Cyclization cascade of the C33-bisnorheptaprenoid catalyzed by recombinant squalene cyclase

Article abstract of DOI:10.1039/b823167b

The enzymatic cyclization reaction of polyprenoid C₃₃ by squalene-hopene cyclase (SHC) was investigated with the intention of creating an unnatural hexacyclic compound. The enzymatic products consisted of mono-, bi-, tri-, tetra- and pentacycli

Full text of DOI:10.1039/b823167b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Cyclization cascade of the C33-bisnorheptaprenoid catalyzed by recombinant
squalene cyclase†
Jun Cheng and Tsutomu Hoshino*
Received 5th January 2009, Accepted 10th February 2009
First published as an Advance Article on the web 11th March 2009
DOI: 10.1039/b823167b
The enzymatic cyclization reaction of polyprenoid C33 by squalene–hopene cyclase (SHC) was
investigated with the intention of creating an unnatural hexacyclic compound. The enzymatic products
consisted of mono-, bi-, tri-, tetra- and pentacyclic skeletons; however, hexacyclic products were not
generated, contrary to our expectations. The absence of a hexacyclic skeleton indicated that the entire
carbon chain of C33 polyprene could not be included in the reaction cavity. Formation mechanisms of
the products having mono- to pentacycles were discussed. Both chair/chair/boat conformation and
chair/chair/chair conformations were formed for a tricycle, and both chair/chair/chair/boat
conformation and chair/chair/chair/chair structures were constructed for a tetracycle. The pentacyclic
product was created from the chair/chair/chair/chair/boat conformation. Squalene was folded in an
all pre-chair conformation inside the reaction cavity to form the hopene skeleton. Therefore, the
formation of a boat structure during the polycyclization reaction indicated that the molecule of
polyprene C33 was folded improperly due to incorrect arrangement/positioning in the reaction cavity.
The creation of the hexacyclic core failed; however, it should be noted that SHC possessed great
potential to tolerate the elongated squalene analog C33, thus leading to the creation of novel
compounds with C33.
shown in Scheme 1. Carbocationic intermediates are involved in
Introduction
multi-reaction steps, and the ring expansion processes (7→8 and
Triterpenes are abundant in nature and have important biological
functions. Polycyclic triterpenes including a steroid scaffold are
9→10) occur during hopene biosynthesis. We have succeeded in
trapping all the tertiary cations (4, 5, 6, 7, 9) by using the squa-
lene analogs with highly nucleophilic hydroxyl group(s).3b,4b,5b,c
The final deprotonation reaction (11→2) exclusively occurs
from the terminal Z-methyl group but not from the E-methyl
group.6
biosynthesized via ring-forming reactions (polycyclization) of
linear molecules of either squalene 1 or 2,3-oxidosqualene with
chain length C30.1 The structural diversity of triterpene skeletons is
remarkable:2 lanosterol from vertebrates and fungi; cycloartenol
and a/b-amyrin from plants; and hopene from prokaryotes are
well known. The polycyclization reactions proceed with complete
regio- and stereospecificity, leading to the formation of new
C–C bonds and chiral centers, seven chiral centers and four
C–C bonds for the lanostane skeleton and nine stereocenters
and five C–C bonds for hopanoid. Site-directed mutations of
squalene–hopene cyclase (SHC) cause early truncation of the
polycyclization cascade1a,b and/or aberrant cyclization products
whose stereochemistry is opposite to those of the normal cycliza-
tion intermediates; this strongly indicates that the stereochemical
destiny during the polycyclization cascade is directed by the steric
bulk size of the active site residues.3 We isolated many aborted
cyclization products from the various site-directed mutants in
which the electronic and steric environments are altered.1b,3,4
Based on the number of rings of the isolated enzymatic products,
i.e. mono-, bi-, tri- and tetracyclic skeletons, we propose the
cyclization pathway of 1 to pentacyclic hopene 2 and hopanol 3, as
We have reported a several studies on the enzymatic con-
versions of squalene analogs by SHC.3b,5–7 Studies conducted
on the norsqualenes that lack a methyl group from squalene
backbone have provided some important information related to
the polycyclization pathway: (1) an isopropylidene moiety at the
terminal side is essential for the initiation of the polycyclization;6,7a
(2) the central methyl group at C(10) plays a crucial role in
the normal polycyclization pathway, and C(10)-norsqualene is
converted into the unprecedented carbocyclic skeleton(s) with a
6/5 + 5/5 + (6) ring system(s);7b (3) loss of one methyl group
in the alternative terminal position creates a 6/6/6/6/6-fused
pentacyclic ring system (tetrahymanol skeleton).6,7a Thus, the
methyl groups at both the left and right terminal sides and the
central methyl group at C(10) are indispensable for the formation
of the hopanoid skeleton. The SHC can also accept truncated
analogs with carbon-chain lengths of C15–C25,5a yielding sesqui-,
di- and sesterterpene skeletons; however, C10 analog, i.e. geraniol,
cannot be recognized as the substrate.5a Recently, enzymatic
conversions of a C20 isoprene unit containing an indole8 or pyrrole
ring9 were reported. In addition, elongated squalene analogs
were tested for the enzymatic reactions. The C31 analogs with a
methylidene appendage were examined to test whether or not they
were converted into the cyclized product(s) or acted as irreversible
Department of Applied Biological Chemistry, Faculty of Agriculture and
Graduate School of Science and Technology, Niigata University, Ikarashi 2,
Nishi-ku, Niigata, 950-2181, Japan
† Electronic supplementary information (ESI) available: Synthetic details,
NMR data. See DOI: 10.1039/b823167b
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Scheme 1 Cyclization pathway of squalene 1 to hopene 2 and hopanol 3.
inhibitors for the SHC activity.10 More recently, Abe et al. reported
that the elongated C35-analog 12 was accepted as the substrate of
the SHC, yielding product 13 with a novel 6/6/6/6/6/5-fused
hexacyclic ring system in a 10% yield as a single product.11 This
report is very interesting because the hexacyclic skeleton has never
been found in nature. However, our review study on its enzymatic
reaction demonstrated that the formation of hexacyclic scaffold 13
from 12 is impossible; instead, a large amount of tri- and tetracyclic
skeletons are produced (Scheme 2).12
heptacycles is very fascinating. We designed substrate 14 with C33
as the candidate for generating a novel hexacyclic compound. The
appendage of allyl group (C3) to 1 is essential to the construction of
hexacyclic scaffold when considering the cyclization mechanism,
as shown in the upper part of Scheme 3. Analog 14 is the bisnor-
compound of 12, suggesting that the entire carbon chain of the
less bulky 14 is likely to be incorporated into the reaction cavity
of SHC. Previously, we have reported that SHC created a six-
membered E-ring in a high yield (60%) from bisnorsqualene 15
(C28) with a vinyl group (bottom part of Scheme 3).6 Therefore,
it is likely that the terminal vinyl group of 14 is subjected to
the annulation reaction to create a 6/6/6/6/6/6-fused hexacyclic
skeleton (Scheme 3). We report herein the enzymatic products
obtained by incubating 14 with SHC. Mono-, bi-, tri-, tetra-
and pentacyclic products were generated. However, hexacyclic
Scheme 2 Polycyclization products of heptapolyprenoid C35 catalyzed by
SHC. Large amounts of tri- and tetracyclic products are generated, but
hexacyclic product 13 is not produced.12
The formation of tri- and tetracyclic compounds without the
production of hexacyclic compound suggests that the entire
carbon framework of analog 12 is not accommodated in the
reaction cavity of SHC. Because compounds comprising a multi-
ring system larger than a pentacycle have not yet been found in
nature, the enzymatic creation of larger cycles such as hexa- and
Scheme 3 Cyclization pathway of C33-polyprene 14 and bisnorsqualene
15 with C28.
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products were not formed, contrary to our expectations. This
result suggests that the C3 appendage cannot be arranged and
folded in the correct orientation and/or at the accurate position
that enable(s) the construction of the hexacyclic skeleton. The
polycyclization mechanisms of 14 are discussed in detail.
(100 : 20), and then the eluted fraction was subjected to GC
analysis. A large number of enzymatic products, up to 16, were
obtained in a high conversion yield (98%), and the yield of
unreacted 14 was 2.0%.
To isolate products 20–35, a large-scale incubation was con-
ducted by using 330 mg of 14 and 1 L of the cell-free homogenates
from 20-L culture of the cloned E. coli. The hexane extract from
the incubation mixture was subjected to a short SiO2 column in
order to remove excess Triton X-100. The eluents were analyzed
by using SiO2 TLC. The TLC of the low polar fraction (performed
using hexane) yielded 9 spots that were named in the descending
order of their Rf values as Fraction 1–9. The TLC of the high
polar fraction (performed using hexane–EtOAc = 100 : 5) yielded
two spots, which was named in a descending order as Fraction 10
and Fraction 11. The unreacted substrate 14 was found between
Fr. 9 and Fr. 10 in SiO2 TLC. Each of the fractions and 14 were
separated by SiO2 column chromatography with hexane as the
eluent; commercially available SiO2 was dried in an oven at 200 ◦C
for 2 h in order to increase its adsorbability prior to use. Fr. 1
contained product 32 (solid), which was isolated in the pure state
by the repeated SiO2 column chromatography eluting with hexane.
Fr. 2 was further subjected to SiO2 chromatography (5% AgNO3)
with step-wise elution (hexane: EtOAc = 100 : 0.5–100 : 5), yielding
pure product 24 (oil). SiO2 chromatographies (5% AgNO3) with
step-wise elution (hexane–EtOAc = 100 : 0.5–100 : 5) of Fr. 3
and Fr. 4 yielded 28 (oil) and 31 (oil), respectively, in the pure
state. Separated Fr. 5 was subjected to SiO2 chromatography (5%
AgNO3) with step-wise elution (hexane–EtOAc= 100 : 1–100 : 5),
yielding pure product 27 (oil) and a mixture of 27 and 33. A
complete separation of 27 and 33 was performed by using an
HPLC (column: Inertsil ODS-3, solvent system: THF: CH3CN:
H2O = 6 : 4 : 2), yielding a pure 33 (oil) and 27. Partially purified Fr.
6 contained two products 29 and 21; the separation was achieved
by means of argentation column chromatography (5% AgNO3)
in a manner similar to the method described above, thus yielding
pure products 29 (oil) and 21 (oil). Fr. 7 contained 23 (oil) and
26 (oil); the separation was achieved by means of argentation
column chromatography. Fr. 8 and 9 contained products 22 (oil)
and 20 (oil), respectively. Fr. 10 was subjected to a thorough SiO2
column chromatography (5% AgNO3) procedure with step-wise
elution (hexane–EtOAc= 100 : 3–100 : 10), yielding pure products
30 (oil) and 35 (oil), Similarly, Fr. 11 was subjected to SiO2
chromatography (5% AgNO3) with step-wise elution (hexane–
EtOAc= 100 : 3–100 : 10), yielding pure products in the following
elution order: 34 (oil) and then 25 (oil).
Results and discussion
Synthesis of 14
The synthesis method is shown in Scheme 4. Allylic alcohol 16
was prepared according to the published method.13 Treatment of
squalene 1 with the mixture of salicylic acid, selenium dioxide and
70% t-BuOOH in CH2Cl2 yielded 16, which was converted into
17 by using PBr3. Bromide 17 was transformed into phenylsulfone
18 by reacting with PhSO2Na. The coupling reaction of 18 with
allyl bromide occurred in the presence of n-BuLi to yield C33-
phenylsulfone 19, followed by the desulfonylation reaction with
the aid of LiBEt3H to yield the desired 14.
Scheme 4 Synthetic scheme of 14.
GC profile and the isolation of the enzymatic products from 14
Fig. 1 shows the gas chromatogram of the incubation mixture
prepared by incubating 14 (1.0 mg) with the cell-free homogenates
(2.0 ml) of E. coli clone (pET3a) encoding the native SHC from
Alicyclobacillus acidocaldarius.4a,c The incubation was carried out
for 16 h under optimal catalytic conditions (60 ◦C and pH 6.0) in
the presence of Triton X-100 (under which 1 was fully converted
into 2 and 3). To the reaction mixture, 5% KOH–MeOH was
added, and the products were extracted with hexane. The Triton
X-100 included in the hexane was removed with a short SiO2
chromatography column eluting with a mixture of hexane–EtOAc
Structures of enzymatic products 20–35
The structures of all the enzymatic products were determined by
means of detailed NMR analyses including DEPT, 1H-1H COSY,
NOESY, HMQC and HMBC spectra (see the ESI†). All the NMR
spectra were measured in C6D6. The C33 analog 14 possessed
seven allylic methyl and one vinyl group. Product 20 consisted
of four allylic methyl protons (dH 1.77, 3H, s; 1.75, 3H, s; 1.73,
3H, s; 1.67, 3H, s) and vinyl protons, suggesting that 20 is a
monocyclic compound. The presence of exomethylene group (H-
29: dH 5.00, s; 4.82, s; C-29: dC 109.4, t) in 20 was confirmed by the
following HMBC correlations: H-29/C-1 (dC 32.76, t), H-29/C-5
(dC 53.84, d) and H-29/C-6 (dC 149.4). Thus, the structure of 20
Fig. 1 Gas chromatogram of the hexane extract from the incubation
mixture of analog 14 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 : 20).
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Fig. 2 Structures of the enzymatic products. Production amounts are shown in parentheses.
was determined to have the core of 3-deoxyachilleol A, as shown in
Fig. 2. In 21, two allylic methyl protons (dH 1.77, 3H, s; 1.68, 3H,
s) and a doublet methyl group (dH 1.00, d, J = 6.0 Hz) were found
together with a vinyl group, which is indicative of the tricyclic core.
HMBC cross peaks of Me-29 (dH 1.13, 3H, s)/C-9 (dC 144.4, s) and
Me-30 (dH 1.21, 3H, s)/C-8 (dC 138.1, s) confirmed that the double
bond was located at C-8 and C-9, as shown in Fig. 2. A prominent
ion peak of m/z 231 in the MS spectrum (ESI†) further supported
the tricyclic skeleton. The tricyclic core of 21 was identical to that
of podiodatriene triterpene. The C(14)-stereochemistry cannot be
determined by means of NMR analyses; however, the cyclization
mechanism discussed later in the next section suggests the 14S-
configuration.
correlations: H-30/C-8 (dC 148.8, s), H-30/C-9 (dC 56.43, d) and
H-30/C-7 (dC 38.74, t). The cyclic structure of 22 was identical
to that of a-polypodatetraene. The detailed HMBC analyses of
23 (ESI†) assigned the tricyclic scaffold for 23. A strong NOE
between Me-30 (dH 1.10, 3H, s) and H-13 (dH 2.31, m), indicated
13b-H orientation. The position of a vinylidene moiety (H-31: dH
4.90, s; 5.16, s; C-31: dC 109.3, t; C-14: dC 154.6, s) was determined
by the HMBC cross peaks of H-31/C-13 (dC 56.98, d), H-13/C-14
and H-13/C-31. The entire structure of 23 is illustrated in Fig. 2.
Detailed HMBC analyses of 26 indicated that 26 had the same
tricyclic structure as 23. A strong NOE between H-13 (dH 2.20,
m) and H-9 (dH 1.28, m) indicated 13a-H configuration. Thus, the
stereochemistry at C-13 was opposite to that of 23. The tricyclic
skeletons of 23 and 26 were identical to that of malabaricane
triterpene. In 24, the presence of an allylic methyl group (Me-33:
dH 1.71, 3H, s) and a doublet methyl group (Me-32: dH 1.14, 3H, d,
J = 6.7 Hz) indicated a tetracyclic skeleton. The detailed HMBC
In 22, three allylic methyl groups (dH 1.74, 3H, s; 1.71, 3H, s;
1.64, 3H, s) were found, suggesting a bicyclic skeleton for 22.
The involvement of exomethylene group (H-30: dH 5.06, s; 4.80, s;
C-30; dC 106.7, t) in 22 was validated by the following HMBC
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analyses (ESI†) showed that the cyclic core was a dammarane
skeleton. The double bond position was determined to be at
C-13-C17, because the HMBC correlations were clearly observed
between Me-32 and C-17 (dC 135.1, s) and between Me-31 (dH 1.31,
3H, s) and C-13 (dC 139.6, s). To determine the stereochemistry
at C-18, the chemical shift of Me-32 (dH 0.908, d, J = 6.8 Hz)
measured in CDCl3 was very close to the published value (dH
0.910, d, J = 6.9 Hz)14 of the corresponding methyl group in 20R-
dammara-13(17)-24-diene, indicating that the stereochemistry at
C-18 of 24 was R. Thus, the entire structure of 24 is shown in
Fig. 2. Product 25 had a monocyclic compound similar as 20, but
this product had a hydroxyl group (dC 73.51, s). In 27, one allylic
Me (Me-33: dH 1.74, 3H, s) and doublet Me (Me-32: dH 1.06, d,
J = 6.5 Hz) was found, indicating that 27 possesses a tetracyclic
skeleton. In the HMBC spectrum, Me-31 (dH 1.07, 3H, s) showed
correlations with C-17 (dC 53.73, d), C-13 (dC 43.95, s) and C-
14 (dC 51.76, s), and Me-30 (dH 1.18, 3H, s) exhibited cross peaks
with C-13, C-14 and C-8 (dC 146.1, s); thus, Me-31 and Me-30 were
positioned at C-13 and C-14, respectively. Me-32 (dH 1.06, 3H, d,
J = 6.5 Hz) also showed HMBC correlation with C-17. The double
bond position in the cyclic core was determined to be at C-7-C-8
by the following HMBC cross peaks: H-7 (dH 5.48, bs)/C-5 (dC
51.76, d) and H-7/C-9 (dC 49.56, d). A definitive NOE between
Me-30 and H-17 (dH 1.64, m) indicated 17b-H stereochemistry.
These findings suggest that the cyclic core of 27 is identical to that
of euphane triterpene. The chemical shift of Me-32 (dH 0.840, d,
J = 6.5 Hz) in CDCl3 was very close to the published value (dH
0.835, d, J = 6.8 Hz)14 of the corresponding Me in 20R-euph-7(8)-
ene, indicating 18R-configuration for 27. Product 31 had a similar
tetracyclic core as that of 27, which was revealed by analyzing the
HMBC spectrum (ESI†); however, the stereochemistry at C-18 was
opposite to that of 27, because the chemical shift of Me-32 (dH
0.870, d, J = 6.5 Hz) in CDCl3 was almost identical to that of the
published value (dH 0.865, d, J = 6.7 Hz)14 of the corresponding
methyl group in tirucall-7(8)-ene with 20S-stereochemsitry. Thus,
the structure of 31 was analogous to that of tirucall-7(8)-ene, and
the entire structure is shown in Fig. 2. Detailed HMBC analyses
of 28 (ESI†) indicated the tetracyclic core. The vinylidene protons
(dH 5.10, s; 5.05, s) had a HMBC correlation with C-17 (dC 48.28).
Thus, 28 had a dammarene cyclic skeleton. A strong NOE between
Me-30 (dH 1.11, 3H, s) and H-13 (dH 1.91, m) demonstrated 13b-H
orientation. Further, a clear NOE between Me-31 (dH 0.971, 3H, s)
and H-17 (dH 2.41, m) indicated 17a-H orientation. Therefore, the
entire structure of 28 can be depicted as shown in Fig. 2. Product
33 exhibited a similar dammarene cyclic skeleton, as revealed by
analyzing the HMBC data. A definitive NOE was found between
H-13 (dH 2.06, m) and H-17 (dH 2.74, m), indicating that H-17 was
arranged in b-orientation. Thus, the structural difference between
28 and 33 was found only in the stereochemistry at C-17. In 29,
two allylic methyl groups were found: Me-32 (dH 1.83, 3H, bs) and
Me-33 (dH 1.74, 3H, s). Detailed HMBC analyses (ESI†) indicated
the tetracyclic core. In the HMBC spectrum, Me-32 correlated
with C-17 (dC 137.1, s), C-18 (dC 126.7, s) and C-19 (dC 34.13,
t), indicating that Me-32 was situated on the tetrasubstituted
double bond. A strong NOE between Me-32 and H-16 (dH 2.25,
m; 2.36, m) demonstrated that the C-17–C-18 double bond had
Z-configuration. Thus, the structure of 29 had the cyclic core
of dammara-17(18)-ene (Fig. 2). The involvement of two allylic
methyl groups (dH 1.76, s and dH 1.66, s) in 30 and the detailed
HMBC analyses allowed us to assign the tricyclic skeleton for
30. Product 30 was suggested to be a hydroxylated compound,
because the Rf value of 30 was smaller than that of substrate 14.
Me-31 (dH 1.31, 3H, s) exhibited HMBC correlations with C-14
(dC 75.49, s) and C-13 (dC 59.11, d). Furthermore, H-13 (dH 1.76,
m) exhibited a HMBC cross peak for C-14. Thus, a hydroxyl group
was located at C-14. The observation of NOE between Me-30 (dH
1.07, 3H, s) and H-13 verified the b-orientation of H-13. Thus,
30 had the cyclic skeleton of malabaricane triterpene (Fig. 2). For
1
product 32, no allylic methyl group was found in the H-NMR;
instead, vinylidene protons (dH 5.00, bs; 5.07, bs) appeared. In
addition, vinyl protons (dH 5.90, m; 5.20, bd, J = 17.1 Hz; 5.13
bd, J = 10.5 Hz) had remained without undergoing the cyclization
reaction. These findings suggest that 32 is a pentacyclic compound.
The stereochemistries of chiral centers determined by the NOESY
spectrum were consistent with the hopene skeleton; however, the
stereochemistry at C-21 (21a-H) was opposite to that of natural
hopene skeleton 2, which was confirmed by the strong NOE
between Me-32 (dH 0.845, 3H, s) and H-21 (dH 2.41, m). Product 34
had a hydroxyl group, which was confirmed by observing the signal
of dC 74.77 (s). The detailed HMBC analyses (ESI†) indicated a
tetracyclic core for 34. A strong NOE between Me-31 (dH 1.02,
3H, s) and H-17 (dH 1.80, m) verified the a-arrangement of H-
17. Product 35 had the same skeleton as that of 34, but only the
stereochemistry at C-17 was different; H-17 was arranged in the
b-orientation because of the absence of NOE between Me-31 (dH
1.22, 3H, s) and H-17 (dH 2.10, m).
Production amounts (%) of the enzymatic products are depicted
in the parentheses of Fig. 2. Mono-, bi-, tri-, tetra- and pentacyclic
products were produced, but no hexacyclic skeleton was detected.
In particular, the yields of tri- (35.3%) and tetracyclic skeletons
(40.3%) were significantly high. No product other than 20–35 was
found during isolating these enzymatic products 20–35 by SiO2
column chromatography. Furthermore, prolonged GC for 240 min
showed that no other peak appeared after the peak 35 (see Fig. 1).
These findings indicate that product(s) other than 20–35 was not
generated from 14.
Cyclization mechanism of 14 into products 20–35 by SHC
Analog 14 has isopropylidene and vinyl moieties at the both the
termini. All the products 20–35 possessed a vinyl moiety in the
side chain, indicating that the polycyclization reaction did not
initiate from the vinyl group site. Previously, we clarified that
the isopropylidene group was essential to the initiation of the
polycyclization.6,7a Thus, the cyclization reaction started from the
isopropylidene site.
The enzymatic cyclization of 14 yielded mono-, bi-, tri-, tetra-
and pentacyclic products. Analog 14 was cyclized to form mono-
cyclic cation 36 (like 5, a chair form, Scheme 5A). Deprotonation
from Me-29 yielded 20, and water attack on the C-6 cation
generated 25. Intermediate 36 was further cyclized to bicyclic
cation 37 (like 6) with a chair–chair conformation (Scheme 5B).
The proton elimination from Me-30 of 37 could yield pro-
duct 22.
The bicyclic cation 37 was further cyclized into tricyclic cations
38 and 40 (like 7). Intermediate 38 had a chair–chair–boat
conformation (Scheme 5C-a), and 40 possessed a chair–chair–
chair structure (Scheme 5C-b). In 38, 1,2-shifts (H-13a →C-14,
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Scheme 5 Formation mechanisms of mono- (A), bi- (B) and tricyclic
products (C).
Me-30→C-13), followed by the deprotonation of H-9, occurred in
antiparallel fashion to yield 21 (path a). The Newman projection
through the C13–C14 axis of 38 is depicted in 39. A small rotation
(ca. 60◦) forms the 14S-configuration, and a large rotation (ca.
120◦) yields 14R-stereochemistry. The least motion is preferable
to the large movement inside the enzyme cavity, because the
substrate is tightly constricted by the cyclase.5b,c,12 Thus, product 21
is presumed to have 14S-stereochemistry. The deprotonation from
Me-31 of 38 yielded 26 (path b). In 40, the proton elimination from
Me-31 yielded 23 (path c), and the attack of water molecule onto
the C-14 cation yielded 30 (path d).
Scheme 6 Formation mechanisms of tetracyclic (A) and pentacyclic
products (B).
As shown in Scheme 6A, the folding of 14 into a chair–chair–
chair–boat conformation could lead to the tetracyclic cation 41
with 17a-H (Scheme 6A-a), and the organization of a chair–
chair–chair–chair conformation could yield the tetracyclic cation
43 having 17b-H (Scheme 6A-b). The production ratio of 41 to
43 was 1.3 : 1. Successive 1,2-shifts of hydrides and methyl groups
(H-17a→C-18, H-13→C-17, Me-31→C-13 and Me-30→C-14),
followed by deprotonation of H-7, yielded 27 and 31, which were
produced from 41 and 43 (like 9), respectively (path a). The
production amount of 27 was significantly higher by ca. 6-fold as
compared to that of 31. This could be attributed to the preference
of antiparallel migration of H-13→C-17 in 41, but its migration in
43 cannot be promoted due to the parallel orientation of H-13 and
H-17. The deprotonation from Me-32 yielded 28 and 33 (path b).
The elimination of H-17 of 41 and/or 43 led to the introduction
of C17–C18 double bond, yielding 29 (path c). The production of
29 having Z-configuration, but not E-geometry, indicated that the
enzymatic reaction proceeded kinetically to form an energetically
unfavourable product. A water attack to C-18 cation of 41 and 43
yielded 34 and 35, respectively (path d). The 18R-configuration
in 27 could be attained via a least motion pathway involving
only a small rotation (60◦) about the C17–C18 axis (see Newman
projection 42). As described above, a large motion (120◦) leading
to 18S was prohibited. Based on the same idea as in the case of 27,
product 31 had 18S-stereochemistry due to a small motion (60◦)
as depicted in Newman projection 44.
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Further cyclization of intermediate 43 into a chair–chair–chair–
chair–boat conformation yielded 6/6/6/6/5-fused pentacyclic
cation 45. The deprotonation from Me-33 yielded 32. It should
be noted that 45 was different from hopanyl cation 11 because the
C-22 stereochemistry of 45 was opposite to that of 11.
Experimental
Analytical method
NMR spectra were measured in C6D6 on a Bruker DMX600 or
DPX400 spectrometer, the chemical shifts being given in ppm
relative to the solvent peak dH 7.280 and dC 128.0 ppm as the
internal reference for 1H- and 13C NMR spectra, respectively. The
chemical shifts in CDCl3 solution were given according to the
internal solvent peaks of dH 7.26 and dC 77.0 ppm. GC analyses
were performed on a Shimadzu GC-8A chromatograph equipped
with 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 290 ◦C (3 C min-1). A Hitachi HPLC apparatus was used
that was composed of L-7100 pump, L-7400 UV detector and
D-2500 chromato-integrator (flow rate, 1 ml min-1; detected at
210 nm). HR-EIMS was measured b◦y direct inlet system. Specific
rotation values were recorded at 25 C with a Horiba SEPA-300
polarimeter.
Conclusion
In this study, mono-, bi-, tri-, tetracyclic and pentacyclic skeletons
were produced.
Abrogation of the polycyclization cascade at mono- (13%)
and bicyclic stages (7%) suggested that 14 was inappropriately
arranged around the formation sites of A and B rings. The tri-
and tetracyclic intermediates 40 and 43 were analogous to 7 and
9, respectively, including the stereochemistries. Thus, 40 and 43
were true cyclic intermediates. However, 38 and 41 were false
intermediates, because the stereochemistries of C-13 and C-17 of
these cyclic cations were opposite to those of 7 and 9. In addition,
the false intermediates were generated in higher yields as compared
to those obtained for the true intermediates; for the tricyclic core,
the false intermediate 38 possessed 26.3% yield, while the true
intermediate 40 comprised 9.0%. In the case of tetracyclic skeleton,
24.4% was obtained for the false intermediate 41, and 18.3% was
obtained for the true intermediate 43. The significantly higher
production of the false tri- and tetracyclic intermediates indicated
that substrate 14 was improperly folded inside the reaction cavity.
Pentacyclic skeleton 32 was produced in a manner similar to that
for squalene substrate 1; however, the C-22 stereochemistry of
E-ring was opposed to that of hopene 2, indicating that cationic
intermediate 45 was a false intermediate. The formation of these
false intermediates was attributed to the appendage of C3 to
squalene (C30). The introduction of extra C3 unit into 1 would
have prohibited the correct placement of the C33 molecule 14
across the entire A–E ring formation sites of the reaction cavity,
leading to the failure of the participation of vinyl group in the
polycyclization reaction. Thus, no hexacyclic scaffold could be
constructed. Pentacyclic compounds were not produced from 1212
but generated from 14, albeit in small amounts (2.2%). In addition,
mono- and bicyclic skeletons were not generated from 12; however,
these cores were produced in 20% yield from 14. Thus, the product
distribution was significantly different between 12 and 14. This
difference would have occurred because the molecular orientation
of 14 inside the reaction cavity differs extensively from that of
12. Substrate 14 lacks two methyl groups of 12. It was surprising
that the presence of two methyl groups at the terminal, albeit
a subtle change between these substrate structures, significantly
influenced the substrate placement inside the reaction cavity, which
could have led to distinct polycyclization pathways between 12
and 14. Furthermore, no production of a hexacyclic compound
from 14 indicated that the reaction cavity was not sufficiently
large to accept appropriately the elongated carbon chain lengths
of C33. Contrary to our expectations, it can be concluded that
hexacyclic terpenoid(s) cannot be created from the elongated C33
molecule 14 as well as from C35 12.12 However, the present and the
previous studies12 show that the elongated C33 and C25 analogs can
be recognized as substrates to yield new cyclic products with C33
and C35 in a high conversion yield (ca. 98%). Thus, SHC exhibits
admirable plasticity to tolerate a variety of truncate and elongated
substrate analogs.
Synthesis of (6E, 10E, 14E, 17E, 21E)-5,9,13,18,
22,26-hexamethyltricosa-1,5,9,13, 17,21-hexanene 14
The synthetic experiments are described in the ESI.†
Incubation of 14 with the wild-type SHC from A. acidocaldarius
The culture condition of the cloned E. coli encoding the native
SHC (pET3a vector) and the preparation of the cell-free extract
were performed according to the method described previously.4–7
The incubation was done at the optimal catalytic conditions that
were described in the previous papers.4
Spectroscopic data of products 20–35
Product 20. 1H-NMR (400 MHz, C6D6): d = 0.972 (Me-28,
3H, s), 1.075 (Me-27, 3H, s), 1.27 (H-3, 1H, m), 1.52 (H-3, 1H,
m), 1.61 (H-2, 2H,m), 1.64 (H-7, m), 1.671 (Me-33, 3H, s), 1.731
(Me-32, 3H, s), 1.746 (Me-31, 3H, s), 1.770 (Me-30 & H-7, 4H, s),
1.86 (H-5, 1H, dd, J = 11.4, 3.0 Hz), 2.05 (H-8, m). 2.10 (H-1, m),
2.17 (H-23, 2H,m), 2.22 (H-1, H-15, H-19 & H-24, 7H, m), 2.23
(H-8, m), 2.27 (H-11 & H-12, 4H,m), 2.30 (H-16 & H-20, 4H,m),
4.82 (H-29, 1H, bs), 5.00 (H-29, 1H, bs), 5.12 (H-26b, 1H, bd,
J = 10 Hz), 5.16 (H-26a, 1H, bdd, J = 16.4, 1.6 Hz), 5.36 (H-21,
1H, t, J = 6.8 Hz), 5.42 (H-17, 1H, t, J = 6.8 Hz), 5.47 (H-10 &
H-13, 2H, m), 5.93 (H-25, 1H, m); 13C NMR (100 MHz, C6D6):
d = 16.01 (C-33, q), 16.10 (C-32, q), 16.19 (C-31, q), 16.28 (C-30,
q), 24.07 (C-2, t), 25.19 (C-7, t), 26.51 (C-28, q), 27.03 (C-20, t),
27.11 (C-16, t), 28.57 (C-27, q), 28.77 (C-11 & C-12, 2C, t), 32.76
(C-1, t), 32.76 (C-24, t), 34.96 (C-4, s), 36.48 (C-3, t), 38.68 (C-8,
t), 39.45 (C-23, t), 40.16 (C-19, t), 40.24 (C-15, t), 53.84 (C-5, d),
109.4 (C-29, t), 114.5 (C-26, t), 124.6 (C-10 & C-17, 2C, d), 124.9
(C-13, d), 125.0 (C-21, d), 134.4 (C-22, s), 134.9 (C-18, s), 135.2 (C-
14, s), 135.7 (C-9, s), 138.8 (C-25, d), 149.4 (C-6, s). The following
13C NMR signals are indistinguishable from each other, due to
very close chemical shifts: C-31/C-32, C-16/C-20 and C-15/C-
19. EIMS: m/z (%): 81(80), 95(46), 109(100), 149(34), 177(21),
341(10), 435(4), 450(M+, 6). HRMS (EI): m/z: calcd for C33H54
This journal is
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Product 23. 1H-NMR (400 MHz, C6D6): d = 0.92 (H-5, 1H,
m), 0.945 (Me-27, 3H, s), 0.969 (Me-28, 3H, s), 0.994 (Me-29, 3H,
s), 1.08 (H-1, 1H, m), 1.101 (Me-30, 3H, s), 1.25 (H-3, 1H, m), 1.44
(H-7, 1H, m), 1.48 (H-3. 1H, m), 1.49 (H-2, 1H, m), 1.49 (H-6,
1H, m), 1.56 (H-11, 1H, m), 1.59 (H-1, 1H, m), 1.65 (H-12, 1H,
m), 1.65 (H-9, 1H, m), 1.670 (Me-33, 3H, s), 1.68 (H-6, 1H, m),
1.70 (H-7, 1H, m), 1.721 (Me-32, 3H, s), 1.76 (H-2, 1H, m), 1.84
(H-12, 1H, m), 2.15 (H-12, 1H, m), 2.18 (H-23, 2H, m), 2.20 (H-
15, 1H, m), 2.20 (H-19, 2H, m), 2.27 (H-20, 2H, m), 2.27 (H-24,
2H, m), 2.30 (H-16, 1H, m), 2.31 (H-13, 1H, m), 2.34 (H-15, 1H,
m), 2.40 (H-16, 1H, m), 4.90 (H-31, 1H, s), 5.11 (H-26b, bd, J =
10.4 Hz), 5.16 (26Ha, 1H, bd, J = 16.8), 5.16 (H-31, 1H, s), 5.35
(H-21, 1H, t, J = 6.4 Hz), 5.42 (H-17, 1H, t, J = 6.4 Hz), 5.93
(H-25, 1H, m); 13C NMR (100 MHz, C6D6): d = 15.80 (C-29, q),
16.04 (C-33, q), 16.12 (C-32, q), 18.83 (C-2, t), 19.63 (C-6, t), 21.06
(C-11, t), 21.63 (C-28, q), 25.01 (C-30, q), 27.00 (C-20, t), 27.38
(C-16, t), 28.17 (C-12, t), 32.77 (C-24, d), 33.14 (C-4, s), 33.62
(C-27, q), 37.05 (C-10, s), 37.10 (C-7, t), 39.45 (C-23, d), 39.81
(C-15, t), 40.13 (C-19, t), 40.74 (C-1, t), 42.68 (C-3, t), 45.86 (C-8,
s), 55.84 (C-9, d), 56.98 (C-13, d), 57.25 (C-5, d), 109.3 (C-31, q),
114.5 (C-26, t), 124.8 (C-21, d), 125.0 (C-17, d), 134.5 (C-22, s),
135.1 (C-18, s), 138.8 (C-25, d), 154.6 (C-14, s). The following 1H-
and 13C NMR signals are indistinguishable from each other, due
to very close chemical shifts: H-2/H-6 and C-2/C-6. EIMS: m/z
(%): 81(55), 95(36), 109(51), 123(19), 137(19), 149(15), 163(12),
177(12), 191(100), 205(14), 231(28), 341(5), 435(5), 450(M+, 10).
HRMS (EI): m/z: calcd for C33H54 (M+) 450.4226, found 450.4227.
[a]2D5= +1.42 (c = 0.2, EtOH). Oil.
Product 24. 1H-NMR (600 MHz, C6D6): d = 0.94 (H-5, 1H,
m), 0.95 (H-1, 1H, m), 0.972 (Me-28, 3H, s), 1.67 (H-7, 1H, m),
1.017 (Me-29, 3H, s), 1.03 (Me-27, 3H, s), 1.105 (Me-30, 3H, s),
1.144 (Me-32, 3H, d, J = 6.7 Hz), 1.27 (H-3, 1H, m), 1.310 (Me-31,
3H, s), 1.43 (H-11, 1H, m), 1.45 (H-2, 1H, m), 1.46 (H-6, 1H, m),
1.50 (H-3, H-19 & H-7, 3H, m), 1.60 (H-9, 1H, bd, J = 12.2 Hz),
1.67 (H-6, 1H, m), 1.70 (H-2 & H-11, 2H, m), 1.79 (H-1, 1H, m),
1.82 (H-19, 1H, m), 2.05 (H-12, 1H, m), 2.11 (H-15, 2H,m), 2.18
(H-20, 1H, m), 2.23 (H-23, 2H.m), 2.27 (H-24, 2H,m), 2.30 (H-16,
1H, m), 2.33 (H-20, 1H, m), 2.48 (H-16, 1H, m), 2.62 (H-12, 1H,
m), 2.67 (H-18, 1H, m), 5.14 (H-26b, 1H, bd, J = 9.7 Hz), 5.23
(H-26a, 1H, bd, J = 17.1 Hz), 5.44 (H-21, 1H, bt, J = 7.1 Hz),
5.97 (H-25, 1H, m); 13C NMR (150 MHz, C6D6): d = 16.03 (C-33,
q), 16.67 (C-29, q), 17.67 (C-30, q), 18.99 (C-2, t), 19.08 (C-6,
t), 20.21 (C-32, q), 21.91 (C-28, q), 22.19 (C-11, t), 23.13 (C-31,
q), 23.41 (C-12, t), 29.49 (C-16, t), 31.09 (C-15, t), 32.27 (C-18,
d), 32.63 (C-20, t), 32.77 (C-24, t), 33.53 (C-4, s), 33.68 (C-27, q),
35.43 (C-19, t), 35.85 (C-7, t), 38.10 (C-10, s), 39.52 (C-23, t), 40.96
(C-1, t), 41.67 (C-8, s), 42.41 (C-3, t), 52.25 (C-9, d), 57.00 (C-14,
s), 57.38 (C-5, d), 114.3 (C-26, t), 125.8 (C-21, d), 134.2 (C-22, s),
135.1 (C-17, s), 137.6 (C-25, d), 139.6 (C-13, s). The following 1H-
and 13C NMR signals are indistinguishable from each other, due to
very close chemical shifts: H-2/H-6 and C-2/C-6. EIMS: m/z (%):
69(67), 81(53), 95(43), 109(27), 121(22), 135(20), 149(48), 161(30),
191(100), 297(34), 341(34), 450(M+, 33). HRMS (EI): m/z: calcd
for C33H54 (M+) 450.4226, found 450.4219. [a]2D5= -35.67 (c = 0.1,
EtOH). Oil.
(M+) 450.4226, found 450.4218. [a]2D5= +8.99 (EtOH, c = 0.2).
Oil.
Product 21. 1H-NMR (600 MHz, C6D6): d = 1.000 (Me-31,
3H, d, J = 6.0 Hz), 1.010 (Me-28, 3H, s), 1.043 (Me-27, 3H, s),
1.132 (Me-29, 3H, s), 1.206 (Me-30, 3H, s), 1.23 (H-12 & H-15,
2H, m), 1.26 (H-1 & H-3, 2H, m), 1.29 (H-3, 1H, m), 1.33 (H-5,
1H, bd, J = 10.7 Hz), 1.49 (H-2 & H-6, 2H, m), 1.54 (H-3, 1H,
m), 1.63 (H-14, m), 1.679 (Me-33, 3H, s), 1.72 (H-1, 1H, m), 1.773
(Me-32, 3H, s), 1.82 (H-15, 1H, m), 1.85 (H-12, 1H, m), 1.97 (H-7,
1H, m), 2.12 (H-7, 1H, m), 2.15 (H-16, m), 2.20 (H-23, 2H,m),
2.20 (H-24, 1H, m), 2.25 (H-19, 2H, m), 2.25 (H-24, 1H, m), 2.27
(H-11, 1H, m), 2.31 (H-16, m)), 2.33 (H-20, 2H, m), 2.36 (H-11,
1H, m), 5.12 (H-26b, 1H, bd, J = 10.1 Hz), 5.17 (H-26a, 1H,
bdd, J = 17.1, 1.6 Hz), 5.38 (H-21, 1H, t, 6.6), 5.46 (H-17, 1H,
t, J = 6.6 Hz), 5.94 (H-25, 1H, m); 13C NMR (150 MHz, C6D6):
d = 15.05 (C-31, q), 16.03 (C-33, q), 16.14 (C-32, q), 19.34 (C-2,
t), 19.54 (C-6, t), 19.69 (C-29, q), 21.75 (C-28, q), 23.75 (C-7, t),
25.52 (C-30, q), 27.04 (C-20, t), 27.16 (C-16, t), 28.31 (C-11, t),
31.28 (C-12, t), 32.27 (C-15, t), 32.78 (C-24, t), 33.27 (C-4, s), 33.56
(C-27, q), 36.29 (C-10, s), 37.83 (C-1, t), 38.86 (C-14, d), 39.46 (C-
23, t), 40.19 (C-19, t), 42.39 (C-3, t), 52.87 (C-5, d), 52.99 (C-13,
s), 114.5 (C-26, t), 125.1 (C-21, d), 125.6 (C-17, d), 134.5 (C-22,
s), 134.7 (C-18, s), 138.1 (C-8, s), 138.9 (C-25, d), 144.4 (C-9, s).
The following 13C NMR signals are indistinguishable from each
other, due to very close chemical shifts: C-2/C-6 and C-20/C-
16. EIMS: m/z (%): 81(12), 95(10), 109(21), 147(8), 231(100),
341(10), 435(1), 450(M+, 1). HRMS (EI): m/z: calcd for C33H54
(M+) 450.4226, found 450.422. [a]2D5= -28.08 (c = 0.073, EtOH).
Oil.
Product 22. 1H-NMR (400 MHz, C6D6): d = 0.831 (Me-29,
3H,s), 0.892 (Me-28, 3H, s), 0.939 (Me-27, 3H, s), 1.05 (H-1, 1H,
m), 1.10 (H-5, 1H, dd, J = 12.8, 2.4 Hz), 1.27 (H-3, 1H, m), 1.39
(H-6, 1H, m), 1.72 (H-6, 1H, m), 1.47 (H-3, 1H, m), 1.53 (H-2,
1H, m), 1.60 (H-2, 1H, m), 1.63 (H-11, 1H, m), 1.64 (Me-33, 3H,
s), 1.71 (Me-32, 3H, s), 1.74 (Me-31, 3H, s), 1.76 (H-1, H-9 &
H-11, 3H, m), 2.08 (H-12, 1H m), 2.17 (H-23, 2H, m), 2.18 (H-7,
1H, m), 2.21 (H-15 & H-19, 4H, m), 2.23 (H-24, 2H, m), 2.31
(H-16 & H-20, 4H, m), 2.40 (H-12, 1H, m), 2.49 (H-7, 1H, m),
4.80 (H-30, 1H, s), 5.06 (H-30, 1H, s), 5.11 (H-26b, 1H, bd, J =
10.0), 5.16 (H-26a, 1H, bdd, J = 16.6, 1.6), 5.33 (H-21, 1H, t,
J = 6.8 Hz), 5.40 (H-17, 1H, m), 5.42 (H-13, 1H, m), 5.90 (H-25,
1H, m); 13C NMR (100 MHz, C6D6): d = 14.76 (C-29, q), 16.04
(C-31, q), 16.14 (C-32, q), 16.19 (C-33, q), 19.77 (C-2, t), 21.92
(C-28, q), 24.22 (C-11, t), 24.77 (C-6, t), 27.06 (C-20, t), 27.14
(C-16, t), 27.37 (C-12, t), 32.77 (C-24, t), 33.67 (C-4, s), 33.75 (C-
27, q), 38.74 (C-7, t), 39.29 (C-1, t)), 39.46 (C-23, t), 39.82 (C-10,
s), 40.21 (C-19, t), 40.26 (C-15, t), 42.43 (C-3, t), 55.65 (C-5, d),
56.43 (C-9, d), 106.7 (C-30, t), 114.5 (C-26, t), 124.9 (C-17, d),
125.0 (C-21, d), 125.7 (C-13, d), 134.4 (C-22, s), 134.9 (C-14, s, &
C-18, s), 138.8 (C-25, d), 148.8 (C-8, s). The following 13C NMR
signals are indistinguishable from each other, due to very close
chemical shifts: C-15/C-19, C-12/C-19/C-20, C-13/C-17/C-21
and C-14/C-18/C-22. EIMS: m/z (%): 69(48), 81(62), 95(48),
109(65), 123(25), 137(33), 149(100), 163(16), 177(21), 217(10),
231(10), 279(10), 341(4), 435(14), 450(M+, 28). HRMS (EI): m/z:
calcd for C33H54 (M+) 450.4226, found 450.4227. [a]2D5= +17.00
(c = 0.20, EtOH). Oil.
Product 25. 1H-NMR (600 MHz, C6D6): d = 0.872 (Me-28,
3H, s), 1.056 (Me-27, 3H, s), 1.15 (H-5, 1H, m). 1.203 (Me-29, 3H,
s), 1.22 (H-3, 1H, m), 1.23 (H-1, 1H, ddd, J = 12.5, 12.5, 3.8 Hz),
1696 | Org. Biomol. Chem., 2009, 7, 1689–1699
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1.35 (H-3, 1H, m), 1.41 (H-2, 1H, m), 1.49 (H-2, 1H, m), 1.57
(H-7, m), 1.673 (Me-33, 3H, s), 1.72 (H-1, 1H, m), 1.734 (Me-32,
3H, s), 1.764 (Me-31, 3H, s), 1.843 (Me-30, 3H, s), 1.86 (H-7, m),
2.23 (H-15, H-19, H-23 & H-24, 8H, m), 2.30 (H-11 & H-12, 4H,
m), 2.32 (H-20, 2H, m), 2.33 (H-8 & H-16, 3H, m), 2.47 (H-8,
m), 5.12 (H-26b, 1H, bd, J = 9.3 Hz), 5.16 (H-26a, 1H, bd, J =
16.6 Hz), 5.37 (H-21, 1H, t, J = 6.6 Hz), 5.42 (H-17, 1H, t, J =
6.4 Hz), 5.47 (H-13, 1H, m), 5.54 (H-10, 1H, m), 5.93 (H-25, 1H,
m); 13C NMR (150 MHz, C6D6): d = 16.02 (C-33, q), 16.11 (C-32,
q), 16.21 (C-31, q), 16.35 (C-30, q), 20.85 (C-2, t), 21.58 (C-28, q),
23.50 (C-29, q), 25.35 (C-7, t), 27.04 (C-20, t), 27.14 (C-16, t), 28.76
(C-12, t), 28.79 (C-11, t), 32.76 (C-24, t), 32.95 (C-27, q), 35.60
(C-4, s), 39.45 (C-23, t), 40.16 (C-15, t), 40.25 (C-19, t), 41.77 (C-3,
t), 43.41 (C-8, t), 43.97 (C-1, t), 56.86 (C-5, d), 73.51 (C-6, s), 114.5
(C-26, t), 124.6 (C-10, d), 124.9 (C-13, d), 124.9 (C-17, d), 125.1
(C-21, d), 134.4 (C-22, d), 134.9 (C-18, s), 135.2 (C-14, s), 136.6
(C-9, s), 138.8 (C-25, d). The following 13C NMR signals are in-
distinguishable from each other: C-11/C-12, C-14/C-18, C-15/C-
19, C-16/C-20 and C-31/C-32. EIMS: m/z (%): 69(90), 81(49),
95(58), 109(60), 119(38), 131(35), 137(31), 149(50), 169(25),
191(100), 231(21), 281(11), 281(12), 301(12), 341(17), 381(8),
435(8), 450(M+-H2O, 28). HRMS (EI): m/z: calcd for C33H54 (M+-
H2O) 450.4226, found 450.4228. [a]2D5= +33.1 (c = 0.036, EtOH).
Oil.
12, 1H, m), 1.98(H-6, 1H, m), 2.04(H-16, 1H, m), 2.18(H-20, 1H,
m), 2.23 (H-23, 2H,m), 2.25(H-6, 1H, m), 2.26 (H-24, 2H,m),
2.33(H-20, 1H, m), 2.43(H-9, 1H, m), 5.11(H-26b, 1H, bd, J =
9.2 Hz), 5.17(H-26a, 1H, bd, J = 17.1 Hz), 5.44(H-21, 1H,bt,
J = 6.9 Hz), 5.48(H-7, 1H, bs), 5.93(H-25, 1H, m); 13C NMR
(150 MHz, C6D6): d = 13.36 (C-29, q), 16.06 (C-33, q), 18.55
(C-2, t), 18.82 (C-32, q), 19.47 (C-11, t), 21.51 (C-28, q), 22.48
(C-31, q), 24.76 (C-6, t), 25.68 (C-20, t), 27.62 (C-30, q), 28.80
(C-16, t), 32.79 (C-24, t), 33.16 (C-27, q), 33.32 (C-4, s), 34.44
(C-12 & C-15, 2C, t), 35.46 (C-10, s), 35.65 (C-19, t), 36.09 (C-18,
d), 39.36 (C-1, t), 39.54 (C-23, t), 42.77 (C-3, t), 43.95 (C-13,
s), 49.56 (C-9, d), 51.76 (C-5, d & C-14, s), 53.73 (C-17, d),
114.5 (C-26, t), 118.5 (C-7, d), 125.8 (C-21, d), 134.2 (C-22,
s), 138.8 (C-25, d), 146.1 (C-8, s). EIMS: m/z (%): 69(52),
81(46), 95(37), 109(48), 121(26), 149(25), 161(11), 191(12), 243(6),
297(10), 311(9), 435(100), 450(M+, 20). HRMS (EI): m/z: calcd
for C33H54 (M+-H2O) 450.4226, found 450.4231. [a]2D5= -19.80 (c =
0.1, EtOH). Oil.
Product 28. 1H-NMR (400 MHz, C6D6): d = 0.872 (H-1, 1H,
ddd, J = 12.0, 12.0, 4.1 Hz), 0.911 (H-5, 1H,dd, J = 12.0, 2.0 Hz),
0.971 (Me-31, 3H, s), 0.984 (Me-28, 3H, s), 1.014 (Me-29, 3H, s),
1.030 (Me-27, 3H, s), 1.109 (Me-30, 3H, s), 1.24 (H-15, 1H, m),
1.27 (H-3, 1H, m), 1.28 (H-11, 1H, m), 1.30 (H-12, 1H, m), 1.37
(H-7, 1H, m), 1.47 (H-9, 1H, m), 1.50 (H-2, H-3 & H-6, 3H, m),
1.63 (H-2, H-6 & H-16, 3H, m), 1.65 (H-11, 1H, m), 1.697 (Me-33,
3H, s), 1.72 (H-1 & H-7. 3H, m), 1.76 (H-15, 1H, m), 1.90 (H-
12, 1H, m), 1.91 (H-13, 1H, m), 2.08 (H-16, 1H, m), 2.18 (H-23,
2H,m), 2.22 (H-24, 2H, m), 2.25 (H-19, 2H, m), 2.38 (H-20, 2H,
m), 2.41 (H-17, 1H, m), 5.05 (H-32, 1H, s), 5.10 (H-32, 1H, s),
5.12 (H-26b, 1H, bd, J = 11.6 Hz), 5.17 (H-26a, 1H, bdd, J =
17.2, 1.6 Hz), 5.41 (H-21, 1H, t, J = 6.4 Hz), 5.93 (H-25, 1H,
m); 13C NMR (100 MHz, C6D6): d = 15.91 (C-33, q), 16.09 (C-
30,q), 16.19 (C-31, q), 16.43 (C-29, q), 18.98 (C-2, t), 19.08 (C-6,
t), 21.61 (C-11, t), 21.79 (C-28, q), 25.49 (C-12, t), 27.39 (C-20,
t), 29.40 (C-16, t), 31.72 (C-15, t), 32.74 (C-24, t), 33.53 (C-4, s),
33.65 (C-27, q), 34.75 (C-19, t), 35.78 (C-7, t), 37.77 (C-10, s),
39.44 (C-23, t), 40.92 (C-1, t), 40.97 (C-8, s), 42.42 (C-3, t), 45.69
(C-13, d), 48.28 (C-17, d), 49.78 (C-14, s), 51.43 (C-9, d), 57.31
(C-5, d), 108.2 (C-32, t), 114.6 (C-26, t), 125.1 (C-21, d), 134.6
(C-22, s), 138.8 (C-25, d), 152.5 (C-18, s). The assignments of
C-2 and C-6 are exchangeable. EIMS: m/z (%): 81(60), 95(57),
109(45), 123(24), 137(25), 149(34), 161(15), 177(11), 191(100),
205(10), 231(13), 244(10), 258(7), 301(7), 367(5), 435(4), 450(M+,
32). HRMS (EI): m/z: calcd for C33H54 (M+-H2O) 450.4226, found
450.4224. [a]2D5= +26.59 (c = 0.135, acetone). Oil.
Product 29. 1H-NMR (600 MHz, C6D6): d = 0.88 (H-1, 1H,
m), 0.91 (H-5, 1H, bd, J = 11.8 Hz), 0.984 (Me-28, 3H, s), 0.991
(Me-29, 3H, s), 1.021 (Me-31, 3H, s), 1.029 (Me-27, 3H, s), 1.102
(Me-30, 3H, s), 1.25 (H-15, 1H, m), 1.27 (H-3, 1H, m), 1.28 (H-11,
1H, m), 1.43 (H-7, 1H, m), 1.47 (H-2, H-3 & H-6, 2H, m), 1.49
(H-9, 1H, m), 1.63 (H-2, H-6 & H-7, 3H, m), 1.65 (H-12 & H-15,
2H, m), 1.68 (H-11, 1H, m), 1.736 (Me-33, 3H, s), 1.75 (H-1, 1H,
m), 1.826 (Me-32, 3H, bs), 2.21 (H-23, 2H, m & H-24, 1H, m),
2.25 (H-16, 1H, m), 2.29 (H-24, 1H, m), 2.30 (H-20, 1H, m), 2.36
(H-16, 1H, m), 2.38 (H-20, 1H, m), 2.41 (H-19, 2H, m), 2.48 (H-12,
1H, m), 2.50 (H-13, 1H, m), 5.12 (H-26b1H, bd, J = 10.0 Hz), 5.18
(H-26a, 1H, bd, J = 17.1), 5.45 (H-21, 1H, m), 5.94 (H-25, 1H,
m); 13C NMR (150 MHz, C6D6): d = 15.92 (C-30, q), 16.08 (C-33,
Product 26. 1H-NMR (400 MHz, C6D6): d = 0.847 (Me-30,
3H, s), 0.88 (H-5, 1H, m), 0.94 (H-1, 1H, m), 0.962 (Me-29, 3H,
s), 0.975 (Me-28, 3H, s), 1.020 (Me-27, 3H, s), 1.27 (H-7, 1H, m),
1.28 (H-3, H-6 & H-9, 3H, m), 1.40 (H-6, 1H, m), 1.45 (H-11, 1H,
m), 1.49 (H-2, 1H, m), 1.52 (H-1 & H-3, 2H, m), 1.60 (H-11, 1H,
m), 1.667 (Me-33, 3H, s), 1.74 (H-2, 1H, m), 1.756 (Me-32, 3H,
s), 1.82 (H-12, 1H, m), 1.92 (H-7, 1H, m), 1.95 (H-12, 1H, m),
2.19 (H-23, 2H, m), 2.20 (H-13, 1H, m), 2.22 (H-19, 2H, m), 2.25
(H-15, 1H, m), 2.26 (H-24, 2H, m), 2.30 (H-20, 2H, m), 2.32 (H-
15, 1H, m), 2.41 (H-16, 2H, m), 5.03 (H-31, 1H, s), 5.12 (H-26b,
bd, J = 10.0 Hz), 5.18 (H-26a, 1H,bdd, J = 16.8, 1.6 Hz), 5.21
(H-31, 1H, s), 5.36 (H-21, 1H, t, J = 6.0 Hz), 5.46 (H-17, 1H, t,
J = 6.4 Hz), 5.93 (H-25, 1H, m); 13C NMR (100 MHz, C6D6):
d = 15.38 (C-30, q), 15.62 (C-29, q), 16.05 (C-33, q), 16.17 (C-32,
q), 18.77 (C-2, t), 19.81 (C-11, t), 19.81 (C-6, t), 21.50 (C-28, q),
25.80 (C-12, t), 26.99 (C-20, t), 27.53 (C-16, t), 32.78 (C-24, t),
33.17 (C-4, s), 33.74 (C-27, q), 37.39 (C-10, s), 38.12 (C-15, t),
39.45 (C-23, t), 40.15 (C-1, t), 40.26 (C-19, t), 41.47 (C-7, t), 42.90
(C-3, t), 43.99 (C-8, s), 57.57 (C-5 & C-13, 2C, d), 63.36 (C-9, d),
110.6 (C-31, t), 114.5 (C-26, t), 124.9 (C-17 & C-21, 2C, d), 134.5
(C-22, s), 135.0 (C-18, s), 138.8 (C-25, d), 149.2 (C-14, s). The
assignments of C-1 and C-19 may be exchangeable. EIMS: m/z
(%): 81(33), 95(35), 109(62), 123(22), 137(25), 149(15), 163(12),
177(12), 191(100), 203(10), 217(12), 231(74), 435(6), 450(M+, 12).
HRMS (EI): m/z: calcd for C33H54 (M+-H2O) 450.4226, found
450.4224. [a]2D5= +19.04 (c = 0.2, EtOH). Oil.
Product 27. 1H-NMR (600 MHz, C6D6): d = 0.955 (Me-29,
3H, s), 0.964 (Me-27, 3H, s), 1.031(Me-28, 3H, s), 1.055(Me-32,
3H, d, J = 6.5 Hz), 1.06(H-1, m), 1.072(Me-31, 3H, s), 1.183(Me-
30, 3H, s), 1.28(H-3, 1H, m), 1.38(H-16, 1H, m), 1.48 (H-5, 1H,
m), 1.50(H-11, 1H, m), 1.52(H-19, 1H, m), 1.55(H-3, 1H, m),
1.58(H-18, 1H, m), 1.62(H-15, 1H, m), 1.64(H-17, 1H, m), 1.65(H-
2, 2H,m), 1.66(H-11, 1H, m), 1.735(Me-33, 3H, s), 1.75(H-1, m),
1.76(H-15, 1H, m), 1.78(H-12, 1H, m), 1.89(H-19, 1H, m), 1.95(H-
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q), 16.58 (C-29, q), 16.90 (C-31, q), 18.93 (C-2, t), 19.05 (C-6, t),
21.08 (C-32, q), 21.74 (C-28, q), 21.78 (C-11, t), 25.49 (C-12, t),
28.39 (C-20, t), 30.05 (C-16, t), 30.53 (C-15, t), 32.77 (C-24, t),
33.51 (C-4, s), 33.59 (C-27, q), 34.13 (C-19, t), 35.89 (C-7, t), 37.62
(C-10, s), 39.53 (C-23, t) \ , 40.36 (C-8, s), 40.87 (C-1, t), 42.44
(C-3, t), 47.27 (C-13, d), 50.25 (C-14, s), 51.08 (C-9, d), 57.33 (C-5,
d), 114.5 (C-26, t), 125.3 (C-21, d), 126.7 (C-18, s), 134.3 (C-22,
s), 137.1 (C-17, s), 138.8 (C-25, d). The assignments of C-2 and
C-6 are exchangeable. EIMS: m/z (%): 69(100), 81(30), 95(31),
109(36), 119(42), 131(43), 149(43), 169(27), 181(28), 191(54),
205(12), 231(18), 281(12), 341(75), 381(7), 450(M+, 20). HRMS
(EI): m/z: calcd for C33H54 (M+) 450.4226, found 450.4240. [a]2D5=
-13.05 (c = 0.082, EtOH). Oil.
Product 30. 1H-NMR (400 MHz, C6D6): d = 0.975 (Me-28,
3H, s), 0.982 (Me-29, 3H, s), 0.99 (H-5, 1H, m), 1.002 (Me-27,
3H, s), 1.04 (H-1, 1H, m), 1.068 (Me-30, 3H, s), 1.25 (H-3, 1H,
m), 1.308 (Me-31, 3H, s), 1.47 (H-2 & H-11, 2H, m), 1.50 (H-3,
1H, m), 1.52 (H-6, 1H, m), 1.54 (H-9, 1H, m), 1.58 (H-1, 1H, m),
1.65 (H-11, 1H, m), 1.662 (Me-33, 3H, s), 1.68 (H-12, 1H, m),
1.70 (H-15, 1H, m), 1.73 (H-2 & H-6, 2H, m), 1.760 (H-13, 1H,
m & Me-32, 3H, s), 1.94 (H-7 & H-12, 2H, m), 2.10 (H-15, 1H,
m), 2.12 (H-19 & H-23, 3H, bt, J = 7.0 Hz), 2.12 (H-7, 1H, m),
2.27 (H-16 & H-20, 4H, m), 2.34 (H-24, 2H, m), 5.13 (H-26b, 1H,
bd, J = 10.0 Hz), 5.21 (H-26a, 1H, bdd, J = 16.8, 2.0 Hz), 5.42
(H-17 & H-21, 2H, m), 5.94 (H-25, 1H, m); 13C NMR (100 MHz,
C6D6): d = 15.93 (C-33, q), 16.04 (C-32, q), 16.57 (C-29, q), 18.89
(C-2, t), 20.17 (C-6, t), 21.54 (C-28, q), 21.70 (C-11, t), 23.23 (C-16,
t), 24.71 (C-12, t), 26.08 (C-31, q), 26.63 (C-20, t), 26.83 (C-30,
q), 32.70 (C-24, t), 33.16 (C-4, s), 33.68 (C-27, q), 37.48 (C-10,
s), 38.32 (C-7, t), 39.59 (C-23, t), 39.67 (C-19, t), 41.13 (C-1, t),
42.31 (C-15, t), 42.69 (C-3, t), 45.04 (C-8, s), 57.13 (C-5, d), 59.11
(C-13, d), 60.39 (C-9, d), 75.49 (C-14, s), 114.3 (C-26, t), 121.9
(C-21, d), 125.4 (C-17, d), 135.0 (C-18, s), 136.5 (C-22, s), 137.6
(C-25, d). The signals of C-15 and C-31 were significantly small.
EIMS: m/z (%): 69(66), 81(78), 95(62), 109(35), 123(28), 137(28),
149(36), 191(100), 205(15), 231(41), 341(15), 435(5), 450(M+-H2O,
18). HRMS (EI): m/z: calcd for C33H54 (M+-H2O) 450.4226, found
450.4240. [a]2D5= -70.80 (c = 0.05, EtOH). Oil.
Product 31. 1H-NMR (400 MHz, C6D6): d = 0.965 (Me-29,
3H, s), 0.971 (Me-27, 3H, s), 1.034 (Me-28, 3H, s), 1.05 (H-1, 1H,
m), 1.058 (Me-31, 3H, s), 1.107 (Me-32, 3H, d, J = 6.0 Hz), 1.186
(Me-30, 3H, s), 1.27 (H-19, 1H, m), 1.29 (H-3, 1H, m), 1.40 (H-16,
1H, m), 1.50 (H-5, 1H, m), 1.55 (H-3 & H-11, 2H, m), 1.58 (H-18,
1H, m), 1.59 (H-19, 1H, m), 1.60 (H-2, 1H, m), 1.63 (H-17, 1H,
m), 1.65 (H-15, 1H, m), 1.68 (H-2, 1H, m), 1.69 (H-11, 1H, m),
1.730 (Me-33, 3H, s), 1.75 (H-1, 1H, m), 1.78 (H-12 & H-15, 2H,
m), 1.89 (H-12, 1H, m), 1.97 (H-6, 1H, m), 2.12 (H-16, 1H, m),
2.17 (H-20, 1H, m), 2.24 (H-23, 2H, m), 2.25 (H-6, 1H, m), 2.31
(H-20, 1H, m), 2.43 (H-9, 1H, m), 5.12 (H-26b, 1H, d, J = 9.5),
5.18 (H-26a, 1H, bd. J = 17.2), 5.42 (H-21, 1H, t, J = 6.4 Hz), 5.47
(H-7, 1H, bs), 5.94 (H-25, 1H, m); 13C NMR (100 MHz, C6D6):
d = 13.36 (C-29, q), 15.99 (C-33, q), 18.49 (C-2, t), 18.63 (C-32,
q), 19.44 (C-11, t), 21.50 (C-28, q), 22.26 (C-31, q), 24.71 (C-6, t),
25.36 (C-20, t), 27.56 (C-30, q), 28.62 (C-16, t), 32.75 (C-24, t),
33.16 (C-27, q), 33.29 (C-4, s), 34.16 (C-12, t), 34.46 (C-15, t), 35.40
(C-10, s), 36.29 (C-18, d), 36.58 (C-19, t), 39.27 (C-1, t), 39.50 (C-
23, t), 42.69 (C-3, t), 43.84 (C-13, s), 49.51 (C-9, d), 51.60 (C-14,
s), 51.67 (C-5, d), 53.43 (C-17, d), 114.6 (C-26, t), 118.5 (C-7, d),
125.9 (C-21, d), 134.1 (C-22, s), 138.8 (C-25, d), 146.0 (C-8, s). The
assignments of the proton and carbon at 12 and 15 positions may
be exchangeable. EIMS: m/z (%): 69(75), 81(55), 95(44), 109(55),
119(17), 135(15), 149(15), 243(10), 257(10), 436(100), 450(M+, 17).
HRMS (EI): m/z: calcd for C33H54 (M+) 450.4226, found 450.4231.
[a]2D5= -15.00 (c = 0.064, EtOH). Oil.
Product 32. 1H-NMR (600 MHz, C6D6): d = 0.845 (Me-32,
3H, s), 0.87 (H-1, 1H, m), 0.91 (H-5, 1H, m), 0.979 (Me-29, 3H, s),
0.989 (Me-28, 3H, s), 1.037 (Me-27, 3H, s), 1.106 (Me-30, 3H, s),
1.114 (Me-31, 3H,s), 1.19 (H-19, 1H, m), 1.25 (H-3, 1H, m), 1.28
(H-17, 1H, m), 1.30 (H-11, 1H, m), 1.33 (H-15, 1H, m), 1.35 (H-7,
1H, m), 1.41 (H-9, 1H, m), 1.42 (H-2 & H-11, 2H, m), 1.51 (H-3 &
H-6, 2H, m), 1.54 (H-15, 1H, m), 1.55 (H-12, 1H, m), 1.58 (H-13
& H-20, 2H, m), 1.61 (H-7, 1H, m), 1.63 (H-2 & H-19, 2H, m),
1.64 (H-12, 1H, m), 1.65 (H-16, 1H, m), 1.72 (H-6 & H-16, 1H,
m), 1.78 (H-1, 1H, m), 2.06 (H-20, 1H, m), 2.24 (H-23, 2H, t, J =
7.7 Hz), 2.37 (H-24, 2H, m), 2.41 (H-21, 1H, m), 5.00 (H-33, 1H,
bs), 5.07 (H-33, 1H, bs), 5.13 (H-26b, 1H bd,10.5 Hz), 5.20 (H-26a,
1H, bd, J = 17.1 Hz), 5.96 (H-25, 1H, m); 13C NMR (150 MHz,
C6D6): d = 15.42 (C-32, q), 16.13 (C-29, q), 16.96 (C-31, q), 17.03
(C-30, q), 19.10 (C-2, t), 19.13 (C-6, t), 21.38 (C-11, t), 21.42 (C-16,
t), 21.85 (C-28, q), 24.35 (C-12, t), 29.03 (C-20, t), 32.96 (C-24,
t), 33.14 (C-15, t), 33.44 (C-4, s), 33.62 (C-27, q), 33.80 (C-7, t),
34.43 (C-23, t), 37.76 (C-10, s), 40.47 (C-19, t), 40.71 (C-1, t),
42.27 (C-14, s), 42.45 (C-3, t), 42.63 (C-8, s), 44.55 (C-18, s), 47.46
(C-21, d), 49.20 (C-13, d), 50.98 (C-9, d), 55.12 (C-17, d), 56.60
(C-5, d), 108.2 (C-33, t), 114.7 (C-26, t), 138.8 (C-25, d), 151.9 (C-
22, s). The following 13C NMR signals are indistinguishable from
each other, due to very close chemical shifts: C-2/C-6, 11/16 and
30/31. EIMS: m/z (%): 69(52), 81(56), 95(51), 109(27), 121(21),
137(20), 149(16), 161(17), 191(100), 229(80), 367(19), 435(23), 450
(M+, 18). HRMS (EI): m/z: calcd for C33H54 (M+) 450.4226, found
450.4213. [a]2D5= -52.39 (c = 0.046, EtOH). Solid.
Product 33. 1H-NMR (600 MHz, C6D6): d = 0.88 (H-1, 1H,
m), 0.910 (H-5, 1H, bd, J = 12.1 Hz), 0.989 (Me-28, 3H, s), 1.003
(Me-29, 3H, s), 1.017 (Me-27, 3H, s), 1.077 (Me-30, 3H, s), 1.131
(Me-31, 3H, s), 1.25 (H-3, 1H, m), 1.27 (H-11, 1H, m), 1.34 (H-
15, 1H, m), 1.40 (H-7, 1H, m), 1.45 (H-12, 1H, m), 1.48 (H-2,
H-6 & H-9, 3H, m), 1.50 (H-3, 1H, m), 1.65 (H-2 & H-6, 2H,
m), 1.68 (H-15, 1H, m), 1.699 (Me-33, 3H, s), 1.70 (H-11, 1H,
m), 1.72 (H-7, 1H, m), 1.75 (H-1, 1H, m), 1.83 (H-12, 1H, m),
1.93 (H-16, 1H, m), 2.74 (H-17, 1H, m), 2.03 (H-16, 1H, m), 2.06
(H-13, 1H, m), 2.20 (H-19 & H-23, 2H, m), 2.21 (H-24, 1H,m),
2.27 (H-24, 1H,m), 2.35(H-20, 1H, m), 2.36 (H-19 & H-23, 2H,
m), 2.42 (H-20, 1H, m), 5.12 (H-26b, 1H, d, J = 10.1 Hz), 5.17
(H-26a, 1H,bd, J = 16.9 Hz), 5.19 (H-32, 1H,s), 5.21 (H-32, 1H,
s), 5.41 (H-21, 1H, bs), 5.93 (H-25, 1H, m); 13C NMR (150 MHz,
C6D6): d = 16.09 (C-30, q), 16.20 (C-33, q), 16.47 (C-29, q), 17.24
(C-31, q), 19.11 (C-2, t, & C-6 t), 21.79 (C-28, q), 22.26 (C-11,
t), 25.52 (C-12, t), 27.68 (C-20, t), 28.82 (C-16, t), 32.76 (C-24,
t), 33.46 (C-15, t), 33.56 (C-4, s), 33.60 (C-27, q), 35.48 (C-7, t),
37.81 (C-10, s), 39.05 (C-19, t), 39.47 (C-23, t), 40.98 (C-1), 41.41
(C-8, s), 42.50 (C-3, t), 44.51 (C-17, d), 45.12 (C-13, d), 50.27
(C-14, s), 51.46 (C-9, d), 57.39 (C-5, d), 109.5 (C-32, t), 114.5
(C-26, t), 125.2 (C-21, d), 134.6 (C-22, s), 138.8 (C-25, d), 152.2
(C-18, s). EIMS: m/z (%): 69(49), 81(54), 95(51), 109(35), 123(21),
137(23), 149(32), 191(100), 231(15), 258(8), 299(8), 341(8), 435(4),
1698 | Org. Biomol. Chem., 2009, 7, 1689–1699
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450(M+, 23). HRMS (EI): m/z: calcd for C33H54 (M+) 450.4226,
found 450.4229. [a]2D5= -51.64 (c = 0.056, EtOH). oil.
C33H54 (M+) 450.4226, found 450.4247. [a]2D5= -18.21 (c = 0.2,
EtOH). Oil.
Product 34. 1H-NMR (600 MHz, C6D6): d = 0.88 (H-1, 1H,
m), 0.93 (H-5, 1H, dd, J = 12.0,1.6 Hz), 0.978 (Me-29, 3H, s), 0.988
(Me-28, 3H, s), 1.022 (Me-31, 3H, s), 1.029 (Me-27, 3H,s), 1.103
(Me-30, 3H, s), 1.19 (H-15, 1H, m), 1.226 (Me-32, 3H, s), 1.27 (H-3
& H-11, 2H, m), 1.32 (H-16, 1H, m), 1.37 (H-7, 1H, m), 1.46 (H-9,
1H, m), 1.48 (H-2, H-3 & H-6, 3H, m), 1.61 (H-11, 1H, m), 1.62
(H-2, H-6 & H-15, 3H, m), 1.66 (H-12 & H-19, 1H, m), 1.68 (H-7,
1H, m), 1.72 (H-12, 1H, m), 1.729 (Me-333H, s), 1.75 (H-1, 1H, m),
1.80 (H-17, 1H, m), 1.81 (H-13, 1H, m), 2.03 (H-16, 1H, m), 2.19
(H-23, 2H, m), 2.22 (H-24, 2H, m), 2.26 (H-20, 2H, m), 5.13 (H-
26b, 1H, bd, J = 10.4 Hz), 5.19 (H-26a, 1H, bd, J = 17.2 Hz), 5.40
(H-21, 1H, t, J = 7.2 Hz), 5.93 (H-25, 1H, m); 13C NMR (150 MHz,
C6D6): d = 15.79 (C-30, q), 16.03 (C-33, q), 16.43 (C-29, q), 16.73
(C-31, q), 19.00 (C-2, t), 19.09 (C-6, t), 21.76 (C-11, t & C-28, q),
22.93 (C-20, t), 25.18 (C-12, t), 25.82 (C-32, q), 28.02 (C-16, t),
31.55 (C-15, t), 32.70 (C-24, t), 33.52 (C-4, s), 33.63 (C-27, q), 35.64
(C-7, t), 37.70 (C-10, s), 39.44 (C-23, t), 40.89 (C-1, t & C-8, s),
40.98 (C-19, t), 42.48 (C-13, d & C-3, t), 50.40 (C-17, d), 50.62 (C-
14, s), 51.20 (C-9, d), 57.34 (C-5, d), 74.77 (C-18, s), 114.6 (C-26,
t), 125.7 (C-21, d), 134.4 (C-22, s), 138.8 (C-25, d). The following
1H- and 13C NMR signals are indistinguishable from each other,
due to very close chemical shifts: H-2/H-6, H-11/H-12/H-16, C-
2/C-6, C-11/C-12/C-16, C-15/C-24, C-1/C-19. EIMS: m/z (%):
69(90), 81(48), 95(58), 109(60), 119(37), 131(35), 149(49), 169(25),
181(24), 191(100), 205(15), 219(12), 231(20), 281(12), 301(12),
341(15), 381(7), 435(7), 450(M+-H2O, 39). HRMS (EI): m/z: calcd
for C33H54 (M+) 450.4226, found 450.4247. [a]2D5= +20.28 (c =
0.036, EtOH). Oil.
Acknowledgements
This work was supported by Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan (Nos.17-05429 and 18380001). One of the
authors (J. C.) worked in this laboratory as a JSPS postdoctoral
fellow for foreign researchers. We thank the Japanese Society for
the promotion of Science (JSPS).
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341(32), 435(4), 450(M+-H2O, 22). HRMS (EI): m/z: calcd for
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