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

Article abstract of DOI:10.1021/acs.joc.6b01313

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

Full text of DOI:10.1021/acs.joc.6b01313

Article
pubs.acs.org/joc
β‑Amyrin Biosynthesis: Promiscuity for Steric Bulk at Position 23 in
the Oxidosqualene Substrate and the Significance of Hydrophobic
Interaction between the Methyl Group at Position 30 and the
Binding Site
Ikki Kaneko and Tsutomu Hoshino*
Department of Applied Biological Chemistry, Faculty of Agriculture and Graduate School of Science and Technology, Niigata
University, Ikarashi 2-8050, Nishi-ku, Niigata, Japan 950-2181
*
S Supporting Information
ABSTRACT: To examine how the sterics at the 23 position of (3S)-2,3-oxidosqualene 1 influence the polycyclization cascade in
β-amyrin biosynthesis, substrate analogues substituted with an ethyl group (10, 11), a hydrogen atom (12, 13), or a propyl
residue (14) at the 23 position were incubated with β-amyrin synthase. The bulkier ethyl group was accepted as a substrate,
leading to formation of the β-amyrin skeleton (42, 43) without truncation of the multiple cyclization reactions. Analogue 13,
possessing a hydrogen atom and an ethyl group at the 23E and 23Z positions, respectively, was also converted into the β-amyrin
skeleton 45. However, the analogue lacking an ethyl group at the 23Z position (12) underwent almost no conversion, strongly
indicating that an alkyl group must exist at the Z position. The cyclization of the analogue with a propyl substituent at the Z
position (14) was poor. Analogue 15 possessing CH OH at the 23E position afforded a new compound 47 in a high yield as a
2
result of trapping of the final oleanyl cation. Conversely, 16 with 23Z-CH OH afforded novel compounds 48−50 in low yields,
2
which resulted from the intermediary dammarenyl and baccharenyl cations. Therefore, the hydrophobic interaction between the
2
3Z-alkyl group and its binding site (possibly via CH/π interaction) is critical for adopting the correct chair−chair−chair−boat−
boat conformation and for the full cyclization cascade.
INTRODUCTION
with the 6/6/6/5-fused A/B/C/D tetracyclic ring system,
which undergoes ring expansion to create the 6/6/6/6-fused
tetracyclic baccharenyl cation 4. Further cyclization occurs to
give the 6/6/6/6/5-fused pentacyclic lupanyl cation 5, which
undergoes further ring expansion to provide the 6/6/6/6/6-
fused pentacyclic oleanyl cation 6. Deprotonation of the H-12α
of 6 confers the final product 2. The enzymatic reactions of
substrate analogues have provided deep insights into how the
polycyclization reaction is affected by specific modifications,
such as alteration of the folding conformation, leading to
different stereochemistry, truncation of the ring-forming
cascade, or different cation-quenching modes. Recently, we
reported the effect of steric bulk at the C-19 and C-23 positions
■
The polycyclization cascades of squalene and (3S)-2,3-
oxidosqualene 1 have been attractive to organic chemists and
biochemists for more than 70 years since Ruzicka and co-
workers proposed the “biogenetic isoprene rule”. The
reactions proceed with complete regiospecificity and stereo-
specificity to yield sterols and triterpenes
structural diversity; more than 100 different carbon frameworks
that exhibit important biological activities can be produced. β-
1
2
−7
with remarkable
8
Amyrin 2 consists of a pentacyclic scaffold with eight chiral
centers and is widely distributed among plants. Recently, we
succeeded in the complete purification of β-amyrin synthase
from Euphorbia tirucalli (EtAS) and in characterizing its
1
6,17
9
of 1 on the cyclization cascade.
In addition to
enzymatic properties. Substrate 1 is folded into a chair−
chair−chair−boat−boat conformation by β-amyrin synthase
1
,10−15
(
Scheme 1).
Proton attack on the epoxide ring initiates
Received: May 31, 2016
the polycyclization reaction to yield the dammarenyl cation 3
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
a
Scheme 1. Cyclization Pathway of (3S)-2,3-Oxidosqualene 1 into β-Amyrin 2
a
The Me-24 and Me-30 groups of compound 1 are regiospecifically converted into the Me-30 and Me-29 groups of compound 2, respectively. The
filled circle shows the destiny of the C-23 atom of compound 1 during the formation of compound 2; atom C-23 of compound 1 is transformed into
atom C-20 of compound 2.
bisnoroxidosqualene 7, we further examined the cyclization
reactions of nor-analogues 8 and 9, which lack the methyl-24
and methyl-30 groups, respectively (Figure 1). We discovered
skeleton without truncation of the multiple cyclization
reactions. Comparing the cyclization yields between (23E)-
ethyl-30-noroxidosqualene 12 and (23Z)-ethyl-24-noroxidos-
qualene 13 clearly demonstrated that the absence of the Z-
configured alkyl group resulted in nearly no cyclization. The
larger propyl-substituted analogue 14 afforded a substantially
decreased conversion yield, even though the alkyl group is Z-
configured in 14. These results indicate that a methyl group is
the appropriate steric bulk and that the alkyl group must be Z-
configured. Little difference in the cyclization yields between 11
and 13 further suggests that the role of the E-Me is minimal.
17
Furthermore, we report here that substrate 15 with E-CH OH
2
(
a hydrophilic group) and Z-Me groups at the 23 position
produced only the oleanyl cation 6-trapped product 47. By
contrast, substrate 16 with a Z-CH OH group and E-Me gave
2
three products: the dammarenyl cation 3-trapped products 48
and 49, and the baccharenyl cation 4-trapped product 50.
These results indicate that Z-Me at the terminus strongly
associates with the binding site possibly via CH/π interaction,
leading to the ordered architecture of the chair−chair−chair−
boat−boat conformation, which further leads to the formation
of final cation 6. A lack of Z-Me (16) resulted in a disordered
folding conformation; thus, trapping of the prematurely
cyclized cations 3 and 4 occurred.
Figure 1. Structures of substrate 1 and its analogues 7−16 and β-
amyrin 2.
RESULTS AND DISCUSSION
that the Me-30 of 1 is critical to accurately folding 1 into a
chair−chair−chair−boat−boat structure that leads to the
pentacyclic scaffold 2. Me-24 and Me-30 of 1 were regiospecifi-
cally converted into Me-30 and Me-29 of 2, respectively, during
■
Syntheses of Analogues 10−16. Scheme 2 outlines the
overall synthetic scheme. The Wittig reagents 18−21 used for
the preparation of these analogues are shown in Scheme 2A.
The starting material 17 with a C -aldehyde was subjected to
17
β-amyrin biosynthesis.
27
In this paper, we describe how the sterics at position 23 of 1
influence the outcome of the polycyclization reaction. (23E)-
Ethyloxidosqualene 10 and (23Z)-ethyloxidosqualene 11 with
the bulkier ethyl group were accepted as a substrate in a
considerably high yield, leading to formation of the β-amyrin
Wittig reactions using each of the phosphorus ylides obtained
by base treatment. Aldehyde 17 was prepared from squalene.
Scheme 2B depicts the subsequent reaction steps. Synthetic
intermediates 22 and 23 were obtained from 18 using NaH as a
base; 24 and 25 were obtained from 19 in the presence of n-
17
B
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
a
Scheme 2. (A) Wittig Reagents Used in This Study and (B) Synthetic Schemes of Analogues 10−16 from 17
a
Reagents: NBS, N-bromosuccinimide; DIBAL-H, diisobutylaluminum hydride; MsCl, methanesulfonyl chloride; LiBEt H, lithium
3
triethylborohydride (super hydride).
BuLi and NaN(SiMe ) , respectively, where the latter base
promoted selective formation of the Z-isomer. Product 26 was
the resulting mixture was heated to 70−80 °C for 30 min. The
lipophilic materials were extracted with hexane, and after
removing TritonX-100 included in the extract by a SiO2
column with hexane/EtOAc (100:10), the hexane extract was
evaporated to dryness. Next, 150 μL of hexane was added to
the residue, and 1.0 μL of the solution was subjected to GC
analysis.
Product profiles are shown in Figure S3 (Supporting
Information). Substrates 10, 11, and 13 afforded substantial
amounts of products 42, 43, and 45, respectively (Figure
S3B,C,E). Figure 2 indicates the relative cyclization yields of
analogues 10−16 compared to that of 1. The cyclization yields
of (3S)-10, 11, and 13 were ca. 40−50% in a comparison to
that (100%) estimated from genuine substrate (3S)-1 (Figure
2). By contrast, the conversion yield of substrate 12 appeared
null (Figure S3D), although a negligible amount of product 44
was detected from the product fraction enriched by partial
3
2
obtained from 20 using NaN(SiMe ) ; 27 and 28 were
3
2
obtained from 21 using NaH as base, yielding a mixture of E-
and Z-isomers. In cases where a mixture of E- and Z-isomers
was produced, separation of the isomers was attained using
SiO column chromatography. Next, 22−28 were converted to
2
the bromohydrin derivatives 29−35 through a reaction with N-
bromosuccinimide (NBS) in a H O/THF solution. The ethyl
2
ester groups of 29, 30, 34, and 35 were reduced by DIBAL-H
reagents to give the corresponding alcohols 36−39. Alcohols
3
6 and 37 were converted into the corresponding mesylates 40
and 41, respectively. The mesylates were treated with K CO to
2
3
give the corresponding epoxides, which were then demesylated
using the hydride reagent LiBEt H, yielding the desired
3
analogues 10 and 11. The epoxide analogues 12−14 were
prepared from 31−33, respectively, by treatment with K CO .
2
3
Using a similar treatment, the desired analogues 15 and 16
were obtained from 38 and 39, respectively.
purification with a SiO column. Other products (peaks A−E
2
and G) were also detected in negligibly small amounts (see
Figure S7.1.1 in Supporting Information). This result suggests
that many products were produced from 12, although the
quantity of each product was extremely small. A non-negligible
amount of product 46 [ca. 4% of the incubation of (3S)-1] was
indicated in the GC trace from the incubation of 14 (Figure
S3F). These findings indicate that only one product was
detected in a substantial amount from the experiments with 10,
Incubation of Substrates 10−16 with β-Amyrin
Synthase EtAS and Product Profiles. A reaction mixture
(2.5 mL, pH 7.4, 100 mM potassium buffer) consisting of
TritonX-100 (0.1%, w/v), 1 [200 μg of a (3R,S)-racemic
mixture], BSA (1 mg/mL), DTT (1 mM), and purified His-
9
tagged EtAS (5 μg) was incubated at 30 °C for 12 h. To this
reaction mixture was added 7.5 mL of 15% KOH/MeOH, and
C
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Supporting Information (Figures S5.3−S5.9). DEPT 45, 90,
and 135 spectra were also measured to differentiate methyl,
methylene, methine, and quaternary carbons. One double bond
1
was observed: δ 5.17 ppm (br s, 1H) in the H NMR (600
H
1
3
MHz, CDCl ) δ 121.7 ppm (d), 145.3 ppm (s) in C NMR
3
C
(
150 MHz, CDCl ). In substrate 10, five olefinic Me groups are
3
involved: δH 1.67 (s, 3H), 1.73 (s, 9H), 1.81 (s, 3H).
Furthermore, two singlet Me groups (δ 1.23, s, 3H; 1.27, s, 3H
H
for Me-1 and Me-25) were found on the epoxide ring, and one
triplet Me (δ 1.073, t, J = 7.6 Hz, 3H for Me-31) exists on the
H
ethyl group (23E-Et) in 10. Product 42 had no olefinic methyl
group, and clear HMBC cross-peaks were observed between
Me-31 (δ 0.764, t, J = 7.4 Hz, 3H) and C-20 (δ 33.3, s),
H
C
between Me-29 (δ 0.781, s, 3H) and C-20 and between H-30
H
Figure 2. Relative cyclization yields of substrate analogues 10−16
against that of 1. Error bars represent the deviation among three
experiments.
(
δH 1.28, m, 1H; 1.35, m, 1H) and C-20. These findings
indicate that full cyclization occurred. The detailed HMBC
analyses (Figure S5.10) further allowed us to propose a β-
amyrin skeleton for product 42. Complete assignments of the
chemical shifts were attained by detailed analyses of the HMBC
contours (Figure S5.9) from the singlet Me groups and by
analyses of the COSY and HOHAHA spectra (Figures S5.5 and
S5.6), as shown in Figure 3 and Figure S5.10. Definitive NOE
1
1, 13, and 14, whereas little was detected for substrate 12.
Substrates 10−14 are lipophilic; by contrast, substrates 15 and
6 have a hydrophilic hydroxyl group. From the incubation
mixture of 15, only one product, 47, was obtained in high yield
1
(
88% of that of substrate 1), whereas three products48, 49,
data for H-18 (δ 1.89, m, 1H)/H-30 clearly indicated that the
H
and 50were detected from the reaction with 16 in a total
yield of ca. 19% of that of (3S)-1. The 48/49/50 product ratio
was 2.9:2.1:1.
stereochemistry at C-20 was in the S-configuration.
This finding leads to an important conclusion that E-Et and
Z-Me at C-23 of 10 are stereospecifically directed toward β-
and α-orientation at the C-20 position, respectively. This
conclusion is in a good agreement with our previous report
Structural Determinations of the Enzymatic Products
4
2−46. EIMS of β-amyrin 2 is shown in the Supporting
Information (Figure S4.1). The characteristic fragment ions, m/
z 218 (100%) and 203 (60%), were observed; the fragment
17
describing the cyclization reaction of 24-noroxidosqualene 8;
13,18,19
Me-30 of 8 is delivered to Me-29 (α-arrangement) of the β-
amyrin skeleton, whereas H-24 is converted into the Me-30
position (β-orientation). Unambiguous NOEs, such as Me-28/
H-18, Me-27/H-9, H-5/H-9, and H-3/H-5, made the overall
stereochemistry of product 42 clear, as depicted in Figure 3.
The EIMS fission pattern of product 43 (Figure S6.1) is
almost identical to that of product 42, suggesting that product
43 also has the β-amyrin core. However, the retention time in
the GC trace (Figure S3) was slightly different, suggesting that
43 and 42 may be diastereomers. Substrate 11 (8 mg) was
incubated in the same manner as 10, and product 43 was
structures are depicted in Figure S4.2.
As shown in
Figure S5.1, a fragment pattern similar to 2 was observed in the
EIMS of product 42: m/z 232 (100%), 217 (25%), 203 (75%),
+
and 440 (M , 3%), indicating that 42 possesses the β-amyrin
scaffold. The proposed EIMS fragment structures are shown in
Figure S5.2. To isolate 42 in an amount sufficient for structural
determination by NMR, we incubated 5 mg of substrate 10
with 1.45 mg of EtAS for 24 h and purified the product by SiO2
column chromatography using hexane/EtOAc (100:10) as the
eluent, which afforded pure 42 (2.1 mg, isolation yield). The
1
13
1
1
NMR spectra, including H, C, H− H COSY, HOHAHA,
NOESY, HSQC, and HMBC spectra, are shown in the
purified using SiO column chromatography, yielding 2.3 mg in
2
Figure 3. Two-dimensional NMR analyses for proposing the structure of product 42 dissolved in CDCl3.
D
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 4. Important 2D NMR data for proposing the structure of product 43 dissolved in CDCl3.
Figure 5. Proposed structures of the products, which were generated from 12 in a negligible amount, and the 2D NMR analyses for product 44
acetate dissolved in C D .
6
6
a pure state. The NMR spectra of product 43 dissolved in
and that the Et group (CH -29 and Me-31) is α-oriented
2
CDCl are shown in Figures S6.2−S6.8. One olefinic proton
(Figure 4). Thus, the stereochemistry at C-20 was determined
to be R-configuration, which is opposite that of 42,
demonstrating that substrate 11 was also subjected to a
cyclization pathway identical to that of 10 (see Scheme 1). That
is, E-Me and Z-Et of 11 were converted in a stereospecific
fashion into the Me-30 position (β-orientation) and into the
Me-29 position (α-orientation) of 2, respectively. The results of
the incubation of 10 and 11 with the enzyme indicate that the
larger steric bulk of the Et group could also be accepted at the
recognition sites, irrespective of being in the E- or Z-
configuration. The cyclization yields decreased compared to
3
1
was observed at δ 5.18 ppm (t, J = 3.6 Hz, 1H) in the H
H
2
NMR spectrum (400 MHz, CDCl ), and two sp carbons
3
assigned for one double bond were observed at δ 121.7 ppm
C
1
3
(
d) and 145.3 ppm (s) in the C NMR spectrum (100 MHz,
CDCl ). All the NMR data, including the 2D NMR spectra of
3
4
3, further supported the assignment of the β-amyrin structure
for 43. However, a strong NOE was observed between H-18
δ 1.97, m, 1H) and β-oriented Me-30 (δ 0.827, s, 3H) but
(
H
H
not between H-18 and H-29 (δH 1.17, q, J = 7.6 Hz, 2H).
These results definitively demonstrate that Me-30 is β-oriented
E
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1
13
that of 1 (Figure 2), but substantial amounts of β-amyrin
homologues were produced.
Substrate 12 underwent almost no conversion (see Figure 2
and Figures S3D and S7.1.1). Removing unreacted substrate 12
for H, 100 MHz for C NMR, C D ) are shown in Figures
6 6
S8.2−S8.8. The analyses of the NMR data are shown in Figure
6 (see also Figure S8.9); these data further indicate that 45 has
using SiO column chromatography (hexane/EtOAc = 100:5)
2
led to detection of the enzymatic product by GC/MS. This
product-enriched fraction was acetylated with Ac O/Py.
2
Negligible amounts of the acetylated form of compounds A−
G were observed, as shown in Figure S7.1.1. The EIMS spectra
of A−G acetates were compared to those of products obtained
17
from the incubation of 30-noroxidosqualene 9, which are
shown in the Supporting Information of ref 17. Identification of
peaks A and C was unsuccessful because EIMS fission patterns
similar to those of the products from 9 could not be found. The
retention times of the products were shorter than that of peak F
(pentacyclic 44), which suggests that the peaks are likely
products of premature termination of the cyclization reaction.
The EIMS of peak B was very similar to that of 27-
1
7
nordammara-20(21),24-diene-3β-ol. Peak D shows the
characteristic ions m/z 393 (100%) and 453 (100%). This
fragment pattern is very similar to that of 27-norbutyrospermol
Figure 6. Two-dimensional NMR analyses for proposing the structure
of product 45 dissolved in C D .
17
6
6
acetate [m/z 379 (100%) and 439 (98%)]; however, the mass
unit increment observed for peak D represents CH (m/z 14),
2
a β-amyrin scaffold similar to that of 44. However, the NOESY
spectrum demonstrated definitively the following NOEs: H-18/
suggesting that the structure of peak D would be a 27-
norbutyrospermol homologue, as shown in Figure 5 (see also
Figure S7.1.1). The difference of the EIMS spectra between
peaks D and E was slight, indicating that compounds D and E
share the same carbocyclic skeleton; the retention time of the
Me-28 and H-18/H-20 (H-18: δ 2.05, dd, J = 12.8, 3.6 Hz,
H
1
H; H-20: δH 1.20, m, 1H; Me-28: δH 1.10, s, 3H). These
findings indicated a 20R-configuration with regard to the
arrangement of the β-H and the α-Et at C-20. No other
products were found at detectable concentrations. Thus, the E-
ring closure occurs in a stereospecific fashion, despite lacking
Me-24 and despite Me-30 being replaced by a larger Et (C1
appendage) at the C-23 position of 1. The cyclization yield of
13 was approximately the same as that of 11 (Figure 2), further
indicating that the substitution of the larger steric bulk (Et
group) or the smaller size (H atom) at the 23E position of 1
had little effect on the cyclization pathway and yield. By
contrast, substitution with a hydrogen atom at the 23Z position
2
0R-form (butyrospermol) is known to be shorter than that of
17
the 20S-form (tirucalladiene). Thus, compound E would be
the homologue of 27-nortirucalla-7(8),24-diene-3β-ol. Peak F
exhibited the fragment ions (m/z 203 and 218) characteristic of
the β-amyrin core. The fragment ion at m/z 189 likely
18,19
corresponds to the structural unit shown in Figure S7.1.2.
Purification by HPLC (normal-phase, hexane/THF = 100:0.5)
led to the successful isolation of the acetate of peak F but in a
very small yield (0.4 mg from 60 mg of 12). Detailed NMR
analyses (Figures S7.2−S7.9) further revealed that peak F was a
β-amyrin analogue (Figure 5). Complete assignments of the
NMR data are shown in Figure S7.9. A clear NOE for H-29/H-
(
comparison of 12 with 13) notably affected the cyclization
pathway and yield, strongly indicating that the presence of an
alkyl group at the Z position is essential for the normal
polycyclization cascade, as we demonstrated in a previous
18 indicates that the β-Et and α-H were substituted at the C-20
position (product 44). Peak G shows the characteristic ions,
such as m/z 177, 189, and 204, that correspond to increments
17
paper.
To examine how the alkyl chain length at the Z position
affects acceptance of a molecule as a suitable substrate, we
incubated substrate 14 (Z-Pr group and E-H atom) with EtAS.
Figure 2 shows that a small amount of product 46 was
produced in ca. 4% yield (see also Figure S3F). The EIMS
spectrum of this peak suggests that product 46 also has the β-
amyrin scaffold; the fragment structures corresponding to the
characteristic ions m/z 232 and 217 are shown in Figure S9.2.
Repeated incubation experiments (3×) with 14 (10 mg) and
of a CH unit (m/z 14) to m/z 163, 175, and 190 observed in
2
17
the EIMS of 29-norgermanicol. Thus, careful inspection of
the EIMS spectra of products from 9 and 12 indicate that
products B, D, E, F, and G are homoderivatives (C1
appendages, Et substituent) of 27-nordammara-20(21),24-
diene-3β-ol, 27-norbutyrospermol (uph-7(8)-24-diene-3β-ol),
27-nortirucalla-7(8)-24-diene-3β-ol, 29-nor-β-amyrin, and 29-
norgermanicol, respectively. These predicted structures and the
2D NMR analyses for 44 (peak F) are depicted in Figure 5.
purification by SiO column chromatography afforded 0.3 mg
By contrast, substrate 13 (the Z-isomer of 12) was well-
2
of 46. Pure 46 was obtained by normal-phase HPLC (hexane/
THF = 100:3). The corresponding NMR spectra (600 MHz,
converted into product (yield: ca. 38%; see Figure 2). The
EIMS spectrum of the acetate of product 45 (Figure S8.1.2)
was almost identical to that of the acetate of product 44; thus,
the carbocyclic core of 45 is likely the same as that of 44.
Interpretation of the major fragment ions are shown in Figures
CDCl
observed at δ
H-30) and 1.37 (m, 2H, H-29). A clear NOE was observed
between H-18 (δ 1.79, m, 1H) and H-20, indicating the
) are shown in Figures S9.3−S9.9. A propyl group was
3
0.863 (t, J = 7.3 Hz, 3H, Me-31), 1.30 (m, 2H,
H
1
8,19
S7.1.2 and S8.1.3.
Product 45 was roughly purified using
H
SiO column chromatography (hexane/EtOAc = 100:10), and
the fraction including 45 was subsequently acetylated with
orientation was 20β-H and 20α-Pr (Figure S9.10). Thus, this
enzymatic product 46 was produced in a stereospecific manner,
as shown in Scheme 1. Notably, the cyclization yield of 14 (ca.
4%) was substantially decreased compared to that of 13 (ca.
2
Ac O/Py. Pure 45 acetate was obtained using normal-phase
2
HPLC (hexane/THF = 100:0.3). The NMR spectra (400 MHz
F
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
8%), indicating that the EtAS enzyme did not accept the
propyl group at the 23 position as well as it accepted the
normal substrate (see Figure 2 and Figure S3F).
Article
3
for C-18, suggesting that a THF ring is involved in 47. One H2-
30 had a clear HMBC correlation with C-18, but the other
exhibited no HMBC correlation (J = 8 Hz was used for
detecting the long-range coupling constant). A three-bond C−
H coupling constant follows a Karplus-type relationship. The
dihedral angles for H −C(30)−O−C(18) and H −C(30)−
O−C(18) are estimated to be 125 and 115°, respectively, from
the corresponding Chem3D models (Figure S10.10), indicating
that H has a clear HMBC correlation with C-18. The definitive
NOEs between Me-27 and H -19 (δ 1.46, m, 1H; 1.62, m,
1H) indicate that the oxygen atom of the THF ring is β-
oriented, and a clear NOE was observed for Me-29/H -30 (δH
3.28, d, J = 7.6 Hz, 1H); thus, Me-29 is determined to be in an
α-arrangement. Additional detailed 2D NMR analyses allowed
us to propose the whole structure of 47, as shown in Figure 7
Interestingly, all the enzymatic products of substrates 10−14,
except that of substrate 12, possessed a β-amyrin skeleton. A
current model that can plausibly explain these results is
proposed as follows: The alkyl group at the 23Z position plays
a critical role in folding the substrate into an ordered chair−
chair−chair−boat−boat conformation. Increasing the steric
bulk at the 23Z position would place the epoxide ring of the
substrates in a location somewhat distant from the DCTA
motif, which is essential for initiating the polycyclization
reaction by providing an acidic proton to activate the epoxide
ring. A substituent with a larger steric size (Me → Et → Pr) at
the 23Z position could move the epoxide ring farther from the
DCTA motif inside the enzyme cavity. This change can explain
the decrease in cyclization yields with increasing steric volume
A
B
A
2
H
B
(
see also Figure S10.9).
(100% → 40% → 4%; see Figure 2); however, the organized
conformation may have still been retained, thereby enabling
formation of the β-amyrin carbocyclic skeleton. Substrate 12,
which is substituted with a hydrogen atom at the 23Z position
and an Et group at the 23E position, exhibited substantially
decreased activity (almost no activity), whereas substrate 13
possessing an Et group at the 23Z position underwent the
normal cyclization in a considerably high yield (ca. 38%). This
difference in reactivity suggests that the hydrophobic
interaction occurs between the Z-alkyl group (Me or Et
group) and its recognition site of the cyclase and further
indicates that the hydrophobic interaction with an alkyl group
in the Z and not the E position is critical to creating the normal
folding architecture. This model is consistent with the results of
the comparative study between the reactions of 8 and 9
Figure 7. Two-dimensional NMR analyses for proposing the structure
of product 47 dissolved in CDCl3.
17
reported in our previous paper. This finding is further
supported by the fact that the reaction yields for substrates 11
and 13 were similar (40% for 11; 38% for 13). Thus, the
contributions of hydrophobic interactions with the 23E-alkyl
group are less important than the interaction with the 23Z-alkyl
group. The importance of this hydrophobic interaction between
the 23Z-alkyl group and its binding site was further established
by the experiments described below involving substrates 15 and
The H (400 MHz) and 13C NMR spectra (100 MHz) of
1
product 48 acetate dissolved in C D showed one olefinic
proton (δH 5.57, br s) and one double bond (δ 125.3, d;
136.8, s). These results suggest that the polycyclization reaction
of 16 terminated at the dammarenyl tetracyclic skeleton. Three
carbons connected to an oxygen atom were observed (δ 80.6,
d, C-3; 80.2, s, C-20; 64.7, t, C-27). In the HSQC spectrum, H-
3 (δ 4.83, dd, J = 11.6, 2.4 Hz) was correlated with C-3. Me-
21 (δ 1.29, s, 3H) had a clear HMBC cross-peak for C-20 and
6
6
C
C
1
6, which have a hydrophilic CH OH group.
Structures of Products 47−50 from 15 and 16 and the
2
H
H
Cyclization Mechanism. The hexane extract obtained from
the incubation mixture of 15 (10 mg) with EtAS (1.12 mg) was
subjected to normal HPLC (hexane/THF = 100:2), yielding ca.
C-17 (δ 45.7, d). Me-26 (δ 1.61, br s, 3H) exhibited strong
C
H
HMBC correlations with C-27, C-25 (δ 136.8, s), and C-24
(δ 125.3, d). In the HOHAHA spectrum, the olefinic proton
H-24 (δ 1.61, br s, 1H) definitively showed the following
H− H network: H-24/H-27 (δ 3.91, br d, J = 16.4 Hz, 1H;
4.24, br d, J = 16.4 Hz, 1H)/Me-26 (δ 1.61, br s, 3H)/H-23
(δ 2.43, m, 1H; 2.17, m, 1H)/H-22 (δ 1.84, m, 1H; 1.72, m,
1H), indicating that a seven-membered ether ring (oxepane
core) is involved in 48. The characteristic fragment ion m/z
125 in the EIMS (Figure S11.1) further supported the presence
of an oxepane moiety (Figure 8). A definitive NOE was
C
C
3
.5 mg of pure 47. Incubation of 16 (10 mg) under the same
H
1
1
conditions as 15 gave crude product mixtures (2.3 mg), which
H
were acetylated with Ac O/Py. The resulting acetate mixtures
2
H
were subjected to normal-phase HPLC (hexane/THF =
H
H
100:0.8), where the acetylated products were eluted in the
order 50 → 48 → 49. The isolation yields of 48−50 were 0.7,
0
.7, and 0.4 mg, respectively.
2
No olefinic proton and no sp carbon were observed in the
1
13
H and C NMR spectra of 47 dissolved in CDCl (Figures
observed between Me-30 and H-17 (δ 2.25, m), indicating
3
H
S10.2 and S10.3), suggesting that substrate 15 underwent the
complete cyclization reaction by EtAS. In the HMBC spectrum
that H-17 is α-oriented. Taking into consideration the analyses
of the other NMR data, we can propose the whole structure of
48, as depicted in Figure 8 (see also Figure S11.9). The EIMS
spectrum of product 49 was almost identical to that of 48 (see
Figures S11.1 and S12.1), indicating that a skeleton similar to
that of 48 is assignable for 49. Observation of the fragment ion
m/z 125 further supported the seven-membered oxepane ring.
This model was again supported by the detailed 2D NMR
(
Figure S10.8), Me-27 (δ 0.914, s, 3H) showed correlations
H
with C-15 (δ 27.2, t) and C-13 (δ 38.1, d), and Me-28 (δ
C
C
H
0
1
.941, s, 3H) showed cross-peaks for C-16 (δ 36.2, t) and C-
C
8 (δ 88.2, s). The chemical shift of C-18 indicates that an
C
oxygen atom is connected to C-18. Furthermore, both H-13
(
δ 2.12, dd, J = 12.4 and 2.8 Hz, 1H) and one of H -30 (δ
H
2
H
3.61, d, J = 7.6 Hz, 1H) showed a definitive HMBC cross-peak
analyses of 49 (Figure S12.2−S12.9). Me-21 (δ 1.27, s, 3H)
H
G
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 9. Two-dimensional NMR analyses for proposing the structure
of product 50 acetate dissolved in CDCl3.
Figure 8. Two-dimensional NMR analyses for proposing the structure
of product 48 or 49 dissolved in C D . Products 48 and 49 are
diastereomers, with their configurations at C-20 being opposite. The
chemical shifts of product 48 are shown.
Figure 10 summarizes the structures of all the products
generated by incubating substrates 10−16 with EtAS enzyme.
Substrates 10−14 afforded only β-amyrin skeleton, except for
6
6
1
2, indicating that the Z-alkyl group at the 23 position was
more strongly bound to the cyclase, as shown in Scheme 3A.
Scheme 3B,C illustrates the product formation mechanism
from substrates 15 and 16. Substrate 15 was folded in the
chair−chair−chair−boat−boat conformation and underwent
the normal cyclization reaction according to Scheme 1,
had clear HMBC correlations with C-20, C-22, and C-17. The
1
1
HOHAHA spectrum revealed the following H− H spin-
coupling network: H-24/Me-26/H-23/H-22/H-27, as observed
in the HOHAHA spectrum of 48. The α-orientation of H-17
was confirmed by the definitive NOE between H-17 and Me-
resulting in the formation of oleanyl cation 6′ with CH OH
2
30. The detailed NMR analyses (Figure S12.9) lead to a
(C-19 cation, ca. 88% yield). A hydride shift of H-18 to C-19
cation afforded the oleanyl cation 6″ with a C-18 cation. The
hydroxyl group subsequently attacked this cation as a
nucleophile, yielding the 6−6−6−6−6−5-fused A−B−C−D−
E−F hexacyclic ring system 47. A nucleophilic attack of
proposed structure identical to that of 48. Product 48 was
separable from 49 by normal HPLC and GC, indicating that 48
and 49 are diastereomers. Careful comparison of the NOESY
spectra of 48 and 49 revealed that the configurations of the
tetracyclic dammarenyl core including H-17 stereochemistry
were identical between 48 and 49, thus demonstrating that the
C-20 stereochemistry is the only difference between the two
isomers. The C-20 stereochemistry for 48 and 49 remained to
be elucidated.
CH OH at the C-19 cation of 6′ would provide a 6−6−6−6−
2
6−4-fused hexacyclic product. The four-membered ring has a
larger steric strain, which triggered the 1,2-shift of hydride of H-
18 to form a less-strained five-membered F-ring. Formation of
6″ likely leads to β-amyrin homologue 2′ according to Scheme
One olefinic proton (400 MHz, C D ; δ 5.76, t, 1H) and
1 and Scheme 3A, but CH OH quickly attacked the generated
6
6
H
2
one double bond (100 MHz, C D ; δ 126.7, d; 132.9, s) were
cation 6″ because of its highly nucleophilic nature, yielding 47.
The 23Z-Me of 15 is anchored by the hydrophobic interaction,
as shown in Scheme 3B, in a manner similar to that of 1
(Scheme 3A). This robust interaction could lead to the
formation of the final cation 6′. However, the reaction of
substrate 16 terminated at the tetracyclic dammarenyl cation 3′
6
6
C
found in 50 acetate, implying that the polycyclization reaction
of 16 terminated at the abortive tetracyclic stage to give 50.
The partial structure of CH −C(Me)CH−CH −CH was
2
2
2
confirmed by the following HOHAHA correlations: H-21 (δH
5
.76, t, J = 8.0 Hz, 1H)/H-20 (δ 1.98, m, 1H; 2.04, m, 1H)/
H
H-19 (δ 1.65, m, 1H; 1.50, m, 1H)/Me-29 (δ 1.56, br s,
in a yield of ca. 19%. The Z-CH OH group then acted as a
H
H
2
3
H)/H-30 (δ 3.85, br d, J = 16.8 Hz, 1H; 4.47, br d, J = 16.8
nucleophile to attack the C-20 cation and produce 48 and 49
[ca. 15% (7 or 8% each)]. The configuration at C-20 of 48 was
H
Hz, 1H), with this partial structure being also observed in the
structure of 48 and 49. Two carbons connected to an oxygen
atom were observed at δ 74.1 (t) and δ 84.5 (d) for 50,
suggesting an ether linkage. This result is in contrast to the
ethereal carbons of 49, which were at δ 64.7 (t) and 79.6 (s).
Furthermore, the multiplicity for one of the two carbons was
different, and the chemical shifts of the ethereal carbons were
distinct between 50 and 49. These data suggest that the
structure of 50 is quite different from that of 48 or 49, which
possess an oxepane ring. Me-28 (δH 1.25, s, 3H) had clear
opposite that of 49, indicating that Z-CH OH of 16 was not
2
anchored by the 23Z recognition site; thus, the side chain of 3′
underwent a free rotation to afford both the 20R- and 20S
-configurations, as shown in Scheme 3C. This finding gives
additional support to the essential nature of the alkyl group at
23Z position for formation of the final oleanyl cation
intermediate 6′. Further cyclization of 3′ led to a baccharenyl
cation 4′ (ca. 4%) in a small amount, which was then converted
into product 50 as a result of the nucleophilic attack of Z-
C
C
C
HMBC correlations with C-16 (δ 36.5, t), C-17 (37.2, s), C-
CH OH at cation 4′. Cation 4′ is the intermediary cation but
C
2
1
8 (84.5, d), and C-19 (45.9, t), indicating that the D-ring of 50
not the final cation 6′. Production of 50 also indicates that the
chair−chair−chair−boat−boat structure was distorted because
of the lack of a 23Z-alkyl group, resulting in the termination at
the intermediary stage. As shown in Figure 2, the cyclization
yield of 15 was outstandingly high (ca. 88%), whereas that of
16 was at most ca. 19%. This fact provides additional evidence
that the alkyl group at the 23Z position is strongly bound to the
binding site through a hydrophobic interaction and is essential
for the normal polycyclization cascade, but this interaction of
is six-membered and not five-membered as in 48 and 49. These
findings indicate that the E-ring of 50 is an eight-membered
ring (oxocane ring), as shown in Figure 9. The strong NOE
between H-18 (δ 3.20, d, J = 10.4 Hz, 1H) and Me-27 (δH
H
0.921, s, 3H) verified that H-18 was arranged in the α-
configuration. Further detailed analyses of 2D NMR made clear
the complete structure of 50, as shown in Figure 9 (see also
Figure S13.9).
H
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 10. Structures of products 42−50 obtained from the incubation experiments with substrates 10−16. The carbon numbering systems are also
shown here to aid in interpretation of the NMR data.
2
3E-alky moiety is not robust. We propose that the robust
The hydrophobic interaction of the Z-alkyl group with the
enzyme is more critical to forming the normal folding
architecture than that of the E-alkyl group. The contribution
of the hydrophobic interaction with the 23E-alkyl group is
much less than that of the 23Z-alkyl group. The importance of
the hydrophobic interaction with the 23Z-alkyl group was
further established by comparing the cyclization reactions of 15
interaction would be caused by CH−π interaction. Our
experimental results clearly demonstrated that this robust
interaction could sufficiently direct the enzymatic reaction
toward pentacyclization, although a binding energy for CH/π
affinity may be considered to be weak. We constructed site-
directed mutants targeting the amino acids surrounding the
terminus of substrate 1 (see Figure S14). The following
residues were mutated into Ala residue: F124, F125, W219,
C260, R261, V263, S412, F413, F552, I555, and C732. None of
the variants produced tetracyclic products but instead produced
only β-amyrin (Figure S15). Therefore, these residues are not
responsible for the interaction with Me-30 (23Z-Me) on 1. We
have reported that the site-directed mutants of F728 afforded
and 16, which possess a hydrophilic CH OH group.
2
Compounds structurally similar 47 have been reported, such
23
24
as 13,28-epoxyoleanan-3β-ol and 19,28-epoxyoleanan-3β-ol;
however, the 19,30-epoxy derivative 47 had not been found
before the present study. Products 48−50 are also new
compounds; no similar structures are found in SciFinder
(American Chemical Sociey, https://scifinder.cas.org/
scifinder/view/scifinder/scifinderExplore.jsf). This study on
the substrate analogues bearing a hydroxyl group has provided
deep insight into the cyclization mechanism in addition to the
creation of new compounds 47−50.
20,21
tetracyclic compounds along with pentacyclic products.
Kushiro et al. revealed that the residues of W259 and Y261
from Panax ginseng β-amyrin synthase stabilize the tetracyclic
and the pentacyclic cation intermediates via cation/π
interaction. The W259 and Y216 from P. ginseng correspond
to W257 and Y259 residues from EtAS, respectively, in the
amino acid alignment. The functions of W257 and Y259
residues from EtAS identified by our experiments were
consistent with those of W259 and Y261 reported by Kushiro
2
2
EXPERIMENTAL SECTION
■
General Analytical Methods. NMR spectra were recorded at 600
or 400 MHz in the solution of CDCl or C D . The chemical shifts (δ)
3
6
6
2
2
et al. These three aromatic residuesF728, W258, and
are given in parts per million relative to the residual solvent peak as the
Y259 (independently or together) may interact with Me-30
internal reference (δ = 7.26 and δ = 77.0 ppm for CDCl ; δ = 7.28
H
C
3
H
of 1 via CH/π interaction (perpendicular to the CH ), as
and δC = 128.0 ppm for C D ; δ = 2.04 and δC = 29.80 ppm for
3
6 6 H
depicted in Scheme 3A, in addition to their function in
acetone-d ). GC analyses were performed by fitting a flame ionization
6
stabilizing the intermediary cation(s) through cation/π
interactions (horizontal to the planar sp carbocation). Further
studies may be necessary to validate this assumption.
detector and a nonpolar DB-1 capillary column (30 m × 0.32 mm);
injection temp = 300 °C; column temp = 235−250 °C (3 °C/min)
and 250−270 °C (0.5 °C/min); flow rate (He gas) = 1.50 mL/min.
Several peaks of substrates 1 and 10−16 indicate that the thermal
degradation of these substrates occurred during the GC analyses. GC/
MS spectra were obtained under electronic impact at 70 eV with a
Zebron ZB-5 ms capillary column (0.25 mm × 30 m), with the oven
temperature being increased from 220 to 270 °C (3 °C/min). High-
resolution mass spectra were obtained on a HRMS-TOF spectrometer.
2
CONCLUSION
■
Through this investigation, we clarified that β-amyrin synthase
tolerates ethyl substituents at position 23 in the substrate, as
evidenced by the high yields of the corresponding products.
I
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
a
Scheme 3. Cyclization Pathway of Substrates 1, 15, and 16 by the Cyclase Enzyme.
a
Robust binding of 23Z-Me of oxidosqualene 1 to the cyclase enzyme but with loose binding of 23E-Me (A). Reaction pathway of substrates 15 and
1
6 to produce 47−50 (B,C).
Synthetic Experiments of Substrate Analogues 10−16. As
the general synthetic method, C -aldehyde 17 was subjected to Wittig
flask, triethyl 2-phosphonobutyrate 18 (1026.7 mg, 6.15 mmol) was
dissolved in Et O (130 mL) and cooled to 0 °C under N atmosphere.
27
2
2
reactions using reagents 18−21. The starting material 17 was prepared
NaH (40% oil, 273.7 mg) was added bit-by-bit into the ethereal
solution. Then, the solution was chilled to −40 °C and stirred for 15
min. Aldehyde 17 (472.9 mg, 1.23 mmol) dissolved in a small amount
as follows. Squalene was converted into the bromohydrin derivative by
NBS treatment, the reaction of which was conducted in H O/THF
2
solution. Subsequently, the epoxide ring was formed in the basic
of Et O was added step-by-step and stirred for 30 min. The reaction
2
1
7
condition using K CO , followed by H IO treatment, affording 17.
was monitored by SiO TLC. To the reaction mixture was added
2
3
5
6
2
Preparations of 22 and 23. Esters 22 and 23 were produced by a
Wittig−Horner reaction of 17 with NaH base. In the three-necked
saturated aq NaCl, and the lipophilic materials were extracted with
hexane (3×), which was dried over Na SO . The dried hexane was
2
4
J
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
evaporated under reduced pressure, yielding a mixture of 22 and 23
NMR (100 MHz, CDCl ) δ 14.4 (q), 15.95 (q), 15.99 (q), 16.02 (q,
3
C
(
571.0 mg, 96.4% yield). The E/Z mixture was separated by SiO2
2C), 17.7 (q), 20.5 (t), 25.7 (q), 25.8 (t), 26.63 (q) 26.66 (t), 26.8 (t),
28.3 (t, 2C), 39.67 (t, 2C), 39.74 (t, 2C), 124.27 (d), 124.30 (d),
124.34 (d), 124.41 (d), 124.48 (d), 128.8 (d), 131.2(s), 131.6 (d),
134.9 (s), 135.05 (s), 135.09 (s).
column chromatography (hexane/EtOAc = 100:5). The repeated
chromatography afforded 85.3 mg of 22 and 100.2 mg of 23 in a pure
state. Yet, an unseparated fraction was available in a yield of 380 mg.
+
1
Compound 22: EIMS; m/z 482 (M , 0.8%); H NMR (400 MHz,
Preparation of 26. The Wittig reaction of 17 with [Ph P-
3
+
−
CDCl ) δ 1.02 (t, J = 7.2 Hz, 3H), 1.30 (t, J = 7.2 Hz), 1.60 (s, 15H,
CH C H ] Br 20 was conducted in the presence of NaN(SiMe ) ,
3
H
2
3
7
3 2
5
× Me), 1.68 (s, 3H, Me), 2.06−1.80 (m, 9 × CH ), 2.25 (q, J = 7.2
selectively affording 31 with Z-configuration, and no contamination of
the E-configuration was found. To the suspension of 20 (1.049 g, 2.63
mmol) in Et O was added 3.57 mL of NaN(SiMe ) (1.10 mol/L)
2
Hz, 2H), 2.50 (q, J = 7.2 Hz, 2H), 4.20 (q, J = 7.2 Hz, 2H), 5.13−5.05
(
5H, m), 5.82 (1H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl ) δ
3
C
2
3 2
1
2
5
1
3.7 (q), 14.3 (q),15.8 (q), 15.98(q), 16.00 (q, 3C), 17.7 (q), 25.7 (q),
drop-by-drop, whereby the suspension was changed into the solution
colored with orange. These treatments were done at 0 °C under the
6.6 (t, 2C), 27.5 (t), 27.9 (t), 28.2 (t, 2C), 39.2 (t), 39.7 (t, 3C),
9.9(t), 124.22(d), 124.24 (d), 124.26 (d), 124.36 (d), 124.9 (d),
31.2 (s), 133.6 (s), 134.1 (s), 134.9 (s), 135.0 (s), 135.1 (s), 140.0
atmosphere of N gas. Next, the temperature was cooled at −78 °C.
2
To the solution was added 17 (101.0 mg, 0.263 mmol), dissolved in a
(
d), 168.3 (s). In the NOESY spectrum, a clear NOE was observed
small amount of Et O, slowly during stirring. After 30 min, the
2
between δ 1.02 and 5.82, indicating that Me of Et at C-23 is oriented
reaction mixture was poured into saturated aq NaCl solution to
quench this reaction, followed by extraction of product with hexane
(3×). The hexane extract was dried over Na SO . Product 26 was
H
in a E-configuration and that CO Et group was in Z-configuration.
2
+
1
Compound 23: EIMS; m/z 482 (M , 6%); H NMR (400 MHz,
2
4
CDCl ) δ 1.00 (t, J = 7.2 Hz, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.60 (s,
purified by a SiO2 column chromatography with hexane/AcOEt
3
H
1
2H, 3 × Me), 1.61 (s, 3H, Me), 1.68 (s, 3H), 2.10−1.90 (m, 10 ×
(100:1) as eluent, yielding 68.2 mg (61.1%).
Compound 26: EIMS m/z 496 (M , 4%); H NMR (400 MHz,
+
1
CH ), 2.02 (m, 4H), 4.17 (q, J = 7.2 Hz, 2H), 5.13−5.05 (m, 5H),
2
13
6
.70 (t, J = 7.2 Hz, 1H); C NMR (100 MHz, CDCl ) δ 13.9
CDCl ) δ 0.904 (t, J = 7.2 Hz, 3H), 1.35 (q, J = 7.2 Hz, 2H), 1.60 (s,
3 C
3
H
(
q),14.3 (q), 16.0 (q, 4Me),17.7 (q), 20.1 (t), 25.7 (q), 26.7 (t, 2C),
15H, 5Me), 1.68 (s, 3H), 2.2−1.9 (m, 22H), 5.12 (m, 5H), 5.36 (m,
2H); 13C NMR (100 MHz, CDCl
) δ 13.3 (q), 15.96 (q), 15.99 (q),
2
6
1
1
6.8 (t), 27.1 (t), 28.3 (t, 2C), 38.6 (t), 39.6 (t), 39.70 (t), 39.74 (t),
0.2 (t), 124.2 (d, 2 × C), 124.4 (d, 2 × C), 125.1 (d), 131.2 (s),
33.8 (s), 133.95 (s), 134.89 (s), 134.96 (s), 135.1 (s), 141.5 (d),
67.9 (s).
3
C
16.02 (q), 17.7 (q), 22.9 (t), 25.7 (q), 25.9 (t), 26.64 (t), 26.66 (t),
26.77 (t), 28.3 (t, 2C), 29.3 (t), 39.65 (t), 39.74 (t, 3C), 124.27 (d),
124.30 (d), 124.33 (d), 124.41 (d), 124.46 (d), 129.59 (d), 129.69
(d), 131.2 (s), 134.6 (s), 134.89 (s), 135.05 (s), 135.09 (s).
Preparations of 24 and 25. A solution of triphenylphosphine
(
17.06 g, 65.04 mmol) and 1-bromopropane dissolved in toluene (50
Preparations of 27 and 28. The synthetic protocols were the
mL) was refluxed for 12 h. The precipitated phosphonium salts were
collected by filtration and washed with toluene (3×) to remove
unreacted materials. Salt 19 thus collected was dried over P O under
same as those of 22 and 23. Ethyl 2-(diethoxyphosphoryl)propanoate
21 (257.0 mg, 1.02 mmol) was suspended in Et
under N gas atmosphere. NaH (40% oil, 61.2 mg, 1.53 mmol) was
O (130 mL) at 0 °C
2
2
5
2
reduced pressure, giving 7.47 g (47.7% yield). Aldehyde 17 was
subjected to a Wittig reaction with 19 in n-BuLi basic condition under
N atmosphere condition. Salt 19 (972.9 mg) was suspended in Et O
slowly added to the suspension. The reaction flask was cooled to −40
°C and stirred for 15 min. To the solution was added aldehyde 17,
dissolved in a small amount of Et
reaction mixture was poured into saturated aq NaCl, and then the
product was extracted with hexane, which was dried over Na SO . The
hexane extract was dried under reduced pressure. The residue (302.0
mg, yield 94.9%) was applied to a SiO column chromatography
O, bit-by-bit. After 30 min, the
2
2
2
(
3
50 mL) at 0 °C in a three-necked flask, to which n-BuLi (1.58 mol/L,
.20 mL) was added in a dropwise, and the solution was gradually
2
4
changed into orange. The temperature was cooled to −78 °C and
stirred for 15 min. Subsequently, an ethereal solution of 17 (193.9 mg,
2
0
.505 mmol) was bit-by-bit added into the orange-colored solution.
(hexane/EtOAc = 100:5) to separate E- and Z-isomers 27 and 28.
Isolation yields of 27 and 28 were 230.1 mg (72.3%) and 52.2 mg
(25.8%), respectively.
The reaction was monitored by TLC. After 30 min, the reaction
mixture was poured into saturated aq NaCl, and the lipophilic
materials were extracted with hexane, which were then dried over
Na SO , followed by purification with a SiO column (hexane/AcOEt
+
1
Compound 27: EIMS m/z 468 (M , 2%); H NMR (400 MHz,
CDCl ) δ 1.29 (t, J = 7.2 Hz, 3H), 1.60 (s, 12H), 1.61 (s, 3H), 1.68
2
4
2
3
H
=
100:10). The purified fraction contained a mixture of E- and Z-
(s, 3H), 1.75 (s, 3H), 2.15−1.96 (m, 18H), 2.26 (q, J = 7.2 Hz, 2H),
4.18 (q, J = 7.2 Hz, 2H), 5.15−5.00 (m, 5H), 6.75 (t, J = 7.2 Hz, 1H);
isomers. Separation of the E- and Z-isomers (24 and 25), which were
produced in the presence of n-BuLi, was difficult even with the
AgNO -impregnated SiO column. Thus, the NMR data of compound
13
C NMR (100 MHz, CDCl ) δ 12.3 (q), 14.3 (q), 15.95 (q), 15.98
3 C
3
2
(q), 16.02 (q, 2C), 17.7 (q), 25.67 (q), 26.7 (t, 2C), 26.8 (t), 27.4 (t),
28.3 (t, 2C), 38.3 (t), 39.6 (t), 39.72 (t), 39.74 (t), 60.3 (t), 124.3 (d,
2C), 124.4 (d, 2C), 125.1 (d), 127.7 (s), 131.2 (s), 133.8 (s), 134.9
(s), 134.95 (s), 135.1 (s), 141.9 (d), 168.2 (s).
2
4 are not given. However, the separation of the epoxides 12 and 13
(
E- and Z-isomers) succeeded by 5% AgNO −SiO column
3
2
chromatography (hexane/EtOAc = 100:5−100:50, stepwise elution).
Bromohydrins 31 and 32 were produced by the reaction of NBS,
followed by treatment of K CO , affording a mixture of 12 and 13
+
1
Compound 28: EIMS m/z 468 (M , 2%); H NMR (400 MHz,
CDCl ) δ 1.28 (t, J = 7.2 Hz, 3H), 1.60 (s, 15H, 5Me), 1.68 (s, 3H),
2
3
3
H
(
NMR data of each product are described below). Argentation
1.88 (s, 3H, s), 2.2−1.9 (m, 18H), 2.55 (q, J = 7.2 Hz, 2H), 5.15−5.00
(m, 5H), 5.90 (t, J = 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl ) δ
chromatography allowed the separation of 12 and 13. The Wittig
reaction of 21 with 17 was conducted using NaN(SiMe ) as base.
Under the basic condition, Z-isomer 25 only was selectively produced.
Phosphonium bromide 19 (649.5 mg, 1.68 mmol) was suspended in
Et O (20 mL) at 0 °C under nitrogen gas in a three-necked flask. A
solution of NaN(SiMe ) (1.01 mol/L, 2.3 mL) was slowly added,
3
C
14.5 (q),16.0 (q), 16.22 (q), 16.26 (q, 2C), 17.9 (q), 20.9 (q), 25.9
(q), 26.9 (t, 2C), 27.0 (t), 28.2 (t), 28.5 (t, 2C), 39.4 (t), 39.95 (t),
40.0 (t, 2C), 60.2 (t), 124.47(d), 124.49 (d), 124.53 (d), 124.61 (d),
124.75 (d), 127.3 (s), 131.5 (s), 134.4 (s), 135.1 (s), 135.27 (s),
135.33 (s), 142.8 (d), 168.3 (s).
3
2
2
3
2
giving a clear orange solution. The reaction flask was cooled to −78 °C
Preparations of 29−35. The protocols for preparing the
bromohydrin derivatives 29−35 were essentially the same between
each of them. As a typical example, the experiments for the synthesis of
34 from 27 are described below. A solution of 27 (90 mg, 0.19 mmol),
dissolved in THF (12 mL), was cooled at 0 °C. To the solution was
added water until the solution became cloudy. Next, a small amount of
THF was added until the transparent solution was formed. To the
solution was added 51.3 mg (0.29 mmol) of NBS bit-by-bit. The
and stirred for 15 min. To the solution was slowly added 17 (64.7 mg,
0
.169 mmol), and the reaction was continued for an additional 30 min.
To the flask was added saturated aq NaCl, and the product was
extracted with hexane, which was dried over Na SO . Pure 25 was
2
4
obtained by SiO column chromatography using a solvent of hexane/
2
EtOAc = 100/1 (47 mg, yield 68%).
Compound 25: EIMS m/z 410 (M , 1%); H NMR (400 MHz,
+
1
CDCl ) δ 0.958 (t, J = 7.2 Hz, 3H), 1.60 (s, 15H, 5Me), 1.68 (s, 3H),
reaction was monitored by SiO TLC. After 30 min, saturated aq NaCl
was added, and the crude product and unreacted material 27 were
3
H
2
13
2
.15−1.96 (m, 11CH ), 5.15−5.00 (m, 5H), 5.37−5.26 (m, 2H);
C
2
K
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1
extracted with hexane (3×), which was dried over Na SO . A SiO
Compound 39: H NMR (400 MHz, CDCl ) δ 1.33 (s, 3H), 1.34
3 H
2
4
2
column chromatography (hexane/EtOAc = 100:30) gave pure 34
(s, 3H), 1.79 (m, 1H), 1.81 (3H, s), 2.17−1.94 (m, 18H), 2.30 (m,
1H), 3.97 (br d, J = 11.2 Hz, 1H), 4.10 (s, 2H), 5.22−5.08 (m, 4H),
5.28 (t, J = 7.0 Hz, 1H).
Preparations of 40 and 41. Protocols for the syntheses of 40 and
41 were essentially the same between them. Therefore, the synthetic
experiment of 40 from 36 only is described here. Compound 36 (25.0
mg, 0.047 mmol) was dissolved in 5 mL of CH Cl . The three-necked
(
51.6 mg, yield 48.0%).
NMR data of 29−35 are given below.
1
Compound 29: H NMR (400 MHz, CDCl ) δ 1.01 (t, J = 7.2
3
H
Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H), 1.32 (s, 3H), 1.34 (s, 3H), 1.60 (s,
2H), 1.80 (m, 1H), 2.16−1.90 (m, 18H), 2.33−2.20 (m, 3H), 2.50
q, J = 7.2 Hz, 2H), 3.98 (br d, J = 11.2 Hz, 1H), 4.20 (q, J = 7.2 Hz,
1
(
2
2
2
H), 5.23−5.10 (m, 4H), 5.82 (t, J = 7.2 Hz, 1H).
flask was cooled to 0 °C and filled with N gas. To the flask was added
2
1
Compound 30: H NMR (400 MHz, CDCl ) δ 0.996 (t, J = 7.2
Et N (70.7 mg, 0.70 mmol), dissolved in a small amount of CH Cl .
3
H
3
2
2
Hz, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.32 (s, 3H), 1.34 (s, 3H), 1.597 (s,
Next, MsCl (80.5 mg, 0.70 mmol), dissolved in a small quantity of
CH Cl , was added slowly and then stirred overnight. The reaction
mixture was washed successively with 5% aq HCl, saturated aq
9
1
7
H), 1.61 (s, 3H), 1.80 (m, 1H), 2.15−1.89 (m, 16H), 3.97 (br d, J =
1.2 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 5.23−5.10 (m, 4H), 6.94 (t, J =
.2 Hz, 1H).
2
2
NaHCO , and saturated aq NaCl, all of which were cooled at 0 °C in
3
Compound 31: The NMR data are not given because a mixture of
advance. The organic layer containing mesylate 40 was dried over
2
7 and 28 was produced by the reaction of 19 and 21 in the presence
Na SO . Mesylates are generally labile, thus the reaction product was
2
4
of n-BuLi. A mixture of 27 and 28 was used for the next reaction
without separation. The NBS treatment afforded a mixture of 36 and
used for the next reaction without purification. Therefore, we did not
measure the H NMR spectrum of the reaction products 40 and 41.
1
3
3
7. The epoxides 12 and 13 only, which were produced from 36 and
Preparations of 10 and 11. The protocols were identical between
them. The MeOH solution (10 mL) of 49 (ca. 10 mg) was added bit-
by-bit into dry MeOH solution of K CO (10 mg/10 mL). After 30
7, were separated by AgNO −SiO2 column chromatography, as
3
described above.
2
3
Compound 32: This compound was synthesized from Z-isomer 25,
min, the reaction mixture was poured into saturated aq NaCl, and the
which was selectively produced from the reaction of 17 and 19 in the
epoxide product was extracted with hexane (3×). The hexane extract
1
presence of NaN(SiMe ) as described above; H NMR (400 MHz,
was subjected to a short SiO column (hexane/EtOAc = 100:10) to
3
2
2
CDCl ) δ 0.955 (t, J = 7.2 Hz, 3H), 1.33 (s, 3H), 1.34 (s, 3H), 1.60
remove just the high-polar impurities found at the origin of TLC and
then employed for the next reaction without further purification. The
epoxide (ca. 10 mg) thus obtained was subjected to reduction with
LiBEt H to convert the −CH OMs into −CH . The epoxide was
3
H
(
s, 12H), 1.78 (m, 1H), 2.13−2.18 (m, 20H), 2.30 (m, 1H), 3.98 (br
d, J = 11.2 Hz, 1H), 5.22−5.01 (m, 4H), 5.33 (m, 2H).
1
Compound 33: The H NMR of 33 was not measured, but the
3
2
3
1
epoxide structure of 14 synthesized from 33 was confirmed by H and
dissolved in Et O (10 mg/10 mL). Under the atmosphere of N gas,
2
2
1
3
C NMR data, which are described below.
the THF solution of LiBEt H (50 μL, 1.0 M) was added in a small
3
1
Compound 34: H NMR (400 MHz, CDCl ) δ 1.29 (t, J = 7.2
portion. The reaction was monitored with TLC. After 2 h, the
mesylate having the epoxide ring disappeared. To the reaction mixture
was added saturated aq NaCl, followed by extraction with hexane
3
H
Hz, 3H), 1.33 (s, 3H), 1.34 (s, 3H), 1.60 (s, 9H), 1.61 (s, 3H), 1.81
(
(
(
m, 1H), 1.82 (s, 3H), 2.21−1.94 (m, 18H), 2.35−2.21 (m, 3H), 3.97
br d, J = 11.2 Hz), 4.17 (q, J = 7.2 Hz, 2H), 5.23−5.12 (m, 4H), 6.74
t, J = 7.2 Hz, 1H).
(3×). The hexane extract was dried over Na SO . Complete
2 4
purification was done by normal-phase HPLC (hexane/THF =
100:0.8, Inertsil, GL Science), giving ca. 7.0 mg of 10.
1
Compound 35: H NMR (400 MHz, CDCl ) δ 1.30 (t, J = 7.2
3
H
+
Hz, 3H), 1.33 (s, 3H), 1.34 (s, 3H), 1.60 (s, 12H), 1.79 (m, 1H), 1.88
s, 3H), 2.15−1.93 (m, 18H), 2.54 (q, J = 7.2 Hz, 2H), 3.98 (br d, J =
1.2 Hz), 4.19 (q, J = 7.2 Hz, 2H), 5.20−5.12 (m, 4H), 5.90 (t, J = 7.2
Compound 10: EIMS m/z 440 (M , 0.6%); HRMS (EI) m/z calcd
for C31
) δ
H
52O 440.40181; found 440.40187; 1H NMR (400 MHz,
(
1
C
6
D
6
H
1.07 (t, J = 7.6 Hz, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.67 (s,
13
Hz); C NMR (100 MHz, CDCl ) δ 14.3 (q), 15.8 (q, 3C), 16.0
3H), 1.73 (s, 9H), 1.81 (s, 3H), 2.33−2.10 (m, 22H), 2.69 (t, J = 6.0
3
C
13
(
q), 16.1 (q), 20.6 (q), 25.8 (q), 26.6 (t), 26.7 (t), 28.0 (t), 28.3 (t,
Hz, 1H), 5.44−5.35 (m, 5H); C NMR (100 MHz, C D ) δ 13.1
6 6 C
2
7
C), 32.2 (t), 38.2 (t), 39.2 (t), 39.6 (t), 39.7 (t), 60.1 (t), 70.9 (s),
2.4 (d), 124.3 (d), 124.4 (d), 124.9 (d), 126.0 (d), 127.1 (s), 132.9
(q), 16.05 (q), 16.13 (q), 16.15 (q, 2C), 18.9 (q), 25.0 (q, 2C), 27.10
(t), 27.14 (t), 28.1 (t), 28.8 (t, 3C), 32.8 (t), 36.9 (t), 40.1 (t), 40.2 (t,
2C), 57.4 (s), 63.5 (d), 123.4 (d), 124.8 (d), 124.9 (d), 125.2 (d),
134.4 (s), 135.0 (s), 135.1 (s), 135.2 (s), 136.7 (s).
(
s),134.2 (s), 134.9 (s), 135.1 (s), 142.5 (d), 168.2 (s).
Preparations of 36−39. The synthetic methods were essentially
+
the same between the syntheses of 36−39. As the typical experiment,
Compound 11: EIMS m/z 440 (M , 0.3%); HRMS (EI) m/z calcd
1
the synthesis of 36 from 29 is described. Bromohydrin 29 (45.9 mg,
for C H O 440.40181; found 440.40206; H NMR (400 MHz,
31
52
0
.079 mmol) was dissolved in Et O (20 mL). The three-necked flask
C D ) δ 1.07 (t, J = 7.2 Hz, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.67 (s,
6 6 H
2
was cooled to −40 °C and was filled with N gas. To the solution was
3H), 1.73 (s, 3H), 1.81 (s, 3H), 2.34−2.10 (m, 22H), 2.70 (t, J = 6.0
13
2
added dropwise DIBAL-H reagent (1.03 M in hexane, 550 μL). The
Hz, 1H), 5.45−5.28 (m, 5H); C NMR (150 MHz, C D ) δ 13.0
6
6
C
reaction was monitored with SiO TLC. After we confirmed that the
(q), 16.0 (q), 16.12 (q), 16.14 (q, 2C), 18.9 (q), 23.0 (q), 24.9 (q),
25.1 (t), 26.8 (t), 27.07 (t), 27.10 (t), 28.0 (t), 28.7 (t, 2C), 36.8 (t),
40.1 (t), 40.2 (t), 40.5 (t), 57.3 (s), 63.5 (d), 124.6 (d), 124.8 (d, 2C),
124.9 (d), 125.1 (d), 134.4 (s), 134.9 (s), 135.0 (s), 135.2 (s), 136.9
(s).
2
starting material 29 disappeared, the reaction was terminated by
adding 10 mL of a mixture of EtOAc and H O and further stirred for 2
2
h, and then 10 mL of saturated aq NH Cl was added and left
4
overnight. The product was extracted with hexane, and the organic
layer was dried over Na SO . Pure 36 was isolated by a SiO column
Preparations of 12−16. The synthetic methods of these epoxide
compounds from the bromohydrin precursors were identical. The
experiment for the synthesis of only 15 is described here. The starting
material 38 (12.0 mg, 0.023 mmol) was dissolved in a small amount of
MeOH. This solution was added into the methanolic suspension (15
mg/10 mL) of K CO and stirred for 30 min. The reaction mixture
2
4
2
chromatography with hexane/EtOAc (100:30) in a yield of 32.2 mg
75.6%).
Compound 36: H NMR (400 MHz, CDCl ) δ 1.03 (t, J = 7.2
(
1
3
H
Hz, 3H), 1.32 (s, 3H), 1.34 (s, 3H), 1.60 (s, 12H), 1.78 (m, 1H),
2
.17−1.93 (m, 20H), 2.31 (m, 1H), 3.98 (br d, J = 11.2 Hz, 1H), 4.12
2
3
(
s, 2H), 5.21−5.07 (m, 4H), 5.28 (t, J = 7.2 Hz, 1H).
was added into saturated aq NaCl solution, and the lipophilic materials
were extracted with hexane (3×). Product 15 was isolated by a SiO2
column chromatography (hexane/EtOAc = 100:5) in a yield of 8.9 mg
(87.4% yield).
1
Compound 37: H NMR (400 MHz, CDCl ) δ 0.997 (t, J = 7.2
3
H
Hz, 3H), 1.32 (s, 3H), 1.34 (s, 3H), 1.60 (s, 12H), 1.78 (m, 1H),
2
.17−1.93 (m, 20H), 2.31 (m, 1H), 3.97 (br d, J = 11.2 Hz, 1H), 4.03
+
(
s, 2H), 5.22−5.08 (m, 4H), 5.36 (t, J = 7.2 Hz, 1H).
Compound 12: EIMS m/z 426 (M , 0.6%); HRMS (EI) m/z calcd
1
1
Compound 38: H NMR (400 MHz, CDCl ) δ 1.32 (s, 3H), 1.34
for C H O 426.38617; found 426.38723; H NMR (400 MHz,
3
H
30 50
(
1
5
s, 3H), 1.60 (s, 12H), 1.66 (s, 3H), 1.78 (m, 1H), 2.15−1.94 (m,
C D ) δ 1.08 (t, J = 7.2 Hz, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.67 (s,
6
6
H
8H), 2.30 (m, 1H), 3.97 (br d, J = 11.0 Hz, 1H), 3.98 (s, 2H), 5.22−
.08 (m, 4H), 5.38 (t, J = 7.0 Hz, 1H).
3H), 1.71 (s, 3H), 1.74 (s, 6H), 2.35−2.08 (m, 22H), 2.69 (t, J = 6.0
13
Hz, 1H), 5.45−5.35 (m, 4H), 5.58 (m, 2H); C NMR (100 MHz,
L
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
C D ) δ 14.2 (q), 16.0 (q), 16.1 (q), 16.2 (q, 2C), 18.9 (q), 25.0 (q),
be exchangeable because the chemical shifts are very similar: MS (EI)
m/z 203 (75%), 217 (22%), 232 (100%), 411 (5%), 440 (5%); [M ];
6
6
C
+
2
4
1
6.0 (t), 27.1 (t, 2C), 28.0 (t), 28.7 (t, 2C), 31.7 (t), 36.9 (t),40.1 (t),
0.2 (t, 2C), 57.3 (s), 63.5 (d), 124.82 (d), 124.84 (d), 124.91 (d),
25.12 (d), 129.3 (d), 132.2 (d), 134.3 (s), 134.8 (s), 135.1 (s), 135.2
HRMS (EI) m/z calcd for C H O 440.40181; found 440.40267;
31
52
[α]D25 = +97.0 (c = 0.115, CHCl ); solid, mp 191−193 °C.
3
1
(
s).
Compound 13: EIMS m/z 426 (M , 0.4%); HRMS (EI) m/z calcd
Product 43: H NMR (400 MHz, CDCl ) δ 0.740 (br d, J = 12.0
3
+
Hz, H-5), 0.792 (s, 3H, Me-24), 0.806 (t, J = 7.6 Hz, 3H, Me-31), 0.82
(m, 1H, H-16), 0.827 (s, 3H, Me-30), 0.834 (s, 3H, Me-28), 0.939 (s,
3H, Me-25), 0.95 (m, 1H, H-15), 0.97 (m, 1H, H-1), 0.970 (s, 3H,
Me-26), 0.998 (s, 3H, Me-23), 1.00 (m, 1H, H-19), 1.10 (m, 1H, H-
21), 1.13 (s, 3H, Me-27), 1.17 (q, J = 7.6 Hz, 2H, H-29), 1.24 (m, 1H,
H-22), 1.28 (m, 1H, H-21), 1.34 (m, 1H, H-7), 1.41 (m, 1H, H-6),
1.42 (m, 1H, H-22), 1.50 (m, 1H, H-15), 1.51 (m, 1H, H-7), 1.54 (m,
1H, H-6), 1.56 (m, 1H, H-9), 1.60 (m, 2H, H-2), 1.62 (m, 2H, H-1,
H-19), 1.62 (m, 1H, H-19), 1.87 (m, 2H, H-11), 1.97 (m, 1H, H-18),
1.98 (ddd, J = 12.8, 12.8, 4.4 Hz, 1H, H-16), 3.22 (dd, J = 11.2, 4.4 Hz,
1
for C H O 426.38617; found 426.38625; H NMR (400 MHz,
30
50
C D ) δ 1.05 (t, J = 7.6 Hz, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.67 (s,
6
6
H
3
H), 1.71 (s, 3H), 1.73 (s, 6H), 2.34−2.10 (m, 22H), 2.69 (t, J = 6.0
13
Hz, 1H), 5.45−5.34 (m, 4H), 5.56 (t-like, J = 6.0 Hz, 2H); C NMR
(
100 MHz, C D ) δ 14.6 (q), 16.0 (q, 2C), 16.1 (q, 2C), 18.9 (q),
6 6 C
2
0.9 (t), 25.0 (q), 27.1 (t, 2C), 28.0 (t), 28.7 (t, 2C), 36.9 (t), 40.09
(
(
t), 40.13 (t), 40.2 (t), 57.3 (s), 63.5 (d), 124.8 (d), 124.9 (d), 125.0
d), 125.1 (d), 129.1 (d), 131.8 (d), 134.3 (s), 134.7 (s), 135.1 (s),
1
35.15 (s).
Compound 14: EIMS m/z 440 (M , 0.4%); HRMS (EI) m/z calcd
+
13
1H, H-3), 5.18 (t, J = 3.6 Hz, 1H, H-12); C NMR (150 MHz,
1
for C H O 440.40182; found 440.40313; H NMR (400 MHz,
CDCl ) δ 7.67 (q, C-31), 15.5 (q, C-25), 15.6 (q, C-24), 16.8 (q, C-
31
52
3
C D ) δ 0.989 (t, J = 7.2 Hz, 3H), 1.21 (s, 3H), 1.26 (s, 3H), 1.45 (q,
26), 18.4 (t, C-6), 20.6 (q, C-30), 23.5 (t, C-11), 26.0 (q, C-27), 26.2
(t, C-15), 27.0 (t, C-16), 27.2 (t, C-2), 28.1 (q, C-23), 28.4 (q, C-28),
32.4 (t, C-21), 32.7 (t, C-7), 32.9 (s, C-17), 33.6 (s, C-20), 36.8 (t, C-
22), 36.9 (s, C10), 38.5 (t, C-29), 38.6 (t, C-1), 38.8 (s, C-4), 39.8 (s,
C-8), 41.7 (s, C-14), 44.7 (t, C-19), 46.9 (d, C-18), 47.6 (d, C-9), 55.2
(d, C-5), 79.0 (d, C-3), 121.7 (d, C-12), 145.3 (d, C-13). Assignment
of C-8 and C-14 and that of C-24 and C-25 may be exchangeable
because their chemical shifts are very similar: MS (EI) m/z 203 (48%),
6
6
H
J = 7.2 Hz, 2H), 1.65 (s, 3H), 1.70 (s, 3H), 1.72 (s, 6H), 2.35−2.08
m, 22H), 2.67 (t, J = 6.0 Hz, 1H), 5.44−5.32 (m, 4H), 5.60−5.50 (m,
H); 13C NMR (100 MHz, C D ) δ 13.9 (q), 16.1 (q), 16.2 (q, 3C),
(
2
1
2
1
6
6
C
8.9 (q), 23.2 (t), 25.0 (q), 26.3 (t), 27.1 (t, 2C), 28.0 (t), 28.7 (q,
C), 29.7 (t), 36.9 (t), 40.1 (t), 40.13 (t), 40.19 (t), 57.3 (s), 63.5 (d),
24.85 (d), 124.92 (d), 124.99 (d), 125.14 (d), 129.9 (d, 2C), 134.4
(
s), 134.7 (s), 135.1 (s), 135.2 (s).
Compound 15: EIMS m/z 424 (M+ − H O, 0.8%), 442 (M ,
+
217 (26%), 232 (100%), 411 (5%), 440 (M , 2%); [M ]; HRMS (EI)
+
+
2
2
5
0
4
1
2
5
1
(
1
(
.2%); HRMS (EI) m/z calcd for C H O 442.38018; found
40.38216; H NMR (400 MHz, C D ) δ 1.23 (s, 3H), 1.28 (s, 3H),
m/z calcd for C H O 440.40181; found 440.40218; [α] = +96.25
3
0
50
2
31 52 D
1
(c = 0.2312, CHCl ); solid, mp: 190−195 °C.
6
6
H
3
1
.67 (s, 3H), 1.69 (s, 3H), 1.72 (s, 3H), 1.737 (s, 3H), 1.743 (s, 3H),
.35−2.13 (m, 20H), 2.70 (t, J = 6.0 Hz), 3.93 (s, 2H), 5.53−5.35 (m,
H); 13C NMR (100 MHz, C D ) δ 13.6 (q), 16.05 (q), 16.09 (q),
6.16 (q, 2C), 18.9 (q), 24.9 (q), 26.7 (t), 27.1 (t, 2C), 28.0 (t) 28.7
t, 2C), 36.8 (t), 39.9 (t), 40.1 (t), 40.2 (t), 57.4 (s), 63.6 (d), 68.7 (t),
24.88 (d), 124.92, 124.94 (d), 125.2 (d), 125.3 (d), 134.8 (s), 135.1
s), 135.2 (s), 135.4 (s), 135.5 (s).
Compound 16: EIMS m/z 424 (M − H O, 1%), 442 (M , 0.1%);
Product 44 Acetate: H NMR (600 MHz, C D ) δ 0.881 (br d, J =
6
6
10.8 Hz, 1H, H-5), 0.94 (m, 1H, H-1), 0.969 (s, 3H, Me-25), 1.00 (m,
H-16), 1.05 (s, 6H, Me-23, Me-24), 1.05 (t, J = 7.2 Hz, 3H, Me-30),
1.08 (s, 3H, Me-28), 1.12 (m, H-15), 1.13 (s, 3H, Me-26), 1.33 (s, 3H,
Me-27), 1.33 (m, 1H, H-22), 1.42 (m, 2H, H-6, H-7), 1.50 (m, 2H, H-
1, H-29), 1.55 (m, 1H, H-21), 1.57 (m, 2H, H-6, H-19), 1.58 (m, 2H,
H-7, H-22), 1.61 (m, 1H, H-29), 1.64 (m, 1H, H-9), 1.71 (m, 2H, H-2,
6
6
C
+
+
H-20), 1.82 (m, 2H, H-2, H-21), 1.88 (s, 3H, CH CO), 1.89 (m, 2H,
2
3
1
HRMS (EI) m/z calcd for C H O 442.38018; found 440.38205; H
H-11), 1.93 (m, 1H, H-15), 2.17 (m, 1H, H-19), 2.18 (m, 1H, H-18),
2.24 (m, 1H, H-16), 4.84 (dd, J = 12.0, 4.8 Hz, 1H, H-3), 5.36 (t, J =
3.4 Hz, 1H, H-12); 13C NMR (150 MHz, C D ) δ 12.6 (q, C-30), 15.7
3
0
50
2
NMR (400 MHz, C D ) δ 1.23 (s, 3H), 1.27 (s, 3H), 1.67 (s, 3H),
6
6
H
1
=
.69 (s, 3H), 1.74 (s, 6H), 1.90 (s, 3H), 2.33−2.10 (m, 20H), 2.69 (t, J
6
6
13
6.0 Hz, 1H), 4.09 (s, 2H), 5.40−5.33 (m, 5H); C NMR (100
(q, C-25), 17.0 (q, 2C, C-24, C-26), 18.6 (t, C-6), 20.8 (q, CH CO),
3
MHz, C D ) δ 16.05 (q), 16.08 (q), 16.14 (q, 2C), 18.9 (q), 21.4 (q),
23.6 (t, C-29), 23.8 (t, C-2), 23.9 (t, C-11), 25.5 (t, C-21), 26.2 (q, C-
27), 26.6 (t, C-15), 27.6 (t, C-16), 28.2 (q, C-23), 28.8 (q, C-28), 32.9
(t, C-7), 33.2 (s, C-17), 35.8 (t, C-22), 35.9 (d, C-20), 37.0 (s, C-10),
37.3 (t, C-19), 37.9 (s, C-4), 38.4 (t, C-1), 40.1 (s, C-8), 42.0 (s, C-
14), 45.8 (d, C-18), 47.8 (d, C-9), 55.6 (d, C-5), 80.6 (d, C-3), 122.1
6
6
C
2
4.94 (q), 26.5 (t), 27.1 (t, 2C), 28.0 (t), 28.7 (t, 2C), 36.8 (t), 40.1
(
(
(
t), 40.2 (t), 40.3 (t), 57.4 (s), 61.4 (t), 63.6 (d), 124.89 (d), 124.92
d), 125.15 (d, 2C), 127.3 (d), 134.4 (s), 134.7 (s), 135.1 (s), 135.14
s), 135.36 (s).
Incubation Experiments of Analogues 10−16. The purifica-
(d, C-12), 145.4 (s, C-13), 169.9 (s, CH CO); MS (EI) m/z 189
3
+
+
tion of EtAS and the incubation conditions were carried out according
(100%), 203 (22%), 218 (84%), 468 (M , 5%); [M ]; HRMS (EI) m/
9
25
D
to the published protocol. The detailed incubation conditions and
z calcd for C H O 468.39673; found 468.39496; [α] = +88.3 (c
32
52
2
GC conditions for the product profiles are described in Figure S3.
= 0.0115, CHCl ); solid.
Product 45 Acetate: H NMR (400 MHz, C D ) δ 0.881 (br d, J =
6 6
3
1
1
Spectroscopic Data for Products 42−50. Product 42: H NMR
(
600 MHz, CDCl ) δ 0.730 (br d, J = 12.5 Hz, 1H, H-5), 0.764 (t, J =
11.6 Hz, 1H, H-5), 0.960 (m, H-1), 0.975 (s, 3H, Me-25), 1.02 (m,
1H, H-16), 1.045 (t, J = 7.0 Hz, 3H, Me30), 1.049 (s, 3H, Me-24),
1.10 (s, 6H, Me-23, Me-28), 1.12 (m, 1H, H-15), 1.14 (s, 3H, Me-26),
1.20 (m, 1H, H-20), 1.22 (m, 1H, H-21), 1.35 (s, 3H, Me-27), 1.38
(m, 1H, H-29), 1.41 (m, 1H, H-22), 1.42 (m, 2H, H-6, H-7), 1.47 (m,
1H, H-29), 1.52 (m, 1H, H-1), 1.57 (m, 1H, H-6), 1.58 (m, 1H, H-7),
1.64 (m, 2H, H-21, H-22), 1.67 (m, 1H, H-9), 1.69 (m, 1H, H-2), 1.73
3
7
0
0
.4 Hz, 3H, Me-31), 0.781 (s, 3H, Me-29), 0.789 (s, 3H, Me-24),
.819 (s, 3H, Me-28), 0.820 (m, 1H, H-16), 0.934 (s, 3H, Me-25),
.960 (m, 1H, H-1), 0.961 (s, 3H, Me-26), 0.970 (m, 1H, H-15), 0.995
(
s, 3H, Me-23), 1.11 (m, 1H, H-19), 1.14 (s, 3H, Me-27), 1.16 (m,
1
7
H, H-22), 1.22 (m, 2H, H-21), 1.28 (m, 1H, H-30), 1.33 (m, 1H, H-
), 1.35 (m, 1H, H-30), 1.38 (m, 1H, H-22), 1.39 (m, 1H, H-6), 1.50
(
m, 1H, H-7), 1.54 (m, 1H, H-9), 1.55 (m, 1H, H-6), 1.58 (m, 1H, H-
(m, 2H, H-19), 1.83 (m, 1H, H-2), 1.88 (s, 3H, CH CO), 1.92 (m,
3
1
1
1
5
9), 1.59 (m, 2H, H-2), 1.62 (m, 1H, H-1),1.76 (dd, J = 14.0, 4.5 Hz,
H, H-15), 1.85 (m, 2H, H-11), 1.89 (m, 1H, H-18), 2.01 (ddd, J =
3.0, 13.0, 4.5 Hz, 1H, H-16), 3.22 (dd, J = 11.4, 4.8 Hz, 1H, H-3),
2H, H-11), 1.95 (m, 1H, H-15), 2.05 (dd, J = 12.8, 3.6 Hz, 1H, H-18),
2.17 (ddd, J = 12.8, 12.8, 4.4 Hz, 1H, H-16), 4.83 (dd, J = 12.0, 4.4 Hz,
1H, H-3), 5.42 (br s, 1H, H-12); 13C NMR (100 MHz, C D ) δ 11.8
6
6
.17 (br s, 1H); 13C NMR (150 MHz, CDCl ) δ 7.75 (q, C-31), 15.4
(q, C-30), 15.7 (q, C-25), 17.1 (q, 2C, C-24, C-26), 18.5 (t, C-6), 20.8
3
(
2
q, C-24), 15.5 (q, C-25), 16.8 (q, C-26), 18.4 (t, C-6), 23.5 (t, C-11),
5.9 (q, C-27), 26.2 (t, C-15), 27.18 (t, C-30), 27.26 (t, C-2), 27.29 (t,
C-16), 28.1 (q, C-23), 28.4 (q, C-28), 29.0 (q, C-29), 32.3 (s, C-17),
2.7 (t, C-7), 32.8 (t, C-21), 33.3 (s, C-20), 36.6 (t, C-1), 37.0 (s, C-
0), 38.6 (t, C-22), 38.8 (s, C-4), 39.8 (s, C-8), 41.8 (s, C-14), 44.6 (t,
(q, CH CO), 23.8 (t, C-2), 23.9 (t, C-11), 26.2 (q, C-27), 26.7 (t, C-
3
15), 27.6 (t, C-16), 28.2 (q, C-23), 28.8 (q, C-28), 28.9 (t, C-21), 30.3
(t, C-29), 32.9 (t, C-7), 33.3 (s, C-17), 37.0 (s, C-10), 37.9 (s, C-4),
38.4 (t, C-1), 40.1 (s, C-8), 40.8 (d, C-20), 41.3 (t, C-19), 41.9 (t, C-
22), 42.0 (s, C-14), 47.8 (d, C-9), 52.1 (d, C-18), 55.5 (d, C-5), 80.5
3
1
C-19), 46.7 (d, C-18), 47.7 (d, C-9), 55.2 (d, C-5), 79.0 (d, C-3),
21.7 (d, C-12), 145.3 (s, C-13). Assignment of C-2 and C-16, that of
C-7 and C-21, that of C-8 and C-14, and that of C-24 and C-25 may
(d, C-3), 122.2 (d, C-12), 145.6 (s, C-13), 169.9 (s, CH CO). The
assignment of C-2 and C-11 and that of C-8 and C-14 may be
exchangeable between the two carbons: MS (EI) m/z 189 (100%),
3
1
M
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
+
+
+
+
2
03 (25%), 218 (82%), 468 (M , 4%); [M ]; HRMS (EI) m/z calcd
484 (M , 0.4%); [M ]; HRMS (EI) m/z calcd for C H O
32 52 3
25
25
for C H O 468.39673; found 468.39405; [α]
= +97.92 (c =
484.39165; found 484.39158; [α]D = +23.6 (c = 0.043, C D );
32
52
2
D
6 6
0
.0534, CHCl ); solid.
Product 46: H NMR (600 MHz, CDCl ) δ 0.742 (br d, J = 11.1
solid, mp 209−213 °C. 1
Product 49 Acetate: H NMR (400 MHz, C D ) δ 0.839 (dd, J =
3
1
3
6
6
Hz, 1H, H-5), 0.791 (s, 3H, Me-24), 0.806 (s, 3H, Me-28), 0.85 (m,
11.6, 2.0 Hz, 1H, H-5), 0.89 (m, 1H, H-1), 0.910 (s, 3H, Me-19),
1.032 (s, 3H, Me-30), 1.036 (s, 6H, Me-18, Me-28), 1.05 (s, 3H, Me-
29), 1.20 (m, 1H, H-11), 1.22 (m, 1H, H-15), 1.27 (s, 3H, Me-21),
1.30 (m, H-12), 1.34 (m, 2H, H-7, H-9), 1.47 (m, 1H, H-6), 1.50 (m,
1H, H-11), 1.56 (m, 1H, H-6), 1.58 (m, 1H, H-7), 1.59 (m, 1H, H-1),
1.61 (br s, 3H, Me-26), 1.65 (m, 1H, H-13), 1.76 (m, 1H, H-2), 1.82
1
0
1
H, H-16), 0.863 (t, J = 7.3 Hz, 3H, Me-31), 0.938 (s, 3H, Me-25),
.971 (s, 3H, Me-26), 0.98 (m, 2H, H-1, H-15), 0.997 (s, 3H, Me-23),
.02 (m, 1H, H-21), 1.13 (m, 1H, H-20), 1.134 (s, 3H, Me-27), 1.21
(
m, 1H, H-22), 1.30 (m, 2H, Me-30), 1.35 (m, 1H, H-7), 1.37 (m, 4H,
H-19, H-29), 1.40 (m, 1H, H-6), 1.43 (m, 1H, H-22), 1.44 (m, 1H, H-
1), 1.51 (m, 1H, H-8), 1.54 (m, 1H, H-6), 1.56 (m, 1H, H-9), 1.60
m, 2H, H-2), 1.63 (m, 1H, H-1), 1.77 (m, 1H, H-15), 1.79 (m, 1H,
2
(
(
m, 3H, H-12, H-15, H-22), 1.85 (m, 1H, H-2), 1.89 (s, 3H, CH CO),
3
1
.92 (m, 1H, H-22), 1.99 (m, 2H, H-16), 2.22 (m, 1H, H-23), 2.27
H-18), 1.88 (m, 2H, H-11), 1.96 (m, 1H, H-16), 3.22 (dd, J = 11.1, 4.1
(
(
m, 1H, H-17), 2.43 (m, 1H, H-23), 4.02 (d, J = 16.4, 1H, H-27), 4.24
Hz, 1H, H-3), 5.21 (br t, J = 3.3 Hz, 1H, H-12); 13C NMR (150 MHz,
d, J = 16.4 Hz, 1H, H-27), 4.83 (dd, J = 11.6, 4.8 Hz, 1H, H-3), 5.59
CDCl ) δ 14.4 (q, C-31), 15.5 (q, C-25), 15.6 (q, C-24), 16.9 (q, C-
13
3
(br s, 1H, H-24); C NMR (100 MHz, C D ) δ 15.6 (q, C-18), 16.4
6
6
2
(
2
(
6), 18.4 (t, C-6), 20.0 (t, C-30), 23.6 (t, C-11), 25.9 (q, C-27), 26.4
t, C-15), 27.29 (t, C-16), 27.33 (t, C-2), 28.1 (q, C-23), 28.5 (q, C-
8), 29.0 (t, C-21), 32.8 (t, C-7), 33.0 (s, C-17), 37.0 (s, C-10), 38.5
d, C-20), 38.7 (d, 2C, C-1, C-3), 38.8 (s, C-4), 39.7 (t, C-29), 39.9 (s,
C-8), 41.3 (t, C-19), 41.8 (s, C-14), 41.8 (t, C-22), 47.7 (d, C-9), 51.8
d, C-18), 55.3 (d, C-5), 121.7 (d, C-12), 145.6 (s, C-13). The
(q, C-19), 16.7 (q, C-30), 16.8 (q, C-29), 18.5 (t, C-6), 20.5 (q, C-21),
2
0.8 (q, CH CO), 21.8 (q, C-26), 21.8 (t, C-11), 23.9 (t, C-23), 24.1
3
(t, C-2), 25.0 (t, C-16), 27.5 (t, C-12), 28.1 (q, C-28), 31.6 (t, C-15),
3
2
5
3
5.5 (t, C-7), 37.2 (s, C-10), 38.1 (s, C-4), 38.8 (t, C-1), 40.3 (t, C-
2), 40.6 (s, C-8), 43.3 (d, C-13), 47.9 (d, C-17), 50.8 (d, 2C, C-9),
0.8 (s, C-14), 56.2 (d, C-5), 64.7 (t, C-27), 79.6 (s, C-20), 80.5 (d, C-
(
assignment of C-2 and C-16 and that of C-24 and C-25 may be
exchangeable: MS (EI) m/z 189 (20%), 203 (25%), 217 (21%), 232
), 125.3 (d, C-24), 136.9 (s, C-25), 169.9 (s, CH CO). The
3
assignments of C-29 and C-30 may be exchangeable: MS (EI) m/z
+
+
(
4
100%), 440 (M , 4%); [M ]; HRMS (EI) m/z calcd for C H O
+
+
3
1
52
1
25 (100%), 343 (8%), 484 (M , 0.4%); [M ]; HRMS (EI) m/z calcd
40.40181; found 440.40177; [α]D25 = +97.92 (c = 0.038, CHCl );
3
25
for C H O 484.39165; found 484.39160; [α] = +16.7 (c = 0.017,
32
52
3
D
solid.
C D ); solid.
1
6
6
Product 47: H NMR (400 MHz, CDCl ) δ 0.678 (br d, J = 11.0
1
3
Product 50 Acetate: H NMR (400 MHz, C D ) δ 0.776 (br d, J =
6
6
Hz, 1H, H-5), 0.762 (s, 3H, Me-24), 0.824 (s, 3H, Me-25), 0.91 (m,
1
1
0.8 Hz, H-5), 0.882 (s, 3H, Me-25), 0.92 (m, 1H, H-1), 0.921 (s, 3H,
H, Me-27), 1.01 (s, 3H, Me-23), 1.03 (s, 3H, Me-24), 1.05 (s, 3H,
1
H, H-15), 0.914 (s, 3H, Me-27), 0.92 (m, 1H, H-1), 0.941 (s, 3H,
Me-28), 0.970 (s, 3H, Me-23), 1.01 (s, 3H, Me-28), 1.05 (s, 3H, Me-
6), 1.13 (m, 1H, H-22), 1.21 (m, 1H, H-9), 1.28 (m, 1H, H-12), 1.30
m, 2H, H-7, H-16), 1.33 (m, 1H, H-11), 1.40 (m, 1H, H-6), 1.41 (m,
H, H-7, H-21), 1.45 (m, 1H, H-2), 1.46 (m, 2H, H-19, H-21), 1.54
m, 1H, H-6), 1.55 (m, 2H, H-11, H-16), 1.57 (m, 1H, H-2), 1.62 (m,
Me-26), 1.17 (m, 1H, H-12), 1.25 (s, 3H, Me-28), 1.32 (m, 1H, H-9),
.35 (m, 1H, H-11), 1.38 (m, 4H, H-7, H-15, 2H for each, and m, 1H
for H-6, total 5H), 1.39 (m, 2H, H-16), 1.50 (m, 1H, H-19), 1.51 (m,
H, H-6), 1.52 (m, 1H, H-11), 1.56 (s, 3H, Me-28), 1.65 (m, 2H, H-1,
H-19), 1.73 (m, 1H, H-2), 1.86 (m, 1H, H-2), 1.88 (s, CH CO), 1.96
2
(
2
(
1
1
3
1
2
H, H-19), 1.68 (m, 1H, H-12), 1.71 (m, 1H, H-1), 1.72 (m, 1H, H-
2), 1.73 (m, 1H, H-15), 2.12 (dd, J = 12.4, 2.8 Hz, 1H, H-13), 3.20
(m, 1H, H-13), 1.98 (m, 1H, H-20), 2.04 (m, 1H, H-20), 2.09 (m, 1H,
H-12), 3.20 (d, J = 10.4 Hz, 1H, H-18), 3.85 (d, J = 16.8 Hz, 1H, H-
(
dd, J = 11.2, 5.2 Hz, H-3), 3.28 (d, J = 7.6 Hz, 1H, H-30), 3.61 (d, J =
13
30), 4.47 (d, J = 16.8 Hz, 1H, H-30), 4.82 (dd, J = 11.6, 4.4 Hz, 1H, H-
7
1
2
.6 Hz, 1H, H-30); C NMR (100 MHz, CDCl ) δ 15.4 (q, C-24),
6.0 (q, C-25), 16.2 (q, 2C, C-26, C-27), 18.4 (t, C-6), 20.8 (t, C-11),
0.9 (t, C-12), 22.5 (q, C-28), 23.7 (q, C-29), 27.2 (t, C-15), 27.4 (t,
3
13
3
), 5.76 (t, J = 8.0 Hz, 1H, H-21); C NMR (100 MHz, C D ) δ 14.9
6
6
(q, C-27), 15.9 (q, C-26), 16.5 (q, C-25), 16.8 (q, C-24), 18.0 (q, C-
2
2
7
4
8), 18.4 (t, C-6), 20.8 (q, CH CO), 21.2 (t, C-20), 21.4 (q, C-29),
3
C-2), 28.0 (q, C-23), 32.6 (t, C-22), 33.3 (t, C-7), 35.5 (t, C-21), 36.2
t, C-16), 37.1 (s, C-10), 38.1 (d, C-13), 38.8 (t, C-1), 38.9 (s, C-4),
2.1 (t, C-11), 24.1 (t, C-2), 25.2 (t, C-12), 28.1 (q, C-23), 33.9 (t, C-
), 36.5 (t, 2C, C-15, C-16), 37.1 (s, C-10), 37.2 (s, C-17), 38.0 (s, C-
), 38.6 (t, C-1), 40.9 (s, C-8), 41.0 (d, C-13), 42.6 (s, C-14), 45.9 (t,
(
3
1
9.2 (s, C-17), 40.6 (s, C-20), 41.9 (t, C-19), 42.1 (s, C-8), 42.5 (s, C-
4), 50.6 (d, C-9), 55.3 (d, C-5), 76.0 (d, C-3), 79.0 (t, C-30), 88.2 (s,
C-19), 50.9 (d, C-9), 55.8 (d, C-5), 74.1 (t, C-30), 80.5(d, C-3), 84.5
d, C-18), 126.7 (d, C-21), 132.9 (s, C-22), 169.9 (s, CH CO); MS
C-18). The assignment of C-8 and C-14 may be exchangeable: MS
(
(
EI) m/z 189 (90%), 203 (68%), 220 (90%), 234 (38%), 411 (100%),
3
+
+
+
(EI) m/z 189 (20%), 402 (100%), 424 (0.9%), 469 (0.03%), 484 (M ,
0.02%); [M ]; HRMS (EI) m/z calcd for C H O 484.39165; found
4
4
42 (M , 18%); [M ]; HRMS (EI) m/z calcd for C H O
30 50 2
+
4
42.38108; found 442.38148; [α]D25 = +45.0 (c = 0.1428, CHCl );
32 52
3
3
25
84.39167; [α]D = +64.2 (c = 0.02, C D ); solid, mp 192−195 °C.
solid, mp 211−214 °C. 1
6
6
Product 48 Acetate: H NMR (400 MHz, C D ) δ 0.853 (dd, J =
6
6
1
1
1
1.6, 2.4 Hz, 1H, H-5), 0.90 (m, 1H, H-1), 0.904 (s, 3H, Me-19),
.040 (s, 3H, Me-28), 1.044 (br s, 3H, Me-30), 1.05 (br s, 3H, Me-29),
.09 (s, 3H, Me-18), 1.22 (m, 1H, H-16), 1.29 (s, 3H, Me-21), 1.35
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
■
*
S
(
m, 1H, H-11), 1.37 (m, 2H, H-7, H-9), 1.38 (m, 2H, H-15), 1.39 (m,
1
1
1
1
1
2
H, H-12), 1.46 (m, 1H, H-6), 1.55 (m, 1H, H-6), 1.60 (m, 1H, H-1),
.61 (br s, 3H, Me-26), 1.62 (m, 1H, H-7), 1.72 (m, 1H, H-22),
.73(m, 1H, H-2), 1.74 (m, 1H, H-11), 1.80 (m, 1H, H-16), 1.84 (m,
ACS Publications website at DOI: 10.1021/acs.joc.6b01313.
EIMS and NMR data of products 10−16, 22−39, and
42−50; product profiles (GC) for the enzymatic
reactions of 10−16 and for those for the mutants
targeting amino acid residues surrounding the E-ring
(PDF)
H, H-22), 1.85 (m, 1H, H-2), 1.88 (s, 3H, CH CO), 1.94 (m, 1H, H-
3
3), 2.17 (m, 1H, H-23), 2.25 (m, 1H, H-17), 2.34 (m, 1H, H-12),
.43 (m, 1H, H-23), 3.91 (d, J = 16.4 Hz, 1H, H-27), 4.24 (d, J = 16.4
Hz, 1H, H-27), 4.83 (dd, J = 11.2, 4.8 Hz, 1H, H-3), 5.57 (br s, 1H, H-
4); 1 C NMR (100 MHz, C D ) δ 15.8 (q, C-18), 16.4 (q, C-19),
3
2
6
6
1
6.6 (q, C-30), 16.8 (q, C-29), 18.5 (t, C-6), 19.1 (q, C-21), 20.8 (q,
3
CH CO), 21.2 (q, C-26), 21.8 (t, C-11), 23.3 (t, C-23), 24.1 (t, C-2),
AUTHOR INFORMATION
■
*
2
6.1 (t, C-16), 27.1 (t, C-12), 28.1 (q, C-28), 31.5 (t, C-15), 35.6 (t,
C-7), 37.2 (s, C-10), 38.1 (s, C-4), 38.8 (t, C-1), 39.7 (t, C-22), 40.7
s, C-8), 43.0 (d, C-13), 45.7 (d, C-17), 49.9 (s, C-14), 51.0 (d, C-9),
6.2 (d, C-5), 64.7 (t, C-27), 80.2 (s, C-20), 80.6 (d, C-3), 125.3 (d,
Corresponding Author
E-mail: hoshitsu@agr.niigata-u.ac.jp
(
5
Notes
C-24), 136.8 (s, C-25), 169.9 (s, CH CO). The assignments of C-29
3
and C-30 may be exchangeable: MS (EI) m/z 125 (100%), 343 (10%),
The authors declare no competing financial interest.
N
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
ACKNOWLEDGMENTS
■
This work was supported in part by Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science,
Nos. 25450150 (C) and 18380001(B).
REFERENCES
■
(
1) Eschenmoser, A.; Ruzicka, L.; Jeger, O.; Arigoni, D. Helv. Chim.
Acta 1955, 38, 1890−1904.
2) Wendt, K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R. Angew. Chem.,
(
Int. Ed. 2000, 39, 2812−2833.
(
(
(
(
3) Hoshino, T.; Sato, T. Chem. Commun. 2002, 2002, 291−301.
4) Yoder, R. A.; Johnston, J. N. Chem. Rev. 2005, 105, 4730−4756.
5) Abe, I. Nat. Prod. Rep. 2007, 24, 1311−1331.
6) Wu, T.-K.; Chang, C.-H.; Liu, Y.-T.; Wang, T.-T. Chem. Rec.
2
(
(
008, 8, 302−325.
7) Nes, W. D. Chem. Rev. 2011, 111, 6423−6451.
8) Xu, R.; Fazio, G. C.; Matsuda, S. P. T. Phytochemistry 2004, 65,
2
(
1
(
7
(
6
(
61−291.
9) Ito, R.; Masukawa, Y.; Hoshino, T. FEBS J. 2013, 280, 1267−
280.
10) Rees, H. H.; Mercer, E. I.; Goodwin, T. W. Biochem. J. 1966, 99,
26−734.
11) Rees, H. H.; Britton, G.; Goodwin, T. W. Biochem. J. 1968, 106,
59−665.
12) Barton, D. H. R.; Mellows, G.; Widdowson, D. A.; Wright, J. J. J.
Chem. Soc. C 1971, 1971, 1142−1148.
(
(
13) Suga, T.; Shishibori, T. Phytochemistry 1975, 14, 2411−2417.
14) Seo, S.; Tomita, Y.; Tori, K. J. Am. Chem. Soc. 1981, 103, 2075−
2
080.
(
15) Seo, S.; Yoshimura, Y.; Uomori, A.; Takeda, K.; Seto, H.;
Ebizuka, Y.; Sankawa, U. J. Am. Chem. Soc. 1988, 110, 1740−1745.
16) Hoshino, T.; Yamaguchi, Y.; Takahashi, K.; Ito, R. Org. Lett.
014, 16, 3548−3551.
17) Hoshino, T.; Miyahara, Y.; Hanaoka, M.; Takahashi, K.; Kaneko,
I. Chem. - Eur. J. 2015, 21, 15769−15784.
18) Budzikiewicz, H.; Wilson, J. M.; Djerassi, C. J. Am. Chem. Soc.
(
2
(
(
1
(
(
963, 85, 3688−3699.
19) Karliner, J.; Djerassi, C. J. Org. Chem. 1966, 31, 1945−1956.
20) Ito, R.; Hashimoto, I.; Masukawa, Y.; Hoshino, T. Chem. - Eur. J.
2
013, 19, 17150−17158.
21) Ito, R.; Masukawa, Y.; Nakada, C.; Amari, K.; Nakano, C.;
Hoshino, T. Org. Biomol. Chem. 2014, 12, 3836−3846.
22) Kushiro, T.; Shibuya, M.; Masuda, K.; Ebizuka, Y. J. Am. Chem.
Soc. 2000, 122, 6816−6824.
23) Narayanan, C. R.; Natu, A. A. J. Org. Chem. 1974, 39, 2639−
641.
24) Dehaen, W.; Mashentseva, A. A.; Seitembetov, T. S. Molecules
011, 16, 2443−2466.
(
(
(
2
(
2
O
DOI: 10.1021/acs.joc.6b01313
J. Org. Chem. XXXX, XXX, XXX−XXX