A new phenolic metabolite, involutone, isolated from the mushroom Paxillus involutus

Article abstract of DOI:10.1139/v02-194

A new optically active metabolite, involutone, was found in methanol, ethanol, or n-butanol extracts of freshly collected Paxillus involutus. The structure of this compound was proved to be 5-(3,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-2-hydroxy-4-cyclopenten-1,3-dione on the basis of the spectral and chemical properties of involutone and its tetraacetyl derivative. In addition, a number of compounds of known structures such as linoleic and crotonic acids, mannitol, ergosterol, involutin, as well as methyl, ethyl, or n-butyl-β-D-glucopyranosides, and methyl, ethyl, or n-butyl linoleates, depending in both cases on the alcohol used in the extraction, were also isolated in a pure state from the mushroom.

Full text of DOI:10.1139/v02-194

118
A new phenolic metabolite, involutone, isolated
from the mushroom Paxillus involutus
Róza Antkowiak, Wieslaw Z. Antkowiak, Izabela Banczyk, Lucyna Mikolajczyk
Abstract: A new optically active metabolite, involutone, was found in methanol, ethanol, or n-butanol extracts of
freshly collected Paxillus involutus. The structure of this compound was proved to be 5-(3,4-dihydroxyphenyl)-2-
(4-hydroxyphenyl)-2-hydroxy-4-cyclopenten-1,3-dione on the basis of the spectral and chemical properties of involutone
and its tetraacetyl derivative. In addition, a number of compounds of known structures such as linoleic and crotonic
acids, mannitol, ergosterol, involutin, as well as methyl, ethyl, or n-butyl-β-D-glucopyranosides, and methyl, ethyl, or
n-butyl linoleates, depending in both cases on the alcohol used in the extraction, were also isolated in a pure state
from the mushroom.
Key words: Paxillus involutus, Brown Roll-rim, metabolite isolation, structure study, quaternary hydroxycyclopenta-
nedione, NMR of mushroom pigments, glucosides and linoleates, enzymic glucosylation and esterification.
Résumé : L’extraction de Paxillus involutus par du méthanol, de l’éthanol ou du butanol a permis d’isoler un nouveau
métabolite optiquement actif, l’involutone. Sur la base des propriétés spectrales et chimiques de l’involutone et de son
dérivé tétraacétylé, on a déterminé sa structure, la 5-(3,4-dihydroxyphényl)-2-(4-hydroxyphényl)-2-hydroxycyclopent-
4-ène-1,3-dione. De plus, les extractions du champignon ont permis d’isolé à l’état pur un certain nombre de composés
de structures connues, dont les acides linoléique et crotonique, du mannitol, de l’ergostérol, de l’involutine ainsi que
des β-D-glucopyranosides de méthyle, d’éthyle ou de butyle et des linoléates de méthyle, éthyle ou butyle suivant, dans
chaque cas, la nature de l’alcool utilisé pour les extractions.
Mots clés : Paxillus involutus, chanterelle brune, isolement d’un métabolite, étude de structure, hydroxycyclopentane-
dione quaternaire, RMN des pigments, glucosides et linoléates du champignon, glucosylation et estérification enzyma-
tique.
[Traduit par la Rédaction] Antkowiak et al. 124
Introduction
nitrogen-containing compounds (including traces of
muscarine and β-acetylcholine) known from their occurrence
in other fungi species (12, 13). In another laboratory,
involutin, an unknown pigment at that time, was isolated
and its structure determined on the grounds of spectral data
and chemical properties (14, 15). In subsequent years, the
supposed presence in P. involutus of two additional com-
pounds, chamonixin (16) and anhydroinvolutin (17), both
with closely related structures to involutin, was suggested.
More recently, studies on the absolute configuration of
involutin and chamonixin were reported (18). Additionally,
the content and metabolism of polyamines occurring in P.
involutus has been of interest in recent years (19).
The usefulness of Paxillus involutus (Brown Roll-rim) as
an edible mushroom has been a matter of controversy for a
long time, which is evident both in the literature and espe-
cially when comparing the opinions of amateur mushroom
gatherers with those of most research reports, and the prob-
lem was the subject of a few review articles (1, 2). The
hemolytic symptoms caused by P. involutus consumption,
which are most often reported as being very serious, were
found in the cases of both laboratory animals (3–5) and of
human beings (6–11), yet the chemical compound responsi-
ble for the mortal toxicity is still unknown.
Research on the chemical composition of the mushroom
carried out at the end of the 1960s and in the early 1970s of
the previous century showed, mainly on the basis of paper
chromatography, electrophoresis and TLC pattern, the pres-
ence of such metabolites as fatty acids, carbohydrates, and
Results and discussion
Research carried out recently in our laboratory confirmed
the occurrence of involutin2 in P. involutus. Moreover, for
Received 23 April 2002. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 31 January 2003.
This paper is dedicated to Dr. O.E. Edwards (NRC) in recognition of his outstanding contribution to the chemistry of natural products.
R. Antkowiak, W.Z. Antkowiak,1 I. Banczyk, and L. Mikolajczyk. Faculty of Chemistry, A. Mickiewicz University,
Grunwaldzka 6, 60-780 Poznan, Poland.
1Corresponding author (e-mail address: wzantk@amu.edu.pl).
2The structure of involutin reported in the literature was confirmed by our HMBC experiments, the results of which are quoted in
the experimental part of this paper.
Can. J. Chem. 81: 118–124 (2003)
doi: 10.1139/V02-194
© 2003 NRC Canada

Antkowiak et al.
119
Scheme 1. The structural relationship between involutin and involutone.
the first time ever, we have isolated from this mushroom
several metabolites in an homogeneous form, namely
D-mannitol, crotonic acid, (–)-ergosterol, and β-methyl,
-ethyl, or -n-butyl-D-glucopyranosides,3 depending on the al-
cohol (MeOH, EtOH, or n-BuOH) used for the extraction at
room temperature. Additionally, the presence in the mush-
room of some fatty acids with linoleic acid as the main com-
ponent was proved, but most of the acid was isolated in the
form of methyl, ethyl, or n-butyl linoleate, depending on the
alcohol (MeOH, EtOH, or n-BuOH) used for the extraction,
respectively; according to GC–MS, the presence of a small
amount of palmitic acid and oleic acid was also identified.
The formation of both glucosides and linoleate esters
from the original metabolites4 suggests enzymatically cata-
lyzed reactions and, consequently, the presence of the suit-
able, active enzymes (glucosidase or glucosyltransferase and
lipase, respectively) as the constituents of P. involutus.
A number of mushrooms are able to produce metabolites
as a response to a mechanical injury of the fruit bodies, but,
as a rule, this involves an enzymatically induced oxidation
process (20). The syntheses of glycosides and fatty acid es-
ters can also be catalyzed by enzymes of fungal origin but
mostly derived from lower fungi (21–23).
The ability of P. involutus to produce some artefacts in the
isolation procedure and, thereby, changing its original chem-
ical composition (especially if transglucosylation and (or)
transesterification takes place), could be responsible for the
hitherto difficulties in finding the right metabolite which
causes the toxic properties of the mushroom.
the product of a simple oxidation of the secondary alcohol
(involutin) to a corresponding ketone (didehydroinvolutin).
Such a transformation would leave the asymmetric benzylic
carbon, to which one hydrogen is attached, as the only chiral
center. According to the literature data (14, 15, 24), the
NMR absorption of a benzylic proton of similar systems was
recorded as being somewhere in the range of 3.98–4.32 ppm.
In the case of involutone, the absence of an absorption in
that region eliminated such a chiral center in the molecule.
Other spectroscopic data recorded for involutone showed
the absence of protons attached to sp3 hybridized carbons
(1H NMR, Distortionless enhancement by polarization
transfer (DEPT) and the presence in the molecule of two
substituted benzene rings, 4-hydroxyphenyl and 3,4-dihy-
droxyphenyl (the aromatic ring protons gave the AA′XX′
and AMX systems, respectively, in the range 7.59–
6.79 ppm). The region of aromatic carbons (158–115 ppm)
contained, apart from the 10 signals originating from the 12
carbons composing the two benzene rings, two additional
peaks of vinyl carbons. One of these carbon atoms, absorb-
ing at 139.57 ppm (C-4), is bonded to a proton of chemical
shift 7.51 ppm (Heteronuclear correlation spectroscopy
(HETCOR) experiment), while the other one (C-5) of the vi-
nyl system gave a resonance absorption, together with the
signals of the carbon atoms of the sp2 hybridization bearing
the OH groups, in the range of 158.19–145.78 ppm.
The presence of the three phenolic groups and one addi-
tional hydroxyl in the molecule was demonstrated by a
broad and intense IR absorption with the maximum at
3350 cm–1, and the strong band at 1250 cm–1. Especially im-
Additionally to the compounds of known structure, we
found and isolated with a high degree of purity an optically
active metabolite of an unknown structure, which melted at
181 to 182°C and showed a specific rotation ([α]D +96.3°),
and was yellow or, when hydrated, orange in colour. The
molecular formula of this metabolite was determined to be
C17H12O6 on the basis of HR-MS. The trivial name involu-
tone was given to that compound, refering both to the mush-
room species as well as to the elemental compositions only
differing from that of involutin by two hydrogens
(Scheme 1).
1
portant in this respect was, however, the H NMR spectrum
run in DMSO-d6 solution, which consisted of three signals
of phenolic OH in the region 9.4–10.0 ppm and of one sin-
glet at 6.60 ppm of the fourth hydroxyl proton separated
from those of aromatic OH by about 3 ppm. Further evi-
dence, which confirmed the presence of four hydroxyl
groups in the molecule, followed from the observations that
four protons are easily exchangeable for deuterium (1H
NMR) and that the metabolite can be easily transformed into
its tetraacetyl derivative.
In spite of the small difference in elemental composition,
the structural analysis showed that the new compound is not
Besides the carbons of the aromatic rings and the vinyl
system, the molecule of involutone contains three additional
3 In the cases of ethyl and n-butyl β-glucosides, the chemical shifts of the diastereotopic protons of the aglycone methylene group attached to
the anomeric carbon through oxygen differ by 0.33 ppm (Jgem = 9.6 Hz), due to the long range influence of the D-glucose molecule’s
chirality.
4 The transformations proceeded in a corresponding alcohol, being diluted with a great deal of water occurring naturally in the mushroom.
© 2003 NRC Canada

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Can. J. Chem. Vol. 81, 2003
carbons, from which two (absorbing at 202.86 and
200.63 ppm) are components of the two carbonyl groups,
giving both a weak and a strong absorption at 1740 and
1692 cm–1 for C=O and C=C-C=O, respectively. The non-
aromatic structural elements, together with the final
remaining carbon, which shows a weak resonance signal at
78.30 ppm, compose a cyclopentenedione skeleton, to which
the two substituted phenyl rings and a hydroxyl group are
attached. This, consequently, gave a system in which the
only sp3 hybridized carbon constitutes a chiral center, bind-
ing either both the differently substituted phenyl rings (while
the nonphenolic hydroxyl is involved in an enol system) or
one of the phenolic rings and the OH group. This problem
was solved by an examination of the intramolecular proton
interactions through the space and the heteronuclear
long-range connectivity through bonds.
spectrum. Already knowing the chemical shifts of aromatic
ring protons at that time, we were able to find the position of
attachment to the phenyl ring of each of the acetoxy groups.
The two-proton signal of H-3′′ and H-5′′ (7.16 ppm) gave a
correlation point with carbonyl carbon absorption at
169.90 ppm, showing on a coupling through C-4′′, to which
that acetoxy group has to be bound. Similarly, the coupling
of H-2′ (7.92 ppm) with the carbonyl carbon giving a signal
at 169.03 ppm made it possible to locate the acetoxy group
at C-3′, while the correlation of the chemical shift of H-5′
(7.40 ppm) with that at 168.80 ppm showed the position C-4′
as the attachment point for the acetoxy group responsible for
that absorption. The assignment of the chemical shift of
146.16 ppm to C-4′ was proved by its correlation with both
H-2′ (7.92 ppm) and H-6′ (7.95 ppm) of the trisubstituted
phenyl ring, while of the value 152.55 ppm to C-4′′, due to
its correlation (3JCH) with H-2′′ and H-6′′ (7.46 ppm). The
assignment of the third aromatic ring carbon bearing the
acetoxy group resulted from the coupling of H-2′ (7.92 ppm)
and H-5′ (7.40 ppm) with C-3′ absorbing at 143.50 ppm
through two and three bonds, respectively.
The assignment of the chemical shifts 127.72 and
129.91 ppm to the aromatic ring carbons C-1′ and C-1′′, re-
spectively, was based on the observation that the 127.72 ppm
value is correlated with the chemical shift of H-5′ and H-4
(3JCH), which proved the C-1′ position for that carbon, while
H-3′′ and H-5′′ is coupled (3JCH) with the carbon absorbing
at 129.91 ppm, which has to be C-1′′. The other pair of pro-
tons of that ring (H-2′′ and H-6′′ (7.46 ppm)) gave a
cross-peak with C-2 (81.70 ppm), which, as an element of
the five-membered ring, binds the 4-acetoxyphenyl group.
The protons (H-2′ and H-6′) mentioned above in connec-
tion with the phenyl carbon – proton relationship elucida-
tion, are additionally correlated with a sp2-hybridized carbon
of a chemical shift 153.55 ppm, which, in turn, gave a strong
cross-peak (2JC-H) with vinyl proton H-4. This observation
led us to the conclusion that the carbon of 153.55 ppm
is C-5 of the vinyl system and constitutes the position of the
attachment of the 3,4-diacetoxyphenyl group to the
five-membered ring.
First, to avoid autooxidation when treated with acetic an-
hydride in the presence of pyridine, involutone was trans-
formed quantitatively into a tetraacetyl derivative, the
chromatography of which gave bright yellow crystals of mp
68–70°C and [α]D +44.2°.
1
A routine analysis of the H and 13C NMR spectra of the
obtained product5 showed, as in the case of nonacetylated
involutone, the presence in the molecule of tri- (1, 3, 4) and
di- (1, 4) substituted phenyl rings, a vinyl system, as well as
two keto carbonyls and, additionally, four acetoxy groups.
On the basis of the HMQC and HMBC experiments
carried out for the CD3CN solutions of the tetraacetyl in-
volutone, the assignments of all carbons in the molecule
were possible. The presence of a five-membered ring was
proved by the observed correlation of the vinyl hydrogen
H-4, that absorbed at 7.70 ppm then and at 7.51 ppm before
acetylation, with the two carbonyl carbons (C-3 and C-1) ab-
sorbing at 197.36 and 196.03 ppm, respectively. The differ-
entiation of these carbonyl carbons was based on the
different intensities of the cross-peaks from H-4 to C-1 and
from H-4 to C-3, the smaller for the former being coupled
through three bonds in comparison with the latter, which
was more intense because of the coupling through two bonds
only. An additional correlation of proton H-4 was found
with both the vinyl carbons C-4 (141.12 ppm) and C-5
(153.55 ppm) by HETCOR and HMBC, respectively, and
with the sp3 hybridized carbon (C-2) absorbing at
81.70 ppm.
The conclusions concerning the location of both the phe-
nolic substituents and the hydroxyl group in the molecule
were confirmed by a NOE difference experiment, which
indicated that a significant interproton contact through the
space of the vinyl hydrogen (H-4) only exists with H-2′ and
H-6′ of the 3,4-diacetoxyphenyl ring (a diagnostic enhance-
ment of signals at δ 7.92 and 7.95 ppm), while the
corresponding protons (H-2′′ and H-6′′) of the monoace-
toxyphenyl ring revealed inertness when the vinyl proton
was irradiated. These observations indicated the presence of
a (AcO)2C6H3-C=C-H fragment in the molecule, thus, ruling
out the previously considered possibility of the immediate,
geminal vicinity of both phenolic substituents.
Apart from the two signals of the ketone function carbons,
the 13C NMR spectrum showed the presence of four ester
carbonyl carbons. The protons of one of the four acetoxy
groups (2.18 ppm) were found to be correlated (4JC-H) with
C-2 (81.70 ppm) being the only sp3 hybridized element of
the five-membered ring. The remaining three (169.90,
169.03, and 168.80 ppm) were recognized as the elements of
acetoxy substituents attached to phenyl rings on the basis of
their carbonyl carbons’ coupling with the suitable aromatic
ring protons6 through four bonds, which gave cross-peaks
(of weak intensities, but easily recognizable) in the HMBC
Based on the presented arguments, the structure of
5-(3,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-2-hydroxy-4-
5 The acetylation of involutone to its tetraacetyl derivative caused the chemical shift of the chiral carbinol carbon to be move downfield from
78.30 to 81.85 ppm, and of the four aromatic carbons (C-2′, C-5′, C-3 ′′, and C-5 ′′) being ortho to OAc positions to be deshielded by about
8 ppm.
6 Besides, there is an evident coupling (2JC-H) with the methyl protons, which, however, appeared to be difficult to analyse, due to the small
differences between the chemical shifts of the methyl protons.
© 2003 NRC Canada

Antkowiak et al.
121
Scheme 2. Selected HMBC (←→) and NOE (← − →) correla-
tions in the structure of tetraacetylinvolutone.
General procedure of the metabolite isolation
Freshly collected fruit bodies of P. involutus were, first
rinsed with water to remove sand and pieces of grass and
leaves, then, either cut into small pieces or ground and, next
treated with methanol, ethanol, or n-butanol (1.5 mL of the
alcohol per 1 g of the mushroom) at room temperature for a
few weeks or months.8 When MeOH or EtOH were used,
some foam appeared on the surface in the first hours of di-
gestion. The resulting dark brown alcohol solution, after sep-
aration from the insoluble, semi-solid material by
decantation and filtration, was concentrated under dimin-
ished pressure until all the alcohol was removed. The re-
maining aqueous residue, containing the naturally occurring
mushroom components, including water (or a solid residue
in the case of the n-butanol extract), was digested succes-
sively with n-hexane (A), benzene (B), ethyl acetate (C), and
a mixture of PhH, AcOEt, and MeCN (1:2:4) (D). When
kept for a week, the aqueous residue separated a light-brown
solid (E) of a carbohydrate character.
Following this procedure in an experiment taken by way
of example, the extraction of 900 g of fresh fruit bodies gave
the individual fractions as follows: 0.84 g (A), 0.52 g (B),
0.53 g (C), 1.39 g (D), and 1.75 g (E). In the case of the ex-
tracts A–D, the soluble material was recovered by drying the
particular solution over Na2SO4, followed by concentrating
it under diminished pressure, and, finally, as a rule, subject-
ing the residue to chromatographic separation on silica gel.
The products of the known structure isolated in a homoge-
neous state, namely methyl, ethyl, or n-butyl linoleate (A),
linoleic acid, ergosterol (B), methyl, ethyl, or n-butyl-β-
D-glucoside, involutin (C), crotonic acid, mannitol (D), were
identified on the basis of their physical properties, including
the comparisons of their EI-MS and NMR spectra with those
reported in the literature (the source of consistent data was
shown in a reference following the spectral method).
cyclopenten-1,3-dione for the new pigment involutone was
assigned.7 The most significant observations for structure
elucidation concerning the scalar coupling (HMBC) and
through-space interactions (1D NOE) are shown in
Scheme 2.
Experimental
Melting points were determined on a Büchi 535 capillary
apparatus and are uncorrected. Optical rotations were deter-
mined with a PerkinElmer 243B digital polarimeter. UV
spectra were taken with a JASCO V-550 spectrophotometer
in an acetonitrile solution. IR spectra were measured on a
1
FT IR Bruker IFS 113v instrument for KBr discs. H and
13C NMR spectra were recorded on Varian Mercury-300 and
Bruker DRX-600 spectrometers. A Bruker DRX-600 spec-
trometer, operating at 600.055 MHz (1H) and 150.898 MHz
(13C), was used for 2D spectra. All spectra were acquired
with a Bruker 5 mm inverse triple resonance gradient probe
(TBI), using standard Bruker pulse sequences for the respec-
tive experiments. Proton and carbon assignments were based
on COSY, NOE, HETCOR, HMQC, and HMBC experi-
ments. MS spectral data were obtained on an AMD 402
mass spectrometer.
Linoleic acid ((Z,Z)-9,12-octadecadienoic acid)
The pure acid was obtained from extract B by an elution
of the silica gel column with PhH-n-hexane (1:1) solution.
1
References: EI-MS (25), H NMR (25), 13C NMR (26).
TLC analyses were carried out using Uniplate™ silica gel
plates GHLF 250 µ (Analtech, Inc.) and PhH–CHCl3–MeOH
(3:6:1) as a developing system. Column chromatography
was carried out using Merck Kieselgel 60 (230–400 mesh)
Linoleic acid esters
The A and B extracts, during the chromatography on sil-
ica gel, gave a methyl, ethyl, or n-butyl ester as a colourless
oil when n-hexane, benzene, and 10% AcOEt-n-hexane were
used as the eluents.
and
a 2:1 mixture of Whatman cellulose-silica gel
(MN-Kieselgel 60, 100–200 mesh) as the stationary phase,
while hexane, benzene, AcOEt, CH2Cl2, CHCl3, and 5%
MeOH in CHCl3 were successively applied as the eluting
solvents.
The fruit bodies of the mushroom P. involutus (growing
near birch trees) were collected in Rybojedzko, a southern
district of Poznan, Poland, every September in recent suc-
cessive years.
Methyl linoleate
References: EI-MS (27), H NMR (28), 13C NMR (29).
1
Ethyl linoleate
References: EI-MS (25), H NMR (25), 13C NMR (25).
1
7 A possible synthetic relationship between involutin and involutone can consist of a process in which the former is subjected both to a water
molecule elimination and the benzylic position oxidation, as suggested by the reviewer, to whom we are very grateful. To preserve the opti-
cal activity in such a biotransformation, when not assisted by an enzyme, the benzylic oxidation should precede the elimination.
8 The treatment of the freshly collected mushrooms (being under nitrogen) with ethyl acetate at room temperature led to the formation of ace-
tic acid, most likely due to enzymic hydrolysis of the solvent.
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Can. J. Chem. Vol. 81, 2003
62.00 (C-6), 20.79 (COCH3), 20.71 (COCH3), 20.67
(COCH3), 20.65 (COCH3), 15.08 (C-2′). EI-MS m/z (%): 375
([M – 1]+), 331, 303, 256, 243, 214, 200, 183, 169, 157, 145,
141, 127, 115, 112, 102, 98, 81, 70, 69, 43 (100).
n-Butyl linoleate
1H NMR (600 MHz, CDCl3): δ 5.40–5.31 (4H, m, H-9,
H-10, H-12 and H-13), 4.07 (2H, t, J = 6.7 Hz, H-1′), 2.77
(2H, t, J = 6.8 Hz, H-11), 2.29 (2H, t, J = 7.5 Hz, H-2),
2.07–2.01 (4H, m, H-8 and H-14), 1.63–1.53 (4H, m, H-3
and H-2′), 1.42–1.25 (16H, m, H-4, H-5, H-6, H-7, H-15,
H-16, H-17 and H-3′), 0.93 (3H, t, J = 7.1 Hz, H-4′), 0.92
(3H, t, J = 7.1 Hz, H-18). 13C NMR of the alcohol moiety
(see ref. (30)). EI-MS m/z (%): 336 ([M]+), 279, 263, 220,
209, 196, 185, 164, 150, 137, 129, 123, 109, 79, 67 (100),
57, 41.
n-Butyl-β-D-glucopyranoside
A colourless solid of mp 52–54°C, [α]D –25.6° (c = 0.55,
1
MeOH). H NMR: see ref. (35). 13C NMR: see ref. (35).
EI-MS m/z (%): 237 ([M + 1]+), 205, 187, 163, 145, 144,
116, 103, 98, 97, 87, 85, 74, 73, 60 (100), 57 (95), 44, 42.
2,3,4,6-Tetra-O-acetyl-1-n-butyl-β-D-glucopyranoside
A colourless solid of mp 54.5 to 55.5°C, [α]D –9.36° (c =
0.435, MeOH). IR (KBr, cm–1): 2967, 2904, 2871, 1759,
1743, 1433, 1378, 1226, 1169, 1099, 1069, 1040, 958, 603.1
H NMR (600 MHz, CDCl3) δ: 5.21 (1H, t, J = 9.6 Hz, H-3),
5.09 (1H, dd, J = 9.8 Hz, J = 9.6 Hz, H-4), 4.99 (1H, dd, J =
9.6 Hz, J = 8.0 Hz, H-2), 4.49 (1H, d, J = 8.0 Hz, H-1), 4.27
(1H, dd, Jgem = 12.3 Hz, J = 4.8 Hz, HA-6), 4.14 (1H, dd,
Ergosterol
The continuing chromatography of fraction B, after re-
moving linoleic acid and its methyl, ethyl, or n-butyl ester,
led to obtaining, by elution with benzene, a colourless solid,
the rechromatography of which, carried out twice, gave crys-
tals of mp 168 to 169°C and [α]D –135° (c = 1, CHCl3). The
spectral data of the product were consistent with those for
ergosterol reported elsewhere (EI-MS (31), IR (32a), 1H
NMR and 13C NMR (33a, 34)).
Jgem = 12.3 Hz, J = 1.9 Hz, HB-6), 3.88 (1H, dq, Jgem
=
9.6 Hz, J = 7.1 Hz, HA-1′), 3.69 (1H, ddd, J = 9.8 Hz, J =
4.8 Hz, J = 1.9 Hz, H-5), 3.48 (1H, dq, Jgem = 9.6 Hz, J =
7.1 Hz, HB-1′), 2.09 (3H, s, COCH3), 2.04 (3H, s, COCH3),
2.03 (3H, s, COCH3), 2.01 (3H, s, COCH3), 1.54 (2H, quin-
tet, J = 7.0 Hz, H-2′), 1.35 (2H, sextet, J = 7.1 Hz, H-3′),
0.90 (3H, t, J = 7.1 Hz, H-4′). 13C NMR (150 MHz, CDCl3)
δ: 170.55 (COCH3), 170.17 (COCH3), 169.24 (COCH3),
169.14 (COCH3), 100.79 (C-1), 72.84 (C-3), 71.70 (C-5),
71.33 (C-2), 69.93 (C-1′), 68.46 (C-4), 62.00 (C-6), 31.44
(C-2′), 20.84 (COCH3), 20.73 (2 × COCH3), 20.70
(COCH3), 19.03 (C-3′), 13.82 (C-4′). EI-MS m/z (%): 403
([M – 1]+), 345, 331, 284, 243, 211, 200, 157, 115, 81, 43
(100).
Alkyl-β-D-glucopyranosides
A suitable (methyl, ethyl, or n-butyl) β-glucoside was ob-
tained when fraction C was chromatographed on silica gel
and the column was washed with CHCl3. With the exception
of the OH proton absorptions, the H and 13C NMR data ob-
1
tained for these glucosides dissolved in DMSO-d6 were
close to the corresponding values reported for D2O solutions
(35). Each of the glucosides was additionally characterized
by the properties of its tetraacetyl derivative obtained when a
solution of the corresponding alkyl-β-glucoside in Ac2O
(supplied with a catalytic amount of pyridine) was kept at
room temperature for 10 h, followed by an ice addition. Af-
ter 30 min, the product was taken up into AcOEt.
Involutin
(5-(3,4-dihydroxyphenyl)-3,4-dihydroxy-2-(4-hydroxyphe
nyl)-2-cyclopenten-1-one)
Methyl-β-D-glucopyranoside
A recrystallization from MeOH gave colourless needles of
mp 110 to 111°C and [α]D –33° (c = 1, MeOH). References
After the elution of glucosides from fraction C, the silica
gel column was washed with a 3% solution of MeOH in
CHCl3, which gave a cream-coloured solid of mp 173–
175°C (with decomp.) and [α]D –24.7° (c = 0.4, AcOEt). An
addition of 10% aq NaOH to a methanol solution of the
product changed its colour to deep purple. IR: see ref. (18).
1H NMR (300 MHz, CD3CN) δ:9 7.68 (2H, d of AA′XX′,
J = 9.0 Hz, H-2′′ and H-6′′), 6.83 (2H, d of AA′XX′, J =
9.0 Hz, H-3′′ and H-5′′), 6.75 (1H, d, J = 8.0 Hz, H-5′),
6.59 (1H, d, J = 2.2 Hz, H-2′), 6.52 (1H, dd, J = 8.0 Hz, J =
2.2 Hz, H-6′), 4.69 (1H, d, J = 6.9 Hz, H-4), 3.95 (1H, d,
J = 6.9 Hz, H-5). 13C NMR (150 MHz, CD3CN) δ: 190.50
(C-1), 174.63 (C-3), 156.91 (C-4′′), 145.25 (C-3′), 144.77
(C-4′), 130.48 (C-2′′,6′′), 128.95 (C-1′), 123.53 (C-1′′),
123.02 (C-6′), 117.60 (C-2′), 116.75 (C-2), 116.21 (C-5′),
115.76 (C-3′′,5′′), 71.88 (C-4), 55.14 (C-5). HMBC
(600 MHz, CD3CN): H-2′′ (3JCH) and H-6′′ (3JCH) ⇒ C-4′′;
H-3′′ (2JCH) and H-5′′ (2JCH) ⇒ C-4′′; H-5′ (3JCH) ⇒ C-3′;
H-2′ (3JCH) and H-6′ (3JCH) ⇒ C-4′; H-5 (2JCH) and H-5′
(3JCH) ⇒ C-1′; H-3′′ (3JCH) and H-5′′ (3JCH) ⇒ C-1′′; H-2′′
1
for: EI-MS (36), H NMR (35, 37), 13C NMR (35, 37).
2,3,4,6-Tetra-O-acetyl-1-methyl-β-D-glucopyranoside
A colourless solid of mp 108 to 109°C and [α]D –18.89°
1
(c = 0.5, MeOH). References for: EI-MS (38), H NMR (38,
37), 13C NMR (37).
Ethyl-β-D-glucopyranoside
A colourless semi-solid of [α]D –35.2° (c= 0.565, MeOH).
1H NMR: see refs. (35, 39). 13C NMR: see ref. (35). EI-MS
m/z (%): 207 ([M – 1]+), 177, 159, 144, 131, 118, 98, 88, 75,
74, 73, 71, 61, 60 (100), 59, 57, 47, 43, 31.
2,3,4,6-Tetra-O-acetyl-1-ethyl-β-D-glucopyranoside
A colourless solid of mp 102.5 to 103.5°C and [α]D –30.6°
(c = 0.20, MeOH). 1H NMR: see ref. (39). 13C NMR
(75 MHz, CDCl3) δ: 170.61 (COCH3), 170.23 (COCH3),
169.31 (COCH3), 169.25 (COCH3), 100.54 (C-1), 72.89
(C-3), 71.75 (C-5), 71.34 (C-2), 68.45 (C-4), 65.66 (C-1′),
1
9 The H and 13C NMR data recorded by us for CD3CN and CD3OD solutions were very similar to each other, but differed to some extent
from those reported in ref. (18). The use of CD3CN as a solvent made it possible to observe the C-1, C-2, and C-3 signals.
© 2003 NRC Canada

Antkowiak et al.
123
(3JCH) and H-6′′ (3JCH) ⇒ C-2; H-2′ (3JCH) and H-6′ (3JCH) ⇒
(1H, s, H-4), 7.59 (1H, d, J = 2.2 Hz, H-2′), 7.53 (1H, dd,
J = 8.2 Hz, J = 2.2 Hz, H-6′), 7.11 (2H, d of AA′XX′, J =
8.8 Hz, H-2′′ and H-6′′), 6.89 (1H, d, J = 8.2 Hz, H-5′),
6.72 (2H, d of AA′XX′, J = 8.8 Hz, H-3′′ and H-5′′), 6.60
(1H, s, OH). 13C NMR (75 MHz, CD3CN) δ: 202.86 (C=O),
200.63 (C=O), 158.19, 156.87, 149.91, 145.78 (C-5, C-3′,
C-4′, C-4′′), 139.57 (C-4), 129.27 (C-1′ or C-1′′), 128.36
(C-2′′ and C-6′′), 124.09 (C-6′), 122.13 (C-1′ or C-1′′),
117.00 (C-2′), 116.69 (C-5′), 115.52 (C-3′′ and C-5′′),
78.26 (C-2). 13C NMR (75 MHz, DMSO-d6) δ: 203.57
(C=O), 201.32 (C=O), 157.61, 155.55, 150.27, 145.92 (C-5,
C-3′, C-4′, C-4′′), 138.39 (C-4), 128.05 (C-1′ or C-1′′),
127.58 (C-2′′ and C-6′′), 122.96 (C-6′), 120.52 (C-1′ or
C-1′′), 116.36 (C-2′), 116.11 (C-5′), 77.39 (C-2). EI-MS
m/z (%): 312 ([M]+), 296, 284, 239, 189, 181, 163, 134, 121
(100), 93, 91, 77, 65. HR-MS calcd. for C17H12O6:
312.06339; found: 312.06170.
C-5. EI-MS: see ref. (18).
D-Mannitol
The crude residue obtained after a concentration of the
PhH–AcOEt–MeCN extract (fraction D) was dissolved in
hot MeOH and the solution allowed to cool. The separated
colourless crystals of mannitol, when recrystallized from
MeOH, melted at 168 to 168.5°C and showed [α]D 0° (c =
1
1, H2O). The spectral data (EI-MS, IR, H NMR, and 13C
NMR) were consistent with those reported elsewhere (40,
32b, 33b).
Hexaacetyl-D-mannitol
Mannitol obtained in the above way, when treated with
Ac2O in the presence of pyridine, gave hexaacetylmannitol
in a yield of above 80%. A recrystallization from AcOEt fur-
nished colourless needles of mp 124 to 125°C and [α]D
+24.83° (c = 1, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 5.45
(2H, d, J = 8.8, H-3 and H-4), 5.08 (2H, ddd, J = 8.8., J =
5.1, J = 2.7, H-2 and H-5), 4.22 (2H, dd, Jgem = 12.5, J =
2.7, HA-1 and HA-6), 4.07 (2H, dd, Jgem = 12.5, J = 5.1,
HB-1 and HB-6), 2.10 (6H, s, COCH3), 2.08 (6H, s, COCH3),
2.06 (6H, s, COCH3). 13C NMR (75 MHz, CDCl3) δ: 170.37
(2 × COCH3), 169.70 (2 × COCH3), 169.49 (2 × COCH3),
71.47 (C-2 and C-5), 67.38 (C-3 and C-4), 61.84 (C-1 and
C-6), 20.94 (2 × COCH3), 20.76 (2 × COCH3), 20.68 (2 ×
COCH3). EI-MS: see ref. (41).
Tetraacetylinvolutone
(5-(3,4-diacetoxyphenyl)-2-(4-acetoxyphenyl)-2-acetoxy-4
-cyclo-penten-1,3-dione)
A solution of involutone ([α]D +96.3°, 6.5 mg) in 1 mL of
Ac2O was supplied with one drop of anhydrous pyridine and
stirred at room temperature for 60 h. The reaction mixture
was worked up in a usual way and the product taken up into
AcOEt. The solution, when washed with aq Na2CO3, dried
over Na2SO4, and concentrated, gave 9.9 mg (99% yield) of
a yellow crude tetraacetate. The pure product was eluted
from silica gel column by 2% AcOEt in PhH and the result-
ing solution, when concentrated, afforded an oil (solidifying
eventually) of Rf 0.76, mp 68–70°C and [α]D +44.2° (c =
0.275, CHCl3). UV (CH3CN) λmax (ε): 231 (18 480), 312
(11 720). IR (KBr, cm–1): 3076 (w), 2935 (w), 2853 (w),
1770 (s), 1743 (s), 1714 (s), 1609 (m), 1574 (m), 1505 (s),
1429 (m), 1372 (s), 1265–1165 (s, b with max at 1202),
Involutone
(5-(3,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-2-hydroxy
-4-cyclopenten-1,3-dione)
The soluble in MeOH material of fraction D, after
mannitol separation by crystallization, contained crotonic
acid in an amount of about 50%, which was sublimed off at
40°C and 0.06 mmHg (1 mmHg = 133.322 Pa); (mp 64 to
1
1118 (m), 1051 (m), 1013 (m), 904 (m), 846 (m). H NMR
1
(600 MHz, CD3CN) δ: 7.95 (1H, dd, J = 8.4 Hz, J = 2.2 Hz,
H-6′), 7.92 (1H, d, J = 2.2 Hz, H-2′), 7.70 (1H, s, H-4), 7.46
(2H, d of AA′XX′, J = 8.8 Hz, H-2′′ and H-6′′), 7.40 (1H,
d, J = 8.4 Hz, H-5′), 7.16 (2H, d of AA′XX′, J = 8.8 Hz,
H-3′′ and H-5′′), 2.29 (3H, s, COCH3), 2.28 (3H, s,
COCH3), 2.23 (3H, s, COCH3), 2.18 (3H, s, 2-OCOCH3). 1H
NMR (300 MHz, CDCl3) δ: 7.92–7.88 (2H, m, H-2′ and H-6
′), 7.46 (2H, d of AA′XX′, J = 9.0 Hz, H-3′′ and H-5′′),
7.43 (s, H-5), 7.35 (1H, d, J = 8.2 Hz, H-5′), 7.11 (2H, d of
AA′XX′, J = 9.0 Hz, H-2′′ and H-6′′), 2.33 (COCH3), 2.32
(COCH3), 2.29 (COCH3), 2.19 (COCH3). 13C NMR
(150 MHz, CD3CN) δ: 197.36 (C-3), 196.03 (C-1), 170.79
(2-OCOCH3), 169.90 (4′′-OCOCH3), 169.03 (3′-OCOCH3),
168.80 (4′-OCOCH3), 153.55 (C-5), 152.55 (C-4′′), 146.14
(C-4′), 143.50 (C-3′), 141.12 (C-4), 129.91 (C-1′′), 128.65
(C-2′′ and C-6′′), 128.47 (C-6′), 127.72 (C-1′), 125.25
(C-2′), 125.22 (C-5′), 123.44 (C-3′′ and C-5′′), 81.70 (C-2),
21.09 (OCOCH3), 20.73 (OCOCH3), 20.65 (OCOCH3),
19.97 (C2-OCOCH3). HMBC–NMR (600 MHz, CD3CN):
see Scheme 2. 13C NMR (75 MHz, CDCl3) δ: 196.33 and
65°C. H NMR (300 MHz, CDCl3) δ: 12.20 (1H, s, COOH),
7.10 (1H, dq, J = 15.5, J = 6.9, H-3), 5.86 (1H, dq, J = 15.5,
J = 1.7, H-2), 1.92 (1H, dd, J = 6.9, J = 1.7, H-4). A nonvol-
atile deep orange residue was subjected to column chroma-
tography on silica gel, which allowed, first, the removal
from it of the remaining crotonic acid by using hexane–ben-
zene (1:1), and, next, the isolation of a product, the
rechromatography of which, when 10% AcOEt in PhH was
used as an eluant, afforded a bright yellow, hygroscopic
solid (Rf 0.23) melting sharply at 181 to 182°C and showing
[α]D +96.3° (c = 0.3, MeOH). Treated with 10% aq NaOH,
the compound changed colour to purple-violet.10 IR
(KBr, cm–1): 3350, 1740, 1692, 1609, 1565, 1512, 1443,
1301, 1250, 1177, 1120, 1060, 875, 833. 1H NMR
(300 MHz, CD3CN) δ: 7.59 (1H, d, J = 2.2 Hz, H-2′), 7.56
(1H, dd, J = 8.2 Hz, J = 2.2 Hz, H-6′), 7.51 (1H, s, H-4),
7.22 (2H, d of AA′XX′, J = 8.8 Hz, H-2′′ and H-6′′), 6.95
(1H, d, J = 8.2 Hz, H-5′), 6.79 (2H, d of AA′XX′, J =
1
8.8 Hz, H-3′′ and H-5′′). H NMR (300 MHz, DMSO-d6) δ:
10.02 (1H, s, OH), 9.59 (1H, s, OH), 9.43 (1H, s, OH), 7.73
10The phenoxide ion, formed as a result of the C(4′)-OH proton abstraction, constitutes a conjugated system, made by 10 π and n electrons
and can be responsible for the colour change.
© 2003 NRC Canada

124
Can. J. Chem. Vol. 81, 2003
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151.73, 145.22, and 142.55 (C-5, C-4′′, C-4′, and C-3′),
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found: 480.10534.
Acknowledgments
W. Z. Antkowiak wishes to thank his students, A. Galek,
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profitable technical assistance in the isolation studies. Spe-
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© 2003 NRC Canada