Enzymatic cyclizations of squalene analogs with threo- And erythro-diols at the 6,7- or 10,11-positions by recombinant squalene cyclase. Trapping of carbocation intermediates and mechanistic insights into the product and substrate specificities

Article abstract of DOI:10.1039/b506590a

In order to trap the carbocation intermediates formed during the squalene cyclization cascade, squalene analogs with threo- and erythro-dioh at the 6,7- and 10,11-positions were incubated with the recombinant squalene cyclase from Alicyclobacillus acidocaldarius, leading to the construction of the triterpenes with tetrahydropyran, octahydrochromene, decahydronaphthalene with a carbonyl group, dodecahydrobenzo[f]chromene, tetradecahydronaphtho[2,1-b]oxepine and malabaricane skeletons, almost of which are novel compounds. These products indicate that 6-membered monocyclic, 6/6-fused bicyclic and 6/6/5-fused tricyclic cations were involved in the cyclization reaction in addition to acyclic cation. All the trapped cations were the stable tertiary cation, but not the secondary one, indicating that the polycyclization reaction proceeds with a Markovnikov closure. The product profiles revealed that the cyclization reactions proceeded with the product and substrate specificities in addition to enantioselectivity. Mechanistic insight into the observed stereochemical specificities indicated that the pre-organized chair-conformation of squalene-diols is tightly constricted by the cyclase and a free motion or a conformational change is not allowed in the reaction cavity, thus, the substrate and product specificities are dominantly directed by the least motion of the nucleophilic hydroxyl group toward the intermediary carbocation; a small rotation of the hydroxyl group afforded the cyclization products in a good yield, but a large rotation of the hydroxyl group gave a marginal or no detectable amount of products. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b506590a

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Enzymatic cyclizations of squalene analogs with threo- and
erythro-diols at the 6,7- or 10,11-positions by recombinant squalene
cyclase. Trapping of carbocation intermediates and mechanistic
insights into the product and substrate specificities†
Takamasa Abe and Tsutomu Hoshino*
Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of
Science and Technology, Niigata University, Ikarashi, Niigata, 950-2181, Japan.
E-mail: hoshitsu@agr.niigata-u.ac.jp; Fax: 81-25-262-6854
Received 11th May 2005, Accepted 21st June 2005
First published as an Advance Article on the web 27th July 2005
In order to trap the carbocation intermediates formed during the squalene cyclization cascade, squalene analogs with
threo- and erythro-diols at the 6,7- and 10,11-positions were incubated with the recombinant squalene cyclase from
Alicyclobacillus acidocaldarius, leading to the construction of the triterpenes with tetrahydropyran,
octahydrochromene, decahydronaphthalene with a carbonyl group, dodecahydrobenzo[f ]chromene,
tetradecahydronaphtho[2,1-b]oxepine and malabaricane skeletons, almost of which are novel compounds. These
products indicate that 6-membered monocyclic, 6/6-fused bicyclic and 6/6/5-fused tricyclic cations were involved in
the cyclization reaction in addition to acyclic cation. All the trapped cations were the stable tertiary cation, but not
the secondary one, indicating that the polycyclization reaction proceeds with a Markovnikov closure. The product
profiles revealed that the cyclization reactions proceeded with the product and substrate specificities in addition to
enantioselectivity. Mechanistic insight into the observed stereochemical specificities indicated that the pre-organized
chair-conformation of squalene-diols is tightly constricted by the cyclase and a free motion or a conformational
change is not allowed in the reaction cavity, thus, the substrate and product specificities are dominantly directed by
the least motion of the nucleophilic hydroxyl group toward the intermediary carbocation; a small rotation of the
hydroxyl group afforded the cyclization products in a good yield, but a large rotation of the hydroxyl group gave a
marginal or no detectable amount of products.
Introduction
The enzymatic cyclizations of acyclic squalene 1 and 2,3-
oxidosqualene to form polycyclic triterpenes have fascinated
chemists and biochemists for over half a century.1,2 X-Ray
crystallographic analysis of prokaryotic squalene–hopene
cyclase (SHC) was reported in 1997.3 More recently, the
three-dimensional structure of human lanosterol synthase was
successfully elucidated.4 SHC catalyses the conversion of 1 into
the pentacyclic triterpenes of hopene 2 and hopanol 3, the ratio
being ca. 5 : 1, respectively (Scheme 1).5,6 This polycyclization
cascade is attained by a single enzyme. 1 is folded in all
pre-chair conformation inside the reaction cavity and cyclized
in a regio- and stereospecific manner through a series of
carbocationic intermediates, leading to the formation of new
five C–C bonds and nine chiral centers. Recent studies on the
SHC from Alicyclobacillus acidocaldarius have revealed that the
polycyclization reaction consists of 8 reaction steps (Scheme 1)5:
(1) 1st cyclization to form A-ring 4 by proton attack on the
terminal double bond, donated by the DXDD motif,7 (2) 2nd
ring closure to give the B-ring (6/6-fused A/B ring system 5),8,9
(3) 3rd cyclization to yield 5-membered C-ring (6/6/5-fused
A/B/C-tricyclic ring system 6) by Markovnikov closure,9,10 (4)
which then undergoes ring expansion to form the 6-membered
C-ring (6/6/6-fused tricyclic ring system 7),10 (5) 5th cyclization
to give the thermodynamically favored 5-membered D-ring
(6/6/6/5-fused A/B/C/D ring system 8, 17-epi-dammarenyl
cation),11–13 (6) followed by the second ring enlargement process
to form the 6-membered D-ring (6/6/6/6-fused A/B/C/D-ring
system, prohopanyl cation 9),11–14 (7) the last ring closure process
to construct the 6/6/6/6/5-fused A/B/C/D/E-ring system
Scheme 1 Polycyclization pathway of squalene 1 to pentacyclic hopene
† Electronic supplementary information (ESI) available: spectroscopic
data. See http://dx.doi.org/10.1039/b506590a
2 and hopanol 3, which is presumed from the abortive cyclization
products accumulated by the site-directed mutants.
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 2 7
©

View Article Online
(10, hopanyl cation),15 and (8) the final deprotonation reaction
from 10 to introduce the double bond. Recently, Rajamani
and Gao have given deeper insight into the polycyclization
mechanism of 1; they proposed that formation of 10 from 5
is achieved by a highly asynchronous concerted reaction and
occurs by a kinetical preference.16 The transient carbocations
formed during the polycyclization cascade are stabilized by
cation–p-interaction.17 Previously, we clarified the roles of all
the Trp residues conserved among prokaryotic SHCs,18 and also
demonstrated that cation–p interaction between p-electrons
of aromatic amino acids and the transient carbocations made
a great contribution to the acceleration of the polycyclization
reaction at lower incubation temperatures. The replacement of
Phe365,8 Phe60110 and Phe60515a by Tyr greatly enhanced the
reaction velocity due to the elevated p-electron density of Tyr.
In addition, a few tyrosine residues were shown to intensify the
cation-p interaction by being placed at correct positions in the
reaction cavity.19–21 The steric bulk size of active site residues is
likely to be responsible for stereochemical control during the
polycyclization reaction; the substitution of Ile with smaller
Ala or Gly at position 261 afforded false intermediates having
13a-H in the 6/6/5-fused tricyclic and 17a-H in the 6/6/6/5-
fused tetracyclic cation,14 the configurations of which are
opposite to those of true intermediates 6 (13b-H) and 8 (17b-H)
(Scheme 1). Replacement of Tyr420 or Leu607 by the bulkier
Trp afforded unnatural monocyclic triterpene having (5R, 6R)-
1,5,6-trimethylcyclohexene ring, named neoachillapentaene;22
this cyclization proceeded via a constrained boat structure. This
is the first example that 1 was folded into a boat structure despite
the SHC adopting all chair structure. These findings indicate
that the steric bulk size of active site residues direct the folding
conformation of 1, i.e., the stereochemical destiny, during
the polycyclization cascade.5 Recently, we have clarified the
catalytic mechanism for the final deprotonation reaction 10 →
2.23,24 Based on the X-ray analysis of A. acidocaldarius SHC,3a
the catalytic base responsible for the deprotonation reaction has
been suggested to be a water molecule (named a “front water”),
the polarization of which is enhanced by other waters (named
“back waters”) that construct the hydrogen-bonding network
by a combination of seven residues T41, E45, E93, R127,
Q262, W133 and Y267.3a,25 The “front water” thus polarized
can store the proton generated from either Me-29 or Me-30 of
hopanyl cation 10 to form 2 (Scheme 6, top), but 3 is produced
if the “front water” adds as hydoxyl to the C-22 cation of 10
instead of accepting the proton.3a,6,25 Recently, we have validate
this proposal by the site-directed mutagenesis experiments.23
The site-directed mutagenesis, targeted for the amino acids
surrounding the “front water” such as Q262 and P263, resulted
in a greatly enhanced production of 3 along with the decreased
formation of 2. A high production of 3 could be explained as
follows. The point mutations could give rise to the perturbation
around the “front water”. This disordered “front water” can
not act correctly as the catalytic base for the deprotonation
reaction to form 2, and in turn could be placed near to the
final hopanyl cation 10, leading to a high production of 3
without forming 2.23 The detailed polycyclization mechanism
of squalene, described above, has been inferred from many
site-directed mutagenesis experiments. Prokaryotic SHC is of
particular note from the aspect of molecular evolution; deletion
of Gly600 from the SHC altered the substrate specificity into
that of eukaryotic oxidosqualene cyclases (OSCs), which is
limited to (3S)-oxidosqualene.26
Scheme 2 Trappings of cationic intermediates 6 (6/6/5-fused tricycle)
and 8 (6/6/6/5-fused tetracycle) by employing truncated squalene
analogs 11 and 13 having a highly nucleophilic hydroxyl group.
Scheme 3 Synthetic scheme of threo squalenediols 15–18 and erythro
diols 19–22. (10R, 11R)-15 and (6R, 7R)-16 were prepared with the chiral
ligand of (DHQD)2-PHAL, while (10S, 11S)-17 and (6S, 7S)-18 were
with that of (DHQ)2-PHAL. (3R)- and (3S)-2,3-squalenediols, which
are produced in the reactions, are omitted in this scheme. To obtain
the erythro diols, squalene 1 was treated with m-chloroperbenzoic acid
(MCPBA) to afford oxidosqualenes having the epoxide at the (2,3)-,
(6,7)- and (10,11)-positions, which are then subjected to hydrolysis
with perchloric acid, giving the desired analogs. The erythro diols thus
obtained are the racemic mixture of (10R, 11S)-19 and (10S, 11R)-20,
and that of (6R, 7S)-21 and (6S, 7R)-22.
intermediates 6 and 8 are involved in the polycyclization
reaction of 1. To extend the trapping experiments of cationic
intermediates, we synthesized threo-squalene diols, e.g., 6,7-
dihydroxy-16 and 18, 10,11-dihydroxysqualenes 15 and 17 in
addition to the corresponding erythro-diols 19–22 (Scheme 3).
By using the diol analogs, cationic intermediates 4, 5 and 6
were successfully trapped. Moreover, these enzymic reactions
were more or less accompanied by product and substrate speci-
ficities. The stereochemical analyses of the enzymic products
demonstrated that the prefolded chair conformation of the
analogs is rigidly constricted by squalene cyclase, thus a free
motion or a conformational change of the folded chair structure
is prohibited in the reaction cavity, once the polycyclization
reaction starts, leading to the stereospecific construction of the
triterpene skeleton. Herein, we describe the structures of the
enzymic products and discuss the mechanism for the formation
In addition, studies on the substrate analogs also have pro-
vided some important information for the reaction mechanism
and the substrate recognition.24,27–32 Previously, we reported
the trapping experiments of the cationic intermediates. The
truncated C22-11 and C27-analogs 13 having an alcoholic group
were efficiently cyclized into the carbocyclic skeleton with 6/6/5
+ tetrahydrofuran (THF) ring 125,10 and 6/6/6/5 + THF ring
14,5,12 respectively (Scheme 2), supporting that carbocationic
3 1 2 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9

View Article Online
of the enzymic products that were generated by incubating threo-
diols 15–18 and erythro-diols 19–22 with squalene cyclase.
Results
Syntheses of threo-diols 15–18 and erythro-diols 19–22
The synthetic method is shown in Scheme 3. The asymmetric
dihydroxylation of 1 was carried out using chiral ligands of
(DHQD)2PHAL and (DHQ)2PHAL, which were devised by
Crispino and Sharpless.33 The former ligand gives 15 and 16
having the (R, R)-stereochemistry, while the latter one affords
17 and 18 with the stereochemistry of (S, S). Isolation of 15–
18 was carried out as follows. 2,3-Dihydroxysqualenes were
concomitantly produced by the asymmetric dihydroxylation
reactions. A SiO2 column chromatography eluting with hexane–
EtOAc (100 : 20) gave unreacted 1 and 2,3-dihydroxysqualenes
in a pure state. A mixture of 15 and 16 was successfully
separated with 5% AgNO3–SiO2 column chromatography by
eluting with hexane–EtOAc (100 : 50). Isolation yields of
2,3-dihydroxysqualene, 15 and 16 were ca. 4, 7 and 6.5%,
respectively. Analogs 17 and 18 were also obtained in similar
yields. The enantiomeric excess (ee) of each diol was determined
to be 88–95% based on the published values of optical rotation
of each compound.34 The erythro-analogs 19–22 were prepared
as follows. 1 was treated with MCPBA and the epoxide rings was
hydrolyzed with aqueous HClO4, giving the racemic mixture of
(10R, 11S)-19 and (10S, 11R)-20, those of (6R, 7S)-21 and (6S,
7R)-22, and (3R, S)-2,3-dihydroxysqualene, the separation of
these erythro-diols was done with a similar method to that of
the threo-diols.
Incubations of 15–22 with wild-type SHC and product profiles
Each of diols 15–22 (1 mg) was incubated with 2 cm3 of the cell-
free homogenates, which were prepared from an E. coli clone
encoding the wild-type SHC. The incubation was carried out
at optimal catalytic conditions (pH 6.0 and 60 ◦C) for 16 h
and terminated by adding methanolic KOH (5%). The hexane
extracts from the reaction mixture were subjected to a short
SO2 column chromatography eluting by hexane–EtOAc (100 :
30) in order to remove an excess of Triton X-100, which was
included in the incubation mixture. Fig. 1 shows GC traces of
the hexane extracts. As depicted in Fig. 1a, four products 23, 24,
25 and 26 were detected from threo-diol 15 in the distribution
ratio of 2.2, 9.2, 61.3 and 19.3%, respectively, and the recovered
15 was 8.0%. Fig. 1b shows that diol 17 gave three products 27,
28 and 29, the product distribution being 61.9, 3.9 and 10.4%,
respectively, along with the recovered 17 (23.8%). The GC peaks
of 17 and 27 were overlapped, thus the distribution ratios were
estimated by the HPLC separation. As seen in Fig. 1c, two
compounds 30 (21.8%) and 31 (8.2%) were obtained from the
reaction mixture of analog 16 in a moderate reaction yield (70%
of 16 recovered). The cyclization reaction of diol 18 was poor
(Fig. 1d) to afford one product 32 (10% yield), almost of 18
remained unchanged. The reaction yield was estimated by the
separation of 32 and 18 with a SiO2 column chromatography,
because their GC peaks were overlapped (Fig. 1d). A mixture of
erythro-diols 19 and 20 afforded 33 and 34 in distribution ratios
of 15.2 and 29.8%, respectively, and the amount of the recovered
analogs was 55.0% (Fig. 1e); as described in the Discussion
section, 33 and 34 were produced only from diol 20 and no
cyclization reaction occurred for diol 19 (Scheme 6). A racemic
mixture of 21 and 22 afforded 35 and 36 in yields of 9.3 and
9.2%, respectively, and the recovered racemic mixture of 21 and
22 were 81.5%. As discussed later, this reaction was active only
to 21, but inert to 22 (Scheme 6).
Fig. 1 Gas chromatograms of the hexane extracts from the incubation
mixture of analogs 15–22 with the wild-type SHC. Triton X-100 included
in the incubation mixture was removed by a short SiO2 column eluting
with hexane–EtOAc (100 : 30). (a) 15, (b) 17, (c) 16, (d) 18, (e) a racemic
mixture of 19 and 20, (f) a racemic mixture of 21 and 22. The quantities
of enzyme and the analogs used in each experiment (a–f) were identical:
2 cm3 of the cell-free homogenates (equivalent to 0.4 mg of the pure
SHC) and 1.0 mg of 15–22. The asterisk symbols indicate the impurities.
As shown in (b), (d) and (e), one of the enzymic products was inseparable
from the diol substrates, thus the relative amounts of the products and
the unreacted analogs were estimated by separating with SiO2 column
chromatography or HPLC.
amount of Triton X-100. Products 23–26 from diol 15 were
partially purified by step-wise gradient elution (hexane–EtOAc,
100 : 3 → 100 : 10). For the complete separation of products
23, 24 and 25, HPLC was carried out eluting with hexane–2-
PrOH (100 : 0.1). Product 26 was purified in a pure state by a
Purification and structures of enzymic products 23–36
All the hexane extracts from the incubation mixture were passed
through a short SiO2 column in order to remove an excess
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 2 9

View Article Online
Fig. 2 Structures of the enzymic products 23–36.
reversed phase HPLC (THF–H2O = 3 : 2). Products from
other diol substrates 16–18 were purified by a combination
of normal and reversed phase HPLC, the composition and
the ratio of the mixed solvent for the HPLC being hexane–
2-PrOH (100 : 0.1) and THF–H2O = 3 : 2, respectively.
Separation of 27 and 29 was attained by the normal phase
HPLC, but 28 was still impure. The complete purification
of 28 was done by the reversed phase HPLC. Pure 31 was
obtained by the normal HPLC, and the repetitive reversed
phase HPLC gave pure 30. Separation of product 32 and diol
18 was performed by the normal phase HPLC. Products 33
and 34, from a racemic mixture of 19 and 20, were purified as
follows. 33 was isolated by the repetitive SiO2 column chromato-
graphy (hexane–EtOAc = 100 : 1) and then the residues were
subjected to the reversed phase HPLC (THF–H2O = 1 : 1) to
afford pure 34. Products 35 and 36 obtained from diols 21 and 22
were homogeneously purified by the normal (hexane–2-PrOH =
100 : 0.05) and reverse phase HPLC (THF–H2O = 1 : 1).
was determined to be at C-13 by a clear HMBC from Me-
27 (dH 1.32, 3H, s). An apparent NOE between Me-25 (dH
0.926, 3H, s) and Me-26 (dH 0.975, 3H, d, J 7.2) indicated the
axial orientation of Me-26. The detailed analyses of HMBC
spectrum established that 24 is composed of a 6/6-fused bicyclic
skeleton having a carbonyl group (Fig. 2). Product 28 had the
same bicyclic skeleton with a carbonyl group as 24, but the
stereochemistry at C14 of 28 should be S in contrast to R of
24, because (10S,11S)-17 was used as the substrate. Tricyclic
structures 25 and 29 were produced as the enzyme products
from 15 and 17, respectively. For 25, an ether bond was found
between C-8 (dC 74.9, s) and C-13 (dC 74.5, d), the assignments
of which were determined by apparent HMBC cross peaks from
Me-26 (dH 1.21, 3H, s) and Me-27 (dH 1.33, 3H, s), respectively.
Detailed NMR analyses indicated that 25 had the 6/6/6-fused
A/B/C tricyclic ring system, where the C-ring consists of a
tetrahydropyran structure, thus dodecahydrobenzo[f ]chromene
skeleton was proposed as shown in Fig. 2. Detailed NMR
analyses of product 29 also indicated the same tricyclic system
as 25. However, the stereochemistry at C-13 of 25 was opposite
to that of 29. A strong NOE between Me-26 (dH 1.21, 3H,
s) and H-13 (dH 3.52, dd, J 11.6, 2.4) was observed for 25,
suggesting the axial orientation of H-13 in the chair structure.
On the other hand, no observation of the NOE between them
(Me-26, dH 1.18, 3H, s; H-13, dH 3.76, 1H, dd, J 12.4, 2.8)
for 29 suggested the equatorial orientation for H-13 of 29, but
the large coupling constant (J 12.4) of the H-13 of 29 does
not support the equatorial disposition. Molecular dynamics by
Chem3D (Ver. 8.0, CambridgeSoft) indicated a boat structure
for the C-ring, in which the dihedral angles between H-12 and
H-13 were estimated to be about 170 and 53◦, these values
being consistent with the observed coupling constants (J 12.4,
2.8). Product 26 had a quite interesting structure consisting
of the 6/6/7-fused A/B/C tricyclic ring system having the
tetradecahydronaphtho[2,1-b]oxepine skeleton (Fig. 2). The 1H
NMR spectrum of 26 in acetone-d6 clearly showed the presence
of alcoholic proton (dH 3.37, d, J 6.0), which had the COSY
cross peaks with H-13 (dH 3.67, t, J 6.0). An ether linkage was
Structures of all the enzymic products were determined by
detailed NMR analyses including DEPT, COSY 45, HOHAHA,
NOESY, HMQC and HMBC. All the substrate analogs 15–22
contain five olefinic protons and seven allylic methyl groups. The
1H NMR spectra of 23 showed the presence of three olefinic
protons (dH 5.51, 5.44, 5.37, each 1H, t, J 6.4 or 7.0) and four
allylic methyl groups (dH 1.69, 1.74, 1.81, 1.84, each 3H, s),
while four methyl groups appeared at higher filed (dH 0.77, 0.90,
1.29 and 1.32, each 3H, s). In the HMBC spectrum, Me-25
(dH 1.32, 3H, s) and Me-26 (dH 1.29, 3H, s) had a correlation
with C-9 (dC 75.4, s) and C-8 (dC 76.0, s), respectively, which
suggested that a tetrahydropyran ring is involved. A clear NOE
was observed between Me-26 and H-5 (dH 1.43, m), but no NOE
of Me-25/Me-26, indicating the equatorial orientation of Me-
26. Thus, 23 was determined to have bicyclic octahydrochromene
structure as shown in Fig. 2. Product 27 also had a similar
bicyclic structure, but the stereochemistry at C-8 was opposite
to that of 23; Me-25 (dH 1.27, 3H, s) of 27 had a strong NOE
cross peak for Me-26 (dH 1.25, 3H, s). Interestingly, product
24 had a carbonyl group (dC 214.3, s), the position of which
3 1 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9

View Article Online
found between C-8 (dC 80.28, s) and C-14 (dC 81.33, s). In the
HMBC spectrum, Me-27 (dH 1.616, 3H, s) had cross peaks
with C-14 and C-13 (dC 75.59, d), indicating that C(11)-OH
of diol substrate 15 was employed for the ether bridge instead
of C(10)-OH. Thus, 26 has the 7-membered oxepane skeleton.
Indeed, the clear connectivity of H-12/H-11/H-9 was found in
the COSY and HOHAHA spectra. Protons of H-13 and Me-
27 were arranged in a-disposition, because strong NOEs were
observed between OH at C-13 and Me-26 (dH 1.336, 3H, s) and
between H-7 (dH 1.57, m) and Me-27, but no NOE between
Me-26 and Me-27. Further detailed NMR analyses proved that
the complete structure of 26 is shown in Fig. 2. It should be
noted that the enzyme product having the 7-membered C-ring
was constructed only from (R, R)-15, but not from the antipode
(S, S)-17.
EI-MS spectrum of 33 was superimposable onto that of 27
(refer to the electronic supplementary information). A clear
NOE between Me-25 (dH 1.31, 3H, s) and Me-26 (dH 1.23, 3H,
s) indicated b-orientation of Me-26, verifying that the chiral
center of C-8 has S-configuration. The proton chemical shift of
H-10 of 33 was significantly different from that of 27, compared
to other proton signals; dH 3.43 for 27, while dH 3.63 for 33,
indicating that 33 has C(10)R-configuration in contrast to that of
27. Thus, product 33 was produced only from (10S, 11R)-20, not
from the corresponding enantiomer (10R, 11S)-19. Product 34
showed the same EI-MS spectrum as 25 including the fragment
ions (see the supplementary information). Detailed analyses of
the NMR spectra in C6D6 verified that 34 has the 6/6/6-fused
tricyclic skeleton (dodecahydrobenzo[f ]chromene structure) like
25. A strong NOE between Me-26 (dH 1.21, 3H, s) and H-13
(dH 3.51, dd, J 11.8, 2.2) showed the axial disposition of H-
13, verifying the same C(13)-R-stereochemistry as that of 25.
This further indicates that the C(14)-stereochemistry must be
S, because the substrate used for the incubation is erythro-diol.
Thus, product 34 was obtained only from 20, but not from 19. It
is inferable that the polycyclization reaction enantioselectively
occurred; diol 20 only was active, but inert to the corresponding
enantiomer 19.
Compounds 30 and 31 were produced from (6R, 7R)-16 as
shown in Fig. 1. The 1H NMR spectrum of 30 dissolved in C6D6
showed four olefinic protons (dH 5.55, 5.45, 5.42, 5.37) and five
allylic methyl protons (dH 1.79, 3H, s; 1.73, 6H, s; 1.69, 3H, s;
dH 1.80, 3H, s), suggesting that one double bond of 16 was used
for the cyclization reaction to afford a monocyclic skeleton. The
chemical shift values of C-4 (dC 71.5, s) and C-5 (dC 75.8, s)
verified that an ether linkage is intervenient between them. The
alcoholic proton (dH 2.76, d, J 4.0) exhibited doublet pattern
due to the spin coupling with H-6 (dH 3.48, m). Both C-5 and
C-6 of 30 must be R-configuration, because (6R, 7R)-16 was
employed as the substrate. Thus, the complete structure of 30
is depicted in Fig. 2. Product 31 had only one double bond (dH
5.45, t, J 6.8, dC 125.7, d; dC 130.9, s). An ether linkage was
found between C-14 (dC 89.95, s) and C-17 (dC 82.03, d), the
assignment of the carbon signals having been determined by
the HMBC cross peaks from Me-27 (dH 1.29, 3H, s) and Me-
28 (dH 1.22, 3H, s), respectively. A strong HMBC correlation
was observed between the hydroxyl proton (dH 2.13, s) and C-
28 (dC 21.99, q). These data strongly support that 31 possesses
5-membered tetrahydrofuran (THF) ring, but does not involve
the 6-membered tetrahydropyran ring. Detailed analyses of the
HMBC spectrum clearly verified that both 6/6/5-fused tricyclic
ring system and THF ring are involved in 31. A strong NOE
between Me-26 (dH 1.00, 3H, s) and H-13 (dH 1.78, m) indicated
the b-orientation of H-13. Stereochemistry of the THF ring was
determined as follows. The chiral center at C-17 must have R-
configuration, because diol substrate 16 used in this experiment
has (R, R)-stereochemistry. A clear NOE between Me-27 and
H-17 (dH 3.72, dd, J 8.4, 6.4) proved that these protons are
arranged in the same geometry, leading to a-orientation of Me-
27 (i.e. 14S-stereochemistry) due to a-orientation of H-17 in the
THF ring (i.e. 17R-configuration), as shown in Fig. 1. From (6S,
7S)-diol 18, only one product 32 was obtained (Fig. 1d). Product
32 also consisted of both a 6/6/5-fused tricyclic ring system and
a THF ring, which was revealed by detailed analyses of the
HMBC spectrum. A clear NOE between Me-26 (dH 1.06. 3H, s)
and H-13 (dH 1.87, m) indicated that H-13 of 32 is arranged in
the same b-orientation as that of 31. However, the orientation of
Me-27 (dH 1.23, 3H, s) is opposite to that of H-17 (dH 3.71, dd,
J 10.4, 4.6), because no NOE was found between them. Since
the diol substrate used in this experiment was (6S, 7S)-18, the
chiral center of C-17 must have S-stereochemistry. Thus, the
stereochemistry at C-14 of 32 was determined to be S as shown
in Fig. 2. When the spin–spin coupling constant of H-17/H-
16 was compared between 31 and 32, the following idea can
be proposed: the C(17)-S configuration gives rise to a pair of
large and small coupling constants (J 10.4, 4.6) as shown in the
1H NMR spectrum of 32, whereas the C(17)-R stereochemistry
provides a set of nearly equivalent values (J 8.4, 6.4) as seen in
that of 31, often leading to triplet-like splitting pattern.35
Products 35 and 36 were obtained by the incubation of
a mixture of erythro-diols, (6R, 7S)-21 and (6S, 7R)-22. EI-
MS spectra of 35 and 36 were identical to those of 30 and
32, respectively, suggesting that 35 and 36 are monocycle
and tetracycle (6/6/5-fused tricycle + THF ring), respectively.
Detailed analyses of the NMR spectra are in full accord with the
proposed structures depicted in Fig. 2. In the NOESY spectrum
of 35, H-2 (dH 1.48, m, ax.) had clear cross peaks with Me-25 (dH
1.20, 3H, s) and Me-24 (dH 1.216, 3H, s), indicating that the Me-
24 and Me-25 are arranged in axial orientation like 30. Thus, the
stereochemistry at the C-5 of 35 is R, leading to S-configuration
at the C-6, because erythro-diol was used as the substrate. In
1
the H NMR spectrum of 36 in acetone-d6, the signal of H-17
(dH 3.31) showed double–doublet splitting pattern (J 11.1 and
3.3), that is, consisted of a pair of large and small coupling
constants. This indicates C(17)-S configuration, as described in
a comparison of 31 with 32, which is further indicative of C(18)-
R, because erythro-diol was used as the substrate. No NOE was
observed between Me-27 (dH 1.37, s) and H-17, suggested that
H-17 and Me-27 was placed in an opposite direction. Thus, the
stereochemistry of the THF ring of 36 was determined to be 14S
and 17S as shown in Fig. 2.
It is noticeable that some signals of the cyclopentane and
the THF rings involved in 31, 32 and 36 did not appear
in the 13C NMR spectrum, suggesting that there was little
tumbling motion of these rings (see spectroscopic data in the
Experimental section and supporting information). Products
31, 32 and 36 are classified as malabaricane triterpenes with a
6/6/5-fused tricyclic ring system. The structure of malabaricol,36
which was isolated from the wood of Ailanthus malabarica,
is quite similar to that of 31; the structural differences are
found only in the following two points: malabaricol has a
carbonyl group at C(3) of 31 and the stereochemistry of C(18)
is opposite to that of 31 (see the structures shown in Fig. 2).
van Tamelen et al. reported the chemical synthesis of the 6/6/5-
fused tricyclic malabaricatriene from 2,3-oxidosqualene by using
a Lewis acid.37 Sharpless also reported the first nonenzymatic
synthesis of malabaricanediol (3b-hydoxymalabaricol); erythro-
18,19-dihydroxysqualene-2,3-oxide was treated with picric acid
in CH3NO2 and then allowed to stand for 22 h at ambient
temperature, giving a malabaricanediol in a yield of 7%.38
The structure of malabaricanediol has the similar fundamental
skeleton as those of 31, 32 and 36, but the configurations of C-
17 and C-18 have not been elucidated. Furthermore, malabari-
canediol was reported to have 14S-stereochemistry by Sharpless,
which was opposite to 14R configuration of malabaricol, 31, 32
and 36.
From the racemic mixture of erythro-diols of (10R, 11S)-19
and (10S, 11R)-20, products 33 and 34 were produced. Detailed
analyses of the HMBC spectrum of 33 led to the bicyclic
octahydrochromene structure like product 27. Furthermore, the
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 3 1

View Article Online
afforded product 26 (path b). The hydride shift of H-11 to the
C15-cation of 37, followed by the deprotonation from 11R-OH,
afforded a carbonyl group (path c) to yield 26. To the C6-cation
of 38, 10R-OH attacked to give 23 (path d). As described above,
the yield of 25 was significantly higher than that of 23 (ca. 28-
fold), suggesting that the nucleophilic attack of axial OH to
cation 37 was more favorable than that of equatorial OH. This
product specificity would be explained in terms of the rotation
angle between the OH and the p-orbital of carbocation. The
equatorial OH of cation 38 must be largely rotated through
C11–C12 axis in the cyclase cavity to form 23 (180◦), whereas,
in case of the axial OH, only a small motion (60◦) is enough for
the production of 25 from cation 37, resulting in a significantly
higher production of 25 than that of 23. Thus, the findings
strongly indicate that a free motion or large conformational
change of the pre-organized chair structures is not allowed in
the reaction cavity due to the tight binding of the folded chair
structures to the cyclase enzyme. A rotation angle of 10R-OH of
37 for the formation of 26 is also smaller compared to that for
the production of 23 and the equatorial OH is close to the C-15
cation, leading to higher production of 26 by ca. 9-fold than that
of 23. However, the yield of 26 having the 7-membered ring was
less by 3-fold than that of 25 with the 6-membered ring, possibly
due to the more favorable formation of 6-membered ring than
that of 7-membered ring. Further evidence that the least motion
directs the product specificity was given also by the cyclization
reaction of (10S, 11S)-diol 17 (Scheme 4B); the yield of bicyclic
27 was highest (61.9%) among all the products 27–29, due to
the least motion (60◦). Both enantiomers of 15 and 17 afforded
the similar octahydrochromene skeleton (23, 27), but the yields
were significantly different; the yield of 23 was negligible (2.2%)
and less by 30-fold than that of 27. This product specificity can
be explained also in terms of the least motion of the OH group;
only a small rotation (60◦) of the axial 10S-OH of cation 40 is
required for the formation of 27 (path h), but a large rotation
(180◦) of 10R-OH of 38 for the formation of 23. The production
of 29 from cation 39 (path e) was not high (10.4%), due to the
large rotation of 11S-OH. Path g led to the formation of 28 via
hydride shift of H-11 to the C15-cation of 39 in a way similar
to the formation of 24. Formation of naphthooxepine skeleton
from 39 is expected to occur via path f , but no detectable amount
of this skeleton was found in the incubation mixture, due to the
requirement of a large motion of the axial 10-OH of 39; 10R-OH
of 37 is close to the C15-cation to give 26, but 10S-OH of 39 is
distant from the C15-cation.
Discussion
Mechanistic insight into the formation of enzymic products
23–32 from threo-diols 15–18
By using threo-diols 15–18, cationic intermediates 4, 5 and 6
were successfully trapped. Products 23 and 27 indicate that
monocyclic cation 4 is involved during the polycyclization
cascade. Products 24–26, 28 and 29 demonstrated the inter-
mediacy of bicyclic cation 5. The presence of tricyclic cation
6 is verified by production of 31 and 32. Furthermore, the
production of 30 showed that acyclic C2-cation 42 (Scheme 5)
is involved in the cyclization reaction, which has not been
demonstrated before. Thus, the trappings of the stable tertiary
cations 4–6 and 40 were successful, but that of secondary
cation 7 failed, suggesting a significantly longer lifetime of
the tertiary cations than that of the secondary one. A ring
expansion from 5- to 6-membered ring (6 → 7) is likely to occur
through cation–p-interaction for the complete polycyclization
cascade,8,15a,17 although another cyclization mechanism has been
proposed that the polycyclization reaction occurs under a
concerted manner without violation of Markovnikov’s rule.16,39
The following results obtained from this study should be
noted.
(1) The conversion yields of squalene-diols 15–18 were
different. 10,11-Dihydroxysqualenes 15 and 17 gave high con-
version ratios of 92 and 76.2%, respectively, but those of 6,7-
dihydroxylsqualenes 16 and 18 were significantly low (10–30%).
(2) The 6/6/7-fused tricycle (26: 6/6-fused bicycle + oxepane
ring, i.e., tetradecahydronaphthooxepine skeleton) was gener-
ated from (10R, 11R)-diol 15, but not from the corresponding
enantiomer (10S, 11S)-diol 17. In addition, the monocycle 30
with a tetrahydropyran ring was constructed from (6R, 7R)-
diol 16, but not from the antipode (6S, 7S)-diol 18. Thus, the
formation of these products was enantioselective.
(3) Both enantiomers of (10R, 11R)-15 and (10S, 11S)-
17, and those of (6R, 7R)-16 and (6S, 7S)-18 gave enzymic
products with the same heterocyclic skeletons, but the product
distribution markedly differed between their enantiomers. The
cyclic skeletons produced from both enantiomers 15 and 17
were as follows: octahydrochromene skeleton (23 and 27),
decahydronaphthalene ring having an additional carbonyl group
(24 and 28), and dodecahydrobenzo[f ]chromene (25 and 29). On
the other hand, 16 and 18 gave 3-deoxymalabaricol skeleton (31
and 32) consisting of both carbocyclic 6/6/5-fused tricycle and
heterocyclic THF ring. However, the product distribution of 15
was quite different from that of the corresponding enantiomer
17. The production ratio of 25, 26, 24 and 23 from 15 was
estimated to be 27.9 : 8.8 : 4.2 : 1, while that of 27, 29and 28
from 17 was 15.9 : 2.7 : 1. Diol 16 gave monocycle 30 and tricycle
31 in a ratio of 2.7 : 1 (higher yield of monocycle 30), while the
corresponding enantiomer 18 afforded only tricycle 32. Thus, it
is inferable that the cyclization reactions of 15–18 exhibited the
product and substrate specificities, which are accompanied by
stereochemical control.
Mechanistic insights into why and how these specificities
occurred provide a better understanding of squalene cyclization
mechanism. Acyclic 1 is folded in all pre-chair conformation
in the reaction cavity, thus substrate analogs 15–18 should be
folded as depicted in Schemes 4 and 5. Previously, we reported
that the isopropylidene moiety is essential to the initiation
of the polycyclization reaction.24,27 Analogs 15–18 have the
isopropylidene double bond at both terminal sides, thus the
cyclization reactions can start from both left side (a) and right
side (b), as shown in Schemes 4 and 5. Scheme 4A shows the
mechanisms for the formations of products 23–26 from 15.
According to Scheme 1, 15 underwent the cyclization reaction to
afford both bicyclic cation 37 (like 5) from 15a and monocyclic
cation 38 (like 4) from 15b. Both 10- and 11-OH of 15 could
attack on cation 37. A nucleophilic attack of the 11R-OH on the
C15-cation of 37 gave 25 (path a), while the attack of 10R-OH
A tetrahydropyran ring 30 was constructed from (6R, 7R)-diol
16, but not from the corresponding enantiomer (6S, 7S)-18 (see
Scheme 5A–B). This enantioselectivity also can be explained in
terms of the degree of motion of the OH group; only a small
motion (60◦) toward C2-cation of acyclic cation 42 (path j),
but a large rotation (180◦) toward C2-cation of cation 44 (path
l), thus, no detectable amount of the monocyclic product with
a tetrahydropyran ring was found in the reaction mixture of
18. The production ratio of 30 to 31 was 2.7 : 1. As shown
in Scheme 5A, 7R-OH of cation 41 must rotate more largely
(180◦, path i) to produce 31 than 6R-OH of cation 42 (60◦,
path j), leading to a less amount of 31. (6S, 7S)-Diol 18 also
afforded product 32 via the least motion (60◦) of 7S-OH of
cation 43. It should be noted that both products 31 and 32
had 14S-stereochemistry (the numbering according to Fig. 2),
strongly indicating that the C10-Me of tricyclic cations 41 and
43 is in the a-orientation during the polycyclization cascade
(Scheme 5).
Insight into the formation of enzymic products 33–36 from
erythro-diols 19–22
Next, we discuss the cyclization mechanism of erythro-diols 19–
22. The formation mechanism of 33 and 34 from the racemate (19
+ 20) is shown in Scheme 6A. The product structures of 33 and
3 1 3 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9

View Article Online
Scheme 4 (A) Mechanisms for the formation of products 23–26 from (10R, 11R)-diol 15 and (B) those of products 27–29 from (10S, 11S)-diol 17.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 3 3

View Article Online
Scheme 5 (A) Mechanism for the formation of products 30 and 31 from (6R, 7R)-diol 16 and (B) that of 32 from (6S, 7S)-diol 18.
34 (Fig. 2) clearly indicate that 20 only underwent the cyclization
reaction, but the corresponding antipode 19 was not active, thus
this reaction enantioselectively proceeded. Monocyclic 45 and
bicyclic cations 46 could be converted to 33 and 34 via paths m
and n, respectively, involving a least motion (60◦), but no product
from 19 was found due to the requirement of large rotation of
the OH, as illustrated in Scheme 6A. The amount of 34 produced
was ca. two-fold higher than that of 33, indicating that bicyclic
cation 46 was more easily formed than monocyclic cation 45.
The reason is not clear, but the steric bulk size of the large OH
group may have affected the conversion yield, as discussed in
the next section. The enantioselective cyclization was also found
for the incubation of a racemic mixture of 21 and 22, 21 was
selectively converted to give 35 and 36 due to a small rotation
(60◦) of the OH (Scheme 6B), but 22 underwent no reaction due
to the large rotation (180◦). Thus, it is inferable that a motion
degree of the nucleophilic OH group, irrespective of threo- and
erythro-diols, dominantly determines the product and substrate
specificities.
Effect of the bulk size of diols on the cyclization yields
Previously, we have demonstrated that the introduction of
methyl group(s) at position(s) of C-7 and C-11 (the numbering
starts from the initiation site of cyclization reaction) of the
squalene backbone strongly prevents the formation of the chair-
folding conformation inside the reaction cavity, leading to no
reaction and/or to the early truncation of the polycyclization
cascade.29,32 This could possibly arise from the disorganization
of the folded chair conformations as a result from the repulsive
interaction of the methyl group(s) with the corresponding
recognition site(s) of the cyclase.29,32 Conformations 15b and
3 1 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9

View Article Online
be published), indicating that, in the case of epoxysqualene, the
formation of tricyclic cation 6 is more preferable than that of
bicyclic cation 5. This opposite feature between epoxide and
diol would be due to the difference of their steric bulk sizes;
the epoxide ring is smaller than that of diol. This finding also
indicates that the bulkier hydroxyl groups more or less affected
the formation of the rigidly held chair conformation, resulting
in decreased cyclization yield. However, we can unambiguously
conclude that the rotation angle of the nucleophilic OH group
substantially directs the substrate and product specificities, as
discussed in the previous sections.
In conclusion, we succeeded in trapping the intermediates of
acyclic C2-cation, monocyclic 4, bicyclic 5 and tricyclic cations
6 by using threo- and erythro-diols of squalene, demonstrat-
ing the significantly longer lifetime of these tertiary cations
(Markovnikov cations) formed during the polycyclization cas-
cade of 1. The cyclization reactions proceeded with the product
specificity and/or substrate specificity, which can be explained
in terms of the motion degree of the nucleophilic hydroxyl
group toward the intermediary carbocation. Thus, we have
now the strong evidence that all the prefolded chair confor-
mation is tightly constricted by the cyclase enzyme, thus, a
free motion or a large conformational change of the folded
chair conformation is not allowed inside the reaction cavity.
Previously, Cornforth proposed that the protosterol cation has
17a-oriented side chain,40 but in order to produce the 20R-
stereochemistry of lanosterol, a 120◦ rotation of the side chain
through the C17–C20 bond is required prior to the hydride
shift from C17 to C20. In contrast, Corey proposed, based
on studies of the substrate analogs, e.g. 20-oxa analog and
(20E)-20,21-dehydro-analog of oxidosqualene, that the side
chain at C17 of protosterol cation must be arranged in b-
orientation, in which only a small rotation (<60◦) around the
C17–C20 bond is required,41,42 thus Cornforth’s rationalization
of the conformational change to explain C20-R configuration
is no longer necessary. Corey’s idea indicates that the pre-
organized chair–boat–chair conformation is tightly bound to
the oxidosqualene cyclase and the conformational change is not
allowed in the reaction cavity, which is in good accordance with
the inference from the present investigations of squalene diols.
Moreover, through these trapping experiments, novel triterpenes
were created that have the skeletons of octahydrochromene, do-
decahydrobenzo[f ]chromene and tetradecahydronaphtho[2,1-
b]oxepine, in addition to the production of natural triterpene
skeleton of 3-deoxymalabaricol.
Scheme 6 (A) Cyclization of racemic mixture of erythro-diols 19 and
20 and (B) that of 21 and 22.
18b have a bulky hydroxyl group at 11- or 7-position, which
is arranged in an axial disposition. In addition to the large
rotation angle of the OH (180◦), which is required for the
formation of 23 from 15b and a terahydropyran ring from 18b,
the steric repulsive interaction of the bulky hydroxyl group with
the cyclase may have disordered the pre-folded chair structure,
resulting in a further decrease of the cyclization yields (2.2%
for 15b and 0% for 18b). When the cyclization reactions of
erythro-diols 20 and 21 were compared, the production of 34
was higher than that of 36 (ca. 3-fold), despite both products
having been produced via a least motion of the OH (60◦) (see
Scheme 6), indicating that bicyclic cation 5 was more easily
formed than tricyclic cation 6. A more preferable formation
of 5 was also found in a comparison of threo-diols of 15 and
18; the production amount of 25 was remarkably larger (ca. 6-
fold) than that of 32, despite both products being generated
via a least motion (60◦) of the OH (see Schemes 4 and 5).
These facts indicate that the cyclization yields depended not
only on the rotation angle of the OH, but also on the steric bulk
size of the substrate analog. The repulsive interaction of the
bulky OH group at C7 of 43 and 48 with the cyclase would be
larger compared to that of C11 of 37 and 46, resulting in higher
production of the bicyclic cation 5 than that of the tricyclic
cation 6. As described above, 10,11-diols 15 and 17 were more
favorably accepted as the substrates than 6,7-diols 16 and 18. On
the other hand, 6,7-epoxysqualene afforded significantly higher
conversion yield compared to 10,11-epoxysqualene (results to
Experimental
Analytical methods
NMR spectra were mainly recorded in C6D6 on a Bruker DMX
600 or DPX 400 spectrometer, the chemical shifts being relative
to the solvent peak dH 7.280 and dC 128.0 ppm as the internal
reference for 1H- and 13C NMR spectra, respectively. Some
synthetic intermediates were measured in CDCl3. The chemical
shifts in CDCl3 solution were given according to the internal
solvent peaks of dH 7.26 and dC 77.0 ppm. To further validate the
proposed structures, the NMR data of some enzymic products
were measured in acetone-d6, the solvent peak being referred
to be dH 2.04, dC 29.8 ppm. The coupling constants J are
given in Hz. GC analyses were done on a Shimadzu GC-8A
chromatograph equipped with a flame ionization detector (DB-
1 capillary column, 0.53 mm × 30 m). GC-MS spectra were
on a JEOL SX 100 spectrometer under electronic impact at 70
eV with a DB-1 capillary column (0.32 mm × 30 m), the oven
temperature being elevated from 220 to 270 ◦C (3 ◦C min−1). HR-
EIMS was measured by direct inlet system. Specific rotation
values were measured at 25 ◦C with a Horiba SEPA-300
polarimeter. FABMS (positive) including HRMS was measured
using glycerol matrix.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 3 5

View Article Online
Incubation condition and purification of enzymatic products
was added 2 cm3 of 3% perchloric acid and stirred for 3 h at
ambient temperature. The reaction mixture was poured into
the ice-water, then the products were extracted with hexane
(500 cm3 × 3), which was washed with 10% NaHCO3 and with
brine, then dried over anhydrous Na2CO3. The three squalene
diols were partially separated by SiO2 column chromatography.
Complete separation was accomplished with 5% AgNO3–SiO2
column chromatography eluting with hexane–EtOAc (100 : 20).
The yields were as follows: 0.28 g for the mixture of (10R, 11S)-
and (10S, 11R)-squalene diol, 0.24 g for the mixture of (6R, 7S)-
and (6S, 7R)-squalene diol, and 0.10 g for the mixture of (2, 3R)-
and (2, 3S)-oxidosqualene. NMR data of the racemic mixture
of (6R, 7S)-21 and (6S, 7R)-22 and that of (10R, 11S)-19 and
(10S, 11R)-20 are given in the ESI.
Standard incubation conditions were performed according to
the published protocols.7a,24 The cell-free homogenates were
prepared as follows. A one liter culture of E. coli encoding the
native SHC was harvested by centrifugation and to the collected
pellets was added 50 cm3 of citrate buffer solution (pH 6.0),
and then subjected to ultrasonication to disrupt the cells. The
supernatant was used for the incubations after removing the cell
debris by centrifugation. One cm3 of the supernatant contains
ca. 200 lg of the pure SHC. The incubation mixtures were
lyophilized, and then the products and the unreacted substrate
analogs were extracted three times with a mixture of hexane
and EtOAc (100 : 10). An excess of Triton X-100 detergent was
removed by passing a short SiO2 column eluting with hexane–
EtOAc (100 : 20). Each product was purified by a combination of
the normal and the reverse phase HPLC, as described in the text.
Spectroscopic data of enzymic products 23–36
Product 23 (oil). NMR data in C6D6 (400 MHz): dH 0.775
(3H, s, H-24), 0.903 (3H, s, H-23), 1.21 (dd, J 12.8, 4.8, H-3),
1.29 (3H, s, H-26), 1.32 (3H, s, H-25), 1.34 (2H, m, H-2 & H-3),
1.39 (m, H-6), 1.43 (1H, m, H-5), 1.48 (m, H-2), 1.49 (m, H-7),
1.50 (2H, m, H-6 & H-11), 1.67 (m, H-11), 1.69 (3H, s, H-29),
1.74 (3H, s, H-28), 1.75 (m, H-1), 1.81 (3H, s, H-30), 1.84 (3H, s,
H-27), 1.89 (m, H-7), 2.21 (2H, t, J 7.2, H-19), 2.22 (2H, t, J
7.2, H-15), 2.27 (2H, t, J 7.2, H-20), 2.33 (2H, t, J 7.2), 2.48
(m, H-1), 2.51 (m, H-12), 2.72 (m, H-12), 3.81 (d, J 10.0, H-10),
5.37 (t, J 6.4, H-21), 5.44 (t, J 6.4, H-17), 5.51 (t, J 7.0, H-13);
dC 16.07 (C-6), 16.11 (C-28), 16.19 (C-27), 17.72 (C-29), 20.34
(C-2), 20.68 (C-24), 23.57 (C-25), 24.29 (C-26), 25.84 (C-30),
26.08 (C-12), 27.23 (C-16), 27.30 (C-20), 31.87 (C-7), 32.14 (C-
23), 32.52 (C-11), 33.54 (C-4), 40.21 (C-15), 40.33 (C-19), 41.72
(C-3), 42.54 (C-1), 51.57 (C-5), 75.39 (C-9), 75.68 (C-10), 76.03
(C-8), 124.88 (C-17), 124.93 (C-21), 125.2 (C-13), 131.1 (C-22),
134.9 (C-18), 135.6 (C-14). The assignments of C-16 and C-20,
and those of C-15 and C-19 are interchangeable. EIMS m/z
(%): 69 (39), 81 (15), 95 (13), 109 (8), 137 (15), 177 (74), 195
(100), 444 (M+, 1). HRFABMS (glycerol) m/z (M + H): calcd.
for C30H53O2, 445.4046; found 445.4122. [a]2D5 = +26.1 (c 0.11,
CHCl3).
Syntheses of threo-squalene diols 15–18
These compounds were synthesized according to the published
protocol. The flask (500 cm3) containing 10 cm3 of BuOH–
t
H2O was well cooled to 0 ◦C. To the well-stirred solution,
(DHQD)2PHAL (40 mg, 5.0 mol%), K3Fe(CN)6 (0.99 g, 3
mmol), K2CO3 (0.42 g, 3 mmol), CH3SO2NH2 (96 mg, 1 mmol),
OsO4 (2.54 mg, 1.0 mol%) were added and further stirred for
30 min until almost of the reagents was dissolved. To the
mixture was added squalene 1 (410 mg, 1 mmol), dissolved in
a small amount of tBuOH. The reaction was further continued
by stirring at 0 ◦C for 24 h, followed by addition of Na2S2O5
(1.5 g, 7.9 mmol), and then warmed to room temperature.
The diol mixture was extracted with EtOAc (50 cm3 × 3).
The organic layer was washed with 6 N aq. NaOH and dried
over anhydrous Na2SO4 and stood overnight. After evaporating
EtOAc under reduced pressure, the residues were subjected to
a column chromatography over SiO2. To isolate the desired
materials, the following solvents were used. First, hexane was
used to remove the unreacted starting material 1. Second, by
eluting with hexane–EtOAc (100 : 10), 2,3(R)-squalene diol
was obtained in pure state, but the separation of 15 and 16
failed. AgNO3-impregnated (5%) SiO2 column chromatography
led to the successful separation, giving the desired products in
the following yields: 16.9 mg (3.8%) for 2,3(R)-squalene diol,
9.0 mg (2.0%) for 15 and 20.2 mg (4.6%) for 16. Sufficient
amounts of 16 and 17 for the present experiments were prepared
by repeated and large-scale experiments. The specific rotations
([a]25), of 15 and 16 were +10.44◦ (c 2.14, CHCl3) and +10.23
(c 1D6.96, CHCl3), respectively, indicating that the optical purities
were 94.9 and 87% ee, respectively.34 To prepare the threo-(S, S)-
diols, (DHQ)2PHAL was used instead of (DHQD)2PHAL, but
the synthetic and isolation methods were essentially the same
as those of the enantiomers. The specific rotations ([a]2D5), for 18
and 17 were +10.33◦ (c 8.51, CHCl3) and +9.75 (c 4.02, CHCl3),
respectively. Thus, the optical purities of 18 and 17 were 88.9
and 87.5% ee, respectively. The NMR and MS spectral data
were indistinguishable between the (R, R)- and the (S, S)-forms,
because of the enantiomeric relationship between them. NMR
data of 15–18 are given in the ESI.
Product 24 (oil). NMR data in C6D6 (400 MHz): dH 0.905
(m, H-5), 0.926 (3H, s, H-25), 0.930 (m, H-1), 0.950 (3H, s, H-
24), 0.975 (d, J 7.2, H-26), 1.01 (3H, s, H-23), 1.12 (m, H-9),
1.25 (ddd, 12.8, 12.8, 3.6, H-3), 1.32 (3H, s, H-27), 1.41 (m,
H-2), 1.47 (2H, m, H-3 & H-6), 1.47 (m, H-3), 1.55 (m, H-2),
1.64 (2H, m, H-6 & H-7), 1.65 (m, H-11), 1.69 (3H, s, H-29),
1.70 (3H, s, H-28), 1.75 (m, H-7), 1.80 (m, H-1), 1.803 (3H, s,
H-30), 1.86 (m, H-15), 2.03 (m, H-11), 2.07 (m, H-16), 2.18 (2H,
t, J 6.8, H-19), 2.26 (m, H-15), 2.29 (2H, t, J 6.8, H-20), 2.30
(m, H-12), 2.39 (m, H-16), 2.43 (m, H-12), 5.31 (t, J 6.8, H-17),
5.34 (t, J 6.8, H-21); dC 15.44 (C-26), 16.04 (C-28), 16.45 (C-
25), 17.71 (C-29), 17.76 (C-6), 18.76 (C-2), 20.52 (C-11), 21.79
(C-24), 22.70 (C-16), 25.75 (C-27), 25.82 (C-30), 27.08 (C-20),
29.82 (C-8), 33.45 (C-4), 33.70 (C-23), 34.45 (C-12), 34.99 (C-
7), 38.75 (C-10), 39.72 (C-1), 40.04 (C-19), 40.16 (C-15), 42.34
(C-3), 53.03 (C-9), 56.87 (C-5), 78.61 (C-14), 124.1 (C-17), 124.8
(C-21), 131.3 (C-22), 135.9 (C-18), 214.3 (C-13). EIMS m/z
(%): 69 (100), 81 (33), 95 (29), 109 (31), 137 (26), 177 (63), 294
(37), 444 (M+, 1). HRFABMS (glycerol) m/z (M + H): calcd.
for C30H53O2, 445.4046; found: 445.4127. [a]2D5 = +8.18 (c 0.275,
CHCl3).
Syntheses of a mixture of erythro-(6R, 7S)-21 and (6S, 7R)-22,
and that of erythro-(10R, 11S)-19 and (10S, 11R)-20
To the cooled solution of squalene (101.3 mg, 0.25 mmol) in
CH2Cl2 (2.5 cm3) at 0 ◦C, MCPBA (62.9 mg, 0.365 mmol) was
added and stirred for 2 h. This reaction mixture was washed
with 20% NaHCO3 (10 cm3 × 3) and with brine (10 cm3 ×
2), and the organic layer was dried over anhydrous Na2SO4.
To remove the unreacted 1, the reaction mixture was passed
through a short SiO2 column to afford 6,7-oxido- and 10,11-
oxido- and 2,3-oxidosqualenes (total yield, 71.7%). To a mixture
of the three oxidosqualenes (1.0 g) dissolved in THF (17.5 cm3),
Product 25 (oil). NMR data in C6D6 (400 MHz): dH 0.738
(3H, s, H-25), 0.780 (ddd, J 12.8,12.8,3.2, H-1), 0.888 (3H, s,
H-24), 0.900 (m, H-5), 0.956 (3H, s, H-23), 1.14 (bd, J 12.0,
H-9), 1.21 (3H, s, H-26), 1.22 (m, H-3), 1.25 (m, H-11), 1.27
(m, H-6), 1.33 (3H, s, H-27), 1.46 (m, H-3), 1.48 (m, H-2), 1.49
(m, H-12), 1.51 (m, H-7), 1.55 (m, H-1), 1.60 (m, H-11), 1.62
(m, H-12), 1.63 (m, H-6), 1.67 (m, H-2),1.69 (3H, s, H-29), 1.80
(m, H-7), 1.81 (2H, m, H-15), 1.811 (3H, s, H-30), 1.82 (3H, s,
H-28), 2.24 (2H, t, J 7.2, H-19), 2.32 (2H, t, J 7.2, H-20), 2.48
3 1 3 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9

View Article Online
(2H, m, H-16), 2.69 (s, OH), 3.52 (dd, J 11.6, 2.4, H-13), 5.38
(t, J 6.8, H-21), 5.50 (t, J 6.6, H-17); dC 15.67 (C-25), 16.10 (C-
28), 17.73 (C-29), 18.34 (C-11), 18.91 (C-2), 20.11 (C-6), 20.80
(C-26), 21.46 (C-24), 21.80 (C-27), 22.77 (C-16), 25.85 (C-30),
27.09 (C-12), 27.22 (C-20), 33.33 (C-4), 33.46 (C-23), 36.79 (C-
10), 39.08 (C-1), 39.76 (C-15), 40.24 (C-19), 42.06 (C-7), 42.34
(C-3), 56.24 (C-5), 57.52 (C-9), 73.04 (C-14), 74.51 (C-13), 74.91
(C-8), 125.0 (C-21), 125.7 (C-17), 131.1 (C-22), 134.7 (C-18).
EIMS m/z (%): 69 (100), 81 (35), 95 (31), 109 (37), 137 (32),
191 (90), 231 (51), 276 (38), 426 (M+ – H2O, 3). HRFABMS
(glycerol) m/z (M + H): calcd. for C30H53O2, 445.4046; found:
445.4070. [a]2D5 = −12.9 (c 0.458, CHCl3).
1.10 (m, H-9), 1.26 (dd, J 12.4, 3.2, H-3), 1.32 (3H, s, H-27),
1.44 (m, H-6), 1.48 (m, H-2), 1.49 (m, H-3), 1.59 (m, H-6), 1.60
(m, H-7), 1.64 (m, H-11), 1.65 (m, H-2), 1.69 (3H, s, H-29),
1.70 (3H, s, H-28), 1.73 (m, H-7), 1.79 (m, H-1), 1.806 (3H, s,
H-30), 1.81 (2H, m, H-15), 1.88 (m, H-8), 2.08 (m, H-16), 2.09
(m, H-11), 2.18 (2H, t, J 6.8, H-19), 2.25 (m, H-12), 2.27 (2H,
m, H-20), 2.40 (m, H-16), 2.46 (m, H-12), 4.06 (s, OH), 5.32 (t,
J 6.6, H-17), 5.35 (t, J 6.8, H-21); dC 15.43 (C-26), 16.02 (C-
28), 16.44 (C-25), 17.71 (C-29), 17.75 (C-6), 18.76 (C-2), 20.44
(C-11), 21.78 (C-24), 22.69 (C-16), 25.78 (C-27), 25.82 (C-30),
27.07 (C-20), 29.79 (C-8), 33.44 (C-4), 33.71 (C-23), 34.38 (C-
12), 34.98 (C-7), 38.71 (C-10), 39.68 (C-1), 40.03 (C-15), 40.15
(C-19), 42.35 (C-3), 52.95 (C-9), 56.86 (C-5), 78.60 (C-14), 124.1
(C-17), 124.8 (C-21), 131.3 (C-22), 135.9 (C-18), 214.2 (C-13).
EIMS m/z (%): 69 (70), 81 (23), 95 (22), 109 (31), 137 (26), 177
(100), 294 (97), 444 (M+, 1). HRFABMS (glycerol) m/z (M +
H): calcd. for C30H53O2, 445.4046; found: 445.4115. [a]2D5 = +32.1
(c 0.42, CHCl3).
Product 26 (oil). NMR data in C6D6 (400 MHz): dH 0.791
(3H, s, H-25), 0.840 (m, H-1), 0.866 (3H, s, H-24), 0.870 (m,
H-5), 0.958 (3H, s, H-23), 1.21 (m, H-3), 1.23 (m, H-6), 1.357
(6H, s, H-26 & H-27), 1.44 (m, H-3), 1.47 (m, H-2), 1.51 (m,
H-11), 1.59 (m, H-15), 1.61 (m, H-9), 1.64 (m, H-2), 1.65 (m,
H-12), 1.68 (m, H-6), 1.70 (3H, s, H-29), 1.72 (m, H-7), 1.74 (m,
H-11), 1.79 (m, H-1), 1.81 (3H, s, H-30), 1.85 (3H, s, H-28), 1.98
(m, H-7), 2.00 (m, H-12), 2.11 (d, J 6.0, OH), 2.16 (dd, J 13.2,
13.2, 4.8, H-15), 2.26 (2H, t, J 6.8, H-19), 2.31 (m, H-16), 2.33
(2H, t, J 6.8, H-20), 2.41 (m, H-16), 3.62 (t, J 5.4, H-13), 5.39
(t, J 6.8, H-21), 5.51 (t, J 7.0, H-17),; dC 16.14 (C-25), 16.16 (C-
28), 17.39 (C-11), 17.75 (C-29), 19.11 (C-2), 21.48 (C-6), 21.50
(C-24), 23.21 (C-16), 24.20 (C-27), 24.69 (C-26), 25.85 (C-30),
27.26 (C-20), 32.97 (C-12), 33.50 (C-4), 33.55 (C-23), 39.39 (C-
10), 40.24 (C-19), 40.43 (C-1), 41.71 (C-15), 42.25 (C-3), 44.00
(C-7), 56.64 (C-5), 59.88 (C-9), 74.73 (C-13), 80.44 (C-8), 80.73
(C-14), 125.0 (C-21), 125.7 (C-17), 131.1 (C-22), 134.6 (C-18).
1H NMR data in acetone-d6 (400 MHz): d 0.794 (3H, s, H-24),
0.807 (3H, s, H-25), 0.880 (3H, s, H-23), 0.910 (m, H-5), 0. 920
(m, H-1), 1.16 (m, H-3), 1.202 (3H, s, H-27), 1.27 (ddd, J 12.4,
12.4, 3.2, H-6), 1.34 (3H, s, H-26), 1.35 (m, H-15), 1.35 (m, H-3),
1.38 (m, H-9), 1.42 (m, H-2), 1.47 (m, H-11), 1.57 (m, H-7), 1.58
(3H, s, H-29), 1.62 (3H, s, H-28), 1.62 (m, H-6), 1.645 (3H, s,
H-30), 1.65 (m, H-2), 1.69 (m, H-12), 1.72 (m, H-11), 1.77 (m,
H-7), 1.83 (2H, m, H-1 & H-15), 1.85 (m, H-12), 1.96 (2H, t, J
6.8, H-19), 2.02 (m, H-16), 2.08 (m, H-16), 2.33 (2H, t, J 7.2,
H-20), 3.37 (d, J 6.0, OH), 3.67 (t, J 6.0, H-13), 5.10 (bt, J 6.8,
H-21), 5.16 (t, J 6.8, H-17). EIMS m/z (%): 69 (100), 81 (47), 95
(42), 109 (51), 136 (47), 191 (90), 231 (18), 235 (13), 250 (11), 276
(18), 426 (M+ – H2O, 1). HRFABMS (glycerol) m/z (M + H):
calcd. for C30H53O2, 445.4046; found: 445.4113. [a]2D5 = −22.9
(c 0.142, CHCl3).
Product 29 (oil). NMR data in C6D6 (400 MHz): dH 0.792
(3H, s, H-25), 0.820 (m, H-1), 0.888 (3H, s, H-24), 0.89 (m, H-5),
0.958 (3H, s, H-23), 1.13 (m, H-6), 1.18 (3H, s, H-26), 1.22 (m,
H-3), 1.29 (3H, s, H-27), 1.36 (m, H-11), 1.46 (m, H-3), 1.47 (m,
H-2), 1.51 (m, H-1), 1.53 (m, H-9), 1.54 (m, H-11), 1.57 (m, H-
7), 1.58 (m, H-6), 1.60 (m, H-12), 1.66 (m, H-2), 1.69 (m, H-12),
1.69 (3H, s, H-29), 1.80 (3H, s, H-30), 1.81 (3H, s, H-28), 1.84
(2H, m, H-15), 1.89 (m, H-7), 2.24 (2H, t, J 7.2, H-19), 2.32 (2H,
m, H-20), 2.51 (2H, m, H-16), 3.76 (dd, J 12.4, 2.8, H-13), 5.38
(t, J 6.8, H-21), 5.52 (t, J 6.8, H-17); dC 14.98 (C-25), 15.82 (C-
11), 16.12 (C-28), 17.73 (C-29), 18.82 (C-2), 20.42 (C-6), 21.31
(C-12), 21.63 (C-24), 21.74 (C-27), 22.83 (C-16), 25.84 (C-30),
26.54 (C-26), 27.24 (C-20), 33.18 (C-4), 33.50 (C-23), 37.24 (C-
10), 39.16 (C-1), 39.75 (C-15), 40.24 (C-19), 42.23 (C-3), 43.40
(C-7), 50.69 (C-9), 56.30 (C-5), 72.98 (C-14), 74.72 (C-13), 74.91
(C-8), 125.0 (C-21), 125.7 (C-17), 131.1 (C-22), 134.8 (C-18).
EIMS m/z (%): 69 (51), 81 (22), 95 (21), 109 (25), 137 (34), 149
(54), 191 (93), 231 (100), 249 (30), 276 (18), 279 (16), 444 (M+,
10). HRFABMS (glycerol) m/z (M + H): calcd. for C30H53O2,
445.4046; found: 445.4070. [a]2D5 = +14.7 (c 0.16, CHCl3).
Product 30 (oil). NMR data in C6D6 (400 MHz): dH 1.19
(6H, s, H-23 & H-24), 1.23 (3H, s, H-25), 1.26 (m, H-3), 1.29
(m, H-1), 1.35 (m, H-3), 1.40 (m, H-8), 1.45 (m, H-2), 1.49
(m, H-1), 1.58 (m, H-2), 1.69 (2H, m, H-7), 1.69 (3H, s, H-29),
1.73 (3H, s, H-27), 1.731 (3H, s, H-28), 1.79 (3H, s, H-26), 1.80
(3H, s, H-30), 2.21 (2H, bt, J 6.8, H-19), 2.21 (2H, m, H-15),
2.27 (4H, m, H-11 & H-12), 2.30 (2H, m, H-16 & H-20), 2.72
(m, H-8), 3.48 (bd, J 10.6, H-6), 5.37 (bt, J 6.8, H-21), 5.42
(m, H-17), 5.45 (m, H-13), 5.55 (bt, J 6.8, H-10); dC 16.10 (C-
27), 16.15 (C-28), 16.30 (C-26), 16.45 (C-2), 17.73 (C-29), 21.22
(C-25), 25.85 (C-30), 27.10 (C-20), 27.22 (C-16), 28.09 (C-24),
28.77 (C-12), 28.79 (C-11), 29.79 (C-7), 31.77 (C-1), 32.79 (C-
23), 36.62 (C-3), 37.34 (C-8), 40.21 (C-15), 40.21 (C-19), 71.47
(C-4), 75.81 (C-5), 78.94 (C-6), 124.8 (C-10), 124.8 (C-13), 124.9
(C-17), 124.9 (C-21), 131.1 (C-22), 134.9 (C-14), 135.1 (C-18),
135.6 (C-9). The following assignments are interchangeable due
to the very close values: C-10/C-13/C-17/C-21, C-11/C-12, C-
14/C-18, C-16/C-20, C-27/C-28. EIMS m/z (%): 69 (32), 81
(16), 109 (56), 127 (100), 444 (M+, 3). HRFABMS (glycerol)
m/z (M + H): calcd. for C30H53O2, 445.4046; found; 445.4029.
[a]2D5 = +5.80 (c 0.50, CHCl3).
Product 27 (oil). NMR data in C6D6 (400 MHz): dH 0.768
(3H, s, H-24), 0.910 (3H, s, H-23), 1.16 (dd, J 12.0, 2.0, H-5),
1.20 (m, H-3), 1.255 (3H, s, H-26), 1.274 (3H, s, H-25), 1.33 (m,
H-3), 1.34 (m, H-1), 1.40 (m, H-6), 1.46 (m, H-7), 1.48 (2H, m,
H-2), 1.50 (m, H-6), 1.59 (m, H-11), 1.69 (3H, s, H-29), 1.71 (2H,
m, H-1 & H-11), 1.73 (3H, s, H-28), 1.74 (m, H-7), 1.81 (3H, s,
H-30), 1.83 (3H, s, H-27), 2.21 (2H, m, H-19), 2.25 (2H, m, H-
15), 2.30 (2H, m, H-20), 2.33 (2H, m, H-16), 2.49 (m, H-12), 2.69
(m, H-12), 2.76 (d, J 5.0, OH), 3.43 (ddd, J 10.4, 5.0, 2.0, H-10),
5.37 (t, J 7.0, H-21), 5.42 (t, J 6.8, H-17), 5.53 (t, J 6.8, H-13); dC
16.11 (C-28), 16.19 (C-27), 16.34 (C-6), 17.73 (C-29), 20.26 (C-
2), 20.87 (C-24), 22.39 (C-26), 23.54 (C-25), 25.62 (C-12), 25.84
(C-30), 27.11 (C-16), 27.23 (C-20), 31.28 (C-11), 32.20 (C-23),
33.39 (C-4), 34.83 (C-7), 40.21 (C-15), 40.26 (C-19), 41.62 (C-3),
41.92 (C-1), 54.37 (C-5), 75.11 (C-9), 75.73 (C-8), 79.63 (C-10),
124.9 (C-17), 125.0 (C-21), 125.3 (C-13), 131.1 (C-22), 134.9 (C-
18), 135.2 (C-14). The assignments of C-16 and C-20 and those
of C-15 and C-19 are interchangeable. EIMS m/z (%): 69 (22),
81 (10), 95 (9), 109 (7), 137 (12), 177 (61), 195 (100), 444 (M+,
3). HRFABMS (glycerol) m/z (M + H): calcd. for C30H53O2,
445.4046; found: 445.4120. [a]2D5 = −9.01 (c 0.233, CHCl3).
Product 31 (solid). NMR data in C6D6 (400 MHz): dH 0.957
(3H, s, H-25), 0.960 (m, H-5), 0.980 (3H, s, H-24), 0.999 (3H, s,
H-26), 1.02 (m, H-1), 1.05 (3H, s, H-23), 1.22 (3H, s, H-28), 1.29
(3H, s, H-27), 1.32 (m, H-3), 1.41 (m, H-11), 1.46 (m, H-2), 1.49
(m, H-6), 1.53 (2H, m, H-3 and H-15), 1.55 (m, H-9), 1.59 (m,
H-1 & H-11), 1.68 (m, H-16), 1.70 (m, H-6), 1.74 (m, H-2), 1.77
(3H, s, H-29), 1.78 (m, H-13 & H-19), 1.79 (2H, m, H-7), 1.82
(m, H-15), 1.83 (3H, s, H-30), 1.93 (m, H-19), 1.96 (2H, m, H-
12), 1.99 (m, H-16), 2.13 (1H, bs, OH), 2.35 (m, H-20), 2.47 (m,
Product 28 (oil). NMR data in C6D6 (400 MHz): dH 0.90
(dd, J 12.8, 3.0, H-5), 0.91 (m, H-1), 0.951 (3H, s, H-24), 0.951
(3H, s, H-25), 0.990 (3H, d, J 7.2, H-26), 1.01 (3H, s, H-23),
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 3 7

View Article Online
H-20), 3.72 (dd, J 8.4, 6.4, H-17), 5.45 (t, J 6.8, H-21); dC 16.25
(C-25), 17.68 (C-29), 18.84 (C-2), 20.03 (C-6), 21.33 (C-11), 21.52
(C-24), 21.99 (C-28), 23.27 (C-20), 24.50 (C-12), 24.50 (C-27),
25.86 (C-30), 26.30 (C-26), 26.45 (C-16), 33.18 (C-4), 33.73 (C-
23), 37.40 (C-10), 38.00 (C-7), 38.49 (C-15), 41.32 (C-1), 41.58
(C-19), 42.75 (C-3), 44.66 (C-8), 57.46 (C-5), 59.23 (C-9), 60.83
(C-13), 72.48 (C-18), 82.03 (C-17), 85.95 (C-14), 125.7 (C-21),
130.9 (C-22). The following carbon signals were particularly
small and did not clearly appear: C-7, C-8, C-9, C-10, C-12, C-
13, C-16, C-17, C-19 and C-27, thus, the chemical shifts (dC) were
determined by the cross peaks of HMQC or HMBC spectra.
EIMS m/z (%): 69 (56), 81 (32), 109 (31), 191 (43), 211 (100),
231 (39), 317 (33), 344 (27), 444 (M+, 10). HREIMS: m/z (M+),
calcd. for C30H52O2, 444.3967; found, 444.3971. [a]2D5 = +1.94
(c 0.283, CHCl3).
2.24 (2H, m, H-19), 2.32 (2H, m, H-20), 2.39 (m, H-16), 2.49
(bs, OH), 2.60 (m, H-16), 3.51 (dd, J 11.8, 2.2, H-13), 5.37 (bt,
6.8, H-21), 5.52 (t, J 7.0, H-17); dC 15.67 (C-25), 16.12 (C-28),
17.72 (C-29), 18.41 (C-11), 18.91 (C-2), 20.11 (C-6), 20.78 (C-
26), 21.46 (C-24), 22.58 (C-16), 23.58 (C-27), 25.83 (C-30), 26.96
(C-12), 27.22 (C-20), 33.33 (C-4), 33.45 (C-23), 36.79 (C-10),
37.40 (C-15), 39.10 (C-1), 40.24 (C-19), 42.08 (C-7), 42.35 (C-3),
56.24 (C-5), 57.60 (C-9), 73.04 (C-14), 74.94 (C-8), 75.91 (C-
13), 125.0 (C-21), 125.8 (C-17), 131.1 (C-22), 134.8 (C-18). The
major fragments and their ion intensities in the EIMS spectrum
were superimposable on those of 25. HRFABMS (glycerol)
m/z (M + H): calcd. for C30H53O2, 445.4046; found: 445.4068.
[a]2D5 = 2.20 (c 0.25, CHCl3).
Product 35 (oil). NMR data in C6D6 (600 MHz): dH 1.153
(3H, s, H-23), 1.200 (3H, s, H-25), 1.216 (3H, s, H-24), 1.23
(m, H-1), 1.24 (m, H-3), 1.35 (m, H-3), 1.48 (m, H-2), 1.63 (m,
H-7), 1.64 (m, H-2), 1.693 (3H, s, H-29), 1.735 (3H, s, H-27),
1.735 (3H, s, H-28), 1.75 (m, H-7), 1.77 (m, H-1), 1.788 (3H, s,
H-26), 1.805 (3H, s, H-30), 2.24 (2H, m, H-15), 2.24 (2H, m,
H-19), 2.27 (2H, m, H-12), 2.27 (2H, m, H-11), 2.32 (2H, H-16),
2.32 (2H, H-20), 2.40 (m, H-8), 2.71 (m, H-8), 2.92 (bs, OH),
3.57 (bd, J 10.4, H-6), 5.37 (bt, J 4.8, H-21), 5.42 (m, H-17),
5.45 (m, H-13), 5.55 (bt, J 4.8, H-10); dC 16.11 (C-27), 16.17
(C-28), 16.25 (C-2), 16.25 (C-26), 17.72 (C-29), 22.67 (C-25),
25.83 (C-30), 27.13 (C-16), 27.24 (C-20), 27.42 (C-24), 27.88 (C-
1), 28.75 (C-11), 28.78 (C-12), 29.00 (C-7), 33.32 (C-23), 36.66
(C-3), 37.52 (C-8), 40.22 (C-15), 40.22 (C-19), 71.73 (C-4), 76.38
(C-5), 77.80 (C-6), 124.87 (C-21), 124.92 (C-13), 124.92 (C-17),
124.97 (C-10), 131.1 (C-22), 134.9 (C-14), 135.1 (C-18), 135.5
(C-9). The following assignments are interchangeable due to the
close values: C-10/C-13/C-17/C-21, C-11/C-12, C-14/C-18,
C-16/C-20, C-27/C-28. The major fragments and their ion peak
intensities in the EIMS spectrum were superimposable on those
of 30. HRFABMS (glycerol) m/z (M + H): calcd. for C30H53O2,
445.4046; found; 445.4032. [a]2D5 = −3.03 (c 0.33, CHCl3).
Product 32 (solid). NMR data in C6D6 (400 MHz): dH 0.960
(m, H-5), 0.985 (3H, s, H-25), 0.998 (3H, s, H-24), 1.02 (m, H-
1), 1.05 (3H, s, H-23), 1.06 (3H, s, H-26), 1.21 (3H, s, H-28),
1.23 (3H, s, H-27), 1.33 (m, H-3), 1.46 (m, H-11), 1.47 (m, H-9),
1.49 (2H, m, H-2 and H-6), 1.53 (2H, m, H-3 & H-15), 1.60
(m, H-16), 1.62 (2H, m, H-1 & H-11), 1.70 (m, H-15), 1.73 (m,
H-6), 1.74 (m, H-19), 1.765 (3H, s, H-29), 1.77 (m, H-2), 1.79
(2H, m, H-7), 1.83 (3H, s, H-30), 1.87 (m, H-13), 1.91 (m, H-19),
1.96 (2H, m, H-12), 2.02 (m, H-16), 2.20 (bs, OH), 2.40 (2H,
m, H-20), 3.71 (dd, J 10.4, 4.6, H-17), 5.44 (t, J 6.8, H-21);
dC 16.37 (C-25), 17.72 (C-29), 18.86 (C-2), 20.04 (C-6), 21.41
(C-11), 21.54 (C-24), 21.92 (C-28), 23.29 (C-20), 24.50 (C-12),
25.19 (C-27), 25.86 (C-30), 26.53 (C-26), 26.54 (C-16), 33.20 (C-
4), 33.75 (C-23), 37.41 (C-10), 37.87 (C-7), 39.10 (C-15), 41.16
(C-1), 41.75 (C-19), 42.70 (C-3), 44.26 (C-8), 57.46 (C-5), 59.56
(C-9), 60.31 (C-13), 71.61 (C-18), 83.96 (C-17), 85.84 (C-14),
125.7 (C-21), 130.9 (C-22). The carbon signals of C-7, C-8, C-
12, C-13, C-14, C-15, C-16, C-17 and C-27 did not appear, thus,
the dC was determined by the cross peaks of HMBC and/or
HMQC spectra. EIMS m/z (%): 69 (37), 81 (20), 95 (21), 109
(31), 191 (32), 211 (100), 231 (30), 317 (25), 344 (15), 444 (M+,
5). HREIMS: m/z (M+), calcd. for C30H52O2, 444.3967; found,
444.3978. [a]2D5 = +4.35 (c 0.09, CHCl3).
Product 33 (oil). NMR data in C6D6 (600 MHz): dH 0.761
(3H, s, H-24), 0.902 (3H, s, H-23), 1.13 (m, H-3), 1.14 (dd, J
12.1, 1.5, H-5), 1.23 (m, H-1), 1.236 (3H, s, H-26), 1.30 (m, H-
3), 1.309 (3H, s, H-25), 1.37 (dt, J 4.8, 13.1, H-7), 1.40 (m, H-6),
1.44 (2H, m, H-2), 1.53 (bd, J 12.3, H-6), 1.61 (2H, m, H-11),
1.694 (3H, s, H-29), 1.72 (m, H-1), 1.729 (3H, s, H-28), 1.812
(3H, s, H-30), 1.823 (3H, s, H-27), 1.99 (dt, J 4.8, 13.1, H-7),
2.23 (2H, m, H-19), 2.25 (2H, m, H-15), 2.32 (2H, m, H-20),
2.33 (2H, m, H-16), 2.53 (m, H-12), 2.73 (m, H-12), 3.19 (bs,
OH), 3.63 (dd, J 9.9, 2.0, H-10), 5.38 (t, J 6.9, H-21), 5.43 (t,
J 6.5, H-17), 5.51 (t, J 6.8, H-13); dC 16.08 (C-6), 16.10 (C-
27), 16.16 (C-28), 17.75 (C-29), 20.22 (C-2), 20.93 (C-24), 23.49
(C-25), 24.02 (C-26), 25.88 (C-30), 25.91 (C-12), 27.06 (C-16),
27.22 (C-20), 30.63 (C-11), 30.77 (C-7), 32.18 (C-23), 33.35 (C-
4), 40.23 (C-15), 40.25 (C-19), 41.36 (C-3), 41.84 (C-1), 54.60
(C-5), 75.51 (C-9), 76.47 (C-8), 77.33 (C-10), 124.9 (C-17), 124.9
(C-21), 125.2 (C-13), 131.1 (C-22), 134.9 (C-18), 135.4 (C-14).
The assignments of C-16 and C-20 and those of C-15 and C-
19 are interchangeable. The major fragments and their peak
intensities in the EIMS spectrum were superimposable to those
of 27. HRFABMS (glycerol) m/z (M + H): calcd. for C30H53O2,
445.4046; found: 445.4122. [a]2D5 = 12.7 (c 0.525, CHCl3).
Product 36 (solid). NMR data in C6D6 (600 MHz): dH 1.014
(3H, s, H-24), 1.032 (3H, s, H-25), 1.066 (3H, s, H-23), 1.100
(3H, s, H-26), 1.17 (m, H-5), 1.20 (m, H-1), 1.271 (3H, s, H-28),
1.371 (very broad singlet, H-27), 1.40 (m, H-3), 1.44 (m, H-11),
1.45 (m, H-16), 1.46 (m, H-13), 1.54 (m, H-2), 1.56 (m, H-3),
1.59 (m, H-6), 1.63 (dt, J 5.3, 12.9, H-19), 1.68 (2H, m, H-1 &
H-9), 1.70 (2H, m, H-15), 1.70 (m, H-16), 1.70 (m, H-1), 1.71
(m, H-11), 1.73 (2H, m, H-12), 1.78 (m, H-2), 1.80 (m, H-6),
1.825 (3H, s, H-29), 1.846 (3H, s, H-30), 1.89 (dt, J 5.3, 12.9,
H-19), 1.99 (very broad, H-7), 2.41 (m, H-20), 2.54 (m, H-20),
3.27 (very broad, H-17), 5.45 (t, J 7.0, H-21); in acetone-d6, the
signal of H-17 clearly appeared as dd (J 11.1 and 3.3), indicating
the stereochemistry of C17 is S, and OH signal was clearly found
at 3.83 (brs); dC 16.77 (C-25), 18.11 (C-29), 19.05 (C-2), 19.66
(C-28), 20.43 (C-6), 21.50 (C-24), 22.13 (C-11), 22.35 (C-20),
25.13 (C-16), 25.84 (C-30), 26.38 (C-27), 27.32 (C-26), 30.25
(C-12), 33.21 (C-4), 33.78 (C-23), 34.10 (C-15), 37.46 (C-10),
38.94 (C-7), 41.25 (C-1), 42.79 (C-3), 43.00 (C-19), 45.26 (C-8),
56.97 (C-5), 60.28 (C-9), 61.95 (C-13), 73.74 (C-17), 76.01 (C-
18), 76.38 (C-14), 126.2 (C-21), 130.7 (C-22). The assignments of
C-11 and C-12 are exchangeable. The carbon signals of C-7, C-8,
C-12, C-13, C-15, C-16, C-27 and C-28 was significantly small
and some peaks did not appear, thus, the dC was determined by
1
the cross peaks of HMBC and/or HMQC spectra. H NMR
data in acetone-d6 (600 MHz): 0.807 (3H, s, H-24), 0.823 (3H, s,
H-23), 0.832 (3H, s, H-25), 0.92 (m, H-5), 0.920 (3H, s, H-26),
0.97 (m, H-1), 1.13 (m, H-3), 1.15 (3H, s, H-28), 1.24 (m, H-11),
1.324 (s, H-27), 1.33 (m, H-3), 1.35 (m, H-2), 1.38 (m, H-13),
1.42 (m, H-11), 1.42 (m, H-19), 1.43 (m, H-6), 1.47 (m, H-1),
1.52 (m, H-9), 1.55 (m, H-16), 1.58 (m, H-6), 1.62 (2H, m, H-2 &
H-6), 1.62 (3H, s, H-29), 1.63 (m, H-12), 1.65 (3H, s, H-30), 1.67
(m, H-19), 1.70 (2H, m, H-15), 1.72 (m, H-12), 1.79 (2H, m,
H-7), 2.13 (m, H-20), 2.23 (m, H-20), 3.31 (dd, J 11.1, 3.3, H-
17), 3.83 (brs, OH), 5.11 (bt, J 6.8, H-22). The proton signal
Product 34 (oil). NMR data in C6D6 (400 MHz): dH 0.735
(3H, s, H-25), 0.77 (m, H-1), 0.889 (3H, s, H-24), 0.89 (m, H-5),
0.957 (3H, s, H-23), 1.14 (dt, 2.0, 12.4, H-9), 1.210 (3H, s, H-26),
1.23 (m, H-3), 1.25 (m, H-11), 1.27 (m, H-6), 1.380 (3H, s, H-27),
1.45 (m, H-3), 1.48 (m, H-2), 1.49 (m, H-12), 1.51 (2H, m, H-1
and H-7), 1.56 (m, H-15), 1.60 (m, H-11), 1.64 (m, H-6), 1.67 (m,
H-2), 1.682 (3H, s, H-29), 1.69 (m, H-12), 1.80 (m, H-7), 1.802
(3H, s, H-30), 1.811 (3H, s, H-28), 1.96 (td, J 12.7, 4.9, H-15),
3 1 3 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9

View Article Online
of Me-27 was significantly small. dC in acetone-d6, 16.86 (C-
25), 18.09 (C-29), 19.24 (C-2), 20.24 (C-28), 20.25 (C-6), 21.54
(C-24), 22.35 (C-11), 22.56 (C-20), 25.46 (C-16), 25.81 (C-30),
26.50 (C-12), 27.10 (C-27), 27.37 (C-26), 33.53 (C-4), 33.84 (C-
23), 34.12 (C-15), 37.82 (C-10), 39.30 (C-7), 41.53 (C-1), 43.09
(C-3), 43.37 (C-19), 45.64 (C-8), 57.29 (C-5), 60.56 (C-9), 62.50
(C-13), 73.59 (C-17), 76.72 (C-18), 76.89 (C-14), 126.7 (C-21),
130.9 (C-22). The carbon signals of C-7, C-8, C-11, C-12, C-13,
C-15, C-16, C-27 and C-28 was significantly small and some
peaks did not appear, thus, the dC was determined by the cross
peaks of HMBC and/or HMQC spectra. EIMS m/z (%): 69
(52), 81 (32), 109 (31), 191 (62), 211 (32), 231 (100), 317 (12),
344 (24), 444 (M+, 4). HREIMS: m/z (M+), calcd. for C30H52O2,
444.3967; found, 444.3974. [a]2D5 = +29.5 (c 0.78, CHCl3).
17 D. A. Dougherty, Science, 1996, 271, 163–168.
18 T. Sato and T. Hoshino, Biosci., Biotechnol., Biochem., 1999, 63,
1171–1180.
19 C. Fu¨ll and K. Poralla, FEMS Microbiol. Lett., 2000, 183, 221–224.
20 T. Sato and T. Hoshino, Biosci., Biotechnol., Biochem., 2001, 65,
2233–2242.
21 C. Fu¨ll, FEBS Lett., 2001, 509, 361–364.
22 T. Sato, S. Sasahara, T. Yamakami and T. Hoshino, Biosci.,
Biotechnol., Biochem., 2002, 66, 1660–1670.
23 T. Sato, M. Kouda and T. Hoshino, Biosci., Biotechnol., Biochem.,
2004, 68, 728–738.
24 T. Hoshino, S. Nakano, T. Kondo, T. Sato and A. Miyoshi, Org.
Biomol. Chem., 2004, 2, 1456–1470.
25 K. U. Wendt, A. Lenhart and G. E. Schulz, J. Mol. Biol., 1999, 286,
175–187.
26 T. Hoshino, K. Shimizu and T. Sato, Angew. Chem., Int. Ed., 2004,
43, 6700–6703.
27 T. Hoshino and T. Kondo, Chem. Commun., 1999, 731–732.
28 T. Hoshino and S. Ohashi, Org. Lett., 2002, 4, 2553–2556.
29 S. Nakano, S. Ohashi and T. Hoshino, Org. Biomol. Chem., 2004, 2,
2012–2022.
References
1 R. B. Woodward and K. Bloch, J. Am. Chem. Soc., 1953, 75, 2023–
2024.
30 (a) H. Tanaka, H. Noguchi and I. Abe, Org. Lett., 2004, 5, 803–806;
(b) H. Tanaka, H. Noguchi and I. Abe, Tetrahedron Lett., 2004, 45,
3093–3096; (c) I. Abe, T. Dang, Y. F. Zheng, B. A. Madden, C. Feil,
K. Poralla and G. D. Prestwich, J. Am. Chem. Soc., 1997, 119, 11333–
11334; (d) Y. F. Zheng, I. Abe and G. D. Prestwich, J. Org. Chem.,
1998, 63, 4872–4873.
2 J. W. Cornforth, R. H. Cornforth, C. Donniger, G. Popjak, Y.
Shimizu, S. Ichii, E. Forchielli and E. Caspi, J. Am. Chem. Soc.,
1965, 87, 3224–3228.
3 (a) K. U. Wendt, K. Poralla and G. E. Schulz, Science, 1997, 277,
1811–1815; (b) D. J. Reinert, G. Ballianoand and G. E. Schulz, Chem.
Biol., 2004, 11, 121–126.
4 R. Thoma, T. Schulz-Gasch, B. D’Arcy, J. Benz, J. Aebi, H. Dehmlow,
M. Hennig, M. Stihle and A. Ruf, Nature, 2004, 432, 118–122.
5 T. Hoshino and T. Sato, Chem. Commun., 2002, 291–301.
6 K. U. Wendt, G. E. Schulz, E. J. Corey and D. R. Liu, Angew. Chem.,
Int. Ed., 2000, 39, 2812–2833.
7 (a) T. Sato and T. Hoshino, Biosci., Biotechnol., Biochem., 1999, 63,
2189–2198; (b) T. Dang and G. D. Prestwich, Chem. Biol., 2000, 7,
643–649.
31 I. Abe, H. Tanaka and H. Noguchi, J. Am. Chem. Soc., 2002, 124,
14514–14515.
32 T. Hoshino, Y. Kumai, I. Kudo, S. Nakano and S. Ohashi, Org.
Biomol. Chem., 2004, 2, 2650–2657.
33 G. A. Crispino and K. B. Sharpless, Tetrahedron Lett., 1992, 33,
4273–4274.
34 J.-L. Abad, J. Casas, F. Sanchez-Baeza and A. Messeguer, J. Org.
Chem., 1995, 60, 3648–3656.
35 W. Aalbersberg and Y. Singh, Phytochemistry, 1991, 30, 921–926; A.
Hisham, M. D. Ajitha Bai, Y. Fujimoto, N. Hara and H. Shimada,
Magn. Reson. Chem., 1996, 34, 146–150.
36 A. Chawla and S. Dev, Tetrahedron Lett., 1967, 4837–4843; W. F.
Paton, I. C. Paul, A. G. Bajaj and S. Dev, Tetrahedron Lett., 1979,
4153–4154; B. N. Ravi, R. J. Wells and K. D. Croft, J. Org. Chem.,
1981, 46, 1998–2001.
8 T. Hoshino and T. Sato, Chem. Commun., 1999, 2005–2006.
9 C. Pale-Grosdemange, T. Merkofer, M. Rohmer and K. Poralla,
Tetrahedron Lett., 1999, 40, 6009–6012.
10 T. Hoshino, M. Kouda, T. Abe and S. Ohashi, Biosci., Biotechnol.,
Biochem., 1999, 63, 2038–2041.
11 C. Pale-Grosdemange, C. Feil, M. Rohmer and K. Poralla, Angew.
Chem., Int. Ed., 1998, 37, 2237–2240.
37 E. E. van Tamelen, J. Willet, M. Schwartz and R. Nadeu, J. Am.
Chem. Soc., 1966, 88, 5937–5938.
38 K. B. Sharpless, J. Am. Chem. Soc., 1970, 92, 6999–7001; K. B.
Sharpless, Chem. Commun., 1970, 1450–1451.
39 B. A., Jr. Hess and L. Smentek, Org. Lett., 2004, 6, 1717–1720.
40 J. W. Cornforth, Angew. Chem., Int. Ed. Engl., 1968, 7, 903.
41 E. J. Corey and S. C. Virgil, J. Am. Chem. Soc., 1991, 113, 4025–4026.
42 E. J. Corey, S. C. Virgil and S. Sarsha, J. Am. Chem. Soc., 1991, 113,
8171–8172.
12 T. Sato, T. Abe and T. Hoshino, Chem. Commun., 1998, 2617–2618.
13 T. Merkofer, C. Pale-Grosdemange, K. U. Wendt, M. Rohmer and
K. Poralla, Tetrahedron Lett., 1999, 40, 2121–2124.
14 T. Hoshino, T. Abe and M. Kouda, Chem. Commun., 2000, 441–442.
15 (a) T. Hoshino, M. Kouda, T. Abe and T. Sato, Chem. Commun.,
2000, 1485–1486; (b) Related paper: S. Schmitz, C. Fu¨ll, T. Glaser,
K. Albert and K. Poralla, Tetrahedron Lett., 2001, 42, 883–885.
16 R. Rajamani and J. Gao, J. Am. Chem. Soc., 2003, 125, 12768–
12781.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 1 2 7 – 3 1 3 9
3 1 3 9