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Botrytis Species: An Intriguing Source of Metabolites with a Wide Range of

Biological Activities. Structure, Chemistry and Bioactivity of Metabolites
Isolated from Botrytis Species...

Article  in  Current Organic Chemistry · December 2000

DOI: 10.2174/1385272003375815

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 Current Organic Chemistry, 2000, 4, 1261-1286 1261

Botrytis Species: An Intriguing Source of Metabolites with a Wide Range of

Biological Activities. Structure, Chemistry and Bioactivity of Metabolites
Isolated from Botrytis Species

I. G. Collado,* J. Aleu, R. Hernández-Galán and R. Durán-Patrón

Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz. República Saharaui s/n, apdo.
40, 11510 Puerto Real, Cádiz. Spain

Abstract: The Botrytis species belong to the most geographically widespread group of plant pathogens
and saprophytes, and cause serious losses to very many commercial crops. These phytopathogenus fungi
are the agents of the disease known as grey mould. They biosynthesise characteristic metabolites that, in
vitro, produce the grey mould symptoms. The recent discovery that oxidative forces are involved when
B. cinerea infects plant and the relevance of toxins for pathogenicity, have increased the interest for these
compounds, in order to determinate the potential links to the plant oxyradical metabolism and the
induction of oxidative stress. This review describes the metabolites isolated from Botrytis species, the
spectroscopic data, grouping together by structural family, synthesis and chemical transformation,
specially those carried out on botryane skeleton. The basic skeleton of botrydial is a bicyclic, non
isoprenoid sesquiterpene system which has provided the stimulus for many investigations into the
biosynthetic pathway to this skeleton. The biosynthetic studies carried out and the approaches to the
asymmetric synthesis on this skeleton are reported in this paper. On the other hand, a detailed study on
biological activity showed for these metabolites and the structure-activity relationship of metabolites with
the botryanes skeleton is included.

INTRODUCTION The Botrytis species belong to the most

The term Botrytis refers to a group of imperfect
fungi of the Moniliales order and the Moniliaceae
family. As is the case with all such fungi, Botrytis
reproduces asexually by means of the formation of
conidia [1].
Despite being considered an imperfect fungus,
Botrytis may present a perfect stage; in other
words, it is capable of both sexual and asexual
reproduction. This, along with the fact that it
exhibits a variety of conditions and forms of
resistance, has resulted in considerable confusion
with regard to its taxonomic classification [2].
geographically widespread group of plant
pathogens and saprophytes. They are found
wherever the host crop is cultivated, from cold
regions such as Alaska and Canada, to subtropical
regions such as Egypt.
These fungi are polyphage parasites; they are
able to live as saprophytes, feeding off
decomposing organic material, or as parasites or
semi-parasites. They may even alternate between
each type of behavior.
Many species belong to this genus, which
causes serious losses to a vast number of
commercial crops; the following are among the
more important:

*Address correspondence to this author at the Departamento de Química
Orgánica, Facultad de Ciencias, Universidad de Cádiz. República
Saharaui s/n, apdo. 40, 11510 Puerto Real, Cádiz. Spain; Ph.: ++956-
016371; Fax: ++956-016193; e-mail: isidro.gonzalez@uca.es
• B. cinerea: a pathogen of more than 235
identified plant species, including grapes,
lettuce, tomatoes, tobacco and strawberries.

1385-2728/00 $19.00+.00 © 2000 Bentham Science Publishers Ltd.

1262 Current Organic Chemistry, 2000, Vol. 4, No. 12
It is the agent responsible for the disease
known as “gray mold”, so named for the
powdery gray mold it produces on the crops
it infects.
• B. squamosa: an onion pathogen which
causes stalk breakage.
• B. allii and B. byssoidea: pathogens of
onions.
• B. fabae: a bean pathogen which causes
epidemic spots on the leaves.
• B. tulipae: a pathogen responsible for burns
on tulip and saffron flowers.
• B. gladioli: a pathogen of gladioli and lilies.
Botrytis cinerea has a number of unique features
in its pathological behavior. The fungus is
primarily a saprophyte that initially establishes
itself on weakened or dead parts of plants, and
later extends to the rest of the healthy plant tissue
[3]. This fungus is also a secondary invader and
attacks plants already infected by other pests [4].
The penetration of the fungus into the host can
be achieved either by germinative conidial tubes,
through natural openings, or by mycelium growing
on dead or deteriorated parts of the plant [5].
In 1992, Goodman monitored the entire process
of infection of B. cinerea using Nuclear Magnetic
Resonance Imaging [6]. Doss replicated this work
several years later using Electron Microscopy [7].
The results of these investigations indicated that
the adhesion of the conidia occurs by hydration of
the conidia themselves, together with relatively
weak adhesive forces and hydrophobic interactions
between the fungal spores and the vegetable
cuticle. After the conidia have been incubated, the
germinative tubes attack the substrate by secreting
a wrapping layer of unknown composition.
The most common symptoms of infection by
B. cinerea are putrefaction and the development of
necrotic lesions. Various compounds are thought
to play a role in the death of plant cells by
necrotrophic fungi, including:
Collado et al.
- Hormones [8].
- Degradation enzymes of cell-walls
(polygalacturonases, pectin lyases, and
cellulases, etc.) or membranes
(phospholipases, lipases, etc.) [9-12].
- Potential toxins [11, 13].
- Oxygen active species [14].
Recently, evidence concerning the role of
exuded toxins in the pathogenesis of the fungus has
also been reported [15,16]. The visible phytotoxic
effects of Botrytis can thus be reproduced in vitro
by metabolites isolated from fermentation broth of
B. cinerea.
Both the recent discovery that oxidative forces
are involved in B. cinerea infections and the
relevance of toxins from B. cinerea for
pathogenicity have increased the interest in these
compounds, not only to determine the potential
links between the oxyradical plant metabolism and
the induction of oxidative stress, but also to
establish the role of these toxins in the infection
mechanism.
Characteristic Metabolites from Botrytis spp
With regard to the metabolites biosynthesized
by the fungi of the Botrytis genus, B. cinerea has
probably been the most widely studied, although
other species have received considerable attention.
B. cinerea, B. squamosa and B. allii produce, in
addition to other minor compounds, characteristic
metabolites which contain the basic botryane
skeleton, principally botrydial (1) and
dihydrobotrydial (2). Both compounds were
isolated by Tschesche et al. [17] from the culture
solution of the fungus B. cinerea.
Botrydial (1) has shown interesting biological
activities, such as antibiotic activity against
Bacillus subtilis and Phytium debaryanum [17]. In
addition, it is very active as a cytotoxic front to an
important number of cellular lines [16].
Furthermore, compounds 1 and 2 have been found
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1263

Table 1. Structures of Compounds 8-23

Natural Products Chemical Transformation Products

OHC CHO HOH2 C CH2 OH CHO

OH OH

H H H
AcO AcO AcO
1 8 9
HO O O O R1 R2 EtO O
OH
OH OH OH

H
H H AcO H
AcO AcO 11 R1=COOH; R2 =CH 2OH R
2 10 12 R1=COOMe; R2 =CH2 OH 15 R=OAc
13 R1=COOMe; R2 =CHO 16 R=OH
14 R1=R2 =COOMe
CHO COOMe

H H
AcO AcO
9 17
HOOC CH2 OH R1 R2
OH OH

H H
AcO AcO
11 12 R1=COOMe; R2 =CH2 OH
13 R1=COOMe; R2 =CHO
14 R1=R2 =COOMe
OHC COOH OHC COOMe
OH OH

H H
HO AcO
18 19
OHC COOH OHC COOMe
OH OH OHC COOMe OHC COOMe

H H
AcO R H
20 AcO 23
19 R=OAc 22
21 R=OH
1264 Current Organic Chemistry, 2000, Vol. 4, No. 12
to produce phytotoxic effects in bioassays on
leaves of tobacco and grape plants [15]. In one
bioassay experiment involving the study of
Botrytis fermentation over a period of several days,
phytotoxic activity was detected by incubating
aliquots of fungus-free culture filtrate on tobacco
leaf discs of 1 cm diameter [15]. The phytotoxic
effect caused by the fungus-free filtrate was the
same as that produced on the leaves by the spores
and the mycelium. Further work was carried out to
isolate the phytotoxic metabolites and to quantify
their effect. When pure metabolites were assayed,
it was found that botrydial (1) and
dihydrobotrydial (2) produced the same
phytotoxic effect as the fungus-free filtrate;
Compound 1 was phytotoxic at just 1 ppm while
compound 2 at 500 ppm. Interestingly, higher
concentrations of purified metabolites were
necessary in order to reproduce the same level of
symptoms as those caused by metabolites from in
vivo production, thus suggesting that these
metabolites, or hitherto undetected ones, may have
a synergistic effect.
The structures of compounds 1 and 2 show that
they do not strictly obey the isoprene rule. Their
structural formulas were elucidated by means of a
series of chemical transformations and by the
HO
10
15
CHO CHO
OH 14
11 1 8
2
5 7
3 6
4 H
13
H OAc
OAc 12
1 2
O O
H O
R
R R
O O
5a R=H
5b R=D
Collado et al.
application of spectroscopic methods. Chemical
transformations of 1 and 2, for example, afforded
compounds 3-7 [17]. Compounds 3a and 3b were
obtained by treating 2 with H2/Pt. Oxidation of 3
followed by catalytic hydrogenation yielded
compounds 4-6. The structure of 2 was confirmed
by means of single crystal X-ray diffraction using
direct methods [18].
Compounds 8-23 [19-21] have all been isolated
either from fungus B. cinerea or by means of
chemical transformation during biosynthetic
studies. Their structures are shown in Table 1.
Marumo et al. found that Botryotinia squamosa
produced a phytotoxic substance when cultured
under blue light [22]. This work led to the isolation
and structural elucidation of botrydienal (24) and
its related metabolites, dehydrobotrydienal (25)
and deacetyldihydrobotrydial (26). Bioassays
were performed on turnip seedlings and the
phytotoxic activity was monitored. The respective
50% inhibitory concentrations (IC 50) of 24, 25 and
26 were 10, 40 and 1000 ppm. Botrydienal (24)
and dehydrobotrydienal (25) also showed
antibacterial activity; the respective MICs against
Escherichia coli B110 were 10 and 20 ppm,
respectively, while those against Staphylococcus
O O
O
OH OH
OH
H
H OR O
3a R=Ac 4
3b R=H
O
O
7
6
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1265

MeO MeO O
HO O O O
CHO CHO CHO CHO
OH OH OH

H H H
OH OAc OH
24 25 26 27 28

aureus IFO 12732, were both 50 ppm. The X-ray
structure of deacetyldihydrobotrydial (26),
isolated from B. squamosa, and its activity as a
growth inhibitor of lettuce seedlings have been
published [23].
In later studies on the metabolites of Botrytis
squamosa, Kimura et al. [24] isolated two new
(10 mg/l) and 50% (300 mg/l), respectively, while
root growth was hardly influenced.
In addition, Kimura’s group has recently
reported two new hypocotyl growth-inhibiting
and root-promoting substances, BSF-A (29) and
botryslactone (30), both from B. squamosa [25].
Compound 29 was determined to be 1-methyl

O
HO
O
CH2 CH COOR1
H
NH CH2 OH

H 3C CH COOR2

29 R1 = CH3 , R2 = H
29a R1 = R2 = CH 3
30

compounds: O-methyldihydrobotrydial (27) and hydrogen 3-phenyl-2,2´-iminodipropionate, which

deacetyl-O-methyldihydro-botrydialone (28). after methylation with diazomethane, produced a
Their biological activity was examined using dimethyl compound 29a. At a concentration of
lettuce seedlings. Both compounds inhibited the 300 ppm, compounds 29 and 29a promoted root
hypocotyl elongation by approximately 70% growth by about 220% compared to that of the

HO OH O HO O O O
MeO O
CHO R OH OH
OH OH

H H
H H H AcO AcO
AcO AcO AcO
31 32 R= CHO 37 38
33 R= CH2 OH 35 β-OMe
34 R= COOH 36 α-OMe
34a R= COOMe
1266 Current Organic Chemistry, 2000, Vol. 4, No. 12
control. The stereochemistry of C-2 and C-2 has
not yet been determined.
Compound 30 was examined for its bioactivity
in lettuce seedlings. At concentrations between 1-
300 mg/l, promotion of root growth was directly
proportional to the level of concentration.
However, this compound actually inhibited root
growth at a concentration of 1000 mg/ml.
A study of the metabolites present in shake and
static cultures from a strain of B. cinerea isolated
from Spanish wine grapes afforded nine new
metabolites (10, 31-38) [21, 26]. The acid 34 was
purified as its methyl ester after treatment with
diazomethane to yield compound 34a.
Comparisons of both cultures revealed a higher
oxidation level in the metabolites isolated from the
shake culture, in accordance with the higher rate of
oxygenation in this culture. The structures were
elucidated by extensive NMR investigations of the
natural compounds and their derivatives.
The tricyclic sesquiterpene 31 is a key
biosynthetic intermediate and its elucidation shed
further light on the final steps of the biosynthesis
of 1 and related compounds [26].
Further studies on the metabolites of B. cinerea
led to the isolation of three new epimer
metabolites of compound 1: 8,9-epibotrydial (39),
1-epibotrydial (40) and 1,8,9-epibotrydial (41)
[16]. The stereochemistry of these compounds
was established by means of NOE experiments. In
addition, six new sesquiterpenoid metabolites (42-
47), together with botryoloic acid methyl ester
(12), have been reported from B cinerea [27].
Secobotrytriendiol (42) exhibits a skeleton not
previously reported in Botrytis species, and which
may result from biodegradation of 1. The methyl
ester of 11 had been previously obtained from the
acid fraction of a fermentation broth of B. cinerea
after treatment with CH2N2 [20]. This was thus
the first time that 12 had been isolated from the
fermentation broth with no previous treatment.
The aromatic compounds 43 and 44 showed
minimum activity on tobacco leaves at
concentrations of 500 and 250 ppm, respectively.
In spite of the lower activity shown by compound
Collado et al.
43 in relation to its derivative 44, we observed that
this product was capable of producing a
phytotoxic effect only after 48 h incubation at a
concentration of 1000 ppm. This effect was not
observed for compound 44 [27].
The proton NMR data of natural compounds 1-
47 are given in Tables 2, 4, 6 and 8. The most
characteristic skeleton signal is that corresponding
to the proton H-4, the resonance of which appears
as a ddd. The chemical shift of this signal is
affected by the presence of an acetyl or hydroxyl
group at C-4; it changes from 5.07 ppm in 2 to
4.21 ppm in the deacetyl derivative (26).
The 1H-NMR spectrum of each compound also
shows the presence of one doublet and three
singlet signals, which correspond to the four
methyl groups of the skeleton. The presence of
two doublets associated to the H-7 protons are
also characteristic and indicate substantial changes
in the oxidation level of C-15.
H-2 appears as a multiplet due to its coupling
with H-3α and H-3β and with a doublet methyl
group (H-11), as seen in the COSY experiment.
The shift of this signal is clearly affected by the
presence of a double bond between C-1 and C-9,
as well as by epimerization at C-9. This
epimerization is also easily recognized due to a
long distance coupling between the signal
corresponding to the hydroxyl group and H-5.
The downfield shift of the signal corresponding
to H-1, which appears as a doublet of doublets, is
associated with an epimerization at C-1. This
change in the stereochemistry also affects the
magnitude of the coupling constant J1-2, which
changes from 12.2 Hz in botrydial (1) to 5.9 Hz in
40.
The proton NMR spectrum of secobotryendiol
(42) shows a skeleton previously unknown in B.
cinerea. The proposed structure was supported
by homo and heteronuclear 2D NMR correlation
experiments. A 2D 1H-13C-shift correlation
indicated that the sequence CH3-CH=CH 2OH
correlated in the long range HETCOR experiment
with the fragment C=C-CH-CH 2.
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1267

OH OH
OHC OHC CHO OHC 10
OH CHO 15
OH OH 14
CHO
9
1 8
2
7
6
11 5
4
H H H
AcO AcO AcO 12 13
3
39 40 41 42
OH OH MeO O OMe HO O O
10 15

R1 OH OH 14
11
1 8
2 9
7
3 5
4 6

R2 H H 13
AcO AcO 12
43 R1 = R2 = CH3
44 R1 = CH2 OH; R2 = CH3 46 47
45 R1 = CH3 ; R2 = CH2 OH

The 13C-NMR data of natural compounds 1-47
are given in Tables 3, 5, 7 and 9. Two of the
skeleton methyl groups, those resonance at 27.2
and 35.9 ppm in dihydrobotrydial (2), remain
relatively unchanged in position irrespective of the
presence of a C-15 lactone, hemiacetal, aldehyde or
ether, and only vary in the presence of double
bonds in the cyclohexane ring. These signals were
assigned to the methyl groups attached to C-6. Of
those two methyl signals, the one at higher field
undergoes a cis 1,3-diaxial interaction with the C-
14 methyl group and is thus shielded with respect
to the methylcyclopentane; it is therefore the α-
oriented methyl group at C-13. The remaining
skeleton signals show substantial changes
depending on both the oxidation level of C-10 and
C-15 and the presence of a double bond between
C-1 and C-9.
The quaternary carbon atoms C-6 and C-8
could be distinguished due to the influence of the
changes at C-15 on the chemical shift of the C-8
resonance.
Biosynthesis of Botryane Skeleton
The biosynthetic pathway to the basic skeleton
of botrydial (1), which is a bicyclic, non-
isoprenoid sesquiterpene system, has provided the
stimulus for many investigations.
In a series of noteworthy papers, Hanson and
co-workers [28] concentrated on defining the
manner in which farnesyl pyrophosphate is folded
in fungal sesquiterpenoids, including the
elucidation of the stereochemical fate of
mevalonoid hydrogen atoms, and on relating
groups with common primary cyclizations. The
problem was analyzed in three stages:
• The origin of the carbon skeleton.
• The fate of the mevalonoid hydrogen atoms.
• The oxidative processes.
The labeling and coupling patterns of
dihydrobotrydial (2), which had been
13 13
biosynthesized from [1- C] and [1,2- C2] acetate
and [4,5-13C2] mevalonate, defined the way in
which farnesyl pyrophosphate is folded to
generate the carbon skeleton of this major
sesquiterpenoid. This led to the biogenetic
proposals shown in Fig. (1). A hydrogen migration
from C-9 to the C-2 of 2 and cleavage to the C(4)-
C(5) bond of farnesyl pyrophosphate with the
formation of the C(10)-C(15) hemiacetal of 2 are
implicit in this scheme.
1268 Current Organic Chemistry, 2000, Vol. 4, No. 12 Collado et al.

1
Table 2. H-NMR Data of Compounds 1, 12, 19, 21 and 39-41 in CDCl3
Proton 1 39 40 41 12 19 21
(400 MHz) (400 MHz) (400 MHz) (400 MHz) (400 MHz) (90 MHz) (90 MHz)
1 2.48 (dd) 2.15 (dd) 2.85 (dd) 2.73 (t) 2.45 (d) — —
2 2.10 (m) 2.49 (m) 2.23 (m) 2.59 (m) 2.04 (m) — —
3α 2.17 (m) 2.26 (ddd) 1.99 (ddd) 2.11 (ddd) 2.14 (ddd) — —

3β 1.12 (br d) 1.00 (ddd) 1.54 (ddd) 1.40 (ddd) 1.04 (m) — —
4 5.03 (ddd) 5.26 (ddd) 4.98 (ddd) 5.31 (ddd) 5.06 (ddd) 5.06 (ddd) 3.95 (ddd)
5 2.00 (d) 1.79 (dd) 2.15 (d) 2.43 (dd) 1.89 (d) — —
7α 1.37 (d) 2.26 (d) 1.36 (d) 2.26 (d) 1.17 (d) — —

7β 2.36 (d) 1.45 (d) 2.36 (d) 1.49 (d) 2.40 (d) — —
10 9.84 (d) 9.73 (d) 9.89 (d) 9.99 (d) 9.86 (d) 9.86 (d)
11 0.96 (d) 0.97 (d) 1.12 (d) 0.92 (d) 0.90 (d) 0.86 (d) 0.90 (d)
12 1.32 (s) 1.20 (s) 1.27 (s) 1.13 (s) a 1.06 (s) 1.33 (s)
1.27 (s)
13 1.30 (s) 1.12 (s) 1.12 (s) 1.12 (s) a 1.30 (s) 1.33 (s)
1.06 (s)
14 1.08 (s) 1.31 (s) 1.06 (s) 1.25 (s) 1.01 (s) 1.36 (s) 1.33 (s)
15 9.58 (s) 9.58 (s) 9.62 (s) 9.52 (s) 3.30 (d)
15' 3.56 (d)
CH 3 -COO 2.04 (s) 2.03 (s) 2.04 (s) 2.05 (s) 2.02 (s) 2.03 (s)
OH 3.92 (s) 3.71 (d) 3.63 (br s) 3.16 (d) 4.54 (br s) — —
COO-C H 3 3.74 (s) 3.70 (s) 3.68 (s)

J (Hz): 1: J1-2 =12.2; J1-10 =2.5; J3α-2=3.2; J3α-3β=12.5; J3α-4=4.8; J4-3$ =11.3; J4-5 =11.3; J7α-7β=13.2; J11-2 =6.3. 39: J1-2 =11.2; J1-10 =3.7; J3α-3β=12.5;
J3α-4=4.7; J3β-4=11.0; J4-5 =11.0; J5-OH=1.6; J7α-7β=13.3; J11-2 =6.6. 40: J1-2 =5.9; J1-10 =3.3; J3α-2=4.5; J3α-3β=13.4; J3α-4=4.6; J3β-2=9.8; J3β-4=8.4;
J4-5 =8.4; J7α-7β=13.6; J11-2 =7.3. 41: J1-10 =5.3; J3α-2=4.2; J3α-3β=13.2; J3α-4=5.2; J3β-2=13.2; J3β-4=10.8; J4-5 =10.8; J5-OH=1.8; J7α-7β=12.9; J11-2 =7.3. 12:
J1-2 =12.4; J3"-2 =3.3; J3"-3$ =12.3; J3"-4 =4.9; J4-3$ =11.3; J4-5 =11.3; J7"-7$ =12.8; J11-2 =6.2; J15-15′ =12.0. 19: J1-10 =3.0; J4-3α=4.5; J4-3β=11.0; J4-5 =11.0; J11 -
a
2 =5.0. 21: J1-10 =3.0; J4-3α=4.5; J4-3β=11.0; J4-5 =11.0; J11-2 =5.0. = interchangeable signals.

13
Table 3. C-NMR Data of Compounds 1, 12, 19, 21 and 39-41 in CDCl3
Carbon 1 39 40 41 12 19 21
(100MHz) (50MHz) (100 MHz) (100 MHz) (100 MHz) (25.15 MHz) (25.15 MHz)
1 66.9 (d) 60.3 (d) 55.6 (d) a 60.9 (d) 68.3 (d) 68.5 (d)
53.3 (d)
2 28.0 (d) 29.1 (d) 29.5 (d) 30.3 (d) 29.2 (d) 28.2(d) 28.5 (d)
3 38.7 (t) 40.2 (t) 34.1 (t) 36.2 (t) 38.7 (t) 38.6 (t) 43.5 (t)
4 72.1 (d) 70.6 (d) 71.3 (d) 71.5 (d) 72.9 (d) 72.9 (d) 70.0 (d)
5 63.4 (d) 58.3 (d) 60.9 (d) a 63.9 (d) 61.7 (d) 66.3 (d)
60.2 (d)
6 39.3 (s) 38.2 (s) 38.8 (s) 37.6 (s) 36.5 (s) 38.6 (s) 38.6 (s)
7 51.6 (t) 50.9 (t) 48.9 (t) 50.3 (t) 52.2 (t) 55.0 (t) 54.9 (t)
8 58.8 (s) 57.2 (s) 59.6 (s) 58.7 (s) 49.7 (s) 55.0 (s) 55.1 (s)
9 89.6 (s) 89.2 (s) 86.9 (s) 86.9 (s) 88.3 (s) 88.0 (s) 88.2 (s)
10 204.7 (d) 207.5 (d) 205.5 (d) 204.3 (d) a 203.5 (d) 204.1 (d)
173.7 (s)
11 20.4 (q) 19.5 (q) 19.5 (q) b 20.8 (q) 20.5 (q) 20.7 (q)
21.0 (q)
12 35.4 (q) 34.2 (q) 34.5 (q) 34.5 (q) b 35.7(q) 36.2 (q)
35.9 (q)
13 27.7 (q) 27.1 (q) 28.0 (q) 27.6 (q) b 27.5 (q) 27.8 (q)
27.8 (q)
14 19.9 (q) 19.2 (q) 18.8 (q) b 21.7 (q) 20.9 (q) 20.7 (q)
18.2 (q)
15 206.8 (d) 207.8 (d) 205.8 (d) 206.4 (d) 67.7 (t) 178.9 (s) 179.0 (s)
CH 3 -COO 21.3 (q) 21.3 (q) 21.5 (q) 21.3 (q) 21.5 (q) 21.4 (q)
CH 3 -COO 170.2 (s) 170.3 (s) 170.2 (s) — a 170.3 (s)
170.4 (s)
COO-CH 3 51.5 (q) 52.6 (q) 52.5 (q)
a, b
= interchangeable signals.
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1269

Table 4. 1 H-NMR Data of Compounds 9, 24-25, 32-34a and 42-45 in CDCl3 .

Proton 9 32 33 34a 24 25 43 44 45 42
(400 MHz) (400 MHz) (400 MHz) (400 MHz) (200 MHz) (400 MHz) (400 MHz) (400 MHz) (400 MHz)
2 2.85 (m) 2.96 (m) 2.94 (m) 2.91 (m) 2.97 (m) 5.73 (q)
3 2.14 (m) 2.16 (m) 2.03 (ddd) 2.10 (m) 2.40 (ddd) 7.22 (d) 7.12 (d) 7.21 (d) 7.14 (d) 5.06 (dd, Hcis)
3 1.38 (m) 1.38 (m) 1.36 (m) 1.40 (ddd) 2.18 (m) 5.40 (dd, Htrans)
4 4.75 (o) 4.93 (ddd) 4.90 (ddd) 4.92 (ddd) 5.87 (ddd) 7.32 (d) 7.02 (d) 7.09 (d) 6.97 (d) 6.20 (dd)
5 2.54 (dd) 2.57 (dd) 2.50 (dd) 2.74 (dd)
7 1.8 (m) 1.52 (d) 1.44 (d) 1.69 (d) 1.62 (d) 1.86 (d) 1.78 (d) 1.79 (d) 1.69 (d) 1.62 (d)
7 1.8 (m) 2.14 (d) 1.91 (d) 2.24 (d) 2.22 (d) 2.21 (d) 2.21 (d) 2.25 (d) 2.48 (d) 2.08 (d)
8 3.2 (m)
10 10.0 (s) 9.70 (s) 10.18 (s) 9.86 (s) 9.72 (s) 10.40 (s) 4.72 (d) 4.76 (d) 4.66 (d) 4.12 (d)
10' 4.78 (d) 4.80 (d) 4.72 (d) 4.29 (dt)
11 1.12 (d) 1.07 (d) 1.05 (d) 1.06 (d) 0.90 (d) 2.68 (s) 2.42 (s) 4.56 (d) 2.41 (s) 1.52 (dd)
11' 4.96 (d)
12 1.25 (s) 1.21 (s) 1.12 (s) 1.17 (s) 1.24 (s) a1.32 (s) 1.29 (s) a1.31 (s) 3.52 (d) a1.34 (s)
12' 3.66 (d)
13 0.75 (s) 0.99 (s) 0.90 (s) 0.94 (s) 1.05 (s) a1.33 (s) 1.29 (s) a1.30 (s) 1.23 (s) a1.33 (s)
14 1.28 (d) 1.52 (s) 1.44 (s) 1.61 (s) 1.52 (s) 1.55 (s) 1.38 (s) 1.38 (s) 1.30 (s) 0.98 (s)
15 9.52 (s) 3.59 (d, H) 9.66 (s) 9.60 (s) 3.63 (d) 3.59 (d) 3.65 (d) 3.27 (d)
15' 3.67 (d, H) 3.92 (d) 3.94 (d) 3.91 (d) 3.56 (d)
CH3 -COO 2.00 (s) 2.07 (s) 2.04 (s) 2.06 (s)
COO-CH3 3.72 (s)

J (Hz): 9: J4-3α=3.0; J4-3β=10.0; J4-5 =9.0; J5-8 =2.0; J11-2 =6.0; J14-8 =7.0. 32: J2-11 =6.8; J3"-2 =6.6; J3α-3β=13.0; J3"-4 =4.1; J3β-4=10.3; J5-2 =3.5; J5-4 =8.9; J7α-
7β=13.2. 33: J2-11 =6.8; J3α-2=6.4; J3α-3β=12.9; J3α-4=4.1; J3β-2=9.5; J3β-4=9.4; J5-2 = 3.1; J5-4 =8.2; J7α-7β=12.9; J15 α-15β=10.5. 34a: J2-11 =6.8; J3α-2=6.5;
J3α-3β=13.1; J3α-4=4.2; J3β-2=9.5; J3β-4=10.0; J5-2 =3.4; J5-4 =8.8; J7α-7β=13.2. 24: J2-4 =0.5; J2-11 =7.0; J3α-2=8.5; J3α-3β=18.6; J3α-4=3.2; J3β-2=1.4; J3β-
4 =6.2; J4-10 =0.7; J7α-7β=13.7. 25 : J3-4 =7.8; J7α-7β=13.5. 43: J3-4 =7.7; J7α-7β=13.2; J10-10′ =11.4; J15-15′ =11.3. 44: J3-4 =7.6; J7"-7$ =13.1; J10-10′ =12.4;
J11-11′ =11.8; J15-15′ =11.7. 45: J3-4 =7.7; J7"-7$ =13.7; J10-10′ =11.7; J12-12′ =10.7; J15-15′ =11.5. 42: J2-11 =6.8; J3cis-3trans =1.7; J3cis-4=11.8; J3trans-4=18.3;
a
J7"-7$ =13.0; J10-10′ =12.9; J10 ′-11 =1.2; J15-15′ =11.2. = interchangeable signals.

13
Table 5. C-NMR Data of Compounds 9, 24-25, 32-34a and 42-45 in CDCl3
Carbon 9 32 33 34a 24 25 43 44 45 42
(50 MHz) (50 MHz) (50 MHz) (25 MHz) 50 MHz) (100 MHz) (50 MHz) (100 MHz) (100 MHz)
1 135.8 (s) 138.2 (s) 131.1 (s) 137.4 (s) 135.7 (s) a
129.7 (s) 134.4 (s) 135.9 (s) 134.9 (s) 136.1 (s)
2 28.3 (d) 29.3 (d) 29.1 (d) 29.3 (d) 24.2 (d) a
140.7 (s) 136.4 (s) 138.5 (s) 137.3 (s) 126.6 (d)
3 37.5 (t) 37.1 (t) 36.7 (t) 37.0 (t) 30.5 (t) b
132.2 (d) 130.4 (d) 129.6 (d) 130.7 (d) 115.3 (t)
4 70.2 (d) 70.5 (d) 71.7 (d) 70.5 (d) 123.5 (d) b
128.3 (d) 123.0 (d) 123.1 (d) 122.9 (d) 131.1 (d)
5 56.0 (d) 58.8 (d) 58.4 (d) 58.4 (d) 149.2 (s) 153.1 (s) 152.3 (s) 155.2 (s) 147.3 (s) 148.6 (s)
6 40.9 (s) 40.0 (s) 39.1 (s) 39.6 (s) 40.4 (s) 42.4 (s) 40.8 (s) 41.1 (s) 46.5 (s) 45.2 (s)
7 49.3 (t) 51.1 (t) 54.1 (t) 55.8 (t) 50.4 (t) 51.3 (t) 54.0 (t) 53.9 (t) 49.3 (t) 52.3 (t)
8 29.1 (d) 68.0 (s) 51.8 (s) 50.9 (s) 56.3 (s) 57.9 (s) 50.3 (s) 50.5 (s) 50.4 (s) 52.5 (s)
9 169.0 (s) 161.2 (s) 162.8 (s) 162.8 (s) 154.9 (s) 141.2 (s) 144.0 (s) 145.1 (s) 145.7 (s) 140.0 (s)
10 190.4 (d) 191.4 (d) 192.4 (d) 191.4 (d) 189.5 (d) 191.3 (d) 58.7 (t) 58.6 (t) 58.4(t) 68.3 (t)
11 20.6 (q) 20.5 (q) 20.8 (q) 20.1 (q) 18.3 (q) 19.2 (q) 18.9 (q) 64.9 (t) 19.0 (q) 14.7 (q)
12 32.5 (q) 29.7 (q) 29.6 (q) 29.5 (q) a c a a 71.7 (t) a
28.7 (q) 30.5 (q) 31.1 (q) 30.8 (q) 30.7 (q)
13 24.2 (q) 23.7 (q) 23.6 (q) 23.7 (q) a c a a 26.8 (q) a
25.3 (q) 31.4 (q) 32.2 (q) 32.1 (q) 29.2 (q)
14 21.9 (q) 29.8 (q) 28.9 (q) 29.5 (q) 30.5 (q) 22.1 (q) 26.3 (q) 26.2 (q) 28.0 (q) 24.0 (q)
15 198.4 (d) 70.3 (t) 176.5 (s) 199.5 (d) 200.5 (d) 71.0 (t) 79.9 (t) 71.7 (t) 70.3 (t)
CH 3 -COO 21.3 (q) 21.3 (q) 21.3 (q) 21.3 (q)
CH 3 -COO 170.1 (s) 170.6 (s) 179.5 (s) 170.2 (s)
COO-CH 3 52.8 (q)
a, b, c
= interchangeable signals.
1270 Current Organic Chemistry, 2000, Vol. 4, No. 12 Collado et al.

1
Table 6. H-NMR Data of Compounds 2, 26-27, 31, 37 and 46
Proton 2 37 26 27 46 31
(CDCl 3 , 400 MHz) (CDCl 3 , 400 MHz) (py-d5 , 200 MHz) (CDCl 3 , 400 MHz) (CDCl 3 , 400 MHz) (CDCl 3 , 400 MHz)
1 1.63 (d) 1.53 (d) 2.03 (d) 1.59 (d) 1.54 (dd) 1.25 (m)
2 1.80 (m) 1.82 (m) 1.85 (qddd) 1.81 (m) 1.80 (m) 1.73 (m)
3α 2.04 (ddd) 2.09 (m) 2.16 (ddd) 2.03 (ddd) 2.04 (ddd) 1.95 (ddd)
3β 1.05 (m) 1.10 (m) 1.44 (ddd) 1.10 (m) 1.05 (m) 1.10 (m)
4 5.07 (ddd) 5.07 (ddd) 4.21 (ddd) 5.08 (ddd) 5.10 (ddd) 5.09 (ddd)
5 1.89 (d) 1.91 (d) 2.25 (d) 1.90 (d) 1.89 (d) 1.70 (d)
7α 1.16 (d) 1.14 (d) 1.24 (d) 1.04 (d) 1.24 (d) 1.20 (d)
7β 1.86 (d) 1.85 (d) 2.20 (d) 1.85 (d) 2.00 (d) 2.00 (d)
10 5.32 (br s) 5.35 (s) 5.74 (d) 4.80 (br s) 4.95 (d) 4.12 (dd)
11 0.97 (d) 0.98(d) 0.96 (d) 0.95 (d) 0.94 (d) 1.02 (d)
a a a
12 1.27 (s) 1.28 (s) 1.63 (s) 1.28 (s) 1.24 (s) 1.29 (s)
a a a
13 1.25 (s) 1.24 (s) 1.67 (s) 1.24 (s) 1.08 (s) 1.16 (s)
14 1.10 (s) 1.10 (s) 1.42 (s) 1.09 (s) 1.11 (s) 1.17 (s)
15" 3.24 (d) 3.25 (d) 3.39 (d) 3.20 (d)
15$ 4.17 (d) 3.91 (d) 4.60 (d) 3.94 (d) 4.86 (s) 4.43 (d)
CH 3 COO 2.02 (s) 2.03 (s) 2.01 (s) 2.01 (s) 2.02 (s)
CH 3 O 3.36 (s) 3.45 (s)
3.49 (s)
OH 3.40 (br s) 2.17 (s) 5.60 (d) 4.11 (s) 3.64 (s) —
3.56 (br s) 3.35 (s) 5.82 (d)
8.31 (s)

J (Hz): 2: J1-2 =12.6; J3"-2 =3.2; J3"-3$ =12.4; J4-3" =4.8; J4-3$ =11.0; J4-5 =11.0; J7"-7$ =11.9; J11-2 =6.4; J15"-15$ =10.6. 37: J1-2 =12.3; J4-3" =4.6; J4-3$ =11.0;
J4-5 =10.6; J7"-7$ =11.9; J11-2 =6.4; J15 α-15β=10.6. 26: J1-2 =12.2; J2-3α=2.7; J2-3β=12.0; J2-11 =6.1; J3α-3β=12.5; J3α-4=4.6; J3β-4=12.0; J4-OH=5.9; J5-4 =9.8;
J7α-7β=11.2; J10-OH =4.4; J15 α-15β=10.3. 27: J1-2 =12.4; J3α-2=3.0; J3α -3$ =12.4; J3"-4 =4.6; J3$-4 =11.0; J4-5 =11.0; J7"-7$ =11.5; J11-2 =6.3; J15 α-15β=10.6. 46:
J1-2 =13.4; J3"-4 =4.7; J3$-4 =11.0; J4-5 =11.0; J7"-7$ =11.9; J10-1 =1.4; J11-2 =6.3. 31: J3"-2 =3.4; J3"-3$ =10.7; J3"-4 =3.8; J3$-4 =10.7; J4-5 =10.7; J7"-7$ =11.7;
a
J10-1 =2.9; J10-15 =5.2; J11-2 =6.4. = interchangeable signals.

13
Table 7. C-NMR Data of Compounds 2, 26-27, 31, 37 and 46
Carbon 2 37 26 27 46 31
(CDCl 3 , 50 MHz) (CDCl 3 , 50 MHz) (CD 3 OD, 50 MHz) (CDCl 3 , 50 MHz) (CDCl 3 , 50 MHz) (CDCl 3 , 100MHz)
1 54.9 (d) 55.0 (d) 57.3 (d) 54.7 (d) 55.0 (d) 58.1 (d)
2 28.6 (d) 29.0 (d) 30.1 (d) 28.6 (d) 29.1 (d) 22.2 (d)
3 39.9 (t) 39.8 (t) 46.0 (t) 40.0 (t) 39.8 (t) 39.6 (t)
4 72.6 (d) 72.6 (d) 70.3 (d) 72.8 (d) 72.7 (d) 72.7 (d)
5 60.0 (d) 59.6 (d) 64.9 (d) 59.4 (d) 59.9 (d) 59.7 (d)
a
6 38.8 (s) 38.8 (s) 39.8 (s) 38.8 (s) 39.2 (s) 46.6 (s)
7 50.4 (t) 50.2 (t) 51.8 (t) 50.4 (t) 50.4 (t) 48.4 (t)
a
8 45.4 (s) 45.5 (s) 46.5 (s) 45.4 (s) 49.3 (s) 57.2 (s)
9 83.4 (s) 82.5 (s) 84.7 (s) 82.5 (s) 86.0 (s) 95.9 (s)
10 92.5 (d) 92.3 (d) 93.3 (d) 98.7 (d) 101.0 (d) 89.1 (d)
11 20.1 (q) 20.1 (q) 20.6 (q) 20.1 (q) 20.1 (q) 20.9 (q)
a
12 35.9 (q) 35.6 (q) 36.5 (q) 35.6 (q) 35.6 (q) 36.4 (q)
a
13 27.2 (q) 27.3 (q) 27.9 (q) 27.3 (q) 27.2 (q) 27.5 (q)
14 25.3 (q) 25.0 (q) 26.0 (q) 25.3 (q) 19.2 (q) 33.5 (q)
15 67.5 (t) 67.7 (t) 68.4 (t) 67.5 (t) 102.8 (d) 84.1 (d)
CH 3 COO 21.4 (q) 21.4 (q) 21.5 (q) 21.4 (q) 21.4 (q)
CH 3 COO 170.5 (s) 170.5 (s) 170.5 (s) 170.5 (s) 170.5 (s)
CH 3 O 54.9 (q) 56.77 (q)
55.34 (q)
a
= interchangeable signals.
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1271

Table 8. 400 MHz 1 H-NMR of Compounds 10, 28, 35-36, 38 and 47 in CDCl3
Proton 10 38 47 35 36 28
1 2.26 (d) 1.94 (m) 1.46 (dd) 1.70 (m) 2.09 (d) 1.71 (dd)
2 1.97 (m) 1.64 (m) 2.13 (m) 1.70 (m) 2.01 (m) 1.61 (qddd)
3α 2.09 (ddd) 2.05 (m) 2.10 (m) 2.08 (m) 2.09 (m) 1.90(ddd)
3β 1.12 (m) 1.17 (ddd) 1.37 (m) 1.16 (m) 1.10 (m) 1.22 (ddd)
4 5.02 (ddd) 4.95 (ddd) 4.85 (ddd) 4.92 (ddd) 4.93 (ddd) 3.80 (ddd)
5 1.86 (d) 1.98 (d) 1.34 (d) 1.97 (d) 1.93 (d) 1.65 (d)
7α 1.33 (d) 1.57 (d) 1.61 (d) 1.55 (d) 1.61 (d) 1.50 (d)
7β 1.90 (d) 2.47 (d) 1.86 (d) 2.46 (d) 2.39 (d) 2.46 (d)
10 α 4.25 (dd) 5.07 (d) 5.19 (d) 5.19 (d)
10 β 4.74 (dd) 5.47 (d)
11 1.27 (d) 0.92 (d) 1.20 (d) 0.99 (d) 0.99 (d) 0.99 (d)
a a a a
12 1.29 (s) 1.28 (s) 0.99 (s) 1.29 (s) 1.25 (s) 1.29 (s)
a a a a
13 1.26 (s) 1.13 (s) 1.14 (s) 1.27 (s) 1.13 (s) 1.25 (s)
14 1.13 (s) 1.38 (s) 1.43 (s) 1.11 (s) 1.51 (s) 1.33 (s)
15 3.92 (d)
15' 4.69 (d)
CH 3 COO 2.03 (s) 2.04 (s) 2.03 (s) 2.03 (s) 2.03 (s)
CH 3 O 3.60 (s) 3.53 (s) 3.58 (s)
J (Hz): 10: J1-2 =11.7; J3"-2 =2.9; J3"-3$ =11.0; J3"-4 =4.1; J4-3$ =11.0; J4-5 =11.0; J7"-7$ =12.5; J11-2 =5.1; J15"-15$ =9.8. 38: J1-2 =12.0; J2-11 = 6.4; J3α-2=3.0; J3α-

3β=13.9; J3α-4=4.1; J3β-2=5.8; J3β-4=11.2; J4-5 =11.3; J7α-7β=13.3; J10 α-1=8.3; J10 α-10β=12.4; J10 β-1=9.6. 47 : J1-2 =1.7; J1-10 =8.5; J3"-4 =11.7; J3$-4 =11.7;
J4-5 =3.2; J7"-7$ =14.6; J11-2 =6.8. 35: J4-3" =4.1; J4-3$ =11.2; J4-5 =11.2; J7"-7$ =13.3; J10-1 =4.5; J11-2 =5.9. 36: J1-10 =7.8; J4-3" =3.9; J4-3$ =11.2; J4-5 =11.2;
J7"-7$ =13.0; J11-2 =5.9. 28: J1-2 =12.0; J1-10 =5.0; J2-3α=3.0; J2-3β=12.0; J2-11 =6.0; J3α-3β=12.0; J3α-4=4.5; J3β-4=12.0; J4-5 =10.5; J7α-7β=13.0.
a
= interchangeable signals.

13
Table 9. C-NMR Data of Compounds 10, 28, 35-36, 38 and 47 in CDCl3

Carbon 10 38 47 35 36 28
(100MHz) (50 MHz) (100 MHz) (100MHz) (100MHz) (50 MHz)
1 58.2 (d) 47.9 (d) 52.8 (d) 56.1 (d) 53.2 (d) 55.6 (d)
2 32.1 (d) 33.7 (d) 28.0 (d) 32.3 (d) 31.2 (d) 32.6 (d)
3 40.7 (t) 38.5 (t) 36.6 (t) 38.6 (t) 39.0 (t) 43.1 (t)
4 72.3 (d) 72.6 (d) 71.6 (d) 72.3 (d) 72.2 (d) 69.5 (d)
5 62.2 (d) 61.0 (d) 63.2 (d) 60.5 (d) 61.4 (d) 64.5 (d)
6 41.5 (s) 39.6 (s) 38.5 (s) 40.0 (s) 39.2 (s) 40.0 (s)
7 49.9 (t) 49.9 (t) 50.6 (t) 49.0 (t) 50.1 (t) 49.0 (t)
8 44.5 (s) 54.3 (s) — 53.8 (s) 54.2 (s) 53.8 (s)
9 84.7 (s) 86.3 (s) 91.8 (s) 83.4 (s) 87.2 (s) 84.4 (s)
10 174.0 (s) 70.9 (t) 99.2 (d) 107.7 (d) 103.6 (d) 108.0 (d)
a
11 22.3 (q) 19.7 (q) 18.2 (q) 20.4 (q) 21.3 (q) 20.4 (q)
a
12 36.9 (q) 36.2 (q) 33.6 (q) 36.0 (q) 36.2 (q) 36.3 (q)
a
13 27.7 (q) 27.3 (q) 25.0(q) 27.4 (q) 27.3 (q) 27.3 (q)
a
14 23.9 (q) 20.0 (q) 25.1 (q) 20.1 (q) 29.3 (q) 20.4 (q)
15 76.7 (t) 173.5 (s) 170.4 (s) 173.9 (s) 174.3 (s) 173.9 (s)
CH 3 COO 22.0 (q) 21.4 (q) 21.3 (q) 21.4 (q) 20.6 (q)
CH 3 COO 169.1 (s) 170.4 (s) 170.4 (s) — 172.0 (s)
CH 3 O 57.4 (q) 57.3 (q) 57.0 (q)
a
= interchangeable signals.
1272 Current Organic Chemistry, 2000, Vol. 4, No. 12
OPP
HO O
10
15
H OH
11
1
9 +
5
OAc
Fig. (1). Hanson biosynthetic route to dihydrobotrydial.
The fate of the mevalonoid hydrogen atoms
provided mechanistic and stereochemical
information about this step. In the proposed
biosynthetic scheme, the rearrangement leads to
the appearance of a hydrogen atom at C-2, thus
generating the secondary methyl group. This could
take the form of two 1,2-shifts (H-9→C-1; H-
1→C-2) or a direct 1,3-shift (H-9→C-2). Feeding
experiments with labeled mevalonates were then
performed. The number of these labels
incorporated into 2 by the fungus was determined
with the aid of 3H:14C ratio studies while the
location of the labels was established by means of
2H NMR methods. The results showed that a 1,3-
hydride shift occurs during the cyclization; 18O
studies subsequently showed that the 9-hydroxy
group arises from water.
Labeling experiments with [2(R)-2-3H, 2-14C]
and [5(R)-5-3H, 2-14C]-mevalonate also provided
information about the formation of the hemiacetal
ring of 2. Oxidation of compound 2, previously
labeled in the [5(R)-5-3H, 2-14C]-mevalonate
feeding experiment, to the C-10 lactone showed
that the C-10 hydrogen atom was derived from the
pro-5(R) position of mevalonic acid. In contrast,
Collado et al.
+
+
H OH H
degradation to the 10,15-dicarboxylic acid ester
indicated that C-15 bore only one [2-3H]-
mevalonoid label, namely a pro-2(R)-hydrogen
atom. Deuterium labeling was used to distinguish
between the two enantiotopic hydrogen atoms at
C-15. The observed labeling pattern indicated that
the formation of the hemiacetal ring proceeded
with overall retention of configuration at both
centers.
A number of mechanisms may be proposed to
account for this. However, since 32% of the
dialdehyde 1 was incorporated into 2, whereas
only 1.09% of 2 was incorporated into 1 in the
reverse experiment, it seems most likely that the
cleavage of the 10:15 bond in the formation of the
hemiacetal occurs through the dialdehyde. This, in
turn, could be formed via a trans-15α,10β-glycol,
as oxidation of a trans-glycol would lead to a
dialdehyde. The subsequent reduction of the
aldehyde at C-15 proceeds with the “re”
stereospecificity typical of a microbial
dehydrogenase.
Recently a tricyclic alcohol 31 [26], a key
biosynthetic intermediate which had previously
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1273

+
+
OPP

H
+

HO HO OH
10 O
15
11 H OH H OH
H OH H
1
9 +
5

OAc OAc

Fig. (2). Biogenetic route to dihydrotrydial.

been proposed by Hanson [28] has been reported.
The isolation of this compound has shed further
light on the final steps of the biosynthesis of
botrydial and related compounds; in fact, a
proposal for these final steps has recently been
published [26] (Fig. (2)).
led to the recognition of 48 and (R)-49 as synthetic
precursors of 1 (Fig. (3)). Welzel et al. have
reported several approaches to 49, but the crucial
problem has, of course, been the generation of the
quaternary carbon center at C-2 by means of an
asymmetric process.

The synthesis of optically active 49 has been

Synthesis of the Botryane Skeleton
To our knowledge, little effort has been made to
synthesize the botryane skeleton. One approach
to the asymmetric synthesis of quaternary carbon
centers as synthetic precursors of 1 has been
reported [29]. Subsequent retrosynthetic analysis
accomplished by asymmetric formation of cyclic
β-keto esters fully substituted at the α-carbon,
followed by chemoselective reduction of the ester
group. Of the asymmetric reactions examined, the
Koga procedure [30] has proven to be the most
selective, providing a 7:93 mixture of the R,S cyclic

CHO CHO HO OH
H O
H H COOR
O
2

+
H CHO
OAc H
OAc
1 48 49 CH2 OH 2-Me
R β α
S α β
Fig. (3). Retrosynthetic analysis of botrydial.
1274 Current Organic Chemistry, 2000, Vol. 4, No. 12
O
O OCO
COCl
(R,S)-49
Fig. (4). Asymmetric synthesis of cyclicβ-keto ester fully substituted at the α-carbon.
β-keto ester (Fig. 4). The results obtained indicate
that the desired R-49 can be prepared with R-
valine as a chiral auxiliary.
Applying a Diels-Alder strategy to the
botryane family based on the cyclohexane 50
reduces the problem to a facile synthesis of diene
51, Fig. (5). The 1-vinylciclopentene 51 was thus
synthesized through the transition metal catalyzed
metathesis of enyne 52. This enyne is a substrate
of unprecedented steric demands in that it bears
quaternary centers adjacent to both the olefin and
acetylene. Contrary to normal expectations, which
predict a high sensitivity of transition metal
catalyzed reactions to steric effects, severe steric
congestion actually enhances the effectiveness of
the reaction. Thus, the palladium or platinum
catalyzed metathesis of enyne 52 led to 51 with
good yield (85% and 79 %, respectively) [31].
The presilphiperfolan-8β-ol (53), a key
intermediate in the biosynthesis of 1 from farnesyl
pyrophosphate, was isolated first from
Eriophyllum staechadifolia and Fluorensia
heterolepis by Bohlmann and associates in 1981
[32]. Its structure and absolute configuration were
CH3 O2 C OR
1
X
50
Fig. (5). Retrosynthetic analysis of botrydial with a Diels-Alder strategy.
Collado et al.
O O
O + O
OCO
O O
recently confirmed by X-ray crystallography and
chemical correlation [33].
The solvolysis of caryophyllen-8β-yl
derivatives has been investigated [34] as a
potential reaction model for the biogenesis of the
tricyclic presilphiperfolanol sesquiterpene, which
is the hydroxyl derivative of the tricyclic
carbocation intermediates (53a) in the biosynthesis
of botrydial (1).
Concurrent investigations of the biosynthesis of
dihydrobotrydial (2) in B. cinerea cultures led
Hanson et al. to propose the same rearrangement-
cyclization mechanism, shown in Fig. (1). The key
initiating step in the Bohlmann-Hanson biogenetic
pathway to 53 is the cyclobutylcarbinyl ring
expansion of the caryophyllen-8-yl ion, Fig. (6).
The products formed from caryophyllen-8-yl and
15-norcaryophyllen-8-yl carbocations having the
natural (4E) double bonds found in kinetically
controlled solvolysis reactions have been
investigated. While caryophyllen-8-yl p-
nitrobenzoate primarily undergoes solvolytic
elimination to β-caryophyllene, Fig. (7), solvolysis
of norcaryophyllen-8-yl tosylate leads to efficient
CH3 O2 C OR OR
CH3 O 2C
R=CH 2Ph
51 52
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1275

+
H H
H H H
+
+

OH
H H H H
+

53 53a

Fig. (6). Bohlmann-Hanson biogenetic pathway to 53.

ring expansion and cyclization to 12-nor-8α- changes may modulate the biological activities
presilphiperfolan-9β-ol (54), Fig. (8) [34]. considerably, and the absolute stereochemistry of

H H H

H2 O/acetone OH
+
+
125 oC
H HO
OpNB OH
(70%) (3%) (10%)

Fig. (7). Solvolysis of caryophellen-8-yl-p-nitrobenzoate.

Structure-activity Relationships of the dialdehydes, as well as their ability to form

Metabolites with Botryane Skeleton
Most saturated and unsaturated dialdehydes
possess potent bioactivities. For instance, they
may be antibiotic [35], fish toxic [36], antifeedant
[37] and, in certain cases, may even be natural
defense metabolites [37].
The biological activities of these compounds
have previously been linked to the dialdehyde
functionality [37]. However, small structural
bioactive autooxidation products during a
bioassay, have both been suggested to be of
importance [38].
In the case of 1 [17] and structurally related
compounds, the 1,5-dialdehyde functionality in
their structures seems to be responsible for their
bioactivity.
The results obtained by Collado et al. have
shown that these metabolites cause the typical

H OH

H2 O/acetone H H H

75 o C
H
OTs
H
54
Fig. (8). Solvolysis of norcaryophyllen-8-yl-tosylate.
1276 Current Organic Chemistry, 2000, Vol. 4, No. 12
necrotic lesions of the fungal infection and that
they play an important role in the pathogenicity of
the organism in vivo [15, 21]. In particular, 1 has
shown phytotoxic activity on tobacco leaves at a
concentration of only 1 ppm [15]. Likewise, this
compound has also displayed significant antibiotic
activity against Bacillus subtilis and Phytium
debaryanum at 400 and 100 ppm, respectively
[17].
During the course of a research project on new
metabolites from B. cinerea, three new epimers of
1 were isolated from the strain B. cinerea 2100
[16]: 8,9-epibotrydial (39), 1-epibotrydial (40) and
1,8,9-epibotrydial (41). Furthermore, metabolites
1 [17] and 2, 10, 24, 27, 32, 33 [17, 21, 22, 24]
were isolated in sufficient amounts to enable us to
perform phytotoxic, antibiotic and cytotoxic
assays. The results shed light on the relationship
between structural changes in the compounds and
the potency of their biological activities.
In order to better define this relationship,
compounds 10, 24, 27, 32, 33, 39-41 were tested
for phytotoxicity and compounds 1, 39-41 and 24,
27, 33 were tested for antibiotic activity. In
addition, 1 and 2, which are the major metabolites
from the fungus, were tested for cytotoxicity.
The results showed that compounds 10, 27 and
33 were inactive while compounds 24, 32, 39-41
displayed phytotoxic activity. The activities
shown by epimer derivatives 39–41 are diverse
and differ significantly from those of 1. While 1
showed high activity at a concentration of 30 ppm,
with the lowest effective concentration being
1 ppm [15], the 8,9-epimer derivative 39 was
active at 100 ppm, and compounds 40 and 41
displayed a minimal effective concentration at 250
and 750 ppm, respectively.
The results suggested that, in addition to the
presence of the dialdehyde functionality, the
phytotoxic activity exhibited by several of these
compounds are strongly correlated with the
stereochemical features of the C-1/C-8 dialdehyde
moieties. The configuration (S) at the C-1
substituent plays a critical role by interacting with
approaching nucleophiles and may thus affect the
binding of the substrate to the receptor site [37].
Collado et al.
The dialdehydes 24 and 32 were more active
than epimers 39-41 and slightly less active than 1.
Botryendial (32) and botrydienal (24) showed a
high activity at concentrations of 125 and 20 ppm,
respectively, with the minimal effective
concentration (MEC) being 25 ppm for 32 and
10 ppm for 24. Interestingly, these compounds
were active after only 24 h at all concentrations
bioassayed. It is important to note that 1 is easily
transformed into derivatives 24 and 32 after
treatment with oxalic acid [21], and quantitatively
transformed into 24 after treatment with Fetizon’s
reagent [39].
On the other hand, when dialdehyde 1 was
oxidized to the diacid, the corresponding dimethyl
ester was found to be inactive. Other methyl ester
derivatives of 1 have been described as non-
phytotoxic [21].
As described above, 1 had been reported to
show significant antibiotic activity while 2 is
inactive. This seems to be related with the
structural differences between these two
compounds. To confirm these data, compounds 1,
39-41 and 10, 24, 27, 33 were tested against
Bacillus subtilis. Compounds 1, 24 and 39 were
active with a minimum inhibitory concentration of
100, 150 and 400 ppm, respectively, while 40, 41
and 10, 27, 33 were not active. Curiously, a
correlation exists between phytotoxic and
antibiotic activities, as demonstrated by the fact
that compounds 1, 24 and 39 are more active as
phytotoxic and antibiotic agents.
In addition, compounds 1 and 2 were both
tested for cytotoxicity. Compound 1 showed high
activity in screens to detect in vitro cytotoxicity
against HUVEC endothelial cells of the umbilical
cord, ASJ-4 prepuce fibroblast, MDA-MB-231
breast adenocarcinoma, HT-1080 fibrosarcoma,
and HT-29 colon adenocarcinoma. The ID50 values
show that 1 is active to values equal to or lower
than 5 µg/ml against tumorous cells, but also that
it is highly cytotoxic against non-tumorous cells.
In contrast, in concentrations below 100 µg/ml,
compound 2 was not active against human cellular
lines ASJ-4, HT-1080 and MDA-MB-231, but it
was cytotoxic for the more sensitive lines HT-29
(ID50 = 40 µg/ml) and HUVEC.
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1277

It has been suggested that α,β-unsaturated
aldehydes react as Michael acceptors with
approaching nucleophiles, such as thiol or amine
groups of proteins, while 1,4-dialdehyde moieties
may form pyrrole derivatives with primary amino
groups [38]. Such pyrrole formation has only been
observed for molecules that have a stereochemical
configuration that permits intramolecular
cyclization [40].
A study using molecular models and molecular
mechanical calculations [41] revealed that
compounds 1 and 39, which showed the highest
activities, presented a similar spatial disposition of
carbonyl groups. In these compounds the formyl
groups are parallel whereas the formyl groups and
the oxygen of the tertiary hydroxyl groups must
be coplanar, as shown in Fig. (9). It can therefore
be concluded that both corresponding carbonyl
moieties at the C-1 and C-8 carbon atoms of
botrydial (1) and related derivatives should be in
close enough proximity to allow intramolecular
cyclization with thiol or amine groups of a
chemoreceptor.These results clearly indicate that
the biological activities of these compounds are
correlated with the oxidation state of the aldehyde
substituents at the C-1 and C-8 carbon atoms.
This is especially apparent when comparing the
structural differences between 1 and its analogous
compounds 2, 10, 27, 33, including the methyl
ester derivatives previously reported [21].
Curiously, compounds 24 and 32, which as
H O
H 3C OH O
H 3C
AcO
H 3C
CH 3
1 39
unsaturated aldehydes should react as Michael
acceptors [38, 42], are less active than the
saturated aldehyde 1. Still, they are more active
than epimers 39-41.
Differences in the biological activities of
compounds 1 and 41 thus support the idea that
bioactivity is strongly correlated with the
stereochemistry of substituents at the C-1, C-8
and C-9 carbon atoms. In particular, the relative
configuration (S) of the C-1 substituent seems to
play a critical role in binding the substrate to the
chemoreceptor.
METABOLITES WITH POLYHYDROXI-
LATED LACTONE SKELETON
In earlier work directed toward the isolation of
metabolites from B. cinerea, in addition to 1 and 2,
a new antibiotic metabolite, namely botrylactone
(55), was isolated by Tschesche et al. [43] from
the culture broth.
Information about the structure of 55 was
obtained from mass spectrometry and 1H- and 13C-
NMR spectroscopy. The absolute configuration
was elucidated by means of X-ray diffraction of
the acetate derivative [43].
Botrylactone 55 is a natural product with
strong antibiotic properties as well as an
interesting chemical structure from the
H CH 3
H CH3
H AcO H
H 3C
H
OH O
H 3C O

OH O H CH 3 O H
O
AcO H
CH3
H3 C H AcO
H 3C H 3C OH H

H 3C O
40 41

Fig. (9). Comparision of the spatial arrangement of carbonyl and hydroxyl groups in compounds 1, 39-41.
1278 Current Organic Chemistry, 2000, Vol. 4, No. 12 Collado et al.

From the malt-yeast medium, p-

HO
O metabolites of B. cinerea cultured on the Czapeck-
O O
55
biosynthetic perspective. In order to study the
biosynthesis of 55, Suga et al.[44], cultured the
fungus with a malt medium following the method
described by Tschesche [43], but compound 55
could not be found in the culture. Further attempts
to grow the fungus on different media, such as
malt-peptone, malt-yeast and Czapeck-Dox media
hydroxybenzaldehyde (63) was isolated. The
O Dox medium were similar to those obtained from
the cultures grown on the malt-peptone medium.
Recently, a convergent approach for the synthesis
of botrylactone (55) from D-glucose was
published [46]. The synthesis was carried out with
an overall yield of 0.74% and a diastereoselectivity
of >95%. However, the resulting synthetic
compound was not identical with the natural
product. The carbon atom C-10, which bears the
OH-group in the natural product 64, was shown to
have S-configuration, with the hydroxyl group in a
pseudoequatorial position. Thus, the originally

CHO
OH O

OH
OH O
HO 57
56 O 63

R (CH2 )6 COOH
CH3 (CH 2)n COOH
n

58: R = (CH 2)3 CH 3; n = 2 60: n=14

59: R = (CH 2)6 CH 3; n = 1 62: n=16
61: R =CH 3; n =3

in order to obtain 55 were fruitless. Although published structure 55 must be renamed 10-epi-
compound 55 was not obtained, a significant botrylactone. Botrylactone (64) is a tricyclic ring
number of metabolites were isolated from the system and must therefore be named
different media. (1S,2S,5S,6R,7R,9R,10S)-10-hydroxy-(1,2,5,7,9)-
pentamethyl-3,11,12-trioxa-tricyclo[5.3.1.12.6]do-
For example, isosclerone (56), a metabolite that decan-4-one.
had previously been isolated from another fungus
[45], was obtained from the chloroform soluble
fraction of the broth when the fungus was grown HO
on the malt-peptone medium. An ethyl acetate O
soluble fraction of the mycelia afforded ergosterol O O O
peroxide (57). The fatty acid fraction was
analyzed by means of GLC and showed the
presence of linoleic acid (58) (50%), oleic acid (59) 64
(28%), plasmatic acid (60) (13%), linolenic acid
(61) (5%) and stearic acid (62)
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1279

H3 C O OH H3 C OH
O OBzl OBzl H3 C
H 3C 5 OH
HO O CH 3 O
7
O OH
HO 1 3 BzlO
H3 C O 9 O
O CH3 CH3 CH 3 CH3 CH3
CH3 CH 3 CH3 CH3

64 69

HO
O O O

TBDMSO R
O
CH 3 O CH3 O
66 R = H
O O
O
TBDMSO 9 7 CH3
5 O
11 O O
HO R 1
R'
CH 3 HO OH
3
O O
HO
H 3C H3 C O O
O O
67 R = H OH
68R = CH3 OH
O
O
O
O

65 R' = S S

Fig. (10). Retrosynthesis of botrylactone.

Key steps in the convergent synthesis of 64 are identical with those described by Tschesche and
outlined in Fig. (10): co-workers [43].

a) the addition of the dithiane 65, which In the course of a screening program to discover
represents C-5 to C-10 of the target natural products with novel biological activities
molecule, to the aldehyde 66 (C-1 to C-4). from microorganisms, a strain of Botrytis cinerea
from raspberry fruit was found to produce a new,
b) stereoselective formation of the tertiary highly substituted lactone, which has been trivially
hydroxyl group at C-7 (67→68), named botcinolide (70) [47,48].
c) stereoselective creation of the
tetrahydropyran ring (68→69), a crucial H
problem because of the two quaternary α- HO 17
O

carbon atoms C-3 and C-7, and HO 4

2
H O
H H
8
d) formation of the tricyclic botrylactone by 18
HO
20

intramolecular acetalization (3-OH→C-9), 6 O H

19
followed by lactonization with participation H 9

of the newly formed acetalic center. O 16

OH
Following this synthetic scheme, botrylactone 70
64 was prepared in excellent yields. The 1H NMR
and 13C-NMR data of synthetic 64 are completely
1280 Current Organic Chemistry, 2000, Vol. 4, No. 12 Collado et al.

H O
O H
R1 R1
R2 HO O
O
H
HO
R3 O
O H
H R2
H R4
O OH
O OH

71 R1 =OH; R2 =OMe; R3 =OH; R4 =(CH2 )3 CH3 73 R1 =OH; R2 =(CH2 )3CH 3

72 R1 =OAc; R2 =OH; R3 =OMe; R4 =(CH2 )3 CH3 74 R1 =OAc; R2 =(CH2 )3CH 3
75 R1 =OH; R2 =OH; R3 =OH; R4=(CH 2 )5CH 3

Botcinolide (70) is a hydroxylated nonalactone However, 70 was inactive in limited antibacterial

that is esterified with 4-hydroxy-2-octenoic acid. and antifungal tests [49].
It significantly inhibited etiolated wheat coleoptile
growth at 10-3 and 10-4 M by 100 and 82%, Preliminary toxicological studies with mice
respectively, relative to controls. In addition, indicate that the acute toxicity of 70 is relatively
greenhouse-grown bean, corn, and tobacco plants low [48], thus making it an interesting candidate
were affected by treatment with 70, exhibiting for development as a biodegradable contact
chlorosis and severe necrosis at 10-2 and 10-3 M. herbicide [49].

1
Table 10. H-NMR Data of Compounds 70-75
Proton 70 71 72 73 74 75
(400 MHz, CD3 OD) (400 MHz, C6 D 6 ) (400 MHz, C6 D 6 ) (400 MHz, CD3 OD) (400 MHz, CDCl3 ) (300 MHz, CD3 OD)
2 2.74 (dq) 2.68 (dq) 3.09 (m) 3.10 (m) 3.15 (dq) —
3 3.57 (d) 3.49 (d) 4.97 (d) 3.97 (d) 5.40 (d) —
5 3.78 (d)) 3.87 (d) 3.09 (dd) 3.83 (d) 3.80 (d) —
6 1.87 (m) 1.89 (m) 1.90 (m) 2.10 (m) 2.18 (ddq) —
7 4.33 (br t) 4.72 (dd) 4.40 (dd) 4.40 (dd) 4.52 (dd) 4.34 (br t)
8 3.60 (m) 3.55 (dq) 3.56 (dq) 3.66 (dq) 3.67 (m) —
10 6.04 (dd) 6.18 (d) 6.03 (bd) 5.94 (dd) 6.05 (bd) 6.03 (dd)
11 6.98 (dd) 7.04 (dd) 6.70 (dd) 7.91 (dd) 7.00 (dd) 6.98 (dd)
12 4.24 (m) 3.77 (m) 4.32 (m) 4.13 (m) 4.33 (m) 4.24 (m)
13 1.54 (m) 1.82 (m) 1.60 (m) 1.45 (m) 1.58 (m) 1.54 (m)
14 1.40 (m) — — 1.25 (m) 1.34 (m) —
15 1.40 (m) — — 1.25 (m) 1.34 (m) —
16 0.92 (t) 0.77 (t) 0.89 (t) 0.81 (t) 0.93 (t) —
17 1.32 (d) 1.30 (d) 1.20 (d) 1.02 (d) 1.10 (d) —
18 1.23 (s) 1.09 (s) 1.33 (s) 1.06 (s) 1.25 (s) —
19 0.97 (d) 1.05 (d) 0.97 (d) 0.90 (d) 1.05 (d) —
20 0.99 (d) 1.09 (d) 1.01 (d) 0.96 (d) 1.08 (d) —
COCH 3 2.20 (s) 2.11 (s)
OCH 3 3.29 (s) 3.64 (s)

J (Hz): 70: J2-3 =2.3; J2-17 =7.1; J5-6 =10.8; J6-7 ∼10.0; J6-19 =4.9; J7-8 =10.8; J8-20 =4.7; J10-11 =15.6; J10-12 =1.6; J11-12 =4.8; J15-16 =7.1. 71: J2-3 =1.9; J2-17 =7.1;
J5-6 =11.0; J6-7 =10.2; J6-19 =6.3; J7-8 =9.6; J8-20 =6.2; J10-11 =15.6; J11-12 =4.4; J15-16 =7.0. 72: J2-3 =3.8; J5-6 =10.5; J6-19 =6.2; J7-6 =10.7; J7-8 =9.6; J8-20 =6.2;
J10-12 =1.7; J11-10 =15.6; J11-12 =4.7; J15-16 =7.1; J17-2 =7.3. 73: J2-3 =9.5; J2-17 =7.1; J5-6 =11.0; J6-7 =10.5; J6-19 =6.3; J7-8 =9.6; J8-20 =6.1; J11-10 =15.6; J11 -

12 =4.6; J10-12 =1.9; J15-16 =5.0. 74: J2-3 =9.6; J2-17 =7.1; J5-6 =10.9; J6-7 =10.7; J6-19 =6.2; J7-8 =9.6; J8-20 =6.2; J10-12 =1.7; J11-10 =15.6; J11-12 =4.7; J15 -
16 =7.1. 75: J7-6 =J7-8 =9.6; J10-11 =15.6; J10-12 =1.6; J11-12 =4.8.
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1281

Recently, four new metabolites with a
botcinolide skeleton have been isolated from the B.
cinerea (strain UCA 992) and characterized [50].
These are 4-O-methylbotcinolide (71), 3-O-acetyl-
5-O-methylbotcinolide (72), 2-epibotcinolide (73)
and 3-O-acetyl-2-epibotcinolide (74). Their
structures have been established by means of
extensive spectroscopic methods (see Tables 10
and 11).
wheat coleoptile growth. Greenhouse-grown bean,
corn and tobacco plants were also affected by
exogenous applications of 75, with severe
chlorosis and necrosis being exhibited in corn [52].
In the wheat coleoptile bioassay compound 75
was an order of magnitude more inhibitory.
Curiously, compound 75 was not phytotoxic in
beans as compared to 70, although it produced the
same growth inhibition of the first internode.

The isolation of these compounds from the acid The relative stereochemistry of compound 75,
fraction suggested that the lactone ring is readily which is a polyhydroxylated nonalactone esterified
cleaved and formed again. However, we were with 4-hydroxy-2-decenoic acid, is identical to that
unable to isolate any hydrolysis products, of 70, based on the results reported [47, 48]. A
probably because of their instability. comparison of the spectroscopic data of 70 and 75
indicated great similarity between the structures of
Homobotcinolide (75), a biologically active the two compounds (see Tables 10 and 11).
natural homologue of 70, has recently been Homonuclear and heteronuclear correlation studies
reported [51] as significantly inhibiting etiolated confirmed the presence of partial structure R1-
13
Table 11. C-NMR Data of Compounds 70-75
Carbon 70 71 72 73 74 75
(100 MHz, CD3 OD) (100 MHz, C6 D 6 ) (100 MHz, CDCl3 ) (100 MHz, CDCl3 ) (100 MHz, CDCl3 ) (75 MHz, CD3 OD)
1 180.1 (s) 176.5 (s) a a a 180.0 (s)
173.9 (s) 173.2 (s) 173.8 (s)
2 39.7 (d) 38.5 (d) 38.8 (d) 38.4 (d) 37.3 (d) 39.3 (d)
3 77.6 (d) 77.5 (s) 78.3 (d) 74.1 (d) 74.2 (d) 77.7 (d)
4 79.9 (s) 79.1 (s) 77.9 (s) 76.3 (s) 75.2 (s) 79.9 (s)
5 72.4 (d) 71.8 (d) 72.1 (d) 78.5 (d) 78.5 (d) 72.5 (d)
6 39.3 (d) 38.4 (d) 37.0 (d) 35.6 (d) 35.4 (d) 39.5 (d)
7 78.4 (d) 77.8 (d) 76.6 (d) 77.2 (d) 76.0 (s) 78.5 (d)
8 69.3 (d) 68.5 (d) 68.1 (d) 68.5 (d) 68.4 (d) 69.3 (d)
9 167.7 (s) 165.8 (s) 165.9 (s) — 165.9 (s) 167.7 (s)
10 120.1 (d) 119.7 (d) 119.5 (d) 119.2 (d) 119.0 (d) 120.2 (d)
11 153.6 (d) 151.7 (d) 151.2 (d) 151.7 (d) 151.8 (d) 153.5 (d)
12 71.5 (d) 70.9 (d) 71.1 (d) 71.1 (d) 71.1 (d) 71.6 (d)
13 37.2 (t) 36.5 (t) 36.3 (t) 36.4 (t) 36.3 (t) 37.5 (t)
14 28.6 (t) a b b b 32.9 (t)
27.6 (t) 27.3 (t) 27.3 (t) 27.3 (t)
15 23.6 (t) a b b b 30.3 (t)
22.8 (t) 22.5 (t) 22.5 (t) 22.5 (t)
16 14.3 (q) 14.1 (q) 13.9 (q) 13.9 (q) 13.9 (q) 26.4 (t)
17 17.4 (q) 17.2 (q) 14.9 (q) 10.3 (q) 10.2 (q) 23.6 (t)
18 14.9 (q) 14.5 (q) 14.2 (q) 10.9 (q) 11.9 (q) 14.4 (q)
19 14.7 (q) 14.2 (q) 16.1 (q) 13.7 (q) 13.7 (q) 17.8 (q)
20 18.1 (q) 18.4 (q) 17.9 (q) 18.2 (q) 18.2 (q) 15.0 (q)
21 14.7 (q)
22 18.2 (q)
COCH 3 20.9 (q) 20.6 (q)
COCH 3 a a a
172.9 (s) 170.1 (s) 170.1 (s)
OCH 3 50.9 (q) 51.5 (q)
a, b
= interchangeable signals
1282 Current Organic Chemistry, 2000, Vol. 4, No. 12
C13H2-C12H(OH)-C11H=C 10H-R2 (a trans double
bond). The additional two methylene signals in the
NMR spectrum of 75 were assigned to an
extended side chain, yielding a structure
homologous to that of 70. The remaining
unassigned carbon signals in the spectrum of 75
(C-1 to C-8 and C-19 to C-22) very closely
matched those of the polyhydroxylated
nonalactone moiety of 70.
A comparison of the structures of botrylactone
(64) [46] and botcinolide (70) [47] suggests a
possible biogenetic relationship between the two.
Compound 70 has a unique structure incorporating
a nine-membered ring lactone, a rare feature in
natural products. An interesting and possibly
fortuitous aspect of the structure of the nine-
membered ring is that it can be dissected into four
propionate units. The elucidation of the
biosynthetic pathway to reveal the addition of
either octenoic acid or decenoic acid as the acyl
chain would be of even further significance. In any
event, synthesis of related homologues should
yield products with interesting biological activities.
STEROL METABOLITES FROM BOTRYTIS
Published reports on the sterol composition of
B. cinerea [53-56] are contradictory and agree only
in that ergosterol is the principal sterol in this
plant pathogenic fungus. B. cinerea was initially
reported [54] to contain fecosterol (76), episterol
(77), eburicol (78) and small amounts of other
methylated sterols, while a later study [55]
indicated the presence only of ergosta-5,8,22-
trienol (79), eburicol (78), ergost-5-enol (80),
HO
H H
R1 R2 76 R1 =R2 =H
86 R1 =H; R2 =CH 3
87 R1 =R2 =CH 3
Collado et al.
stigmasterol (81) and sitosterol (82), and ergosterol
(83). It was subsequently reported [56] that
ergosterol (83), episterol (77) and zymosterol (84)
were the only sterols in B. cinerea. Since then,
however, all the sterols of mycelia of B. cinerea
grown in liquid culture have been studied [53] and
the results of the earliest report [54] concerning
the sterol composition of the fungus were more or
less confirmed. The major exceptions were that an
ergostatetraenol, ergost-8-enol (85), as well as 4-
methylfecosterol (86) and 4,4-dimethylfecosterol
(87) were also isolated. Compounds 80, 81, and 84
were absent, although traces of 82 were presumed
to be present from mass spectral data.
These results indicate that the plant pathogenic
fungus B. cinerea produces only sterols bearing a
C9 side-chain (ergostane skeleton) [54].
MISCELLANEOUS METABOLITES
Overeem and Van Dijkman [57] observed that
during the growth of Botrytis allii a zone containing
small yellow spots is formed in the agar medium.
The substance responsible for the yellow color
was isolated. The compound, botrallin (88), was
shown to be a benzoquinone substituted with a
methyl, a methoxyl and a 2-carboxy-3-hydroxy-5-
methoxy-phenyl group. Compound 88, in a
concentration of 100 ppm, had no inhibitory effect
on the growth of the fungi B. allii, Penicillium
italicum, Cladosporium cucumerinum, or
Aspergillus niger. In addition, no effect was
observed on Escherichia coli or Bacillus subtilis.
Since Botrytis allii is a parasite of onions, tests
were performed to determine whether 88 had a
HO
77
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1283

HO 78 HO 79

80 81
HO HO

HO 82 83
HO

HO
H 84 85
HO

toxic effect on onion scales. No effect was
observed.
Sporogenic Metabolites
Many authors have considered it worthwhile to
perform in-depth studies on the structure of the
conidium and its processes of formation and
germination; in recent years, a number of papers
have been published on this subject [2]. The factor
most frequently studied has been the effect of light
in stimulating the formation of the conidia. The
results of such studies indicate that light radiation
in the near UV band and the distant red band
stimulate sporulation, while blue and red light
inhibit it [58].
As an explanation for this behavior in B.
cinerea, two models have been proposed based on
the existence of a photoreceptor [59] called
mycochrome, which is involved in a process of
1284 Current Organic Chemistry, 2000, Vol. 4, No. 12 Collado et al.

HO CO 2H O O O
OMe OMe
OMe
HO CH2 OH HO CH2 OH
MeO Me O HOH2 C NH HOH2 C N
CH2 OH

88 89 90 O

photoinduction leading to the formation of the
conidium. Satisfactory results regarding the nature
of the photoreceptors involved have not been
obtained, although some progress has been made in
the study of the presence of chemical substances
with sporogenic activity. Leach [60], working with
cultures of Ascochyta pisi, isolated a substance
called P310, which absorbs UV light in the 260-340
nm range with an absorption maximum at 310 nm.
Favre-Bonvin elucidated the structure of this
substance, which had been isolated from material
extracted from Stereum hirsutum, and designated it
mycosporine 1 (89) [61]. In addition, Favre-
Bonvin isolated a new metabolite from B. cinerea,
namely mycosporine 2 (90) [62].
Marumo et al.[63] have reported the microbial
production of abscisic acid (ABA), a well-known
plant hormone, from B. cinerea. Irradiating the
fungus with blue light during the culture
remarkably enhanced the production of ABA;
which may play an important role in spore
formation and/or germination processes.
During the search for biologically active natural
products from fungi that possessed plant growth
regulatory activity, Cutler et al.[64] isolated the
compound cinereain (91) from a strain of B.
cinerea found on the surface of sunflower seeds.
O bioassay.
O
N
HN
N O
91
Compound 91 significantly inhibited the growth of
etiolated wheat coleoptiles at 10-3 and 10-4M in
bioassays. No effects were noted on treated intact
greenhouse-grown bean and tobacco plants, but
there was mild necrosis and chlorosis in corn.
Furthermore, Botrytis sp. isolated from the
inner bark of the Pacific yew, Taxus brevifolia,
was shown to produce ramulosin (92), 6-
hydroxyramulosin (93) and the new compound 8-
dihydroramulosin (94). The structure of 94 was
deduced from its NMR spectra and confirmed by
chemical conversion from (92) [65].
OH O OH O
H
8
O O
6
R
H H
92 R = H 94
93 R = OH
The fungus Botrytis sp. was grown in
mycological broth for 21 days and extracted with
CH2Cl2. The resulting extract had strong antibiotic
activity. It was then fractionated and the antibiotic
activity of the resulting fractions was monitored.
This process yielded the three aforementioned
antimicrobial compounds 92-94, which showed
moderate antimicrobial activity in the disk
ACKNOWLEGEMENT
This research was supported by grants from
1FD97-0668-C06-01 and FAIR 5-PL97-3361.
Botrytis Species Current Organic Chemistry, 2000, Vol. 4, No. 12 1285

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