Asymmetric preparation of antifungal 1-(4′-chlorophenyl)-1- cyclopropyl methanol and 1-(4′-chlorophenyl)-2-phenylethanol. Study of the detoxification mechanism by Botrytis cinerea

Article abstract of DOI:10.1016/j.molcatb.2011.02.005

Chiral alcohols are important as bioactive compounds or as precursors to such molecules. On the basis of the different antifungal properties of the enantiopure alcohol derivatives of 4′-chlorophenyl cyclopropyl ketone and benzyl 4′-chlorophenyl ketone, their enantioselective synthesis by chemical and biocatalytic methods was studied. The detoxification pathways by the phytopathogen fungus Botrytis cinerea are reported.

Full text of DOI:10.1016/j.molcatb.2011.02.005

Journal of Molecular Catalysis B: Enzymatic 70 (2011) 61–66
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Asymmetric preparation of antifungal 1-(4ꢀ-chlorophenyl)-1-cyclopropyl
methanol and 1-(4ꢀ-chlorophenyl)-2-phenylethanol. Study of the detoxification
mechanism by Botrytis cinerea
Cristina Pinedo-Rivilla, Antonio J. Bustillo, Rosario Hernández-Galán, Josefina Aleu∗, Isidro G. Collado∗
Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, República Saharaui s/n, Apdo. 40, 11510 Puerto Real, Cádiz, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Chiral alcohols are important as bioactive compounds or as precursors to such molecules. On the basis of
the different antifungal properties of the enantiopure alcohol derivatives of 4ꢀ-chlorophenyl cyclopropyl
ketone and benzyl 4ꢀ-chlorophenyl ketone, their enantioselective synthesis by chemical and biocatalytic
methods was studied. The detoxification pathways by the phytopathogen fungus Botrytis cinerea are
reported.
Received 20 October 2010
Received in revised form 27 January 2011
Accepted 2 February 2011
Available online 24 February 2011
© 2011 Elsevier B.V. All rights reserved.
Keywords:
Botrytis cinerea
Lipase
Baker’s yeast
1-(4ꢀ-Chlorophenyl)-1-cyclopropyl
methanol
1-(4ꢀ-Chlorophenyl)-2-phenylethanol
1. Introduction
have been gaining in popularity [11]. In this paper we report on the
enantioselective synthesis of 1-(4ꢀ-chlorophenyl)-1-cyclopropyl
Botrytis cinerea has been identified as a pathogenic fungus
associated with over 235 plant species including grapes, lettuce,
tomatoes, tobacco, and strawberries. It grows as a grey mould
causing serious economic losses [1,2]. Since several strains have
developed resistance to some commercial fungicides [3], there is an
important need to develop novel antifungal agents that are active
against this organism.
On the basis of previous results indicating that the presence of
a hydroxyl group was fundamental for the expression of antifun-
gal activity against the phytopathogenic fungus B. cinerea [4–7], we
showed that the alcohol derivatives of 4ꢀ-chlorophenyl cyclopropyl
ketone (1) and benzyl 4ꢀ-chlorophenyl ketone (2), compounds anal-
ogous to several phytoalexins [8,9], are active fungistatic agents
against B. cinerea and Colletotrichum gloeosporioides [10].
methanol (3) and 1-(4ꢀ-chlorophenyl)-2-phenylethanol (4) in both
absolute configurations by chemical and biocatalytic methods.
Since several phytopathogenic fungi have been reported to
detoxify phytoalexins [12] producing less toxic degradation prod-
ucts than the parent compounds [13], we decided to study the
biotransformation of 1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol
(3) by B. cinerea as a part of the fungal detoxification mechanism.
Previous studies of the detoxification of 1-(4ꢀ-chlorophenyl)-2-
phenylethanol (4) by B. cinerea and C. gloeosporioides suggest that
it is not likely to persist in the environment for long periods post-
application [14].
2. Experimental
The development of highly effective systems for the synthesis
of chiral alcohols is not only of interest to the academic world but
also has attracted the attention of industrial scientists. Because
of their environmentally benign reaction conditions and unpar-
alleled chemo, regio, and stereoselectivities, enzymatic protocols
2.1. General experimental procedures
Optical rotations were determined with a Perkin–Elmer 241
polarimeter. IR spectra were recorded on a Mattson Genesis spec-
trophotometer, series FTIR. 1H and 13C NMR measurements were
obtained on Varian Unity 400 and Varian Unity 600 NMR spec-
trometers with SiMe4 as the internal reference. Mass spectra were
recorded on a GC–MS Thermoquest spectrometer (model: Voy-
ager), and a VG Autospec-Q spectrometer. HPLC was performed
with a Hitachi/Merck L-6270 apparatus equipped with a UV-VIS
∗
Corresponding author. Tel.: +34 956 016368; fax: +34 956 016193.
E-mail addresses: josefina.aleu@uca.es (J. Aleu), isidro.gonzalez@uca.es
(I.G. Collado).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.02.005

62
C. Pinedo-Rivilla et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 61–66
detector (L 6200) and a differential refractometer detector (RI-
71). TLC was performed on Merck Kiesegel 60 F254, 0.2 mm thick.
Silica gel (Merck) was used for column chromatography. Purifica-
tion by means of HPLC was accomplished with a silica gel column
(Hibar 60, 7 m, 1 cm wide, 25 cm long). Chemicals were produced
by 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 (CRL, Sigma, Type VII,
950 U/mg), Pseudomonas cepacia lipase (PSL, Amano Pharmaceu-
ticals Co., Japan) and porcine pancreas lipase (PPL, Sigma, Type II).
an additional 1 h, water (20 mL) was added, the organic solvent
was removed by means of rotary evaporation and the remaining
aqueous phase was extracted with diethyl ether. The combined
organic layers were then dried over magnesium sulphate. Removal
of the solvent by means of rotary evaporation and subsequent
filtration through a short silica gel column afforded 153.9 mg
(98.1%) of the (S)-alcohol ([˛]2D0 = −9.1◦ (c 2.9 in CHCl3), 40.1% ee).
2.2.2.2. (R)-(+)-1-(4ꢀ-chlorophenyl)-1-cyclopropylmethanol
(R)-3
and (R)-(+)-1-(4ꢀ-chlorophenyl)-2-phenylethanol (R)-4. Under an
argon atmosphere, an oven-dried Schlenk tube was charged with
(R)-methyl oxazaborolidine (0.086 mmol, 100 ␮L of 1 M solution
in toluene). The solvent was removed under high vacuum condi-
tions (0.1 mbar) at room temperature and tetrahydrofuran (THF,
11 mL) was added. The solution was then cooled to 0 ◦C, treated
with a borane–THF complex (1.0 M, 1.49 mL) and stirred at room
temperature for 2 h. This reagent and a solution of 4ꢀ-chlorophenyl
cyclopropyl ketone (1) (180 mg, 0.996 mmol) or benzyl 4ꢀ-
chlorophenyl ketone (2) (230 mg, 1.0 mmol), respectively, in THF
(10 mL), were then added simultaneously from two syringes into
an oven dried, round-bottomed flask at a temperature of 30 ◦C
over a period of 16 h. The work-up procedure was essentially the
same as that described above affording 175.3 mg (96.3%) of (R)-3
([˛]2D0 = +9.4◦ (c 2.1 in CHCl3), 45.2% ee) and 227 mg (98%) of (R)-4
([˛]2D0 = +23.3◦ (c 1.2 in CHCl3), >99% ee), respectively.
2.2. Chemical transformations
2.2.1. Synthesis of racemic substrates
Compounds 4ꢀ-chlorophenyl cyclopropyl ketone (1) (2 g,
0.012 mol) and benzyl 4-chlorophenyl ketone (2) (2 g, 0.008 mol)
were treated with NaBH4 (0.8 g, 0.02 mol) in methylene chlo-
ride:methanol 1:1 (400 mL) at room temperature stirring for
24 h. Distillation under reduced pressure to eliminate the solvent
led to
a crude mixture that was neutralised with aque-
ous HCl 10% and extracted with ethyl acetate. The organic
layer was dried over Na2SO4 and the solvent was eliminated
by means of distillation under reduced pressure. The reduc-
tion mixture was chromatographed on
eluting with hexane–ethyl acetate mixtures to give
(4ꢀ-chlorophenyl)-1-cyclopropyl methanol (3) (1.98 g, 98%) and
a
silica gel column
(
)-1-
(
)-1-(4ꢀ-chlorophenyl)-2-phenylethanol (4) (1.85 g, 91%), respec-
2.2.3. Treatment of 1-(4ꢀ-chlorophenyl)-1-cyclopropylmethanol
tively. The 1H NMR spectra of these products were in agreement
with those found in the literature [15,16].
(3) in Czapek-Dox medium
The
compound
(
)-1-(4ꢀ-chlorophenyl)-1-
After purification, compounds
3 (1.44 g, 0.006 mol) or 4
cyclopropylmethanol (3) (10 mg, 0.055 mmol) in 100 mL of
Czapek-Dox medium at pH 4 was stirred at room temperature for
120 h without the fungus B. cinerea. No reaction products were
obtained.
(1.5 g, 0.008 mol) were dissolved in dry pyridine and acetic
anhydride (50 mL) was added dropwise. The reaction mixtures
were stirred for 20 h. The solvent was then removed and
the crude reaction product chromatographed to give
(4ꢀ-chlorophenyl)-1-cyclopropyl methyl acetate (5) (94.1%) and
)-1-(4-chlorophenyl)-2-phenylethyl acetate (6) [14] (93.2%).
)-1-(4ꢀ-chlorophenyl)-1-cyclopropyl methyl acetate (5).
( )-1-
2.3. General procedure for the 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. The
substrate (2 g), dissolved in the 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 filtered through
a large Büchner 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 dried over Na2SO4, and the
solvent then evaporated under reduced pressure to dryness. The
residue obtained was purified by means of column chromatogra-
phy.
Obtained as a colourless oil. IR ꢀmax (film): 2925, 1738, 1615, 1371,
1090, 818; 1H NMR (400 MHz, CDCl3): ı 0.36 (m, 2H, H-3), 0.54
(m, 2H, H-4), 1.25 (m, 1H, H-2), 2.09 (s, 3H, –COOCH3), 5.17 (d, 2H,
J1–2 = 8.7 Hz, H-1), 7.31 (bs, 4H, Ar–H); 13C NMR (100 MHz, CDCl3):
ı 3.0 (t, C-4), 4.1 (t, C-3), 16.4 (d, C-2), 21.2 (c, –COOCH3), 78.4 (d,
C-1), 127.9 (d, C-2ꢀ, C-6ꢀ), 128.5 (d, C-3ꢀ, C-5ꢀ), 133.5 (s, C-4ꢀ), 138.8
(s, C-1ꢀ), 170.2 (s, C O). MS (m/z, %): 226 (M++2, 2), 224 (M+, 5),
164 (M+−60, 71), 138 (100), 77 (50).
Enantiomeric excesses were determined by means of HPLC
analyses on
a chiral column (Chiralcel OD, Daicel, Japan):
254 nm, 0.5 mL/min, hexane:isopropanol (95:5) (R)-3 tR = 18.6 min,
(S)-3 tR = 21.6 min; 0.8 mL/min, hexane:isopropanol (97:3) (S)-4
tR = 20.3 min, (R)-4 tR = 29 min; 0.7 mL/min, hexane:isopropanol
(99.5:0.5) (R)-5 tR = 9 min, (S)-5 tR = 14 min.; 0.7 mL/min, hex-
ane:isopropanol (99.5:0.5) (S)-6 tR = 8 min, (R)-6 tR = 10 min.
2.3.1. 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 (2 g)
was dissolved in 8 mL of allyl alcohol (67 mmol) and 12 mL of hex-
ane (1.2%) and was then added dropwise to the mixture [18]. 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.
2.2.2. Synthesis of enantiomeric alcohols
2.2.2.1. (S)-(−)-1-(4ꢀ-chlorophenyl)-1-cyclopropylmethanol (S)-3.
Under an argon atmosphere, an oven-dried Schlenk tube was
charged with (S)-methyl oxazaborolidine (0.086 mmol, 86 ␮L of
1 M solution in toluene) [17]. The solvent was removed under high
vacuum conditions (0.1 mbar) at room temperature and tetrahy-
drofuran (THF, 11 mL) was added. Once the solution was cooled to
0 ◦C, it was treated with a borane–THF complex (1.0 M, 1.29 mL)
and stirred at room temperature for 2 h. This reagent and a solution
of 4ꢀ-chlorophenyl cyclopropyl ketone (1) (155 mg, 0.997 mmol) in
THF (10 mL) were then added simultaneously from two syringes
into an oven dried, round-bottomed flask at a temperature of 30 ◦C
over a period of 14 h. After the reaction mixture was stirred for
2.4. Lipase-mediated reactions
2.4.1. Lipase-mediated acetylations
A mixture of racemic alcohol 3 or 4 (50 mg), lipase (50 mg), and
vinyl acetate in tert-butylmethyl ether (TBME) (2 mL) was stirred at
room temperature. The residue obtained upon evaporation of the
filtered reaction mixture was chromatographed on a silica gel col-

C. Pinedo-Rivilla et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 61–66
63
Table 1
Results of baker’s yeast-mediated reduction.
Substrate
Allyl alcohol (mM)
Hexane (%)
Conversiona (%)
ee (%)
Time (h)
−
67
−
−
5.0
12.0
1.0
15.3
2.0
27
96
50
67
99
94
72
96
72
96
168
240
1
2
1.2
−
−
−
67
67
1.2
1.2
3.7
a
% of product in the recovered material.
Table 2
Results of lipase-mediated acetylation.
Substrate
Enzyme
Vinyl acetate (mmol)
Temp. (◦C)
Time (h)
Yieldp (%)a
eep (%)
Yields (%)
ees (%)
Conversion (%)
E
PSL
PSL
PSL
PPL
PSL
PSL
PSL
PPL
2.3
0.23
10
RT
RT
RT
RT
RT
RT
48
40
72
47.5
240
240
96
168
264
264
18.0
1.0
29.0
10.0
2.7
15.3
10.0
10.0
97
74
90
>99
54
67
78.4
85.6
84.0
93.0
87.3
80.0
94.0
94.0
88
47.6
51.3
16.5
6.6
31.0
38.5
6.0
190
16
24
>200
4
8
6
73
78
18
7
24
42
6
3
10
0.23
0.23
10
4
70
97
10
6
6.0
a
Yield 100% at 50% conversion after hydrolysis of the acetate derivative.
umn and eluted with hexane:ethyl acetate (95:5). The first eluted
2.5. Microorganism culture and antifungal assays
fractions provided the acetate derivatives (R)-5 ([˛]2D0 = +1 (c 1.1 in
CHCl3), >99% ee) and (R)-6 ([˛]20 = +5 (c 1.7 in CHCl3), 97% ee) while
The culture of B. cinerea employed in this work, B. cinerea
UCA992, was obtained from grapes from the Domecq vineyard,
Jerez de la Frontera, Cádiz, Spain. This culture is deposited in the
Mycological Herbarium Collection (Universidad de Cádiz).
D
the last afforded the unreacted starting material. Detailed results
of the enzyme-mediated acetylations are reported in Table 2.
Treatment of acetate derivatives (R)-5 (300 mg, 1.32 mmol)
and (R)-6 (375 mg, 1.4 mmol), respectively, with KOH (100 mg,
2 mmol) in methanol solution at room temperature for 2 h afforded
239.4 mg (97.8%) of the compound (R)-(+)-1-(4ꢀ-chlorophenyl)-
1-cyclopropyl methanol (R)-3 ([˛]2D0 = +21◦ (c 2.7 in CHCl3),
>99% ee) and 321.2 mg (98.8%) of (R)-(+)-1-(4ꢀ-chlorophenyl)-
2-phenylethanol (R)-4 ([˛]2D0 = +22◦ (c 1.6 in CHCl3), 97% ee),
respectively.
Antifungal activity was determined by means of a conidial ger-
mination assay. The test compound was dissolved in the minimum
amount of ethanol, and water was then added 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 strain were added to a solution of the test com-
pound (15 ␮L) and PDB (potato dextrose broth) medium (2 ␮L)
on ELISA plates. The final ethanol concentration was identical in
control and treated cultures. Three replicates were made per com-
pound and the viability of conidia was estimated by measuring
germination just after incubation at 25 ◦C for 24 h.
2.4.2. Lipase-mediated hydrolysis
Lipase (90 mg of PSL, 60 mg of PPL or CRL) was added to a
mixture of racemic acetates 5 or 6 (0.075 mmol) in 1.5 mL of sol-
vent (mixture of 1,4-dioxane and buffer KH2PO4:KOH 0.1 M pH
7). The solution 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 hex-
ane:ethyl acetate (95:5). The first eluted fractions provided the
unreacted starting material and the last eluted fractions afforded
the alcohol derivatives (R)-(+)-1-(4ꢀ-chlorophenyl)-1-cyclopropyl
methanol (R)-3 ([˛]2D0 = +20◦ (c 1.3 in CHCl3), 98% ee) and (R)-(+)-
1-(4ꢀ-chlorophenyl)-2-phenylethanol (R)-4 ([˛]2D0 = +4.8◦ (c 1.0 in
CHCl3), 21.3% ee), respectively. Detailed results are reported in
Table 3.
2.6. Biotransformation of ( )-1-(4ꢀ-chlorophenyl)-1-cyclopropyl
methanol (3) by B. cinerea UCA992
B. cinerea UCA992 was grown as a surface culture in Roux
bottles at 25◦ C on a Czapek-Dox medium (150 mL per bottle)
for 2 days. Substrate 3 was dissolved in ethanol and then dis-
tributed over 17 Roux bottles (100 ppm per bottle). Fermentation
continued for a further period of 5 days. The mycelium was fil-
tered and washed with brine and ethyl acetate. The broth was
extracted three times with ethyl acetate and the extract dried
over anhydrous sodium sulphate. The solvent was then evapo-
rated and the residue was chromatographed first on a silica gel
Table 3
Results of lipase-mediated hydrolysis.
Substrate
Enzyme
Buffer (%)
Time (h)
Yieldp (%)
eep (%)
Yields (%)
ees (%)
Conv. (%)
E
PPL
PSL
CRL
CRL
CRL
PPL
PSL
CRL
83
83
67
67
20
20
20
20
192
192
480
240
288
288
288
360
38.0
66.0
76.0
85.5
42.0
17.0
46.7
10.0
96
73
71
98
38
36
47
21.3
65.0
28.0
21.0
13.0
40.0
71.0
40.0
75.6
88
70
40
87
10
23
33
12
47.7
49.0
36.0
47.0
20.6
39.0
41.0
36.8
142
13
9
>200
5
2
3
4
2
6
Conv., conversion.

64
C. Pinedo-Rivilla et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 61–66
column and then with HPLC with an increasing gradient of ethyl
acetate to petroleum ether. The following compounds were iso-
lated: recovered ( )-1-(4ꢀ-chlorophenyl)-1-cyclopropylmethanol
(3) (207.5 mg), 4ꢀ-chlorophenyl cyclopropyl ketone (1) (2 mg) and
di(4ꢀ-chlorophenyl cyclopropyl methyl)ether (7) (4 mg).
Di(4ꢀ-chlorophenyl cyclopropyl methyl)ether (7). Obtained as
a colourless oil, anti/syn 50:50 as determined by 1H NMR spec-
troscopy. IR ꢀmax (film): 2923, 1490, 1062, 817. MS (m/z, %): 346
(M+, >0.1), 183 (2), 181 (5), 167 (32), 165 (100), 132 (2), 130 (35).
m/z HRMS (EI, 70 eV) calcd. for C20H20OCl2: 346.08912 [M]+; found:
346.0913. syn-isomer: 1H NMR (400 MHz, CDCl3): ı 0.16 (m, 2H, H-
3), 0.42 (m, 4H, H-3, H-4), 0.66 (m, 2H, H-4), 1.09 (m, 2H, H-2), 3.86
(d, 2H, J = 7.9 Hz, H-1), 7.19 (d, 4H, J = 8.3 Hz, H-2ꢀ, H-6ꢀ), 7.25 (d,
4H, J = 8.3 Hz, H-3ꢀ, H-5ꢀ). 13C NMR (100 MHz, CDCl3): ı 1.6 (t, C-3),
5.0 (t, C-4), 17.3 (d, C-2), 81.7 (d, C-1), 128.2 (d, C-2ꢀ, C-6ꢀ), 128.4
(d, C-3ꢀ, C-5ꢀ), 133.0 (s, C-1ꢀ), 141.1 (s, C-4ꢀ). anti-isomer: 1H NMR
(400 MHz, CDCl3): ı 0.08 (m, 2H, H-3), 0.21 (m, 2H, H-4), 0.36 (m,
2H, H-3), 0.60 (m, 2H, H-4), 1.13 (m, 2H, H-2), 3.25 (d, 2H, J = 8.2 Hz,
H-1), 7.17 (d, 4H, J = 8.3 Hz, H-2ꢀ, H-6ꢀ), 7.31 (d, 4H, J = 8.3 Hz, H-3ꢀ,
H-5ꢀ). 13C NMR (100 MHz, CDCl3): ı 2.5 (t, C-3), 4.6 (t, C-4), 18.3 (d,
C-2), 82.2 (d, C-1), 128.3 (d, C-2ꢀ, C-6ꢀ), 128.6 (d, C-3ꢀ, C-5ꢀ), 133.3 (s,
C-1ꢀ), 140.4 (s, C-4ꢀ). HPLC (Chiralcel OD, Daicel, Japan, hexane/IPA
9.8:0.2, 0.6 mL/min): anti-tR 7.3 min (major) and 9.7 min (minor);
syn-tR 6.7 min (minor) and 7.2 min (major).
Scheme 1.
the transformation and the ee, we therefore tried to inhibit the
reducing enzymes selectively by adding inhibitors such as allyl
alcohol and a small amount of organic solvent [21] to dissolve the
substrate (see Table 1). The addition of allyl alcohol and hexane
improved the ee of both alcohols significantly, but the conversion
itself was diminished for (S)-4: (S)-(−)-1-(4ꢀ-chlorophenyl)-1-
cyclopropyl methanol (S)-3 (12% conversion, [˛]2D0 = −20.1◦ (c 0.9
in CHCl3), 96% ee) and (S)-1-(4ꢀ-chlorophenyl)-2-phenylethanol
(S)-4 (2% conversion, [˛]D20 = −22◦ (c 1.8 in CHCl3), 99% ee), respec-
tively. The spectroscopic data of these products corresponded to
those found in the literature [15,16].
3.2.2. Lipase-mediated transformations
In order to study new methods for the preparation of the enan-
tiomerically pure alcohols 3 and 4, we focused on the kinetic
resolution of the racemic secondary alcohols and acetates, obtained
as described in the experimental section. Lipases are versatile bio-
catalysts acting with high efficiency in acylation or hydrolysis
[11,22].
3. Results and discussion
3.1. Chemical transformations
The chemical preparation of the (S)-enantiomer of 1-(4ꢀ-
chlorophenyl)-2-phenylethanol (S)-4 was previously reported with
excellent yield and enantiomeric excess [14]. Thus, in order to
obtain (R)-1-(4ꢀ-chlorophenyl)-2-phenylethanol (R)-4 and both
enantiomers of 1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol (3)
[15], we employed the same chemical experimental procedure con-
sisting of treatment of 4ꢀ-chlorophenyl cyclopropyl ketone (1) or
benzyl 4ꢀ-chlorophenyl ketone (2) with BH3–THF and (R)- or (S)-
methyl oxazaborolidine [17]. Alcohol (R)-4 was obtained with >99%
ee and 98% yield but results in obtaining (R)- or (S)-3 were moderate
(45.2% ee for (R)-3 and 40.1% ee for (S)-3). Enantiomeric excesses
were determined by HPLC analysis using a Chiralcel OD column.
3.2.2.1. Lipase-mediated acetylations. The starting material 3 or 4
was added to different media with vinyl acetate as an acyl donor
and tert-butyl methyl ether as the organic solvent and three dif-
ferent lipases: lipase PS (from Pseudomonas cepacia), PPL (porcine
pancreas lipase) and CRL (Candida rugosa lipase). The lipases inves-
tigated converted the R enantiomers of 3 and 4 in accordance with
Kazlauskas’ empirical rule for secondary alcohols (Scheme 2) [23].
The results are summarised in Table 2.
The lipase PPL appeared to be more efficient for the enzymatic
acetylation of these substrates affording the highest enantioselec-
tivities. However, this enhanced selectivity was accompanied by a
moderate reaction rate. CRL was rejected as a possible mediator
due to the low selectivity and slow conversions observed when it
was used. Lipase PSL showed high enantioselectivity and yield for
compound 3 but low selectivity for alcohol 4.
In an attempt to increase the low conversion rate and enan-
tioselectivity showed for substrate 4 with all the lipases at room
temperature, we also studied the effect of temperature on reaction
conditions. We found that transesterification at the optimal tem-
perature of each enzyme increased both parameters, conversion
and enantiomeric excesses. This indicates that temperature may
accelerate the kinetics of the enzyme. Different reaction times and
vinyl acetate concentrations were also tested (see Table 2).
3.2. Biocatalytic transformations
3.2.1. Baker’s yeast reduction
Based on the well known ability of baker’s yeast fermentation
to reductively transform a variety of ketones into optically active
alcohols with (S)-configuration (Prelog specificity family) [18], we
decided to use a baker’s yeast-mediated approach to enantiopu-
rify (S)-(−)-1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol (S)-3 and
(S)-(−)-1-(4ꢀ-chlorophenyl)-2-phenylethanol (S)-4, using the cor-
responding ketones 1 and 2 as substrates. The results are shown in
Table 1.
A major limitation of this methodology is the difficulty in
extracting the product from the fermentation broth; consequently,
often only moderate yields of products are obtained, presumably
due to absorption of the starting material and/or product within
the larger quantity of yeast cells [19,20].
As shown in Table 1, after the incubation of ketones 1 and 2 in
fermenting baker’s yeast following the conventional procedure (see
Section 2), a single transformation product was isolated, (S)-(−)-
1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol (S)-3 (5% conversion,
[˛]2D0 = −5.7◦ (c 1.9 in CHCl3), 27% ee) and (S)-1-(4ꢀ-chlorophenyl)-
2-phenylethanol (S)-4 (15.3% conversion, [˛]2D0 = −15.4◦ (c 1.5 in
CHCl3), 67% ee), respectively (Scheme 1). In order to improve
OH
OH
OAc
R
Lipase
R
R
+
TBME/vinyl acetate
Cl
Cl
Cl
3 R=
(S)-3 R=
(R)-5 R=
4
R= -CH2Ph
(S)-4 R= -CH2Ph
(R)- R= -CH2Ph
6
Scheme 2.

C. Pinedo-Rivilla et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 61–66
65
OAc
R
OAc
R
OH
Lipase
R
+
1,4-dioxane/buffer
Cl
Cl
Cl
5 R=
6 R= -CH2Ph
(S)-5 R=
(R)-3 R=
Scheme 4.
(S)-6 R= -CH2Ph
(R)- R= -CH2Ph
4
Scheme 3.
extracted with ethyl acetate and the extract dried over anhy-
drous sodium sulphate. In addition to the starting material (see
Scheme 4), the purification of the organic dry extract afforded
two compounds: 4ꢀ-chlorophenyl cyclopropyl ketone (1), and com-
pound 7, a mixture of diastereoisomers 50:50 of the corresponding
dimer of the biotransformation starting material which could be
separated by HPLC giving syn-7 (>99% ee) and anti-7 (30% ee). The
presence of these compounds as biotransformation products shows
the interesting enzymatic potential of the fungus B. cinerea UCA992
as a biocatalyst.
As mentioned above, apart from the ketone 1, the new com-
pound 7 was isolated from the biotransformation. Many of the
signals in their 1H and 13C NMR spectra were similar to those
of 1-(4ꢀ-chlorophenyl)-1-cyclopropylmethanol (3). However, the
signal corresponding to the hydroxyl group on C-1 was absent
while the signal assigned to H-1 was more deshielded, indicat-
ing that the hydroxy group of 3 had been etherificated with
another molecule of the alcohol. In addition, the HRMS of the
compound confirmed the structure of the new product as di(4ꢀ-
chlorophenyl cyclopropyl methyl)ether (7). To confirm that this
compound was a biotransformation product and not a condensa-
tion product from the chemical reaction between two molecules
of the starting material of the biotransformation, 3, we treated 1-
(4ꢀ-chlorophenyl)-1-cyclopropylmethanol (3) in the culture broth
under the same conditions as those of the biotransformation, but
without the fungus. No condensation compound was obtained.
The antifungal activity of the biotransformation products
against the fungus B. cinerea was studied and proved to be less
active than the alcohol used as substrate (for product 1 [10]) or
completely inactive (compound 7). This fact confirms the existence
of a detoxification mechanism by the phytopathogen.
The best results for 3 and 4 were obtained with lipase PPL giv-
ing acetate derivatives with yields of 10% and >99% and 97% ee,
respectively, using 10 mmol of vinyl acetate as the acyl donor.
After hydrolysis of the acetate derivatives with potassium
hydroxide in methanol, the enantiopure alcohols were identified as
(R)-(+)-1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol (R)-3 ([˛]2D0
=
+21◦ (c 2.7 in CHCl3), >99% ee) and (R)-(+)-1-(4ꢀ-chlorophenyl)-
2-phenylethanol (R)-4 ([˛]2D0 = +22◦ (c 1.6 in CHCl3), 97% ee),
respectively. In general, the results obtained for alcohol (R)-4 are
moderate owing to its low solubility which decreases the transfor-
mation.
3.2.2.2. Lipase-mediated hydrolysis. Herein we report on the
enzyme mediated enantioselective hydrolysis of acetates 1-
(4ꢀ-chlorophenyl)-1-cyclopropyl methyl acetate (5) and 1-(4-
chlorophenyl)-2-phenylethyl acetate (6) [14].
The racemic acetate derivatives were prepared by adding
the corresponding alcohols to a solution of acetic anhydride in
dichloromethane:methanol (1:1). The acetates were then trans-
formed by lipases dissolved in a mixture of 1,4-dioxane and
different concentrations of buffer solution (see Scheme 3). The
results of the enzymatic hydrolysis are summarized in Table 3.
The 1-(4ꢀ-chlorophenyl)-1-cyclopropyl methyl acetate (5)
was transformed to (R)-(+)-1-(4ꢀ-chlorophenyl)-1-cyclopropyl
methanol (R)-3 with
a high ee of 98% with a conversion
of 47% using CRL as catalyst and 67% of buffer. For 1-(4ꢀ-
chlorophenyl)-2-phenyethanol (4) the enantiomer obtained was
(R)-(+)-1-(4ꢀ-chlorophenyl)-2-phenyethanol (R)-4 in only one case,
using CRL as biocatalyst with 20% aqueous buffer. The low solubil-
ity of this compound makes it very difficult for the reaction to take
place with moderate amounts of water (necessary for the hydroly-
sis reaction). Thus, the final product (R)-4 with high enantiomeric
excess could not be obtained by means of this methodology.
5. Conclusions
The studies described above have demonstrated the potential of
several methods, biocatalytic and chemical, to obtain enantiomeri-
cally pureforms of1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol(3)
and 1-(4ꢀ-chlorophenyl)-2-phenylethanol (4), which had already
proven to be highly active against B. cinerea [10].
The best selectivity-yield relationship for the reduction of ben-
zyl 4ꢀ-chlorophenyl ketone (2) was observed after treatment with
oxazaborolidine [17], yielding compounds (R)-4 and (S)-4 [14], with
>99% ee and 98% yield. However, results for ketone 1 were only
moderate using this methodology.
4. Detoxification by B. cinerea
The biotransformation of the fungistatic agent 1-(4ꢀ-
chlorophenyl)-2-phenyethanol (4) by the phytopathogen B.
cinerea has been studied in a previous report [14]. In incorporation
experiments of 4 at a concentration of 150 ppm, B. cinerea was
unable to detoxify this compound. When biotransformation was
carried out at a lower concentration, the ketone derivative was
obtained as the only product, benzyl 4ꢀ-chlorophenyl ketone (2),
which showed less antifungal activity than the corresponding
alcohol 4 against the fungus B. cinerea [10], suggesting that the
fungus has its own detoxification mechanism.
We were thus able to obtain enantiomerically pure alcohols
of 1-(4ꢀ-chlorophenyl)-1-cyclopropyl methanol (3) by means of
biocatalytic methods. The best result for the preparation of (S)-3
(96% ee) was observed after fermentation of 4ꢀ-chlorophenyl cyclo-
propyl ketone (1) with baker’s yeast cells in the presence of allyl
alcohol and hexane, while the best R selectivity-yield relationship
for compound 3 was after lipase-mediated hydrolysis of 1-(4ꢀ-
chlorophenyl)-1-cyclopropyl methyl acetate (5) to give (R)-3 with
98% ee.
Thus, in order to investigate the detoxification mechanisms for
antifungal compound 3, the biotransformation of that alcohol by B.
cinerea UCA992 was studied.
4.1. Detoxification of ( )-1-(4ꢀ-chlorophenyl)-1-cyclopropyl
methanol (3) by B. cinerea UCA992
Compounds (R)- and (S)-1-(4ꢀ-chlorophenyl)-2-phenylethanol
(R)- and (S)-4 were also prepared by enzymatic methods with excel-
lent enantioselectivity results. Thus, (S)-4 was obtained with 99%
ee by means of baker’s yeast reduction in the presence of addi-
Biotransformation was carried out on surface cultures using
Roux bottles for five days, after which the mycelium was filtered,

66
C. Pinedo-Rivilla et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 61–66
tives while the best result for (R)-4 (97% ee) was observed after
lipase-mediated acetylation with PPL followed the hydrolysis of
the acetate.
Cristina Pinedo Rivilla studied biochemistry at the Uni-
versity of Sevilla, where she graduated in 2004. After
studying a M.S. in microbial biotechnology in 2008, she
received her Ph.D. degree in 2009 under the supervi-
sion of Prof. Collado and Prof. Aleu Casatejada on the use
of filamentous fungi as biocatalysts in the preparation
of enantiomerically pure compounds and the enantiose-
lective biocatalytic synthesis of bioactive molecules. She
is currently a postdoctoral student at the University of
Cádiz, Department of Organic Chemistry, and her cur-
rent research interests are directed towards the putative
role of the phytotoxins excreted by Botrytis cinerea in
the infection mechanism and the design of new selective
Based on previous results pointing to the high antifungal activ-
ity of enantiopure alcohols 3 and 4 [10] against the phytopathogen
B. cinerea, and the detoxification mechanism exhibited by 4 [14],
we also carried out the biotransformation of 1-(4ꢀ-chlorophenyl)-
1-cyclopropyl methanol (3) by the fungus. Biotransformation
products have been shown to be less toxic to fungal growth than
3, confirming the existence of a detoxification mechanism by the
phytopathogen. The main detoxification reaction involved oxida-
tion of the starting material. It is also interesting to note the
isolation of a new dimer compound of ( )-1-(4ꢀ-chlorophenyl)-1-
cyclopropylmethanol (3) as a detoxification product by B. cinerea
UCA992.
fungicides.
Antonio J. Bustillo Pérez studied chemistry at the Uni-
versity of Cádiz, where he graduated in 1999. He received
his Ph.D. degree in 2006 under the supervision of Prof.
Aleu and Prof. Hernández-Galán on biocatalytic synthesis
of selective fungicides. He is currently working at Bodegas
Garvey (Jerez de la Frontera) as Head of the laboratory of
chemistry.
Acknowledgements
This work was supported by grants from MICINN (AGL2009-
13359-C02-01) and from Junta de Andalucía (P07-FQM-02689).
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