Studies on biotransformation of (±)-1-(4′-chlorophenyl)-2- phenylethanol

Article abstract of DOI:10.1016/j.tetasy.2003.08.026

The metabolism of (±)-1-(4′-chlorophenyl)-2-phenylethanol 1 by the phytopathogenic fungi Colletotrichum gloeosporioides and Botrytis cinerea has been studied. Regioselective monooxygenase-catalyzed hydroxylation of C-H bonds yielded the corresponding diols, the ratios of which showed a clear preference for the C-H benzylic bond. m-Hydroxylation in the substituted aromatic ring, a rarely described phenomenon, has been observed and with good enantioselectivity. The chemistry involved in determining the configuration of the biotransformation products is also described.

Full text of DOI:10.1016/j.tetasy.2003.08.026

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3755–3760
Studies on biotransformation of
( )-1-(4%-chlorophenyl)-2-phenylethanol
Antonio J. Bustillo, Carlos M. Garc´ıa-Pajo´n, Josefina Aleu, Rosario Herna´ndez-Gala´n and
Isidro G. Collado*
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias, Universidad de Ca´diz, Apdo. 40, 11510 Puerto Real, Ca´diz, Spain
Received 29 July 2003; accepted 27 August 2003
Abstract—The metabolism of ( )-1-(4%-chlorophenyl)-2-phenylethanol 1 by the phytopathogenic fungi Colletotrichum gloeospori-
oides and Botrytis cinerea has been studied. Regioselective monooxygenase-catalyzed hydroxylation of CꢀH bonds yielded the
corresponding diols, the ratios of which showed a clear preference for the CꢀH benzylic bond. m-Hydroxylation in the substituted
aromatic ring, a rarely described phenomenon, has been observed and with good enantioselectivity. The chemistry involved in
determining the configuration of the biotransformation products is also described.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
and plants.5,6 Based on previous results, from which the
presence of a hydroxyl group was determined to be
fundamental for the expression of the antifungal activ-
ity of the compounds studied,7,8 we have thus chosen to
study the resveratrol analogue ( )-1-(4%-chlorophenyl)-
Over the past few years, our research programme has
been directed towards the rational design of fungicides
for use against fungal infections of commercial crops.
Since phytoalexins are known to exhibit significant
antifungal activity,1 we have undertaken a screening of
analogous compounds2 in order to find substrates with
similar antifungal properties.
2-phenylethanol 1, which contains
a
secondary
hydroxyl group in its structure.
2. Results and discussion
There are, however, many reports on the degradation
of plant anti-microbial compounds by phytopathogenic
fungi via a variety of mechanisms; for example, isolates
of Botrytis cinerea have been shown to detoxify the
grapevine phytoalexin resveratrol.3 In many cases the
degradation products are actually less toxic to fungal
growth than the parent compounds.4 Some fungi have
even been reported to detoxify more than one plant
compound.
One of our main objectives was to elucidate the
metabolism of ( )-1-(4%-chlorophenyl)-2-phenylethanol
1 by C. gloeosporioides and B. cinerea (Scheme 1) and
to characterize and, where possible, to assign the stereo-
chemistry to the metabolites.
Compound 1, obtained by means of chemical reduction
of commercial benzyl 4%-chlorophenyl ketone 2, was
incubated for nine days on shaken cultures of either C.
gloeosporioides or B. cinerea at a concentration of 150
ppm on a Czapek-Dox medium and then extracted with
ethyl acetate. As a control, cultures of only the fungi or
a medium containing only compound 1 were also incu-
bated under similar conditions and extracted. These
control extracts were compared with those from the
biotransformation experiments by means of TLC.
Our investigations have thus focused on the metabolism
of diverse compounds that are analogous to the phy-
toalexins produced by the agriculturally important
pathogens Botrytis cinerea and Colletotrichum gloeospo-
rioides. These two species are highly destructive plant-
pathogenic fungi, responsible for diseases such as grey
mould in grapes and anthracnose in strawberry fruits
In addition to the starting material, the biotransforma-
tion of ( )-1-(4%-chlorophenyl)-2-phenylethanol 1 by C.
* Corresponding author. Tel.: +34-956-016368; fax: +34-956-016193;
e-mail: isidro.gonzalez@uca.es
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.08.026

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A. J. Bustillo et al. / Tetrahedron: Asymmetry 14 (2003) 3755–3760
less for predicting the enantiofacial selectivity of the
dihydroxylation process for different alkene types.11,12
Second, comparison of the specific rotations of the
obtained diols with those described in the literature for
(1R,2R)-1-(4%-chlorophenyl)-2-phenylethane-1,2-diol13
and (1R,2R) and (1S,2S)-1,2-diphenyl-1,2-ethane-1,2-
diol supported this assignment.14
Asymmetric dihydroxylation of (E)-1-(4%-chlorophenyl)-
2-phenylethene 10, prepared from 1 by acidic treatment
using a and b AD-mix (H2O:t-BuOH, 20°C) led to
(1S,2S)-3 and (1R,2R)-3, respectively. The enan-
tiomeric excess values observed for these diols were
found to be over 99%.
Further metabolism of diol 2 led to the ketoalcohol 4
(wmax 1679 cm−1, lC 198.6 ppm; wmax 3418 cm−1, lC 75.4
ppm), which was found to be present with an excess of
the (R) enantiomer (7.5% ee). The absolute configura-
tion was tentatively assigned as (R) after comparison of
its specific rotation sign with those of commercial (R)-
and (S)-benzoin. This transformation seems to be due
to the action of a dehydrogenase enzyme.
Scheme 1. Biotransformation of 1 by C. gloeosporoides.
gloeosporioides afforded four compounds: (1RS,2SR)-1-
(4%-chlorophenyl)-2-phenylethane-1,2-diol 2, (1S,2S)-1-
(4%-chlorophenyl)-2-phenylethane-1,2-diol 3, (R)-2-
hydroxy-2-(4%-chlorophenyl)acetophenone 4 and benzyl
4%-chlorophenyl ketone 5. The acetylation of the polar
fraction with acetic anhydride–pyridine led to the isola-
tion of an additional three compounds: (1R)-2-(2%-ace-
toxyphenyl)-1-(4¦-chlorophenyl)ethyl acetate 6, (1R)-
2-(3%-acetoxyphenyl)-1-(4¦-chlorophenyl)ethyl acetate 7
and ( )-2-(4%-acetoxyphenyl)-1-(4¦-chlorophenyl)ethyl
acetate 8.
Isolation of ketone 5 from the fermentation broth
confirmed the presence of a dehydrogenase. The spec-
troscopic data of 5 were identical to those of the
commercial product benzyl 4%-chlorophenyl ketone 5.
It is interesting to note that while the hydroxylation of
aromatics in the o- and p-positions to existing sub-
stituents is frequently achieved by means of monooxy-
genases, m-hydroxylation is rarely observed.15
The recovered starting material showed [h]2D0=−0.2
(CHCl3, c=1.8): with 14.1% ee, indicating that the
fungus exhibited a slight preference for the enantiomer
(1R)-(+)-1-(4%-chlorophenyl)-2-phenylethanol (R)-1 in
the biotransformation. The absolute configuration of
the residual alcohol was proposed to be S after its
specific rotation sign was compared with that described
for (1R)-1-(4-fluorophenyl)-2-phenylethan-1-ol, which
was obtained by means of a chemical reaction via
boronic esters.9 In addition, alcohol (2S)-(−)-1 was
obtained with 99% ee and 98% yield, by means of
treatment of benzyl 4%-chlorophenyl ketone 2 with BH3–
THF and (S)-methyl oxazaboralidine.10
During the metabolism of 1, however, we observed the
hydroxylation in all three positions. Thus the respective
spectroscopic data of compounds 6, 7 and 8, which
were isolated from the fermentation broth after acetyla-
tion, not only pointed to hydroxylation in the o-, m-
and p-positions of the aromatic ring without a chloro
substituent, but also confirmed that these compounds
were in fact 2-(2%-acetoxyphenyl)-1-(4¦-chlorophenyl)-
ethyl
acetate
6,
2-(3%-acetoxyphenyl)-1-(4¦-
chlorophenyl)ethyl acetate 7 and 2-(4%-acetoxyphenyl)-
1-(4¦-chlorophenyl)ethyl acetate 8. Compounds 6 and 7
showed [h]2D0=−3.6 (CHCl3, c=0.8): 92.7% ee, and
[h]2D0=−4.0 (CHCl3, c=0.5): 94.8% ee, respectively,
indicating that monooxygenase exhibited a high prefer-
ence for the (R) enantiomer. Compound 8 was found to
be racemic. The absolute configurations of alcohols 6
and 7 were both assigned as (R) after their specific
rotation signs were compared with those obtained for
(1S)-(−)-1-(4-chlorophenyl)-2-phenylethyl acetate 9.
Compound 9 was obtained after acetylation of alcohol
(1S)-(−)-1.
Diols 2 and 3 were the two major biotransformation
products. Comparison of their spectra with that of
starting material 1 indicated that these compounds were
two
diasteromers
of
1-(4%-chlorophenyl)-2-
phenylethane-1,2-diol [h]2D0=0 (CHCl3, c=0.7): for 2
and [h]2D0=−100.4 (CHCl3, c=4.2): with 91.3% ee for 3.
The hydroxylation on C-2 of 1 indicates that this
biotransformation is most likely due to the action of a
monooxygenase.
In contrast, no biotransformation products were
obtained during the feeding experiment of B. cinerea
with ( )-1-(4%-chlorophenyl)-2-phenylethanol 1 at 150
ppm. Mycelial growth was not observed during the
biotransformation and study of the fermentation broth
only led to the isolation of fatty acids, indicating that
B. cinerea was unable to detoxify this compound. One
The absolute configuration of compound
3 was
assigned as (1S,2S) on the basis of two considerations.
First, this assignment is in agreement with both the
preferred configurations of diols produced using a- and
b-forms of AD-mix and the model developed by Sharp-

A. J. Bustillo et al. / Tetrahedron: Asymmetry 14 (2003) 3755–3760
3757
reason for this may be that the concentration of the
starting material was too high for the fungus, and thus
inhibited fungal growth.
1 cm wide, 25 cm long). Chemicals were products of
Fluka or Aldrich. All solvents used were freshly dis-
tilled. Enantiomeric excesses were determined by means
of HPLC analyses on a chiral column (Chiralcel OD,
Daicel, Japan).
To confirm this, ( )-1-(4%-chlorophenyl)-2-phenyl-
ethanol 1 was incubated at a lower concentration (100
ppm) under the same conditions on shaken cultures of
B. cinerea. In this case, in addition to the starting
4.2. Biotransformation by Colletotrichum
gloeosporioides
material,
the
biotransformation
of
( )-1-(4%-
chlorophenyl)-2-phenylethanol 1 afforded benzyl 4%-
chlorophenyl ketone 4. The remaining alcohol 1 was
found to be of low enantiopurity (6% ee) and of the
(1R) configuration.
The culture of Colletotrichum gloeosporioides employed
in this work, 20122-CECT, was obtained from the
‘Centro Espan˜ol de Cultivos Tipo’ (CECT), Facultad
de Biolog´ıa, Universidad de Valencia, Spain, where a
culture of this strain is deposited.
3. Conclusions
C. gloeosporioides was grown on an agar-malta-peptone
medium in a Petri dish. Ten-day-old mycelium was used
to inoculate 28 (500 ml) flasks, each containing 250 ml
of Czapek-Dox medium: glucose (50 g), yeast extract (1
g), KH2PO4 (5 g), NaNO3 (2 g), MgSO4 (0.5 g) and
FeSO4 (10 mg), per litre of distilled water. The pH was
adjusted to 7.0 with aqueous NaOH. For 2 days, flasks
were incubated at 25°C and stirred at 180 rpm; the
mycelium was then filtered and transferred into 26 (500
ml) flasks containing 250 ml of Czapek-Dox medium
(with only 25 g of glucose) and the substrate (( )-1-(4%-
chlorophenyl)-2-phenylethanol 1, 150 ppm per flask).
The remaining two flasks were used as the control. 9
days after inoculation, the mycelium was filtered and
the fermentation broth was extracted three times with
ethyl acetate. The extract was dried over anhydrous
sodium sulphate and the solvent was then evaporated.
Fractionation of the extract (870 mg) was carried out
by means of column chromatography on silica gel
(SiCC), eluting with petroleum ether: ethyl acetate mix-
tures containing increasing percentages of ethyl acetate
to give 5 fractions (A–E). Final purification of fraction
A (40 mg, petroleum ether) was carried out by means of
semi-preparative HPLC (hexane:ethyl acetate 97:3; 2.5
mL min−1) to afford benzyl 4%-chlorophenyl ketone 5 (5
mg). Compounds 1 and 4 were obtained after further
purification of fraction B (409 mg, petroleum ether:
ethyl acetate 85:15) with SiCC under conditions similar
to those described above. Final purification was carried
out by means of semi-preparative HPLC (hexane:ethyl
acetate 90:10; 3.2 mL min−1) to afford recovered 1-(4%-
chlorophenyl)-2-phenylethanol 1 (211 mg) and (2R)-2-
(4%-chlorophenyl)-2-hydroxy-1-phenylethanone 4 (3 mg,
The biotransformation of ( )-1-(4-chlorophenyl)-2-
phenylethanol 1 by C. gloeosporioides led to hydroxyla-
tion and further oxidation at C-1 and C-2, as well as to
hydroxylation of the aromatic ring. The lack of hydrox-
ylation of the 4-chloro aromatic ring indicated the
regioselectivity of the monooxygenase.
In incorporation experiments of ( )-1-(4-chlorophenyl)-
2-phenylethanol 1 at a concentration of 150 ppm, B.
cinerea was not able to detoxify this compound. When
the biotransformation was carried out at a lower con-
centration, a ketone derivative was obtained as the only
product.
In conclusion, fungal biodegradation of ( )-1-(4-
chlorophenyl)-2-phenylethanol 1 using C. gloeospori-
oides and B. cinerea has generally been found to
proceed via a monooxygenase-catalyzed hydroxylation
of CꢀH bonds to yield compounds 2, 3, 6, 7 and 8. The
compound ratios indicate a clear preference for the
CꢀH benzylic bond. m-Hydroxylation in the substituted
aromatic ring, which is rarely described, has been
observed with high enantioselectivity (94.8% ee). In
addition, dehydrogenase-catalyzed oxidation occurred
at secondary benzylic groups to yield ketones 4 and 5.
4. Experimental
4.1. General experimental procedures
Optical rotations were determined with a Perkin–Elmer
241 polarimeter. IR spectra were recorded on a
Mattson Genesis spectrophotometer, series FTIR. H
7.5% ee). Diols
2
(RS,SR)-1-(4%-chlorophenyl)-2-
phenylethane-1,2-diol (98 mg) and 3 (1S,2S)-1-(4%-
chlorophenyl)-2-phenylethane-1,2-diol (96 mg, 91.3%
ee), eluted in fraction C (280 mg, petroleum ether: ethyl
acetate 70:30), were obtained via purification on semi-
preparative HPLC (hexane:ethyl acetate 70:30; 3.1 mL
min−1).
1
and 13C NMR measurements were obtained on Varian
Gemini 200 and Varian Unity 400 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 4250)
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 chro-
matography. Purification by means of HPLC was
accomplished using a silica gel column (Hibar 60, 7 m,
Acetylation of the polar fractions (D–E, 51 mg) fol-
lowed by final purification with semi-preparative HPLC
(hexane:ethyl acetate 88:12; 3.3 mL min−1) led to the
isolation of the following compounds: (1R)-2-
(2%-acetoxyphenyl)-1-(4¦-chlorophenyl)ethyl acetate
6
(10 mg, 92.7% ee), (1R)-2-(3%-acetoxyphenyl)-1-(4¦-
chlorophenyl)ethyl acetate 7 (16 mg, 94.8% ee) and

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A. J. Bustillo et al. / Tetrahedron: Asymmetry 14 (2003) 3755–3760
( )-2-(4%-acetoxyphenyl)-1-(4¦-chlorophenyl)ethyl ace-
tate 8 (7 mg).
i-PrOH 90:10 as eluent, flow rate 1.0 mL/min, tR/min=
7.4 ((R)-4), 10.12 ((S)-4).
4.2.1. (1S)-1-(4%-Chlorophenyl)-2-phenylethanol 1.16
White solid; mp 46–48°C; [h]2D0=−0.2 (CHCl3, c=1.8):
14.1% ee. The enantiomeric excess was measured by
means of chiral HPLC with n-hexane/i-PrOH 97:3 as
eluent, flow rate 0.8 mL/min, tR/min=20.3 ((S)-1), 29.0
((R)-1).
4.2.5. Benzyl (4%-chlorophenyl) ketone 5. White solid; mp
102–103°C; IR wma1x (film): 3093, 3027, 2898, 1687, 1589,
1090, 1071 cm−1; H NMR (400 MHz, CDCl3): l 4.24
(2H, s, H-2), 7.25 (3H, ddd, J=7.1, 7.1, 2.9 Hz, H-2¦,
H-6¦, H-4¦), 7.33 (2H, ddd, J=7.2, 7.1, 2.9 Hz, H-3¦,
H-5¦), 7.41 (2H, d, J=8.7 Hz, H-3%, H-5%), 7.93 (2H, d,
J=8.8 Hz, H-2%, H-6%); 13C NMR (100 MHz, CDCl3): l
45.5 (t, C-2), 126.9 (d, C-4¦), 128.6 (d, C-3%, C-5%), 128.8
(d, C-3¦, C-5¦), 129.3 (d, C-2¦, C-6¦), 129.9 (d, C-2%,
C-6%), 134.0 (s, C-1%), 134.6 (s, C-1¦), 139.5 (s, C-4%),
196.2 (s, C-1); EIMS m/z: 230 (M+, 5), 165 (15); 141
(95), 139 (100), 113 (50), 111 (90), 91 (93), 75 (80).
4.2.2.
(1RS,2SR)-1-(4%-Chlorophenyl)-2-phenylethane-
1,2-diol 2. White solid; mp 119–121°C; [h]2D0=0 (CHCl3,
c=0.7): IR wmax (film): 3358, 3296, 2901, 1028 cm−1; H
1
NMR (400 MHz, CDCl3): l 2.29 (1H, s, OH), 2.34
(1H, s, OH), 4.81 (2H, bs, H-1, H-2), 7.12 (2H, d,
J=8.4 Hz, H-2%, H-6%), 7.17–7.21 (2H, m, H-6¦, H-2¦),
7.24 (2H, d, J=8.5 Hz, H-3%, H-5%), 7.27–7.31 (3H, m,
H-3¦, H-5¦, H-4¦); 13C NMR (100 MHz, CDCl3): l 77.
3 (d, C-1), 77.9 (d, C-2), 126.9 (d, C-2¦, C-6¦), 128.1 (d,
C-2%, C-6%, C-4¦), 128.2 (d, C-3¦, C-5¦), 128.3 (d, C-3%,
C-5%), 133.6 (s, C-4%), 138.0 (s, C-1%), 139.2 (s, C-1¦);
EIMS m/z: 232 (M+−18+2, 2), 230 (M+−18, 8), 214
(M+−18−18+2, 28), 212 (M+−18−18, 5), M+−36; 179
(40), 178 (43), 139 (77), 111 (48), 105 (86), 77 (100);
HREIMS m/z 248.0600 [M+] (calcd. for C14H13O2Cl,
248.0626).
4.2.6.
(1R)-2-(2%-Acetoxyphenyl)-1-(4¦-chlorophenyl)-
ethyl acetate 6. Colourless oil; [h]2D0=−3.6 (CHCl3, c=
0.8): 92.7% ee; IR wmax (film): 3023, 2925, 2853, 1739,
1235 cm−1; H NMR (400 MHz, CDCl3): l 2.03 (3H, s,
1
CH3COO), 2.35 (3H, s, CH3COO), 2.85 (1H, dd, J=
13.6, 7.4 Hz, H-2a), 3.20 (1H, dd, J=13.6, 6.8 Hz,
H-2b), 5.87 (1H, dd, J=7.4, 6.8 Hz, H-1), 7.01–7.07
(3H, m, H-3%, H-5%, H-4%), 7.14 (2H, d, J=8.5 Hz, H-2¦,
H-6¦), 7.20–7.23 (1H, m, H-6%), 7.25 (2H, d, J=8.4 Hz,
H-3¦, H-5¦); 13C NMR (100 MHz, CDCl3): l 21.0 (c,
C6 H3COO), 21.1 (c, C6 H3COO), 37.5 (t, C-2), 74.7 (d,
4.2.3. (1S,2S)-1-(4%-Chlorophenyl)-2-phenylethane-1,2-
diol 3. White solid; mp 100–102°C; [h]2D0=−100.4
(CHCl3, c=4.2): 91.3% ee; IR wmax (film): 3400, 3061,
3031, 2930, 1493, 1091, 1017 cm−1; 1H NMR (400
MHz, CDCl3): l 3.13 (1H, s, OH), 3.27 (1H, s, OH),
4.53–4.61 (2H, m, H-1, H-2), 6.94–6.99 (2H, m, H-2%,
H-6%), 7.02–7.08 (2H, m, H-6¦, H-2¦), 7.13–7.18 (2H, m,
H-3%, H-5%), 7.20–7.25 (3H, m, H-3¦, H-5¦, H-4¦); 13C
NMR (100 MHz, CDCl3): l 78.4 (d, C-1), 79.1 (d, C-2),
126.9 (d, C-2¦, C-6¦), 128.0 (d, C-4¦), 128.08 (d, C-2%,
C-6%), 128.1 (d, C-3¦, C-5¦), 128.2 (d, C-3%, C-5%), 133.4
(s, C-4%), 138.1 (s, C-1%), 139.3 (s, C-1¦); EIMS m/z: 214
(M+−36, 58), 179 (70), 178 (83), 139 (75), 111 (58), 105
(94), 77 (100); HREIMS m/z 248.0619 [M+] (calcd. for
C14H13O2Cl, 248.0604). The enantiomeric excess was
determined to be diacetate (see general procedure),
measured by means of chiral HPLC with n-hexane/i-
PrOH 99.7:0.3 as eluent, flow rate 0.8 mL/min, tR/
min=34.0 ((S,S)-3Ac), 49.2 ((R,R)-3Ac).
C-1), 122.4 (d, C-3%), 125.8 (d, C-5%), 127.9 (d, C-4%),
128.1 (d, C-6%), 128.5 (d, C-2¦, C-6¦), 131.6 (d, C-3¦,
C-5¦), 133.8 (s, C-1%, C-4¦), 138.1 (s, C-1¦), 149.4 (s,
C-2%), 169.5 (s, CH3C6 OO), 170.0 (s, CH3C6 OO); EIMS
m/z: 274 (M+−60+2, 9), 272 (M+−60, 14), 232 (38), 230
(93), 195 (18), 165 (51), 141 (26), 107, (100), 77 (20);
HREIMS m/z 332.0811 [M+] (calcd. for C18H17O4Cl,
332.0837). The enantiomeric excess was measured by
means of chiral HPLC with n-hexane/i-PrOH 90:10 as
eluent, flow rate 0.8 mL/min, tR/min=10.6 ((R)-6), 16.5
((S)-6).
4.2.7.
(1R)-2-(3%-Acetoxyphenyl)-1-(4¦-chlorophenyl)-
ethyl acetate 7. Colourless oil; [h]2D0=−4.0 (CHCl3, c=
0.5): 94.8% ee; IR wmax (film): 3044, 2930, 2860, 1763,
1209 cm−1; H NMR (400 MHz, CDCl3): l 2.01 (3H, s,
1
CH3COO on C-3%), 2.27 (3H, s, CH3COO on C-1), 3.0
(1H, dd, J=13.7, 6.3 Hz, H-2a), 3.15 (1H, dd, J=13.8,
7.7 Hz, H-2b), 5.87 (1H, dd, J=7.5, 6.5 Hz, H-1), 6.83
(1H, bs, H-4%), 6.92 (2H, bd, J=7.9 Hz, H-2%, H-6%),
7.17 (2H, d, J=8.5 Hz, H-2¦, H-6¦), 7.22 (1H, dd,
J=7.7, 7.7 Hz, H-5%), 7.27 (2H, d, J=8.4 Hz, H-3¦,
H-5¦); 13C NMR (100 MHz, CDCl3): l 21.1 (c,
4.2.4. (2R)-2-Hydroxy-2-(4%-chlorophenyl) acetophenone
4. White solid; mp 88–90°C; [h]2D0=−0.8 (CHCl3, c=
0.3): 7.5% ee; IR wmax (film): 3418, 3063, 2917, 2858,
1
1679, 1595, 1087 cm−1; H NMR (400 MHz, CDCl3): l
4.54 (1H, s, OH), 5.92 (1H, s, H-2), 7.26 (2H, d, J=8.3
Hz, H-2%, H-6%), 7.29 (2H, d, J=8.1 Hz, H-3%, H-5%),
7.40 (2H, ddd, J=7.7, 7.7, 3.3 Hz, H-3¦, H-5¦), 7.54
(1H, ddd, J=7.4, 7.4, 2.5 Hz, H-4¦), 7.88 (2H, bdd,
J=8.0, 3.4 Hz, H-2¦, H-6¦); 13C NMR (100 MHz,
CDCl3): l 75.4 (d, C-2), 128.8 (d, C-3¦, C-5¦), 129.1 (d,
C-2¦, C-6¦, C-3%, C-5%), 129.3 (d, C-2%, C-6%), 133.2 (s,
C-4%), 134.1 (d, C-4¦), 134.5 (s, C-1%), 137.5 (s, C-1¦),
198.6 (s, C-1); EIMS m/z: 139(28), 141 (45), 111 (38),
105 (100), 77 (60); HREIMS m/z 246.0473 [M+] (calcd.
for C14H11O2Cl, 246.0506). The enantiomeric excess
was measured by means of chiral HPLC with n-hexane/
C6 H3COO), 21.2 (c, C6 H3COO), 42.6 (t, C-2), 75.6 (d,
C-1), 119.8 (d, C-4%), 122.6 (d, C-2%), 126.9 (d, C-6%),
127.8 (d, C-2¦, C-6¦), 128.5 (d, C-3¦, C-5¦), 129.2 (d,
C-5%), 133.7 (s, C-4¦), 138.10 (s, C-1¦), 138.1 (s, C-1%),
150.4 (s, C-3%), 169.3 (s, CH3C6 OO), 169.9 (s, CH3C6 OO);
EIMS m/z: 274 (M+−60+2, 17), 272 (M+−60, 46), 232
(48), 230 (65), 195 (26), 165 (77), 150 (100), 141 (95),
107 (88), 77 (52); HREIMS m/z 332.0814 [M+] (calcd.
for C18H17O4Cl, 332.0837). The enantiomeric excess
was measured by means of chiral HPLC with n-hexane/
i-PrOH 90:10 as eluent, flow rate 0.8 mL/min, tR/min=
10.12 ((R)-7), 15.32 ((S)-7).

A. J. Bustillo et al. / Tetrahedron: Asymmetry 14 (2003) 3755–3760
3759
4.2.8. ( )-2-(4%-Acetoxyphenyl)-1-(4¦-chlorophenyl)ethyl
acetate 8. Colourless oil; [h]2D0=0 (CHCl3, c=0.4): IR
it was treated with a borane–THF complex (1.0 M, 1.0
mL) and stirred at room temperature for 2 h. This
reagent and a solution of benzyl 4%-chlorophenyl ketone
5 (155 mg, 0.671 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 1 h. After the reaction mixture was
stirred for 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 150 mg (98%) of the (S) alcohol,
[h]2D0=−23.0 (CHCl3, c=2.9): 99% ee.
1
wmax (film): 3037, 2928, 2854, 1739, 1234, 1196 cm−1; H
NMR (400 MHz, CDCl3): l 2.02 (3H, s, CH3COO),
2.27 (3H, s, CH3COO), 2.99 (1H, dd, J=13.8, 6.5 Hz,
H-2a), 3.16 (1H, dd, J=13.8, 7.5 Hz, H-2b), 5.86 (1H,
dd, J=6.9, 6.9 Hz, H-1), 6.96 (2H, d, J=8.7 Hz, H-3%,
H-5%), 7.06 (2H, d, J=8.6 Hz, H-2%, H-6%), 7.17 (2H, d,
J=8.5 Hz, H-2¦, H-6¦), 7.27 (2H, d, J=8.5 Hz, H-3¦,
H-5¦); 13C NMR (100 MHz, CDCl3): l 21.2 (c,
2C6 H3COO), 42.2 (t, C-2), 75.8 (d, C-1), 121.3 (d, C-3%,
C-5%), 127.9 (d, C-2%, C-6%), 128.5 (d, C-2¦, C-6¦), 130.4
(d, C-3¦, C-5¦), 133.7 (s, C-4¦), 134.0 (s, C-1%), 138.2 (s,
C-1¦), 149.3 (s, C-4%), 169.4 (s, CH3C6 OO), 169.9 (s,
CH3C6
OO); EIMS m/z: 274 (M+−60+2, 8), 272 (M+−60,
20), 232 (36), 230 (100), 195 (12), 165 (48), 150 (34), 141
(42), 107 (94), 77 (30); HREIMS m/z 332.0800 [M+]
(calcd. for C18H17O4Cl, 332.0815).
4.4.3. (E)-1-(4%-Chlorophenyl)-2-phenylethene 10. After
dissolution in 1,4-dioxane (15 ml), 3.01 g (12.95 mmol)
4.3. Biotransformation by B. cinerea
of ( )-1-(4%-chlorophenyl)-2-phenylethanol
1
were
placed in a 500 ml round-bottom flask and 5 ml of
H2SO4:H2O 2:1 was added slowly to the reaction mix-
ture. The solution was then stirred at room temperature
for 10 min. After this period, it was neutralised with
sodium carbonate and extracted with ethyl acetate. The
residue was purified by means of chromatography with
hexane as eluent to give the compound (E)-1-(4%-
The culture of Botrytis cinerea employed in this work,
B. cinerea 2100, was obtained from the ‘Centro Espan˜ol
de Cultivos Tipo’ (CECT), Facultad de Biolog´ıa, Uni-
versidad de Valencia, Spain, where a culture of this
strain is deposited.
1
Biotransformation
of
( )-1-(4%-chlorophenyl)-2-
chlorophenyl)-2-phenylethene 10 (2.53 g, 91%). The H
phenylethanol 1 by B. cinerea was carried out as
described above for the Colletotrichum experiment (at a
concentration of 150 ppm), but in this case, the fungus
was inoculated by conidia. No biotransformation prod-
ucts were obtained.
NMR spectrum of this product was in agreement with
that found in the literature.17,18
4.4.4. Sharpless dihydroxylation
4.4.4.1. (1S,2S)-1-(4%-Chlorophenyl)-2-phenylethane-
1,2-diol (1S,2S)-3. Distilled water (1.2 ml) and tert-
butanol (1.2 ml) were added to AD mix a (0.681 g) in
a 10 ml round-bottom flask and the resulting suspen-
sion was stirred at 0°C. Stirring was maintained during
addition of the substrate (E)-1-(4%-chlorophenyl)-2-
phenylethene 10 (0.098 g, 0.46 mmol) in tert-
butanol:H2O 1:1 (1 ml). Then, methanesulphonamide
(0.044 g, 0.46 mmol) was added. The reaction mixture
was stirred vigorously in the dark at room temperature.
After 50 h, anhydrous sodium sulphite (0.731 g, 5.8
mmol) was added and stirring continued for 1 h. After
extraction with ethyl acetate (3×20 ml), purification on
silica gel afforded the compound (1S,2S)-1-(4%-
chlorophenyl)-2-phenylethane-1,2-diol (1S,2S)-3 (0.085
g, 75%), which showed [h]2D0=−114.2 (CHCl3, c=1.9).
This product exhibited >99% ee upon chiral HPLC
analysis.
However, at a lower substrate concentration (100 ppm
per flask), in addition to compounds characteristic of B.
cinerea, the following biotransformation products were
also isolated from the extract (847 mg): recovered 1-(4%-
chlorophenyl)-2-phenylethanol 1 (410 mg, [h]2D0=+3.1
(CHCl3, c=0.5): 6% ee and benzyl 4%-chlorophenyl
ketone 2 (44 mg).
4.4. Chemical transformations
4.4.1. ( )-1-(4%-Chlorophenyl)-2-phenylethan-1-ol 1. To
1000 mg of benzyl 4-chlorophenyl ketone dissolved in a
mixture of dichlomethane:methanol 1:1, 400 mg of
NaBH4 were added. The reaction mixture was stirred at
room temperature for 1 h. Distillation under reduced
pressure to eliminate the solvent led to a crude mixture
that was neutralised with aqueous 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 to yield
925.6 mg (91%) of 1.16
4.4.4.2. (1R,2R)-1-(4%-Chlorophenyl)-2-phenylethane-
1,2-diol (1R,2R)-3. AD mix b (0.890 g) was added to
distilled water (1.5 ml) and tert-butanol (1.5 ml). The
mixture was stirred at 0°C. Stirring was maintained
during
addition
of
the
substrate
(E)-1-(4%-
4.4.2.
(1S)-(−)-1-(4%-Chlorophenyl)-2-phenylethan-1-ol
chlorophenyl)-2-phenylethene 10 (0.116 g, 0.54 mmol)
in tert-butanol:H2O 1:1 (1.2 ml). Then, methane-
sulphonamide (0.057 g, 0.54 mmol) was added. The
reaction mixture was stirred vigorously in the dark at
room temperature. After 24 h, anhydrous sodium
sulfite (0.956 g, 7.6 mmol) was added and stirring
continued for 1 h. After extraction with ethyl acetate
(−)-1. Under an argon atmosphere, an oven-dried
Schlenk tube was charged with (S)-methyl oxazabo-
ralidine (0.84 mmol, 0.84 ml of 1 M solution in tolu-
ene). The solvent was removed under high vacuum (0.1
mbar) at room temperature and tetrahydrofuran (THF,
11 ml) was added. After the solution was cooled to 0°C,

3760
A. J. Bustillo et al. / Tetrahedron: Asymmetry 14 (2003) 3755–3760
(3×20 ml), purification on silica gel afforded the com-
pound (1R,2R)-1-(4%-chlorophenyl)-2-phenylethane-1,2-
diol (1R,2R)-3 (0.101 g, 75%), which showed
[h]2D0=+105 (CHCl3, c=2.8). This compound exhib-
ited >99% ee upon chiral HPLC analysis.
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(1S)-(−)-1-(4-Chlorophenyl)-2-phenylethyl
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J=8.0 Hz, H-2¦, 6¦), 7.17 (2H, d, J=8.6 Hz, H-2%,
6%), 7.20–7.26 (3H, m, H-3¦, 5¦, 4¦), 7.26 (2H, d, J=
8.4 Hz, H-3%, 5%); 13C NMR (100 MHz, CDCl3): l
21.12 (c, C6 H3CO), 42.80 (t, C-2), 75.96 (d, C-1),
126.69 (d, C-4¦), 128.02 (d, C-2¦, 6¦), 128.30 (d, C-3¦,
5¦), 128.53 (d, C-2%, 6%), 129.49 (d, C-3%, 5%), 133.72 (s,
C-4%), 136.51 (s, C-1%), 138.48 (s, C-1¦), 170.01 (s,
CH3C6 O); EIMS m/z: 216 (28), 214 (75), 179 (85) 178
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This work was supported by grants from DGICYT
AGL2000-0635-C02-01 and AGL2002-04388-C02-01.