Botrylactone: New interest in an old molecule - Review of its absolute configuration and related compounds

Article abstract of DOI:10.1016/j.tet.2010.11.022

The absolute configuration of botrylactone, a unique compound with an interesting polyketide lactone skeleton with two oxirane bridges previously isolated from Botrytis cinerea and described as a powerful antibiotic, has been reviewed on the basis of sign of the optical rotation, NOE experiments and NMR method. The isolation of 7-deoxybotrylactone and 5-hydroxy-7-(4-hydroxydec-2(3)- enoyl) botrylactone enables us to characterize an intriguing new family of compounds with this interesting polyketide skeleton. A common biosynthetic origin with botcinin derivatives is proposed. Copyright

Full text of DOI:10.1016/j.tet.2010.11.022

Tetrahedron 67 (2011) 417e420
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Botrylactone: new interest in an old moleculedreview of its absolute
configuration and related compounds
*
ꢀ
ꢀ
ꢀ
ꢀ
Javier Moraga, Cristina Pinedo, Rosa Duran-Patron, Isidro G. Collado, Rosario Hernandez-Galan
ꢀ
ꢀ
Departamento de Química Organica, Facultad de Ciencias, UCA, Polígono Rio San Pedro s/n, 11510 Puerto Real, Cadiz, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The absolute configuration of botrylactone, a unique compound with an interesting polyketide lactone
skeleton with two oxirane bridges previously isolated from Botrytis cinerea and described as a powerful
antibiotic, has been reviewed on the basis of sign of the optical rotation, NOE experiments and NMR
method. The isolation of 7-deoxybotrylactone and 5-hydroxy-7-(4-hydroxydec-2(3)-enoyl) botrylactone
enables us to characterize an intriguing new family of compounds with this interesting polyketide
skeleton. A common biosynthetic origin with botcinin derivatives is proposed.
Received 9 September 2010
Received in revised form 3 November 2010
Accepted 4 November 2010
Available online 11 November 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
metabolites produced by it. Additionally, in the course of our in-
vestigation on biotransformation with a wild strain of B. cinerea (UCA
Botrytis cinerea is a well-known pathogen affecting a number of
commercial crops, which produces several structurally unique
metabolites. Botrytis produces two series of phytotoxic metabolites:
a family of characteristic sesquiterpene metabolites with the basic
botryane skeleton, principally botrydial and dihydrobotrydial and
a family of polyketide lactones. The first isolated polyketide was
reported by Cutler in 1993 who called it botcinolide (1), proposing
a highly hydroxylated nonanolactone structure.1 Based on botci-
nolide structures and in addition to other botcinolide metabolites,
we described2 new metabolites and investigated the biosynthesis
of the botcinolide skeleton.3
In 2005, Nakajima’s group reported the isolation of a group of
antifungal metabolites, which they designated as botcinins.4 The
absolute configuration of botcinin A (2) was determined through
the modified Mosher method. A careful reinvestigation of the
spectroscopic data reported for botcinolide analogues allowed
them to revise the structures of botcinolide derivatives to botcinic
(3) and botcineric (4) acids and their cyclized derivatives, botcinins
AeF.5 Ultimately, the revised structures of this group of natural
products were unequivocally determined by total synthesis6 and
a revision comparing the botcinolides with their corresponding
botcinin structures has been reported.7
992), a newmetabolitewithabotrylactoneskeletonwasisolated. This
paperfocusesonthecharacterizationandbiosyntheticrouteproposal
ofanintriguingnewfamilyofcompounds, botrylactones,whichcould
be an intermediate in the biosynthesis to botcinins. Additionally, the
absolute configuration of botrylactone (6) and the structure of 2-
epihomobotcinolide (5) are revised.
2. Results and discussion
Botrylactone is a unique C-9 polyhydroxylated lactone described
as a powerful antibiotically active compound reported by Welmar
et al.9 Several unsuccessful attempts to isolate this intriguing
compound have been made and some of them reported.10 Recently
we isolated it from the B. cinerea cat 2 strain.3
Isolation of botrylactone together with botcinins,3 and the
similarity of their spectroscopic data, led us to believe that they are
closely related metabolites. However, comparison of the stereo-
chemistry proposed for botrylactone (6) and that proposed for
botcinin A (2) turned out be diametrically opposed. These data,
together with the overproducer mutant
conduct a study of 6 focusing on botcinic acid derivatives.
The Botrytis cinerea mutant, bcbot2, was cultured on malt agar
Dbcbot2, prompted us to
D
Recently, a genetically modified strain ofB. cinerea, Dbcbot2, which
medium at 24e26 ꢀC for 7 days. The fermentation broth, after fil-
tration and extraction was purified following the methodology
described in the Experimental section. In addition to botcinin A (2)
and B (7), (þ)-botrylactone (11) and 3-acetylbotcineric acid (8),
a metabolite whose spectroscopic and physical constants were
identical to those described for 2-epihomobotcinolide (5)3 and
a new compound with a botrylactone skeleton, 5-hydroxy-7-(4-
hydroxydec-2(3)-enoyl)botrylactone (12), were isolated.
is unable to produce botryanes but significantly overproduces bot-
cinic acid (3) and its derivatives was constructed.8 The higher pro-
duction capacities of this strain prompted us to reinvestigate the
* Corresponding author. Tel.: þ34 956016371; fax: þ34 956 016193; e-mail
ꢀ
ꢀ
address: rosario.hernandez@uca.es (R. Hernandez-Galan).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.11.022

418
J. Moraga et al. / Tetrahedron 67 (2011) 417e420
A careful spectroscopic study of the physical constants of 5 and
signals of H-6, C6eMe, C4eMe and H-5a. A quasi-boat conformation
its comparison with those of botcinin derivatives described in the
literature showed that the 1H NMR and 13C NMR of 5 were very
similar to those of botcinin E (9).5 The 13C NMR, including DEPT
data, showed that 5 has two more methylenes than 9. The fragment
ion assignable to the fatty acyl portion was detected at m/z 169,
which is characteristic of a C10H16O. Thus, 5 differ from 9 in the
length of the acyl portion. The key NOE correlations were identical
to those of 9 indicating that 5 and 9 share the same relative ste-
reochemistry. Therefore, the structure of 2-epihomobotcinolide (5)
should be revised to 10 and renamed as botcinin G.
was established for pentanolide ring on the basis of NOE enhance-
ment of C2eMe, C4eMe and H-3 observed when H-2 was irradiated.
These data coincide with a configuration where H-2, C4eMe and
C8eMe were on the
the
was definitively established as 2R, 3S, 4S, 6S, 7R, 8R, 9R.
b face and H-3, H-7, C6eMe and C9eCH3 were on
. The absolute stereochemistry of natural botrylactone, (þ)- 11,
a
Compound 12 was isolated as an oil whose molecular formula
was established as C24H38O8 by HRMS and 13C NMR data requiring 6ꢀ
of unsaturation. Its 1H NMR spectrum was very similar to that of
botcinins however the presence of a signal in 13C NMR at
Botrylactone was reported for the first time by Welmar et al.9
who initially proposed structure 6a based on spectroscopic data.
Although they used the term, ‘absolute configuration’ in the text of
the paper, they did not determine the absolute configuration but its
relative configuration by X-ray diffraction of its acetate derivative
(6b).9 Later, Redlich et al. synthesized it and revised the originally
d 104.2 ppm, attributable to a ketal group plus an additional methyl
group, should correspond to an additional C2 unit resulting in
a structure similar to that of botrylactone (11). However, the 1H NMR
showed signals at H 7.00, 6.07 and 4.33 ppm characteristic of the fatty
acyl portion on C-7 of botcinins. The fragment ion detected at m/z
169 was characteristic of a fatty acyl chain with molecular formula
C10H16O. The HMBC experiment performed on 12 showed correla-
tions between the quaternary ketalic carbon signal (dC 104.2 ppm),
two methyl singlet groups (dH 1.53 and 1.10 ppm), which were fur-
ther correlated with a quaternary oxygenated carbon at dC 79.2 ppm
and two signals at dH 4.93 and 3.52 ppm attributable to H-7 and H-3,
respectively (Fig. 2). H-3 was further correlated with signal at dC
171.0 ppm corresponding to carbon C-1 while H-7 exhibited corre-
lationwith the carbon signal at dC 165.4 ppm corresponding to the C-
1 of the fatty acyl portion. Additional correlations were observed
between carbon C-3 (dC 80.9 ppm) and two methyl groups at dH 1.46
(d) and 1.14 (s) ppm. The COSY experiment showed correlations
betweenthe signal doubletat dH 4.93 ppm(H-7) anda multiplet atdH
1.86 ppm, which was further correlated with two doublets at dH
3.67 ppm and 1.02 ppm, pointing to the presence of a fragment
OeCHeCH(CH3)eCHeO in the molecule. All these data are consis-
tent with a structure of 5-hydroxybotrylactone bearing a fatty acyl
chain on C-7 for this compound. The stereochemistry was de-
termined by N.O.E experiments and was consistent with the ste-
reochemistry assigned to compound 11 confirming the structure of
2R, 3S, 4S, 5S, 6S, 7R, 8R, 9R-5-hydroxy-7-(4-hydroxydec-2(3)-enoyl)
botrylactone for compound 12.
published structure to 7-OHa
-botrylactone (6).11
The optical rotation of natural botrylactone had not been pre-
viously reported. Welmar et al. prepared acetyl botrylactone and
reported its optical rotation as þ88 (c 1, CHCl3),9 while Redlich et al.
described it for synthetic botrylactone (6) as L31 (c 0.14, MeOH).11
The isolation of a sufficient amount of natural botrylactone from
Dbcbot2 has enabled us tomeasureits optical rotation determined as
þ19 (c 2.6, CHCl3), the opposite sign of that described for the syn-
thetic compound. Acetylation of it led us to a product whose spec-
troscopic data were identical to those described for natural acetyl
botrylactone whose optical rotation proved to be þ73 (c 2.4 mg,
CHCl3), coinciding with the sign described by Welmar et al.9 As
a result, it can be inferred that the structure synthesized by Redlich
et al. should be the enantiomeric to the natural compound,
(ꢁ)-botrylactone (6). In order to confirm this hypothesis we de-
termined the absolute configuration of natural (þ)-botrylactone
using the NMR method. (þ)-Botrylactone was treated with R (ꢁ) and
S (þ)-MPA acid yielding the corresponding R and S-MPA esters 11a
and 11b, respectively. Comparison of the chemical shifts in 1H NMR
spectra of the two compounds showed a negative DdRS value for
C8eCH3 and C9eCH3, (ꢁ0.57 and ꢁ0.26 ppm, respectively), while the
DdRS for H-6 and C6eCH3 were positive (0.11 and 0.47 ppm). Appli-
cation of the Mosher rule12 showed a 7R configuration for ester-
ificated products 11a and hence for (þ)- botrylactone (11).
The stereochemistry of the rest of the chiral carbons was con-
firmed on the basis of NOE data (Fig. 1). Irradiation of the H-7 signal
of compound 11a produced NOE enhancement of H-5a, C6eCH3 and
C9eCH3, establishing a boat conformation for this tetrahydropyran
ring, which was confirmed by irradiation of H-5b that enhanced the
Fig. 2. Selected HMBC-COSY correlations for 12 and 13.
Furthermore, during the course of our biotransformation ex-
periments with B. cinerea UCA 99213 we found a new compound 13
with an NMR pattern very similar to that of (þ)-botrylactone (11),
the principal difference being the absence of the characteristic H-7
signal in its 1H NMR spectrum and one of the carbon bearing to
oxygen and the presence of a signal corresponding to one more
methylene in the 13C NMR spectrum. Therefore, 13 differ from 11 in
the absence of the hydroxyl group on C-7. HRMS confirms this
showing an ion assignable to C13H22O3 [MꢁCO]þ at m/z 226.1573.
The botrylactone skeletonwas confirmed by the 2D NMR datawhere
correlation in the HMBC experiment between the characteristic
Fig. 1. NOEs interactions in 11a.

J. Moraga et al. / Tetrahedron 67 (2011) 417e420
419
quaternary carbon signal (dC 104.8 ppm) and signals corresponding
to H-3, H2-7 and two methyl singlet groups, which were further
correlated with C-8 were observed. The key NOE correlations
were identical to those of 11a, indicating that 11 and 13 share the
same relative stereochemistry. On the basis of these data the
structure of 13 was suggested to be 2R, 3S, 4S, 6S, 8R, 9R-7-
dehydroxybotrylactone.
The occurrence of botrylactone derivatives and botcinins in the
same strain, their structural homology, the resulting absolute
configuration of natural botrylactone (11) therefore being identical
to that assigned to botcinins by Nakajima4 except in C-8, seems to
indicate that both compounds may be biogenetically related.
Incorporation studies with 13C and 2H-labelled precursors con-
ducted by our group3 indicated that 3-O-acetylhomobotcinolide
(14) was an acetate derived polyketide whose methyl groups
originate from C-methyl methionine. This conclusion is applicable
to its revised structure, 8, but an important question, the presence
of the methyl group derived from C-methyl methionine on the C-8
carbon atom, the alleged carbon two of the starter-acetate unit, has
yet to be resolved.
column chromatography. TLC was performed on Merck Kiesegel 60
254, 0.25 mm thick.
F
4.2. Microorganism
B. cinerea mutant strain, bcbot2D, was supplied by Dr. Muriel
Viaud of the UMR BIOGER, INRA (Versailles, France). The strain was
maintained viable on mycelia discs of 0.5 cm diameter submerged
in 80% glycerol at ꢁ40 ꢀC.
B. cinerea (UCA 992) was obtained from grapes from Domecq
vineyard, Jerez de la Frontera, Cadiz, Spain. This culture is deposited
at the Universidad de Cadiz, Facultad de Ciencias Mycological
Herbarium Collection (UCA).
4.3. Culture conditions
bcbot2D was grown on malt agar medium (20 g of D-glucose,10 g
of malt extract, 20 g of agar, pH 6.57 per liter of water) at 25 ꢀC and
used to inoculate Roux bottles or Erlenmeyer flasks. For surface
culture, mycelium was grown in 1 L Roux bottles containing 150 mL
of modified Czapek-Dox medium (50 mg of D-glucose, 1 g of yeast
extract, 5 g of KH2PO4, 2 g of NaNO3, 0.5 g of MgSO4$7H2O and 0.01 g
of FeSO4$7H2O, pH 6.57.0 perliterof water) at room temperature. For
shaken cultures, mycelium was grown in Erlenmeyer flasks con-
taining 200 mL of the same medium agitated on an orbital shaker at
140 rpm at 25 ꢀC. Each Roux bottle or Erlenmeyer flask was in-
oculated with mycelium on six small slices of agar (1 cm).
3. Conclusions
A plausible explanation for this, which has been previously
reported for other natural products, such as aurovertin,14 is that B.
cinerea biosynthesis involves a C10-polyketide, which is methylated
at activated methylene groups, followed by the loss of the starter-
acetate unit through a retro-Claisen type CeC bond cleavage with
inversion of configuration at C-8 (Scheme 1).
4.4. Extraction and isolation of metabolites
O
O
O
After 7 days of incubation under fluorescent light, the culture
media were filtered, saturated with NaCl, extracted with ethyl ac-
etate (3ꢂ0.5 vol) and washed with water (3ꢂ0.25 vol). The organic
extracts were dried over Na2SO4 and concentrated to dryness.
Preliminary fractionation of the extracts was achieved by col-
umn chromatography eluting with petroleum ether/ethyl acetate
mixtures containing increasing percentages of ethyl acetate
(10e100%) to give 10 fractions F1eF10. Final purification of each
fraction was carried out by means of semi-preparative or analytical
HPLC. Botcinin A (2), botcinin B (2a), botcinin G (10), 3-O-ace-
tylbotcineric acid (8), (þ)-botrylactone (11) and 5-hydroxy-7-(4-
hydroxydec-2(3)-enoyl)botrylactone (12) were obtained.
O
O
O
O
O
SCOA
SCOA
SCOA
H+
H
HO
OH
HO
O
O
O
H
O
OH
O
O
O
O
O
HO
OH
O
O
H
H
O
15
O
O
HO
O
O
O
H
O
O
O
O
O
4.4.1. Botcinin A (2) and B (2a)4. Compounds 2 and 2a were
obtained from purification of fractions F4 and F5 as a colourless oil.
Semi-preparative HPLC: hexaneeethyl acetate 70:30; flow
3 mL minꢁ1; tR around 29 and 35 min, respectively.
Scheme 1. Biosynthetic route proposal for botrylactones and botcinins.
This hypothesis is in agreement with the common biosynthetic
origin of botrylactone (11) and botcinins, where a hypothetical bi-
cyclic acid intermediate 15 could be the branching point to give
botrylactone or botcinin derivatives as shown in Scheme 1. This
proposal explains the presence of the methyl group on C-8 of
botcinins and the different stereochemistry in this carbon.
4.4.2. Botcinin G (10). Compound 10 was obtained from purifica-
tion of fractions F7 as a colourless oil. Semi-preparative HPLC:
35
hexaneeethyl acetate 75:25; flow 3 mL minꢁ1; tR¼7.0 min [
a]
D
ꢁ34ꢀ (c 2.0, CHCl3); IR ymax (film) 3452, 2933, 2870, 1723, 1652,
1456, 1166 cmꢁ1; 1H NMR (400 MHz, CD3OD)
d
7.02 (dd, 1H, J¼15.5,
4.9 Hz, H-30), 6.05 (dd, 1H, J¼15.5, 1.7 Hz, H-20), 4.50 (dd, 1H, J¼10.5,
9.7 Hz, H-7), 4.25 (m, 1H, H-40), 4.08 (d, 1H, J¼9.4 Hz, H-3), 3.94 (d,
1H, J¼10.9 Hz, H-5), 3.77 (dq, 1H, J¼9.7, 5.9 Hz, H-8), 3.20 (dq, 1H,
J¼9.4, 7.3 Hz, H-2), 2.21 (m, 1H, J¼10.5, 6.4 Hz, H-6), 1.49 (m, 2H, H-
50), 1.31 (m, 2H, H-50), 1.17 (m, 6H, H-70, H-80, H-900), 1.17 (s, 3H,
C4eCH3), 1.11 (d, 3H, J¼5.9 Hz, C8eCH3), 1.13 (d, 3H, J¼7.3 Hz,
C2eCH3), 1.07 (d, 3H, J¼6.4 Hz, C6eCH3), 0.89 (t, 3H, J¼5.0 Hz, H-
100); 13C NMR (100 MHz, CD3OD) 177.4 (s, C-1), 167.6 (s, C-10), 154.0
(d, C-30), 119.9 (d, C-20), 79.7 (d, C-5), 77.9 (d, C-7), 78.1 (s, C-4), 75.0
(d, C-3), 71.6 (d, C-12), 69.6 (d, C-8), 39.6 (d, C-2), 37.5 (t, C-50), 36.8
(d, C-6), 32.9 (t, C-80), 30.2 (t, C-70), 26.4 (t, C-60), 23.6 (t, C-90), 18.6
(q, C8eCH3), 14.3 (q, C-100), 13.9 (q, C6eCH3), 11.5 (q, C4eCH3), 10.4
(q, C2eCH3); EIMS m/z (rel int.) 394 [MꢁH2O]þ (9), 226 (26),169 (9),
4. Experimental section
4.1. General experimental procedures
1H and 13C NMR measurements were recorded on Varian Unity
400 MHz and Varian Inova 600 MHz spectrometers with SiMe4 as
the internal reference. Chemical shifts were referenced to CDCl3 (dH
7.25, dC 77.0). HPLC was performed with a Hitachi/Merck L-6270
apparatus equipped with a differential refractometer detector (RI-
7490). A Lichrofer Si 60 (5
and a Lichrofer Si 60 (10
used in isolation experiments. Silica gel (Merck) was used for
m
m
m) LichoCart (250 mmꢂ4 mm) column
m) LichoCart (250 mmꢂ10 mm) were

420
J. Moraga et al. / Tetrahedron 67 (2011) 417e420
140(23), 124 (83), 109 (100); HREIMS calcd for C22H34O6 [MꢁH2O]þ
1.42(d, 3H, J¼7.3 Hz, C2eCH3), 1.12 (s, 3H, C4eCH3), 1.04 (s, 3H,
C8eCH3), 0.44 (d, 3H, J¼6.4 Hz, C6eCH3).
394.2355, found 394.2353.
4.4.3. 3-O-Acetylbotcineric acid (8). Compound 8 obtained from
4.7. Biotransformation of 2-benzylideneindan-1-one
fractions F9 and F10. Semi-preparative HPLC: hexaneeethyl acetate
20
95:5; flow 3 mL minꢁ1; tR¼13.2 min. Colourless oil; [
a]
ꢁ5.9ꢀ (c
Botrytis cinerea UCA 992 was grown at 25 ꢀC on a Czapeck-Dox
medium (200 mL per flask). The shaken culture was incubated in an
orbital shaker at 140 rpm under fluorescent light. 2-Benzylide-
neindan-1-one was dissolved in ethanol and then distributed over
12 flasks (150 ppm per flask) and the fermentation continued for 5
days in six flasks and 10 days in the others. The mycelium was then
filtered and the broth was extracted as described below. The sol-
vent was then evaporated and the residue was purified first on
a silica gel column and then with HPLC with an increasing gradient
of ethyl acetate to petroleum ether.
D
1.4, ethyl acetate).15
4.4.4. (þ)-Botrylactone (11). Compound 11 obtained from fractions
F5. Analytical HPLC: hexaneeethyl acetate 65:35; flow 1 mL minꢁ1
;
25
tR¼28.98 min. Colourless oil; [
a
]
D
þ19ꢀ (c 2.3, CHCl3).9,11
4.4.5. 2R, 3S, 4S, 6S, 8R, 9R-5-Hydroxy-7-(4-hydroxydec-2(3)-enoyl)
botrylactone (12). Compound 12 obtained from fractions F9 and
F10. Semi-preparative HPLC: hexaneeethyl acetate 95:5; flow
20
3 mL minꢁ1; tR¼15.1 min. Colourless oil; [
a
]
þ10.6ꢀ (c 0.9, ethyl
Chromatography of the extract fermented for 5 days produced
2-benzylideneindan-1-one (4 mg), O-methyldihydrobotrydial
(3.2 mg), botrydial (4 mg), dihydrobotrydial (3 mg), 7-deoxybo-
D
acetate); IR ymax (film) 3445, 2931, 2859, 1727, 1652, 1456,
1116 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.00 (dd, 1H, J¼15.6, 4.5 Hz,
H-30), 6.07 (dd, 1H, J¼15.6, 1.6 Hz, H-20), 4.93 (d, 1H, J¼10.9 Hz, H-7),
4.33 (m, 1H, H-40), 3.67 (d, 1H, J¼11.1 Hz, H-5), 3.52 (s, 1H, H-3), 2.75
(q, 1H, J¼7.4 Hz, H-2), 1.99 (m, 1H, H-6), 1.60 (m, 2H, H-50), 1.53 (s,
3H, C9eCH3), 1.46 (d, 3H, J¼7.4 Hz, C2eCH3), 1.28 (m, 8H, H-60, H-70,
H-80, H-90), 1.14 (s, 3H, C4eCH3), 1.10 (s, 3H, C8eCH3), 1.02 (d, 3H,
J¼6.4 Hz, C6eCH3), 0.87 (t, 3H, J¼6.8 Hz, H-100); 13C NMR (100 MHz,
CDCl3) 171.0 (s, C-1), 165.4 (s, C-10), 151.9 (d, C-30), 119.0 (d, C-20),
104.2 (s, C-9), 80.9 (d, C-3), 79.2 (s, C-8), 76.2 (s, C-4), 75.1 (d, C-5),
74.2 (d, C-7), 71.1 (d, C-40), 36.7 (d, C-50), 36.5 (t, C-60), 34.5 (d, C-2),
31.6 (t, C-80), 29.1 (t, C-70), 25.2 (t, C-60), 22.5 (t, C-90), 21.7 (q,
C9eCH3), 18.5 (q, C2eCH3), 18.2 (q, C4eCH3), 18.1 (q, C8eCH3), 14.0
(q, C-100), 13.8 (q, C6eCH3); EIMS m/z (rel int.) 454 [M]þ (1), 426
[MꢁCO]þ (7), 410 [MꢁCO2]þ (6); HREIMS calcd for C23 H38O7
[MꢁCO]þ 426.2618, found 426.2641.
trylactone (13,2.8 mg) and 2-(p-hydroxyphenylmethyl)indan-1-
20
one (50 mg) ([
a
]
D
þ6ꢀ (c 0.1, MeOH), 36% ee).
Chromatography of the extract fermented for 10 days produced
2-benzylideneindan-1-one (14 mg), botrydial (2 mg), dihydrobo-
trydial (3 mg), 7-deoxybotrylactone (13, 15.3 mg), botcinic acid (3,
60 mg) and 2-(p-hydroxyphenylmethyl)-7-hydroxyindan-1-one
20
(17 mg) [
a
]
ꢁ3.7ꢀ (c 0.1, MeOH, 7% ee).
D
4.7.1. 7-Deoxybotrylactone(13). Compound 13 obtained from fraction
F3. Column chromatography: hexaneeethyl acetate 80:20. Amor-
phous solid; [
a
]
25 þ2.45ꢀ (c 0.3 CHCl3); IR ymax (film) 2925, 1735, 1459,
D
1103, 950 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 3.28 (s, 1H, H-3), 2.70 (q,
1H, J¼7.3 Hz, H-2), 1.75 (m, 1H, H-6), 1.63 (m, 1H, H-5), 1.57 (m, 2H, H-
7) 1.44 (s, 3H, C9eCH3), 1.40 (d, 3H, J¼7.3 Hz, C2eCH3), 1.37 (m, 1H, H-
50),1.26 (m, 1H, H-70), 1.14 (s, 3H, C4eCH3),1.18 (s, 3H, C8eCH3), 0.95 (d,
3H, J¼6.7 Hz, C6eCH3); 13C NMR (100 MHz, CDCl3) 171.7 (s, C-1), 104.8
(s, C-9), 81.7 (d, C-3), 75.5 (s, C-8), 71.6 (s, C-4), 75.6 (d, C-5), 71.6 (d, C-
12), 41.9 (t, C-5), 39.9 (t, C-7), 34.5 (d, C-2), 26.17 (q, C4eCH3), 25.0 (q,
C8eMe), 23.4 (d, C-6), 21.6 (q, C9eCH3), 20.5 (q, C6eCH3), 18.6 (q,
C2eCH3); EIMS m/z (rel int.) 226 [MꢁCO]þ (26), 166 (36),123 (83), 109
(100); HREIMS calcd for C13H22O3 [MꢁCO]þ 226.1569, found 226.1573.
4.5. Acetylation of botrylactone
Botrylactone (11, 10 mg) was dissolved in dry pyridine (1 mL,
0.001 mmol) and acetic anhydride (2.4 mL, 24,6 mmol) was added
dropwise. The reaction mixture was stirred for 24 h. Then the solvent
wasremovedandthecrudereactionproductchromatographedtogive
botrylactone acetate (11c). Colourless oil; [
a
]
25 þ73, (c 2.4 mg, CHCl3).9
D
Acknowledgements
4.6.
a
-Methoxyphenylacetyl ester of botrylactone
This work was supported by grants from MICINN (AGL2009-
13359-C02-01) and from Junta de Andalucía (P07-FQM-02689). We
gratefully acknowledge Dr. Muriel Viaud from the UMR BIOGER,
A solution of the botrylactone (11, 10 mg, 0.037 mmol) in dry
dichloromethane CH2Cl2 (1.5 mL) was treated with DMAP (9.05 mg,
2.0 equiv) and (þ)-(2S)- or (ꢁ)-(2R)-2-methoxy-2-phenylacetic
acid MPA (13.85 mg, 2.25 equiv). After 15 min stirring at room
temperature, EDC (14.91 mg, 2.1 equiv) was added. Stirring was
maintained for 24 h. The solvent was stirred under reduced pres-
sure. Residue purification was achieved by flash column chroma-
tography on silica gel (elution with 60:40 hexane/ethyl acetate).
INRA (Versailles, France) for supplies B. cinerea mutant bcbot2D.
References and notes
1. Cutler, H. G.; Jacyno, J. M.; Harwood, J. S.; Dulik, D.; Goodrich, P. D.; Roberts, R.
G. Biosci., Biotechnol., Biochem. 1993, 57, 1980e1982.
ꢀ
ꢀ
2. Collado, I. G.; Aleu, J.; Hernandez-Galan, R.; Hanson, J. R. Phytochemistry 1996,
42, 1621e1624.
ꢀ
ꢀ
ꢀ
ꢀ
3. Reino, J. L.; Duran-Patron, R. M.; Daoubi, M.; Collado, I. G.; Hernandez-Galan, R.
-Methoxyphenylacetyl ester of botrylactone (11a). 1H
J. Org. Chem. 2006, 71, 562e565.
4.6.1. (R)-
NMR (400 MHz, CDCl3)
a
4. Tani, H.; Koshino, H.; Sakuno, E.; Nakajima, H. J. Nat. Prod. 2005, 68, 1768e1772.
5. Tani, H.; Koshino, H.; Sakuno, E.; Cutler, H. G.; Nakajima, H. J. Nat. Prod. 2006, 69,
722e725.
6. Fukui, H.; Shiina, I. Org. Lett. 2008,10, 3153e3156; Fukui, H.; Hitomi, S.; Suzuki, R.;
Ikeda, T.; Umezaki, K.; Tsuji, K.; Shiina, I. Tetrahedron Lett. 2008, 49, 6514e6517.
7. Shiina, I.; Fukui, H. Chem. Commun. 2009, 385e400.
8. Pinedo, C.; Wang, Ch.-M.; Pradier, J. M.; Dalmais, B.; Choquer, M.; Le Pecheur, P.;
Morgant, G.; Collado, I. G.; Cane, D. E.; Viaud, M. ACS Chem. Biol. 2008, 3, 791e801.
9. Welmar, K.; Tschesche, R.; Breitmaier, E. Chem. Ber. 1979, 112, 3598e3602.
10. Suga, T.; Hirata, T.; Utsumi, R.; Yoshioka, T. J. Sci. Hiroshima Univer., Ser. A 1984,
48, 75e79.
0
d
7.40 and 7.33 (m, 2H and 3H,C2 ePh), 4.87
0
0
(d, 1H, J¼10.8 Hz, H-7), 4.75(s, 1H, H-2 ), 3.39 (s, 3H, C2 eOMe), 3.27
(br s, 1H, H-3), 2.63 (q, 1H, J¼7.3 Hz, H-2), 1.90 (m, 1H, H-6), 1.71 (dd,
J¼4.7, 13.5 Hz, H-5
b
), 1.55 (dt, J¼2.7, 13.2 Hz, H-5a), 1.39(d, 3H,
J¼7.3 Hz, C2eCH3),1.26 (s, 3H, C9eCH3),1.12 (s, 3H, C8eCH3), 0.91 (d,
3H, J¼6.4 Hz, C6eCH3), 0.86 (t, 3H, J¼7.2 Hz, H-100).
4.6.2. (S)-
a
-Methoxyphenylacetyl ester of botrylactone (11b). 1H
0
11. Bruns, W.; Horns, S.; Redlich, H. Synthesis 1994, 335e342.
12. Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Assymetry 2001, 12, 2915e2925.
13. Unpublished results
14. Steyn, P. S.; Vieggaar, R.; Wessels, P. L. J. Chem. Soc., PerkinTrans.11981,1298e1308.
15. Sakuno, E.; Tani, H.; Nakajima, H. Biosci., Biotechnol., Biochem. 2007, 71, 2592e2595.
NMR (400 MHz, CDCl3)
d
7.44 and 7.38 (m, 2H and 3H,C2 ePh), 4.81
~
0
0
(d, 1H, J¼10.8 Hz, H-7), 4.76(s, 1H, H-2 ), 3.40 (s, 3H, C2 eOMe), 3.27
(br s, 1H, H-3), 2.67 (q,1H, J¼7.3 Hz, H-2), 1.79 (m, 1H, H-6), 1.62 (dd,
J¼5.0, 13.5 Hz, H-5
b), 1.55 (t, J¼13.2 Hz, H-5
a), 1.52 (s, 3H, C9eCH3),