Biocatalytic preparation and absolute configuration of enantiomerically pure fungistatic anti-2-benzylindane derivatives. Study of the detoxification mechanism by Botrytis cinerea

Article abstract of DOI:10.1039/c003938a

Enantiomerically pure 2-benzylindane derivatives were prepared using biocatalytic methods and their absolute configuration determined. (1R,2S)-2-Benzylindan-1-ol ((1R,2S)-2) and (S)-2-benzylindan-1-one ((S)-3) were produced by fermenting baker's yeast. Li

Full text of DOI:10.1039/c003938a

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Biocatalytic preparation and absolute configuration of enantiomerically pure
fungistatic anti-2-benzylindane derivatives. Study of the detoxification
mechanism by Botrytis cinerea
Cristina Pinedo-Rivilla,a Josefina Aleu,a Manuel Grande Benitob and Isidro G. Collado*a
Received 9th March 2010, Accepted 3rd June 2010
First published as an Advance Article on the web 23rd June 2010
DOI: 10.1039/c003938a
Enantiomerically pure 2-benzylindane derivatives were prepared using biocatalytic methods and their
absolute configuration determined. (1R,2S)-2-Benzylindan-1-ol ((1R,2S)-2) and
(S)-2-benzylindan-1-one ((S)-3) were produced by fermenting baker’s yeast. Lipase-mediated
esterifications and hydrolysis of the corresponding racemic substrates gave rise to the enantiopure
compounds (1S,2R)-2-benzylindan-1-ol ((1S,2R)-2) and (1R,2S)-2-benzylindan-1-ol ((1R,2S)-2),
respectively. The antifungal activity of these products against two strains of the plant pathogen Botrytis
cinerea was tested. The metabolism of anti-( )-2-benzylindan-1-ol (anti-( )-2) by B. cinerea as part of
the fungal detoxification mechanism is also described and revealed interesting differences in the genome
of both strains.
formation of anti-( )-2-benzylindan-1-ol (anti-( )-2), which had
Introduction
shown the highest antifungal activity in previous studies, using B.
The Botrytis species belong to the most geographically widespread
cinerea as a part of the fungal detoxification mechanism.
group of plant pathogens and saprophytes.1 Grey mould, a
grapevine disease caused by the fungus Botrytis cinerea results
in crop losses and low quality wines from infected grapevines
affecting many wine-producing countries.2
Results and discussion
Baker’s yeast reduction
Because the fungus develops a rapid tolerance to commercial
fungicides, these compounds must be used in increasingly higher
quantities. This in turn leads to the emergence of highly resistant
strains in Botrytis cinerea populations while also polluting both
soil and water. Consequently, there is interest in the development
of novel antifungal agents that are active against this organism.
We screened compounds analogous to various phytoalexins3
with a view to finding substrates with antifungal properties against
this fungus and were successful in identifying several different
benzylindane derivatives which exhibited strong fungicidal activity
against B. cinerea.
The preparation of optically active forms of benzylindane
derivatives is not only interesting in terms of their antifungal
properties but also because they could serve as synthetic precursors
to other natural products playing an important role in the
development of pharmaceuticals and for medicinal chemistry.4
Thus, based on previous results,5 we selected biocatalytic methods
to pursue enantiomerically pure forms of benzylindane derivatives
from the ketone (E)-2-benzylideneindan-1-one (1) in order to
compare their antifungal activities.
Based on previous results8 and the well known ability of baker’s
yeast to reduce a high number of ketones into optically active
alcohols,9 we decided to employ a baker’s yeast-mediated approach
to enantiopure alcohol derivatives using the corresponding ketone
(E)-2-benzylideneindan-1-one (1) as substrate. The results are
shown in Table 1. The diastereomeric ratio and enantiomeric
excesses were determined by HPLC analysis using a Chiralcel OD
column.
Table 1 Results of baker’s yeast-mediated reduction
(1R,2S)-2
Allyl alcohol Hexane Conv. a
(S)-3
Conv. a
Time
ee (%) (h)
(mM)
(%)
(%)
ee (%) ed (%) (%)
—
—
67
—
—
1.2
0.24
0.5
2.0
89
36
74
96
94
97
9
16
—
52
13
—
48
240
96
a Conv.: Conversion is the % of product in the recovered material.
As shown in Table 1, after the incubation of 1 in fermenting
baker’s yeast following the conventional procedure (see Experi-
mental section), two transformation products were isolated, (S)-
(+)-2-benzylindan-1-one ((S)-3) (52% ee, [a]2D0 +162 (c 0.1 in
CHCl3)) and (1R,2S)-(+)-2-benzylindan-1-ol (1R,2S)-2 (89% ee,
96% ed, [a]2D0 +6.8 (c 1 in CHCl3)) (Scheme 1). The spectroscopic
data of these products corresponded to those found in the
literature.8,10 The ed was very high for alcohol 2 in all the cases
(>94%). The absolute configuration of (1R,2S)-2 was determined
by means of circular dichroism.
Since several phytopathogenic fungi have been reported to
detoxify phytoalexins,6 producing less toxic degradation products
than the parent compounds,7 we decided to study the biotrans-
aDepartamento de Qu´ımica Orga´nica, Facultad de Ciencias, Universidad
de Ca´diz, Repu´blica Saharaui s/n, Apdo. 40, 11510 Puerto Real, Ca´diz,
Spain. E-mail: isidro.gonzalez@uca.es; Fax: +34-956-016193; Tel: +34-
956-016368
bDepartamento de Qu´ımica Orga´nica, Facultad de Ciencias Qu´ımicas,
Universidad de Salamanca, E-37008 Salamanca, Spain
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Table 2 Results of lipase-mediated acetylation of anti-( )-2-benzylindan-1-ol (anti-( )-2)
Enzyme Vinyl acetate (eq) Time/h Yield aof (1S,2R)-4 (%) ee of (1S,2R)-4 (%) Yield aof (1R,2S)-2 (%) ee of (1R,2S)-2 (%) Conv. b (%)
E
PS
CRL
PS
PS
PS
10
10
1
1
1
67
67
120
43
160
12
8
34
12
67
>99
>99
>99
>99
>99
87
92
63
91
51
>99
>99
>99
>99
>99
50
50
50
50
50
>400
>400
>400
>400
>400
a Yield 100% at 50% conversion after hydrolysis of the acetate derivative. b Conv.: conversion.
Pseudomonas cepacia), PPL (porcine pancreas lipase) and CRL
(Candida rugosa lipase). The results are summarised in Table 2. The
lipase PS appeared to be more efficient in the enzymatic acetylation
of these substrates producing the highest enantioselectivities and
yields while lipase PPL showed no enantioselection.
Different reaction times and vinyl acetate concentrations (1 and
10 eq) were tested in an attempt to increase the conversion rate
(Table 2). Enantiomeric excesses were very high in all the cases
(>99%).
The best result for enantiomer (1S,2R)-4 was with lipase PS
yielding 67% acetate derivative. Following hydrolysis of the acetate
derivative with potassium hydroxide in methanol, the enantiopure
alcohol was identified as (1S,2R)-2-benzylindan-1-ol ((1S,2R)-2)
which was obtained with a high ee >99% ([a]2D0 -11.6 (c 0.1 in
CHCl3)) and a conversion of 50%, using 1 eq of vinyl acetate as
the acyl donor.
Scheme 1 Biological reduction of (E)-2-benzylideneindan-1-one (1).
The circular dichroism curves of alcohol 2 show two positive
Cotton effects (CE) related to the 1Lb (De272 +0.22) and 1La (De221
+1.5) bands. The low intensity of these bands rules out an exciton
coupling between both aromatic rings so the CE should be caused
by the benzylic chiral center. According to the benzene sector
rules11 applied to the most stable conformation of the indanol
(equatorial –OH and –CH2Ph groups), a positive CE of the
1Lb band agrees with the 1R configuration. In addition, the CD
spectrum of 2 is quite similar to the mirror CD curve published
for S-indan-1-ol.12
The best result for the enantiomer (1R,2S)-2-benzylindan-1-ol
((1R,2S)-2) was with lipase CRL yielding 92%, ee >99% ([a]D20
+11.8 (c 0.1 in CHCl3)) and a conversion of 50%, using 10 eq of
vinyl acetate as the acyl donor.
The absolute configuration of (S)-3 was determined from
oxidation of the alcohol (1R,2S)-2 whose absolute configuration
was known, and by comparing their optical values.
It has been reported that inhibition of certain type of enzymes
in the baker’s yeast reaction may control the enantioselectivity of
the reduction.13 We therefore tried to inhibit the reducing enzymes
selectively by adding inhibitors such as allyl alcohol and a small
amount of organic solvent14 to dissolve the substrate (see Table 1)
in order to improve the transformation. The addition of allyl alco-
hol and hexane improved the stereoselectivity and conversion of
the alcohol (1R,2S)-(+)-2-benzylindan-1-ol ((1R,2S)-2) (97% ed,
[a]2D0 +6.5 (c 0.1 in CHCl3)) but ee was diminished (74% ee). Ketone
3 was not isolated from the broth containing the additives.
Lipase-mediated hydrolysis
Here we report on the enzyme mediated enantioselective hydrolysis
of anti-( )-2-benzylindan-1-yl acetate (anti-( )-4). Of the enzymes
screened only CRL displayed high selectivity.
The racemic acetate derivative was prepared by adding the
corresponding alcohol to a solution of acetic anhydride in
dichloromethane–methanol (1 : 1). The acetate was then trans-
formed by lipases dissolved in a mixture of 1,4-dioxane and
different concentrations of buffer solution. The results of the
enzymatic hydrolysis are summarized in Table 3.
Lipase-mediated transformations
In this case, the enzymatic-mediated hydrolysis afforded
(1R,2S)-2-benzylindan-1-ol ((1R,2S)-2) at a yield of 30%, 93%
ee ([a]2D0 +10.4 (c 0.1 in CHCl3)) and a conversion of 49% using
lipase CRL with a mixture of 1,4-dioxane and buffer 30%. We
were unable to obtain a high yield which could be due to the low
solubility of the starting material meaning that there would be less
substrate available for the lipase reaction.
We focused on the kinetic resolution of the racemic secondary
alcohols and acetates, obtained as described in the experimental
section toexplore new methods for the preparation ofenantiomeri-
cally pure alcohols from ketone 1. Lipases are versatile biocatalysts
acting with high efficiency in acylation or hydrolysis.5,15
Lipase-mediated acetylations
The racemic secondary alcohol anti-( )-2 prepared by reacting
the ketone 1 with LiAlH4 was added to different media with vinyl
acetate as an acyl donor and tert-butyl methyl ether (TBME) as
the organic solvent and three different lipases: lipase PS (from
Antifungal assays
The antifungal properties of all the compounds obtained in
this work were determined based on their ability to inhibit
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Table 3 Results of lipase-mediated hydrolysis of anti-( )-2-benzylindan-1-yl acetate (anti-( )-4)
Enzyme Buffer (%) Time/h Yield aof (1R,2S)-2 (%) ee of (1R,2S)-2 (%) Yield aof (1S,2R)-4 (%) ee of (1S,2R)-4 (%) Conv. b (%)
E
CRL
CRL
CRL
83
67
30
144
96
96
52
26
30
84
87
93
61
75
72
80
68
90
49
44
49
29
28
84
a Yield 100% at 50% conversion after hydrolysis of the acetate derivative. b Conv.: conversion.
Table 4 Results of conidial germination bioassays of 2-
benzylideneindan-1-one (1)
Detoxification of anti-( )-2-benzylindan-1-ol (anti-( )-2) by
Botrytis cinerea 2100 and UCA992
Compound
% of inhibition SDa
The biotransformation of racemic alcohol anti-( )-2 by
BcUCA992 and Bc2100 was studied to identify fungal detoxifi-
cation mechanisms and differences in their enzymatic potential to
degrade xenobiotic substrates.
[ppm]
BcUCA992
Bc2100
100
0
100
50
0
100
25
0
100
12.5
0
100
Biotransformation was done in surface cultures using Roux
bottles for five days after which the mycelium was filtered,
extracted with ethyl acetate and the extract dried over anhydrous
sodium sulfate. Purification of the dry organic extract afforded
several compounds (see Scheme 2).
a % of inhibition using PDB medium as control. Each value is the
percentage of the media of three experiments standard deviation.
Table 5 Results of conidial germination bioassays of (1R,2S)-(+)-2 and
((1S,2R)-(-)-2
% of inhibition SDa
Compound (1R,2S)-(+)-2
(1S,2R)-(-)-2
[ppm]
BcUCA992 100 94
Bc2100 100 100
100 50
25
34
100
12.5
17
100
100 50 25
100 100 84
100 100 100
12.5
19
100
1
2
3
1
2
a % of inhibition using PDB medium as control. Each value is the
percentage of the media of three experiments standard deviation.
Scheme 2 Biotransformation products of anti-( )-2-benzylindan-1-ol
(anti-( )-2) by Botrytis cinerea.
the growth of the two different strains of B. cinerea, Bc2100
and BcUCA992, using the conidial germination assay.16 In
general, compounds (E)-2-benzylideneindan-1-one (1) and anti-
2-benzylindan-1-ol ((1R,2S)-(+)-2 and (1S,2R)-(-)-2) were the
most active products against both strains of B. cinerea. The
alcohol (1S,2R)-(-)-2 exhibited a more pronounced antifungal
effect. The results of the antifungal assays for these compounds
are summarized in Tables 4 and 5.
In addition to the starting material, the biotransformation of
anti-( )-2-benzylindan-1-ol (anti-( )-2) by BcUCA992 afforded
the following compounds: syn-2-benzyl-2,3-dihydroxyindan-1-
one (5), anti-2-benzyl-3-hydroxyindan-1-one (6), and (R)-2-(p-
hydroxybenzyl)-7-hydroxyindan-1-one (7). The starting material
recovered exhibited [a]D20 -3.5 (c 2.3 in CHCl3) with 25% ee, in
favour of the configuration (1S,2R).
Ketone 1 and alcohols 2 at all concentrations, even at 12.5 ppm,
completely inhibited the germination (100%) of B. cinerea 2100.
Ketone 1 was completely inactive against B. cinerea UCA992
but when treated with anti-2-benzylindan-1-ol ((1R,2S)-(+)-2 and
(1S,2R)-(-)-2), conidia and germlings of B. cinerea UCA992 ger-
minated and grew to produce stunted, distorted, and abnormally
branched germlings. Compound (1S,2R)-(-)-2-benzylindan-1-ol
((1S,2R)-2) was the most active against B. cinerea UCA992. To
confirm the results three independent experiments were carried
out.
On the other hand, anti-( )-2-benzylindan-1-ol (anti-( )-2) was
incubated at a lower concentration (50 ppm) on surface cultures
of Bc2100 producing only one biotransformation product, (R)-
(-)-2-benzylindan-1-one ((R)-3) at a yield of 60% and 75% ee. The
higher potential of the strain BcUCA992 to biotransform anti-( )-
2-benzylindan-1-ol (anti-( )-2) is in accordance with those results
obtained from antifungal assays. So, the antifungal assays and
results obtained from biotransformation reveal genetic differences
between both strains that could be associated with their adaptive
lifestyles and their different host ranges. The strain BcUCA992, a
wild strain isolated from a vineyard in 1992, seems to have genes
which codify for oxidases involved in the detoxification of (anti-
( )-2), which would not be present in the genome of strain Bc2100.
It is worth noting that acetyl derivatives 4 were inactive for both
strains, indicating that the hydroxyl group plays an important role
in the inhibitory mechanism.
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These data clearly reveal adaptive resources for the wild strain with
respect to strain Bc2100 from CECT lab collection.
Based on these results we also studied the biotransformation
of anti-( )-2-benzylindan-1-ol (anti-( )-2) by B. cinerea as part of
the fungal detoxification mechanism. Biotransformation products
have been shown to be less toxic to fungal growth than anti-( )-2.
The different behaviour of both strains indicates that the enzymatic
system of Bc2100 is not able to detoxify totally the compound
anti-( )-2, which produces a total inhibition on the fungus. These
data reveal significant differences in the genome of these strains,
specially in genes which would codify for the oxidases involved in
the detoxification mechanism of 2.
Compound 5 was isolated as a colourless oil with the molecular
formula C16H14O3, determined from its HRMS and corroborated
1
by 13C NMR data. The H NMR and 13C NMR spectra were
close to that of 2-benzylindan-1-one (3), but the appearance of a
signal at 4.81 and 77.1 ppm (H-3 and C-3, respectively) indicated
the presence of a secondary hydroxyl group. Another signal at
77.1 ppm in the 13C NMR spectrum (C-2) showed that 5 was a
diol derivative. The stereochemistry of both chiral carbons was
determined by nOe experiments, which showed an enhancement
for the signal corresponding to H-10 when the signal assigned to H-
3 was irradiated. Compound syn-2-benzyl-2,3-dihydroxyindan-1-
one (5) is reported here for the first time. The stereoselectivity of the
compound 5 was very high, giving only one diastereoisomer but
in the absence of at least one enantiomer of known configuration,
we had no way of determining which enantiomer was which.
Spectroscopic data of compound 6 coincided with those found
in the literature.4 That compound was obtained as a mixture of
diastereoisomers 78 : 22 in favour of the anti-configuration and
61% ee.
Experimental
General experimental procedures
Optical rotations were determined with a Perkin–Elmer 241
polarimeter. IR spectra were recorded on a Mattson Genesis
spectrophotometer, series FTIR. 1H and 13C NMR measurements
were obtained on Varian Unity 400 and Varian Unity 600
NMR spectrometers with SiMe4 as the internal reference. Mass
spectra were recorded on a GC-MS Thermoquest spectrometer
(model: Voyager), and a VG Autospec-Q spectrometer. HPLC
was performed with a Hitachi/Merck L-6270 apparatus equipped
with a UV–VIS detector (L 6200) and a differential refractometer
detector (RI-71). CD spectra were recorded on a Jobin–Ybon
Mark III Dichrograph in ethanol as solvent. TLC was performed
on Merck Kiesegel 60 F254, 0.2 mm thick. Silica gel (Merck) was
used for column chromatography. Purification by means of HPLC
was accomplished with a silica gel column (Hibar 60, 7 m, 1 cm
wide, 25 cm long). Chemicals were products of Fluka or Aldrich.
All solvents used were freshly distilled. Baker’s yeast was obtained
from a local store. The following enzymes were used in this work:
Candida rugosa Lipase (Sigma, Type VII, 950 U mg-1), Pseu-
domonas cepacia Lipase (Amano Pharmaceuticals Co., Japan) and
Porcine Pancreas Lipase (Sigma, Type II).
Compound 7 was isolated as a yellow oil, with an M+ peak at
m/z 254 and gave rise to a 13C NMR spectrum consistent with
1
the molecular formula C16H14O3. The H NMR and 13C NMR
spectra showed that it retained the benzylindanone skeleton, but
the appearance of two signals at 156.9 and 158.5 ppm in its
13C NMR spectrum (C-4¢ and C-7, respectively) indicated two
hydroxylations on the aromatic rings, according to the product 2-
(p-hydroxybenzyl)-7-hydroxyindan-1-one (7). Furthermore, struc-
ture 7 was supported by homo- and heteronuclear-2D-correlation
experiments. Its absolute configuration was determined by com-
parison of its specific rotation value ([a]2D0 -27.6, 81% ee) with that
of (R)-2-benzylindan-1-one ((R)-3) ([a]2D0 -211.2, 75% ee), whose
absolute configuration was known. Both compounds presented
a negative value, indicating the same configuration on the C-2.
Compound (R)-2-(p-hydroxybenzyl)-7-hydroxyindan-1-one (7) is
reported here for the first time.
The antifungal activity of the detoxification products against
B. cinerea was also studied using the ‘inhibition of conidial
germination’ technique. These products have been shown to be
less toxic to fungal growth than anti-( )-2-benzylindan-1-ol (anti-
( )-2) thus confirming that B. cinerea has a mechanism to detoxify
compound anti-( )-2 by hydroxylating several positions.
General procedure for baker’s yeast transformation
A mixture composed of baker’s yeast (250 g), D-glucose (100 g),
and tap water (1 L) was stirred in a 2 L beaker at 50 ◦C for 30 min.
Substrate 1 (5.93 mmol), which had been dissolved in a minimum
amount of ethanol, was then added dropwise. At the end of the
reaction period 1 L of ethyl acetate was added and the crude
reaction mixture was filtered through a large Bu¨chner funnel on
a Celite pad which was later washed with the same solvent. The
aqueous phase was extracted twice with 0.5 L of ethyl acetate,
the organic phase was dried over Na2SO4 and the solvent was
then evaporated under reduced pressure to dryness. The residue
obtained was purified by means of column chromatography.
Conclusions
The economic problems arising from phytopathogen fungi make
the rational control of these diseases a very interesting focus
of organic chemistry. Moreover, biocatalysis shows high regio-
and stereo-specificity in nature under mild and environmentally
friendly reaction conditions. In this work we have reported how
new antifungal compounds were obtained by means of biocatalytic
methods based on the use of baker’s yeast and enzymes which
could actually be re-used thanks to their remarkable stability and
biodegradability.
The studies described above demonstrate the biocatalytic po-
tential of the lipases in obtaining enantiomerically pure (1R,2S)-
and (1S,2R)-2-benzylindan-1-ol (2) which have proven to be highly
active against Botrytis cinerea.
Baker’s yeast transformation in the presence of several additives.
A mixture of baker’s yeast (250 g), water (1 L), and D-glucose
(100 g, 0.505 mol) was stirred at 50 ◦C for 30 min. The substrate
1 (5.93 mmol) was dissolved in allyl alcohol (8 mL, 67 mmol)
and hexane (12 mL, 1.2%) and was then added dropwise to the
mixture. The reaction was then stirred for a further period of time.
The work-up procedure was essentially the same as that described
above. The results are shown in Table 1.
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Chemical transformations
temperature. The residue obtained upon evaporation of the filtered
reaction mixture was chromatographed on a silica gel column and
eluted with hexane–ethyl acetate (95 : 5). The first eluted fractions
provided the unreacted starting material and the last eluted
fractions afforded the alcohol derivative (1S,2R)-2-benzylindan-
1-ol ((1S,2R)-2), 93% ee, [a]2D0 +10.4 (c 0.1 in CHCl3). Detailed
results are reported in Table 3.
Synthesis of racemic substrates. Reduction of (E)-2-
benzylideneindan-1-one10 (1) (5 g, 0.02 mol) with LiAlH4 (8 g, 0.2
mol) in 10 mL dry THF at room temperature in N2 atmosphere
gave anti-( )-2-benzylindan-1-ol (anti-( )-2) (4.2 g, 82.5%, 100%
ed). The 1H NMR data of this product coincided with those found
in the literature.8,10 HPLC (n-hexane/i-PrOH 9 : 1, 0.6 mL min-1):
tR 14 min (S,R) and 30 min (R,S).
Microorganism cultures and antifungal assays
After purification, compound anti-( )-2 (0.1 g, 0.44 mmol)
was dissolved in dry pyridine and acetic anhydride (50 mL) was
added dropwise. The reaction mixture was stirred for 20 h. The
solvent was then removed and the crude reaction product chro-
matographed to give anti-( )-2-benzylindan-1-yl acetate (anti-( )-
4) (84.5%), obtained as a white solid; dH(400 MHz; CDCl3) 2.00
(3 H, s, H-11), 2.65 (1 H, dd, J 6.4 and 16.0, H-3a), 2.71 (1 H, dd,
J 9.1 and 12.9, H-10a), 2.78 (1 H, m, H-2), 2.98 (1 H, dd, J 6.1 and
12.9, H-10b), 3.10 (1 H, dd, J 7.6 and 16.0, H-3b), 6.08 (1 H, d, J
5.0, H-1), 7.21 (4 H, dd, 4Harom), 7.25 (3 H, m, 3Harom), 7.29 (2
H, d, 2Harom); dC(100 MHz; CDCl3) 21.1 (q, C-11), 35.7 (d, C-3),
39.1 (d, C-10), 47.4 (d, C-2), 82.1 (d, C-1), 124.9 (d, Carom), 125.4
(d, Carom), 126.1 (d, Carom), 126.8 (d, Carom), 128.4 (d, Carom),
128.8 (d, 2Carom), 128.9 (d, 2Carom), 140 (d, Carom), 140.7 (s, C-
8), 142.8 (s, C-9), 189.4 (CO). MS (m/z, %) 206 (M+ - 60, 92), 115
(78), 91 (100), 77 (26), 43 (61). HPLC (n-hexane/i-PrOH 95 : 5,
0.7 mL min-1): tR 12 min (S,R) and 17 min (R,S).
The cultures of Botrytis cinerea employed in this work were
obtained from grapes from the Domecq vineyard, Jerez de la Fron-
tera, Ca´diz, Spain (BcUCA992) and from the Coleccio´n Espan˜ola
de Cultivos Tipo (CECT), Facultad de Biolog´ıa, Universidad de
Valencia, Spain (Bc2100).
Antifungal activity was determined by means of a conidial
germination assay. The test compound was dissolved in ethanol–
water mixtures to give a final compound concentration of 10–
150 mg L-1. Two microlitres of a conidial suspension in water
containing about 5 ¥ 104 conidia/mL of the B. cinerea strains
were added to a solution of the test compound (15 mL) and PDB
(potato dextrose broth) medium (2 mL) on ELISA plates. The final
ethanol concentration was identical in control and treated cultures.
Three replicates were made per compound and the viability of
conidia was estimated by measuring their germination just after
incubation at 25 ◦C for 24 h.
Oxidation of (1R,2S)-(+)-2-benzylindan-1-ol (1R,2S)-2. The
alcohol (1R,2S)-(+)-2-benzylindan-1-ol ((1R,2S)-2) (5 mg, 0.022
mmol) was oxidised with manganese(IV) oxide (1.5 eq) in methy-
lene chloride solution (1.5 mL) at room temperature. After
purification on a silica gel column, eluting with hexane–ethyl
acetate (9 : 1), the derivative (S)-(+)-2-benzylindan-1-one ((S)-3)
(98% yield) was afforded.
General culture conditions
Fungi were grown at 25 ◦C on a Czapeck–Dox medium (150 mL
per Roux bottle) on surface culture for 2 days. The substrate dis-
solved in ethanol was added to each Roux bottle and fermentation
continued for a further period of 5 days. The mycelium was filtered
and washed with brine and ethyl acetate. The broth was extracted
three times with ethyl acetate and the extract dried over anhydrous
sodium sulfate. The solvent was then evaporated and the residue
was chromatographed first on a silica gel column and then with
HPLC with an increasing gradient of ethyl acetate to petroleum
ether.
Lipase-mediated reactions
Lipase-mediated acetylations. A mixture of the racemic al-
cohol anti-( )-2 (51 mg, 0.23 mmol), lipase (51 mg), and vinyl
acetate (0.23 mmol or 2.3 mmol) in tert-butyl methyl ether (2 mL)
was stirred at room temperature. The residue obtained upon
evaporation of the filtered reaction mixture was chromatographed
on a silica gel column and eluted with hexane–ethyl acetate
(95 : 5). The first eluted fractions provided the acetate derivative
(1S,2R)-4 ([a]2D0 -89 (c 0.2 in CHCl3), >99% ee) and the last
eluted fractions afforded the unreacted starting material (1R,2S)-
(+)-2-benzylindan-1-ol ((1R,2S)-2), [a]2D0 +11.8 (c 0.1 in CHCl3),
ee >99%, De272 +0.22; De221 +1.5 (c 7.7 ¥ 10-3 M in EtOH).
Detailed results of the enzyme-mediated acetylations are reported
in Table 2.
Biotransformation of anti-( )-2-benzylindan-1-ol (anti-( )-2) by
Botrytis cinerea 2100
B. cinerea 2100 was grown as a surface culture in Roux bottles on
a Czapeck–Dox medium. The substrate anti-( )-2-benzylindan-1-
ol (anti-( )-2) dissolved in ethanol was distributed over 17 Roux
bottles (50 ppm per bottle). After the work-up procedure described
above the compound (R)-(-)-2-benzylindan-1-one ((R)-3) (60 mg)
([a]2D0 -172.3 (c 0.1 in CHCl3), 75% ee) was isolated. This product
was obtained as a colourless oil and its spectroscopic data coincide
with those found in the literature.10,17 Enantiomeric excess was
measured by means of chiral HPLC with n-hexane/i-PrOH 9 : 1
as eluent, flow rate 0.4 mL min-1: tR = 17 min (R) and 18.4 min
(S).
Treatment of acetate derivative (1S,2R)-4 (5 mg, 0.02 mmol)
with KOH (2 mg, 0.04 mmol) in methanol solution at room
temperature for 2 h afforded 3.8 mg (90.2%) of the compound
(1S,2R)-(-)-2-benzylindan-1-ol ((1S,2R)-2), [a]2D0 -11.6 (c 0.3 in
CHCl3): >99% ee, De272 -0.22; De221 -1.3 (c 6.2 ¥ 10-3M in EtOH).
Biotransformation of anti-( )-2-benzylindan-1-ol (anti-( )-2) by
Botrytis cinerea UCA992
Lipase-mediated hydrolysis. Lipase (60 mg) was added to
a mixture of the racemic acetate anti-( )-4 (20 mg, 0.075
mmol) in 1.5 mL of solvent (mixture of 1,4-dioxane and buffer
KH2PO4 : KOH 0.1 M pH 7). The solution was stirred at room
B. cinerea UCA992 was grown as
a surface culture in
Roux bottles on Czapeck–Dox medium. The substrate
a
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anti-( )-2-benzylindan-1-ol (anti-( )-2) dissolved in ethanol was
distributed over 17 Roux bottles (100 ppm per bottle). After
the work-up procedure described above the following compounds
were isolated: recovered (1S,2R)-2-benzylindan-1-ol ((1S,2R)-2)
(36 mg) ([a]2D0 -3.5 (c 2.3 in CHCl3), 25% ee), syn-2-benzyl-2,3-
dihydroxyindan-1-one (5) (4 mg) ([a]2D0 -10.2 (c 0.1 in CHCl3), 16%
ee), anti-2-benzyl-3-hydroxyindan-1-one (6) (2 mg) (61% ee), and
(R)-2-(p-hydroxybenzyl)-7-hydroxyindan-1-one (7) (5 mg) ([a]D20
-27.6 (c 0.1 in MeOH), 81% ee).
8.2, H-6), 7.27 (1 H, bd, J 8.5, H-4); dC(100 MHz; CD3OD) 32.1
(t, C-3), 37.2 (t, C-10), 51.2 (d, C-2), 108.9 (d, C-5), 116.2 (d, C-3¢,
C-5¢), 125.0 (d, C-6), 128.5 (d, C-4), 130.9 (d, C-2¢, C-6¢), 131.5 (s,
C-1¢), 138.8 (s, C-9), 147.0 (s, C-8), 156.9 (s, C-4¢), 158.5 (s, C-7),
211.0 (s, C-1); MS (m/z, %): 254 (M+, 64), 236 (3), 148 (69), 107
(100), 91 (13), 77 (34); m/z HRMS (EI, 70 eV) calcd for C16H14O3:
254.0943 [M]+; found: 254.0927; HPLC (n-hexane/i-PrOH 97 : 3,
0.5 mL min-1): tR 30.1 min (R) and 50.9 min (S).
References
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Obtained as a colourless oil; [a]2D0 -10.2 (c 0.1 in CHCl3), 100%
ed, 16% ee; nmax(film)/cm-1 3417, 2924 and 1631; dH(600 MHz;
CDCl3) 3.05 (2 H, d, J 7.6, H-10), 4.81 (1 H, bs, H-3), 7.25 (3 H,
m, 3Harom), 7.33 (6 H, m, 6Harom); dC(150 MHz; CDCl3) 37.2
(t, C-10), 77.1 (d, C-3), 77.7 (s, C-2), 123.9 (d, Carom), 125.4 (d,
Carom), 126.7 (d, Carom), 128.7 (d, 2Carom), 128.9 (d, 2Carom),
129.0 (d, Carom), 129.5 (d, Carom), 139.6 (s, Carom), 139.8 (s,
Carom), 143.4 (s, Carom), 214.6 (s, C-1); MS (m/z, %): 254 (M+,
25), 236 (19), 147 (100), 77 (48); m/z HRMS (EI, 70 eV) calcd
for C16H14O3: 254.0943; found: 236.0861 [M - H2O]+. HPLC (n-
hexane/i-PrOH 95 : 5, 0.8 mL min-1): tR 16.8 min (major) and
23.8 min (minor).
anti-2-Benzyl-3-hydroxyindan-1-one (6)
Obtained as a colourless oil, 61% ee, anti/syn 78 : 22 as de-
1
termined by H NMR spectroscopy. The assignments of prod-
uct structure were done attending to the spectroscopic data
reported in literature.4 HPLC anti-isomer (n-hexane/i-PrOH 9 : 1,
0.6 mL min-1): tR 13.7 min (major) and 14.4 min (minor).
(R)-2-(p-Hydroxybenzyl)-7-hydroxyindan-1-one (7)
Obtained as a yellow oil; [a]2D0 -27.6 (c 0.1 in MeOH), 81% ee;
n
max.(film)/cm-1 3343, 2356 and 1688; dH(400 MHz; CD3OD) 2.61
(1 H, dd, J 13.8 and 9.5, H-10a), 2.74 (1 H, dt, J 16.8 and 3.0,
H-3a), 2.94 (1 H, m, H-2), 3.04 (1 H, dd, J 16.8 and 7.4, H-3b), 3.13
(1 H, dd, J 13.8 and 4.4, H-10b), 6.67 (2 H, d, J 8.5, H-3¢, H-5¢),
7.03 (1 H, m, H-5), 7.04 (2 H, d, J 8.5, H-2¢, H-6¢), 7.07 (1 H, bd, J
17 J. M. Lamarche, J. Vebrel and B. Laude, Spectrochim. Acta, Part A,
1979, 35, 673–677.
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