Chromogenic meroterpenoids from the mushrooms Russula ochroleuca and R. viscida

Article abstract of DOI:10.1002/ejoc.200500714

The spirodioxolactone ochroleucin A₁ (1) is responsible for the red colour produced when the stalk base of Russula ochroleuca and R. viscida is treated with aqueous KOH. The labile chromogen rearranges easily into the isomeric dilactone ochrole

Full text of DOI:10.1002/ejoc.200500714

FULL PAPER
DOI: 10.1002/ejoc.200500714
Chromogenic Meroterpenoids from the Mushrooms Russula ochroleuca and
R. viscida
Bernd Sontag,[a][‡] Matthias Rüth,[a][‡‡] Peter Spiteller,[a] Norbert Arnold,[a]
Wolfgang Steglich,*[a] Matthias Reichert,[b] and Gerhard Bringmann*[b]
Dedicated to Professor Dieter Enders on the occasion of his 60th birthday
Keywords: Natural products / Meroterpenoids / Mushrooms / Rearrangements / Colour reaction
The spirodioxolactone ochroleucin A1 (1) is responsible for
oxidative condensation of two monomeric units. One of them,
the red colour produced when the stalk base of Russula ochro- 2,5-dihydroxy-4-(3-methylbut-3-en-1-ynyl)benzaldehyde (6),
leuca and R. viscida is treated with aqueous KOH. The labile
chromogen rearranges easily into the isomeric dilactone
ochroleucin A2 (2). Ochroleucin A1 is accompanied by the
biosynthetically related hemiacetal ochroleucin B (5). The
new compounds, whose structures were established by MS
and NMR methods, appear to be derived biosynthetically by
was detected in the crude toadstool extract by GC/MS com-
parison with a synthetic sample. The absolute configurations
of the ochroleucins A1 and B have been determined by quan-
tum chemical calculation of their CD spectra.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Introduction
Results and Discussion
Russula ochroleuca (Pers.) Fr. (German: Ockertäubling)
is a medium-sized agaric with ochre to yellow cap, whitish
gills and a white stem. The base of the stem is covered with
ochraceous fibers, which can be interpreted as rest of the
velum universale. This very common mushroom grows on
sour soil in coniferous and deciduous forests and is in some
regions the most abundant Russula species. It can be iden-
tified by a colour reaction, first reported by the Italian my-
cologist Galli: [1] If a drop of aqueous KOH solution is ap-
plied to the yellowish base of the toadstool, an intense red
spot is immediately formed, which changes to brown within
a few minutes. This behaviour is only known from R. ochro-
leuca, R. viscida, R. messapica, and R. insignis.[1] In this
publication, we report on the isolation, structural elucida-
tion and biosynthetic formation of the chromogenic prin-
ciple from the first two species.
Isolation of the Metabolites
To reveal the chemical structure of the chromogen, about
12 kg of fresh fruit bodies of R. ochroleuca were collected
in spruce forests around Munich. Only the lower third of
the stipes was extracted with EtOAc, resulting in a yellow
solution which still displayed the red colour reaction after
the addition of base. After removal of the solvent, the resi-
due was fractionated by flash chromatography on silica gel.
Fractions showing the colour reaction were combined and
further purified by preparative HPLC on reversed phase.
Two structurally related yellow pigments were isolated,
which were named ochroleucins A1 (1) and B (5). The latter
is chemically stable and shows no discolouration with
KOH, whereas ochroleucin A1 is responsible for the red col-
our reaction. From 4 kg of fresh stipes, 18 mg of ochroleu-
cin A1 (1) and 36 mg of ochroleucin B (5) were obtained.
Chromogen 1 is very labile and rearranges rapidly into a
stable red isomer, ochroleucin A2 (2). As a result of this
rearrangement the purification of 1 by HPLC was difficult.
Fractions containing pure 1 change their colour within a
few minutes at room temperature from yellow to red and
have therefore to be cooled to –78 °C immediately.
[a] Department Chemie der Ludwig-Maximilians-Universität,
Butenandtstr. 5–13, 81377 München, Germany
Fax: +49-89-218077756
E-mail: wos@cup.uni-muenchen.de
[b] Institut für Organische Chemie, Universität Würzburg,
Am Hubland, 97074 Würzburg, Germany
Fax: +49-931-8885323
E-mail: bringman@chemie.uni-wuerzburg.de
[‡] Present address: Discovery Partners International GmbH,
Waldhofer Str. 104, 69123 Heidelberg, Germany
[‡‡] Present address: Roche Diagnostics GmbH,
Medicinal Chemistry,
Ochroleucins A1 and A2
Nonnenwald 2, Building 231/380, 82372 Penzberg, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Due to the instability of ochroleucin A1 (1), no molecular
ion peak could be detected by any of the available ioni-
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Figure 1. UV/Vis spectra of ochroleucin A1 (1) in (a) MeOH and (b) MeOH + KOH.
sation modes (EI, FAB, ESI, APCI). Instead, the molecular benzaldehyde can be proposed (Figure 2). In a similar fash-
composition of 1, C23H14O6, was derived indirectly from ion the HMBC correlations originating from the proton sig-
the highly resolved molecular ion m/z = 386.0790 of the nal at δH = 7.58 ppm reveal a cylopentenedione unit B car-
stable isomer 2. The UV/Vis spectrum of ochroleucin A1 (1) rying the second methylbutenynyl residue. Connection of
in MeOH shows three main maxima at λmax = 233, 314, the two units A and B with the so far unconsidered CO
and 369 nm. On addition of base, the latter is shifted to group (signal at δC = 166.1 ppm) leads to spirolactone
452 nm, thus explaining the red colour reaction (Figure 1). structure 1 for ochroleucin A1 (Figure 3), in agreement with
In the IR spectrum (KBr) a characteristic band of medium the IR absorption at 1809 cm–1. The structure is supported
intensity at 2198 cm–1 indicates the presence of a doubly by comparison of the NMR spectroscopic data of ochro-
1
substituted triple bond. The surprisingly simple H NMR leucin A1 with those of simple cyclopent-4-ene-1,3-diones[2]
spectrum of 1 shows two singlets at δH = 7.07 and 7.58 ppm and the mutadiones, structurally related spirodioxolactones
in the aromatic region, an additional singlet at δH
=
from the polypore Hapalopilus mutans.[3]
9.65 ppm for an aldehyde proton, and four doublets of qua-
druplets at δH = 5.47/5.56 and 5.63/5.71 ppm, revealing two
olefinic methylene groups, connected by allylic coupling (4J
= 0.6 Hz) with methyl groups at δH = 2.01 and 2.04 ppm,
respectively. In addition, a broad signal at δH Ϸ 9.4 ppm
can be assigned to a phenolic OH group.
The 13C NMR spectrum of 1 exhibits signals for 23 car-
bon atoms in agreement with the molecular formula
C23H14O6. According to the DEPT spectrum, two methyl,
two methylene, three methine, and 16 quaternary carbon
atoms are present. COSY and HMBC experiments indicate
that the methyl and methylene groups form part of two 3-
Figure 2. Partial structures A and B for ochroleucin A1 (1) derived
from HMBC experiments.
To determine the absolute configuration of ochroleucin
methylbut-3-en-1-ynyl residues, in agreement with the sig- A1, quantum chemical circular dichroism (CD) calcula-
1
nals in the H NMR spectrum. The aromatic proton signal tions[4,5] were performed. Arbitrarily starting with the (R)
at δH = 7.07 ppm shows HMBC correlations with four car- enantiomer of 1, a conformational analysis on the semiem-
bon atoms of a benzene ring, an aldehyde carbon atom (δC pirical PM3[6] level was carried out, resulting in five mini-
= 9.65 ppm), and the α-alkynyl carbon atom of one of the mum structures within the energetically relevant range of 3
side chains. From this information and chemical shift con- kcal/mol[7] above the global minimum. For each of these
siderations, the partial structure A of a 2,5-dioxygenated geometries a single CD spectrum was computed by means
Figure 3. Structures of ochroleucin A1 (1) and ochroleucin A2 (2).
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Figure 4. Attribution of the absolute configuration to ochroleucin A1 (1) by CNDO/S-CI-CD calculations based on a) a conformational
analysis and b) an MD simulation.
of the CNDO/S[8] Hamiltonian. The resulting CD curves uously determined the absolute configuration of ochro-
were summed up weighted, following the Boltzmann statis- leucin A1 (1) as (R) (Figure 3).
tics, i.e., according to the heat of formation of the respective
The rearrangement of ochroleucin A1 (1) into ochro-
conformer. The overall CD spectrum thus obtained was leucin A2 (2) occurs even in the solid state at –20 °C under
subsequently UV-corrected[9] and compared with the exper- argon. The less polar, deeply red ochroleucin A2 (2) can be
imental one of ochroleucin A1 (1), revealing a fairly good easily separated from unchanged 1 by preparative HPLC.
agreement with the simulated CD curve of (R)-1 (Figure 4a, Ochroleucin A2 (2) exhibits a molecular ion peak at m/z
left) and a nearly mirror-image behaviour as compared to = 386 in the high-resolution EIMS, corresponding to the
1
the one calculated for (S)-1 (Figure 4a, right).
molecular formula C23H14O6. The H NMR spectrum re-
In order to confirm this result, additional CD calcula- sembles that of ochroleucin A1 (1); however, the signals in
tions were performed for (R)-1, this time based on a mol- the aromatic region exhibit a low-field shift, most pro-
ecular dynamics (MD) simulation using the TRIPOS[10] nounced for the aldehyde proton, shifting from δH = 9.65
force field at a virtual temperature of 500 K and a simula- to 10.80 ppm, and for the olefinic proton, shifting from δH
tion length of 500 ps. For the geometries extracted every = 7.58 to 8.73 ppm. The phenolic proton, in the case of 1
0.5 ps from the trajectory of motion, single CD spectra occurring as a broad signal at δH Ϸ 9.4 ppm, now appears
were computed using again the CNDO/S-CI[8] method. as a sharp singlet at δH = 12.24 ppm, indicating the forma-
Summation of the resulting 1000 calculated CD curves and tion of a strong hydrogen bond.[11] Considering the HMBC
subsequent UV correction[9] delivered the overall simulated correlations in the benzene part of the molecule, the partial
CD spectrum, which again matched well with the measured structure AЈ can be proposed for ochroleucin A2 (2, Fig-
one of 1 (Figure 4b, left), while the curve predicted for (S)- ure 5). More severe changes are discernible in the cyclopen-
1 was once again found to be virtually opposite (Figure 4b, tenedione part of ochroleucin A1 (1). The signals of the
right). Consequently, both theoretical approaches unambig- spirocarbon atom at δC = 66.4 ppm and of the two carbonyl
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groups are missing, and three new signals for quaternary C dilactone moiety, supporting the (E) configuration given for
atoms at δC = 105.1, 155.8, and 163.0 ppm are visible in- ochroleucin A2 in structure 2. This was confirmed by DFT
stead. From HMBC experiments and selective decouplings calculations using the B3LYP[15] hybrid functional with a 6-
1
in the H-coupled 13C NMR spectrum, partial structure BЈ 31G* basis set, favouring the (E) diastereomer over (Z) by
can be proposed (Figure 5). An alternative structure with 7.9 kcal/mol. This result can be explained by the mutual
the side chain at C-4 of the 2(5H)-furanone ring is excluded hydrogen bond formation in (E)-2 between the hydrogen
3
by the large JH,C coupling of 13 Hz between the ring pro- atom at the aldehyde functionality and the lactone oxygen
ton and the lactone carbonyl group. Based on partial struc- atom at position 1Ј, and between 4Ј-H and the carbonyl
tures AЈ and BЈ and the remaining atoms as formulated in oxygen atom at C-2, leading to a planar minimum geometry
CЈ (Figure 5), the dilactone structure 2 can be assigned to of ochroleucin A2 (2). By contrast, (Z)-2 suffers from a ste-
ochroleucin A2 (Figure 3).
ric hindrance between the aldehyde hydrogen atom and 4Ј-
H, and from an electronical interference between O-1Ј and
2-O, resulting in a twisted minimum structure, and therefore
in an significantly higher heat of formation.
Ochroleucin A1 (1) is also responsible for the red colour
reaction of Russula viscida Kudrna (German name: Leder-
ˇ
stiel-Täubling) on treatment with aqueous base. In this case,
small amounts of ochroleucin A2 (2), but no ochroleucin
B were detected. Preliminary experiments indicate that the
chromogens of R. insignis Quél. differ from those of the two
other species.
Figure 5. Partial structures AЈ, BЈ, and CЈ for ochroleucin A2 (2)
derived from HMBC experiments.
The rearrangement of 1 into 2 can be explained by open-
ing of the spirodione ring at C-2Ј through traces of water
and recyclization of the resulting enol carboxylic acid 3 into
the thermodynamically more stable dilactone 2 (Figure 6).
Interestingly, only the sterically less hindered of the two
possible regioisomers of 2 is formed. Possibly, hydrolytic
cleavage at C-5Ј also takes place; however, in this case recy-
clization of the enol carboxylic acid to the lactone is pro-
hibited because of the strong steric interaction of the meth-
ylbutenynyl substituent with the coplanar carbonyl group
of the neighbouring furanone ring. This regioselectivity
agrees with the observation that the rearrangement of 1 into
2 proceeds only with low yield. Formation of an anion from
3 would lead to the highly delocalized species 4, explaining
the red colour reaction of ochroleucin A1 (1) with base.
Ochroleucin B
The second metabolite, ochroleucin B (5), shows a mol-
ecular ion peak at m/z = 390 in the high-resolution EIMS
corresponding to the molecular formula C24H22O5. In the
NMR spectra, again signals for two 3-methylbut-3-en-1-
1
ynyl residues can be identified. The H NMR spectrum of
ochroleucin B (5) resembles that of 1; however, it lacks the
signals for the aldehyde and phenolic protons. In addition
two singlets at δH = 6.80 and 6.45 ppm (δC = 113.2 and
135.5 ppm), two methoxy signals at δH = 3.48/4.10 ppm,
and an AB quadruplet for a diastereotopic OCH2 group
2
(δH = 4.43/4.92 ppm, J = 16 Hz; δC = 59.2 ppm) can be
recognized. Furthermore, two singlets at δH = 4.02 (δC
=
49.5 ppm) and 3.21 ppm can be attributed to an aliphatic
methine proton, and a tertiary OH group, respectively. In
the 13C NMR spectrum 24 signals can be identified, which,
according to the DEPT spectrum, belong to four methyl,
three methylene, and three methine groups and 14 quar-
ternary carbon atoms. Only one carbonyl signal is visible at
δC = 198.7 ppm. From HMBC measurements and selective
1
decouplings in the H-coupled 13C NMR spectrum (Fig-
ure 7), gross structure 5 can be assigned to ochroleucin B.
Of special diagnostic value are the HMBC correlations of
the methylene protons at C-5 as well as those of the angular
proton 9b-H, which indicate a connection of the benzene
ring with the cyclopentenone unit.
For a completion of the structural elucidation of 5, the
absolute configuration at the stereogenic centers C-3a and
C-9b had to be determined. For this reason, quantum
chemical CD calculations were performed, as in the case of
Figure 6. Rearrangement of ochroleucin A1 (1) into ochroleucin A2
(2) and formation of the red anion 4.
Structurally, ochroleucin A2 (2) resembles other fungal ochroleucin A1 (1). Due to the possible existence of four
dilactones like variegatorubin,[12] gomphilactone,[13] and stereoisomers, two independent conformational analyses,
bovilactone-4,4.[14] All of these metabolites are red pig- one for the (3aS,9bS) and one for the (3aS,9bR) dia-
ments and exhibit similar NMR spectroscopic data for the stereomer of 5, were launched using the semiempirical
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FULL PAPER
While the theoretical spectra for both, the (3aS,9bS) and
the (3aR,9bS) diastereomers, match the experiment quite
well (Figure 8, top), the CD curves for (3aR,9bR)-5 and
(3aS,9bR)-5 both behave almost oppositely as compared to
the experimental one (Figure 8, bottom). Therefore, only
the stereogenic centre at C-9b can unambiguously be as-
signed as (S) by the calculations. From thermodynamic
considerations, however, one should expect C-3a to be (S)-
configured, too, since the (S,S) diastereomer of 5, with its
relative cis configuration, is drastically favoured over the
trans isomer, by 16.7 kcal/mol, according to the PM3 calcu-
lations. This assignment is confirmed by considerations on
the biosynthetic formation of 5 (see below).
Figure 7. Selected HMBC correlations and structure of ochroleucin
B (5).
Biosynthetic Considerations
PM3[6] method, resulting in 50 and 41 minimum geometries,
respectively.[7]
The structures of the ochroleucins and their co-occur-
rence suggest a close biogenetic relationship. Ochroleucin
These structures were submitted to CD calculations, as A1 (1) could be formed by oxidative coupling of two mono-
described above, this time only by means of the OM2[16] meric precursors 6 and 7. After conversion into the corre-
instead of the CNDO/S[8] Hamiltonian. The comparisons sponding benzoquinones 8 and 9, they might undergo a
of the four UV-corrected[9] overall CD spectra thus ob- Michael addition to form the dimer 10, which should then
tained with the measured curve are displayed in Figure 8: be oxidized to the tetraketone 11. Rearrangement of this
Figure 8. Comparison of the OM2-predicted CD spectra for the four possible diastereomers of ochroleucin B (5) with the experimental
CD curve.
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intermediate as indicated by the arrows would afford ochro- mine the stereogenic center at C-9b as (S), the absolute con-
leucin A1 (1).[17] Since only the (3R) isomer is formed, the figuration of ochroleucin B (5) can now clearly be assigned
stereochemistry of the rearrangement appears to be con- as (3aS,9bS).
trolled by an enzyme (Scheme 1).
Meroterpenoids with 3-methylbut-3-en-1-yl side chains
similar to the potential precursors 6 and 7 are known as
fungal metabolites, e.g., eutypine, a plant pathogen from
Eutypa lata,[20] siccayne from Helminthosporium siccans,[21]
asperpentyne from Aspergillus duricaulis,[22] and sterehirsut-
inal from Stereum hirsutum.[23]
Synthesis of the Presumed Ochroleucin Precursors
In order to obtain experimental evidence for the pro-
posed biosynthesis, the suggested precursors 6 and 7 were
synthesized (Scheme 3). The synthesis of 6 starts from 1,4-
diiodo-3,5-dimethoxybenzene (16),[24] which was efficiently
transformed into the iodo aldehyde 17 according to a
known procedure.[25] Demethylation of 17 with BBr3 gave
the dihydroxy aldehyde 18, which was converted into the
bis(tert-butyldimethylsilyl) (TBS) derivative 19. Sonoga-
shira coupling of 19 with 3-methylbut-3-en-1-yne[26] af-
forded the alkenynyl derivative 20 and small quantities of
Scheme 1. Proposal for the biosynthesis of ochroleucin A1 (1).
The further pathway from 1 to ochroleucin B (5) involves the monodesilylated product 21 in excellent yields. Desi-
reduction of the aldehyde group followed by regioselective lylation of this mixture with n-tetrabutylammonium fluo-
attack of the resulting carbinol at the 2Ј-carbonyl group ride in acetic acid provided the desired monomer 6, which
to yield the cyclic hemiacetal 12 with (2ЈS) configuration. could be converted into the trihydroxy compound 7 by Da-
Hydrolytic opening of the lactone ring would afford the β- kin reaction with sodium percarbonate. Catalytic hydrogen-
oxo acid 13, and, after decarboxylation, enol 14. Proton- ation of precursor 6 yielded the perhydro derivative 22. The
ation of 14 from the Re face, followed by dimethylation of overall yields of compounds 6, 7, and 22 were 38, 32, and
the resulting diphenol 15, would lead to ochroleucin B (5) 18%, respectively.
with the more favourable cis configuration. While the (S)
configuration at C-3a of 5 is directly determined by the
mechanistic considerations displayed in Scheme 2, leading
to the correct constitution of ochroleucin B (5) as deduced
by NMR experiments (see above), the absolute configura-
tion at C-9b can mechanistically not be assigned a priori,
since the protonation of 14 could, in principle, take place
from both sides. But in combination with the results of the
CD calculations (see above), which unambiguously deter-
Scheme 3. Synthesis of the monomeric precursors 6 and 7; reagents
and conditions: a) nBuLi, Et2O, then DMF; b) BBr3; c) TBSCl,
imidazole, DMAP; d) 3-methylbut-3-en-1-yne, cat. Pd(PPh3)2Cl2,
CuI, THF; e) nBu4NF, AcOH; f) Na2CO3 ×1.5 H2O2, THF; g) H2,
PtO2.
With the potential precursors 6 and 7 at hand, experi-
Scheme 2. Mechanistic proposal for the biosynthetic formation of
ochroleucin B (5) from ochroleucin A1 (1).
ments were performed to detect these compounds in the
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Scheme 4. Detection of the monomeric precursor 6 in the crude extract of R. ochroleuca by GC/MS.
mushroom extract. Accordingly, fresh stipes of R. ochro-
leuca were treated with MeOH, and the extract was prepuri-
Experimental Section
General: Optical rotations: Perkin–Elmer 214. UV/Vis: Perkin–El-
mer Lambda 16. IR: Perkin–Elmer FT-IR 1000. NMR: Bruker
ARX-300, AMX-600 and Varian VXR-400 S, in CDCl3 with the
solvent peak as internal standard. Multiplets due to 1J(C,H) coup-
lings are indicated by capital letters. MS: Finnigan MAT 90 and
MAT 95Q. Solid-phase extractions: Chromabond C18 cartridges
fied by preparative HPLC on RP-18. Fractions with the
same retention times as synthetic 6 and 7 were collected
separately, per(trimethylsilyl)ated with N-methyl-N-(tri-
methylsilyl)trifluoroacetamide (MSTFA), and subjected to
GC/MS analysis. The aldehyde 6 was thus identified as the
expected bis(trimethylsilyl) derivative 23 and its MSTFA (Macherey–Nagel). TLC: Merck, silica HPTLC plates Kiesel-
gel 60 F254 S, 0.2 mm, solvent system (v/v): EtOAc/hexanes, 1:2.
Analytical HPLC: Waters 600 E Pump and System Controller with
Photodiode Array Detector 990+. Knauer Vertex columns
4×250 mm, packing material Nucleosil 100 C18, 5 µm. Eluent A:
H2O/MeCN, 9:1; eluent B: MeCN. Linear gradient: 0 min: A
100%, 30 min: B 100%; flow rate 1 mL/min, detection range 200–
400 nm. Preparative HPLC for the isolation of 1, 2, and 5: Merck
Hitachi L 6200 Intelligent Pump and 655A Variable Wavelength
UV Monitor. Knauer Vertex column 16×250 mm, packing mate-
rial Nucleosil 100 C18, 7 µm. Eluent A: H2O/MeCN, 9:1; eluent B:
MeCN. Gradient: 0 min: A 100%, B 30 min: 100%; flow rate:
7 mL/min, detection at 300 nm. Preparative HPLC for the identifi-
cation of 6: Pumps: Waters 590 EF. Injector: U6K. Detector:
Knauer Variable Wavelength Monitor. Detection at 300 nm. Col-
umn: 16×250 mm, Nucleosil 100 C18, 7 µm (Knauer). Gradient:
0 min: 100% H2O, 40 min: 100% MeOH; flow rate: 6 mL/min. GC/
MS: Finnigan MAT 90 double focussing mass spectrometer,
equipped with an EI ion source operating at 70 eV. For GC-MS a
Varian GC 3400 gas chromatograph with a fused silica DB-5ms
capillary column (30 m×0.25 mm, coated with a 0.25 µm layer of
liquid phase) and helium as carrier gas was used for sample separa-
tion. The injector temperature was kept at 300 °C, injection vol-
umes were 0.2–0.4 µL of a 1–2% (m/v) solution. Temperature pro-
gramme: 2 min isothermal at 50 °C, then 10 °C/min up to 300 °C,
finally 10 min isothermal at 300 °C. Retention indices Ri were de-
termined by co-injection of a 0.2 µL sample of a standard mixture
of saturated straight-chain alkanes C10–C36.
adduct 24 (Scheme 4). Both derivatives appeared at the
same retention times and showed the same characteristic
mass spectrometric fragmentation patterns as the silylation
products derived from synthetic aldehyde 6. This result was
confirmed by catalytic hydrogenation of the crude fungal
extract, followed by trimethysilylation with MSTFA and
subsequent GC/MS analysis. By comparison with an au-
thentic sample of the hexahydro derivative 22, subjected to
the same procedure, the two silyl derivatives 25 and 26
could be identified unambiguously.
Attempts to identify the benzenetriol 7 by the same pro-
tocol failed, probably due to its instability. Even synthetic 7
could only be characterized after immediate transformation
into the per(trimethylsilyl) derivative. The identification of
the TMS derivatives of homogentisic acid, gentisic acid
(trace), gentisaldehyde (trace), and gentisyl alcohol in the
crude extract points to tyrosine as biosynthetic precursor of
the ochroleucins.[27]
Conclusion
In summary, the paper shows the interplay of biological
observations, biosynthetic considerations, synthetic studies,
and modern techniques of isolation and structural elucida-
tion including efficient quantum chemical calculations. The
ochroleucins 1 and 5 and their biogenetic precursor 6 repre-
sent the first meroterpenoids from a Russula species. Simple
prenylated derivatives of benzene-1,4-diol are known from
the related genus Lactarius (Russulaceae).[28,29]
Mushrooms: R. ochroleuca was collected in autumn 1996, 1997, and
1998 in spruce forests around München (leg./det. B. Sontag). Two
fruit bodies of the rare R. viscida were collected in August 1998
under Picea near Herrsching, Bavaria (leg./det. N. Arnold).
Isolation Procedure: In a typical workup, 4 kg of the lower third of
the frozen stipes (corresponding to 12 kg of fresh fungi) was ex-
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tracted with EtOAc (5×3 L), until the resulting solution was
Ochroleucin B (5): Yellow gum. Rf (TLC) = 0.70, yellow spot,
colourless. After removal of the solvent, the strongly smelling, brown with KOH. [α]2D9 = –17 (c = 0.33, CHCl3). UV (CHCl3): λmax
brown, oily residue (ca. 50 g) was dissolved in hexanes/EtOAc (2:1)
and purified by chromatography on silica gel with the same solvent
mixture. The first eluting yellow fraction contained ochroleucin B
(2), the slower moving, weakly orange fraction mainly ochroleucin
A1 (1). After removal of the solvent under reduced pressure, each
fraction was further purified by preparative HPLC on a reversed
phase column [Nucleosil-100 C-18, 7 µm, 16×250 mm (Knauer);
solvent A: H2O/MeCN, 9:1; solvent B: MeCN; gradient: start
100% A, linear in 30 min to 100% B; flow rate: 7 mL/min] to afford
ochroleucin B (5, 36 mg, 0.0003%) and ochroleucin A1 (1, 18 mg,
0.00015%). In the case of R. viscida, the yellow stipes from two
specimens (30 g) were extracted overnight with EtOAc (100 mL).
Direct analysis of this extract by TLC and analytical HPLC re-
vealed the presence of 1 and 2. Ochroleucin B (5) could not be
detected.
(log ε) = 286 (3.07), 312 (2.92) nm. CD (MeCN): λ (∆ε) = 230
(–0.004), 237 (–0.06), 251 (+0.10), 247 (+0.02), 275 (–0.27), 314
(–0.04) nm. IR (KBr): ν = 3434 (s, br), 2923 (m), 2194 (m), 1711
˜
(s), 1615 (m), 1483 (w), 1460 (m), 1406 (m), 1232 (s), 1073 (m), 904
1
(w) cm–1. H NMR (600 MHz, CDCl3): δ = 2.02 (dd, J = 1.8, 0.6
Hz, 3 H, 5ЈЈ-CH3), 2.03 (dd, J = 1.8, 0.6 Hz, 3 H, 5Ј-CH3), 3.78 (s,
3 H, 6-OCH3), 4.02 (s, 1 H, 9b-H), 4.10 (s, 3 H, 9-OCH3), 4.43 (d,
J = 16 Hz, 1 H, 5-H), 4.92 (d, J = 16 Hz, 1 H, 5-H), 5.32 (m, 1 H,
4ЈЈ-H), 5.42 (m, 1 H, 4ЈЈ-H), 5.52 (m, 1 H, 4Ј-H), 5.60 (mq, 1 H, 4Ј-
H), 6.45 (s, 1 H, 2-H), 6.80 (s, 1 H, 7-H) ppm. 13C NMR (150 MHz,
CDCl3): δ = 22.9 (Qdd, J = 129, 11, 6 Hz, C-5Ј), 23.3 (Qdd, J =
129, 11, 6 Hz, C-5ЈЈ), 49.5 (Dm, J = 134 Hz, C-9b), 55.7 (Q, J =
144 Hz, 6-OCH3), 59.2 (Td, J = 149, 2 Hz, C-5), 62.0 (Q, J = 145
Hz, 9-OCH3), 79.2 (d, J = 5 Hz, C-1Ј), 85.1 (d, J = 6 Hz, C-1ЈЈ),
95.5 (m, C-2ЈЈ), 101.1 (m, C-3a), 108.9 (m, C-2Ј), 113.2 (D, J = 162
Hz, C-7), 115.3 (d, J = 1 Hz, C-8), 121.9 (Tq, J = 159, 6 Hz, C-
4ЈЈ), 123.4 (m, C-9a), 124.1 (m, C-5a), 125.7 (qdd, J = 6, 2, Ͻ1 Hz,
C-3Ј), 126.2 (Tq, J = 161, 6 Hz, C-4Ј), 126.9 (qdd, J = 6, 2, Ͻ1
Hz, C-3ЈЈ), 135.5 (D, J = 177 Hz, C-2), 150.2 (m, C-6), 152.1 (s, C-
3), 154.4 (m, C-9), 198.7 (dd, J = 7, 4 Hz, C-1) ppm. EI MS (DI,
220 °C, 70 eV): m/z (%) = 390 (100) [M]+, 362 (14), 347 (13), 317
(11), 315 (11), 151 (20), 149 (52), 135 (16), 121 (20), 109 (22), 107
(21), 95 (42), 91 (37), 81 (46), 69 (42), 57 (47), 55 (58). (+)-FAB-
MS: m/z (%) = 391 (32) [M + H]+, 390 (29) [M+]. C24H22O5: calcd.
390.1454; found 390.1453 (HR EIMS).
Ochroleucin A1 (1): Orange-yellow gum. Rf (TLC) = 0.44, orange-
yellow spot, red with KOH. [α]2D9 = –200 (c = 0.002, MeOH). UV
(MeOH): λmax (log ε) = 233 (4.38), 314 (4.17), 369 (3.84) nm. UV
(MeOH + 100 µl 4% KOH in MeOH): λmax (log ε) = 210 (4.44),
290 (4.36), 337 sh (3.88), 452 (3.71) nm. CD (MeOH): λ (∆ε) = 221
(+0.64), 246 (+0.08), 251 (+0.10), 279 (–0.23), 297 (–0.17), 313
(–0.18), 349 (–0.06), 377 (–0.12) nm. IR (KBr): ν = 3432 (s, br),
˜
2928 (m), 2198 (m), 1809 (m), 1716 (s), 1676 (w), 1619 (m), 1419
(m), 1284 (w), 1252 (w), 1216 (w), 990 (m) cm–1. 1H NMR
(600 MHz, CDCl3): δ = 2.01 (dd, J = 1.8, 0.6 Hz, 3 H, 5ЈЈ-CH3),
2.04 (dd, J = 1.8, 0.6 Hz, 3 H, 5ЈЈЈ-CH3), 5.47 (m, 1 H, 4ЈЈ-H), 5.56
(m, 1 H, 4ЈЈ-H), 5.63 (m, 1 H, 4ЈЈЈ-H), 5.71 (m, 1 H, 4ЈЈЈ-H), 7.07
(s, 1 H, 6-H), 7.58 (s, 1 H, 4Ј-H), 9.40 (br., 1 H, 5-OH), 9.65 (s, 1
H, 8-H) ppm. 13C NMR (150 MHz, CDCl3): δ = 22.5 (C-5ЈЈЈ), 22.9
(C-5ЈЈ), 66.4 (C-3), 77.7 (C-1ЈЈЈ), 80.3 (C-1ЈЈ), 101.1 (C-2ЈЈ), 114.9
(C-2ЈЈЈ), 116.4 (C-3a), 121.7 (C-7), 122.4 (C-6), 125.4 (C-4ЈЈ), 125.5
(C-3ЈЈЈ), 125.8 (C-3ЈЈ), 128.4 (C-4ЈЈЈ), 128.5 (C-4), 147.3 (C-4Ј),
147.4 (C-3Ј), 149.2 (C-7a), 157.7 (C-5), 166.1 (C-2), 188.7 (C-5Ј),
188.8 (C-8), 190.7 (C-2Ј) ppm. MS: No [M+], [M + H]+ or [M –
H]– were observed in any ionisation mode.
4-Iodo-2,5-dimethoxybenzaldehyde (17): To a suspension of 1,4-di-
iodo-2,5-dimethoxybenzene (16)[23] (2.0 g, 5.13 mmol) in dry Et2O
(60 mL) was added dropwise at 0 °C a solution of n-BuLi (2.5  in
n-hexane, 2.05 mL, 5.13 mmol) in dry Et2O (7 mL). To the resulting
clear solution a mixture of DMF (0.6 mL, 7.69 mmol) and dry
Et2O (5 mL) was slowly added, and the stirring continued at room
temperature for 3 h. Then, the slightly yellow suspension was
quenched with water (200 mL). The organic phase was separated
and the aqueous phase extracted with Et2O (3×200 mL). The com-
bined organic phases were washed with water (200 mL) and brine
(200 mL), and dried (MgSO4). Concentration under reduced pres-
sure yielded yellow crystals, which were purified by flash
chromatography (hexanes/EtOAc, 9:1) to yield 17 (0.98 g, 65%) as
a colourless solid. M.p. 109 °C. Rf (TLC) = 0.42 (hexanes/EtOAc,
5:1). UV (MeOH): λmax (log ε) = 206 (4.18), 229 (4.05), 273 (3.90),
Rearrangement of 1 into 2: A solution of 1 (12 mg) in MeCN (5 mL)
was stirred at room temperature for 12 d and the resulting mixture
of 1 and 2 was separated by preparative HPLC as described above.
Yield: 2 mg of 2.
Ochroleucin A2 (2): Red gum. Rf (TLC) = 0.89, red spot. UV
(MeCN): λmax (log ε) = 201 (4.00), 300 (3.82), 311 sh (3.78), 402
1
351 (3.70) nm. H NMR (CDCl3, 300 MHz): δ = 3.88, 3.90 (each
s, 3 H, OMe), 7.23 (s, 1 H), 7.47 (s, 1 H), 10.40 (s, 1 H, CHO) ppm.
13C NMR (CDCl3, 75.5 MHz): δ = 56.4, 56.9 (each CH3), 95.9
(Cq), 108.0, 123.7 (each CH), 125.1, 152.8, 156.2 (each Cq), 189.0
(CHO) ppm. MS (EI): m/z (%) = 292 (100) [M+], 277 (27) [M –
CH3]+, 246 (10). C9H9IO3 (292.07): calcd. C 37.01, H 3.11; found
C 36.77, H 3.00.
(3.54), 489 (3.39) nm. IR (KBr): ν = 3435 (s, br), 2924 (s), 2852 (s),
˜
2197 (w), 1782 (w), 1735 (w), 1654 (w), 1458 (w), 1382 (w), 1261
(w), 1178 (w), 912 (w) cm–1. 1H NMR (600 MHz, CDCl3): δ = 2.02
(m, 6 H, 5ЈЈ-CH3 + 5ЈЈЈ-CH3), 5.47 (m, 1 H, 4ЈЈ-H), 5.56 (m, 1 H,
4ЈЈ-H), 5.57 (m, 1 H, 4ЈЈЈ-H), 5.68 (m, 1 H, 4ЈЈЈ-H), 7.15 (s, 1 H,
6-H), 8.73 (s, 1 H, 4Ј-H), 10.80 (s, 1 H, 8-H), 12.24 (s, 1 H, 5-OH)
ppm. 13C NMR (150 MHz, CDCl3): δ = 22.3 (Qdd, J = 130, 11, 4
Hz, C-5ЈЈЈ), 23.2 (Qdd, J = 130, 11, 4 Hz, C-5ЈЈ), 78.5 (d, J = 4
2,5-Dihydroxy-4-iodobenzaldehyde (18): To a solution of 17 (1.0 g,
3.42 mmol) in dry CH2Cl2 (50 mL) was slowly added at –78 °C a
Hz, C-1ЈЈЈ), 80.3 (d, J = 6 Hz, C-1ЈЈ), 100.5 (m, C-2ЈЈ), 105.1 (s, C- solution of BBr3 (4.32 g, 17.1 mmol) in dry CH2Cl2 (10 mL). The
3), 108.1 (m, C-2ЈЈЈ), 113.1 (ddd, J = 21, 6, 6 Hz, C-4), 116.5 (d, J
= 2 Hz, C-3a), 120.3 (s, C-7), 121.8 (d, J = 2 Hz, C-3Ј), 124.9 (Ddd,
cooling bath was removed and the reaction mixture stirred at room
temperature for 12 h. Then, the mixture was quenched with ice-
J = 167, 8, 2 Hz, C-6), 125.3 (Tq, J = 159, 6 Hz, C-4ЈЈ), 125.6 cold water (100 mL), and after phase separation, the aqueous phase
(qdd, J = 7, 2, Ͻ1 Hz, C-3ЈЈ), 125.9 (qdd, J = 7, 2, Ͻ1 Hz, C-3ЈЈЈ), was extracted with CH2Cl2 (3×100 mL). The combined organic
127.6 (Tq, J = 154, 6 Hz, C-4ЈЈЈ), 139.0 (D, J = 189 Hz, C-4Ј), phases were subsequently washed with water (100 mL) and brine
147.9 (d, J = 10 Hz, C-7a), 155.8 (d, J = 8 Hz, C-5Ј), 160.0 (ddd, (100 mL), and dried (MgSO4). Concentration in vacuo and purifi-
J = 5, 5, 3 Hz, C-5), 163.0 (d, J = 13 Hz, C-2Ј), 165.8 (s, C-2), cation of the residue by flash chromatography on silica gel (hex-
196.6 (D, J = 186, C-8) ppm. EI MS (DI, 220 °C, 70 eV): m/z (%)
anes/EtOAc, 3:2) yielded 18 (0.62 g, 70%) as a yellow solid. M.p.
= 386 (100) [M+], 358 (7) [M – CO]+, 330 (10) [M – 2 CO]+, 240 102 °C. Rf (TLC) = 0.51 (hexanes/EtOAc, 3:2). UV (MeOH): λmax
(14), 135 (14), 98 (26), 83 (20), 69 (26), 55 (26). C23H14O6: calcd. (log ε) = 211 (4.03), 234 (3.93), 278 (3.97), 365 (3.59) nm. 1H NMR
386.0790; found 386.0792 (HR EI-MS).
([D6]DMSO, 300 MHz): δ = 7.03, 7.47 (each s, 1 H), 9.99, 10.16,
1030
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1023–1033

Chromogenic Meroterpenoids from the Mushrooms Russula ochroleuca and R. viscida
10.17 (each s, 1 H) ppm. 13C NMR ([D6]DMSO, 75.5 MHz): δ =
(4.3 mL). After cooling to 0 °C, tetra-n-butylammonium fluoride
FULL PAPER
96.1 (Cq), 111.0 (CH), 123.0 (Cq), 127.4 (CH), 149.9, 153.8 (each (8.96 mL, 1  in THF, 8.96 mmol) was added and the mixture
Cq), 190.2 (CHO) ppm. MS (EI): m/z (%) = 264 (100) [M+], 263 stirred at 0 °C for 3 h. The mixture was then quenched with water
(31) [M – CH3]+, 246 (3), 236 (10), 218 (3). C7H5IO3·0.5H2O (150 mL) and extracted with Et2O (3×150 mL). The combined or-
(264.02): calcd. C 30.79, H 2.22; found C 30.80, H 2.49.
ganic phases were washed with brine (100 mL), dried (MgSO4), and
the solvents evaporated in vacuo. The residue was purified by flash
chromatography on silica gel (hexanes/EtOAc, 4:1) to yield 6
(0.90 g, 95.8%) as yellow crystals. M.p. 100 °C. Rf (TLC) = 0.24
(hexanes/EtOAc, 4:1). HPLCprep: Rt = 36.23 min. UV (MeOH):
λmax (log ε) = 207 (4.27), 221 (sh, 4.24), 299 (4.23), 384 (3.91) nm.
2,5-Bis(tert-butyldimethylsilyloxy)-4-iodobenzaldehyde (19): 18
(2.0 g, 7.6 mmol), imidazole (2.1 g, 30.4 mmol), tert-butyldimethyl-
silyl chloride (2.9 g, 19 mmol), and a small quantity of DMAP were
dissolved in anhydrous DMF (60 mL) and heated at 50 °C for 18 h.
After cooling, the reaction mixture was poured into water (200 mL)
and extracted with Et2O (5×150 mL). The combined organic
phases were washed with water (250 mL) and brine (250 mL), and
dried (MgSO4). After concentration in vacuo, the brownish residue
was purified by flash chromatography on silica gel (hexanes/EtOAc,
10:1) to yield 19 (3.51 g, 94%) as a colourless solid. M.p. 84 °C. Rf
(TLC) = 0.78 (hexanes/EtOAc, 10:1). 1H NMR (CDCl3, 300 MHz):
δ = 0.26, 0.28 (each s, 6 H, 2 CH3), 1.01, 1.05 (each s, 9 H, 3 CH3),
7.16, 7.35 (each s, 1 H), 10.32 (s, 1 H, CHO) ppm. 13C NMR
(CDCl3, 75.5 MHz): δ = –4.4, –4.1 (each 2 CH3), 18.3 (2 Cq), 25.7,
25.9 (each 3 CH3), 100.2 (Cq), 114.6 (CH), 127.5 (Cq), 131.3 (CH),
149.9, 152.6 (each Cq), 189.4 (CHO) ppm. MS (EI): m/z (%) = 477
(2) [M – CH3]+, 436 (26) [M – C4H8]+, 435 (100) [M – C4H9]+, 309
(11), 307 (7), 251 (11). C19H33IO3Si2 (492.54): calcd. C 46.33, H
6.75; found C 46.95, H 6.72.
IR (KBr): ν = 3340 (s, br), 3054 (m), 2880 (m), 2197 (w), 1804 (w),
˜
1643 (ss), 1548 (s), 1489 (ss), 1461 (s), 1395 (m), 1344 (s), 1249 (ss),
1207 (m), 1159 (ss), 1012 (w), 896 (s), 864 (s), 804 (s), 782 (s), 657
(m), 515 (m) cm–1. 1H NMR ([D6]DMSO, 300 MHz): δ = 1.94 (dd,
4JH,H = 1.4, 1.1 Hz, 3 H, 5Ј-CH3), 5.39, 5.41 (each m, 1 H, 4Ј-
CH2), 6.88, 7.09 (each s, 1 H), 9.68, 10.10 (each s, 1 H, OH), 10.18
(s, 1 H, CHO) ppm. 13C NMR ([D6]DMSO, 75.5 MHz): δ = 23.0
(CH3), 84.9, 97.0 (each Cq), 112.7 (CH), 118.0 (Cq), 120.5 (CH),
122.8 (Cq), 123.2 (=CH2), 126.2, 150.8 (each Cq), 150.6, 152.2,
153.0 (each Cq), 190.0 (CHO) ppm. EI-MS m/z (%) = 203 (23) [M
+ H]+, 202 (100) [M+], 201 (98) [M – H]+, 174 (6), 173 (11), 156
(12), 117 (7), 115 (18), 91 (13). C12H10O3 (202.06): calcd. C 71.28,
H 4.98; found C 71.23, H 4.96.
Per(trimethylsilyl)ation of 6: MSTFA (10 µL) was added to 1 µg of
6 and incubated at 40 °C for 2 h to yield 23 and 24. 23: GC/MS:
Ri = 2045; m/z (%) = 346 (20) [M]+, 331 (100) [M – CH3]+, 315 (2),
301 (1), 273 (1), 259 (1), 75 (3), 73 (9). 24: GC/MS: Ri = 2189;
m/z (%) = 545 (60) [M+], 530 (21) [M – CH3]+, 456 (30) [M –
OSi(CH3)3]+, 419 (66) [M – N(CH3)COCF3]+, 346 (18), 331 (100),
315 (2), 184 (4), 147 (1), 134 (7), 130 (4), 77 (13), 75 (5), 73 (25),
45 (3).
2,5-Bis(tert-butyldimethylsilyloxy)-4-(3-methylbut-3-en-1-ynyl)benz-
aldehyde (20): To a solution of 19 (6.0 g, 12.2 mmol), 3-methylbut-
3-en-1-yne (3.4 mL, 36.3 mmol), bis(triphenylphosphane)palla-
dium dichloride (0.200 g, 0.28 mmol) and CuI (0.042 g, 0.22 mmol)
in dry THF (200 mL) was added triethylamine (102 mL,
0.73 mmol). The mixture was refluxed for 12 h, whereby a colour-
less deposit formed. After cooling, the suspension was filtered
through Celite and concentrated in vacuo. The resulting solid resi-
due was dissolved in Et2O (250 mL), washed with brine
(3×200 mL), and dried (MgSO4). Evaporation of the solvent under
reduced pressure yielded an oily residue that was flash-chromato-
graphed on silica gel (hexanes/EtOAc, 15:1) to yield 20 (4.22 g,
81%) and the 2-O-desilylated analogue 21 (0.47 g, 12%).
1,2,5-Trihydroxy-4-(3-methylbut-3-en-1-ynyl)benzene (7): To a solu-
tion of 6 (100 mg, 0.49 mmol) in degassed water (2 mL) and THF
(5 mL) was added sodium percarbonate (80 mg, 0.50 mmol) under
argon. The mixture was sonicated in an ultrasound bath for 2 h,
the resulting brown solution treated with 2  HCl (1 mL), and ex-
tracted rapidly with Et2O (3×50 mL) (protecting gas!). The com-
bined extracts were carefully concentrated in vacuo at 20 °C to
yield 7 (80 mg, 86%) as a brownish, very unstable powder. Rf
(TCL) = 0.75 (hexanes/EtOAc, 3:1). 7 was characterized by GC/MS
after immediate silylation of the reaction product with MSTFA.
Tris(trimethylsilyl) derivative of 7: Ri = 2019, m/z (%) = 406 (48)
[M+], 391 (4), 318 (2), 303 (4), 229 (2), 147 (8), 123 (3), 97 (2), 77
(1), 75 (9), 73 (100), 59 (1), 45 (8).
20: Yellowish solid. M.p. 75 °C. Rf (TLC) = 0.84 (hexanes/EtOAc,
15:1). UV (MeOH): λmax (log ε) = 205 (4.45), 223 (sh, 4.41), 293
1
(4.28), 360 (4.00) nm. H NMR (CDCl3, 300 MHz): δ = 0.24, 0.26
(each s, 6 H, 2 CH3), 1.01, 1.02 (each s, 9 H, 3 CH3), 1.99 (dd,
4JH,H = 1.5, 1.1 Hz, 3 H, 5Ј-CH3), 5.35, 5.43 (each m, 1 H, 4Ј-
CH2), 6.87, 7.20 (each s, 1 H), 10.34 (s, 1 H, CHO) ppm. 13C NMR
(CDCl3, 75.5 MHz): δ = –4.4 (4 CH3), 18.2, 18.3 (each Cq), 23.3
(CH3), 25.7 (6 CH3), 85.0, 97.6 (each Cq), 117.0 (CH), 123.0
(=CH2), 123.4 (Cq), 124.4 (CH), 126.7, 127.2, 150.6, 152.2 (each
Cq), 189.3 (CHO) ppm. MS (EI): m/z (%) = 415 (2) [M – CH3]+,
373 (100) [M – C4H9]+. C24H38O3Si2·H2O (430.23): calcd. C 64.24,
H 8.98; found C 64.53, H 8.89.
2,5-Dihydroxy-4-(3-methylbutyl)benzaldehyde (22): To a solution of
the aldehyde 6 (100 mg, 0.5 mmol) in dry MeOH (7 mL) was added
a small portion of PtO2. Then, a stream of H2 was bubbled through
the solution for 1.5 h, whereby the yellow colour disappeared. Fil-
tration of the mixture through Celite and concentration of the fil-
trate in vacuo gave a bright yellow solid that was purified by flash
chromatography (hexanes/EtOAc, 4:1) to yield 22 (50 mg, 48%).
M.p. 75 °C. Rf (TLC) = 0.32 (hexanes/EtOAc, 4:1). 1H NMR
(CDCl3, 300 MHz): δ = 0.95 (d, J = 6.6 Hz, 6 H, 4Ј-CH3), 1.50 (m,
2 H, 2Ј-CH2), 1.62 (sept., J = 6.6 Hz, 1 H, 3Ј-CH), 2.63 (t, J = 8
Hz, 2 H, 1Ј-CH2), 5.11 (s, 1 H, OH), 6.79, 6.90 (each s, 1 H, CH),
9.74 (s, 1 H, OH), 10.63 (s, 1 H, CHO) ppm. 13C NMR (CDCl3,
75.5 MHz): δ = 22.4 (2 CH3), 27.9 (CH), 28.5, 38.2 (each CH2),
117.4, 118.4 (each CH), 118.5 (Cq), 141.8, 146.8, 156.0 (each Cq),
195.3 (CHO) ppm. MS (EI): m/z (%) = 208 (81) [M+], 153 (18),
152 (100) [M – C4H8]+, 151 (63) [M – C4H9]+, 124 (15), 123 (58).
HRMS (EI): m/z = 208.1104 [M+] (calcd. for C12H16O3: 208.1099).
C12H16O3 (208.25): calcd. C 69.21, H 7.74; found C 69.09, H 7.72.
1
21: Yellow oil. Rf (TLC) = 0.63 (hexanes/EtOAc, 15:1). H NMR
(CDCl3, 300 MHz): δ = 0.24 (s, 6 H, 2 CH3), 1.04 (s, 9 H, 3 CH3),
1.99 (“s”, 3 H, CH3), 5.37, 5.44 (each “s”, 1 H, =CH2), 6.92, 7.00
(each s, 1 H), 9.78 (s, 1 H, OH), 10.55 (s, 1 H, CHO) ppm. 13C
NMR (CDCl3, 75.5 MHz): δ = –4.4 (2 CH3), 18.2 (Cq), 23.2 (CH3),
25.7 (3 CH3), 84.9, 98.3, 120.1 (each Cq), 121.8, 122.2 (CH), 123.4
(=CH2), 125.6, 126.5, 148.8, 155.4 (each Cq), 195.4 (CHO) ppm.
MS (EI) m/z (%) = 316 (7) [M+], 261 (8), 260 (23), 259 (100) [M –
C4H9]+, 219 (11). HRMS (EI): m/z = 316.1051 [M+] (calcd. for
C18H24O3Si: 316.1495). C18H24O3Si·0.5H2O (316.47): calcd. C
66.42, H 7.74; found C 66.73, H 8.30.
2,5-Dihydroxy-4-(3-methylbut-3-en-1-ynyl)benzaldehyde (6): To a
solution of 20 (2.0 g, 4.65 mmol) in dry THF was added AcOH
Eur. J. Org. Chem. 2006, 1023–1033
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1031

B. Sontag, M. Rüth, P. Spiteller, N. Arnold, W. Steglich, M. Reichert, G. Bringmann
FULL PAPER
[10] SYBYL, Tripos Associates, 1699 Hanley Road, Suite 303, St.
Per(trimethylsilyl)ation of 22: MSTFA (10 µL) was added to 1 µg
of 22 and incubated at 40 °C for 2 h to yield 25 and 26. 25: GC/
MS: Ri = 1965; m/z (%) = 352 (18) [M+], 337 (100) [M – CH3]+,
265 (10), 75 (20), 73 (60). 26: GC/MS: Ri = 2065; m/z (%) = 551
(40) [M+], 536 (16) [M – CH3]+, 462 (25) [M – OSi(CH3)3]+, 425
(100) [M – N(CH3)COCF3]+, 392 (2), 353 (3), 352 (3), 351 (2), 337
(17), 279 (4), 265 (4), 184 (4), 147 (2), 134 (4), 110 (3), 77 (2), 73
(18).
Louis, MO 63144, USA.
[11]
It should be mentioned that the minimum geometry of ochro-
leucin A1 (1), possessing a hydrogen bond between the alde-
hyde oxygen atom and the o-phenolic hydrogen atom, was cal-
culated to be energetically favoured over the respective mini-
mum structure without this bonding by 7.2 kcal/mol on DFT
level (B3LYP/6-31G*). In contrast, no indication of a hydrogen
bond was found in the corresponding NMR experiment.
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[12]
[13]
Computational Methods: The conformational analyses were per-
formed with a Linux AMD MP 2800+ workstation, in the cases of
1 and 5 by means of the semiempirical PM3[6] method, in the case
of 2 by the use of a DFT approach (B3LYP/6-31G*),[15] as im-
plemented in the program package Gaussian 98,[30] starting from
preoptimized geometries generated by the TRIPOS[10] force field
as part of the molecular modeling package SYBYL 7.0.[10] The
molecular dynamics simulation for 1 was performed at a virtual
temperature of 500 K using the TRIPOS[10] force field. The overall
simulation time was 500 ps, the single geometries were extracted
every 0.5 ps yielding 1000 conformers. The wave functions required
for the computation of the rotational strengths for the electronic
transitions from the ground state to excited states were obtained in
the case of 1 by CNDO/S[8] calculations followed by SCI computa-
tions including 784 singly occupied configurations and the ground-
state determinant, and in the case of 5 by OM2[17] calculations
followed again by SCI computations, now including 900 singly oc-
cupied configurations as well as the ground-state determinant.
These calculations were also carried out with a Linux AMD MP
2800+ workstation using the BDZDO/MCDSPD[31] program pack-
age, and by the use of the MNDO99[32] software package. The sin-
gle CD spectra were summed up weighted according to the Boltz-
mann statistics, i.e., to the respective heats of formation. The rota-
tional strengths were transformed into ∆ε values and for a better
visualization superimposed with a Gaussian band-shape function.
a) E. Jägers, B. Steffan, R. von Ardenne, W. Steglich, Z. Natur-
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[17]
W. Weber, W. Thiel, Theor. Chem. Acc. 2000, 103, 495–506.
A sequence 11 Ǟ 1 Ǟ 2 could also explain the Posternak re-
arrangement,[18] in which a diquinone of type 11 is heated in a
mixture of MeOH and diluted H2SO4. Under these reaction
conditions, a spirodioxolactone of type 1 is expected to be con-
verted into the more stable dilactone isomer 2. Similar path-
ways can be suggested for the formation of dilactones from
suitable benzoquinones.[13,14,19]
[18]
[19]
a) T. Posternak, W. Alcalay, R. Huguenin, Helv. Chim. Acta
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G. Tsoupras, P. de Angelis, J. Fallot, R. Tabacchi, Phytochemis-
try 1991, 30, 471–473.
Acknowledgments
The work was supported by the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie. We thank Dr. Werner
Spahl for various MS experiments and Dr. Bert Steffan for exten-
sive NMR measurements and fruitful discussions.
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1023–1033

Chromogenic Meroterpenoids from the Mushrooms Russula ochroleuca and R. viscida
FULL PAPER
M. W. Wong, J. L. Andres, C. Gonzalez, M. Head-Gordon,
E. S. Replogle, J. A. Pople, Gaussian 98, revision A.7, Gaussian,
Inc., Pittsburgh, PA, USA, 1998.
[32] W. Thiel, Software Package MNDO99, version 6.0, Max-
Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1,
45470 Mülheim, Germany.
[31] J. W. Downing, Program Package BDZDO/MCDSPD, Depart-
ment of Chemistry and Biochemistry, University of Colorado,
Boulder, CO, USA; modified by J. Fleischhauer, W. Schleker,
B. Kramer; ported to Linux by K.-P. Gulden.
Received: September 20, 2005
Published Online: December 12, 2005
Eur. J. Org. Chem. 2006, 1023–1033
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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