Structural and biosynthetic studies on eremophilenols related to the phytoalexin capsidiol, produced by Botrytis cinerea

Article abstract of DOI:10.1016/j.phytochem.2018.06.010

A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopathogenic fungi. The biosynthesis of these compounds has been studied using ²H and ¹³C labelled acetate.

Full text of DOI:10.1016/j.phytochem.2018.06.010

Phytochemistry 154 (2018) 10–18
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Structural and biosynthetic studies on eremophilenols related to the
phytoalexin capsidiol, produced by Botrytis cinerea
a
a
a,c
a
a
Ivonne Suárez , Gesiane da Silva Lima , Raphael Conti , Cristina Pinedo , Javier Moraga ,
a
a,c
a
a
Javier Barúa , Ana Ligia L. de Oliveira , Josefina Aleu , Rosa Durán-Patrón ,
Antonio J. Macías-Sánchez , James R. Hanson , Mônica Tallarico Pupo ,
Rosario Hernández-Galán , Isidro G. Collado
Departamento de Química Orgánica, Facultad de Ciencias, Campus Universitario Río San Pedro s/n, Torre sur, 4a planta, Universidad de Cádiz, 11510, Puerto Real,
Cádiz, Spain
a
b
c
a
a,∗
a
b
Department of Chemistry, University of Sussex, Brighton, Sussex, BN1 9QJ, United Kingdom
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, Brazil
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified
Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These
metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to
that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that
they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopatho-
Botrytis cinerea
Sclerotiniaceae
Biosynthesis
Cryptic metabolites
Eremophilenols
2
13
genic fungi. The biosynthesis of these compounds has been studied using H and C labelled acetate.
1. Introduction
with necrotizing activities that are effective against a range of plant
species. Thus B. cinerea produces specific terpenoid and polyketide
The ascomycete Botrytis cinerea Pers. Fr, classified within the family
metabolites such as the sesquiterpene botrydial (1) and related com-
pounds (Colmenares et al., 2002), botrylactone (2) and the botcinic (3)
and botcineric acids (4) as well as their cyclic relatives, the botcinins (5,
6) (Tani et al., 2005, 2006). The sesquiterpenoid abscisic acid (7)
(Marumo et al., 1982) which mediates leaf fall and the polyketide,
cinbotolide (8) have also been isolated from the fungus (Fig. 1)
(Botubol et al., 2014; Collado and Viaud, 2016).
It is now clear that the development of pathogenesis by B. cinerea is
much more complex than previously believed. Disease progression is
tightly regulated throughout the infection process and the fungus un-
dergoes developmental adaptions that coincide with the different stages
of the infection. The interactions between the fungus and the plant are
mediated by compounds which affect both the fungus and the plant
(González et al., 2016). One class of such compounds are the effectors.
These compounds are secreted by a microbial pathogen in order to
inhibit the plant defensive system (Maffei et al., 2012). In many plant-
pathogen interactions, effectors are key pathogenicity determinants
that modulate the plant's intrinsic immunity to facilitate the develop-
ment of the parasitic infection (Oliva et al., 2010). Thus the discovery of
the fungal effectors produced by B. cinerea and the identification of
Sclerotiniaceae, is the causative agent of a grey mould disease which
infects at least 1400 plant species (Elad et al., 2016), including crops of
economic importance such as grapes and strawberries (Dean et al.,
2
012). The fungus is a typical necrotroph whose infective cycle includes
the induction of plant cell death followed by the maceration of the plant
tissue and then reproduction by forming asexual spores on the de-
composing plant material. The emergence of the disease symptoms
depends on the host plant, the infected part of the plant and the
weather conditions (van Kan, 2006; Williamson et al., 2007). It has
recently been reported that B. cinerea can also systemically colonize
plants without causing disease symptoms (van Kan et al., 2014). In
general, B. cinerea is responsible for severe economic losses that are
either due to the damage to the growing plants in the field or to the
subsequent decay of the harvested fruits, flowers and vegetables during
storage under cold and humid conditions (van Kan, 2006; Williamson
et al., 2007).
The wide host range of B. cinerea in contrast to other Botrytis species
that are specialized on a single host species, e.g. B. elliptica on lilies,
may be due to the formation of phytotoxic metabolites and proteins
∗
Corresponding author.
E-mail address: isidro.gonzalez@uca.es (I.G. Collado).
https://doi.org/10.1016/j.phytochem.2018.06.010
Received 9 March 2018; Received in revised form 11 June 2018; Accepted 12 June 2018
0031-9422/ © 2018 Published by Elsevier Ltd.

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
Fig. 1. Some metabolites isolated from B. cinerea.
their plant targets holds promise for the improvement of plant tolerance
to this serious pathogen.
and, as a result, a significant number of metabolites remain to be dis-
covered. Consequently B. cinerea is a valuable organism with which to
study the role of orphan biosynthetic genes and cryptic metabolites
(Scherlach and Hertweck, 2009). In this context we have recently de-
scribed the induction of a silent eremophilene biosynthetic pathway
using sub-lethal doses of copper ions and characterized an undescribed
family of eremophilenols (9–15) as cryptic metabolites (Pinedo et al.,
2016).
The recent sequencing of the B. cinerea genome has provided an
opportunity to discover novel biosynthetic gene clusters involved in the
biosynthesis of novel fungal metabolites. Forty-four genes that encode
key enzymes have been identified including those involved in the bio-
synthesis of terpenes, polyketides and peptides that are specific to this
phytopathogen (Amselem et al., 2011). The genome has shown that the
biosynthetic potential of this microorganism has been barely explored
These metabolites, which have sporogenic activity, possess a (+)-5-
11

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
epi-aristolochene or (+)-4-epi-eremophil-9-ene carbon skeleton and are
enantiomeric to the phytoalexin capsidiol and its derivatives (Pinedo
et al., 2016). This paper deals with the isolation and elucidation of the
structure of five cryptic eremophilenols from B. cinerea and the bio-
synthetic pathway leading to their formation from C and H-labelled
acetates.
the nOe interactions (see Table S1 in the supplementary data). Irra-
diation of H-15β produced nOe enhancements of the signals corre-
sponding to H-1β, H-2, H-3β, H-6β, and H−7β whilst irradiation at H-
1
1
14α produced a nOe effect at H-3α and H-6α. Analysis of the H- H
COSY, NOE and 1H- C heteronuclear correlation spectra (HSQC and
HMBC) led to the assignment of all the proton and carbon signals
confirming the relative stereochemistry and structure of compound 16
as (4R,5R, 7S)-eremophil-9-ene-4,11-diol.
1
3
2
13
2. Results and discussion
The 1H and C NMR spectra of compound 17, C15
13
26 2
H O , had a
pattern of signals that were characteristic of an eremophilene. There
A mixture of the major cryptic metabolites (11–13) from the group
13
were C NMR signals at δ 75.2 (s, C-11), 30.1 (q, C-12) and 23.7 (q, C-
of eremophilenols (9–15) which we had isolated previously, was sub-
mitted to conidiation-inducing experiments (Pinedo et al., 2016). The
increase in the formation of conidiophores was 5–6 times greater with
the strain B. cinerea B05.10 and three fold greater with B. cinerea UCA
13), which were assigned to a gem-dimethyl group attached to a hy-
droxylated carbon at C-11. In the HMBC spectrum there were correla-
tions between H-12 and H-13 and the signals corresponding to the
carbons C-7, C-11, C-12/C-13. There was a 1H NMR signal at δ 4.25
9
92 when compared to controls (Pinedo et al., 2016). This led us to
(
1H, ddd, J = 9.3, 3.3 and 1.4 Hz). This was assigned to a secondary
conclude that the reported eremophilenols were involved in conidiation
and in the self-regulation of the production of asexual spores (Pinedo
et al., 2016). Consequently we have carried out a more detailed study of
the chemical induction of the silent eremophilene biosynthetic
pathway.
The strains B. cinerea UCA992, B05.10 and the double-mutant from
the B05.10 strain, BcBOT2ΔBcBOA6Δ, were grown on a modified
Czapek-Dox medium containing 5 ppm copper sulphate. The broths
from the different fermentations were extracted with EtOAc and the
extracts separated by column chromatography. The fractions containing
eremophilene derivatives were submitted to HPLC chromatography to
obtain pure compounds, which were characterized by extensive NMR
experiments. In addition to the known compounds indicated in the
experimental section (Pinedo et al., 2016), the following products were
isolated: compounds 16–18 were obtained from B. cinerea UCA992, 16
and 19 from B05.10 and compound 20 was obtained from
BcBOT2ΔBcBOA6Δ (Fig. 2).
alcohol which was located at C-8 because in the two-dimensional
spectrum there were HMBC correlations between this resonance and C-
9
4
and C-10 and between H-6 and C-7 and C-8. The stereochemistry at C-
, C-7 and C-8 followed from the magnitudes of the 1H- H coupling
1
constants. Thus H-4 (δ 1.6), with α disposition, was a quartet of double-
doublets, (J = 7.1, 4.8 and 2.2 Hz) and H-7 (β) (δ 1.78) was doublet of
double-doublets, (J = 13.8, 9.3 and 3.5 Hz). The signal assigned to H-8
showed a coupling to H-7 (J = 9.3 Hz) and to H-9 (J = 1.4 Hz).
Molecular models reveal the possibility of a hydrogen bond involving
the hydroxyl groups at C-8 and C-11. The consequent reduction in the
rate of exchange of the hydroxyl hydrogen, which appear superimposed
at δ 2.25, as deduced from the observed correlation in the COSY ex-
periment, leads to the additional coupling of H-8 as 3.3 Hz.
Furthermore the presence of this hydrogen bond restricts the rotation of
the C-7/C-11 bond placing the methyl groups C-12 and C-13 in different
13
environments. This is reflected in their C NMR chemical shifts (δ 23.7
and 30.1) when compared to 16 (δ 27.0 and 27.2) where there is no
hydrogen bond. The coupling constants of 17 were identical to those for
the same signals in 13 whose absolute configuration had been de-
termined by Mosher's method (Pinedo et al., 2016). A detailed analysis
of the COSY, NOESY, HSQC and HMBC spectra (Table S2, supplemen-
tary data) led to the assignment of all the proton and carbon signals and
confirmed the structure of 17 as (4S,5S,7R,8R)-eremophil-9-ene-8,11-
diol.
The molecular formula for compound 16 was established as
1
3
15 26 2
C H O by HRMS. This was consistent with the C NMR and HSQC
1
3
data and implied three degrees of unsaturation. The C NMR revealed
the presence of four methyl, five methylene, two methine groups and
1
four quaternary carbons, two of which bore an oxygen function. The H
NMR spectrum contained a signal at δ 5.49 which was characteristic of
one proton on a trisubstituted double bond, together with four three-
1
13
proton singlet signals at δ 1.23, 1.20, 1.19 and 1.13. The H and
C
26 3
Compound 18 had a molecular ion corresponding to C15H O and
NMR spectra were very similar to those reported previously for
possessed signals in its 1H NMR spectrum which resembled those of
(
+)-4α,11-dihydroxy-4-epi-eremophil-9-en-8-one (15) except for the
compound 17 except for the presence of four three-proton singlets in
place of the three singlets and one doublet observed in 17. This sug-
gested that compound 18 had a structure corresponding to 17 with an
additional hydroxyl group geminal to the methyl group at C-4. This
structure was confirmed by the COSY, HSQC and HMBC experiments
1 13
absence of the C-8 carbonyl signal. In its place there were H and
C
NMR signals at δ 2.05 and 25.7 in accord with structure 16.
The stereochemistry of the asymmetric carbons was confirmed by
(
Table S3). The couplings observed in the HSQC and COSY spectra led
13
to the assignment of all fifteen signals in the C NMR spectrum. The
HMBC correlations between H-6 and C-4, C-5, C-7, C-8, C-11 and C-14
and between H-12/H-13 and C-7, C-11, C-12/C-13 located the two
tertiary hydroxyl groups at C-4 and C-11 and the secondary hydroxyl
group at C-8. The stereochemistry of the hydroxyl groups at C-4 and C-8
followed from nOe studies and the coupling constants for H-8. Thus
irradiation of the methyl group H-15β produced nOe enhancements at
H-6β and H-7 consistent with the α-orientation of the hydroxyl group at
C-4 whilst irradiation of the signal for H-8 led to the enhancement of H-
1
3
1
2/H-13. The coupling pattern of the H-8 signal (δ 4.26, ddd, J = 9.3,
.4 and 1.5 Hz) was identical to that of compound 13 and compound
7. The hydroxyl group at C-8 was therefore assigned the β-config-
uration with a hydrogen bond to the hydroxyl group at C-11 accounting
for the additional coupling of 3.4 Hz. Consequently compound 18 had
the structure (4R,5R,7R, 8R)-eremophil-9-ene-4,8,11-triol.
Compound 19 had a molecular ion corresponding to C15
H
24
O
3
. The
C NMR spectrum revealed the presence of four methyl, three me-
thylene and four methine groups together with four quaternary
13
Fig. 2. (+)-4-epi-eremophilene derivatives isolated from B. cinerea strains.
12

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
carbons, one of which at δ 203.0 ppm corresponded to a carbonyl
fungus (Pinedo et al., 2016). Their enantiomeric relationship to the
phytoalexin capsidiol, raises the interesting possibility that they may
also be involved in the fungal response to the antifungal activity of the
phytoalexin by, for example, regulating the biosynthesis of the phy-
toalexins in the host plant.
1
group. The H NMR spectrum showed, in addition to the signals for four
methyl groups at δ 1.12 (3H, d, J = 7.3 Hz), 1.21 (6H, s) and 1.34 (3H,
s), a pattern of signals that were characteristic of an eremophilene
derivative with two hydroxyl groups. The spectra were very similar to
those for compound 11 which possessed two hydroxyl groups at C-2
and C-11. The principal difference was the presence in the 13C NMR
spectrum of the signal for the carbonyl group at δ 203.0 ppm and the
2.1. Biosynthesis of (+)-4-epi-eremophil-9-en-11-ols
1
deshielding in the H NMR spectrum of the signal for H-9 to δ 5.92. This
9
,10
Aristolochene is a sesquiterpenoid diene which occurs naturally in
two enantiomeric forms. (−)-Aristolochene has been isolated from
plants including the roots of Aristolochia indica (Govindachari et al.,
indicated the presence of a Δ
8-keto-α,β-unsaturated ketone. The
proposed structure 19 was supported by the correlations which were
observed in the COSY, HSQC and HMBC spectra (Table S4). The ste-
reochemistry was assigned by extensive nOe experiments. Irradiation of
the proton geminal to the hydroxyl group at C-2 produced nOe en-
hancements of the signals corresponding to H-1β and H-15β. Irradiation
of the signals for H-7β and H-15β produced nOe effects at H-12, H-13
and H-15β, and H-2β and H-7β, respectively. This data, together with
the large number of correlations in the HMBC experiment (Table S4)
were in accord with the structure and stereochemistry shown for
compound 19 as (2S,4S,5S,7S)-2,11-dihydroxy-eremophil-9-en-8-one.
1970) and from insect sources (Baker et al., 1981) whilst (+)-aris-
tolochene is of fungal origin (Aspergillus terreus) (Cane et al., 1987).
Previous biosynthetic studies have established that (+)-aristolochene is
formed by the cyclization of (E,E)-farnesyl diphosphate (FDP) via the
intermediate (S)-(−)-germacrene A as the U,U-conformer (C-14 and C-
15 methyls Up) (Faraldos et al., 2010). The protonation of the germa-
crene A leads to cyclization and the formation of the bicyclic trans-fused
eudesmane cation. Successive 1,2-hydride and methyl group re-
arrangements followed by the loss of the pro-S hydrogen from C-9
generates (+)-aristolochene (Fig. 4, route a) (Faraldos et al., 2010).
1
13
Compound 20 also showed a pattern of H and C NMR signals
characteristic of an eremophilenol containing two secondary and one
tertiary hydroxyl groups. A preliminary study of these spectra indicated
a similarity to capsidiol (21) (Fig. 3) except for the absence of the
signals corresponding to the protons and carbons of the isopropenyl
(
(
R)-Germacrene A exists as three NMR-distinguishable conformers UU
Up C10-Me, Up C4-Me), UD (Up C10-Me, Down C4-Me) and DU (Down
C10-Me, Up C4-Me), (Fig. 4), in a ratio of 5:3:2 at or below 25 °C
Faraldos et al., 2007). Under similar conditions the related sesqui-
1
(
group. Instead the H NMR spectrum contained two methyl group
1
3
terpene alcohol, hedycaryol (Fig. 4) exists in soln. in the same three
forms, except that the UD and DU conformers predominate over the UU
alternative due to the larger steric bulk of the hydroxypropyl sub-
stituent (Ōsawa et al., 1979; Terada and Yamamura, 1979; Wharton
et al., 1973). Hence it is plausible that, through the action of a hitherto
unidentified enzyme, (E,E)-FDP is transformed to (S)-hedycaryol whose
DU conformer would lead via an acid-catalysed cyclization to an epi-
eudesmyl cation with the correct stereochemistry to act as a precursor
to the (+)-5-epi-aristoloch-9-en-11-ols, also known as (+)-4-epi-ere-
mophil-9-en-11-ols (Fig. 4, route b). In order to explore this hypothesis,
singlets at δ 1.18 and 1.20 and a signal in the C NMR spectrum at δ
2.7 characteristic of a 2-hydroxypropan-2-yl group at C-7. The mole-
cular formula, C15 , deduced from the HRMS and the data ob-
7
26 3
H O
tained from the COSY, NOESY, HSQC and HMBC experiments were
consistent with the structure 20 of an 11-hydroxy relative of capsidiol
(
21) (Table S5).
In order to correlate the compounds 20 and 21, natural capsidiol
21) was isolated from green peppers treated with aq. cellulase fol-
(
lowing a modification to the procedures of Whiteheah et al. (1987) and
Zhao et al. (2004) (see experimental section). Capsidiol (21) was
acetylated with acetic anhydride in pyridine to give the known diace-
tate 21a which possessed identical data to that reported in the literature
biosynthetic experiments with sodium [1–13C]-, [2– C]- and [ H
13
2
3
]-
acetates have been carried out with resting cell cultures of the B. cinerea
mutant BcBOT2Δ.
(
Zhao et al., 2004). This was then submitted to an oxymercuration-
The optimum times for feeding the precursors to resting cell cultures
of BcBOT2Δ have been established as result of the time-course study for
the production of (+)-4-epi-eremophil-9-en-2α,11-diol (11).
Consequently 5 days old resting cell cultures of this mutant were fed
with the labelled sodium acetates and the resultant (+)-4-epi-eremo-
demercuration reaction (Blay et al., 2001) to give compound 22 in good
yield. Compound 20 was also acetylated by the conventional method to
yield the diacetate 20a (Table S6). Both compounds had identical
spectroscopic data but their optical rotations were of opposite sign, 20a
21
21
[
α] -20°, 22 [α] +27.5°. This data confirmed that the phytopathogenic
D D
1
3
fungus B. cinerea has afforded a family of eremophilenes (16–20) with
phil-9-en-2α,11-diols (11) were extracted, purified and analyzed by
C
and 2H NMR spectroscopy (Table 1).
skeleton that are enantiomeric to those of the phytoalexin, capsidiol
(
21) and its relatives.
We have shown that the induction of conidiation and spore for-
Significant enrichments were observed for C-2, C-4, C-6, C-8, C-10
and C-11 from the sodium [1–13C]-acetate whilst the sodium [2– C]-
acetate led to enhanced signals for C-1, C-3, C-7, C-9, C-12, C-13 and C-
15. Furthermore the enhanced signals for C-5 and C-14 showed char-
13
mation in B. cinerea as a result of the stress of sub-lethal concentrations
of copper ions is mediated by the formation of eremophilenols by the
13
13
acteristic satellite signals arising by
C- C spin-spin coupling
Fig. 3. Capsidiol (21) and derivatives.
13

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
Fig. 4. Proposed biosynthetic route to (+)-4-epi-eremophilenols. Labelling pattern resulting from feeding experiments with sodium [2–13C]- and [ H
2
3
]-acetates.
(
Table 1). This coupling indicated that a 1,2-methyl migration had
occurred suggesting that the biosynthesis of the compound 11 from
E,E)-FDP had followed the folding outlined in Fig. 4 (route b).
D-4, D-7, D-9 and to the methyl groups D-12, D-13, D-14 and D-15 were
unambiguously identified. The presence of a deuterium atom at C-4
corresponded to a 1,2-hydride migration from C-5 to C-4 of the eu-
desmane cation during the biosynthesis (Fig. 4).
(
2
3
A further feeding experiment with sodium [ H ]-acetate gave ad-
ditional evidence to support this hypothesis. The incorporation of the
labelled precursor into 11 was analyzed by H NMR. Although there
was only a low incorporation, the signals corresponding to D-1β, D-3,
These data are consistent with the biosynthetic pathway outlined in
Fig. 4 (route b) in which the (+)-4-epi-eremophil-9-en-11-ols are bio-
synthesised by cyclization of (E,E)-FDP via (S)-hedycaryol. Protonation
2
14

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
Elmer, Spectrum BX FTIR System, and reported as wave number
Table 1
−
1
1
13
Isotopic enrichment of carbons and coupling constants in (+)-4-epi-eremophil-
(cm ). H and C NMR measurements were recorded on Agilent 500
and 600 MHz spectrometers with SiMe as the internal reference.
Chemical shifts were referenced to CDCl (δ 7.25, δ 77.0). NMR as-
1
3
13
9
-en-2α,11-diol (11) after feeding sodium [1– C]- and [2– C]-acetates to
4
BcBOT2Δ.
3
H
C
Carbon atom (N)
δ
C
(CDCl
3
)
Atom % 13C excess a
1JCC (Hz)
signments were made using a combination of 1D and 2D techniques.
High-Resolution Mass Spectroscopy (HRMS) was performed in a QTOF
mass spectrometer, (Water, Synapt G2), in positive or negative ion ESI
or APCI modes.
[
1–13C]-acetate
[2–13C]-acetate
1
2
3
4
5
6
7
8
9
41.64
67.69
39.685
41.38
37.925
38.39
42.98
25.96
123.38
137.89
72.62
0.15
3,63
0.27
3.12
0.59
3.16
−0.02
3.52
−0.06
1.95
2.38
0.03
0.17
0.18
0
6.95
−0.19
8.30
−0.20
4.00
0
7.91
−0.20
9.28
−0.55
−0.68
7.43
10.06
4.26
4.2. Labelled precursors
38
Sodium [1–13C]- (99%), [2–13C]- (99%), and [ H
2
3
]-acetates (99%)
were obtained from Cambridge Isotope Laboratories, Inc. (Andover,
MA, USA). All samples were dissolved in water and sterilized through
.22 μm Millex GP (Millipore) filters before being added to the fer-
0
10
11
12
13
14
15
mentation flasks.
b
27.12
27.08
30.26
18.60
b
4.3. Microorganisms
38
7.78
4
The genus Botrytis Pers. is classified within the family
a
Atom % 13C excess = {(R
Sclerotiniaceae Whetz. Four strains were used in this work: wild types
B. cinerea UCA 992 and B05.10, and the mutants BcBOT2ΔBcBOA6Δ
and BcBOT2Δ. B. cinerea UCA 992 was obtained from grapes from the
Domecq vineyard, Jerez de la Frontera, Cádiz, Spain. This culture is
deposited in the University of Cádiz, Mycological Herbarium Collection
N
/RN(UL) x 1.1} – 1.1, where R
N
is the ratio of the
peak intensity at N-position in labelled compound calculated on the basis of the
peak intensity at the C-15-position in [1– C]-acetate and C-6-position in
2– C]-acetate. Similarly, RN(UL) is the ratio of the peak intensity at N-position
13
1
3
[
13
in unlabeled compound. Value 1.1 is the theoretical C natural abundance
atom %).
(
UCA). Strain B05.10 of B. cinerea Pers. Fr. was isolated from a Vitis
(
b
field sample (Quidde et al., 1998). The double mutant
BcBOT2ΔBcBOA6Δ (Dalmais et al., 2011), and the mutant BcBOT2Δ
Interchangeable signals.
(
Pinedo et al., 2008) were supplied by Dr. Muriel Viaud, and are
at C-1 of the DU conformer of (S)-hedycaryol and attack by the C-4/C-5
pi) bond on the resulting C-10 carbocation leads to the cis-fused eu-
desmane cation. This generates the (+)-4-epi-eremophil-9-en-11-ols
after successive 1,2-hydride and methyl group migrations followed by
the loss of the pro-R hydrogen from C-9.
maintained in the UMR BIOGER strain collection, INRA (Grignon,
France). Conidial stock suspensions of all of these strains were main-
tained viable in 80% glycerol at −40 °C.
(
4
.4. Culture conditions and isolation of eremophilenols
3
. Conclusions
5
B. cinerea strains were grown at 25 °C in Roux culture bottles
200 ml), each containing 150 ml of modified Czapek-Dox medium
50.0 g glucose, 1.0 g yeast extract, 5.0 g K HPO , 2.0 g NaNO , 0.5 g
7H O, 0.01 g FeSO 7H O, 0.005 g CuSO ·5H O, pH 7, 1 L of
(
(
Five undescribed eremophilenol derivatives 16–20 have been iso-
2
4
3
lated from 27 days old cultures of the strains of B. cinerea UCA992,
B05.10 and the double mutant from the B05.10 strain,
BcBOT2ΔBcBOA6Δ. Their structures have been established and, inter-
estingly, these compounds have been shown to possess an underlying
carbon skeleton which is enantiomeric to that of the phytoalexin cap-
sidiol (21), which is produced by green peppers when they are infected
by phytopathogenic fungi. Indeed the metabolite of B. cinerea, com-
pound 20, is ent-11-hydroxydihydrocapsidiol. This suggests that the
eremophilenols of B. cinerea may also be effectors inhibiting the plant
defences or modulating the plant immunity in order to facilitate the
progress of the infection. Finally, using labelled acetates, a biosynthetic
pathway has been established involving the probable transformation of
MgSO
4
2
4
2
4
2
6
water) (Pinedo et al., 2016). Each bottle was inoculated with 1,1 × 10
fresh conidia obtained from a 18 days old culture of B. cinerea on Agar-
Malt medium, and then incubated for 27 days at 24–26 °C on surface
cultures under white light (day light lamp) for metabolite production.
The broths (3l) from each of the strains (B. cinerea UCA992, B05.10
and BcBOT2ΔBcBOA6Δ), were filtered, satd. with NaCl and extracted
with EtOAc (x4). The organic extracts were washed with H
then dried over dry Na SO . Evaporation of the solvent at reduced
pressure gave the extracts as yellow oils, 180 mg (from B. cinerea UCA
2
O (x3) and
2
4
9
92), 140 mg (B05.10) and 160 mg (BcBOT2ΔBcBOA6).
A preliminary fractionation of the extracts was achieved by column
(
E,E)-FDP via (S)-hedycaryol and cyclization of its DU conformer to a
chromatography on silica gel eluting with n-hexane-EtOAc mixtures
containing increasing percentages of EtOAc (9:1 → 0:1). Final pur-
ification of the fractions of interest were carried out by means of
semipreparative HPLC using n-hexane-EtOAc-acetone (7:2:1) as the
mobile phase and flow rate 2.5 ml/min.
cis-fused eudesmane cation and thence the (+)-4-epi-eremophilenol
derivatives.
4
. Experimental
The broth of B. cinerea UCA992 gave, in addition to the known
compounds 9 (1.0 mg), 11 (1.1 mg), 12 (3.2 mg), and 13 (2.0 mg), the
metabolites 16 (1.1 mg), 17 (1.2 mg), and 18 (0.7 mg). Strain B05.10
broth afforded the known compounds 11 (3.1 mg), 12 (2.8 mg), and 13
(3.0 mg), and the specialized metabolites 16 (0.7 mg), and 19 (1.0 mg).
The mutant BcBOT2ΔBcBOA6 yielded, in addition to the metabolites
11–15 (1.0, 0.8, 1.5, 1.3, 1.1 mg, respectively), an undescribed meta-
bolite 20 (0.8 mg).
4
.1. General experimental procedures
TLC was performed on Merck Kiesegel 60 F254, 0.25 mm thick. Silica
gel 60PF254 (60–100 mesh, Merck) was used for column chromato-
graphy. HPLC was performed with a Hitachi/Merck L-6270 apparatus
equipped with an UV-VIS detector (L 4250) and a differential re-
fractometer detector (RI-7490). Lichrosfer Si 60 (5 μm) LichoCart
(
(
250 mm × 4 mm) and Lichrosfer Si 60 (10 μm) LichoCart
250 mm × 10 mm) columns were used for isolation experiments.
4.4.1. (4R, 5R, 7S)-eremophil-9-ene-4,11-diol (16)
2
1
Infrared spectra were recorded on a FT-IR spectrophotometer, Perkin
Colourless oil, [α] -3.7° (0.07c, CHCl
3
). IR νmax (cm−1): 3420,
D
15

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
1
2932, 1372, 1103, 925. H NMR (600 MHz, CDCl
3
): δ 0.91 (t, 1H,
4.4.5. (1S, 3S, 4R, 5S, 7S)-eremophil-9-ene-1,3,11-triol (20)
∗
21
). IR νmax (cm−1): 3463, 2972,
): δ 0.85 (d, 3H, J = 7.0 Hz,
J = 13.8 Hz, H-6α), 1.13 (s, 3H, H-14), 1.19 (s, 3H, H-12 ), 1.20 (s, 3H,
Colourless oil,1[α]
D
-6° (0.07c, CHCl
3
∗
H-13 ), 1.23 (s, 3H, H-15), 1.34 (m, 1H, H-1β), 1.34 (m, 1H, H-2β),
2934, 1370, 772. H NMR (500 MHz, CDCl
3
∗
∗
1
1
2
.50 (m, 1H, H-3β), 1.69 (m, 1H, H-7β), 1.69 (m, 1H, H-2α), 1.87 (td,
H, J = 13.6, 4.6 Hz, H-3α), 1.96 (m, 1H, H-1α), 2.05 (m, 2H, H-8),
.24 (dt, J = 13.8, 2.5 Hz, H-6β), 5.49 (dt, 1H, J = 6.7, 1.7 Hz, H-9).
H-15), 1.18 (s, 3H, H-13 ), 1.20 (s, 3H, H-12 ), 1.22 (t, 1H, J = 13.7 Hz,
H-6), 1.36 (s, 3H, H-14), 1.62 (m, 1H, H-7β), 1.63 (m, 1H, H-2), 1.72
(m, 1H, H-8), 1.74 (m, 1H, H-4α), 1.80 (dt, 1H, J = 13.7, 3.1 Hz, H-6′),
1.92 (m, 1H, H2′), 2.14 (ddt, 1H, J = 16.0, 7.0, 3.5 Hz, H-8′), 4.34 (dd,
1H, J = 3.8, 2.4 Hz, H-1β), 4.60 (dt, 1H, J = 12.3, 4.6 Hz, H-3α), 5.91
1
3
C NMR (150 MHz, CDCl
3
): δ 24.3 (CH
, C-15), 27.0 (CH , C-12 ), 27.2 (CH
, C-6), 38.5 (CH , C-3), 43.3 (CH, C-7), 43.6 (C, C-
2 3
, C-2), 25.4 (CH , C-14), 25.7
∗
∗
(
(
CH
CH
2
, C-8), 25.9 (CH
, C-1), 33.4 (CH
3
3
3
, C-13 ), 31.0
(dd, 1H, J = 7.0, 1.9 Hz, H-9). 13C NMR (125 MHz, CDCl
): δ 9.1 (CH
,
,
2
2
2
3
∗
3
∗
5
), 72.7 (C, C-11), 75.6 (C, C-4), 121.0 (CH, C-9), 142.0 (C, C-10),
C-15), 26.3 (CH
C-14), 36.2 (CH
47.8 (CH, C-4), 65.4 (CH, C-3), 72.4 (C, C-11), 74.9 (CH, C-1), 128.9
2
, C-8), 27.0 (CH
3
, C-12 ), 27.3 (CH
3
, C-13 ), 32.3 (CH
3
∗
+
interchangeable assignments. HRMS(ESI ): calcd. for C15
+
H
26
O
2
Na [M
2 2
, C-2), 38.9 (C, C-5), 40.7 (CH , C-6), 43.6 (CH, C-7),
+
+
Na] 261.1830, found 261.1831; calcd. for C15
H
H
25O [M+H-H
23 [M+H-2H
17 [M+H-H O-(CH
2
O]
O]
CO]
+
+
∗
+
2
2
1
21.1905, found 221.1901; calcd. for
03.1800, found 203.1800; calcd. for C12
61.1330, found 161.1330.
C
15
2
(CH, C-9), 140.4 (C, C-10), interchangeable assignments. HRMS(ESI ):
+
H
2
3
)
2
calcd. for C15
for C15 21 [M+H-3H
3 2
15 [M+H-2H O-(CH ) CO] 159.1174, found 159.1175.
H
23O [M+H-2H
2
O] 219.1749, found 219.1751; calcd.
+
H
2
O] 201.1643, found 201.1644; calcd. for
+
C
12
H
2
4
.4.2. (4S, 5S, 7R, 8R)-eremophil-9-ene-8,11-diol (17)
21
). IR νmax (cm−1): 3331,
): δ 1.00 (d,
Colourless oil, [α] +8.2° (0.13 c, CHCl
3
D
4.5. Acetylation of (1S, 3S, 4R, 5S, 7S)-eremophil-9-en-1,3,11-triol (20)
1
2
3
1
2
932, 1464, 1377, 1172, 1034. H NMR (500 MHz, CDCl
3
H, J = 7.1 Hz, H-15), 1.05 (t, 1H, J = 14 Hz, H-6) 1.14 (s, 3H, H-14),
Eremophilenetriol (20) (5.0 mg, 0.020 mmol) was dissolved in
∗
∗
.21 (s, 3H, H-13 ), 1.31 (s, 3H, H-12 ), 1.30 (m, 1H, H-3′), 1.50 (m,
H, H-2, H-2′), 1.50 (dd, 1H, J = 14, 3.5 Hz, H-6′), 1.60 (qdd, 1H,
pyridine (0.5 ml) and stirred with acetic anhydride (1.0 ml, 0.011 mol)
for 18 h. Then the reaction mixture was concentrated and purified by
column chromatography to afford 5.0 mg of the diacetate derivative
J = 7.1, 4.8, 2.2 Hz, H-4α), 1.78 (ddd, 1H, J = 13.8, 9.3, 3.5 Hz, H-7β),
1
1
5
.98 (m, 1H, H-3), 2.00 (m, 1H, H-1′), 2.25 (m, 1H, H-1), 2.22–2.26 (m,
(
20a).
H(superimposed), C8-OH), 4.25 (ddd, 1H, J = 9.3, 3.3, 1.4 Hz, H-8α),
1
3
.40 (t, 1H, J = 1.8 Hz, H-9). C NMR (125 MHz, CDCl
3
): δ 17.6 (CH
, C-3), 30.1 (CH
3
,
,
∗
C-15), 21.8 (CH
C-12 ), 30.4 (CH
2
, C-2), 23.7 (CH
, C-14), 31.4 (CH
), 41.2 (CH, C-4), 49.3 (CH, C-7), 69.7 (CH, C-8), 75.2 (C, C-11), 126.0
3
, C-13 ), 30.0 (CH
2
3
4.5.1. (1S, 3S, 4R, 5S, 7S)-1,3-diacetoxyeremophil-9-en-11-ol (20a)
∗
21
1
3
2
, C-1), 38.3 (CH
2
, C-6), 38.8 (C, C-
Amorphous solid, [α]
CDCl
D
-20° (0.05c, CHCl
3
). H NMR (500 MHz,
∗
5
3
): δ 0.87 (d, 3H, J = 7.1 Hz, H-15), 1.18 (s, 3H, H-13 ), 1.19 (s,
∗
+
∗
(
CH, C-9), 141.8 (C, C-10), interchangeable assignments. HRMS(ESI ):
3H, H-12 ), 1.22 (t, 1H, J = 13.6 Hz, H-6), 1.31 (s, 3H, H-14), 1.61 (m,
1H, H-7β), 1,73 (m, 1H, H-8′), 1.76 (m, 1H, H-6′), 1.79 (m, 1H, H-2’),
+
calcd. for C15
H
27
O
2
[M+H] 239.2011, found 239.1992.
1
3
3
.88 (m, 1H, H-2), 1.92 (m, 1H, H-4α), 2.02 (s, 3H, CH CO), 2.04 (s,
H, CH CO), 2.15 (m, 1H, H-8), 5.39 (dd, 1H, J = 4.0, 2.4 Hz, H-1β),
3
4
.4.3. (4R, 5R, 7R, 8R)-eremophil-9-ene-4,8,11-triol (18)
21
). IR νmax (cm−1): 3363, 2933,
Colourless oil, [α] -3.8° (0.15c, CHCl
3
5.53 (dt, 1H, J = 12.6, 4.6 Hz, H-3α), 6.08 (dd, 1H, J = 7.1, 1.9 Hz, H-
D
1
13
1
1
3
1
3
1
674, 1464, 1368, 1067.5, 924.5. H NMR (500 MHz, CDCl
3
): δ 0.80 (t,
9). C NMR (125 MHz, CDCl
135.3 (C, C-10), 132.0 (CH, C-9), 75.4 (CH, C-3), 72.3 (C, C-11), 69.3
(CH, C-3), 44.1 (CH, C-4), 43.2 (CH, C-7), 40.2 (CH , C-6), 38.9 (C, C-
5), 31.0 (CH , C-2), 31.0 (CH , C-14), 27.1 (CH , C-12 ), 27.0 (CH
13 ), 26.3 (CH , C-8), 21.6 (CH , eCOCH ), 21.3 (CH , eCOCH ), 10.1
, C-15), interchangeable assignments. HRMS(ESI ): calcd. for
3
) δ 170.3 (C, -OAc), 170.1 (C, -OAc),
∗
H, J = 14.5 Hz, H-6α), 1.11 (s, 3H, H-14), 1.23 (s, 3H, H-12 ), 1.30 (s,
∗
H, H-15), 1.35 (s, 3H, H-13 ), 1.42 (m, 1H, H-2′), 1.51 (m, 1H, H-3β),
2
∗
.72 (m, 1H, H-2), 1.80 (dd, 1H, J = 9.3, 3.5 Hz, H-7β), 1.84 (m, 1H, H-
α), 2.04 (m, 1H, H-1β), 2.10 (dd, 1H, J = 14.5, 3.5 Hz, H-6β), 2.26 (m,
H, H-1α), 4.26 (ddd, 1H, J = 9.3, 3.4, 1.5 Hz, H-8), 5.38 (t, 1H,
2
3
3
3
, C-
∗
2
3
3
3
3
∗
+
(CH
3
1
3
∗
+
J = 1.8 Hz, H-9). C NMR (125 MHz, CDCl
3
): δ 23.6 (CH
3
, C-12 ), 24.0
19 30 5 23
C H O Na [M+Na] 361.1991, found 361.1990; calcd. for C15H O
[M+H-2AcOH] 219.1749, found 219.1750; calcd. for C15
∗
+
(
(
CH
CH
2
, C-2), 25.6 (CH
, C-1), 32.4 (CH
3
, C-14), 25.7 (CH
, C-6), 38.5 (CH
3
, C-15), 30.3 (CH
3
, C-13 ), 30.4
H
21 [M+H-
+
2
2
2
, C-3), 43.5 (C, C-5), 49.3 (CH, C-
2AcOH-H
3 2
2AcOH-(CH ) CO] 159.1174, found 159.1175.
2
O] 201.1643, found 201.1644; calcd. for C12H15 [M+H-
+
7
), 69.3 (CH, C-8), 75.5 (C, C-11), 75.5 (C, C-4), 126.4 (CH, C-9), 142.5
∗
+
(
C, C-10), interchangeable assignments. HRMS(ESI ): calcd. for
+
15 26 3 23
C H O Na [M+Na] 277.1780, found 277.1778; calcd. for C15H O
4.6. Isolation of capsidiol (21) from green peppers
+
[
M+H-2H
2
O] 219.1749, found 219.1749; calcd. for C15
H
21 [M+H-
O-
+
3H
2
O] 201.1643, found 201.1642; calcd. for C12 15 [M+H-2H
H
2
After removing the uppermost section of 23 green bell pepper fruits
Capsicum annuum) with a knife, the fruits were placed upright into
trays and the exposed cavities filled with a 3 mg/l soln. of cellulase
Trichoderma viride) in deionized water. The fruits were incubated at
5 °C for 72 h after which time the aq. soln. was filtered through Nytal,
+
(
3 2
CH ) CO] 159.1174, found 159.1173.
(
4
.4.4. (2S, 4S, 5S, 7S)-2,11-dihydroxyeremophil-9-en-8-one (19)
(
2
21
). IR νmax (cm−1): 3369,
): δ 1.12
Colourless oil, [α] +9.9° (0.04c, CHCl
3
D
1
2
924, 2853, 1741, 1652, 1464, 772. H NMR (400 MHz, CDCl
3
pH adjusted to 6.7–7, and extracted with CH
water soln.). Concentration of the combined CH
2
Cl
2
(2 × 0.25 ml/l of
2
layers and pur-
∗
∗
(
(
4
d, 3H, J = 7.3 Hz, H-15), 1.21 (s, 3H, H-13 ), 1.21 (s, 3H, H-12 ), 1.34
s, 3H, H-14), 1.62 (t, 1H, J = 14.7 Hz, H-6β), 1.74 (dd, 1H, J = 12.2,
2
Cl
ification by column chromatography (n-hexane-EtOAc as eluents) pro-
vided 57.0 mg of pure capsidiol (21) (Whiteheah et al., 1987; Zhao
et al., 2004).
.4 Hz, H-3α), 2.00 (m, 1H, H-1α), 2.01 (m, 1H, H-3β), 2.03 (m, 1H, H-
α), 2.03 (m, 1H, H-6α), 2.52 (d, 1H, J = 8.8 Hz, H-1β), 2.63 (dd, 1H,
4
13
J = 14.7, 6.2 Hz, H-7β), 3.90 (m, 1H, H-2β), 5.92 (s, 1H, H-9). C NMR
∗
(
100 MHz, CDCl
3
): δ 18.3 (CH
, C-14), 36.7 (CH
, C-1), 42.1 (CH, C-4), 52.6 (CH, C-7), 67.9 (CH, C-2), 72.6 (C,
3
, C-15), 24.1 (CH
3
, C-12 ), 28.1 (CH
3
, C-
∗
1
4
3 ), 31.1 (CH
2.1 (CH
3
2
, C-6), 39.3 (C, C-5), 39.4 (CH
2
, C-3),
4.7. Acetylation of capsidiol (21)
2
∗
C-11), 127.4 (CH, C-9), 164.3 (C, C-10), 203.0 (C, C-8), interchange+-
A soln. of capsidiol (21) (21.0 mg, 0.089 mmol) in pyridine (1 ml)
and acetic anhydride (2.5 ml, 0.026 mol) was stirred for 18 h, then the
reaction mixture was concentrated and purified by column chromato-
graphy affording 24.0 mg of the diacetate derivative 21a (Zhao et al.,
2004).
+
able assignments. HRMS(ESI ): calcd. for
C
H
15
23
21
H
25
O
3
[M+H]
+
+
2
2
2
53.1804, found 253.1807; calcd. for
35.1698, found 235.1699; calcd. for
17.1592, found 217.1595.
C
15
O
2
[M+H-H
[M+H-2H
2
O]
O]
C
15
H
O
2
16

I. Suárez et al.
Phytochemistry 154 (2018) 10–18
4.8. Preparation of (1R, 3R, 4S, 5R, 7R)-1,3-diacetoxyeremophil-9-en-11-
4.9.2. Feeding of sodium [2–13C]-acetate to BcBOT2Δ
7
ol (22) from capsidiol diacetate (21a)
Fourteen Erlenmeyer flasks, each subcultured with 10 fresh conidia
in Czapek-Dox medium without glucose, were pulse fed on day 5 with
an aseptic aq. soln. of sodium [2–13C]-acetate to a final concentration of
500 ppm. Extraction of the broth 3 days post-feeding yielded a extract
(50.0 mg, 3.6 mg/flask) which was purified as described in the general
method to afford 11 (0.8 mg).
Capsidiol diacetate (21a) (11.4 mg, 0.042 mmol) was dissolved in
0
(
3
.5 ml of a THF-H
17.5 mg, 0.055 mmol). The reaction mixture was stirred at rt for
0 min. To quench the reaction it was first diluted with ether (0.2 ml),
cooled to 0 °C and it was treated drop wise with 3 M NaOH (0.1 ml) and
.5 M NaBH (0.1 mg) and 41.5 mg of NaCl. Then the reaction mixture
2 2
O mixture (1:1), and treated with Hg(OAc)
2
0
4
4.9.3. Feeding of sodium [ H
3
]-acetate to BcBOT2Δ
was stirred at 0 °C for 10 min, diluted with ether and water and ex-
tracted in the usual way (Blay et al., 2001). The extract was purified by
column chromatography, using n-hexane-EtOAc as eluent, yielding
In accordance with the above procedure, twenty subcultured
Erlenmeyer flasks with conidia of BcBOT2Δ in Czapek-Dox medium
without glucose, were pulse fed with a filter-sterilized soln. of sodium
2
7.0 mg of pure 1β,3α-diacetoxy derivative (22).
[ H
3
]-acetate in H
days post-feeding, the culture medium and mycelia were separated by
filtration. Extraction of the broth yielded a eremophilenes extract
121.1 mg, 6.1 mg/flask), which was purified as described in the gen-
2
O on day 5 to a final concentration of 500 ppm. After
3
4
.8.1. (1R, 3R, 4S, 5R, 7R)-1,3-diacetoxyeremophil-9-en-11-ol (22)
(
21
1
Colourless oil, [α] +27.5° (0.43 c, CHCl
3
). H NMR (500 MHz,
D
eral method to afford the compound 11 (2.5 mg).
CDCl
3
1
CH
3
): δ 0.86 (d, 3H, J = 7.5 Hz, H-15), 1.16 (s, 3H, H-13), 1.17 (s,
H, H-12), 1.22 (m, 1H, H-6β), 1.31 (s, 3H, H-14), 1.56 (m, 1H, H-7β),
.71–1.80 (m, 3H, H-2α, H-8α, H-4α), 1.90 (m, 1H, H-6α), 2.01 (s,
Acknowledgments
3
), 2.02 (m, 1H, H-2β), 2.03 (s, CH ), 2.12 (m, 1H, H-8β), 5.38 (dd,
3
This research was supported by grant from MINECO-FEDER
AGL2015-65684-C2-1-R) and CAPES-DGU collaborative grant between
Brazil and Spain. Use of NMR facilities at the Servicio Centralizado de
Ciencia y Tecnología (SCCYT) of the University of Cádiz is acknowl-
edged.
1
H, J = 2 Hz, H-1β), 5.53 (dt, 1H, J = 12.5 Hz, J = 3 Hz, H-3α), 6.08
(
(
dd, 1H, J = 4.5 Hz, J = 2 Hz, H-9). HRMS(ESI+): calcd. for
+
19 30 5 23
C H O Na [M+Na] 361.1991, found 361.1994; calcd. for C15H O
+
[
M+H-2AcOH] 219.1749, found 219.1744; calcd. for C15
H21 [M+H-
+
2
2
AcOH-H
3 2
AcOH-(CH ) CO] 159.1174, found 159.1171.
2
O] 201.1643, found 201.1639; calcd. for C12H15 [M+H-
+
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.phytochem.2018.06.010.
4.9. Feeding experiments with labelled precursors
B. cinerea was grown at 25 °C on shake culture at 180 rpm in 500 ml
Erlenmeyer flasks, each containing 200 ml of modified Czapek-Dox
medium (30.0 g glucose, 1.0 g yeast extract, 1.0 g KH PO , 2.5 g
NaNO , 0.5 g MgSO 7H O, 0.001 g FeSO 7H O, 0.5 g KCl, 0.005 g
CuSO 5H O and distilled water to 1.0 l). The pH of the medium was
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3
4
2
4
2
4
2
adjusted to 7.0 with an aq. soln. 1 M NaOH. Each flask was inoculated
7
with 10 fresh conidia coming from a 14–21 days old culture of B. ci-
nerea on Tomato-Agar medium (0.5 l tomato juice, 20.0 g agar, and
distilled water to 0.5 l).
After 5 days of incubation (see detailed experiments below) the
mycelia was transferred into the same number of 500 ml Erlenmeyer
flasks, each containing 200 ml of Czapek-Dox medium without glucose
or with a reduced dose of glucose (15.0 g/l) and a filter-sterilized aq.
soln. of the labelled precursor. Flasks were incubated under the same
conditions described above for 3 days. Then, the culture medium and
mycelia were separated by filtration. The broth was satd. with NaCl,
and extracted with EtOAc (× 3). The EtOAc extract was washed with
distilled H O (× 3) and then dried over dry Na SO . The organic ex-
2 2 4
tract which was obtained, was evaporated at reduced pressure to dry-
ness.
The extract was subjected to column chromatography on silica gel
with an increasing gradient of EtOAc in n-hexane as eluent. Final pur-
ification was carried out by normal phase HPLC using a Lichrosfer Si 60
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.9.1. Feeding of sodium [1–13C]-acetate to BcBOT2Δ
Twenty subcultured Erlenmeyer flasks with conidia of the BcBOT2Δ
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1
3
[
1– C]-acetate in H
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2.3 mg).
2
O on day 5 to a final concentration of 500 ppm.
1
(
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