Retipolides - Unusual spiromacrolactones from the mushrooms Retiboletus retipes and R. ornatipes

Article abstract of DOI:10.1002/ejoc.200700579

Mushrooms of the genus Retiboletus contain the retipolides A-D (1, 6-8), unusual 14-membered spiromacrolides with a biphenyl ether linkage. The structures of these metabolites suggested a biogenetic sequence starting from retipolide E (16), which could fu

Full text of DOI:10.1002/ejoc.200700579

FULL PAPER
DOI: 10.1002/ejoc.200700579
Retipolides – Unusual Spiromacrolactones from the Mushrooms
Retiboletus retipes and R. ornatipes
Karl Justus,[a] Rupert Herrmann,[a] Jörg-Dieter Klamann,[a] Gertraud Gruber,[b]
Veronika Hellwig,[b] Andrea Ingerl,[b] Kurt Polborn,[b][‡] Bert Steffan,[b] and
Wolfgang Steglich*[b]
Dedicated to Professor Wolfgang Beck on the occasion of his 75th birthday
Keywords: Natural products / Retipolides / Macrolactones / Spiro compounds / Oxepinones / Basidiomycetes
Mushrooms of the genus Retiboletus contain the retipolides
A–D (1, 6–8), unusual 14-membered spiromacrolides with a
biphenyl ether linkage. The structures of these metabolites
suggested a biogenetic sequence starting from retipolide E
(16), which could furnish the unique 2(3H)-oxepinone unit of
retipolide C (7) via an oxidative enlargement of the 4-hy-
droxyphenyl ring. A subsequent O/C-acyl shift would then
lead to the cyclopenta[c]pyran system of retipolide A (1). The
A (1) was accomplished after transformation into a meth-
oxyfulvene derivative 2a, and the absolute (R) configuration
of 1 was assigned by a single-crystal X-ray analysis of the
corresponding (S)-sec-butoxyfulvene analogue 2b. Some
samples of R. retipes/ornatipes contained isoretipolide A (9),
an isomer of 1 with a highly strained 12-membered lactone
ring incorporating two C,C-coupled 4-hydroxyphenyl units.
A comparison of the CD spectrum of retipolide A (1) with
proposed precursor 16 was synthesized and subsequently de- those of the other retipolides and isoretipolide A (9) indicates
tected in the fungal extract in addition to the probable bio-
synthetic intermediates butyrolactone II (13), tyrosol (14), and
secoretipolide E (15). The structural elucidation of retipolide
that all of these macrolides possess R configuration.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
Introduction
Results and Discussion
Isolation of the Retipolides A–D
Retiboletus retipes (Berkeley & Curtis) Binder & Bresin-
sky is a showy mushroom with yellow pores and flesh, eas-
ily recognized by a conspicuous reticulated stalk. It occurs
together with a twin species, R. ornatipes (Peck) Binder &
Bresinsky, the Ornate-stalked Bolete,[1] in mixed or decidu-
ous woods in eastern North America, Japan, and Yunnan
(China). The mushrooms are unpalatable because of their
bitter taste and stain the fingers yellow on handling. On
cutting or bruising the affected areas develop yellow-orange
to rusty brown colors. We were attracted by the bright color
of these remarkable fungi and now report the structural elu-
cidation of a biosynthetically and structurally unique set of
fungal metabolites, named retipolides A–E.
Our first attempts to isolate the pigments from MeOH
extracts of R. retipes/ornatipes[1] were unsuccessful, due to
rapid degradation of the metabolites in this solvent re-
sulting in the formation of dark brown to black materials.
This problem could be bypassed by column chromatog-
raphy of an EtOAc extract from freeze-dried fruit bodies of
R. retipes/ornatipes on Sephadex LH-20 with acetone/tolu-
ene at 8 °C (Scheme 1). This procedure yielded four frac-
tions, which on further chromatographic purification af-
forded the retipolides A–D.
Retipolides A (1) and B (6)
[a] Kekulé Institut für Organische Chemie und Biochemie der Uni-
versität Bonn,
Retipolide A was obtained as a yellowish, amorphous
powder, which appears on TLC plates as a pale yellow spot.
On exposure to HCl (gaseous) the spot turns colorless and
changes to yellow-orange with ammonia. Retipolide A is
optically active, [α]2D0 = +160, and exhibits UV maxima at
λmax = 208, 316, 390, and 428 nm. Its rather simple EI-MS
shows a molecular peak at m/z = 476, corresponding to
Gerhard-Domagk-Str. 1, 5300 Bonn 1, Germany
[b] Department Chemie, Ludwig-Maximilians-Universität
München,
Butenandtstr. 5–13, 81377 München, Germany
Fax: +49-89-218077756
E-mail: wos@cup.uni-muenchen.de
[‡] Crystal structure determination.
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Retipolides – Unusual Spiromacrolactones from Mushrooms
Scheme 1. Procedures outlining the isolation of retipolides A–D.
C26H20O9 on high resolution. In addition to peaks indicat-
ing the loss of OH, H2O, and CO2 from the molecular ion,
only the base peak at m/z = 225 (C15H13O2) is remarkable.
1
In the H NMR spectrum of retipolide A all 20 protons
including those of two exchangeable OH groups are clearly
discernible. Characteristic is an aromatic ABCD system at
δH = 7.00, 7.24, 7.35, and 7.67 ppm, whose signals are each
split into a doublet of doublets with J = 8.5 and 2.2 Hz.
This is in accord with a sterically fixed 1,4-disubstituted
benzene ring with strongly hindered rotation around the
para-axis. In addition, a 1,2,4-trisubstituted benzene ring
Figure 1. Partial structure A of retipolide A and formation of the
base ion m/z = 225 under electron impact.
can be recognized with signals at δH = 4.83, 6.51, and with δC = 127.0, 131.7, 143.9, 157.6, and 163.4 ppm. At this
6.70 ppm. The first signal occurs at unusually high field, stage, the completion of the structural determination was
suggesting a highly shielded position in a macrocycle. In the prevented by strong line broadening of several 13C NMR
aliphatic region signals for 10 additional protons are pres- signals,[2] missing connectivities for the quaternary carbons
ent, which belong to an isolated methylene group (δH = 3.32 in the 2D spectra, and the propensity of retipolide A to
and 3.89, AB-q, J = 13.8 Hz) and two -CH2CH2- units. Ac- undergo decomposition.
cording to the HMBC spectra, one of these units (δH
=
These problems could be solved by conversion of retipol-
2.62/2.85 and 4.00/4.12 ppm) connects the trisubstituted ide A (1) into a red, stable methoxyfulvene, anhydroretipol-
benzene ring with an oxygen atom carrying the lactone car- ide A 6-O-methyl ether (2a), by heating with 2,2-dimeth-
bonyl group at δC = 167.8 ppm. Noticeable are the very oxypropane/p-TsOH in toluene at 75 °C (Scheme 2). In con-
different vicinal couplings of the individual protons indicat- trast to the natural product, the new compound exhibited
ing a fixed conformation. The two isolated methylene pro- sharp NMR signals for all hydrogen and carbon atoms and
tons at C-13Ј exhibit HMBC correlations with a neigh- meaningful cross peaks in the COLOC and HMBC spectra
boring quaternary carbon atom at δC = 86.8, three C atoms (Figure 2, a). In addition to the unchanged NMR signals of
of the p-phenylene unit at δC = 131.6, 132.4, and 133.1, and the macrolide ring, signals for a cyclopenta[c]pyran[3] unit
the lactone carbon at δ = 167.8 ppm. From these results carrying a methoxy substituent were clearly discernible. Of
partial structure A containing a 14-membered macrolide importance is the signal for 5-H at δH = 8.51 (d, J = 1.1 Hz,
ring incorporating a biphenyl ether moiety can be pos- δC = 146.0) exhibiting a long-range coupling to 8-H at δH
tulated for retipolide A. Structure A explains the formation = 7.12 (dd, J = 3.1, 1.1 Hz, δC = 116.9), itself coupled with
of the base peak at m/z = 225 (C15H13O2) in the mass spec- the vicinal 7-H proton at δH = 6.57 (d, J = 3.1 Hz, δC
=
trum (Figure 1) and the strong shielding of the aromatic 110.5 ppm). The NMR spectroscopic data and chemical
19Ј-hydrogen atom located under the para-disubstituted shifts are in accordance with those reported in the litera-
benzene ring.
ture.[3]
The remaining part of the structure shows NMR signals
Combining the two partial structures with the remaining
for a -CH2CH2CO- unit (δCO = 203.5), a hemiacetal group lactone carbonyl at δC = 163.0 leads to structure 2a for the
at δH = 6.49 (δC = 93.9), and five quaternary carbon atoms red methoxy derivative. The assignment of the individual
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= 203.5 ppm disappear, and new signals for olefinic hydro-
gen and carbon atoms are visible. From these spectral
changes and the appearance of a new methoxy group, struc-
ture 1 can be proposed for retipolide A.
The first intermediate in the conversion of retipolide A
(1) into methoxyfulvene 2a is methyl acetal 3, obtained in
high yield by treatment of 1 with 2,2-dimethoxypropane
and catalytic amounts of p-TsOH at room temperature. On
heating this mixture to 75 °C, acetal 3 is converted into the
stable methoxyfulvene 2a. In addition to retipolide A,
methyl acetal 3 could also be obtained as an extraction arte-
fact from the methanol extract of R. retipes/ornatipes. Ac-
cording to the NMR spectra, acetal 3 exists as a 2:1 mixture
of the two epimers.
Scheme 2. Conversion of retipolide A into anhydroretipolide A 6-
O-alkyl ethers 2.
Treatment of retipolide A (1) with acetic anhydride in the
presence of catalytic amounts of concd. H2SO4 led to the
formation of a complex mixture of acetylation products,
from which a yellow diacetate and a triacetyl compound
could be separated by preparative TLC. The similarity of
the NMR spectroscopic data of these compounds to those
of anhydroretipolide A 6-O-methyl ether (2a) allowed as-
signment of the fulvene structures 4 and 5 to these deriva-
tives. In the triacetyl compound 5 an additional C-acylation
at C-8 had occurred, the position of which follows from the
5
small J coupling of 0.3 Hz between protons 5-H and 7-
H.[3] The acetyl residue causes a remarkable down field shift
of the 13Ј-Ha signal from δH = 3.93 in diacetate 4 to
5.40 ppm in the case of 5. Molecular models indicate that
proton 13Ј-Ha is strongly exposed to the deshielding region
of the acetyl carbonyl group attached at C-8.
The high tendency to form anhydro derivatives could be
used to determine the absolute configuration of retipolide
A (1). For this purpose, the latter was heated with 2,2-
bis[(S)-sec-butoxy]propane,[4] preformed by stirring acetone
Figure 2. Selected HMBC (a) and NOE (b) relationships for anhyd-
roretipolide A 6-O-methyl ether (2a).
protons was secured by NOE experiments (Figure 2, b). Of and (S)-sec-butanol in the presence of p-TsOH and 5-Å mo-
special importance were the NOE effects observed for 19Ј- lecular sieves. The resulting (S)-sec-butoxy derivative 2b
H, which relate this H atom to all protons of the second crystallized from an acetone/MeOH mixture as orange-red
benzene ring and to 8Ј-Hb and 9Ј-Ha of the neighboring needles suitable for a single-crystal X-ray structural analysis
dimethylene unit, thereby confirming the conformation (Figure 3). From the known S configuration of the intro-
given in Figure 2. An NOE between 13Ј-Ha and 8-H at the duced ether residue, the absolute configuration of fulvene
cyclopenta[c]pyran system establishes the relative stereo- 2b could be unambiguously determined as 1R. Since the
chemistry, and NOE correlations between 13Ј-Ha and 18Ј- conversion of retipolide A (1) into 2b does not affect the
H as well as between 13Ј-Hb and 15Ј-H allow the assign- stereogenic center at C-1, retipolide A (1) must possess the
ment of these protons in the sterically fixed 1,4-disubsti- same stereochemistry.
tuted benzene ring.
The molecular structure of fulvene 2b in the crystal (Fig-
During the transformation of retipolide A (1) into anhy- ure 3) shows that the highly strained macrolide ring adopts
1
dro derivative 2a, the signals of the hemiacetal group as the same conformation as deduced from the H NMR pa-
3
well as of the -CH2CH2- unit and the carbonyl group at δC rameters in solution. Thus, the J coupling of 11.0 Hz be-
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Retipolides – Unusual Spiromacrolactones from Mushrooms
Figure 3. ORTEP plot derived from a single-crystal X-ray analysis of anhydroretipolide A 6-O-(S)-sec-butyl ether (2b).
tween 8Ј-Hb and 9Ј-Ha is in accord with an antiplanar posi-
Retipolide B (6) exhibits the same yellow color as retipol-
tion of these protons, whereas 8Ј-Ha and 9Ј-Ha exhibit no ide A (1) upon treatment with bases and shows nearly iden-
visible coupling, suggesting a dihedral angel near 90°. Due tical NMR spectra. The high resolution EI-MS indicates
to the ring strain of the macrocycle the p-phenylene ring is the molecular formula C26H20O10, containing one more
considerably bent.[5] The conformation shown in Figure 3 oxygen atom than retipolide A. The position of the ad-
also explains the strong shielding of aromatic proton 19Ј-H ditional oxygen could be located as a phenolic OH group
by the second benzene ring.
at C-5Ј, accounting for the expected NMR changes in the
The structure of retipolide A (1) accounts for the devel- benzene ring. The highly shielded proton 19Ј-H gives rise
opment of the orange-yellow color upon treatment with to a doublet of doublets at δH = 4.35 (J = 2.1, 1.0 Hz),
bases. Opening of the cyclic hemiacetal unit yields enol 1a, coupling with the meta-proton 6Ј-H, which occurs at δH
=
which forms the highly delocalized yellow anion 1b on de- 6.20 ppm (dd, J = 2.1, 0.5 Hz). The small splittings of 1.0
protonation (Scheme 3).[6] Even solutions of retipolide A and 0.5 Hz are due to J couplings with the benzylic pro-
4
(1) in acetic acid remain yellow due to an equilibrium con- tons 8Ј-Hb and 8Ј-Ha, respectively. The additional OH
centration of anion 1b. The color disappears on addition of group in the biaryl ether portion of retipolide B (6) ac-
one drop of concd. aqueous HCl, thus showing that the counts for the strong MS fragment at m/z = 241
acidity of enol 1a is comparable to that of AcOH. An equi- (C15H13O3), containing one more oxygen than that formed
librium concentration of anion 1b in the cells of R. from retipolide A (Figure 1). From the optical rotation of
retipes/ornatipes seems therefore responsible for the yellow [α]2D0 = +61 and the close agreement of the CD spectrum
color of the fruit bodies.
with that of retipolide A (1), the (1R) configuration can be
assigned to retipolide B (6).
Retipolides C (7) and D (8)
Retipolide C is a colorless, amorphous solid with a spe-
cific optical rotation of [α]2D0 = +112 and UV absorptions
at λmax = 233, 240, and 275 nm. The IR spectrum (KBr)
shows strong bands at 3410 (OH) and 1255 (C–O), and two
intense bands at 1760 and 1725 cm–1 in the carbonyl region.
The molecular formula was determined as C26H20O9 by
high-resolution electron ionization (EI) mass spectrometry.
The molecular ion appeared at m/z = 476, and the charac-
teristic fragment ion at m/z = 225 (C15H13O2) implied the
Scheme 3. Formation of the delocalized yellow anion 1b from reti-
polide A (1).
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presence of the same partial structure A as in retipolide A in the short-wavelength region of the CD spectra (Figure 5).
(1) (Figure 1). The 1H NMR spectrum of retipolide C exhi- In support of this, all of these compounds are dextrorota-
bits only 18 of the expected 20 proton signals, suggesting tory.
the presence of two exchangeable protons as OH groups. In
Surprisingly, the retipolides C and D contain a simple
the 13C NMR spectrum all 26 carbon signals are visible, 2(3H)-oxepinone residue, a heterocycle, found for the first
which can be assigned to 4 methylene, 10 methine, and 12 time in a natural product. The basic ring system has only
quaternary carbons, three of the latter representing lactone recently been synthesized,[7] and the 2-oxepinoxy radical
carbons at δC = 166.8, 167.1, and 168.5 ppm. Comparison has aroused interest as a possible intermediate in the com-
with the 1H and 13C NMR spectra of the isomeric retipolide bustion of benzene.[7]
A (1) confirmed the presence of partial structure A (Fig-
ure 1). The unknown portion of the structure contains a
Isoretipolide A (9)
disubstituted double bond, whose H atoms at δH = 6.54
and 6.83 ppm are coupled with 3J = 6.9 Hz, indicating their
position on a ring system. In addition, signals for a frag-
ment –CH2–CH=C(R)– were visible, whose olefinic proton
Several collections of R. retipes/ornatipes contain in ad-
dition to the retipolides a constituent,[8] which undergoes
rapid decomposition in solution with the formation of
black material. Extraction of the air-dried mushrooms with
acetone followed by preparative HPLC on RP 18 yields the
new compound as a pale yellow powder. This new metabo-
lite is optically active, [α]2D0 = +144, and exhibits UV/Vis
maxima resembling those of retipolide A (1). In the NMR
spectra a set of signals can be observed, which is nearly
identical to that of the cyclopenta[c]pyran moiety of retipol-
ide A (1). In contrast, the signals for the macrolide portion
exhibit characteristic differences. Again, two substituted
benzene rings are present, which, however, are both 1,2,4-
trisubstituted. A closer inspection of the HMBC spectra re-
veals that instead of the biphenyl ether group, present in all
the other retipolides, the molecule contains a 2,2Ј-dihydroxy-
biphenyl moiety embedded in the macrolide ring. Most
important for this assignment are the correlation signals be-
tween 17Ј-H and C-2Ј as well as between 18Ј-H and C-1Ј.
This leads to structure 9 for the new metabolite, which is
appropriately named isoretipolide A. The assignment of the
H atoms was established by the NOE relationships depicted
in Figure 6.
3
is coupled with J = 7.0 Hz to the protons of the allylic
2
methylene group (δH = 3.28 and 3.29 ppm, J = 12 Hz). In
addition, signals due to a tetrasubstituted double bond and
two ester groups can be recognized. HMBC experiments
(Figure 4) allowed the determination of the connectivities
of these fragments, leading to structure 7 for retipolide C.
Figure 4. Selected HMBC relationships for the oxepinone moiety
of retipolide C (7).
Retipolide D (8), [α]2D0 = +128, exhibits a molecular ion
signal at m/z = 492, corresponding to the molecular formula
C26H20O10. Comparison of the NMR spectra of 8 with
those of retipolide C (7) revealed the presence of an ad-
ditional OH group at C-5. All other spectroscopic data were
in excellent agreement with those of retipolide C, which al-
lowed the assignment of structure 8 to retipolide D. The
absolute configuration of retipolides C (7) and D (8) was
assumed to be (R), as per retipolide A (1), based on the
observation that a strong positive exciton couplet is present
Figure 6. Selected NOE relationships for isoretipolide A (9).
Structure 9 is in accordance with the formation of an
orange anhydroisoretipolide A 6-O-methyl ether (10) under
similar conditions used for the preparation of methoxyful-
vene 2a. Treatment of isoretipolide A with N-methyl(tri-
Figure 5. CD spectra of retipolides A (1) (––) and C (7) (···)
(MeCN).
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Retipolides – Unusual Spiromacrolactones from Mushrooms
methylsilyl)trifluoroacetamide (MSTFA) yields a fulvene Biosynthetic Considerations
derivative 11 containing five TMS residues. The molecular
Comparison of the structures of the different retipolides
composition C41H60O9Si5 was determined by high-resolu-
tion EI-MS. Under identical conditions, retipolide A
forms an analogous tetrakis(trimethylsilyl) derivative,
C38H52O9Si4, indicating the different kind of coupling of
the 4-hydroxyphenyl residues.
leads to the suggestion, that all of these compounds might
be formed by a biosynthetic sequence starting from the hy-
pothetical 4-hydroxyphenyl derivative 16, named retipolide
E (Scheme 4). Formally, a mechanism can be proposed, in
which this phenol is converted into an epoxide 17, which
then undergoes ring enlargement to yield the 2(3H)-ox-
epinone derivative retipolide C (7). Ring contraction of the
oxepinone ring by an O/C-acyl shift could afford formylcy-
clopentenone 18, which should rearrange into the conju-
gated isomer 1a, the ring-opened form of retipolide A (1).
The suggested precursor 16 could be formed by oxidative
phenol coupling[10] of secoretipolide E (15), which could in
turn be derived from butyrolactone II (13)[11] by transesteri-
fication with tyrosol (14). There is ample evidence that lac-
tone 13 is formed in Aspergillus terreus from two units of
3-(4-hydroxyphenyl)pyruvic acid (12), a reaction that could
According to a MacroModel[9] calculation, the 12-mem-
bered ring system of isoretipolide A (9) adopts a conforma-
tion in which the two benzene rings are less twisted than in
retipolide A (1) (Figure 7). This causes only a weak shield-
ing effect of the neighboring benzene ring upon proton 18Ј-
H (δH = 7.31 ppm). The positive optical rotation, biosyn-
thetic considerations and the similarity of the CD spectra
of isoretipolide A and retipolide A indicate that compound
9 also possesses the R configuration.
be mimicked by treatment of methyl 3-(4-hydroxyphenyl)-
[11b]
pyruvate with aqueous Na2CO3
or triethylamine.[12]
In order to test this proposal, we set out to isolate retipo-
lide E (16) from the mushroom extract. For this purpose we
synthesized retipolide E in both racemic and optically pure
form[13] and studied its behavior on column chromatog-
raphy and HPLC. Careful chromatography of the extract
from R. retipes/ornatipes and collection of all fractions with
the same Rf value as the synthetic compound finally yielded
a small amount of retipolide E (16), in every respect iden-
tical with the synthetic product, including optical rotation
[α]2D0 = +83 and CD spectrum. In addition to 16, a consider-
able amount of tyrosol (14) could be isolated.
The presence of the precursors butyrolactone II (13) and
secoretipolide E (15) in the crude mushroom extract was
confirmed using synthetic racemic mixtures. After chroma-
Figure 7. Conformation of isoretipolide A (9) according to Macro-
Model calculations.
Scheme 4. Proposal for the biosynthesis of butyrolactone II (13), secoretipolide E (15), retipolide E (16), retipolide C (7), and retipolide
A (1).
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(very strong), s (strong), m (medium), and w (weak). UV/Vis spec-
tra: Varian Cary 17 spectrophotometer. CD spectra: Jobin Yvon
Instruments S.A. CD-6-Dichrograph. NMR: Bruker instruments
AMX 600, WM 400, ARX 300, AC 200 and WH 90, in CDCl3
and [D6]acetone with solvent peak as internal standard. Multiplets
due to 1J(C,H) couplings are indicated by capital letters, 3J and
other couplings in small letters. MS: A.M.E. Kratos MS 50 with
data system DS 50, Finnigan MAT 90 and 95 Q (direct inlet,
70 eV). X-ray diffraction: Enraf–Nonius CAD4 diffractometer at
293(2) K using Mo-Kα (λ = 0.71069 Å) radiation. All solvents were
tographic enrichment of the mushroom extract, compounds
13 and 15 could be identified by analytical HPLC and
showed identical Rt values and UV spectra with the syn-
thetic references as well as no differences upon co-
chromatography. This supports the biosynthetic sequence
depicted in Scheme 3.
Interestingly, Shibata et al.[14] isolated the macrolactone
ornatipolide (19) from a Japanese collection of R. ornatipes.
This metabolite may be considered as an alternative precur-
sor of retipolide E (16), with formation of this spiro com- distilled before use. Evaporation of the solvents was performed un-
der reduced pressure using a rotary evaporator. Analytical TLC:
Silica gel 60 F254 aluminium foils (Merck); solvent system A (v/v):
toluene/HCO2Et/HCO2H, 10:5:3; B: hexanes/EtOAc, 1:1; C: hex-
anes/EtOAc, 2:1; D: hexanes/EtOAc, 3:1; E: hexanes/EtOAc/
AcOH, 60:40:1; F: hexanes/EtOAc/AcOH, 50:50:1; G: CHCl3/
MeOH, 10:1. Preparative TLC: TLC glass plates with silica gel 60
F254 2 mm (Merck). Column chromatography: Silica gel 60, 40–
63 µm (Merck), Sephadex LH-20 (Pharmacia), acetylated poly-
amide-6 (MN Polyamide SC 6-AC, Macherey–Nagel). Analytical
HPLC: Waters 600 E Pump and System Controller with Photodi-
ode Array Detector 990+. Preparative HPLC separations: Waters-
Millipore with gradient controller M 680, two M 590 EF pumps
and U 6 K injector equipped with a Knauer variable-wavelength
monitor with a super-preparative flow cell. Pre-filtration of the
solutions over SepPak RP18 cartridges (Waters). A Nucleosil 100
C18 (7 µm) pre-packed HPLC column 250ϫ20 mm with a pre-
column 30ϫ20 mm (Macherey–Nagel) and gradient systems with
MeOH/H2O mixtures were used.
pound by condensation with an equivalent of 3-(4-hy-
droxyphenyl)pyruvic acid (12), a reaction that has ample in
vitro precedence.[13] Experiments to detect ornatipolide (19)
in the North American collections of R. retipes/ornatipes by
HPLC comparison with a synthetic reference compound[13]
were, however, unsuccessful.
From a specimen of R. retipes/ornatipes with an un-
usually dark brown cap skin, we were able to identify xero-
comic acid (20)[15] by HPLC comparison. Pulvinic acids such
as 20 are characteristic pigments of the genus Boletus,[16]
and their occurrence in Retiboletus signals the close rela-
tionship of both genera. More detailed chemotaxonomic in-
vestigations of the genus Retiboletus and the detection of
retipolides in Retiboletus flavoniger (Halling) Binder & Bre-
sinsky from Costa Rica will be reported in a separate publi-
cation.
Mushrooms: R. retipes/ornatipes was collected in September 1986
and in August/September 1990 and 1991 around the “Ball Creek
area”, Coweeta Hydrologic Laboratory, Macon County, NC, USA
(leg. et det. W. Steglich). For the isolation of isoretipolide A, collec-
tions from the White Mountains National Park, NH, were used
(leg. et det. N. Arnold, 1999).
Isolation Procedure for the Retipolides A–D: Air- and freeze-dried
fruit bodies of R. retipes/ornatipes (50 g) were pulverized and defat-
ted with 3ϫ400 mL of petroleum ether (60–80 °C). Extraction of
the material with EtOAc (3ϫ400 mL) yielded a deep orange solu-
tion. The volume of the combined extracts was reduced to 200 mL
with a rotary evaporator (30 °C, 20 Torr) and the resulting solution
washed with water (100 mL), dried (MgSO4), and concentrated to
yield a brown residue, which was triturated with hexanes (50 mL).
The crude extract (1.20 g) was prepurified by gel chromatography
on Sephadex LH-20 at 8 °C with acetone/toluene (9:1) as eluent to
yield 4 fractions. Fraction 1 (86 mg) contained crude retipolide C
as a brown solid; purification by column chromatography at 8 °C
Conclusions
The chemical investigation of Retiboletus retipes/ornat-
ipes revealed the presence of several spiromacrolides, thus
underlining the unique position of this genus in Boletales
taxonomy. All of these compounds are biosynthetically
closely related, including an unprecedented example for the
biochemical conversion of a 4-hydroxyphenyl ring into a on acetylated polyamide with acetone gave retipolide C (7) (56 mg,
0.11% relative to dry weight). Fraction 2 (230 mg) yielded retipol-
ide A (1) as a microcrystalline powder (0.46%). Fraction 3 (73 mg)
contained a mixture of retipolide D with retipolide A, which was
separated by preparative TLC on silica gel (hexanes/EtOAc/AcOH,
30:60:1) to give retipolide D as a yellow glass (22 mg). Rechromato-
graphy of the latter on a Sephadex LH-20 column at 8 °C with
acetone yielded pure retipolide D (8) (7 mg, 0.014%). Fraction 4
(138 mg) contained mainly retipolide B (6), which was obtained
in pure form (52 mg, 0.10%) by rechromatography on acetylated
polyamide with acetone at 8 °C.
2(3H)-oxepinone moiety. The proposed biosynthetic pre-
cursor retipolide E (16) and its congeners 13–15 were iden-
tified in the fungal extract in small amounts by comparison
with synthetic samples. This technique for establishing bio-
synthetic relationships might also be useful in other
cases.[17]
Experimental Section
The retention times and UV data for an HPLC analysis of a retipo-
lide mixture under standardized conditions are given in Table 1
[Nucleosil 100 C18, 5 µm, 250ϫ4.6 mm (Merck); solvent A: H2O/
MeCN, 9:1 + 0.5% TFA; solvent B: H2O/MeCN, 1:9; gradient:
General: Melting points (uncorrected): Reichert Thermovar hot-
stage. Optical rotations: Perkin–Elmer 241. IR: Perkin–Elmer 1420
Ratio Recording Infrared Spectrometer. Intensity of the bands: ss
5566
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 5560–5572

Retipolides – Unusual Spiromacrolactones from Mushrooms
start 90% A + 10% B, 55 min: 10% A + 90% B, 65 min: 10% A
and 90% B, 75 min: 90% A and 10% B; flow rate 1 mL/min; diode
array detection, 210–500 nm].
Retipolide B, (1R,5RS)-7,8-Dihydro-4Ј,5,5Ј-trihydroxyspiro[cyclo-
penta[d]furo[3,4-b]pyran-1,12Ј(5H)-[2,10]dioxatricyclo[12.2.2.13,7]-
nonadeca[1(16),3,5,7(19),14,17]hexaene]-3,6,11Ј-trione (6): Yellow-
ish, amorphous powder; easily soluble in acetone, sparingly soluble
in CHCl3, decomposes in MeOH; m.p. Ͼ 360 °C (the sample dark-
ens above 190 °C). Rf (TLC) = 0.39 (solvent system A), colorless
spot, + NH3 red-orange. [α]2D0 = +61 (c = 0.1, CHCl3). UV/Vis
(MeCN): λmax (log ε) = 195 (4.71), 215 sh (4.53), 317 (3.91), 436
(3.19) nm. CD (MeCN): λmax (∆ε) = 218 (0), 228 (+26.49), 270
Table 1. HPLC analysis of a retipolide mixture.
Retipolide
UV/Vis spectrum
Retention time
λmax (% rel. int.)
tR [min]
Retipolide B (6)
Retipolide D (8)
Retipolide A (1)
219 (100), 327 (33)
213 (100), 276 (27), 305 (sh, 5)
216 (100), 327 (35)
20.79
22.99
25.14
26.74
(+4.42), 380 (0) nm. IR (KBr): ν = 3400 (s, br), 2980 (m), 2940
˜
(m), 2880 (w), 1790 (ss), 1740 (s), 1700 (s), 1650 (m), 1620 (m),
1600 (m), 1540 (m), 1530 (m), 1510 (m), 1450 (m), 1370 (m), 1340
(m), 1320 (m), 1270 (s), 1220 (s), 1190 (s), 1170 (s), 1150 (s), 1110
(s), 1030 (s), 970 (w), 950 (w), 920 (w), 900 (w), 880 (w), 840 (w),
820 (w), 790 (w), 760 (w), 740 (w), 720 (w), 710 (w), 690 (w), 680
Retipolide E (16) 213 (100), 229 (sh, 81),
289 (sh, 35), 306 (36)
Retipolide C (7)
213 (100), 238 (sh, 64), 278 (29) 27.24
1
(w), 640 (w), 610 (w) cm–1. H NMR (400 MHz, [D6]acetone): δ =
2.49 (dddd, J = 17.3, 5.1, 0.7, 0.5 Hz, 1 H, 8Ј-Ha), 2.58 (m, 2 H,
7-H), 2.80 (dddd, J = 17.3, 10.9, 1.8, 1.0 Hz, 1 H, 8Ј-Hb), 3.26+
(ddd, J = 18.5, 4.5, 4.5 Hz, 1 H, 8-Ha), 3.30 (d, J = 13.9 Hz, 1 H,
13Ј-Hb), 3.38+ (br. m, 1 H, 8-Hb), 3.87 (d, J = 13.9 Hz, 1 H, 13Ј-
Ha), 3.96 (ddd, J = 11.4, 5.1, 1.8 Hz, 1 H, 9Ј-Hb), 4.13 (ddd, J =
11.4, 10.9, 0.7 Hz, 1 H, 9Ј-Ha), 4.35 (dd, J = 2.1, 1.0 Hz, 1 H, 19Ј-
H), 6.20 (dd, J = 2.1, 0.5 Hz, 1 H, 6Ј-H), 6.50 (br. s, 1 H, 5-H),
7.00 (dd, J = 8.5, 2.4 Hz, 1 H, 17Ј-H), 7.21 (dd, J = 8.5, 2.4 Hz, 1
H, 16Ј-H), 7.31 (dd, J = 8.5, 2.2 Hz, 1 H, 18Ј-H), 7.63 (dd, J = 8.5,
Retipolide A, (1R,5RS)-7,8-Dihydro-4Ј,5-dihydroxyspiro[cyclopenta-
[d]furo[3,4-b]pyran-1,12Ј(5H)-[2,10]dioxatricyclo[12.2.2.13,7]nona-
deca[1(16),3,5,7(19),14,17]hexaene]-3,6,11Ј-trione (1): White to yel-
lowish, amorphous powder; easily soluble in acetone, sparingly sol-
uble in CHCl3, decomposes in MeOH; m.p. Ͼ 360 °C (the sample
darkens above 200 °C). Rf (TLC) = 0.47 (solvent system A), color-
less spot, + NH3 orange-yellow. [α]2D0 = +160 (c = 0.1, CHCl3). UV/
Vis (MeCN): λmax (log ε) = 208 sh (4.20), 238 sh (3.71), 288 (3.44),
316 (3.59), 390 (3.04), 428 (3.13) nm; (MeCN + NH3): 244 (3.96),
284 (3.50), 312 (3.40), 392 (3.62), 429 (3.71) nm; (MeOH + HCl):
208 sh (4.21), 237 sh (3.86), 270 (3.52), 284 (3.49), 322 (3.59) nm.
CD (MeCN): λmax (∆ε) = 215 (0), 227 (+25.90), 268 (+4.70), 280
1
2.2 Hz, 1 H, 15Ј-H) ppm (+ assignments are interchangeable). H-
coupled 13C NMR (100.6 MHz, [D6]DMSO/[D6]acetone, 3:2): δ =
26.1 (br. Tm, J = 136 Hz, C-8), 32.1 (Tm, J = 127 Hz, C-8Ј), 35.8
(Tm, J = 132 Hz, C-7), 43.9 (br. Tm, J = 134 Hz, C-13Ј), 69.7 (Td,
J = 150, 9 Hz, C-9Ј), 87.6 (d, J = 7 Hz, C-1), 94.7 (br. Dm, J =
180 Hz, C-5), 107.7 (Dt, J = 160, 7 Hz, C-19Ј), 109.6 (Dm, J =
158 Hz, C-6Ј), 125.2 (Dd, J = 162, 5 Hz, C-16Ј), 125.4 (Dd, J =
162, 5 Hz, C-17Ј), 128.0 (br. m, C-8b), 131.5 (m, C-7Ј), 132.4§ (br.
m, C-4Ј), 132.5§ (br. m, C-3a), 132.5 (m, C-14Ј), 133.1 (Dq, J =
161, 6 Hz, C-18Ј), 133.8 (Dtd, J = 163, 8, 4 Hz, C-15Ј), 144.6 (d, J
= 6 Hz, C-5Ј), 146.6 (m, C-5a), 152.9 (d, J = 3 Hz, C-3Ј), 158.6 (br.
m, C-8a), 159.9 (tt, J = 10, 5 Hz, C-1Ј), 163.9 (s, C-3), 168.7 (br.
m, C-11Ј), 203.8 (m, C-6) ppm (§ assignments are exchangeable).
EI-MS (DI, 180 °C): m/z (%) = 492 (1) [M+], 242 (11), 241 (17,
C15H13O3), 121 (12), 120 (20), 118 (8), 107 (31), 85 (17), 83 (26),
44 (100, CO2). C26H20O10 [M+]: calcd. 492.1056; found 492.1039.
(0), 285 (–1.03), 291 (0), 302 (+1.47), 385 (0) nm. IR (KBr): ν =
˜
3400 (m, br), 2920 (m), 2860 (w), 1785 (ss), 1730 (s), 1700 (s), 1650
(m), 1590 (m), 1520 (m), 1500 (m), 1430 (m), 1380 (m), 1370 (w),
1340 (w), 1270 (s), 1210 (m), 1180 (w), 1160 (m), 1140 (m), 1100
(m), 1030 (m), 1000 (w), 980 (m), 930 (w), 920 (w), 880 (w), 840
(w), 820 (w), 780 (w), 750 (w), 740 (w), 610 (w) cm–1. 1H NMR
(400 MHz, [D6]acetone): δ = 2.58 (m, 2 H, 7-H), 2.62 (ddd, J =
17.1, 5.2, 0.8 Hz, 1 H, 8Ј-Ha), 2.85 (dddd, J = 17.1, 11.0, 1.8,
0.9 Hz, 1 H, 8Ј-Hb), 3.29 (dddd, J = 18.5, 4.5, 4.5, 1.0 Hz, 1 H, 8-
Ha), 3.32 (d, J = 13.8 Hz, 1 H, 13Ј-Hb), 3.38 (ddd, J = 18.5, 4.5,
4.5 Hz, 1 H, 8-Hb), 3.89 (d, J = 13.8 Hz, 1 H, 13Ј-Ha), 4.00 (ddd,
J = 11.1, 5.2, 1.8 Hz, 1 H, 9Ј-Hb), 4.12 (dd, J = 11.1, 11.0 Hz, 1
H, 9Ј-Ha), 4.83 (dd, J = 2.1, 0.9 Hz, 1 H, 19Ј-H), 6.49 (br. s, 1 H,
5-H), 6.51 (ddd, J = 8.0, 2.1, 0.8 Hz, 1 H, 6Ј-H), 6.70 (d, J = 8.0 Hz,
1 H, 5Ј-H), 7.00 (dd, J = 8.5, 2.2 Hz, 1 H, 17Ј-H), 7.19 (br. s, 1 H,
4Ј- or 5-OH), 7.24 (dd, J = 8.5, 2.2 Hz, 1 H, 16Ј-H), 7.35 (dd, J =
8.5, 2.2 Hz, 1 H, 18Ј-H), 7.67 (dd, J = 8.5, 2.2 Hz, 1 H, 15Ј-H),
7.97 (br. s, 1 H, 4Ј- or 5-OH) ppm. 1H-coupled 13C NMR
Retipolide C, (R)-3Ј-(2,3-Dihydro-2-oxo-5-oxepinyl)-4,4Ј-dihydroxy-
spiro[2,10-dioxatricyclo[12.2.2.13,7]nonadeca-1(16),3,5,7(19),14,17-
hexaene-12,2Ј(5ЈH)-furan]-11,5Ј-dione (7): Colorless amorphous so-
lid; easily soluble in acetone, sparingly soluble in CHCl3; m.p. 100–
(100.6 MHz, [D6]DMSO/[D6]acetone, 3:2): δ = 25.3 (br. Tm, J = 105 °C. Rf (TLC) = 0.51 (solvent system A), colorless spot, + NH3
135 Hz, C-8), 30.9 (Tm, J = 126 Hz, C-8Ј), 35.2 (Tm, J = 133 Hz, pink. [α]2D0 = +112 (c = 0.1, CHCl3). UV/Vis (MeCN): λmax (log ε)
C-7), 42.7 (br. Tm, J = 132 Hz, C-13Ј), 69.9 (Td, J = 150, 9 Hz, C-
9Ј), 86.8 (d, J = 8 Hz, C-1), 93.9 (br. Dm, J = 180 Hz, C-5), 115.2
(Ddd, J = 158, 7, 6 Hz, C-19Ј), 115.9 (Dd, J = 155, 4 Hz, C-5Ј),
= 233 sh (4.31), 240 sh (4.23), 275 (4.03) nm. CD (MeCN): λmax
(∆ε) = 250 (+21.04), 313 (0), 330 (–0.61), 355 (–0.37), 390 (–0.72),
460 (0) nm. IR (KBr): ν = 3410 (s, br), 2460 (w), 2420 (w), 1760
˜
121.3 (Ddm, J = 158, 6 Hz, C-6Ј), 124.4 (Dd, J = 162, 5 Hz, C- (s, br), 1725 (s), 1670 (w), 1630 (w), 1590 (w), 1515 (m), 1500 (m),
16Ј), 124.5 (Dd, J = 162, 6 Hz, C-17Ј), 127.0 (br. m, C-8b), 130.6 1455 (w), 1430 (w), 1415 (w, sh), 1390 (w), 1345 (w), 1310 (w, sh),
(m, C-7Ј), 131.6 (m, C-14Ј), 131.7 (br. m, C-3a), 132.4 (Dq, J = 1255 (s), 1205 (m), 1160 (m), 1105 (m), 1090 (m, sh), 1060 (w, sh),
162, 6 Hz, C-18Ј), 133.1 (Dtd, J = 163, 8, 4 Hz, C-15Ј), 143.5 (ddd,
J = 7, 6, 2 Hz, C-4Ј), 143.9 (m, C-5a), 151.5 (dd, J = 8, 4 Hz, C-
3Ј), 157.6 (br. m, C-8a), 158.4 (tt, J = 10, 4 Hz, C-1Ј), 163.4 (s, C-
3), 167.8 (br. d, J = 8 Hz, C-11Ј), 203.5 (m, C-6) ppm. EI-MS (DI,
1030 (w), 1005 (w, sh), 975 (w), 945 (w), 920 (w), 875 (w), 840 (w),
1
770 (w), 720 (w), 615 (w) cm–1. H NMR (400 MHz, [D6]acetone):
δ = 2.58 (dd, J = 17.2, 5.1 Hz, 1 H, 8-Ha), 2.82 (dddd, J = 17.2,
11.0, 1.8, 0.8 Hz, 1 H, 8-Hb), 3.16 (d, J = 13.8 Hz, 1 H, 13-Hb),
200 °C): m/z (%) = 477 (4) [M + 1]+, 476 (15) [M+], 459 (14), 458 3.28/3.29 (each dd, J = 12.0, 7.0 Hz, 1 H, 3ЈЈ-Ha and 3ЈЈ-Hb), 3.90
(43), 432 (13), 430 (5), 414 (6), 386 (5), 227 (50), 226 (80), 225 (100)
[C15H13O2], 212 (11), 120 (45) [C8H8O], 107 (37) [C7H7O], 91 (42) 9-Hb), 4.15 (dd, J = 11.1, 11.0 Hz, 1 H, 9-Ha), 4.77 (dd, J = 2.0,
C7H7], 84 (48), 82 (63). C26H20O9 [M+]: calcd. 476.1107; found
0.8 Hz, 1 H, 19-H), 6.49 (dd, J = 8.0, 2.0 Hz, 1 H, 6-H), 6.54 (d, J
476.1111. C26H20O9 (476.4): calcd. C 65.55, H 4.32; found C 65.65, = 6.9 Hz, 1 H, 6ЈЈ-H), 6.64 (t, J = 7.0 Hz, 1 H, 4ЈЈ-H), 6.70 (d, J
(d, J = 13.8 Hz, 1 H, 13-Ha), 3.92 (ddd, J = 11.1, 5.1, 1.8 Hz, 1 H,
H 4.45.
= 8.0 Hz, 1 H, 5-H), 6.83 (d, J = 6.9 Hz, 1 H, 7ЈЈ-H), 6.95 (dd, J
Eur. J. Org. Chem. 2007, 5560–5572
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5567

W. Steglich et al.
FULL PAPER
= 8.4, 2.5 Hz, 1 H, 17-H), 7.19 (dd, J = 8.4, 2.5 Hz, 1 H, 16-H),
7.30 (dd, J = 8.4, 2.4 Hz, 1 H, 18-H), 7.61 (dd, J = 8.4, 2.4 Hz, 1
175 °C. Rf (TLC) = 0.61 (solvent system A), 0.52 (solvent system
B), 0.38 (solvent system C), orange spot. tR (HPLC) = 38.03 min,
H, 15-H) ppm. 1H-coupled 13C NMR (100.6 MHz, [D6]DMSO/ conditions as given for Table 1. [α]2D0 = –196 (c = 0.14, CHCl3). UV/
[D6]acetone, 3:2): δ = 32.1 (Tm, J = 127 Hz, C-8), 36.9 (Td, J =
136, 5 Hz, C-3ЈЈ), 43.9 (Tt, J = 134, 4 Hz, C-13), 69.1 (Td, J = 150,
8 Hz, C-9), 88.8 (d, J = 8 Hz, C-12), 114.4 (Dt, J = 166, 10 Hz, C-
6ЈЈ), 115.9 (Ddd, J = 159, 7, 6 Hz, C-19), 116.3 (Dd, J = 156, 3 Hz,
Vis (MeCN): λmax (log ε) = 250 (4.44), 272 sh (4.06), 374 sh (3.95),
387 (4.00) nm. CD (MeCN): λmax (∆ε) = 228 (+8.43), 234 (0), 242
(–14.37), 261 (0), 283 (+3.08), 309 (0), 385 (–2.20), 450 (0) nm. IR
(KBr): ν = 3420 (s, br), 1770 (ss), 1720 (s), 1620 (w), 1590 (s), 1550
˜
C-5), 122.3 (Dddd, J = 156, 10, 6, 4 Hz, C-6), 124.8 (Dd, J = 163, (m), 1510 (m, sh), 1500 (s), 1495 (m, sh), 1420 (m), 1400 (w), 1370
5 Hz, C-16), 124.9 (Dd, J = 162, 5 Hz, C-17), 126.9 (Dm, J = (w), 1350 (w), 1260 (s), 1200 (m), 1100 (m), 1070 (m), 1040 (w),
170 Hz, C-4ЈЈ), 127.0 (m, C-3Ј), 130.0 (m, C-5ЈЈ), 132.4 (m, C-7), 1010 (s), 970 (m), 870 (m), 795 (m) cm–1. 1H NMR (400 MHz, [D6]-
132.9 (m, C-14), 133.7 (Dq, J = 162, 6 Hz, C-18), 134.4 (Dtd, J = acetone): δ = 2.59 (dddd, J = 17.1, 5.1, 0.8, 0.7 Hz, 1 H, 8Ј-Ha),
162, 8, 4 Hz, C-15), 141.8 (m, C-4), 142.0 (Dd, J = 198, 6 Hz, C- 2.82 (dddd, J = 17.1, 11.2, 1.8, 1.0 Hz, 1 H, 8Ј-Hb), 3.32 (d, J =
7ЈЈ), 144.5 (m, C-4), 152.3 (m, C-3), 159.3 (tt, J = 10, 4 Hz, C-1),
166.8 (m, C-2ЈЈ), 167.1 (br. s, C-5Ј), 168.5 (dd, J = 10, 3 Hz, C-11)
ppm. EI-MS (DI, 230 °C): m/z (%) = 477 (0.2) [M + 1]+, 476 (0.4)
[M+], 300 (8), 226 (12), 225 (20), 122 (5), 121 (10), 120 (10), 107
(14), 91 (14), 44 (100). C26H20O9 [M+]: calcd. 476.1107; found
476.1107.
13.8 Hz, 1 H, 13Ј-Hb), 3.96 (s, 3 H, OMe), 3.93 (ddd, J = 11.5, 5.1,
1.8 Hz, 1 H, 9Ј-Hb), 3.99 (d, J = 13.8 Hz, 1 H, 13Ј-Ha), 4.17 (ddd,
J = 11.5, 11.2, 0.7 Hz, 1 H, 9Ј-Ha), 4.87 (dd, J = 2.1, 1.0 Hz, 1 H,
19Ј-H), 6.51 (ddd, J = 8.0, 2.1, 0.8 Hz, 1 H, 6Ј-H), 6.57 (d, J =
3.1 Hz, 1 H, 7-H), 6.71 (d, J = 8.0 Hz, 1 H, 5Ј-H), 7.04 (dd, J =
8.5, 2.5 Hz, 1 H, 17Ј-H), 7.12 (dd, J = 3.1, 1.1 Hz, 1 H, 8-H), 7.26
(dd, J = 8.5, 2.5 Hz, 1 H, 16Ј-H), 7.45 (dd, J = 8.5, 2.5 Hz, 1 H,
18Ј-H), 7.71 (dd, J = 8.5, 2.5 Hz, 1 H, 15Ј-H), 8.02 (br. s, 4Ј-OH),
8.51 (d, J = 1.1 Hz, 1 H, 5-H) ppm. 1H-coupled 13C NMR
(100.6 MHz, [D6]acetone): δ = 31.9 (Tm, J = 127 Hz, C-8Ј), 44.8
(Tt, J = 134, 4 Hz, C-13Ј), 58.1 (Q, J = 145 Hz, OMe), 69.5 (Td,
J = 150, 8 Hz, C-9Ј), 87.5 (d, J = 8 Hz, C-1), 110.5 (Dd, J = 168,
6 Hz, C-7), 110.5 (ddd, J = 10, 6, 3 Hz, C-8a), 116.1 (Dt, J = 158,
8 Hz, C-19Ј), 116.4 (Dd, J = 156, 3 Hz, C-5Ј), 116.9 (Dd, J = 174,
5 Hz, C-8), 119.7 (q, J = 8 Hz, C-5a), 122.2 (Dm, J = 156 Hz, C-
6Ј), 125.1 (Dd, J = 163, 5 Hz, C-16Ј), 125.2 (Dd, J = 163, 5 Hz, C-
17Ј), 131.9 (d, J = 7 Hz, C-3a), 132.3 (m, C-7Ј), 132.8 (m, C-14Ј),
133.5 (Dq, J = 161, 6 Hz, C-18Ј), 134.0 (Dtd, J = 163, 8, 3 Hz, C-
15Ј), 137.0 (d, J = 2 Hz, C-8b), 144.6 (m, C-4Ј), 146.0 (Dd, J =
203, 2 Hz, C-5), 152.4 (m, C-3Ј), 154.4 (m, C-6), 159.6 (tt, J = 10,
4 Hz, C-1Ј), 163.0 (s, C-3), 168.0 (d, J = 8 Hz, C-11Ј) ppm. EI-MS
(DI, 200 °C): m/z (%) = 474 (6) [M + 2]+, 473 (32) [M + 1]+, 472
(100) [M+], 445 (13), 444 (47), 429 (7), 401 (8), 249 (11), 236 (6),
226 (10), 225 (32), 173 (8), 120 (28), 119 (7), 107 (11), 91 (26), 44
(13), 32 (64). C27H20O8 [M+]: calcd. 472.1158; found 472.1153.
Retipolide D, (R)-3Ј-(2,3-Dihydro-2-oxo-5-oxepinyl)-4,4Ј,5-trihy-
droxyspiro[2,10-dioxatricyclo[12.2.2.13,7]nonadeca-1(16),3,5,7(19),
14,17-hexaene-12,2Ј(5ЈH)-furan]-11,5Ј-dione (8): Colorless amorph-
ous solid; easily soluble in acetone, sparingly soluble in CHCl3;
m.p. 135–140 °C. Rf (TLC) = 0.46 (solvent system A), 0.28 (solvent
system F), colorless spot, + NH3 pink. [α]2D0 = +128 (c = 0.14,
CHCl3). UV/Vis (MeCN): λmax (log ε) = 272 (3.92), 280 sh (3.89),
295 sh (3.69) nm. CD (MeCN): λmax (∆ε) = 255 (+16.21), 356 (0),
385 (–0.08), 470 (0) nm. IR (KBr): ν = 3430 (s, br), 3100 (w, sh),
˜
2940 (w), 1760 (s, br), 1730 (s), 1610 (w), 1590 (m), 1535 (w, sh),
1520 (w), 1500 (m), 1440 (w), 1390 (w), 1250 (m), 1200 (m), 1160
(m), 1100 (m), 1075 (w, sh), 1010 (m), 970 (w), 875 (w), 835 (w),
830 (w, sh), 810 (w), 785 (w), 770 (w), 760 (w, sh), 740 (w), 650 (w)
cm–1. 1H NMR (400 MHz, [D6]acetone): δ = 2.48 (dd, J = 17.2,
5.3 Hz, 1 H, 8-Ha), 2.78 (ddd, J = 17.2, 11.2, 1.5 Hz, 1 H, 8-Hb),
3.13 (d, J = 13.5 Hz, 1 H, 13-Hb), 3.25/3.29 (each dd, J = 12.1,
7.3 Hz, 1 H, 3ЈЈ-Ha and 3ЈЈ-Hb), 3.89 (d, J = 13.5 Hz, 1 H, 13-Ha),
3.88 (ddd, J = 11.3, 5.3, 1.5 Hz, 1 H, 9-Hb), 4.17 (dd, J = 11.3,
11.2 Hz, 1 H, 9-Ha), 4.30 (d, J = 2.0 Hz, 1 H, 19-H), 6.20 (d, J =
2.0 Hz, 1 H, 6-H), 6.55 (d, J = 7.1 Hz, 1 H, 6ЈЈ-H), 6.64 (t, J =
7.3 Hz, 1 H, 4ЈЈ-H), 6.83 (d, J = 7.1 Hz, 1 H, 7ЈЈ-H), 6.95 (dd, J =
Preparation of Methoxyfulvene 2a from the Crude Mushroom Ex-
tract: The crude extract from Retiboletus retipes/ornatipes (600 mg)
8.5, 2.5 Hz, 1 H, 17-H), 7.16 (dd, J = 8.5, 2.5 Hz, 1 H, 16-H), 7.27 was dissolved in MeOH (2 mL), treated with a 1% solution of
(dd, J = 8.5, 2.5 Hz, 1 H, 18-H), 7.58 (dd, J = 8.5, 2.5 Hz, 1 H,
TsOH·H2O in 2,2-dimethoxypropane (2.5 mL), and heated with
stirring for 20 min to 75 °C (bath temperature). After cooling to
room temperature, the solution was immediately flash chromato-
graphed on silica gel (hexanes/EtOAc, 1:1) to yield a red oil. Re-
1
15-H) ppm. H-coupled 13C NMR (100.6 MHz, [D6]acetone): δ =
32.3 (Tm, J = 128 Hz, C-8), 36.9 (Td, J = 135, 5 Hz, C-3ЈЈ), 43.9
(Tt, J = 134, 5 Hz, C-13), 69.0 (Td, J = 150, 8 Hz, C-9), 88.7 (d, J
= 7 Hz, C-12), 107.5 (Dtd, J = 160, 8, 2 Hz, C-19), 109.6 (Dm, J peated flash chromatography (hexanes/EtOAc, 2:1) afforded 2a
= 157 Hz, C-6), 114.4 (Dt, J = 164, 10 Hz, C-6ЈЈ), 124.8 (Dd, J =
162, 6 Hz, C-16), 125.0 (Dd, J = 162, 5 Hz, C-17), 126.5 (Dm, J =
164 Hz, C-4ЈЈ), 126.5 (m, C-3Ј), 130.2 (m, C-5ЈЈ), 131.7 (m, C-7),
132.5 (m, C-4), 132.8 (m, C-14), 133.5 (Dq, J = 162, 6 Hz, C-18),
134.2 (Dtd, J = 163, 8, 3 Hz, C-15), 141.9 (Dd, J = 198, 6 Hz, C-
7ЈЈ), 142.4 (m, C-4Ј), 146.6 (m, C-5), 152.9 (m, C-3), 159.6 (tt, J =
10, 4 Hz, C-1), 166.8 (m, C-2ЈЈ), 167.3 (s, C-5Ј), 168.5 (d, J = 8 Hz,
C-11) ppm. EI-MS (DI, 230 °C): m/z (%) = 492 (0.9) [M+], 107 (8),
44 (100). C26H20O10 [M+]: calcd. 492.1056; found 492.1060.
(118 mg).
Synthesis of Anhydroretipolide A 6-O-(S)-sec-Butyl Ether (2b): A
mixture of acetone (167 mg, 210 µL, 2.88 mmol), (S)-2-butanol
(850 mg, 1050 µL, 11.5 mmol), cyclohexane (4.3 mL), p-toluenesul-
fonic acid mono hydrate (109 mg, 0.57 mmol), and activated pul-
verized molecular sieves (5 Å) (1.1 g) was stirred for 2 h at 0 °C.
After removal of the molecular sieves by filtration and concentra-
tion with a rotary evaporator (20 °C, 20 Torr), the remaining dark-
violet solution was treated with 1 (100 mg, 0.210 mmol) and stirred
Synthesis of Anhydroretipolide A 6-O-Methyl Ether (2a): A stirred for 1 h at 80 °C (bath temperature). The mixture was cooled to
solution of 1 (86 mg, 0.181 mmol) in MeOH (0.5 mL) was treated
with a 1 % solution of TsOH·H2O in 2,2-dimethoxypropane
(2.5 mL) and heated for 20 min at 75 °C (bath temperature). After
cooling to room temperature, the dark red solution was immedi-
ately flash chromatographed (hexanes/EtOAc, 1:1) to yield a red
oil (97 mg). Purification by chromatography on Sephadex LH-20
with acetone afforded 2a (52 mg, 61%) as an orange-red crystalline
solid, which was easily soluble in acetone or chloroform; m.p. 170–
room temperature and flash chromatographed (hexanes/EtOAc,
1:1) to yield a red oil (100 mg), which was adsorbed on silica gel
(0.40 g). Flash chromatography on silica gel (hexanes/EtOAc, 3:1)
afforded 2b (22 mg, 20%) as orange red crystals, easily soluble in
acetone and CHCl3; m.p. 305 °C. Crystals suitable for the X-ray
crystal structure determination were obtained by dissolving 2b
(22 mg) in acetone (2 mL), adding MeOH (2 mL), and keeping the
probe for 4 weeks at room temperature, which allowed the acetone
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Retipolides – Unusual Spiromacrolactones from Mushrooms
to slowly evaporate. Rf (TLC) = 0.72 (solvent system A), 0.77 (sol-
vent system B), 0.39 (solvent system D), orange spot. [α]2D0 = –160
(c = 0.43, CHCl3). UV/Vis (MeCN): λmax (log ε) = 241 (4.26), 273
(200 MHz, CDCl3): δ = 2.37, 2.40 (each s, 3 H, CH3CO), 2.58
(ddm, J = 17.1, 5.2 Hz, 1 H, 8Ј-Ha), 3.04 (ddm, J = 17.1, 10.6 Hz,
1 H, 8Ј-Hb), 3.30 (d, J = 13.8 Hz, 1 H, 13Ј-Hb), 3.88 (ddd, J =
(4.00), 391 (3.97) nm. CD (MeCN): λmax (∆ε) = 236 (+25.31), 244 11.5, 5.2, 1.5 Hz, 1 H, 9Ј-Hb), 3.93 (d, J = 13.8 Hz, 1 H, 13Ј-Ha),
(0), 253 (–21.11), 263 (0), 283 (+3.81), 324 (0), 392 (–2.14), 465 4.28 (dd, J = 11.5, 10.6 Hz, 1 H, 9Ј-Ha), 4.97 (dd, J = 2.1, 0.9 Hz,
(–0.04), 490 (–0.16), 550 (0) nm. 1H NMR (400 MHz, [D6]acetone):
1 H, 19Ј-H), 6.60 (ddd, J = 8.1, 2.1, 0.4 Hz, 1 H, 6Ј-H), 6.89 (d, J
δ = 1.00 (t, J = 7.5 Hz, 3 H, 4ЈЈ-H), 1.37 (d, J = 6.2 Hz, 3 H, 1ЈЈ- = 8.1 Hz, 1 H, 5Ј-H), 7.04 (dd, J = 8.3, 2.4 Hz, 1 H, 17Ј-H), 7.21
H), 1.70/1.80 [each dqd, J = 14.3, 7.5, 6.0 (6.4) Hz, 2 H, 3ЈЈ-H],
2.59 (dd, J = 17.3, 5.2 Hz, 1 H, 8Ј-Ha), 2.87 (dddd, J = 17.3, 11.0,
1.7, 0.8 Hz, 1 H, 8Ј-Hb), 3.30 (d, J = 13.8 Hz, 1 H, 13Ј-Hb), 3.94
(ddd, J = 11.1, 5.2, 1.7 Hz, 1 H, 9Ј-Hb), 3.99 (d, J = 13.8 Hz, 1 H,
13Ј-Ha), 4.18 (dd, J = 11.1, 11.0 Hz, 1 H, 9Ј-Ha), 4.44 (dqd, J =
6.4, 6.2, 6.0 Hz, 1 H, 2ЈЈ-H), 4.87 (dd, J = 2.1, 0.8 Hz, 1 H, 19Ј-
H), 6.51 (dd, J = 8.0, 2.1 Hz, 1 H, 6Ј-H), 6.55 (d, J = 3.2 Hz, 1 H,
7-H), 6.71 (d, J = 8.0 Hz, 1 H, 5Ј-H), 7.04 (dd, J = 8.4, 2.5 Hz, 1
H, 17Ј-H), 7.12 (dd, J = 3.2, 1.1 Hz, 1 H, 8-H), 7.26 (dd, J = 8.4,
2.5 Hz, 1 H, 16Ј-H), 7.44 (dd, J = 8.4, 2.3 Hz, 1 H, 18Ј-H), 7.71
(dd, J = 8.4, 2.3 Hz, 1 H, 15Ј-H), 8.02 (br. s, 4Ј-OH), 8.50 (d, J =
1.1 Hz, 1 H, 5-H) ppm. 13C NMR (100.6 MHz, [D6]acetone): δ =
(dd, J = 3.3, 1.2 Hz, 1 H, 8-H), 7.25 (dd, J = 3.3, 0.4 Hz, 1 H, 7-
H), 7.26 (dd, J = 8.5, 2.4 Hz, 1 H, 16Ј-H), 7.34 (dd, J = 8.3, 2.3 Hz,
1 H, 18Ј-H), 7.68 (dd, J = 8.5, 2.3 Hz, 1 H, 15Ј-H), 8.33 (dd, J =
1.2, 0.4 Hz, 1 H, 5-H) ppm. 13C NMR (100.6 MHz, [D6]acetone):
δ = 20.8 (CH3), 21.1 (CH3), 31.9 (CH2), 44.9 (CH2), 68.6 (CH2),
87.2 (C), 112.9 (C), 115.4 (CH), 116.7 (CH), 119.5 (C), 121.5 (CH),
122.3 (CH), 124.4 (CH), 124.6 (CH), 124.8 (CH), 131.1 (C), 132.4
(C), 132.5 (CH), 133.3 (CH), 136.0 (C), 137.4 (C), 138.4 (C), 140.2
(C), 145.5 (CH), 154.8 (C), 158.9 (C), 162.4 (C), 167.1 (C), 168.2
(C), 169.1 (C) ppm. EI-MS (DI, 240 °C): m/z (%) = 542 (13) [M+],
501 (29), 500 (100, C28H20O9), 472 (17), 459 (17), 458 (64), 431 (8),
430 (33), 249 (10), 226 (16), 225 (37, C15H13O2), 212 (9), 120 (21),
9.9 (C-4ЈЈ), 19.2 (C-1ЈЈ), 29.7 (C-3ЈЈ), 32.0 (C-8Ј), 44.8 (C-13Ј), 69.5 107 (17), 91 (21). C30H22O10 [M+]: calcd. 542.1213; found 542.1249.
(C-9Ј), 78.7 (C-2ЈЈ), 87.5 (C-1), 110.0 (C-8a), 111.5 (C-7), 116.1 (C-
8-Acetylanhydroretipolide A 4Ј,6-Di-O-acetate (5): Yellow glass,
m.p. Ͼ 200 °C (decomp.). Rf (TLC) = 0.75 (toluene/HCO2Et/
HCO2H, 10:5:3), yellow spot. [α]2D0 = –169 (c = 0.09, CHCl3). UV/
Vis (MeCN): λmax (log ε) = 231 (sh, 4.26), 278 (3.42), 370 (3.11)
nm. CD (MeCN): λmax (∆ε) = 225 (+5.56), 235 (0), 248 (–4.51),
272 (–0.19), 277 (+0.12), 280 (0), 289 (+1.11), 305 (0), 321 (–0.86),
19Ј), 116.4 (C-5Ј), 117.2 (C-8), 120.7 (C-5a), 122.2 (C-6Ј), 125.1 (C-
16Ј), 125.2 (C-17Ј), 131.8 (C-3a), 132.3 (C-7Ј), 132.9 (C-14Ј), 133.5
(C-18Ј), 134.1 (C-15Ј), 137.1 (C-8b), 144.6 (C-4Ј), 146.0 (C-5), 152.4
(C-3Ј), 152.7 (C-6), 159.6 (C-1Ј), 163.1 (C-3), 168.1 (C-11Ј) ppm.
EI-MS (DI, 200 °C): m/z (%) = 515 (18) [M + 1]+, 514 (70) [M+],
459 (28), 458 (100), 431 (16), 430 (74), 250 (6), 249 (11), 229 (7),
227 (5), 226 (17), 225 (42), 232 (11), 120 (20), 107 (12), 91 (11).
C30H26O8 [M+]: calcd. 514.1628; found 514.1640.
460 (0) nm. IR (KBr): ν = 2920 (s), 1780 (ss), 1730 (s), 1680 (m),
˜
1200 (ss) cm–1. 1H NMR (200 MHz, [D6]acetone): δ = 2.28, 2.32
(each s, 3 H, CH3CO), 2.60 (s, 3 H, CH3CO), 3.13 (d, J = 14.0 Hz,
1 H, 13Ј-Hb), 3.94 (m, 2 H, 9Ј-H), 5.03 (dd, J = 2.0, 0.8 Hz, 1 H,
19Ј-H), 5.40 (d, J = 14.0 Hz, 1 H, 13Ј-Ha), 6.62 (ddd, J = 8.0, 2.0,
0.9 Hz, 1 H, 6Ј-H), 6.88 (d, J = 8.0 Hz, 1 H, 5Ј-H), 7.02 (dd, J =
8.3, 2.5 Hz, 1 H, 17Ј-H), 7.20 (dd, J = 8.5, 2.5 Hz, 1 H, 16Ј-H),
7.45 (d, J = 0.3 Hz, 1 H, 7-H), 7.49 (dd, J = 8.3, 2.2 Hz, 1 H, 18Ј-
H), 7.67 (dd, J = 8.5, 2.2 Hz, 1 H, 15Ј-H), 9.01 (d, J = 0.3 Hz, 1
H, 5-H) ppm; signals for 8Ј-H obscured. EI-MS (DI, 220 °C): m/z
(%) = 585 (5), 584 [M+] (12), 543 (18), 542 (53), 501 (30), 500 (100,
C28H20O9), 472 (12), 458 (8), 455 (9), 226 (26), 225 (46, C15H13O2),
212 (10), 107 (14), 91 (28), 77 (8), 71 (9), 69 (8), 57 (13), 44 (12),
43 (44). C32H24O11 [M+]: calcd. 584.1318; found 584.1318.
Synthesis of Retipolide A 5-O-Methyl Ether (3): To a solution of 1
(2.0 mg, 4.2 µmol) in acetone (50 µL) was added a 1% solution of
TsOH in 2,2-dimethoxypropane (50 µL). The mixture was kept for
24 h at room temperature and then separated by preparative TLC
on silica gel (solvent system C) to yield 3 (1.4 mg, 68%) as a color-
less, amorphous solid, and less than 0.1 mg of 2a.
3 (Mixture of Epimers): M.p. 110–112 °C. Rf (TLC) = 0.56 (solvent
system A), 0.24 (solvent system B), 0.31 (solvent system F). tR
(HPLC) = 29.84 min, conditions as given for Table 1. UV/Vis
(MeCN): λmax (log ε) = 212 (4.12), 290 sh (3.39), 318 (3.58), 438
1
(2.09) nm. H NMR (400 MHz, [D6]acetone): δ = 3.54 (s, 1.95 H,
OMe), 3.57 (s, 1.05 H, OMe), 6.18 (d, J = 1.2 Hz, 0.65 H, 5-H),
6.19 (d, J = 1.2 Hz, 0.35 H, 5-H) ppm. EI-MS (DI, 200 °C): m/z
(%) = 491 (28) [M + 1]+, 490 (84) [M+], 462 (8), 459 (14), 458 (5),
257 (12), 256 (5), 228 (12), 227 (74), 226 (100), 225 (44), 212 (5),
211 (10), 120 (51), 119 (10), 107 (36), 92 (10), 91 (58), 85 (10), 57
(28), 45 (22), 44 (62), 32 (74), 31 (44). C27H22O9 [M+]: calcd.
490.1264; found 490.1272.
Isolation Procedure for Tyrosol (14) and Retipolide E (16): Air- and
freeze-dried fruit bodies of R. retipes/ornatipes (102 g) were pulver-
ized and defatted with hexanes (3ϫ500 mL). Extraction of the ma-
terial with EtOAc (3ϫ500 mL) yielded a solution, which was used
for the production of the retipolides A–D (see above). The residue
was treated with MeOH (1 L), 2  HCl (1 mL), and ascorbic acid
(0.5 g), and extracted for one week to yield a black solution. The
extract was concentrated under reduced pressure and then dis-
solved in EtOAc (200 mL). The solution was washed with acidified
water (200 mL, adjusted to pH 3 with 2  HCl), dried (MgSO4),
and concentrated under reduced pressure. The resulting black solid
was flash chromatographed on silica gel (CHCl3/acetone, 4:1),
whereby all fractions with Rf = 0.30–0.40 were combined and con-
centrated to yield a brown oil (1.04 g), which was purified by a
second flash chromatography on silica gel (hexanes/EtOAc/AcOH,
60:40:1). All fractions with Rf = 0.35–0.45 were combined and con-
centrated to yield a brown solid (102 mg). Preparative HPLC [Nu-
cleosil C18, 7 µm, 20ϫ250 mm (Knauer); solvent A: H2O/MeOH,
9:1; solvent B: H2O/MeOH, 1:9; gradient: start 70% A + 30% B,
Acetylation of Retipolide A (1): A solution of 1 (30 mg, 0.063 mmol)
in acetic anhydride (30 mL) was treated with one drop of concd.
H2SO4 and kept for 65 h at room temperature. The reaction mix-
ture was then stirred for 1 h with water (200 mL) and the aqueous
phase subsequently extracted with EtOAc (2ϫ200 mL). The com-
bined extracts were dried (Na2SO4) and concentrated. The complex
reaction mixture was chromatographed on a silica gel column (hex-
anes/EtOAc, 2:1), and the two acetates were purified by preparative
TLC on silica gel with the same solvent mixture to yield the diace-
tyl and triacetyl derivatives 4 (15 mg) and 5 (5 mg), respectively.
Both compounds are rather sensitive and decompose on standing
in solution.
Anhydroretipolide A 4Ј,6-Di-O-acetate (4): Yellow glass, m.p. 130 °C 45 min: 10% A + 90% B, 50 min: 0% A + 100% B, 55 min: 70%
(decomp.). Rf (TLC) = 0.50 (solvent system C), yellow spot. IR A + 30% B; flow rate: 3.38 mL/min, probe 1000 µL (MeOH), de-
(KBr): ν = 2920 (m), 1780 (s), 1730 (m), 1200 (s) cm–1. H NMR tection at 225 nm] yielded 14 (64 mg, 0.063 %, relative to dry
1
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W. Steglich et al.
FULL PAPER
weight) and 16 (2.2 mg, 2.2ϫ10–3 %). Retention times: tR = 15 min
(14), tR = 38 (16), tR = 40 (unidentified compound).
tem A). [α]2D0 = +144 (c = 0.1, MeCN). UV/Vis (MeCN): λmax (log
ε) = 211 (3.54), 302 (3.14) nm. CD (MeCN): λmax (∆ε) = 203
(–2.703), 219 (0), 234 (2.503), 266 (1.464), 296 (2.502) nm. IR
Tyrosol (14): Colorless solid, m.p. 92 °C. Rf (TLC) = 0.40 (solvent
system E), 0.44 (solvent system F), 0.35 (CHCl3/acetone, 4:1). The
spectroscopic and chromatographic data are in agreement with
those of tyrosol purchased from Fluka.
(KBr): ν = 3436 (ss, br), 1779 (m), 1728 (m), 1684 (m), 1637 (m),
˜
1
1512 (w), 1266 (w), 1209 (w), 1026 (w) cm–1. H NMR (600 MHz,
[D6]acetone): δ = 2.56 (br., 2 H, 7-H), 2.79 (d, J = 16.3 Hz, 1 H,
7Ј-H), 3.03 (d, J = 15.6 Hz, 1 H, 12Ј-Hb), 3.14 (br. m, 2 H, 8-H),
3.24 (m, 1 H, 7Ј-H), 4.21 (m, 1 H, 8Ј-H), 4.24 (d, J = 15.6 Hz, 1
H, 12Ј-Ha), 5.03 (m, 1 H, 8Ј-H), 6.53 (br. s, 1 H, 5-H), 6.79 (d, J
= 8.1 Hz, 1 H, 4Ј-H), 6.83 (d, J = 8.1 Hz, 1 H, 15Ј-H), 7.02 (br., 1
H, 17Ј-H), 7.05 (dd, J = 8.1, 1.8 Hz, 1 H, 14Ј-H), 7.06 (dd, J = 8.1,
1.8 Hz, 1 H, 5Ј-H), 7.31 (br., 1 H, 18Ј-H), 8.35 (br., OH) ppm. 13C
NMR (150.9 MHz, [D6]acetone): δ = 25.6 (C-8), 32.2 (C-7Ј), 35.8
(C-7), 41.2 (C-12Ј), 70.1 (C-8Ј), 87.7 (C-1), 94.8 (C-5), 115.3 (C-
4Ј), 115.9 (C-15Ј), 126.8 (C-13Ј), 127.4 (C-8b), 127.5 (C-2Ј), 128.0
(C-1Ј), 129.2 (C-5Ј), 130.4 (C-14Ј), 130.8 (m, C-6Ј), 132.5 (C-3a),
139.2 (C-17Ј), 140.6 (m, C-18Ј), 144.1 (C-5a), 153.6 (C-3Ј, C-16Ј),
158.0 (C-8a), 164.4 (C-3), 171.4 (C-10Ј), 203.5 (C-6) ppm. EI-MS
of pertrimethylsilyl derivative 11 (DI, 200 °C): m/z (%) = 477 (4)
[M + 1]+, 476 (15) [M+], 459 (14), 458 (43), 432 (13), 430 (5), 414
(6), 386 (5), 227 (50), 226 (80), 225 (100) [C15H13O2], 212 (11), 120
(45), 107 (37), 91 (42), 84 (48), 82 (63).
Retipolide E, (R)-4,4Ј-Dihydroxy-3Ј-(4-hydroxyphenyl)spiro[2,10-di-
oxatricyclo[12.2.2.13,7]nonadeca-1(16),3,5,7(19),14,17-hexaene-
12,2Ј(5ЈH)-furan]-11,5Ј-dione (16): Colorless, amorphous solid; eas-
ily soluble in acetone, sparingly soluble in CHCl3; m.p. 195–197 °C.
Rf (TLC) = 0.51 (solvent system A), 0.40 (solvent system E). [α]2D0
= +83 (c = 0.17, MeCN). UV/Vis (MeCN): λmax (log ε) = 223 sh
(4.28), 288 (3.97), 305 (3.98), 350 sh (3.10) nm. CD (MeCN): λmax
(∆ε) = 243 (+12.96), 284 (+0.88), 294 (+1.76), 314 (0), 342 (+0.88),
365 (0) nm. IR (KBr): ν = 3380 (ss, br), 2950 (w), 2920 (w), 1750
˜
(s), 1725 (s), 1600 (m), 1580 (m), 1510 (s), 1495 (m), 1450 (w, sh),
1430 (m), 1380 (m), 1355 (w), 1300 (w, sh), 1250 (s), 1200 (m), 1180
(w, sh), 1155 (m), 1110 (m), 1100 (m), 1070 (w), 1060 (w, sh), 1020
(m), 1000 (w, sh), 970 (m), 915 (m), 870 (m), 840 (m), 820 (m), 770
(w), 760 (w), 740 (w), 620 (m) cm–1. 1H NMR (400 MHz, [D6]-
acetone): δ = 2.59 (dd, J = 17.1, 5.2 Hz, 1 H, 8-Ha), 2.84 (dddd, J
= 17.1, 11.2, 1.2, 0.9 Hz, 1 H, 8-Hb), 3.11 (d, J = 13.8 Hz, 1 H, 13-
Hb), 3.88 (ddd, J = 11.3, 5.2, 1.2 Hz, 1 H, 9-Hb), 3.97 (d, J =
13.8 Hz, 1 H, 13-Ha), 4.22 (dd, J = 11.3, 11.2 Hz, 1 H, 9-Ha), 4.80
(dd, J = 2.1, 0.9 Hz, 1 H, 19-H), 6.50 (dd, J = 8.0, 2.1 Hz, 1 H, 6-
H), 6.70 (d, J = 8.0 Hz, 1 H, 5-H), 6.93 (3ЈЈ/5ЈЈ-H, J = 8.9 Hz, 2
H, AAЈBBЈ-d), 6.97 (dd, J = 8.5, 2.5 Hz, 1 H, 17-H), 7.20 (dd, J
= 8.5, 2.5 Hz, 1 H, 16-H), 7.36 (dd, J = 8.5, 2.5 Hz, 1 H, 18-H),
7.65 (dd, J = 8.5, 2.5 Hz, 1 H, 15-H), 8.07 (2ЈЈ/6ЈЈ-H, J = 8.9 Hz,
2 H, AAЈBBЈ-d) ppm. 13C NMR (100.6 MHz, [D6]acetone): δ =
32.2 (C-8), 44.5 (C-13), 68.7 (C-9), 89.1 (C-12), 115.8 (C-19), 115.9
(2ϫC-3ЈЈ/5ЈЈ), 116.3 (C-5), 122.3 (C-6), 123.0 (C-1ЈЈ), 124.6 (C-16),
124.7 (C-17), 128.2 (C-3Ј), 131.7 (2ϫC-2ЈЈ/6ЈЈ), 132.5 (C-7), 133.3
(C-14), 134.0 (C-18), 134.6 (C-15), 141.2 (C-4Ј), 144.6 (C-4), 152.4
(C-3), 158.4 (C-4ЈЈ), 159.2 (C-1), 167.8 (C-5Ј), 169.0 (C-11) ppm.
EI-MS (DI, 220 °C): m/z (%) = 460 (0.2) [M+], 417 (8), 416 (34),
226 (12), 225 (7), 207 (7), 198 (8), 188 (10), 121 (8), 120 (9), 91
(13), 44 (100). C26H20O8 [M+]: calcd. 460.1158; found 460.1143. In
all properties identical with a synthetic sample.[13]
Synthesis of Anhydroisoretipolide A 6-O-Methyl Ether (10): 9
(0.6 mg) was dissolved in 3 drops of MeOH and immediately
treated with a solution of p-TsOH (1%) in 2,2-dimethoxypropane
(1 mL). The mixture was refluxed for 25 min at 75 °C, cooled to
room temperature, and concentrated under reduced pressure. The
orange-brown residue was separated with EtOAc/hexanes (1:1)
using a Pasteur pipette filled with silica gel. The orange-colored
fraction was concentrated to yield 10 (0.3 mg) as an orange oil. Rf
(TLC) = 0.40 (solvent system B). UV/Vis (MeCN): λmax = 216,
285, 385 nm. EI-MS (DI, 200 °C): m/z (%) = 472 (3) [M+], 225 (9),
73 (100). C27H20O8 [M+]: calcd. 472.1158; found 472.1137.
rac-Butyrolactone II, Methyl (RS)-4-Hydroxy-2-(4-hydroxybenzyl)-
3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylate [(RS)-
13]: The compound was prepared according to ref.[11b] Colorless
powder; m.p. 202–205 °C, ref.[11b] 206–208 °C. Rf (TLC) = 0.25 (sol-
vent system C). 1H NMR (400 MHz, [D6]acetone): δ = 3.50 (s, 2
H, CH2), 3.79 (s, 3 H, OMe), 6.60, 6.69 (d, J = 8.3 Hz, 2 H, 17-
H), 6.69 (d, J = 8.3 Hz, 2 H, 16-H), 6.98 (d, J = 8.7 Hz, 2 H, 8-
H), 7.65 (d, J = 8.7 Hz, 2 H, 7-H), 8.16, 8.87 (each s, OH), 9.06 (s,
OH) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 39.2, 53.7, 86.0,
115.5, 116.7, 122.7, 124.9, 128.3, 130.2, 132.3, 139.1, 157.4, 158.9,
168.6, 170.8 ppm.
Isolation Procedure for Isoretipolide A (9): Collections of R. retipes/
ornatipes containing 9 in larger quantities (HPLC screening)[8] were
cut and extracted in 2-g portions with acetone containing a small
amount of HCl under argon atmosphere. The dark yellow to
brownish black extracts were concentrated, treated with water and
extracted with EtOAc (3ϫ). After removal of the solvent under
reduced pressure, the black residue was dissolved in H2O/MeCN
(1:1), filtered through a SepPak RP18 cartridge, and immediately
separated by preparative HPLC [Nucleosil 100 C18, 7 µm,
250 ϫ 16 mm (Macherey–Nagel); solvent A: H2O/MeCN, 9:1 +
0.5% TFA; solvent B: MeCN; gradient: start 80% A + 20% B,
50 min: 45% A + 55% B, 55 min: 100% B, flow rate 6.80 mL/min;
detection at 300 nm]. The fractions were stored at –10 °C before
further treatment. Concentration under reduced pressure at room
temperature and removal of the residual water by lyophilization
yielded compounds 9 (15.8 mg, 0.047% of the dry weight) and 1
(78 mg, 0.23%) in pure form. Retention times: tR = 48.6 min (9)
and 50.4 min (1). Isoretipolide A (9) was kept at –10 °C in the
freezer.
Synthesis of rac-Secoretipolide E, 2-(4-Hydroxyphenyl)ethyl (RS)-4-
Hydroxy-2-(4-hydroxybenzyl)-3-(4-hydroxyphenyl)-5-oxo-2,5-dihy-
drofuran-2-carboxylate [(RS)-15]: Lactone (RS)-13 (530 mg,
1.49 mmol), tyrosol (415 mg, 3.00 mmol), and 1,3-dichlorotetrabu-
tyldistannoxane (552 mg, 1.00 mmol)[18] were suspended in dry tol-
uene (30 mL) and refluxed for 16 h. The mixture was concentrated
and the residue purified by flash chromatography (CHCl3/MeOH,
10:1) to yield (RS)-15 (494 mg, 69%) as a beige solid; m.p. 97 °C.
R (TLC) = 0.18 (solvent system G). IR (KBr): ν = 3424 (s, br),
˜
f
3025 (w), 2975 (w), 2920 (w), 1740 (s), 1700 (w), 1653 (w), 1636
(w), 1611 (m), 1580 (m), 1516 (s), 1444 (m), 1385 (m), 1265 (m, sh),
1242 (m), 1180 (m), 1130 (w), 1106 (w), 1068 (w), 1032 (w), 838
(m) cm–1. 1H NMR (300 MHz, [D6]acetone): δ = 2.80 (br. t, J =
7.6 Hz, 2 H), 3.46 (s, 2 H), 4.33 (m, 2 H), 6.58 (d, J = 8.1 Hz, 2 H,
Isoretipolide A, (1R,5RS)-7,8-Dihydro-3Ј,5,16Ј-trihydroxyspiro[cyclo- Ar-H), 6.67 (d, J = 8.5 Hz, 2 H, Ar-H), 6.71 (d, J = 8.1 Hz, 2 H,
penta[d]furo[3,4-b]pyran-1,11Ј(5H)-9-oxatricyclo[11.3.1.12,6]- Ar-H), 6.94 (d, J = 8.7 Hz, 2 H, Ar-H), 7.00 (d, J = 8.5 Hz, 2 H,
octadeca[1(17),2,4,6(18),13,15]hexaene]-3,6,10Ј-trione (9): Pale yel-
low powder, m.p. 120 °C (decomp.). Rf (TLC) = 0.55 (solvent sys-
Ar-H), 7.62 (d, J = 8.7 Hz, 2 H, Ar-H), 8.14, 8.16, 8.87 (each s,
OH), 9.05 (br. s, OH) ppm. 13C NMR (75.5 MHz, [D6]acetone): δ
5570
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Eur. J. Org. Chem. 2007, 5560–5572

Retipolides – Unusual Spiromacrolactones from Mushrooms
= 34.6, 39.2, 68.1, 86.1, 115.5, 116.1, 116.6, 122.8, 125.0, 128.3,
129.1, 130.2, 130.8, 132.3, 139.2, 157.0, 157.3, 158.9, 168.6, 170.2
ppm. EI-MS (DI, 190 °C): m/z (%) = 462 (2) [M+], 280 (5), 147 (9),
138 (18), 121 (10), 120 (8), 108 (9), 107 (100), 91 (8), 77 (15), 44
(72). C26H22O8 (462.5)ϫMeOH: calcd. C 65.58, H 5.30; found C
65.20, H 5.31.
facilities and Dr. Norbert Arnold for his help in collecting the
mushrooms. The investigations were financially supported by the
Bundesministerium für Bildung und Forschung and the Fonds der
Chemischen Industrie. J.-D. K. would like to thank the Stiftung
Volkswagenwerk and K. J. the Stiftung Stipendien-Fonds des Ver-
bandes der Chemischen Industrie for Kekulé Ph. D. grants. The
authors gratefully acknowledge Dr. Hans-Christoph Weiß for pre-
paring the X-ray plot of compound 2b, Dr. Gustav Penzlin for his
help in the nomenclature of the retipolides, and Dr. Kay Greenfield
for linguistic improvements.
Identification of Butyrolactone II (13) and Secoretipolide E (15) in
the Mushroom Extract: Air- and freeze-dried fruit bodies of R. re-
tipes/ornatipes (135 g) were pulverized and defatted with hexanes
(1.5 L). The residue was treated with MeOH (1.5 L), 2  HCl
(1.5 mL), and ascorbic acid (1 g), and extracted for 1 day to yield
a brown solution. The extract was concentrated under reduced
pressure and then dissolved in EtOAc (500 mL). The solution was
washed with acidified water (300 mL), adjusted to pH 3 with 2 
HCl, dried (MgSO4), and concentrated under reduced pressure.
The resulting black solid (6.8 g) was flash chromatographed on sil-
ica gel (CHCl3/acetone, 5:2), whereby all fractions with Rf = 0.25–
0.45 were combined and concentrated to yield a yellow-brown solid
(0.80 g). Purification by a second flash chromatography on silica
gel (hexanes/EtOAc/AcOH, 50:50:1) yielded two fractions. Fraction
1 consisted of impure 13 (77 mg, brown solid), fraction 2 of en-
riched 15 (96 mg, brown solid). The identity of 13 and 15 with the
corresponding synthetic compounds was established by co-HPLC
and UV comparison. 13: Nucleosil 5 C18, 5 µm, 250 ϫ 4.6 mm,
(Merck); solvent A: H2O/MeCN, 9:1; solvent B: H2O/MeCN, 1:9;
gradient: start 100% A, 60 min: 100% B, flow rate 1.0 mL/min;
Diode array detection; tR = 26.99 min. 15: same column and condi-
tions as above, but MeOH instead of MeCN; tR = 44.48 min.
[1] There is a long-standing dispute, if the two species, formerly
named Boletus retipes and B. ornatipes, are autonomous or syn-
onyms for only one quite variable species (see, for example,
E. E. Both, The Boletes of North America, Buffalo Museum of
Science Buffalo NY, 1996). Bender and Bresinsky have recently
created the genus Retiboletus from molecular biological evi-
dence and the presence of retipolides as chemotaxonomical
markers (M. Binder, A. Bresinsky, Feddes Repertorium 2002,
113, 30–40). The authors state that R. retipes and R. ornatipes
cannot be differentiated by morphological and anatomical
characters. As a consequence, our collections probably contain
mixtures of both fungi. A HPLC comparison of several indi-
vidual collections from the Eastern USA indicates the presence
of retipolides in all cases. These investigations together with
molecular biologic studies will be reported in a separate publi-
cation.
[2] The line broadening is most pronounced for the hemiacetal
group and atoms in its surrounding. This can be explained by
rapid ring opening and re-closure of the hemiacetal in solution,
leading to averaged NMR signals for the two epimers.
[3] E. Güllük, E. Bogdan, M. Christl, Eur. J. Org. Chem. 2006,
531–542 and references cited therein.
[4] D. P. Roelofsen, H. van Bekkum, Synthesis 1972, 419–420.
[5] a) S. Tamai, M. Kaneda, S. Nakamura, J. Antibiot. 1982, 35,
1130–1136; b) M. Kaneda, S. Tamai, S. Nakamura, T. Hirata,
Y. Kushi, T. Suga, J. Antibiot. 1982, 35, 1137–1140.
[6] A solution of retipolide A (1) in acetonitrile exhibits UV/Vis
maxima at λmax = 208, 316, 390, and 428 nm. On addition of
ammonia, the weak yellow color intensifies, and the intensity
of the maxima at λmax = 390 and 428 nm increases consider-
ably. After addition of HCl, the solution becomes colorless,
and the maxima at 390 and 428 nm disappear completely.
[7] S. M. Kroner, M. P. DeMatteo, C. M. Hadad, B. K. Carpenter,
J. Am. Chem. Soc. 2005, 127, 7466–7473, and literature cited
therein.
Identification of Xerocomic Acid (20): An air-dried, defatted sample
(1 g) from a specimen of R. retipes/ornatipes with brown cap skin,
collected in August 1991 in the ‘Ball Creek area’, was extracted
with MeOH (50 mL) with the addition of 2  HCl (0.2 mL) and
ascorbic acid (0.1 g). The solution was concentrated under reduced
pressure, and the resulting residue dissolved in EtOAc (50 mL),
washed with H2O (50 mL, pH 3), dried (MgSO4) and subjected to
HPLC analysis (same column as for the separation of 13 and 15;
solvent A: H2O/MeCN, 9:1 + 0.5% TFA; solvent B: H2O/MeCN,
1:9; gradient: start 90% A and 10% B, 55 min: 10% A and 90%
B, 65 min: 10% A and 90% B, 75 min: 90% A and 10% B, flow
rate 1.0 mL/min; Diode array detection): tR = 17.24 min (20), 26.44
(1), 28.49 (7), 31.19 (3). Retention time tR and UV spectrum agreed
with those of authentic 20.
Crystallographic Data for Anhydroretipolide A 6-O-(S)-sec-Butyl
Ether (2b): C3 0 H2 6 O8 , M = 514.54, crystal dimensions
0.23ϫ0.37ϫ0.67 mm, space group P212121, unit cell dimensions
and volume: a = 11.382, b = 14.820, c = 15.231 Å, V = 2569.1 Å3,
Z = 4, Dcalcd. = 1.329 g/cm3, F(000) = 1080, µ = 0.097 mm–1, radia-
tion: Mo-Kα, wavelength λ = 0.71073 Å, 2Θmax = 45.9°, hmin/hmax
= –12/12, kmin/kmax = 0/16, lmin/lmax = 0/16, No. of measured reflec-
tions: 3886, No. of unique reflections: 3485, No. of observed reflec-
tions: 3282 [I Ͼ2σ(I)], No. of parameters 343, R factor 0.0445,
wR(F2) = 0.1207, Goodness of fit: 1.094, structure solution with
SHELXLS-86,[19] refinement by SHELXTL-PLUS.[20]
[8] For the chemotaxonomic significance of this substance see V.
Hellwig, Dissertation, Ludwig-Maximilians-Universität,
München, 1999.
[9] The force-field calculations were carried out with MacroModel
5.5 (Merck Molecular Force Field).
[10] D. H. R. Barton, T. Cohen, in Festschrift A. Stoll, Birkhäuser,
117–143 (1957).
[11] a) K. Arai, T. Yoshimura, Y. Itatani, Y. Yamamoto, Chem.
Pharm. Bull. 1983, 31, 925–933; b) K. Nitta, N. Fujita, T. Yosh-
imura, K. Arai, Y. Yamamoto, Chem. Pharm. Bull. 1983, 31,
1528–1533; c) K. V. Rao, A. K. Sadhukhan, M. Veerender, V.
Ravikumar, E. V. S. Mohan, S. D. Dhanvantri, M. Sitaramku-
mar, J. Moses Babu, K. Vyas, G. Om Reddy, Chem. Pharm.
Bull. 2000, 48, 559–562.
CCDC-167856 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
[12] M. F. Braña, M. L. García, B. López, B. de Pascual-Teresa, A.
Ramos, J. M. Pozuelo, M. T. Domínguez, Org. Biomol. Chem.
2004, 2, 1864–1871, and literature cited therein.
[13] A. Ingerl, K. Justus, V. Hellwig, W. Steglich, Tetrahedron 2007,
63, 6548–6557.
Acknowledgments
[14] H. Shibata, T. Fukuda, T. Wada, Y. Morita, T. Hashimoto, Y.
Asakawa, Biosci. Biotechnol. Biochem. 1998, 62, 1432–1434.
[15] W. Steglich, W. Furtner, A. Prox, Z. Naturforsch., Teil B 1968,
23, 1044–1050.
We thank Dr. Richard C. Bruce, Executive Director, Highlands
Biological Station, Highlands, NC, USA, for providing research
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W. Steglich et al.
FULL PAPER
[16] M. Gill, W. Steglich, Prog. Chem. Org. Nat. Prod. 1987, 51, 1–
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[17] For a recent example, see B. Sontag, M. Rüth, P. Spiteller, N.
Arnold, W. Steglich, M. Reichert, G. Bringmann, Eur. J. Org.
Chem. 2006, 1023–1033.
[18] a) J. Otera, T. Yano, A. Kawabata, H. Nozaki, Tetrahedron
Lett. 1986, 27, 2383–2386; b) J. Otera, S. Ioka, H. Nozaki, J.
Org. Chem. 1989, 54, 4013–4014; c) B. Jousseaume, C. Laporte,
M.-C. Rascle, T. Toupance, Chem. Commun. 2003, 1428–1429.
[19] G. M. Sheldrick, SHELXS-86, Program for the Solution of
Crystal Structures, University of Göttingen, 1990.
[20] G. M. Sheldrick, SHELXTL-Plus, rel. 4.1, Siemens Analytical
X-RAY Instruments Inc., Madison, WI, 1990.
Received: June 22, 2007
Published Online: September 25, 2007
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Eur. J. Org. Chem. 2007, 5560–5572