukaryotic OSCs suggests that OSCs lack
Gly600, which is conserved among prokaryotic SHCs. Therefore, we created the G600
variant and incubated 1, 4 and 7 with the G600-AaSHC variant.10 No reaction occurred
for substrates 1 and 7, but (3S)-4 underwent the polycyclization reaction to yield mono-
and tricyclic products with the 3-hydroxyl group.10 This is the first report of the
successful transformation of the prokaryotic AaSHC into a eukaryotic OSCs. Here, we
describe the incubation experiments of 1, 4 and 7 with two double mutants
(G600F/F601G and F601G/P602F) to generate detailed information as to why the
G600 variant only accepts (3S)-4. The G600 variant generated mono- and tricyclic
products, but did not furnish further cyclized products, such as tetra- and pentacyclic
scaffolds. We attempted to generate tetra- and pentacyclic products with the 3-
hydroxyl group and/or develop the potential of the AaSHC to create unnatural
compounds. As shown in Fig. S1, the Trp169 residue is in proximity to the C/D-ring
formation sites, while the Gly600 and Phe601 residues are located primarily around the
D/E-ring sites in the reaction cavity.
Here, we report that rare and novel triterpene scaffolds, which possess the 7-
oxabicyclo[2.2.1]heptane ring and the novel 6,6,6,6,6-fused pentacyclic scaffold (named
neogammacerane scaffold), are generated by the enzymatic reactions of 4 and 7 with
mutated SHCs (W169L and F601G/P602F) and discuss the formation mechanisms of
these new products from 4 and 7.
3

Organic & Biomolecular Chemistry
Page 4 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Results
We constructed 6 site-specific mutants: W169V, W169L, W169F, W169Y,
G600F/F601G and F601G/P602F. Primers used for the preparation of these mutants are
shown in Table S1.The preparation of cell-free extracts and incubation conditions are
described in the Experimental section. Incubation experiments for 1 with the W169V
and W169L variants gave no enzymatic product (Fig. S2), although Phe and Tyr
mutants with aromatic -electrons afforded 17-epi-dammara-20(21),24-diene.9 The two
double mutants, G600F/F601G and F601G/P602F, also generated no enzymatic product
in an incubation reaction with 1 (Fig. S12.2), as we reported in the enzymatic reaction
of 1 with the G600 mutant. 10 However, the enzymatic reactions of oxidosqualene (4
and 7) with all variants tested here yielded products in good conversion, as seen in the
Figs. S3 and S4 and Figs. S12.2 and S12.3.
Isolation of products 10─16 from W169X variants.
Fig. S3 shows the product distributions obtained from the incubation of the racemic
mixture of 4 and 7 with native SHC and the W169X variants. Peaks, 5, 6, 8 and 9, were
confirmed to be 3-hydroxyhopene, 3-hydroxyhopanol, 3-hydroxyhopene and 3-
hydroxyhopanol, respectively, as isolated in our previous paper.11 The new prominent
peaks 10─16 were isolated to determine the structures. A racemic mixture of 4 and 7
(100 mg) was incubated with cell-free homogenates (200 mL) prepared from a 2.3 L-
culture of the W169L variant at 55C (pH 6.0) for 20 h. After heating the reaction
mixture at 70-80C for 20 min after adding 15% KOH/MeOH (100 mL), lipophilic
materials were extracted with hexane. The hexane extract was subjected to SiO2 column
chromatography and eluted with hexane/EtOAc (100:0─100:2) to afford approximately
8 fractions, one of which contained almost pure product 12 (18.2 mg). Separation of
other products was unsuccessful. The fractions containing product 13 were again
applied to SiO2 column chromatography (hexane/EtOAc=100:0─100:5), followed by
normal phase HPLC (hexane/2-PrOH=100:0.05─100:0.15), yielding pure 13 (1.2 mg).
The fractions including products 10 and 11 were combined and subjected to normal
phase HPLC (hexane/2-PrOH=100:0.05) to afford 10 (1.1 mg) and 11 (4.2 mg) in a pure
state. The isolation yields did not correspond to the production yields (see the GC
profile in Fig. S3), as there were still many fractions that required further purification.
In a similar way, incubation of the racemic mixture of 4 and 7 (100 mg) with cell-free
homogenates (200 mL) from the W169V mutants was performed. SiO2 column
chromatography (hexane/EtOAc=100:0.1─100:6) afforded two fractions: one contained
a mixture of 14, 16 and 5 and the other fraction was a mixture of 15 and 8. Compounds
4

Page 5 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
5 and 8 were insoluble in MeOH. By washing the two fractions with MeOH, 5 and 8
were removed by filtration. Products 14 and 16 were purified via normal-phase HPLC
(hexane/2-PrOH=100:0.1) and pure 15 was also isolated using the same HPLC
conditions.
Structures of products 10─16 from W169X variants
Substrates 4 and 7 possess 8 vinylic Me groups, which usually appear at H
1.8─1.5. The 1H-NMR spectrum of product 10 (600 MHz, CDCl3、Fig. S5.2) indicated
the presence of 5 vinylic Me groups, which appeared at H 1.68 (s, 3H) and 1.60 (s,
12H) and the presence of 3 aliphatic Me groups (H 1.06, s, 3H, Me-24; 0.90, s, 3H, Me-
25; and 1.37 (s, 3H, Me-26). These chemical shifts imply that 10 is a monocyclic
product, as shown in the monocyclic structure of product 12 (Fig. 1). However, two
oxygenated carbon signals were observed at C 86.26 (d, C-3) and 88.08 (s, C-6) in the
13C-NMR spectrum (150 MHz, CDCl3), suggesting that 10 has an oxygen-bridged
monocyclic structure that differs from the typical monocyclic skeleton (like 12), which
possesses one oxygenated carbon. H-3 (H 3.77, d, J=5.0 Hz, 1H) was correlated with
C-3 in the HMQC spectrum (Fig. S5.7), and the following HMBC cross peaks were
clearly found: H-3/C-6, H-3/C-4 (C 41.76, s), H-3/C-5 (C 56.27, d), Me-26/C-6, Me-
26/C-1 (C 28.75, t), Me-26/C-5, Me-24/C-5 and Me-25/C-5. Furthermore, the COSY
and TOCSY spectra showed that the 1H-1H spin coupling network was observed
between the following three protons: H-3/H-2/H-1. These NMR data unambiguously
indicate that 10 has a 7-oxabicyclo[2.2.1]heptane moiety, as shown in Fig. S5.9. The
1H-NMR spectrum of product 11 (600 MHz, CDCl3、Fig. S6.2) also indicated the
presence of 5 olefinic Me groups (H 1.68, s, 3H) and 1.60 (s, 12H), the H of which
were identical to those of product 10, but the H of three aliphatic Me groups (H 1.04, s,
3H, Me-24; H 1.01, s, 3H, Me-25; and H 1.32, s, 3H, Me-26) were somewhat
different from those of 10. Furthermore, two oxygenated carbons (C 86.06, d, C-3; C
86.71, s, C-6) were also found in the 13C-NMR spectrum of 11 (150 MHz, CDCl3), and
the C of Me-26 was different between 11 and 10. These NMR data indicate that the
side chains of 11 and 10 were identical, but the stereochemistry of monocyclic ring are
different, thus 11 and 10 are diastereomers. Detailed NMR analyses, including 2D NMR
(Fig. S6.9) for product 11 demonstrated that product 11 also had a 7-
oxabicyclo[2.2.1]heptane moiety, which is further supported by the same EIMS spectra
between 10 and 11 (compare Fig. S5.1 with S6.1).
Products 10 and 11 are diastereomers because the complete separation of the two
compounds was successfully achieved. The R- and S-stereochemistry at the three
5

Organic & Biomolecular Chemistry
Page 6 of 33
View Article Online
DOI: 10.1039/C8OB02009D
stereocenters were determined as follows. Authentic (3S)-4 was supplied from cultures
of Saccharomyces cerevisiae, which produces only (3S)-4, but not (3R)-7,2f, 2j and 4 was
isolated via SiO2 column chromatography using hexane/EtOAc (100:0.5─100:20). The
racemic mixture of 4 and 7 was synthesized by treatment of 1 with mPCBA, 6i,j and
(3R)-7 was successfully obtained by separating the racemic mixture of 4 and 7 with a
CHIRAL PACK IA (Daicel Industries LTD) using hexane/THF (100:0.05). Fig. S4
shows gas chromatograms (GC) of hexane extracts obtained by incubating the racemic
mixture of 4 and 7 (A), pure 7 (B) and pure 4 (C) with the W169L variant. Fig. S4C
indicates that products 11, 12 and 13 were produced from 4, but no detectable amount
of substrate 4 was found in the GC trace, indicating that 4 was almost fully converted
into products. By contrast, only product 10 was generated from 7 (Fig. S4B), but the
yield was significantly low. Therefore, the stereocenters at C-3 of products 10 and 11
must have R- and S-configurations, respectively. Careful comparison of the NOESY
spectra (600 MHz, CDCl3) of 10 and 11 indicated that 10 had a distinct NOE between
Me-25 and H-2 (Fig. S5.6.2), but 11 did not exhibit a clear NOE between Me-25 and H-
2 (Fig. S6.6.2), even though a strong NOE was observed between Me-24 and H-2 (Fig.
S5.6.2). This clear difference in NOE between 10 and 11 coincides with the
conformational structures illustrated in Fig. S5.10 and Fig. S6.10. Consequently, the
complete structures of products 10 and 11, including the stereochemistry, are depicted
in Fig. 1.
This bridged bicyclic ether (7-oxabicyclo[2.2.1]heptane) moiety of 11 was reported
to be generated by the enzymatic reactions of (3S)-11-fluoro- and (3S)-14-
fluorooxidosqualenes with native SHC (Fig. S6.11).12 Robustell et al. reported 1H-NMR
chemical shifts for fluorinated compounds A and B (Fig. 1C), but did not report all of
the 1H NMR data (especially with respect to the H of H-5). Furthermore, they did not
assign the 1H- and 13C-signals of the oxygen-bridged monocycle and the configuration
at C-5. 12 Through detailed NOESY analyses of 10 and 11, we unambiguously propose
the S-stereochemistry at C-5; strong NOEs of H-5/ Me-24 and H-5/Me-26 for 10 (Fig.
S5.6.2) and distinct NOEs of H-5/H-1 and H-5/H-2 for 11 (Fig. S6.6.2). Notably,
compounds A and B, which have the same oxygen-bridged monocycle as that of 11, are
produced from fluoro-derivatives of 4 (not genuine 4). Fluorine is a strong electron-
withdrawing atom, and thus, the -electron density on the fluorine-substituted double
bond significantly decreased, leading to the termination of the polycyclization reaction
at the mono- (A, B) and bicyclic stages (C, D) without completion of the
polycyclization reaction. We further generated a new product, 10, in addition to 11,
although the production yield of 10 was relatively lower than that of 11 (see Fig. S3 and
6

Page 7 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
S4). Moreover, compound 11 from (3S)-4 has not been found in nature. This is the first
report of conversion of a genuine oxidosqualene into the oxygen-bridged monocyclic
triterpenes 10 and 11 by an enzymatic reaction of triterpene cyclases (including
prokaryotic SHCs and eukaryotic OSCs). The variant W169L also generated 12, which
was first isolated from Achillea odorata and named achilleol A 13 and was prepared
from site-directed variants from OSCs14a, b and SHCs.10, 14c The MS and NMR data are
given in Fig. S7. We also isolated product 13 from the enzymatic reaction of 4 with the
A306F variant.7 The MS and NMR data are shown in Fig. S8.
The 1H-NMR spectrum of product 14 (600 MH, CDCl3, Fig. S9.2) indicated four
aliphatic Me groups, H 0.956 (s, 3H, Me-23); 0.779 (s, 3H, Me-24); 0.852 (s, 3H, Me-
25); 0.975 (s, 3H, Me-26), and further showed three olefinic Me groups, H 1.595 (s,
3H, Me-28); 1.681 (s, 3H, Me-29); and 1.600 (s, 3H, Me-30). One of the 8 Me groups
involved in 4 and 7 was missing in the 1H-NMR spectrum; however, in turn,
methylidene protons (H 4.59, s, 1H, H-27; and 4.87, s, 1H, H-27) were observed, and
the methylidene group was further confirmed by the DEPT pulse sequences (C 108.8, t,
C-14; 154.5, s, C-14). Detailed HMBC analyses (Fig. S9.4) indicated that 14 was
composed of a 6,6,5-fused tricyclic system, as shown in Fig. 1. A strong NOE was
observed between Me-26 and H-13 (H 2.12, m, 1H), indicating the -orientation for H-
13. H-3 (H 3.20, 1H) exhibited a double-doublet splitting pattern (J=11.0 and 5.2 Hz),
and thus, the OH function at C-3 is -oriented. Consequently, the whole structure is as
shown in Fig. 1.
The 1H-NMR spectrum of product 15 (600 MHz, CDCl3, Fig. S10.2) showed two
olefinic Me groups (H 1.68, s, 3H, Me-26; 1.60, s, 3H, Me-27), which were correlated
with H-24 (H 5.11, bt, J=6.8 Hz) in the COSY spectrum, indicating that the
isopropylidene group is present in 15. Methylidene protons (H-21) were also found at
the following chemical shifts: H 4.88, s, 1H and H 4.91, s, 1H. Five aliphatic Me
groups were observed as follows:H 0.935 (s, 3H, Me-28); H 0.834 (s, 3H, Me-29);H
0.845 (s, 3H, Me-19); H 0.942 (s, 3H, Me-18); and H 0.901 (s, 3H, Me-30). The
detailed HMBC analyses (Fig. S10.4) demonstrated that 15 consisted of a 6,6,6,5-fused
tetracyclic skeleton. Definitive NOEs were found between H-13 (H 2.02, m, 1H) and
H-17 (H 2.63, m, 1H), between Me-30 and H-9 (H 1.43, m, 1H) and between Me-18
and H-13, demonstrating the stereochemistry of 13-H, 17-H and 30-Me. The H-3
proton (H 3.41, 1H) gave a broad singlet, indicating that the OH group is arranged in -
disposition. The whole structure of 15 is as shown in Fig. 1.
The structural determination of 16 was conducted in a similar manner to the method
for 15. Detailed 2D analyses were as shown in Fig. S11.4. The stereochemistry in the
7

Organic & Biomolecular Chemistry
Page 8 of 33
View Article Online
DOI: 10.1039/C8OB02009D
tetracyclic skeleton was identical to that of 15, but, contrary to 15, the OH group at C-3
was determined to be -oriented (H 3.13, dd, J=10.9, 5.2 Hz, 1H), and thus, the
structure of 16 is as shown in Fig. 1.
Isolation of products 16, 20 and 22─24 from the F601G/P602F mutant.
Cell-free homogenates (750 mL) were prepared from a 30 L culture of the double
mutant F601G/P602F. A mixture of 4 and 7 (300 mg) was incubated with the
homogenates, prepared as above, at 45C (pH 6.0) for 20 h. After terminating the
enzymatic reaction with 15% KOH/MeOH, lipophilic materials were extracted with
hexane. Triton X-100 was removed using a short SiO2 column and eluted with
hexane/EtOAc (100:30). SiO2 column chromatography (hexane/EtOAc=100:0─100:20)
afforded 20 (7.8 mg) in an almost pure state. A mixture of 22, 23, 16 and 24 was loaded
on a SiO2 column impregnated with 5% AgNO3, followed by elution with
hexane/EtOAc=100:0─100:5 to afford 16 (8.0 mg) and 22 (5.6 mg) in an almost pure
state. A fraction containing a mixture of 22, 23 and 24 was acetylated with Ac2O/Py,
and the acetate mixture was subjected to 5% AgNO3-SiO2 column chromatography and
eluted with hexane/EtOAc (100:0─100:5) to yield the acetates of 23 (1.8 mg) and 24
(0.7 mg) that contained some purities, but the purities were good enough for structural
determination (see Figs. S14.9 and S15.9). Compounds 12 and 17─21 were produced
from the G600 and double mutant G600F/F601G variants. Previously, we reported
that compounds 12 and 17─21 were produced by incubating the racemic mixture of 4
and 7 with the G600 variant.10 Product 22 was also isolated from the enzymatic
reaction of a mixture of 4 and 7 with the A306T7 and A306V variants.7 The NMR data
are shown in Fig. S13. Products 23 and 24 are new metabolites that have not been
reported before.
Structures of products 16, 20 and 22─24 from the F601G/P602F mutant.
In the 1H NMR spectrum of 23-acetate (600 MHz, C6D6, Fig. S14.2), no olefinic
Me protons were found, suggesting that 23 is a fully cyclized product. Further, no
olefinic proton was found. Detailed HMBC analyses (Fig. S14.9) indicated that 23
consists of a 6,6,6,6,5-fused pentacyclic skeleton, similar to 2, but the double bond
position was different from that of hop-22(29)-ene 2. The presence of an isopropyl
residue was demonstrated by distinct COSY correlations; the two aliphatic methyl
protons of Me-29 (H 1.14, d, J=6.8 Hz, 3H) and Me-30 (H 1.18, d, J=6.8 Hz, 3H) were
correlated with allylic proton H-22 (H 2.87, m, 1H). Me-29 and Me-30 both had clear
HMBC cross peaks with C-21 (C 136.5, s), and Me-28 (H 1.13, s, 3H) showed a
8

Page 9 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
definitive HMBC correlation with C-17 (C 140.4, s). Thus, the tetra-substituted double
bonds are positioned at C-17 and C-21. Further HMBC analyses are shown in Fig.
S14.9. The splitting pattern of H-3 (H 4.83, dd, J=11.6 and 4.4 Hz, 1H) and a distinct
NOE between H-5 (H 0.811, bd, J=11.6 Hz, 1H) and H-3 (Fig. S14.6) indicates that the
3-OH is -oriented. Therefore, product 23 was determined to be 3-hydroxy-neohop-
17(21)-ene, as shown in Fig. 2 and Fig. S14.9.
Product 24 had two vinylic Me groups (H 1.79, s, 3H for Me-29; H 1.97, s, 3H for
Me-30), suggesting that an isopropylidene residue remains in 24 without participating in
the polycyclization reaction. However, this possibility was denied because the DEPT
data clearly indicated the presence of a tetra-substituted double bond (C 120.7, s and C
135.7, s; C-21 and C-22 ). The distinct HMBC correlations between the two vinylic Me
signals (Me-29 and Me-30) and the two sp2 carbons (C-21 and C-22) were observed
(Fig. S15.8), and thus, both Me-29 and Me-30 are located at both C-21 and C-22 (Fig.
S15.9). Distinct NOEs were observed between Me-27 (H 1.10, s, 3H) and Me-28 (H
0.855, s, 3H) and between H-13 (H 1.54, m, 1H) and H-17 (H 1.84, m, 1H), indicating
that Me-27 and Me-28 are -oriented, whereas H-13 and H-17 are placed in the -
orientation in the D-ring of 24. A distinct HMBC cross peak was found for Me-28/C-19
(C 39.48, t)/C-17 (C 56.23, d). H-19 (H 1.13, m, 1H and H 1.70, m, 1H), which was
assigned by the HMQC spectrum, had a COSY correlation with H-20 (H 2.23, m, 1H
and H 2.40, m, 1H). The chemical shifts of H-20 indicate that H-20 is in the allylic
position. A clear spin-coupling between Me-30 and H-17 was observed through
homoallylic spin coupling (see the COSY and TOCSY spectra, Fig. S15.4 and Fig.
S15.5), and a distinct NOE for Me-30/H-16 allowed the assignment of Me-30. In
addition, Me-30 showed a HMBC cross peak for C-17. All of the NMR data described
above suggests that the E-ring of 24 is composed of a 6-membered ring, but not the 5-
membered ring from the hopane skeleton. Product 24 is different from the gammacerane
scaffold E and tetrahymanol F consisting of the 6,6,6,6,6-fused pentacyclic skeleton
(see Fig. 2). Thus, to our knowledge, 24 is a novel triterpene (i.e., in a ACS SciFinder
search). We propose the name neogammacerane (compound G, Fig. 2) for the
fundamental carbon framework of 24, in which the two Me groups are positioned at C-
21 and C-22.
Discussion
Formation mechanisms and distribution of products 10─16 by the W169X variants
9

Organic & Biomolecular Chemistry
Page 10 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Scheme 2 shows the formation mechanisms of products 10─16 by the W169X
mutants. Scheme 2A shows the cyclization mechanisms for the formation of products
10─13 by the W169L variant; (3S)-4 folds into a chair conformation to give monocyclic
25 bearing a C-6 cation. Proton elimination from Me-25 of 25 affords 12. The chair
structure of 25 underwent a ring flip to give a boat structure 26, in which the OH group
is rearranged into a flagpole position of 26 from the equatorial position of 25. The
flagpole OH was approximately located at the C-6 cation of 26, leading to a
nucleophilic attack of the OH on the C-6 cation to afford product 11 with (1R,2S,4S)-
configurations (IUPAC numbering). (3R)-7 was also converted into the oxygen-bridged
monocycle 10. As described in Scheme 1, (3R)-7 folds into a boat structure to give 27,
where the OH group is arranged at the bowsprit position, and therefore, the OH group of
27 is very far from the C-6 cation. Cation 27 underwent a ring flip to afford the chair
structure 28 with -OH. The highly nucleophilic OH group attacked the C-6 cation,
yielding 10 with (1S,2S,4R)-stereochemistry (IUPAC numbering). However, the
distance between the OH and C-6 cation of 28 is longer than that in 26, which may have
led to the lower yield of 10 than 11 of approximately 3-fold (see Table 1). The W169L
variant also folds 4 into a chair-chair conformation to give bicyclic 29 with the C-8
cation. The deprotonation of Hax-7 generated 13 with a C-7─C-8 double bond.
Scheme 2B depicts the cyclization mechanism of the conversion of 4 and 7 into
14─16 by the W169V variant. (3S)-4 is folded in a chair-chair-chair conformation in the
reaction cavity of this variant to generate a 6,6,5-fused tricyclic cation 30, which
undergoes proton elimination from Me-27, yielding 14. The folding of 4 and 7 into a
chair-chair-chair-chair conformation and a boat-chair-chair-chair conformation affords
the 17-epi-damamrenyl cations 31 with 3-OH and 32 with 3-OH, respectively. The
deprotonation reactions from the Me-21 of 31 and 32 affords 16 and 15, respectively.
In our preceding paper,9 we reported that the W169F and W169Y variants afforded
tetracyclic 17-epi-dammara-20(21),24-diene (3-deoxy-15 and 16) from the enzymatic
reactions of 1 in addition to 2 and 3, but the W169V variant was completely inactive to
1, and thus, we propose that W169 functions to stabilize the tetracyclic intermediary
cation via cation- interactions.9 By contrast, the reactions of oxidosqualenes (4 and 7)
with the Phe and Tyr variants only afforded the normal products 5, 6, 8 and 9, but no
detectable amount of the premature products with the tetracyclic dammarenyl skeleton
was found (Fig. S3). The -electron densities increase as follows: Phe<Tyr<Trp. Thus,
the richest -electron densities (Trp) are necessary for the full cyclization reaction of 1,9
but in the case of 4 and 7, the highest-electron densities, such as those for Trp, are not
necessary for complete polycyclization. Moreover, squalene 1 underwent no reaction
10

Page 11 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
with the two aliphatic Val and Leu mutants (Fig. S2), but 4 and 7 underwent
polycyclization (Fig. S3). The epoxide rings of 4 and 7 have significantly higher
nucleophilicity toward the DXDD motif (proton donor) than the double bond of 1,
easily triggering polycyclization of 4 and 7. Otherwise, the orientation of substrate 4 or
7 inside the reaction cavity may be somewhat different from that of 1, in which the
substrate heads of 4 or 7 may be located near the DXDD motif, but the head of 1 may
be not close to the acidic motif.
<Scheme 2 and Table 1, here>
Table 1 shows that the Val mutant generated the prematurely cyclized tri- 14 and
tetracycles 15 and 16. Production of the abortive tetracyclic 14─16 indicated that -
electrons are required for complete cyclization to generate pentacyclic products.
However, the Leu variant afforded a significantly large amount of monocyclic products
(approximately 72 %) and a small amount of bicyclic product (approximately 7%) but
did not produce tri- and tetracyclic 14─16. In other words, the product distribution is
distinct for the Val and Leu variants, even though there is only a single CH2 unit
difference between the two amino acid residues. For the Val variant, 4 and 7 could be
folded into somewhat extended or stretched conformations (chair-chair-chair, chair-
chair-chair-chair or boat-chair-chair-chair structures) in the reaction cavity, and thus, the
polycyclization reaction terminated at the later reaction stages of tri- and tetracyclic
30─32. As shown in Fig. S3, the Val mutant also produced pentacyclic 5 and 6. The
somewhat large reaction cavity of the Val mutant may have led to the final products 5
and 8. However, the Leu mutant constrained 4 and 7 in an unextended or curled
conformation inside the reaction cavity, in which only mono- and bicyclic
conformations (ca 10:1) are organized, and thus, the polycyclization cascade mainly
stopped at the earlier reaction stage 25─28 (monocyclic intermediates). We surmise that
the reaction cavity of the Leu variant, which is responsible for the cyclization reaction
of 4 and 7, is fairly smaller than that of the Val variant. Therefore, the organization of
chair or boat structures may be mainly limited to the monocyclic stages. To ascertain
this theory, the X-ray crystal structures, which are complexed with 4 and 7, should be
resolved.
Formation mechanisms and distribution of products 22─24 by the G600F/F601G
and F601G/P602F double mutants
Fig. S12.3A shows the GC profile, which was obtained from the incubation
experiment, of racemic 4 and 7 with the G600 variant. As described previously,
11

Organic & Biomolecular Chemistry
Page 12 of 33
View Article Online
DOI: 10.1039/C8OB02009D
products 12 and 17─21 were found.10 The G600F/F601G mutant yielded the same
products, 12 and 17─21, as the G600 variant, despite the somewhat different yields of
each product reported between the two mutants (Table 2 and compare Figs. S12.3A
with Fig. S12.3B). Fig. S12.1 shows the amino acid alignments of the mutated and wild-
type AaSHCs. The G600F/F601G variant was constructed by exchanging Gly at 600 for
Phe and Phe at 601 for Gly (see Fig. S12.1). The G600 and G600F/F601G mutants
both have the same Phe residue at position 600, which may have led to their identical
production of mono- 12 and tricycles 17─21; the large steric bulk of the Phe residue15 at
position 600 may have prevented full cyclization. This theory prompted us to create the
double mutant F601G/P602F, which may enable the generation of tetra- and pentacyclic
products because the Gly residue, which has the smallest steric size,15 is substituted at
position 601; two Gly residues are aligned at the positions of both 600 and 601of the
F601G/P602F mutant (see Fig. S12.1). As shown in Table 2 and Fig. S12.3, the
F601G/P602F variant successfully produced tetra- (16 and 22, yield: 25%) and
pentacyclic products (23 and 24, yield 6.5%) in addition to tricycle (20, yield:13%). It is
to be noted that all of the products possessed a 3-hydroxyl group, indicating that (3S)-4
only reacted with three mutants, but 1 and 7 were inactive (see Fig. S12.2 and S12.3);
thus, the three mutated SHCs are categorized as OSCs. Currently, it is unknown why the
three mutants can only accept 4 as the substrate. One possible answer is that these
mutations may bring about either looser or tighter packing at the local sites of the
reaction cavity, and thus, substrates 1, 4 and 7 could be arranged in different geometries
in the reaction cavities of the three mutants. The epoxide head of 4 may be arranged in
proximity to the DXDD motif, but the heads of 1 and 7 may have failed to access the
motif. Further studies are necessary, including X-ray crystallographic analyses, to
determine the exact altered substrate specificities and product distribution differences
between the G600F/F601G and F601G/P602F variants.
Scheme 3 illustrates the formation mechanisms of 5 and 6 from 4 by the native
SHC; 1 and 4 fold into a chair-chair-chair conformation to provide the 6,6,5-fused
tricyclic (13-H)-malabaricanyl cation 30. Further cyclization of 30 into a chair-chair-
chair-chair conformation affords the 6,6,6,5-fused tetracyclic 17-epi-dammarenyl cation
31. Subsequently, ring expansion and further cyclization of 31 yields the 6,6,6,6,5-fused
pentacyclic hopanyl cation 33. In our preceding paper,6j we revealed that a robust
hydrophobic interaction occurs between Me-24 (E-Me at C-23 of 4) and the D/E ring
formation sites (marked with a pink crescent shape, the E-Me binding site), and this
strong interaction enforces the correct positioning of Me-30 (Z-Me at C-23 of 4) at its
binding site, where a highly polarized water molecule is located in proximity to the C-
12

Page 13 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
22 cation of 33 (marked with a blue crescent). The polarized water molecule (a strong
base) abstracts the proton from the Z-Me (Me-29 of 33) to yield 5 (path a). Product 6 is
produced via the nucleophilic attack of the polarized water molecule on the C-22 cation
of 33 (path b). This mechanism was proposed by Wendt et al.16 The Phe601 and the
Gly600 residues are located primarily at the D/E-ring formation site in the reaction
cavity (Fig. S1). Consequently, the strong hydrophobic interaction between the E-Me
(Me-24) and D/E-ring sites is critical to the formation of cation 33 with the 5-membered
E-ring, as well as for smooth deprotonation. Perturbation near the C/D/E-ring formation
site may prevent the construction of an organized chair conformation at each local
site(s) in the reaction cavity, leading to an altered product distribution.
<Scheme 3 here>
Scheme 4 depicts the mechanisms of formation of products 12 and 17─21 by the
G600 and the G600F/F601G variants. As described above, the two variants have Phe
residues at position 600 (see Fig. S12.1). The Phe residue has significantly larger steric
bulk than the Gly residue (van der Waal’s volumes (nm3): 0.00279 for Gly; 0.55298 for
Phe).15 The amino acid residue positioned at 600 is located in close proximity to the
C/D-ring formation sites (Fig. S1). The embedded Phe, with a large steric volume at
position 600, collides with substrate 4 due to tight packing, as represented by the grey
crescent shape. This perturbation at the local site(s) could prevent the organization of
the normal all-chair folding conformation. Only the initially cyclized A-ring (cation 34)
could be organized to give 12, which resulted from deprotonation of Me-25. A chair-
chair-chair fold could afford the tricyclic cation 30, followed by the nucleophilic attack
of a water molecule on the C-14 cation of 30 to give the 6/6/5-fused tricycle 20 with
13-H. The tight packing at the local site(s) could exert a further influence on the
folding conformation to afford the chair-chair-boat folding conformation 35, leading to
the 6/6/5-fused tricyclic cation 36 with 13-H. Deprotonation from Me-27 could afford
19, and a water attack of the C-14 cation could give 21. A series of 1,2-shift reactions—
H-13 to the C-14 cation, Me-26 to C-13, and then deprotonation of H-9—could give 17.
Consecutive rearrangement reactions—H-13 to C-14 cation, Me-26 to C-13, and H-9 to
C-8, followed by the proton elimination of H-11—furnish 18.
<Scheme 4, here>
Scheme 5 shows the formation mechanisms of products 16 and 22─24 by the
F601G/P602F variant. Contrary to variants G600 and G600F/F601G, the local domain
at positions 600 and 601(Gly600Gly601) of the F601G/P602F variant is more loosely
13

Organic & Biomolecular Chemistry
Page 14 of 33
View Article Online
DOI: 10.1039/C8OB02009D
packed than that of wild type because Gly residues are present at positions 600 and 601.
As shown in Fig. S1, the domain is located near the D/E-ring site (represented by a
purple crescent). The loosely packed domain could afford an unconstrained
conformation 37, near the D/E-ring formation sites (yield 30%) because no or little
hydrophobic interaction occurs between the purple crescent and 37. The dotted black
line, depicted in 37, indicates the long axis along the all-chair folding conformation for
the formation of A─E rings inside the reaction cavity of the wild-type SHC (see also
Scheme 3). The long axis is far from the purple crescent near the D/E-ring sites. This
perturbation halted polycyclization at the tricyclic 30 (13% yield, from a chair-chair-
chair conformation) and tetracyclic stages 31 (17% yield, from a chair-chair-chair-chair
structure) to yield 20 and 16, respectively. As described above, 20 was also produced by
the variants G600 and G600F/F601G (Scheme 4A), and 16 was generated from the
W169V variants (Scheme 2). Furthermore, the folding conformation of 4 was altered
into a chair-chair-chair-boat structure (8% yield), yielding the tetracyclic cation 39 with
17-H, which underwent the hydride shift of H-17 to the C-20 cation, and the
deprotonation of H-13 gave product 22. This conformational change at the D-ring site
of 4 would have resulted from the geometry of 38, which is distinct from that of 37. The
long axis (dotted line) shown in 37 was largely rotated to the right (red line in 38),
where the D-ring site of 4 was near the purple crescent, possibly leading to a collision
between them, which would cause a boat structure for the D-ring formation site of 39
(like 30 in Scheme 4). However, 4 was, in part, folded into an all-chair conformation via
the small rotation of the dotted line (yield: 6.4%), albeit in a somewhat distorted
conformation, to yield the pentacyclic intermediate 40. Intermediate 40 could be
generated from the geometry shown by the red line in 40, in which somewhat
appropriate, but not perfect, distances between the D/E-ring site and the purple crescent
could be set up for the hydrophobic interaction to give a chair-chair-chair-chair-chair
conformation to form pentacyclic 40, albeit at a low yield (6.4%). Then, 40 was
converted into two different intermediates: 33 (hopanyl cation), consisting of a
6,6,6,6,5-fused pentacyclic skeleton, and 41 consisting of a 6,6,6,6,6-fused pentacyclic
skeleton (gammacerane skeleton). The imperfect hydrophobic interaction of the D/E-
ring formation site with the E-Me (Me-24) led to failure to place the polarized water at
the accurate position, and hence, the distance between the Z-Me (Me-30 of 4, i.e., Me-
29 of 33) and the polarized water molecule is large. Thus, the deprotonation reaction
from the Me-29 of 33 and a water attack on the C-22 cation did not occur; instead, the
hydride shift of H-21 to the C-22 cation, followed by the proton elimination of H-17,
afforded the neohopane scaffold 23. This imperfect hydrophobic interaction could
14

Page 15 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
further afford 41 with 6-membered E-ring, because of less steric strain of 6-membered
chair structure than that of 5-membered structure 33. A polarized water molecule is also
far from the C-21 cation of 41, and thus, the nucleophilic attack of a water molecule at
the C-21 cation did not occur. Therefore, tetrahymanol scaffold (compound H: 3-
hydroxytetrahymanol) was not produced. Instead, the migration of Me-29 of cation 41
to the C-21 cation, followed by the elimination of H-21, resulted in the production of
24. An alternative biosynthetic pathway to form 41 may be possible (green arrow): a
ring expansion of 33 to 41 through stabilization of the secondary C-21 cation of 41 via
cation-interactions with aromatic amino acids;17a for example, Phe602 from the
F601G/P602F variant or the Phe605 residue of the native SHC,17a,b which may possibly
be near the C-21 cation of 41. To confirm the cyclization mechanisms described above,
the X-ray crystallographic analyses of the G600, G600F/F601G and F601G/P602G
mutants complexed with 4 will be necessary.
<Scheme 5 and Table 2, here>
In conclusion, we present the first enzymatic syntheses of triterpenes 10 and 11
bearing the 7-oxabicyclo[2.2.1]heptane moiety and novel neogammacerane triterpene
scaffold 24 by employing mutated SHCs. The oxygen-bridged monocyclic skeleton has
been reported via chemical syntheses, such as those found in the cyclization of 14,15-
oxidogeranylgeranyl acetate with SnCl418. and in the cyclization of 2,3-oxidosqualene
with Ph4BF4/HEIP.19. However, these fundamental scaffolds, 10, 11 and 24, have not
been identified in nature. These new triterpenes were only produced from the enzymatic
cyclization reactions of 2,3-oxidosqualene, but not from squalene cyclization. The Val
mutant accepts almost equal amounts of 4 and 7 to afford 3- and 3-hydroxy-cyclic
triterpenes, but the W169L mutant has significantly high selectivity for (3S)-4 (73 %),
while (3R)-7 was used at a very small amount (6 %). The double mutants G600F/F601G
and F601G/P602F also showed high substrate selectivity; only (3S)-4 was recognized,
yielding 3-hydroxy-derivatives of cyclic triterpene scaffolds. SHC has excellent
potential for creating new terpenoid compounds2b, 2i, 4,5 and is also a promising tool for
generating non-terpenoid scaffolds by selecting substrates and employing further
mutated SHCs.
Experimental
General analytical methods: NMR spectra were recorded in CDCl3 on a Bruker DMX
600 or DPX 400 spectrometer, and the chemical shifts () are given in ppm relative to
15

Organic & Biomolecular Chemistry
Page 16 of 33
View Article Online
DOI: 10.1039/C8OB02009D
the residual solvent peak H 7.26 and C 77.0 as the internal reference for 1H- and 13C-
NMR spectra, respectively. In the case of C6D6 solutions, the chemical shifts are given
in ppm relative to the solvent peak (H 7.28; C 128.0). The coupling constants J are
given in Hz. GC analyses were performed on a Shimadzu GC-8A or GC2014
chromatograph equipped with a flame ionization detector (DB-1 capillary column, 0.53
mm  30 m). GC-MS spectra were performed on a JEOL SX 100 spectrometer under
electronic impact at 70 eV with a DB-1 capillary column (0.32 mm x 30 m). HR-EIMS
was performed by direct inlet system. Specific rotation values were measured at 25°C
with a Horiba SEPA-300 polarimeter.
Site-directed mutagenesis
The details are shown in Table S1; primers used for mutagenesis and protocols are
described.
Incubation conditions and GC analysis
The SHC described here is from A. acidocaldarius.11, 20 The standard culture of the cloned
Escherichia coli and incubation conditions were performed according to our published
protocols.11, 20 Cell-free homogenates were used as the enzyme sources. The cell-free
extracts were prepared as follows. The E. coli BL 21 strains encoding the mutated SHCs,
W169X, G600F/F601G and F601G/P602F, were cultured in 100 mL of a LB medium.
To the cultured cells, a buffer solution (Tris, pH 8.0, 10 mL) containing 1% Triton X-100
was added, and suspensions were subjected to ultrasonication and centrifugation. The
homogenates were used as the enzymatic sources. To 1.0 mg of 1 or a racemic mixture
of 4 and 7 emulsified in a distilled water containing Triton X-100 (20 mg), 2 mL of the
cell-free homogenates (equivalent to 40 g of the pure SHC) and 0.5 M citrate buffer (pH
6.0, 3 mL) were added and incubated at 55C for 20 h. Then, deactivation of the enzyme
activities was achieved by adding 15% KOH/MeOH and heating at 70C for 45 min. The
lipophilic materials from the reaction mixture were extracted with hexane. The Triton X-
100 included in the hexane extract was removed by a short SiO2 column by eluting with
hexane/EtOAc (100:20), followed by GC analyses, shown in Figs. S2─S4 for W169X
variants and in Figs. S12.2─S12.3 for G600, G600F/F9601G and F601G/P602F variants.
The GC conditions are also described in the supporting information.
Spectroscopic data of enzymatic products
We have isolated products 12, 13, 17-21and 22 from some mutants and reported the NMR
data. Compound 12 and 13 were isolated from G600 variant,10 and from A306F variant,
16

Page 17 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
6 respectively. These products were identified by the NMR data (see Figs. S7 for 12, S8
for 13 and S13 for 22). Products 17-21 were isolated from G600 mutant and these NMR
data were reported in the ref. 10. Product 22 was isolated from A306T variant,7 but we
have not isolated products 10, 11, 14, 15, 16, 23 and 24 before. The NMR and EIMS data
of these products are given below.
Products 10. []D25= -5.5 (c=0.096, EtOH); 1H NMR (600 MHz,CDCl3): =0.901 (s,
3H, Me-25), 1.06 (s, 3H, Me-24), 1.16 (m, 1H, H-1), 1.22 (m, 1H, H-5), 1.24 (m, 1H,
H-7), 1.37 (m, 1H, H-7), 1.38 (s, 3H, Me-26), 1.58 (m, 1H, H-2), 1.60 (s, 12H, Me-27,
Me-28, Me-29 and Me-30, total 4 x Me), 1.62 (m, 1H, H-1), 1.68 (s, 3H, Me-23), 1.80
(m, 1H, H-2), 1.89 (m, 1H, H-8), 1.98 (m, 1H, H-8; m, 2H, H-15; m, 2H, H-19, total
5H), 2.03 (m, 2H, H-11; m, 2H, H-12, total 4H), 2.06 (m, 2H, H-16; m, 2H, H-20, total
4H), 3.77 (d, J=5.0 Hz, 1H, H-3), 5.11 (m, 1H, H-10; m, 1H, H-13; m, m, 1H, H-17; m,
1H, H-21, total 4H); 13C NMR (150 MHz, CDCl3):=16.18 (q, C-29), 16.21 (q, C-28),
16.27 (q, C-27), 17.67 (q, C-30), 19.87 (q, C-25), 21.41 (q, C-26), 25.68 (q, C-23; t, C-
7 ), 26.66 (t, C-20), 26.73(t, C-16), 26.77 (t, C-2), 28.24 (t, C-11; t, C-12), 28.75 (t, C-
1), 32.44 (q, C-24), 39.62 (t, C-19), 39.72 (t, C-15), 39.75 (t, C-8), 41.76 (s, C-4), 56.27
(d, C-5), 86.26 (d, C-3), 88.08 (s, C-6), 124.2 (d, C-21), 124.3 (d, C-17), 124.4 (d, C-
13), 124.7 (d, C-10), 131.2 (s, C-22), 134.9 (s, C-18), 135.2 (s, 2C, C-9 and C-14).
Since the chemical sifts are close to each other, assignments of the following carbons
may be exchangeable: between C-2, C-16 and C-20, between C-8, C-15 and C-19,
between C-10, C-17 and C-21, and between C-9, C-14, C-18 and C-21. MS (EI) see Fig.
S15.1. HRMS (EI): m/z 426 (M+): calcd. for C30H50O: 426.3862; found: 426.3888.
Product 11. []D25= -8.2 (c=0.445, EtOH); 1H NMR (600 MHz,CDCl3): =1.01 (s, 3H,
Me-25), 1.04 (s, 3H, Me-24), 1.17 (dd, J=8.6,5.8 Hz, 1H, H-5), 1.32 (s, 3H, Me-26),
1.37 (m, 1H, H-7), 1.41 (m, 1H, H-7), 1.43 (m, 1H, H-1), 1.50 (m, 1H, H-1), 1.60 (s,
12H, Me-27, Me-28, Me-29, Me-30, total 4 x Me ), 1.68 (m, 1H, H-2; s, 3H, Me-23,
total 4H), 1.89 (m, 1H, H-8), 1.91 (m, 1H, H-2), 1.97 (m, 1H, H-8; m, 2H, H-15; m, 2H,
H-19, total 5H), 2.02 (m, 2H, H-11; m, 2H, H-12, total 4H), 2.06 (m, 2H, H-16; m, 2H,
H-20, total 4H), 3.71 (d, J=5.4, 1H, H-3), 5.09 (t, J=7.0 Hz, 1H, H-21), 5.12 (m, 3H, H-
10, H-13 and H-17); 13C NMR (150 MHz, CDCl3):=15.95 (q, C-29), 15.99 (q, C-28),
16.05 (q, C-27), 17.66 (q, C-30), 18.88 (q, C-26), 23.38 (q, C-25), 25.67 (q, C-23),
25.75 (t, C-2), 26.08 (q, C-24), 26.16 (t, C-7), 26.67 (t, C-20), 26.77 (t, C-16), 28.21 (t,
C-12), 28.25 (t, C-11), 39.01 (t, C-1), 39.72 (t, C-19), 39.75 (t, C-15), 39.82 (t, C-8),
45.23 (s, C-4), 55.19 (d, C-5), 86.06 (d, C-3), 86.71(s, C-6), 124.2 (d, C-21; d, C-17,
17

Organic & Biomolecular Chemistry
Page 18 of 33
View Article Online
DOI: 10.1039/C8OB02009D
total 2C), 124.4 (d, C-13), 124.5 (s, C-10), 131.2 (s, C-22), 134.9 (s, C-18), 135.2 (s, C-
14), 135.4 (s, C-9). Since the chemical sifts are close to each other, assignments of the
following carbons may be exchangeable: between C-8, C-15 and C-19, between C-11
and C-12, between C-16 and C-20, between C-27, C-28 and C-29, between C-10, C-13,
C-17 and C-21, and between C-14 and C-18. MS (EI) see Fig. S6.1. HRMS (EI): m/z
426 (M+): calcd. for C30H50O: 426.3862; found: 426.3866.
Product 14. []D25= -5.6 (c=0.151, EtOH); 1H NMR (600 MHz,CDCl3): =0.67 (dd,
J=12.0, 2.1 Hz, 1H, H-5), 0.779 (s, 3H, Me-24), 0.852 (s, 3H, Me-25), 0.956 (s, 3H,
Me-23), 0.975 (s, 3H, Me-26), 1.08 (td, J=12.4, 4.1 Hz, 1H, H-1; td, J=12.4, 4.1 Hz,
1H, H-7, total 2H), 1.30 (dd, J=12.5, 7.7Hz, 1H, H-9), 1.41 (m, 1H, H-6), 1.44 (m, 1H,
H-11), 1.51 (m, 1H, H-1), 1.52 (m, 1H, H-11), 1.54 (m, 1H, H-6), 1.55 (m, 1H, H-7),
1.56 (m, 1H, H-12), 1.57 (m, 2H, H-2), 1.59 (s, 3H, Me-28), 1.60 (s, 3H, Me-30), 1.68
(s, 3H, Me-29), 1.87 (m, 1H, H-15), 1.98 (m, 1H, H-12), 2.00 (m, 1H, H-16), 2.02 (m,
1H, H-15 ), 2.05 (m, 2H, H-20), 2.12 (m, 1H, H-13), 2.15 (m, 1H, H-16), 2.18 (m, 2H,
H-19), 3.20 (dd, J=11.0, 5.2 Hz, H-3), 4.59 (s, 1H, H-27), 4.87 (s, 1H, H-27), 5.09 (br t,
J=6.7 Hz, H-21), 5.12 (br t, J=6.7 Hz, 1H, H-17); 13C NMR (150 MHz,
CDCl3):=15.30 (q, C-24), 15.60 (q, C-25), 16.02 (q, C-28), 17.69 (q, C-30), 18.98 (t,
C-6), 20.68 (t, C-11), 24.68 (q, C-26), 25.70 (q, C-29), 26.74 (t, C-20), 26.87 (t, C-16),
27.41 (t, C-2), 27.62 (t, C-12), 28.03 (q, C-23), 36.44 (s, C-10), 36.47 (t, C-7), 38.68 (t,
C-1), 38.71 (s, C-4), 39.24 (t, C-15), 39.70 (t, C-19), 45.23 (s, C-8), 55.37 (d, C-9),
55.68 (d, C-5), 56.28 (d, C-13), 79.17 (d, C-3), 108.8 (t, C-27), 124.2 (d, C-17), 124.4
(d, C-21), 131.3 (s, C-22), 135.1 (s, C-18), 154.5 (s, C-14). The assignments of C-2 and
C-12 and those of C-16 and C-20 may be exchangeable. MS (EI) see Fig. S9.1. HRMS
(EI): m/z 426 (M+): calcd. for C30H50O: 426.3862; found: 426.3830.
Product 15. []D25= -10.3 (c=0.165, EtOH); 1H NMR (600 MHz,CDCl3): =0.834 (s,
3H, Me-29), 0.845 (s, 3H, Me-19), 0.901 (s, 3H, Me-30), 0.935 (s, 3H, Me-28), 0.942
(s, 3H, Me-18), 1.22 (m, 2H, H-12 and H-15), 1.24 (m, 2H, H-5 and H-11), 1.28 (m,
1H, H-1), 1.30 (m, 1H, H-7), 1.41 (m, 2H, H-6), 1.42 (m, 1H, H-1), 1.43 (m, 1H, H-9),
1.55 (m, 1H, H-2), 1.57 (m, 1H, H-15), 1.59 (m, 1H, H-11), 1.60 (br s, 3H, Me-27),
1.63 (m, 1H, H-7), 1.67 (m, 1H, H-12), 1.68 (br s, 3H, Me-26), 1.79 (m, 2H, H-16),
1.96 (m, 1H, H-2), 1.98 (m, 1H, H-22), 2.02 (m, 1H, H-13), 2.08 (m, 1H, H-23), 2.13
(m, 1H, H-22), 2.16 (m, 1H, H-23), 2.63 (m, 1H, H-17), 3.41 (br s, 1H, H-3), 4.88 (s,
1H, H-21), 4.91 (s, 1H, H-21), 5.11 (br t, J=6.8 Hz, H-24); 13C NMR (150 MHz,
CDCl3):=15.82 (q, C-18), 16.04 (q, C-19), 16.92 (q, C-30), 17.70 (q, C-27), 18.27 (t,
18

Page 19 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
C-6), 21.84 (t, C-11), 22.11 (q, C-29), 24.94 (t, C-12), 25.39 (t, C-2), 25.70 (q, C-26),
27.32 (t, C-23), 28.26 (t, C-16), 28.27 (q, C-28), 33.05 (t, C-15), 33.62 (t, C-1), 34.91 (t,
C-7), 37.27 (s, C-10), 37.63 (s, C-4), 38.59 (t, C-22), 41.03 (s, C-8), 44.02 (d, C-17),
44.70 (d, C-13), 49.53 (d, C-5), 49.98 (s, C-14), 50.66 (d, C-9), 79.29 (d, C-3), 108.8 (t,
C-21), 124.4 (d, C-24), 131.4 (s, C-25), 152.3 (s, C-20). MS (EI) see Fig. S10.1. HRMS
(EI): m/z 426 (M+): calcd. for C30H50O: 426.3862; found: 426.3846.
Product 16. []D25= + 14.2 (c=0.359, EtOH); 1H NMR (600 MHz,C6D6): =0.764 (dd,
J=11.3, 2.0 Hz, 1H, H-5), 0.89 (m, 1H, H-1), 0.921 (s, 3H, Me-19), 0.931 (s, 3H, Me-
29), 1.027 (s, 3H, Me-18), 1.09 (s, 3H, Me-30), 1.13 (s, 3H, Me-28), 1.23 (m, 2H, H-1
and H-6), 1.33 (m, 1H, H-15), 1.36 (m, 1H, H-9), 1.37 (m, 1H, H-7), 1.48 (m, 1H, H-
12), 1.50 (m, 1H, H-6), 1.58 (m, 1H, H-11), 1.59 (m, 1H, H-1), 1.63 (m, 1H, H-7), 1.65
(m, 1H, H-1), 1.66 (m, 1H, H-15), 1.76 (s, 3H, Me-27), 1.82 (s, 3H, Me-26), 1.88 (m,
1H, H-12),, 1.92 (m, 1H, H-16), 1.98 (m, 1H, H-11), 2.01(m, 1H, H-16), 2.02 (m, 1H,
H-13), 2.19 (m, 1H, H-22), 2.35 (m, 1H, H-23), 2.36 (m, 1H, H-22), 2.43 (m, 1H, H-
23), 2.75 (m, 1H, H-17), 3.13 (dd, J=10.9, 5.2 Hz, 1H, H-3), 5.19 (s, 1H, H-21), 5.20 (s,
1H, H-21), 5.40 (br t, 1H, H-24); 13C NMR (150 MHz, C6D6):=15.66 (q, C-29), 16.02
(q, C-18), 16.43 (q, C-19), 17.13 (q, C-30), 17.74 (q, C-27), 18.66 (t, C-6), 22.26 (t, C-
2), 25.37 (t, C-12), 25.82 (q, C-26), 27.83 (t, C-11), 27.91 (t, C-23), 28.20 (q, C-28),
28.69 (t, C-16), 33.43 (t, C-15), 35.39 (t, C-7), 37.33 (s, C-10), 39.09 (t, C-22), 39.18 (s,
C-4), 39.30 (t, C-1), 41.05 (s, C-8), 44.39 (d, C-17), 45.02 (d, C-13), 50.11 (s, C-14),
51.22 (d, C-9), 56.27 (t, C-5), 78.75 (d, C-3), 109.4 (t, C-21), 124.9 (d, C-24), 131.3 (s,
C-25), 152.2 (s, C-20). The assignments of C-11 and C-23 may be exchangeable. MS
(EI) see Fig. S11.1. HRMS (EI): m/z 426 (M+): calcd. for C30H50O: 426.3862; found:
426.3845.
Product 23 acetate. []D25= + 132 (c=0.27, CHCl3); 1H NMR (400 MHz,C6D6):
=0.811 (br d, J=11.6 Hz, 1H, H-5), 0.868 (s, 3H, Me-25), 0.88 (m, 1H, H-1), 1.00 (s,
3H, Me-26), 1.03(s, 6H, Me-23 and Me-24), 1.13 (s, 3H, Me-28), 1.14 (d, J=6.8 Hz,
3H, Me-29), 1.18 (d, J=6.8 Hz, 3H, Me-30), 1.20 (s, 3H, Me-27), 1.27 (m, 1H, H-11),
1.30 (m, 1H, H-9), 1.37 (m, 1H, H-6), 1.40 (m, 1H, H-7), 1.46 (m, 2H, H-15), 1.52 (m,
1H, H-6), 1.54 (m, 1H, H-11), 1.55 (m, 2H, H-7 and H-12), 1.60 (m, 1H, H-19), 1.64
(m, 1H, H-1), 1.65 (m, 1H, H-13), 1.68 (m, 1H, H-12), 1.72 (m, 1H, H-2), 1.86 (m, 1H,
H-2), 1.88 (s, 3H, acetyl CH3), 1.93 (m, 1H, H-19), 2.18 (m, 1H, H-16), 2.36 (m, 1H, H-
20), 2.45 (m, 1H, H-20), 2.52 (m, 1H, H-16), 2.87 (m, 1H, H-22), 4.83 (dd, J=11.6, 4.4
Hz, 1H, H-3); 13C NMR (100 MHz, C6D6):=15.23 (q, C-27), 16.40 (q, C-26), 16.43 (q,
19

Organic & Biomolecular Chemistry
Page 20 of 33
View Article Online
DOI: 10.1039/C8OB02009D
C-25), 16.83 (q, C-24), 18.55 (t, C-6), 19.42 (q, C-28), 20.24 (t, C-16), 20.86 (q, acetyl
CH3), 21.50 (q, C-29), 21.63 (t, C-11), 22.10 (q, C-30), 24.11 (t, C-12), 24.33 (t, C-2),
26.81 (d, C-22), 27.94 (t, C-20), 28.10 (q, C-23), 32.27 (t, C-15), 33.73 (t, C-7), 37.21
(s, C-10), 37.98 (s, C-4), 38.56 (t, C-1), 41.92 (s, C-14), 42.09 (t, C-19), 42.29 (s, C-8),
49.78 (d, C-13), 50.19 (s, C-18), 50.92 (d, C-9), 55.55 (d, C-5), 80.53 (d, C-3), 136.5 (s,
C-21), 140.4 (s, C-17), 169.9 (s, acetyl CO). The assignments of proton signals Me-29
and Me-30 may be exchangeable. Since the chemical sifts are close to each other,
assignments of the following carbons may be exchangeable: between C-2 and C-12,
between C-8 and C-14 and between C-25 and C-26. MS (EI) see Fig. S14.1. HRMS
(EI): m/z 468 (M+): calcd. for C32H52O2: 468.3967; found: 468.3932.
Product 24 acetate. []D25= + 24.1 (c=0.23, CHCl3) ; 1H NMR (600 MHz,CDCl3):
=0.81 (br d, J=11.6 Hz, H-5), 0.855 (s, 3H, Me-28), 0.88 (m, 1H, H-1), 0.897 (s, 3H,
Me-25), 1.037 (s, 3H, Me-23), 1.037 (s, 3H, Me-26), 1.049 (s, 3H, Me-24), 1.104 (s,
3H, Me-27), 1.13 (m, 1H, H-19), 1.27 (m, 1H, H-9), 1.29 (m, 1H, H-11), 1.34 (m, 2H,
H-7 and H-15), 1.44 (m, 1H, H-6), 1.48 (m, 1H, H-15), 1.53 (m, 1H, H-11), 1.54 (m,
1H, H-13), 1.55 (m, 2H, H-2 and H-12), 1.57 (m, 1H, H-6), 1.60 (m, 1H, H-1), 1.70 (m,
2H, H-7 and H-19), 1.74 (m, 1H, H-2), 1.78 (m, 1H, H-16), 1.793 (s, 3H, Me-29), 1.84
(m, 1H, H-17), 1.885 (s, 3H, acetyl CH3), 1.965 (s, 3H, Me-30), 2.23 (m, 1H, H-20),
2.33 (m, 1H, H-16), 2.40 (m, 1H, H-20), 4.83(dd, J=11.8, 4.6 Hz, 1H, H-3); 13C NMR
(150 MHz, CDCl3):=14.99 (q, C-28), 16.10 (q, C-25), 16.70 (q, C-26), 16.84 (q, C-
24), 16.87 (q, C-27), 18.58 (t, C-6), 19.65 (q, C-30), 20.85 (q, acetyl CH3), 21.29 (t, C-
11), 23.00 (q, C-29), 23.69 (t, C-12), 23.94 (t, C-16), 24.10 (t, C-2), 28.14 (q, C-23),
28.77 (t, C-20), 33.12 (t, C-15), 33.50 (t, C-7), 37.16 (s, C-10), 37.99 (s, C-4), 38.45 (t,
C-1), 39.48 (t, C-19), 41.69 (s, C-8), 41.87 (s, C-14), 44.55 (s, C-18), 48.43 (d, C-13),
50.44 (d, C-9), 55.45 (d, C-5), 56.23 (d, C-17), 80.56 (d, C-3), 120.7 (s, C-21), 135.7 (s,
C-22), 169.9 (s, acetyl CO). The assignments of the following carbons may be
exchangeable between C-8 and C-14 and between C-2, C-12 and C-16, due to the very
close values. The discrimination of C-21 and C-22 are impossible by the various NMR
techniques including 2D NMR analyses. MS (EI) see Fig. S15.1. HRMS (EI): m/z 468
(M+): calcd. for C32H52O2: 468.3967; found: 468.3932
Conflicts of Interest
There are no conflicts of interest to declare.
Acknowledgements
20

Page 21 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
This work was supported in part by Grant-in-Aid for Scientific Research from Japan
Society for the Promotion of Science, Nos. 25450150 (C) and 18380001(B).
Keywords: oxidosqualene; enzyme catalysis; hopene; neogammacerane; oxygen-
bridged monocycle.
References
1. A. Eschenmoser, L. Ruzicka, O. Jeger, D. Arigoni, Helv. Chim. Acta 1955, 38,
1890–1904.
2. For a review of squalene, oxidosqualene cyclases, see: a) K. U. Wendt, G. E.
Schulz, E. J. Corey and D. R. Liu, Angew. Chem. Int. Ed. 2000, 39, 2812–2833; b)
T. Hoshino, T. Sato, Chem. Commun. 2002, 291–301; c) R. A. Yoder and J. N.
Johnston, Chem. Rev. 2005, 105, 4730–4756; d) M. J. R. Segura, B. E. Jackson and
S. P. T. Matsuda, Nat. Prod. Rep., 2003, 20, 304-317; e) I. Abe, Nat. Prod. Rep.
2007, 24, 1311–1331; f) T. K. Wu, C. H. Chang, Y. T. Liu and T. T. Wang, Chem.
Rec. 2008, 8, 302–325; g) W. D. Nes, Chem. Rev. 2011, 111, 6423–6451; h) G.
Siedenburg and D. Jendrossek, Appl. Environ. Microbiol., 2011, 77, 3905-3915; i)
P.-O. Syrén, S. Henche, A. Eichler, B. M. Nestl and B. Hauer, Curr. Opin. Struct.
Biol. 2016, 41, 73-82; j) T. Hoshino, Org. Biomol. Chem., 2017, 15, 2869-2891.
3. R. Xu, G. C. Fazio, and S. P. T. Matsuda, Phytochemistry 2004, 65, 261–291.
4. a) M. Rohmer, C. Anding, G. Ourisson, Eur. J. Biochem., 1980, 112, 541-547; b) P.
Bouvier, Y. Berger, M. Rohmer, G. Ourisson, Eur. J. Biochem., 1980, 112, 549-556;
c) R. Rohmer, P. Bouvier, G. Ourisson, Eur. J. Biochem., 1980, 112, 557-560; d) T.
Hoshino, S. Nakano, T. Kondo, T. Sato and A. Miyoshi, Org. Biomol. Chem., 2004,
2, 1456-1470.
For example, the Hauer group reported the following papers: a)
5.
M. Seitz, P. O.
b) S. C.
Syrén, M. Seitz, B. M. Nestle, B. Hauer, ChemBioChem, 2013, 14, 436-439;
Hammer, A. Marjanovic, J. M.
Dominicus, B. M. Nestle, B. Hauer, Nat. Chem.
Kühnel, B. M. Nestl and B. Hauer,
Biol., 2015, 11, 121-126; c) L. C.
ChemBioChem, 2017, 18, 2222-2225; d) S. A. Bastian, S. C. Hammer, N. Kreß, B.
M. Nestle, B. Hauer, ChemCatChem., 2017, 9, 4364-4368.
6. a) T. Hoshino and S. Ohashi, Org. Lett., 2002, 4, 2553-2556; b) S. Nakano, S.
Ohhashi and T. Hoshino, Org. Biomol. Chem., 2004, 2,2012-2022; c) T. Hoshino, I.
Kudo, S. Nakano and S. Ohashi, Org. Biomol. Chem., 2004, 2, 2650-2657; d) T.
Abe and T. Hoshino, Org. Biomol. Chem., 2005, 3, 3127-3139; e) T. Hoshino, Y.
21

Organic & Biomolecular Chemistry
Page 22 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Yonemura, T. Abe and Y. Sugino, Org. Biomol. Chem., 2007, 5, 792-801; f) T.
Hoshino, Y. Kumai and T. Sato, Chem. Eur. J., 2009, 15, 2091-2100; g) J. Cheng
and T. Hoshino, Org. Biomol. Chem., 2009, 7, 1689-1699; h) Y. Yonemura, Y.
Ohyama and T. Hoshino, Org. Biomol. Chem., 2012, 10, 440-446; i) K. Takahashi,
Y. Sasaki and T. Hoshino, Eur. J. Org. Chem.,2018, 1477-1490; j) I. Kaneko, Y.
Terasawa and T. Hoshino, Chem. Eur. J., 2018, 24, 11139-11157; k) T. Abe and T.
Hoshino, Org. Biomol. Chem., 2005, 3, 3127-3139; (l) H. Tanaka, H. Noguchi and I.
Abe, Org. Lett., 2004, 6, 803-806. (m) H. Tanaka, H. Noguchi and I. Abe, Org.
Lett., 2005, 7, 5873-5876. (n) H. Tanaka, N. Noguchi and I. Abe, Tetrahedron Lett.,
2004, 45, 3093-3096.
7. N. Ideno, S. Umeyama, T. Watanabe, M. Nakajima, T. Sato and T. Hoshino,
ChemBioChem, 2018, in press, DOI: 10.1002/cbic.201800281.
8. a) T. Sato and T. Hoshino, Biosci, Biotechnol. Biochem., 1999, 63, 2189-2198; (b)
T. Dang, G. D. Prestwich, Chem. Biol., 2000, 7, 643–649.
9. T. Sato, T. Abe and T. Hoshino, Chem. Commun. 1998, 2617-2618.
10. T. Hoshino, K. Shimizu and T. Sato, Angew. Chem. Int. Ed., 2004, 43, 6700-6703.
11. a) T. Sato and T. Hoshino, Biosci. Biotechnol. Biochem, 1999, 63, 1171-1180; b) T.
Sato and T. Hoshino, Biosci. Biotechnol. Biochem., 1999, 63, 2189-2198.
12. a) B. Robustell, I. Abe and G. D. Prestwich, Tetrahedron Lett., 1998, 39, 957-960; b)
B. Robustell, I. Abe and G. D. Prestwich, Tetrahedron Lett., 1998, 39, 9385-9388.
13. A. F. Barrero, E. J. Alvarez-Manzaneda and R. Alvarez-Manzaneda, Tetrahedron
Lett., 1989, 30, 3351-3352.
14. a) C-H. Chang, H-Y. Wen, W-S. Shie, C-T. Lu, M-E. Li, Y-T. Liu, W-H. Li and T-
K. Wu, Org. Biomol. Chem., 2013, 11, 4214-4219; b) S. P. T. Matsuda, L. B. Darr, E.
A. Hart, J. B.R. Herrera, K. E. McCann, M. M. Meyer, J. Pang, H. G. Schepmann,
Org. Lett., 2000, 2, 2261-2263; c) T. Sato and T. Hoshino, Biosci. Biotechnol.
Biochem., 1999, 63, 2189-2198.
15. Z-h Lin, H-x Long, Z. Bo, Y-q Wang, , Y-z Wu, Peptides, 2008, 29, 1798-1805.
16. a) K. U. Wendt, K. Poralla, G. E. Schulz, Science, 1997, 277, 1811-1815; b) K. U.
Wendt, A. Lenhart, G. E. Schulz, J. Mol. Biol., 1999, 286, 175-187; c) D. J. Reinert,
G. Balliano, G. E. Schulz, Chem. Biol., 2004, 6, 1717-1720.
17. a) M. Morikubo, Y. Fukuda, K. Ohtake, N. Shinya, D. Kiga, K. Sakamoto, M.
Asanuma, H. Hirota, S. Yokoyama , T. Hoshino, J. Am. Chem. Soc., 2006, 128,
13148-13194; b) T. Hoshino, M. Kouda, T. Abe and T. Sato, Chem. Commun., 2000,
1485-1486.
18. E.E. van Tamlen and R. G. Nadeau, Biorg. Chem., 1982, 11, 197-218.
22

Page 23 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
19. Y. Tian, X. Xin, L. Zhang and J. Qu, Org. Lett., 2016, 18, 268-271.
20. a) T. Sato, T. Hoshino, Biosci, Biotechnol. Biochem., 1999, 63, 2189-2198; b) T. Sato,
S. Sasahara, T. Yamakami, T. Hoshino, Biosci, Biotechnol. Biochem., 2002, 66, 1660-
1670.
23

Organic & Biomolecular Chemistry
Page 24 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Figure and Scheme Legends
Scheme 1. (A) Polycyclization pathways of squalene 1 into hopene 2 and hopanol 3 by
squalene-hopene cyclase. Squalene 1 folds in an all-prechair conformation in the
reaction cavity. (B) Polycyclization reactions of (3S)-2,3-oxidosqualene 4 and (3R)-2,3-
oxidosqualene 7. Here, 4 folds into an all-prechair structure, similar to the
polycyclization reaction of 1, leading to 3-hydroxyhopene 5 and 3-hydroxyhopanol 6.
However, 7 folds into a boat-chair-chair-chair-chair conformation, resulting in the
production of 3-hydroxyhopene 8 and 3-hydroxyhopanol 9.
24

Page 25 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
Figure 1. Product structures of 10─16, generated by incubating a mixture of 4 and 7
with W169X variants. The W169L variant afforded 10─13 (A), and the W169V variant
gave 14─16 (B). Compounds A─D are enzymatic products obtained by incubation of
(3S)-11-fluoro- and (3S)-14-fluorooxidosqualenes with wild-type SHC (C). The
structure of the 7-oxabicyclo[2.2.1]heptane moiety of 11 is identical to those of A and
B, including configurations at C-3 and C-6, but their C-5 stereochemistry was not
previously determined.12 Compounds C and D were reported to be produced only from
(3S)-14-fluorooxidosqualene.
Compound names:10: (1S,2S,4R)-1,3,3-trimethyl-2-((3E,7E,11E)-3,8,12,16-
tetramethylheptadeca-3,7,11,15-tetraen-1-yl)-7-oxabicyclo[2.2.1]heptane; 11:
(1R,2S,4S)-1,3,3-trimethyl-2-((3E,7E,11E)-3,8,12,16-tetramethylheptadeca-3,7,11,15-
tetraen-1-yl)-7-oxabicyclo[2.2.1]heptane; 12: achilleol A; 13: polypoda-7(8), 13,17,21-
tetraen-3-ol (3-hydroxy--polypodatetraene); 14: (13-H)-malabarica-14(27), 17, 21-
trien-3-ol; 15: 17-epi-dammara-20(21), 24-dien-3-ol; 16: 17-epi-dammara-20(21),
24-dien-3-ol.
25

Organic & Biomolecular Chemistry
Page 26 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Figure 2. Product structures of 17─24, which are produced from enzymatic reactions
of a racemic mixture of 4 and 7 with SHC mutants of G600 and double mutants of
G600F/F601G and F601G/P602F. Products 12 and 16, generated by W169X mutants
(see Fig. 1 and Scheme 2), were also furnished from these site-specific mutants. G600
26

Page 27 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
and G600F/F601F variants afforded products 12, 17, 18, 19, 20 and 21.
F601G/P602Fgenerated products 16, 20, 22, 23 and 24.
Compound names. 17: podioda-8(9),17,21-trien-3-ol; 18: podioda-9(11),17,21-trien-
3-ol; 19: (13-H)-malabarica-14(27), 17, 21-trien-3-ol; 20: (13-H)-malabarica-17,
21-dien-3-diol; 21: (13-H)-malabarica-17, 21-dien-3-diol; 22: (20R)-
dammara-13(17),24-dien-3-ol; 23: hop-17(21)-en-3-ol; 24: novel carbocyclic
triterpene. Gammacerane skeleton consists of a 6.6.6.6.6-fused pentacycle, as
exemplified by compounds C and D. We name neogammacerane skeleton for 24, in
which Me-29 at C-22 in gammacerane skeleton is migrated to C-21, and thus
neogammacera-21(22)-en-3-ol is proposed for 24.
27

Organic & Biomolecular Chemistry
Page 28 of 33
View Article Online
DOI: 10.1039/C8OB02009D
Scheme 2. Cyclization mechanisms of 4 and 7 into monocyclic products 10─12 and 13
(A) by the W169L variant and into 14─16 (B) by the W169V variant.
28

Page 29 of 33
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB02009D
Scheme 3. Cyclization mechanism of (3S)-oxidosqualene 4 to form 3-
hydroxyhopene 5 and 3-hydroxyhopanol 6 by native SHC.6j
A pink crescent represents a robust hydrophobic interaction between the E-Me on
the terminal double bond of 4 and the binding site of the reaction cavity. This strong
interaction leads to the 5-membered E-ring of hopanyl cation 33 and contributes to the
placement of Me-30 (Z-Me) of 4 at the correct position of the recognition site (a blue
crescent), which plays a crucial role for the deprotonation reaction from Z-Me to
generate 5 (path a) and for the nucleophilic attack of a water molecule to afford 6 (path
b). The highly ordered hydrogen-bonding networks generate a strongly polarized water
molecule, which is in proximity to Z-Me and abstracts the proton of Z-Me.6j,16 This
scheme indicates that the Ｚ-Me (Me-30) of 4 is led to the Me-29 of cation 33. The
dotted line indicates the long axis along the normally folded conformation (all chair
structures).
29