Isolation and asymmetric total synthesis of fungal secondary metabolite hygrophorone B12

Article abstract of DOI:10.1002/ejoc.201403455

Hygrophorone B¹², a new antifungal constituent from the fruiting bodies of Hygrophorus abieticola, has been isolated and subsquently synthesized in enantiomerically pure form. The total synthesis includes a Sharpless asymmetric dihydroxylation

Full text of DOI:10.1002/ejoc.201403455

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FULL PAPER
DOI: 10.1002/ejoc.201403455
Isolation and Asymmetric Total Synthesis of Fungal Secondary Metabolite
1
2
Hygrophorone B
[
a]
[a]
[a]
[b]
[a]
Eileen Bette, Alexander Otto, Tobias Dräger, Kurt Merzweiler, Norbert Arnold,*
Ludger Wessjohann,*[ and Bernhard Westermann*
a,c]
[a,c]
Dedicated to Professor Dr. Dr. h. c. W. Steglich on the occasion of his 80th birthday
Keywords: Total synthesis / Asymmetric synthesis / Natural products / Fungal constituents / Configuration determination /
Structure elucidation
Hygrophorone B12, a new antifungal constituent from the
fruiting bodies of Hygrophorus abieticola, has been isolated
and subsquently synthesized in enantiomerically pure form.
The total synthesis includes a Sharpless asymmetric di-
hydroxylation protocol as the stereodifferentiating step,
followed by two diastereoselective aldol-type reactions. The
approach allows the unambiguous control of all three
stereogenic centres, and, furthermore, unequivocal determi-
nation of the relative and absolute configuration of antibiotic
hygrophorones B for the first time.
are rarely infected by parasitic fungi.[1,2] These ecological
observations triggered the exploration of a molecular basis
Introduction
Field observations by curious, woodcreeping natural for this phenomenon. Petroleum ether extracts of the fruit-
product chemists suggested that fruiting bodies of the genus ing bodies of H. persoonii and H. olivaceoalbus, both of
Hygrophorus (German: Schnecklinge, English: wax caps) which are characterized by a very slimy pileus surface,
Figure 1. Structures of isolated natural products and related natural antibiotics.
[
[
[
a] Leibniz Institute of Plant Biochemistry, Dept. of Bioorganic
Chemistry,
revealed a series of structurally related constituents. These
included new trihydroxy-functionalized cyclopentenones,
termed hygrophorones An (1) and hygrophorones Bn (2;
Weinberg 3, 06120 Halle, Germany
b] Institute of Inorganic Chemistry, Martin Luther University
Halle-Wittenberg,
Kurt-Mothes-Straße 2, 06120 Halle, Germany
c] Institute of Organic Chemistry, Martin Luther University
Halle-Wittenberg,
Figure 1), in which n denotes the length of the side-chain
[2,3]
connected to C-6.
The endocyclic diol has a trans
Kurt-Mothes-Straße 2, 06120 Halle, Germany
E-mail: bernhard.westermann@ipb-halle.de
http://www.ipb-halle.de/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403455.
relationship in hygrophorones A, and a cis relationship in
[2,4]
hygrophorones B.
The different aliphatic side-chains (n)
found for both diastereomers appear to be related to dif-
ferent fatty acids 5a–5e as biosynthetic precursors, which
Eur. J. Org. Chem. 0000, 0–0
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N. Arnold, L. Wessjohann, B. Westermann et al.
FULL PAPER
can also be isolated from Hygrophorus fruiting bodies.[5] Al- Synthesis and Stereochemical Assignment of Hygrophorone
1
2
though the biosynthesis has not yet been clarified, it seems
to be most likely that fatty acids 5a–5e are connected to the
synthesis of the hygrophorones. Very recently, Stratakis et
al. discussed the synthesis of hygrophorones[ as a product
of a singlet-oxygen-mediated oxidation of furans,[ which
may be formed as condensation products of these fatty
acids in vivo.
B
(2)
The very compact functionalization at the cyclo-
pentenone moiety in 2 sets some synthetic challenges. The
structural elements make up a 1,2,3-trihydroxy unit, with
two of the hydroxy groups at tertiary centres, and the other
one at a quaternary centre. The relative configuration of
the two hydroxy groups attached to the ring is known from
structure elucidation of isolated natural products, as de-
scribed above. But the overall relative configuration in-
cluding the exocyclic hydroxy group is not known, and nor
is the absolute configuration of the three stereogenic
centres.
Cyclopentenones 1 and 2 have a pronounced lability un-
der certain conditions: the quaternary centre α to the carb-
onyl functionality supports retro-Dieckmann type reactions.
This must be considered when planning the synthesis.
Therefore, in our retrosynthetic approaches, the formation
of the cyclopentenone was set to occur at a very late stage
6]
7]
The cyclopentenone moieties and the endocyclic diol
fragment of the hygrophorones closely resemble the natural
[
2]
products (–)-epipentenomycin (3) and (–)-pentenomycin
8]
(
4).[ But the addition of the lipophilic side-chain in the
hygrophorones makes the molecules amphiphilic, and cre-
ates an additional stereogenic centre, which is significant for
their pronounced biological activities, especially their fungi-
[
5]
cidal activities.
The biological profile, the densely functionalized head-
group, and the ill-defined relative and unknown absolute
configuration of these natural products prompted us to
undertake an asymmetric total synthesis.
[
9]
by cis-elimination from A (Scheme 1). For the construc-
tion of carbocycle A, two consecutive aldol-type reactions
(starting from C and D) can be considered. The second,
intramolecular, aldol reaction (from intermediate B) would
be prompted by a functional group conversion.
Results and Discussion
This retrosynthetic analysis suggests the following se-
quence in the forward sense: for a partial formation of the
Isolation of Hygrophorone B12
1,2,3-trihydroxylation pattern and to define the absolute
Frozen fruiting bodies of H. abieticola were extracted configuration of the final product early, a Sharpless asym-
with ethyl acetate. The slightly yellow crude extract was metric dihydroxylation (AD) protocol was envisioned as the
purified by adsorption chromatography (silica gel) and size- starting point (Scheme 1). Readily available trans-olefinic
exclusion chromatography (Sephadex LH 20). The final pu- α,β-unsaturated carboxylic esters serve as starting materials,
rification was carried out by preparative HPLC to give pure and the absolute stereochemistry of the two newly gener-
1
2
hygrophorone B (2) as a colourless powder.
ated stereocentres in C is controlled by the use of the chiral
The molecular formula was determined by HRMS [FT- ligands (DHQ) -PHAL (hydroquinine 1,4-phthalazinediyl
2
ICR (ion-cyclotron resonance); negative ion mode] to be diether; AD-mix-α) or (DHQD) -PHAL (hydroquinidine
2
–
1
13
[10]
C H O : m/z = 311.2224 ([M – H] ). The H and
C
1,4-phthalazinediyl diether; AD-mix-β). The first of two
1
8
32
4
NMR spectra of 2 are very similar to spectra of hygro- subsequent aldol-type reactions establishes the 1,2,3-tri-
[2]
phorones of the B series isolated earlier. In contrast to hydroxy pattern under regime of self-regenerating
1
4
16
[11]
known hygrophorones B and B , the alkyl side-chain of stereogenic centres.
was determined to be –C H , which was further con- cyclization, is responsible for the formation of the cyclo-
The second one, a Dieckmann-type
2
1
2
25
1
1
firmed by mass spectrometrical data. Also, H, H-COSY, pentanone scaffold. Overall, it is the exocyclic stereocentre
H, C-HMBC, and NOE correlations of the cyclic moiety at C-1 of the final product (β position in C) that controls
1
13
β
of 2 are consistent with those of other hygrophorones B of the stereochemical outcome of the asymmetric synthesis of
the homologous series. Therefore, the structure of 2 could 2.
be assigned as (+)-cis-4,5-dihydroxy-5-(1-hydroxytridecyl)-
Following this plan, the asymmetric total synthesis of
cyclopent-2-enone, and the compound was named hygro- hygrophorone B12 (Scheme 2) was begun with the synthesis
1
2
phorone B .
of trans-α,β-unsaturated fatty acid esters 6. To establish the
Scheme 1. Retrosynthetic analysis of 2.
2
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Fungal Secondary Metabolite Hygrophorone B12
absolute configuration, the double bond was transformed methods revealed an optical purity of Ͼ99% ee; no trace
into its dihydroxy congener by Sharpless asymmetric di- of the other enantiomer could be detected in either series,
[
12]
hydroxylation.
–)-7 and (2S,3R)-(+)-7] were formed in high yields (98 and only the synthesis of the AD-mix-α-derived series starting
5%, respectively), and with high stereoselectivities. HPLC from (–)-7 is described in this paper; (+)-7 is redundant
The dihydroxylated esters [i.e., (2R,3S)- starting either with AD-mix-α or AD-mix-β. For brevity,
(
9
Scheme 2. Asymmetric total synthesis of hygrophorones. TFA = trifluoroacetic acid.
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N. Arnold, L. Wessjohann, B. Westermann et al.
FULL PAPER
(
Scheme 2). To have high diastereoselectivities in subse- portantly, an elimination was made possible.[14] In practical
quent aldol-type reactions, transformation/protection of the terms, the oxidation of sulfides 10 and 11 was carried out
acyclic diol into a cyclic acetal was thought to be a pre- with sodium periodate to give sulfoxides 12 and 13 in yields
requisite. Acetalization to 8 was carried out with diethoxy- of 93 and 71%, respectively. Subsequently, a Dieckmann-
propane in order to avoid any transesterified products, and type cyclization of each of the sulfoxides was carried out in
this led to the cyclic product in 94% yield. For the aldol presence of LDA, which led to diastereomeric mixtures of
[
13]
reaction, thiophenol-derived aldehyde 9 was used.
The spirocyclic compounds 14 (66%) and 15 (67%). In both
formation of the ester enolate from 8 by treatment with cases, a 1:1 diastereomeric composition was obtained, with
2
LDA (lithium diisopropylamide) led to a prochiral sp cen- the sulfoxide equally cis and trans oriented, as determined
tre at the α position. The remaining stereocentre at the β by NMR spectroscopy. Although the diastereomers could
position controls the diastereoselectivity of the aldol reac- be separated by column chromatography for analysis, the
tion with aldehyde 9. The aldol reaction was diastereoselec- diastereomeric mixtures could be used for the next step,
tive, leading to 10 and 11 in a 4:1 ratio in an overall yield since the sulfoxide moiety is eliminated, the stereocentre is
of 80%. The aldol products 10 and 11 could be separated destroyed, and both diastereomers give the desired product.
by column chromatography. The three hydroxy groups in This elimination proceeded as expected in toluene under
the major product (i.e., 10) had an α,β-anti, α,βЈ-syn config- reflux to give cyclopentenones 16 (81%) and 17 (73%). Fi-
uration, and the hydroxy groups in the minor diastereomer nally, cleavage of the acetals with trifluoroacetic acid led to
(i.e., 11) had an α,β-anti, α,βЈ-anti conformation. Support hygrophorones (–)-2 (43%) and (+)-1 (64%), respectively.
for this stereochemical assignment is provided by an X-ray The deprotection must be carried out under solvent-free
structure of a closely related intermediate with a relative conditions under ice cooling, because higher temperatures
configuration identical to that of the major diastereomer or the use of solvents (e.g., methanol) led to decomposition
(see Supporting Information). Only two of four possible and side-reactions.
diastereomers are formed in the aldol reaction, both of NMR spectroscopic analysis revealed a trans relationship
which originate from attack of the Re face of the (E)-config- for the endocyclic dihydroxy groups of stereoisomer (+)-1,
ured ester enolate onto aldehyde 9. A Zimmerman–Traxler and a cis relationship for the same moieties in (–)-2. Data
transition state predicts that the formation of diastereomer obtained in earlier isolation studies strongly confirm this
[
2]
1
0 is favoured over 11, which is consistent with the experi- result. Further evidence for the relative configuration of
mental results. the hygrophorones comes from X-ray studies. Since no
For the following steps, the sulfide moiety had to be crystals could be obtained from compound (–)-2, acetyl de-
changed from a protecting group to a reactive group. This rivatives were synthesized (Scheme 3). Therefore, cyclo-
was achieved by altering the oxidation state to sulfoxide (12 pentenone 16 was acetylated with acetic anhydride and pyr-
and 13), which enabled this newly transformed functional idine, and the resulting acetonide (i.e., 18) was deprotected
group to act in two ways: the latent CH-acidity of the with trifluoroacetic acid to give acetate 19 and its acetate-
neighbouring methylene moiety was unveiled, and most im- migration isomer 20 as a regioisomeric mixture in a 2:1 ra-
Scheme 3. Synthesis of acetylated derivatives of (–)-2.
Figure 2. Molecular structure of acetylated hygrophorone 19 from X-ray crystallography.
4
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Fungal Secondary Metabolite Hygrophorone B12
1
12
Figure 3. Comparison of H NMR spectra and optical rotation values of isolated hygrophorone B (spectrum a) with synthetic hygro-
phorones (+)-2 (b) and (–)-2 (c).
tio. The regioisomers were separated by preparative HPLC, phorone B12 is identical to that of synthetic hygrophorone
and crystals of 19 suitable for X-ray diffraction analysis (+)-2, which is (4S,5S,6R) configured.
were obtained from a solution of the compound in ethyl
acetate overlayed with n-pentane. The crystal structure of
Conclusions
acetylated hygrophorone 19 shows the relative alignment of
the three hydroxy groups (Figure 2). As deduced from
NMR spectroscopic studies, the two endocyclic hydroxy
groups have a cis relationship, whereas the exocyclic
hydroxy group at C-6 has an anti configuration relative to
hydroxy group at C-5. Because the configuration of carbon
atom C-6, which carries the exocyclic hydroxy group being
known from the Sharpless dihydroxylation, the absolute
configuration of acetylated hygrophorone 19 can be deter-
mined to be (4R,5R,6S) based on the crystal structure.
Thus, it can be concluded that the synthesized hygro-
phorone (–)-2 has a (4R,5R,6S) configuration, and its
enantiomer (+)-2 is configured (4S,5S,6R).
Hygrophorone B12 was isolated, and its total synthesis
was accomplished in nine steps with an overall yield of
12%. The formation of all of the stereocentres could be
controlled by the stereogenic centre at the β position of
starting dihydroxy carboxylate (i.e., 8). This allowed us to
determine the relative and absolute configurations of hygro-
phorones B for the first time. The approach presented in
this paper offers several other opportunities. The stereodif-
ferentiating step can be carried out with AD-mix-α, leading
to (–)-2, or AD-mix-β, leading to the corresponding
enantiomer (+)-2. This offers the possibility of accessing
both stereoisomers for biological studies. Diastereomers are
also formed, and the use of different aldol protocols (open
shell) may allow the synthesis of the trans-configured di-
hydroxy congeners as the major diastereomers.
For the elucidation of the relative configuration of the
1
2
isolated hygrophorone B , the NMR spectra of the syn-
thetic hygrophorones were compared to those of the natural
1
product (Figure 3). This comparison revealed that H and
1
3
1
1
C chemical shifts as well as H, H coupling constants of
synthetic (+)-2 and (–)-2 are in good agreement with those
Experimental Section
1
2
of natural hygrophorone B . Nevertheless, for an unambig-
General Information: Unless otherwise stated, all chemicals and sol-
uous determination of the absolute configuration of hygro-
vents were obtained commercially and were used without further
1
2
phorone B , a comparison of the optical rotation values
was necessary. The optical rotation value obtained for (+)-
1
13
purification. H and C NMR spectra were recorded with a Varian
1
13
Mercury-VX 400 system at 400 MHz ( H) and 100 MHz ( C). 2D
NMR spectra (HSQC, HMBC, COSY, NOESY) were obtained
2
is in the same range as the corresponding value of the
isolated natural product, whereas (–)-2 shows an opposite
optical rotation (see also Figure 3). Therefore, it can be con- 150 MHz ( C). Chemical shifts are reported as δ values (ppm),
1
with an Agilent VNMRS 600 system at 600 MHz ( H) and
1
3
cluded that the absolute configuration of natural hygro- and are referenced to tetramethylsilane as an internal standard (δ
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N. Arnold, L. Wessjohann, B. Westermann et al.
FULL PAPER
1
(δ = 77.0 ppm, 13C). High-resolution ESI
=
0 ppm, H) or CDCl
3
and stirring was continued at 0 °C for 20 h until TLC indicated
that the reaction was complete. The reaction was quenched with
saturated aq. sodium sulfite (100 mL), and the mixture was stirred
for a further 30 min and then extracted with dichloromethane (5ϫ
200 mL). The combined organic extracts were dried with sodium
sulfate, filtered, and evaporated to dryness. The residue was puri-
fied by column chromatography (petroleum ether/ethyl acetate, 1:1)
to give (–)-7 (6.82 g, 98%) as a colourless solid, m.p. 71 °C. TLC
mass spectra were obtained with a Bruker Apex III Fourier trans-
form ion-cyclotron resonance (FT-ICR) mass spectrometer (Bruker
Daltonics, Billerica, USA) equipped with an Infinity™ cell, a
7.0 Tesla superconducting magnet (Bruker, Karlsruhe, Germany),
an RF-only hexapole ion guide, and an external electrospray ion
source (Agilent, off axis spray). The sample solutions were intro-
duced continuously using a syringe pump with a flow rate of
2
1
120 μL/h. Melting points were determined with a Leica DM LS2
(petroleum ether/ethyl acetate, 2:1): R
f
= 0.67. [α]
1.41, MeOH). H NMR (400 MHz, CDCl ): δ = 4.29 (q, J =
7.1 Hz, 2 H, OCH CH ), 4.08 (d, J = 2.0 Hz, 1 H, 2-H), 3.88 (td,
J = 2.0, 7.1 Hz, 1 H, 3-H), 3.12 (br. s, 1 H, OH), 2.03 (br. s, 1 H,
OH), 1.61 (m, 2 H, CH ), 1.46 (m, 2 H, 5-H), 1.35–1.21 (m, 21 H,
9 CH + OCH CH ), 0.88 (t, J = 6.8 Hz, 3 H, CH
NMR (100 MHz, CDCl ): δ = 173.7 (C=O), 73.0 (C-2), 72.5 (C-
3), 62.0 (OCH CH ), 33.8, 31.9, 29.6, 29.6, 29.6, 29.5, 29.5, 29.5,
29.3, 25.7, 22.6 (11 CH ), 14.1 (OCH CH ), 14.1 (CH ) ppm.
[M + Na] 325.2349; found
D
= –9.1 (c =
1
microscope. Specific optical rotations were determined with a Jasco
DIP-2000 digital polarimeter. Data for X-ray diffraction analysis
of single crystals were collected with a Stoe-IPDS diffractometer at
3
2
3
2
00 K using Mo-K
α
radiation (λ = 0.71073 Å, graphite mono-
2
1
3
chromator). Crude products were purified by column chromatog-
raphy on silica gel 60 (230–400 mesh, 0.040–0.063 mm, Merck) or
Sephadex LH 20 (Fluka), and TLC was carried out on silica-gel-
coated aluminium plates (silica gel 60 F254 with fluorescence indi-
cator, Merck). Preparative HPLC was carried out with a Knauer
system equipped with a WellChrom K-1001 pump and a Well-
Chrom K-2501 UV detector using a ODS-A column with fully end-
capped C18 phase (5 μm, 150ϫ10 mm ID, YMC, USA), using
2
2
3
3
) ppm.
C
3
2
3
2
2
3
3
+
+
HRMS (ESI): calcd. for C17
325.2348.
H34NaO
4
Ethyl (2S,3R)-2,3-Dihydroxypentadecanoate [(+)-7]: Potassium os-
mate(VI) dihydrate (40 mg, 0.11 mmol), hydroquinidine 1,4-phthal-
H
2
O (A) and CH
3
CN (B) as eluents (linear gradient: 0–20 min, 55–
azinediyl diether [(DHQD)
2
PHAL; 169 mg, 0.22 mmol], methane-
70% B, flow rate of 3.5 mL/min).
sulfonamide (2.25 g, 23.7 mmol), potassium carbonate (9.82 g,
7
7
1.1 mmol), and potassium hexacyanoferrate(III) (23.4 g,
1.1 mmol) were sequentially added to tBuOH/H O (1:1; 150 mL),
Fungal Material: Fruiting bodies of Hygrophorus abieticola
Krieglst. ex Gröger & Bresinsky were collected under Abies alba
near Kelheim, Bavaria, Germany (October 11, 2007, leg./det. A.
Bresinsky). A voucher specimen (coll. 57/07) has been deposited at
the Leibniz Institute of Plant Biochemistry (IPB), Halle, Germany.
2
and the resulting mixture was cooled to 0 °C with stirring. After-
wards, a solution of ethyl (2E)-pentadec-2-enoate (6; 6.36 g,
3.6 mmol)[ in tBuOH/H
15]
O (1:1; 150 mL) was added dropwise,
2
2
and stirring was continued at 0 °C for 20 h until TLC indicated
that the reaction was complete. The reaction was quenched with
saturated aq. sodium sulfite (100 mL), and the mixture was stirred
for a further 30 min and then extracted with dichloromethane (5ϫ
Extraction and Isolation of Hygrophrone B12 (+)-2: Frozen fruiting
bodies of H. abieticola (2.5 kg) were macerated using a blender,
and extracted with ethyl acetate (3ϫ 3 L). The slightly yellow solu-
tion was evaporated to dryness in vacuo. The crude extract (20.2 g)
was separated by column chromatography on silica gel
200 mL). The combined organic extracts were dried with sodium
sulfate, and evaporated to dryness. The residue was purified by col-
umn chromatography (petroleum ether/ethyl acetate, 1:1) to give
(450ϫ45 mm; chloroform/methanol, 100:0Ǟ0:100) to give 10
fractions (A1–A10, each 50 mL). Fraction A6 (3.05 g) was further
purified by size-exclusion chromatography on Sephadex LH 20
(
+)-7 (6.830 g, 95%) as a colourless solid, m.p. 69 °C. TLC (petro-
21
leum ether/ethyl acetate, 2:1): R
f
= 0.67. [α]
MeOH). H NMR (400 MHz, CDCl ): δ = 4.29 (q, J = 7.1 Hz, 2
H, OCH CH ), 4.08 (d, J = 2.0 Hz, 1 H, 2-H), 3.88 (t, J = 6.4 Hz,
H, 3-H), 3.11 (d, J = 4.9 Hz, 1 H, OH), 1.98 (br. s, 1 H, OH),
.60 (m, 2 H, CH ), 1.48 (m, 2 H, CH ), 1.35–1.23 (m, 21 H, 9
+ OCH CH ), 0.88 (t, J = 6.8 Hz, 3 H, CH
100 MHz, CDCl ): δ = 173.7 (C=O), 73.0 (C-2), 72.5 (C-3), 62.1
OCH CH ), 33.8, 31.9, 29.6, 29.6, 29.6, 29.5, 29.5, 29.5, 29.3, 25.7,
2.7 (11 CH ), 14.1 (OCH CH ), 14.1 (CH ) ppm. HRMS (ESI):
[M + Na]+ 325.2349; found 325.2348.
calcd. for C17
D
= 9.0 (c = 0.945,
(400ϫ40 mm, dichloromethane/methanol, 1:1) to give seven frac-
1
3
tions (B1–B7). Fraction B5 (1.4 g) was further purified by silica gel
column chromatography (450ϫ45 mm; n-hexane/ethyl acetate, 1:1)
to give 230 fractions (each 20 mL). Fractions 173–180 were com-
bined (32.3 mg), and finally this material was purified by prepara-
2
3
1
1
CH
(
(
2
2
2
13
2
2
3
3
) ppm. C NMR
tive HPLC (t
R
= 14.2 min) to give (+)-2 as a colourless solid
= 0.29.
= 20.7 (c = 0.135, MeOH). H NMR (400 MHz, CDCl ): δ =
.64 (dd, J = 6.1, 2.3 Hz, 1 H, 3-H), 6.30 (dd, J = 6.1, 1.2 Hz, 1
3
(
20.1 mg), m.p. 84 °C. TLC (n-hexane/ethyl acetate, 1:1): R
f
2
3
27
1
[α]
D
3
2
2
3
3
7
+
H
34NaO
4
H, 2-H), 4.73 (d, J = 6.0 Hz, 1 H, 4-H), 3.78 (m, 1 H, 6-H), 3.66
s, 1 H, 6-OH), 3.02 (d, J = 7.0 Hz, 1 H, 4-OH), 2.14 (br. d, J =
Ethyl (4R,5S)-2,2-Dimethyl-5-dodecyl-1,3-dioxolane-4-carboxylate
8): Compound (–)-7 (0.80 g, 2.64 mmol) and 2,2-diethoxypropane
(
(
6
1
.6 Hz, 1 H, 5-OH), 1.65–1.54 (m, 2 H, CH
2
), 1.39–1.20 (m, 20 H,
) ppm. 13C NMR (100 MHz,
(1.7 mL, 10.6 mmol) were dissolved in dichloromethane (70 mL),
and p-toluenesulfonic acid monohydrate (50 mg, 0.26 mmol) was
added. The resulting solution was stirred at room temperature for
0 CH ) 0.88 (t, J = 7.0 Hz, 3 H, CH
): δ = 207.2 (C-1), 163.5 (C-3), 133.5 (C-2), 75.9 (C-5), 73.3
C-6), 71.4 (C-4), 31.9, 31.3, 29.6, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3,
2
3
CDCl
(
3
24 h. After TLC indicated that the reaction was complete, saturated
2
2
6.1, 22.7 (11 CH ), 14.1 (C-18) ppm. HRMS (ESI): calcd. for
–
–
aq. NaHCO (7 mL) and ethyl acetate (50 mL) were sequentially
3
18 31 4
C H O [M – H] 311.2228; found 311.2224.
added. The organic phase was separated, and the aqueous layer
was extracted with ethyl acetate (2ϫ 25 mL). The combined or-
ganic extracts were dried with sodium sulfate, and filtered, and the
Ethyl (2R,3S)-2,3-Dihydroxypentadecanoate [(–)-7]: Potassium os-
mate(VI) dihydrate (40 mg, 0.11 mmol), hydroquinine 1,4-phthal-
azinediyl diether [(DHQ)
2
PHAL; 169 mg, 0.22 mmol], methane- volatiles were removed under reduced pressure. The residue was
sulfonamide (2.19 g, 23.0 mmol), potassium carbonate (9.55 g, purified by column chromatography (petroleum ether/ethyl acetate,
6
6
9.1 mmol), and potassium hexacyanoferrate(III) (22.8 g,
9.1 mmol) were sequentially added to tBuOH/H O (1:1; 150 mL),
5:1) to give 8 (0.85 g, 94%) as a pale yellow oil. TLC (petroleum
2
1
2
ether/ethyl acetate, 5:1): R
f
= 0.80. [α]
H NMR (400 MHz, CDCl ): δ = 4.24 (m, 2 H, OCH
(m, 2 H, 4-H + 5-H), 1.80–1.59 (m, 2 H, CH ), 1.55–1.36 (m, 2 H,
CH ), 1.47 (s, 3 H, CH acetal), 1.44 (s, 3 H, CH acetal), 1.36–
D
= –40.4 (c = 0.056, MeOH).
1
and the resulting mixture was cooled to 0 °C with stirring. After-
wards, a solution of ethyl (2E)-pentadec-2-enoate (6; 6.18 g,
2
3
2
CH ), 4.11
3
2
[
15]
3.0 mmol)
in tBuOH/H
2
O (1:1; 150 mL) was added dropwise,
2
3
3
6
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O43455
/KAP1
Date: 09-02-15 13:54:45
Pages: 10
Fungal Secondary Metabolite Hygrophorone B12
1
.21 (m, 21 H, 9 CH
CH
) ppm. 13C NMR (100 MHz, CDCl
C(CH acetal], 79.2 (C-4), 79.1 (C-5), 61.2 (OCH
1.9, 29.6 (3 CH ), 29.6 (2 CH ), 29.5, 29.5, 29.5, 29.3 (4 CH
7.2 (CH acetal), 25.7 (CH acetal), 25.6, 22.7 (2 CH ), 14.1
CH ), 14.1 (CH ppm. HRMS (ESI): calcd. for
38NaO [M + Na] 365.2662; found 365.2663.
2
+ OCH
2
CH
3
), 0.88 (t, J = 6.8 Hz, 3 H,
): δ = 171.0 (C=O), 110.7
3
CH ), 33.5, hexane/ethyl acetate, 2:1) to give sulfoxide 12 or 13.
that the reaction was complete, the solvent was removed in vacuo,
and the crude product was purified by column chromatography (n-
3
3
[
3
2
3
)
2
2
2
2
2
),
Ethyl (4S,5S)-5-Dodecyl-4-[(1R)-1-hydroxy-3-(phenylsulfinyl)prop-
3
3
2
yl]-2,2-dimethyl-1,3-dioxolane-4-carboxylate (12): Colourless oil
(OCH
2
3
3
)
24
(
=
557 mg, 93%). TLC (n-hexane/ethyl acetate, 2:1): R
f
= 0.17. [α]
–5.1 (c = 0.54, MeOH). H NMR (400 MHz, CDCl
(m, 2 H, o-H Ph), 7.51 (m, 3 H, m-H + p-H Ph), 4.38 (m, 1 H, 5-
dimethyl-1,3-dioxolane-4-carboxylate (10) and Ethyl (4S,5S)-5-Do H), 4.17 (m, 2 H, OCH CH ), 3.92 (dd, J = 26.3, 8.9 Hz, 1 H,
decyl-4-[(S)-1-hydroxy-3-(phenylthio)propyl]-2,2-dimethyl-1,3-di- CHOH), 3.22–2.87 (br. m, 2 H, CH CH S), 2.04 (s, 1 H, OH),
oxolane-4-carboxylate (11): Diisopropylamine (0.71 mL,
2.02–1.75 (m, 2 H, CH ), 1.63–1.47 (m, 4 H, 2 CH ), 1.42 (s, 3 H,
.96 mmol) was dissolved in dry THF (150 mL), and n-butyllithium
CH acetal), 1.39–1.20 (m, 24 H, 9 CH + OCH CH + CH
2.5 m in hexane; 2.0 mL, 4.96 mmol) was added at 0 °C. The mix-
acetal), 0.88 (t, J = 6.8 Hz, 3 H, CH ) ppm. C NMR (100 MHz,
CDCl ): δ = 171.5, 171.4 (C=O), 143.2, 142.9 (i-C Ph), 131.0, 131.0
(p-C Ph), 129.2, 129.2 (m-C Ph), 124.2, 124.1 (o-C Ph), 109.9, 109.9
[C(CH acetal], 88.5, 88.4 (C-4), 79.1, 79.0 (C-5), 69.3, 68.9
(COH), 61.3, 61.2 (OCH CH ), 53.7, 53.2 (CH CH S), 31.9, 30.6,
29.6, 29.6, 29.6, 29.5, 29.5, 29.3 (8 CH ), 26.9, 26.9 (CH CH S),
26.8, 26.8 (CH acetal), 26.5 (CH acetal), 26.4, 25.8, 22.7 (3 CH ),
14.2, 14.1 (OCH CH + CH ) ppm. HRMS (ESI): calcd. for
[M + Na]+ 547.3064; found 547.3064.
D
+
+
C
20
H
4
1
3
): δ = 7.61
Ethyl (4S,5S)-5-Dodecyl-4-[(R)-1-hydroxy-3-(phenylthio)propyl]-2,2-
2
3
2
2
2
2
4
(
3
2
2
3
3
1
3
3
ture was stirred for 30 min. The reaction mixture was then cooled
to –78 °C and stirred for a further 30 min, after which a solution
of acetal 8 (851 mg, 2.48 mmol) in dry THF (2 mL), was added
dropwise. The mixture was stirred for a further 30 min, then a solu-
tion of aldehyde 9 (618 mg, 3.72 mmol) in dry THF (2 mL) was
slowly added. Stirring was continued for 3 h at –78 °C, then the
3
3 2
)
2
3
2
2
2
2
2
3
3
2
reaction was quenched with saturated aq. NaHCO
3
(5 mL). After-
2
3
3
+
wards, ethyl acetate (20 mL) was added, the organic phase was sep-
29 6
C H48NaO S
arated, and the aqueous layer was extracted with ethyl acetate (3ϫ
Ethyl (4S,5S)-5-Dodecyl-4-[(1S)-1-hydroxy-3-(phenylsulfinyl)prop-
yl]-2,2-dimethyl-1,3-dioxolane-4-carboxylate (13): Colourless oil
20 mL). The combined organic extracts were dried with sodium
sulfate, and the solvents were evaporated to dryness. The residue
was purified by column chromatography (dichloromethane) to give
two diastereomers 10 and 11 in a 4:1 ratio.
24
(
=
(
113 mg, 71%). TLC (n-hexane/ethyl acetate, 2:1): R
f
= 0.14. [α]
4.8 (c = 0.37, MeOH). H NMR (400 MHz, CDCl
m, 2 H, o-H Ph), 7.52 (m, 3 H, m-H + p-H Ph), 4.25 (m, 2 H,
OCH CH ), 4.14 (m, 1 H, 5-H), 3.87 (ddd, J = 48.8, 10.2, 1.9 Hz,
1 H, CHOH), 3.25–2.86 (br. m, 2 H, CH CH S), 2.04 (s, 1 H, OH),
D
1
3
): δ = 7.61
Data for 10: Pale yellow oil (860 mg, 65%). TLC (dichlorometh-
2
3
1
ane): R
f
= 0.59. H NMR (400 MHz, CDCl
3
): δ = 7.37–7.22 (m, 4
2
2
1
.89–1.71 (m, 2 H, CH
CH acetal), 1.38–1.20 (m, 24 H, 9 CH
acetal), 0.88 (t, J = 6.7 Hz, 3 H, CH ) ppm. C NMR (100 MHz,
CDCl ): δ = 172.5, 172.3 (C=O), 143.2, 142.5 (i-C Ph), 131.0, 131.0
p-C Ph), 129.2, 129.2 (m-C Ph), 124.3, 124.1 (o-C Ph), 109.6, 109.5
[C(CH ) acetal], 86.5, 86.4 (C-4), 80.8, 80.7 (C-5), 71.7, 71.5
2
), 1.68–1.42 (m, 4 H, 2 CH
2
), 1.40 (s, 3 H,
H, o-H + m-H Ph), 7.16 (m, 1 H, p-H Ph), 4.31 (m, 1 H, 5-H),
3
2
+ OCH
2
CH + CH
3
3
4
3
1
.10 (m, 2 H, OCH
.23–2.97 (br. m, 2 H, CH
.85–1.72 (m, 2 H, CH ), 1.65–1.48 (m, 2 H, CH
acetal), 1.40 (s, 3 H, CH acetal), 1.37–1.17 (m, 23 H, 10
+ OCH CH ), 0.88 (t, J = 6.8 Hz, 3 H, CH
100 MHz, CDCl ): δ = 171.1 (C=O), 136.1 (i-C Ph), 129.2 + 128.8
o-C + m-C Ph), 125.9 (p-C Ph), 109.8 [C(CH acetal], 88.6 (C-
), 32.7, 31.9, 30.6, 30.3,
9.6, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 27.0 (12 CH ), 26.8 (CH
acetal), 22.7 (CH ), 14.1 (OCH CH ), 14.0
) ppm. HRMS (ESI): calcd. for C29
[M + Na]+
31.3115; found 531.3104.
2 3
CH
), 4.01 (td, J = 10.0, 3.1 Hz, 1 H, CHOH),
CH S), 1.96 (d, J = 10.4 Hz, 1 H, OH),
CH S), 1.59 (s, 3
1
3
3
2
2
3
2
2
2
(
H, CH
CH
(
(
3
3
1
3
) ppm. C NMR
3 2
2
2
3
3
(
2
(
(
COH), 61.6, 61.5 (OCH
9.6, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1 (9 CH
CH CH S), 27.1, 26.9 (CH acetal), 26.3, 26.0 (CH
CH acetal), 22.7 (CH ), 14.2, 14.1 (OCH CH + CH
[M + Na]+ 547.3064;
2
CH
3
), 54.0, 53.3 (CH
2
CH
2
S), 31.9, 29.7,
), 27.4, 27.3
3
3 2
)
2
2
2
3
2
), 24.7. 24.7
) ppm.
2 3
4), 79.1 (C-5), 69.7 (COH), 61.2 (OCH CH
2
2
3
3
2
2
3
3
+
HRMS (ESI): calcd. for C29
found 547.3068.
6
H48NaO S
acetal), 26.6 (CH
CH
3
2
2
+
3
(
5
3
5
H48NaO S
Cyclization of 12 and 13 with LDA: n-Butyllithium (2.5 m in n-hex-
ane; 3.2 equiv.) was slowly added to an ice-cold solution of diiso-
propylamine (3.2 equiv.) in dry THF (5 mL/mmol diisopropyl-
amine), and the mixture was stirred for 30 min. The mixture was
then cooled to –78 °C, and a solution of sulfoxide 12 or 13
Data for 11: Pale yellow oil (191 mg, 15%). TLC (dichlorometh-
1
ane): R
H, o-H + m-H Ph), 7.16 (m, 1 H, p-H Ph), 4.26 (m, 2 H,
OCH CH ), 4.09 (dd, J = 10.2, 2.9 Hz, 1 H, 5-H), 4.01 (t, J =
.6 Hz, 1 H, CHOH), 3.25–3.01 (br. m, 2 H, CH CH S), 2.32 (d,
J = 10.0 Hz, 1 H, OH), 2.07–1.44 (br. m, 6 H, 3 CH ), 1.42 (s, 3
H, CH acetal), 1.40 (s, 3 H, CH acetal), 1.34–1.22 (m, 21 H, 9
CH + OCH CH ), 0.88 (t, J = 6.8 Hz, 3 H, CH
100 MHz, CDCl ): δ = 172.8 (C=O), 136.2 (i-C Ph), 128.9 + 128.9
o-C + m-C Ph), 125.8 (p-C Ph), 109.6 [C(CH acetal], 86.5 (C-
), 32.4, 31.9, 30.1, 29.7,
9.6, 29.6, 29.6, 29.5, 29.4, 29.3, 29.0, 27.3 (12 CH ), 26.9 (CH
acetal), 22.7 (CH ), 14.1 (OCH CH + CH
S [M + Na] 531.3115;
f 3
= 0.21. H NMR (400 MHz, CDCl ): δ = 7.37–7.23 (m, 4
2
3
(
1 equiv.) in dry THF (5 mL/mmol of 12 or 13) was added drop-
9
2
2
wise. The resulting mixture was stirred for 2 h. The reaction mix-
ture was then allowed to warm slowly to room temperature, and
2
3
3
1
3
stirring was continued for 2 h. Saturated aq. NaHCO (5 mL/mmol
) ppm. C NMR
3
2
2
3
3
of 12 or 13) and ethyl acetate (30 mL/mmol of 12 or 13) were se-
quentially added, the organic phase was separated, and the aqueous
layer was extracted with ethyl acetate (3ϫ 30 mL/mmol of 12 or
(
(
3
3 2
)
2 3
4), 80.9 (C-5), 71.7 (COH), 61.6 (OCH CH
13). The combined organic extracts were dried with sodium sulfate,
2
2
3
and filtered, and the solvents were evaporated to dryness. The resi-
due was purified by column chromatography (n-hexane/ethyl acet-
ate, 3:2) to give two diastereomers of cyclopentanone 14 or 15.
acetal), 24.7 (CH
ppm. HRMS (ESI): calcd. for C29
found 531.3102.
3
2
2
3
3
)
+
+
H48NaO
5
(
1
4S,5S,9R)-4-Dodecyl-9-hydroxy-2,2-dimethyl-7-(phenylsulfinyl)-
,3-dioxaspiro[4.4]nonan-6-one (14)
Sulfoxidation of 10 and 11 with Sodium Periodate: Aldol product
0 or 11 (1 equiv.) was dissolved in methanol/water (19:1; 10 mL/
mmol of 10 or 11) and the solution was cooled to 0 °C. Sodium Data for first diastereomer of 14: Yellow oil (168 mg, 35%). TLC
periodate (1.1 equiv.) was added, and the reaction mixture was
stirred for 10 min. The mixture was then allowed to warm to room
temperature, and stirred for a further 24 h. After TLC indicated
1
2
5
(n-hexane/ethyl acetate, 3:2): R
f
= 0.49. [α]
MeOH). H NMR (400 MHz, CDCl ): δ = 7.58 (m, 2 H, o-H Ph),
7.52 (m, 3 H, m-H + p-H Ph), 4.21 (dd, J = 3.9, 2.9 Hz, 1 H, 4-
D
= –308.7 (c = 0.99,
1
3
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O43455
/KAP1
Date: 09-02-15 13:54:45
Pages: 10
N. Arnold, L. Wessjohann, B. Westermann et al.
FULL PAPER
H), 4.12 (m, 1 H, 9-H), 3.48 (t, J = 9.5 Hz, 1 H, 7-H), 2.62 (ddd, 15), and the resulting mixture was heated at reflux for 1 h. After
J = 14.4, 10.1, 4.4 Hz, 1 H, 8-Ha), 2.04 (s, 1 H, OH), 1.84 (ddd, J TLC indicated that the reaction was complete, the volatile material
=
1
1
14.4, 9.0, 2.7 Hz, 1 H, 8-Hb), 1.59 (s, 3 H, CH
.48 (m, 2 H, CH ), 1.43 (s, 3 H, CH acetal), 1.35–1.21 (m, 20 H, by column chromatography (n-hexane/ethyl acetate, 7:3) to give cy-
0 CH ), 0.88 (t, J = 6.8 Hz, 3 H, CH clopentenone 16 or 17.
) ppm. 13C NMR (100 MHz,
): δ = 207.3 (C=O), 141.6 (i-C Ph), 131.3 (p-C Ph), 129.3
3
acetal), 1.54– was removed under reduced pressure, and the residue was purified
2
3
2
3
CDCl
3
(
4S,5S,9R)-4-Dodecyl-9-hydroxy-2,2-dimethyl-1,3-dioxaspiro[4.4]-
(
(
m-C Ph), 123.8 (o-C Ph), 111.4 [C(CH acetal], 89.6 (C-5), 81.4
C-4), 72.2 (C-9), 68.0 (C-7), 31.9, 31.0, 29.7, 29.6, 29.6, 29.6, 29.5,
3 2
)
non-7-en-6-one (16): Colourless solid (216 mg, 81%), m.p. 59 °C.
24
f D
TLC (n-hexane/ethyl acetate, 7:3): R = 0.71. [α] = –39.2 (c = 0.15,
2
2
9.3, 29.2 (9 CH
1.9 (2 CH + C-8), 14.1 (CH
2
), 28.0 (CH
3
acetal), 27.6 (CH
) ppm. HRMS (ESI): calcd. for
[M + H] 479.2826; found 479.2823.
3
acetal), 26.5, 22.7,
1
MeOH). H NMR (400 MHz, CDCl
3
): δ = 7.57 (dd, J = 6.2,
2
3
2.3 Hz, 1 H, 8-H), 6.26 (dd, J = 6.2, 1.2 Hz, 1 H, 7-H), 4.47 (d, J
+
+
C
27
H
43
O
5
S
=
6.9 Hz, 1 H, 9-H), 4.22 (dd, J = 9.1, 4.1 Hz, 1 H, 4-H), 2.91 (d,
Data for second diastereomer of 14: Yellow oil (147 mg, 31 %).
J = 7.3 Hz, 1 H, OH), 1.75 (br. m, 2 H, CH
acetal), 1.48 (s, 3 H, CH acetal), 1.43–1.15 (m, 20 H, 10 CH
0.88 (t, J = 6.9 Hz, 3 H, CH
.56 (m, 3 H, m-H + p-H Ph), 4.15–4.03 (m, 2 H, 4-H + 9-H), 3.40 δ = 202.6 (C=O), 161.9 (C-8), 134.5 (C-7), 111.5 [C(CH
dd, J = 9.9, 4.5 Hz, 1 H, 7-H), 2.49 (ddd, J = 15.7, 7.9, 4.0 Hz, 1 82.6 (C-4), 80.6 (C-2), 72.2 (C-4), 31.9, 29.6, 29.6, 29.6, 29.5, 29.5,
H, 8-Ha), 2.19 (ddd, J = 15.3, 10.0, 5.0 Hz, 1 H, 8-Hb), 2.04 (s, 1 29.4, 29.3, 29.1 (9 CH ), 27.3 (CH acetal), 26.6 (CH ), 26.2 (CH
H, OH), 1.61 (s, 3 H, CH acetal), 1.56–1.48 (m, 2 H, CH ), 1.46 acetal), 22.7 (CH ), 14.1 (CH ) ppm. HRMS (ESI): calcd. for
s, 3 H, CH acetal), 1.33–1.19 (m, 20 H, 10 CH ), 0.88 (t, J =
[M + Na]+ 375.2506; found 375.2504.
.9 Hz, 3 H, CH ): δ = 207.3
) ppm. 13C NMR (100 MHz, CDCl
2
), 1.60 (s, 3 H, CH
3
= 0.27. [α]2
5
= 186.4 (c = 1.27,
): δ = 7.65 (m, 2 H, o-H Ph),
),
TLC (n-hexane/ethyl acetate, 3:2): R
MeOH). H NMR (400 MHz, CDCl
f
D
3
2
1
13
3
3
) ppm. C NMR (100 MHz, CDCl
3
):
7
(
3 2
) acetal],
2
3
2
3
3
2
2
3
+
(
3
2
21 4
C H36NaO
6
3
3
(
4S,5S,9S)-4-Dodecyl-9-hydroxy-2,2-dimethyl-1,3-dioxaspiro[4.4]-
(C=O), 141.6 (i-C Ph), 131.6 (p-C Ph), 129.4 (m-C Ph), 124.3 (o-C
non-7-en-6-one (17): Colourless oil (48.3 mg, 73%). TLC (n-hexane/
Ph), 111.4 [C(CH acetal], 90.5 (C-5), 80.5 (C-4), 71.1 (C-9), 68.3
3
)
2
24
1
ethyl acetate, 7:3): R
NMR (400 MHz, CDCl
H), 6.30 (dd, J = 6.3, 1.6 Hz, 1 H, 7-H), 4.98 (s, 1 H, 9-H), 4.50
dd, J = 9.8, 2.8 Hz, 1 H, 4-H), 2.26 (s, 1 H, OH), 1.65 (s, 3 H,
f
= 0.54. [α]
D
= 19.2 (c = 0.15, MeOH). H
(
C-7), 31.9, 31.0, 29.6, 29.6, 29.5, 29.4, 29.4, 29.3, 29.3 (9 CH ),
2
3
): δ = 7.43 (dd, J = 6.3, 2.0 Hz, 1 H, 8-
2
7.4 (CH
3
acetal), 26.5 (CH
3
acetal), 26.8, 26.0, 22.6 (2 CH
2
+ C-
[M +
+
8), 14.1 (CH
3
) ppm. HRMS (ESI): calcd. for C27
H
43
O
5
S
(
+
H] 479.2826; found 479.2824.
4S,5S,9S)-4-Dodecyl-9-hydroxy-2,2-dimethyl-7-(phenylsulfinyl)-
,3-dioxaspiro[4.4]nonan-6-one (15)
CH
CH
CDCl
3
acetal), 1.46 (s, 3 H, CH
), 0.88 (t, J = 6.9 Hz, 3 H, CH
): δ = 203.1 (C=O), 159.5 (C-8), 134.4 (C-7), 109.8 [C(CH
acetal], 89.2 (C-5), 78.3 (C-4), 75.0 (C-9), 31.9, 30.3, 29.6, 29.6,
29.6, 29.5, 29.5, 29.4, 29.3 (9 CH ), 27.3 (CH acetal), 27.0 (CH ),
26.5 (CH acetal), 22.7 (CH ), 14.1 (CH ) ppm. HRMS (ESI):
calcd. for C21
[M + Na]+ 375.2506; found 375.2504.
3
acetal), 1.42–1.19 (br. m, 22 H, 11
1
3
(
1
2
3
) ppm. C NMR (100 MHz,
3
3 2
)
Data for first diastereomer of 15: Yellow oil (21.0 mg, 32%). TLC
= 0.76. [α]2
5
2
3
2
(
n-hexane/ethyl acetate, 3:2): R
f
D
= 128.4 (c = 0.40,
1
3
2
3
MeOH). H NMR (400 MHz, CDCl
.51 (dd, J = 10.9, 3.1 Hz, 1 H, 4-H), 4.04 (d, J = 3.5 Hz, 1 H, 9-
H), 3.77 (dd, J = 11.0, 2.0 Hz, 1 H, 7-H), 2.55 (ddd, J = 15.3, 11.1, Deprotection of Cyclopentenone 16 or 17 with Trifluoroacetic Acid:
3
): δ = 7.57 (m, 5 H, H Ph),
+
H
36NaO
4
4
4
1
.0 Hz, 1 H, 8-Ha), 2.04 (s, 1 H, OH), 1.94 (m, 1 H, 8-Hb), 1.72–
.60 (m, 2 H, CH ), 1.60–1.48 (m, 2 H, CH ), 1.39 (s, 3 H, CH
+ CH acetal), 0.88 (t, J =
) ppm. 13C NMR (100 MHz, CDCl
C=O), 140.3 (i-C Ph), 131.5 (p-C Ph), 129.6 (m-C Ph), 124.0 (o-C
Ph), 110.2 [C(CH acetal], 88.0 (C-5), 76.6 (C-4), 69.0 (C-9), 68.1
C-7), 31.9, 31.0, 29.7, 29.6, 29.6, 29.6, 29.6, 29.5, 29.3 (9 CH ),
A solution of 16 or 17 in trifluoracetic acid (2.5 mL/mmol 16 or
17) was stirred for 2 h at 0 °C. Then, all the volatiles were removed
in vacuo, and the residue was purified by column chromatography
2
2
3
acetal), 1.36–1.20 (m, 21 H, 9 CH
.8 Hz, 3 H, CH
2
3
6
(
3
3
): δ = 207.3 (n-hexane/ethyl acetate, 1:1) to give hygrophorones (–)-2 or (+)-1.
4R,5R)-4,5-Dihydroxy-5-[(S)-1-hydroxytridecyl]cyclopent-2-enone
(–)-2]: Colourless solid (55.4 mg, 43%), m.p. 88 °C. TLC (n-hex-
(
3 2
)
[
(
2
27
ane/ethyl acetate, 1:1): R
f
= 0.29. [α]
H NMR (400 MHz, CDCl ): δ = 7.64 (dd, J = 6.1, 2.3 Hz, 1 H,
-H), 6.30 (dd, J = 6.1, 1.1 Hz, 1 H, 2-H), 4.73 (d, J = 6.4 Hz, 1
D
= –18.8 (c = 0.13, MeOH).
28.9 (CH
3
acetal), 26.3 (CH
3
acetal), 28.1, 26.3, 22.7 (2 CH
2
+ C-
[M +
1
3
+
8), 14.1 (CH
3
) ppm. HRMS (ESI): calcd. for C27
H
43
O
5
S
3
+
H] 479.2826; found 479.2817.
H, 4-H), 3.77 (d, J = 4.1 Hz, 1 H, 6-H), 3.69 (s, 1 H, OH), 3.05 (d,
J = 7.2 Hz, 1 H, OH), 2.17 (d, J = 5.8 Hz, 1 H, OH), 1.62–1.53
Data for second diastereomer of 15: Yellow oil (23.5 mg, 35%).
= 0.45. [α]2
.38, MeOH). H NMR (400 MHz, CDCl ): δ = 7.61 (m, 2 H, o- 3 H, CH
H Ph), 7.52 (m, 3 H, m-H + p-H Ph), 4.36 (dd, J = 10.0, 4.1 Hz, 1 163.5 (C-3), 133.5 (C-2), 75.9 (C-5), 73.3 (C-6), 71.4 (C-4), 31.9,
H, 4-H), 4.33 (t, J = 3.4 Hz, 1 H, 9-H), 3.57 (t, J = 9.0 Hz, 1 H, 31.2, 29.6, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 26.1, 22.7 (11 CH ),
5
= –306.1 (c =
(m, 2 H, CH
), 0.88 (t, J = 6.8 Hz,
): δ = 207.3 (C-1),
TLC (n-hexane/ethyl acetate, 3:2): R
f
D
2 2
), 1.41–1.20 (m, 20 H, 10 CH
1
13
0
3
3
) ppm. C NMR (100 MHz, CDCl
3
2
–
–
7
-H), 2.83 (ddd, J = 13.7, 9.4, 4.2 Hz, 1 H, 8-Ha), 2.09 (br. s, 1 H,
OH), 1.83 (ddd, J = 13.9, 8.7, 2.8 Hz, 8-Hb), 1.78–1.67 (m, 2 H,
CH ), 1.62–1.51 (m, 2 H, CH ), 1.45 (s, 3 H, CH acetal), 1.35 (s,
H, CH acetal), 1.32–1.20 (m, 18 H, 9 CH ), 0.88 (t, J = 6.8 Hz,
H, CH ): δ = 206.8 (C=O),
) ppm. 13C NMR (100 MHz, CDCl
42.5 (i-C Ph), 131.3 (p-C Ph), 129.2 (m-C Ph), 124.2 (o-C Ph),
10.5 [C(CH acetal], 86.5 (C-5), 77.5 (C-4), 72.3 (C-9), 68.1 (C-
), 31.9, 30.5, 29.6, 29.6, 29.6, 29.6, 29.5, 29.5, 29.3 (9 CH ), 28.8
acetal), 26.1 (CH acetal), 26.8, 26.4, 22.7 (2 CH + C-8),
4.1 (CH ) ppm. HRMS (ESI): calcd. for C27
[M + H]+
79.2826; found 479.2821.
14.1 (CH
3 31 4
) ppm. HRMS (ESI): calcd. for C18H O [M – H]
311.2228; found 311.2225.
2
2
3
(
4S,5R)-4,5-Dihydroxy-5-[(S)-1-hydroxytridecyl]cyclopent-2-enone
3
3
1
1
7
3
2
[
(+)-1]: Colourless solid (15.5 mg, 64%), m.p. 69 °C. TLC (n-hex-
3
3
27
ane/ethyl acetate, 1:1): R
f
= 0.27. [α]
H NMR (400 MHz, CDCl ): δ = 7.59 (dd, J = 6.1, 1.9 Hz, 1 H,
-H), 6.31 (dd, J = 6.1, 1.3 Hz, 1 H, 2-H), 4.91 (s, 1 H, 4-H), 4.29
s, 1 H, OH), 3.87 (br. s, 2 H, 6-H + OH), 3.08 (d, J = 6.5 Hz, 1
D
= 54.1 (c = 0.145, MeOH).
1
3
3 2
)
3
(
2
(CH
3
3
2
H, OH), 1.59–1.47 (m, 2 H, CH
2
), 1.39–1.17 (m, 20 H, 10 CH
2
),
):
+
1
4
3
43 5
H O S
) ppm. 13C NMR (100 MHz, CDCl
0.88 (t, J = 6.8 Hz, 3 H, CH
3
3
δ = 205.4 (C-1), 162.3 (C-3), 132.5 (C-2), 83.9 (C-5), 79.1 (C-6),
73.8 (C-4), 31.9, 31.5, 29.7 (3 CH ), 29.6 (2 CH ), 29.6, 29.6, 29.4,
29.3, 26.0, 22.7 (6 CH ), 14.1 (CH ) ppm. HRMS (ESI): calcd. for
[M – H] 311.2228; found 311.2226.
Elimination of the Sulfoxide Moiety with Calcium Carbonate: Cal-
cium carbonate (1.1 eqiuv.) was added to a solution containing
cyclopentanone 14 or 15 (1 equiv.) in toluene (20 mL/mmol 14 or
2
2
2
3
–
–
18 31 4
C H O
8
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Job/Unit: O43455
/KAP1
Date: 09-02-15 13:54:45
Pages: 10
Fungal Secondary Metabolite Hygrophorone B12
4S,5R,6R)-4-Dodecyl-2,2-dimethyl-9-oxo-1,3-dioxaspiro[4.4]non-7-
3 3
3 H, CH ): δ = 205.1 (C-1),
) ppm. 13C NMR (100 MHz, CDCl
(
en-6-yl Acetate (18): Cyclopentenone 16 (156 mg, 0.44 mmol) was 170.0 (C=O OAc), 162.9 (C-3), 133.0 (C-2), 75.5 (C-5), 73.7 (C-6),
dissolved in pyridine (6 mL). The solution was cooled to 0 °C, and
acetic anhydride (3 mL) was slowly added. After 10 min, the mix-
ture was allowed to warm to room temperature, and stirring was
continued for 3 h. The volatiles were removed under reduced pres-
sure, and the residue was purified by column chromatography (n-
hexane/ethyl acetate, 5:1) to give acetylated cyclopentenone 18
71.5 (C-4), 31.9, 29.6, 29.6, 29.6, 29.5, 29.4, 29.4, 29.3, 29.2, 25.8,
22.7 (11 CH ), 20.8 (CH OAc), 14.1 (CH ) ppm. HRMS (ESI):
calcd. for C20
[M + Na]+ 377.2298; found 377.2296.
2
3
3
+
H
34NaO
5
Supporting Information (see footnote on the first page of this arti-
cle): Asymmetric total synthesis of hygrophorones (+)-2 and (–)-1;
X-ray structure of aldol product 30 (related intermediate to 11);
NMR spectra of intermediates and final products.
(
165 mg, 95%) as a colourless oil. TLC (n-hexane/ethyl acetate,
8
1
5
:1): R
f
= 0.44. [α]2
): δ = 7.46 (dd, J = 6.3, 2.8 Hz, 1 H, 3-H), 6.41 (dd, J = 6.3,
.3 Hz, 1 H, 8-H), 5.65 (dd, J = 2.8, 1.3 Hz, 1 H, 6-H), 4.39 (dd,
J = 9.4, 3.2 Hz, 1 H, 4-H), 2.15 (s, 3 H, CH OAc), 1.62 (s, 3 H,
CH acetal), 1.51 (m, 2 H, CH ), 1.36 (s, 3 H, CH acetal), 1.33–
.20 (m, 20 H, 10 CH ), 0.88 (t, J = 6.9 Hz, 3 H, CH
NMR (100 MHz, CDCl ): δ = 203.9 (C=O), 170.3 (C=O OAc),
56.3 (C-7), 136.9 (C-8), 111.3 [C(CH acetal], 82.8 (C-5), 81.8
C-4), 73.2 (C-6), 31.9, 29.6, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3
9 CH ), 27.4 (CH acetal), 26.7 (CH ), 25.7 (CH acetal), 22.7
), 20.6 (CH OAc), 14.1 (CH ) ppm. HRMS (ESI): calcd. for
[M + Na] 417.2611; found 417.2611.
D
= –6.2 (c = 0.14, MeOH). H NMR (400 MHz,
CDCl
1
3
Acknowledgments
3
3
2
3
The authors want to express their gratitude to the late Gisela
Schmidt for her valuable input on the synthesis.
1
3
1
2
3
) ppm.
C
3
1
3 2
)
[
1] M. Bon, Flore Mycologique d’Europe 1: Les Hygrophores
Hygrophoraceae Lotsy, in: Documents Mycologiques Mémoire
Hors Série 1, CRDP Amiens, France, 1990, p. 1–99.
(
(
(
2
3
2
3
CH
2
3
3
[2] T. Lübken, J. Schmidt, A. Porzel, N. Arnold, L. A.
Wessjohann, Phytochemistry 2004, 65, 1061–1071.
+
+
C
23
H
38NaO
5
(
4
4R,5R)-5-Hydroxy-5-[(S)-1-hydroxytridecyl]-4-oxocyclopent-2-en-
-yl Acetate (19) and (4R,5S)-5-Hydroxy-5-[(S)-1-(acetyloxy)tri-
[3] Hygrophorones A could only be isolated as monoacetates
(acetylated at the 4 or 6 position) and as the 4,6-diacetate, see
ref.[
2]
decyl]-1-oxocyclopent-2-enone (20): To remove the diol protecting
group, a solution of 18 (132 mg, 0.33 mmol) in trifluoroacetic acid/
methanol (5:1; 6 mL) was stirred for 30 min at 0 °C, then it was
allowed to warm to room temperature, and stirred for a further
[
4] V. Schmidts, M. Fredersdorf, T. Lübken, A. Porzel, N. Arnold,
L. A. Wessjohann, C. M. Thiele, J. Nat. Prod. 2013, 76, 839–
844; T. Lübken, N. Arnold, L. Wessjohann, C. Böttcher, J.
Schmidt, J. Mass Spectrom. 2006, 41, 361–371.
5] A. Teichert, T. Lübken, J. Schmidt, A. Porzel, N. Arnold, L. A.
Wessjohann, Z. Naturforsch. B 2005, 60, 25–32.
2
h. The reaction mixture was evaporated to dryness, and the resi-
[
due was purified by column chromatography (n-hexane/ethyl acet-
ate, 3:2) to give acetylated hygrophorones 19 and 20 in a 2:1 ratio.
The regioisomeric mixture could be separated by preparative
[6] C. Gryparis, I. N. Lykakis, C. Efe, I. P. Zaravinos, T. Vidali, E.
Kladou, M. Stratakis, Org. Biomol. Chem. 2011, 9, 5655–5661.
[7] T. Montagnon, M. Tofi, G. Vassilikogiannakis, Acc. Chem. Res.
HPLC [H
5 min, 60–90% B, flow rate of 3.5 mL/min].
Data for 19: White solid (78.6 mg, 67%), m.p. 99 °C. TLC (n-hex-
2 3
O (A) and CH CN (B) as eluents; linear gradient: 0–
2
008, 41, 1001–1011.
1
[
8] D. J. Aitken, H. Eijsberg, A. Frongia, J. Ollivier, P. P. Piras,
Synthesis 2014; 46, 1–24; W. Phutdhawong, S. G. Pyne, A. Bar-
amee, D. Buddhasukh, B. W. Skelton, A. H. White, Tetrahedron
Lett. 2002, 43, 6047–6049.
ane/ethyl acetate, 3:2): R
f
= 0.35. [α]2
D
= –16.4 (c = 0.13, MeOH).
7
1
H NMR (400 MHz, CDCl ): δ = 7.58 (dd, J = 6.1, 2.7 Hz, 1 H,
3
3
1
7
1
-H), 6.43 (dd, J = 6.2, 1.2 Hz, 1 H, 2-H), 5.73 (dd, J = 2.6, 1.3 Hz,
H, 4-H), 3.82 (m, 1 H, 6-H), 3.20 (s, 1 H, OH), 2.24 (d, J =
.7 Hz, 1 H, OH), 2.16 (s, 3 H, OAc), 1.61–1.45 (m, 2 H, 7-H),
[9] Adopted from: M. Pohmakotr, S. Popuang, Tetrahedron Lett.
1991, 32, 275–278.
[
10] A. B. Zaitsev, H. Adolfsson, Synthesis 2006, 1725–1756.
11] D. Seebach, A. R. Sting, M. Hoffmann, Angew. Chem. Int. Ed.
Engl. 1996, 35, 2708–2748; Angew. Chem. 1996, 108, 2881–
[
.39–1.19 (m, 20 H, 10 CH
2
), 0.88 (t, J = 6.8 Hz, 3 H, CH
C NMR (100 MHz, CDCl ): δ = 206.6 (C-1), 170.1 (C=O OAc),
58.2 (C-3), 135.9 (C-2), 77.0 (C-5), 73.9 (C-6), 73.0 (C-4), 31.9,
1.2, 29.6, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 26.1, 22.7 (11 CH ),
0.7 (CH OAc), 14.1 (CH ) ppm. HRMS (ESI): calcd. for
34NaO +
[M + Na] 377.2298; found 377.2297.
3
) ppm.
1
3
3
2921.
1
3
2
[
[
[
12] R. A. Fernandes, A. B. Ingle, V. P. Chavan, Tetrahedron: Asym-
metry 2009, 20, 2835–2844.
13] C. Donald, L. Pengfei, M. Tanya, N. T. H. Tholen, Tetrahedron
2010, 66, 6376–6382.
2
3
3
+
C
20
H
5
14] S. Chooprayoon, C. Kuhakarn, P. Tuchinda, V. Reutrakul, M.
Pohmakotr, Org. Biomol. Chem. 2011, 9, 531; K. Supakeat, K.
Chutima, T. Patoomratana, M. Pohmakotr, Synthesis 2010,
Data for 20: Colourless oil (38.5 mg, 33%). TLC (n-hexane/ethyl
acetate, 3:2): R
= 0.35. [α]2
8
1
f
D
= –16.6 (c = 0.14, MeOH). H NMR
(400 MHz, CDCl
3
): δ = 7.64 (dd, J = 6.1, 2.4 Hz, 1 H, 3-H), 6.29
1453–1458.
(dd, J = 6.1, 1.2 Hz, 1 H, 2-H), 5.19 (dd, J = 10.1, 2.8 Hz, 1 H, 6-
[
15] R. A. Fernandes, P. Kumar, Synthesis 2003, 129–135; R. A.
Fernandes, P. Kumar, Tetrahedron 2002, 58, 6685–6690.
Received: November 10, 2014
H), 4.80 (d, J = 6.0 Hz, 1 H, 4-H), 3.39 (s, 1 H, OH), 2.99 (d, J =
.2 Hz, 1 H, OH), 1.99 (s, 3 H, OAc), 1.85 (m, 1 H, 7-Ha), 1.62
m, 1 H, 7-Hb), 1.37–1.20 (m, 20 H, 10 CH ), 0.88 (t, J = 6.9 Hz,
7
(
2
Published Online: ᭿
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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9

Job/Unit: O43455
/KAP1
Date: 09-02-15 13:54:45
Pages: 10
N. Arnold, L. Wessjohann, B. Westermann et al.
FULL PAPER
Total Synthesis
Hygrophorone B12 was isolated from the
fruiting bodies of Hygrophorus abieticola,
and was synthesized in enantiomerically
pure form using a Sharpless asymmetric di-
hydroxylation and two diastereoselective
aldol-type reactions as the key steps. This
approach enabled the unambiguous deter-
mination of the relative and absolute con-
figuration of hygrophorones B for the first
time.
E. Bette, A. Otto, T. Dräger,
K. Merzweiler, N. Arnold,*
L. Wessjohann,* B. Westermann* ... 1–10
Isolation and Asymmetric Total Synthesis
of Fungal Secondary Metabolite Hygro-
1
2
phorone B
Keywords: Total synthesis / Asymmetric
synthesis / Natural products / Fungal con-
stituents / Configuration determination /
Structure elucidation
10
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