Hydride transfer versus electron transfer in the reduction of 4-phenyl-3-halo-3-buten-2-ones mediated by Pichia stipitis

Article abstract of DOI:10.1016/j.molcatb.2011.07.001

Reductions of (Z)-C₆H₅CHCXC(O)CH₃ (X = Cl, Br) mediated by Pichia stipitis gave 4-phenylbutan-2-one through dehalogenation of intermediaries 3-halo-4-phenylbutan-2-one by an electron transfer mechanism. The addition of 1,3-dinitrobenzene avoids the dehalogenation and thus the corresponding (2S,3S)-halohydrins were obtained in excellent enantiomeric excesses by a hydride transfer mechanism. Irganox 1010 and 1076 were also used to inhibit the electron transfer mechanism. The obtained halohydrins are important chiral building blocks to obtain optically active epoxides and aminoalcohols.

Full text of DOI:10.1016/j.molcatb.2011.07.001

Journal of Molecular Catalysis B: Enzymatic 72 (2011) 289–293
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Hydride transfer versus electron transfer in the reduction of
4-phenyl-3-halo-3-buten-2-ones mediated by Pichia stipitis
Dávila S. Zampieri, Luiz A. Zampieri, J. Augusto R. Rodrigues, Bruno R.S. de Paula, Paulo J.S. Moran∗
Institute of Chemistry, University of Campinas, C.P. 6154, 13084-971 Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Reductions of (Z)-C6H5CH CXC( O)CH3 (X = Cl, Br) mediated by Pichia stipitis gave 4-phenylbutan-
2-one through dehalogenation of intermediaries 3-halo-4-phenylbutan-2-one by an electron transfer
mechanism. The addition of 1,3-dinitrobenzene avoids the dehalogenation and thus the corresponding
(2S,3S)-halohydrins were obtained in excellent enantiomeric excesses by a hydride transfer mecha-
nism. Irganox® 1010 and 1076 were also used to inhibit the electron transfer mechanism. The obtained
halohydrins are important chiral building blocks to obtain optically active epoxides and aminoalcohols.
© 2011 Elsevier B.V. All rights reserved.
Received 20 April 2011
Received in revised form 24 June 2011
Accepted 4 July 2011
Available online 13 July 2011
Keywords:
Haloenones
Halohydrins
␣-Haloketones
Enantioselective bioreduction
Pichia stipitis
Electron transfer mechanism
1. Introduction
In this work, we found in the preliminary experiments that the
reduction of haloenones (Z)-3-chloro-4-phenyl-3-buten-2-ones 1a
The enones have been investigated as potential substrates for
biocatalytic reductions that can lead to the introduction of 1, 2
or 3 new chiral centers into an achiral structure [1–3]. Therefore,
the bioreduction of C C and C O conjugated double bonds is an
important method for preparing chiral building blocks. The Baker’s
yeast (Saccharomyces cerevisiae) is the most used microorganism
to mediate the reduction of enones [2,4–9], although some other
microorganisms such as Beauveria sulfurescens ATCC 7159 [10],
Pichia stipitis CCT 2617 [11], Pyrococcus furiosus DSM 3638 [12],
Geotrichum candidum, Mortierella isabellina, Rhodotorula rubra [13],
have also been used for this purpose.
The enantioselective reductions of C C and C O bonds of ␣-
haloenones are very useful to give halohydrins in high ee, that can
be converted to enantioenriched epoxides. The later compounds
are very useful synthetic intermediates and were used in the prepa-
ration of a number of biological active natural products such as
aminoalcohols derivatives [14]. There are some papers describing
the ␣-haloenones bioreduction including the reduction of a series
of 3-fluor-3-alken-2-ones [15], 3-chloro-3-alken-2-ones [16] and
␣-bromoenones [6] both mediated by Baker’s yeast giving the cor-
responding halohydrins.
and (Z)-3-bromo-4-phenyl-3-buten-2-ones 1b mediated by P. stipi-
tis gave the dehalogenated product 4-phenylbutan-2-one 2 instead
of the corresponding halohydrins (Scheme 1).
The
methylcinnamaldehyde mediated by Baker’s yeast giving
2-methyl-3-phenylpropan-1-ol was proposed in cascade
dehalogenation
mechanism
of
3-chloro-2-
a
reaction sequence: reduction of C C bond to provide 3-chloro-
2-methyl-3-phenylpropanaldehyde followed by a spontaneous
elimination of HCl, and finally the reduction of C C and C O bonds
[17]. A similar spontaneous elimination of HCl was suggested for
dehalogenation of 3-chloropropiophenone during experiments
of Baker’s yeast reduction to give propiophenone [18]. Taking
into account those cascade reaction sequence, the reduction of
haloenones 1a and 1b will provide at first the corresponding
␣-haloketone due to reduction of C C bond. Since it is known that
the ␣-bromoketone does not spontaneously eliminate HBr [19]
as does ␤-haloaldehyde and ␤-haloketone, the obtaining dehalo-
genated product 4-phenylbutan-2-one (Scheme 1) stimulates us
to study the mechanism of 1a and 1b biotransformation.
The dehalogenation of ␣-haloketones mediated by microor-
ganisms is an interesting reaction that generally is observed
in bioreductions of ␣-iodoacetophenone [20], ethyl 2-
chloroacetoacetate [21,22], 2-chloro-3-oxoesters [23,24] and ethyl
4-chloroacetoacetate [25]. The dehalogenation of ␣-haloketones
by microorganisms might be associated with glutathione [22] or
by a mechanism that involve a radical NAD species [20]. The ␣-
∗
Corresponding author. Tel.: +55 19 3788 3048; fax: +55 19 3788 3023.
E-mail address: moran@iqm.unicamp.br (P.J.S. Moran).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.07.001

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D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 289–293
O
O
70
Pichia stipitis
72 h, 30 o
C
60
X
2
1a X=Cl
1b X=Br
50
40
30
Scheme 1.
haloketones have been used as mechanistic probe in the reduction
reactions of NADH-dependent horse liver alcohol dehydrogenase
(NADH/HLADH) [26], in reductions mediated by Baker’s yeast [20],
in reductions using 1,4-dihydronicotinamides [27–29] and even
for identification of reductants in sediments [30]. Dehalogenated
ketone is the product obtained by a multistep electron transfer (e−,
H•, or e−, H+, e− as has been suggested) [31,32] while halohydrin
is obtained by hydride transfer process in which the mechanism
is discussed elsewhere [33]. Generally, a small quantity of 1,3-
dinitrobenzene (DNB), an efficient radical ion scavenger [34], is
added in order to inhibit the free radical process [20,26–29]. If
an enzyme mediates a hydride transfer process, the product is
an optically active halohydrin since the DNB did not hinder the
enzyme controlled formation of halohydrin [26]. Therefore, in
this work, the reduction reaction of the haloenones 1a and 1b
mediated by P. stipitis is investigated and the results are discussed
in the light of the above mentioned concepts.
20
10
0
10
20
30
40
50
60
70
80
time reaction (h)
Fig. 2. Bioreduction of (Z)-3-bromo-4-phenyl-3-buten-2-one (ꢀ) by P. stipitis in
aqueous medium giving 3-bromo-4-phenyl-2-butanone (᭹) as transient and 4-
phenyl-2-butanone (ꢁ) as final product.
temperature was kept 270 ◦C and detector was kept at 280 ◦C.
The column temperature was held at 80 ◦C for 3 min and then
increase to 280 ◦C at a rate of 10 ◦C/min. 1 ␮L of a compound solu-
tion (1 mg/mL) in ethyl acetate was injected and the retention
times in min are reported for each compound; GC/MS(B) analy-
ses were obtained on an Agilent 6890 Series GC System and mass
spectra were recorded with a Hewlett-Packard 5973 Mass Selec-
tive Detector (70 eV) using a HP-5MS fused silica capillary column
(crosslinked 5% phenyl ethyl siloxane, 30 m × 0.25 m ID × 0.25 ␮m
film thickness) and helium as a carrier gas (1.2 mL/min). The split
ratio was 1:30. The injector temperature was kept 270 ◦C and detec-
tor was kept at 280 ◦C. The column temperature was held at 80 ◦C
for 3 min and then increase to 180 ◦C at a rate of 20 ◦C/min and
from 180 ◦C to 290 ◦C at a rate of 30 ◦C/min and then kept con-
stant for 3 min. 1 ␮L of a compound solution (1 mg/mL) in ethyl
acetate was injected and the retention times in min are reported
for each compound. Chiral GC/FID analyses were obtained on an
Agilent 6850 Series GC System, using a Hydrodex chiral capillary
column (30 m × 0.25 mm × 0.25 ␮m). Hydrogen was used as a car-
rier gas (1 mL/min), the injector temperature was 200 ◦C and the
detector temperature was 220 ◦C. The column temperature was
held at 80 ◦C and then increase to 180 ◦C at a rate of 1 ◦C/min,
and then kept constant for 5 min. 1 ␮L of a compound solution
(1 mg/mL) in ethyl acetate was injected and the retention times
in min are reported for each compound. Thin-layer chromatog-
raphy (TLC) analyses were performed with precoated aluminum
sheets (silica gel 60 Merck), and flash column chromatography
was carried out on silica (200–400 mesh, Merck). trans-4-Phenyl-
3-buten-2-one, Oxone® and triethylamine were purchased from
Aldrich. Sodium chloride, sodium bromide and dichloromethane
were purchased from Synth. 4-Phenylbutan-2-one was obtained by
reduction of trans-4-phenyl-3-buten-2-one with H2/Pd-C in ethyl
acetate, and 4-phenylbutan-2-ol was obtained by reduction of 4-
phenylbutan-2-one with NaBH4 in EtOH. All reagents were used
as received. The yeast strain P. stipitis CCT 2617 is stored at André
Tosello Research Foundation (Campinas-SP, Brazil) [35].
2. Experimental
2.1. Materials and methods
1H NMR spectra were determined at 250 MHz (Varian Gemini
250) or 500 MHz (INOVA 500). 13C NMR spectra were deter-
mined at 62.5 MHz (Varian Gemini 250) or 125.7 MHz (INOVA
500). Chemical shifts are reported in ppm relative to tetram-
ethylsilane (TMS, ı = 0.00 for 1H) in CDCl3. IR spectra were
recorded on a FT-IR BOMEM MB-100 from Hartmann and Braun
and absorptions are reported in cm−1. GC/MS(A) analyses were
obtained on a QP 5000-Shimadzu (to provide data presented in
Figs. 1 and 2) using a silica capillary column DB1 from J&W Sci-
entific (30 m × 0.25 m ID × 0.25 ␮m film thickness) and helium as
a carrier gas (1.0 mL/min). The split ratio was 1:30. The injector
70
60
50
40
30
20
10
0
10
20
30
40
50
60
70
80
2.2. Growth conditions of P. stipitis
time reaction (h)
P. stipitis CCT 2617 was cultivated in YM (yeast-malt extract)
nutrient broth (1000 mL) under aseptic conditions, incubation at
30 ◦C on an orbital shaker (180 rpm) for 1 day just before use. All
Fig. 1. Bioreduction of (Z)-3-chloro-4-phenyl-3-buten-2-one (ꢀ) by P. stipitis in
aqueous medium giving 3-chloro-4-phenyl-2-butanone (᭹) as transient and 4-
phenyl-2-butanone (ꢁ) as final product.

D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 289–293
291
materials and medium were sterilized in an autoclave at 121 ◦C
before use and the yeast was manipulated in a laminar flow cabinet.
with sodium sulfate, the solvent evaporated, and the products were
purified by column chromatography (hexane/ethyl acetate 9:1).
2.3. Procedure for the P. stipitis mediated bioreduction of
trans-4-phenyl-3-buten-2-one
2.7. General procedure for the epoxide ring closure from 4a and
4b
trans-4-Phenyl-3-buten-2-one (20 mg) dissolved in 0.5 mL of
ethanol was added to an Erlenmeyer containing 20 mL of slurry
of growing P. stipitis cells. The reaction mixture was incu-
bated in an orbital shaker (200 rpm, 30 ◦C) for 72 h. Then,
the product was extracted with dichloromethane. The solvent
was dried with sodium sulfate, evaporated, and the products
were analyzed by GC/MS giving 24.3% of 4-phenylbutan-2-one
(retention time = 5.9 min), 1.6% of 4-phenylbutan-2-ol (retention
time = 6.1 min) and 73.9% of trans-4-phenyl-3-buten-2-one (reten-
tion time = 7.2 min).
An aqueous solution of NaOH (2 M, 1.2 mL) was added drop wise
to a round-bottom flask containing a solution of 4a or 4b (4 mmol)
in diethyl ether (5 mL). The reaction mixture was maintained under
magnetic stirring for 6 h. After that, the organic layer was sepa-
rated, the aqueous layer was extracted with dichloromethane and
the combined organic layers were dried under sodium sulfate. The
solvent was evaporated and the product was purified by column
chromatography (hexane/ethyl acetate 9:1).
2.8. (Z)-3-Chloro-4-phenyl-3-buten-2-one (1a)
2.4. General procedure for the preparation of ˛-haloenones 1a
and 1b [36]
Following the general procedure for preparation of ␣-
haloenones, compound 1a was obtained in 20% yield as pale yellow
oil; retention time on GC/MS(A): 7.68 min, GC/MS(B): 8.57 min EI-
MS m/z (relative intensity): 51 (14), 63 (6), 75 (15), 102 (55), 115
(5), 129 (20), 137 (24), 145 (28), 165 (25), 179 (100), 180 (M+, 81),
181 (42), 182 (27); IR (film) 3353, 3055, 2928, 1686, 1596, 1573,
1491, 1447, 1359, 756, 690; 1H NMR (250 MHz, CDCl3): ı 2.56 (s,
3H), 7.41–7.43 (m, 3H), 7.75 (s, 1H), 7.83–7.87 (m, 2H); 13C NMR
(62.5 MHz, CDCl3): ı 26.81, 128.65, 130.19, 130.39, 130.86, 132.74,
135.62; 193.37.
Oxone® (25.2 g, 40 mmol) and NaCl (2.4 g, 40 mmol) or NaBr
(4.1 g, 40 mmol) were added to an ice bath cooled mixture of
dichloromethane (100 mL) and water (20 mL) in a 250 mL in
a round-bottom flask. The mixture was stirred for 40 minutes,
and after that trans-4-phenyl-3-buten-2-one (3.0 g, 20 mmol) was
added and the mixture was stirred for 4 h. After that, triethylamine
(8.6 mL, 30 mmol) was added with a dropping funnel, and the reac-
tion mixture was stirred for 1 h. After that, the reaction mixture
was neutralized with a 1 M HCl solution, the organic layer was sepa-
rated, and the aqueous phase was extracted with dichloromethane.
The combined organic fractions were dried with sodium sulfate and
the solvent was evaporated. The product 1a was purified by prepar-
ative TLC (silica gel containing 10% AgNO3 in mass; elution solvent
as hexane/ethyl acetate 9:1) and the 1b product was purified by
preparative TLC (silica gel and hexane/ethyl acetate 9:1).
2.9. (Z)-3-Bromo-4-phenyl-3-buten-2-one (1b)
Following the general procedure for preparation of ␣-
haloenones, compound 1a was obtained in 78% yield as yellow oil;
retention time on GC/MS(A): 8.21 min, GC/MS(B): 9.19 min, EI-MS
m/z (relative intensity): 51 (23), 63 (13), 77 (23), 102 (98), 145 (100),
181 (10), 183 (9), 210 (9), 212 (8), 224 (M+, 53), 226 (52); IR (film):
3102, 30,29, 2922, 2855, 1685, 1602, 1480, 1446, 1356, 1219, 1171,
1073; 1H NMR (500 MHz, CDCl3): ı 2.60 (s, 3H), 7.43–7.45 (m, 3H),
7.85–7.87 (m, 2H), 8.03 (s, 1H); 13C NMR (125 MHz, CDCl3): ı 27.02,
123.25, 128.50, 130.42, 130.47, 133.71, 139.92, 193.13.
2.5. General procedure for the P. stipitis mediated bioreduction of
˛-haloenones 1a and 1b
Procedure A (to collect data shown in Figs. 1 and 2): compound
1a or 1b (10 mg) dissolved in 1 mL of ethanol was added in each of
8 Erlenmeyer flasks containing 10 mL of slurry of growing P. stipi-
tis cells. The reaction mixtures were incubated in an orbital shaker
(200 rpm at 30 ◦C). After the appropriate intervals, one Erlenmeyer
was collected and the products were extracted with ethyl acetate,
dried with sodium sulfate, the solvent was evaporated and the
products analyzed by GC/MS. The results are shown in Fig. 1 for
1a and Fig. 2 for 1b.
Procedure B (for isolation the reaction product): compound 1a
or 1b (100 mg) dissolved in 1 mL of ethanol was added in an Erlen-
meyer flask containing 200 mL of slurry of growing P. stipitis cells.
The reaction mixture was incubated in an orbital shaker (200 rpm
at 30 ◦C) for 72 h. Then, the product was extracted in a continuous
extractor with dichloromethane for 2 days. The solvent was dried
with sodium sulfate, evaporated, and the products were purified
by column chromatography (hexane/ethyl acetate 9:1).
2.10. 4-Phenyl-2-butanone (2)
Following the general procedure for bioreduction of haloenones
1a and 1b mediated by P. stipitis (Procedure B), after 72 h at
30 ◦C compound 2 was obtained as colorless oil; retention time on
GC/MS(A): 7.30 min, GC/MS(B): 6.79 min, EI-MS m/z (relative inten-
sity): 51 (12), 65 (9), 77 (23), 91 (64), 105, (92), 133 (19), 148 (M+,
100); IV (film): 3085, 3062, 3027, 3002, 2922, 1718, 1602, 1583,
1496, 1453, 1408, 1357, 1283, 1228, 1161, 1080, 749, 699; 1H NMR
(250 MHz, CDCl3): ı 2.14 (s, 3H), 2.77 (t, 2H, J = 8 Hz), 2.91 (t, 2H,
J = 8 Hz), 7.16–7.31 (m, 5H); 13C NMR (62.5 MHz, CDCl3): ı 29.72,
30.07, 45.16, 126.10, 128.49, 148.29, 140.97, 207.95.
2.11. 3-Chloro-4-phenyl-2-butanone (3a)
2.6. General procedure for the P. stipitis mediated bioreduction of
˛-haloenones 1a and 1b with addition of m-dinitrobenzene
Following the general procedure for bioreduction of haloenone
1a mediated by P. stipitis (Procedure A), compounds 1a, 2 and 3a
were detected by GC/MS in each Erlenmeyer flasks (data shown
in Fig. 1). Retention time of compound 3a on GC/MS(A): 7.78 min,
GC/MS(B): 8.63 min, EI-MS m/z (relative intensity): 43 (100), 63
(14), 77 (25), 91 (4), 103 (22), 115 (5), 129 (28), 145 (21), 182 (5),
184 (2).
DNB (1 mg) was added to an Erlenmeyer flask containing 200 mL
of slurry of growing P. stipitis cells and the mixture was stirred for
40 min in an orbital shaker (200 rpm, 30 ◦C). Thus, 100 mg 1a or 1b
dissolved in 1 mL of ethanol were added. The stirring was continued
for 24 h. Then, the products were extracted in a continuous extrac-
tor with dichloromethane for 2 days. The organic phase was dried

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D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 289–293
O
O
O
2
OH
Pichia stipitis
DNB
X
X
3a: X = Cl
3b: X = Br
1a: X = Cl
1b: X = Br
X
(2S,3S)-4a: X = Cl
(2S,3S)-4b: X = Br
Scheme 2.
2.12. 3-Bromo-4-phenyl-2-butanone (3b)
show the same retention time and spectra data: retention time on
GC/MS(A): 6.10 min EI-MS m/z (relative intensity): 51 (12), 65 (13),
78 (58), 91 (100), 105 (37), 131 (92), 148 (M+, 3); 1H NMR (250 MHz,
CDCl3): ı 1.41 (d, 3H, J = 6 Hz), 2.81 (dd, 1H, J1 = 14 Hz, J2 = 6 Hz), 2.95
(dd, 1H, J1 = 14 Hz, J2 = 6 Hz), 3.13–3.18 (m, 2H), 7.08–7.36 (m, 5H);
13C NMR (62.5 MHz, CDCl3): ı 13.43, 34.01, 52.90 (C-2), 57.31 (C-3),
126.54, 128.62, 129.20, 137.80.
Following the general procedure for bioreduction of haloenone
1b mediated by P. stipitis (Procedure A), compounds 1b, 2 and 3b
were detected by GC/MS in each Erlenmeyer flasks (data shown
in Fig. 1). Retention time of compound 3b on GC/MS(A): 7.93 min,
GC/MS(B): 8.27 min, EI-MS m/z (relative intensity): 43 (100), 63
(56), 77 (95), 91 (27), 103 (80), 115 (29), 129 (98), 145 (96), 193
(6), 196 (6), 211 (3), 213 (2), 226 (8), 228 (8).
3. Results and discussion
2.13. (2S, 3S)-4-phenyl-3-chloro-2-butanol (4a)
The enones 1a and 1b were prepared by reaction of trans-4-
phenyl-3-buten-2-one with Oxone® and the salts NaCl and NaBr
respectively. Subsequently, triethylamine was added to the result-
ing mixture following the procedure published elsewhere [36]. In
both cases, the major isomers obtained have (Z)-structure and were
isolated by column chromatography. The assign of the stereoiso-
mers structure are supported by the appearance of vinylic hydrogen
singlet in their NMR spectra that is down field for (Z) relatively to
(E)-isomers [37].
Both reduction of 1a and 1b mediated by P. stipitis yielded 4-
phenyl-2-butanone 2 as the only product after 72 h in an orbital
shaker at 200 rpm and 30 ◦C (Scheme 1). In order to investi-
gate those biotransformations, the reactions were monitorated
by GC/MS giving the reaction profiles presented in Figs. 1 and 2,
that are typical for a consecutive reaction, showing the transient
3-halo-4-phenyl-2-butanones 3a and 3b as intermediaries in the
production of 2. Generally, the C C bond of enones is reduced by
an enoate reductase [3]. The microorganism P. stipitis used in this
work has enoate reductases since trans-4-phenyl-3-buten-2-one
was reduced by this microorganism to give 4-phenylbutan-2-one
as the major product after 72 h at 30 ◦C. An electronegative group
bound to the ␣-carbon of ketones 3a–b like bromo and chloro
should activate the C O bond to be reduced producing the cor-
responding halohydrins, as has been observed in reduction of 1b
mediated by Baker’s yeast [6]. But, it is clear in this work that the
dehalogenation reaction of ␣-haloketones 3a–b prevail upon the
reduction of C O bond.
Taking into account that the production of ketone is due to
dehalogenation of ␣-haloketone that may be proceed by a free rad-
ical chain process, a small quantity of 1,3-dinitrobenzene (DNB)
was added in order to inhibit the free radical process. It is worth to
mention that the addition of small quantity of DNB was effective to
avoid dehalogenation of ␣-haloacetophenones in reduction reac-
tion using isolated enzyme NADH/HLADH [26] and dehalogenation
of ␣-iodoacetophenone in the reduction reaction mediated by
Baker’s yeast [20].
The experiments were carried out in duplicate with haloenones
1a and 1b varying quantities of DNB in the reaction mixture from
0.1 mg to 8.0 mg of DNB added to 200 mL of growing P. stipitis
slurry. The best result in terms of conversion and inhibition of the
Following the general procedure for bioreduction of haloenones
mediated by P. stipitis with addition of DNB, after 24 h at 30 ◦C com-
pound 4a was obtained in 38% yield as pale yellow oil; [˛]2D5 −20 (c
1.0, CHCl3); Chiral GC/FID analyses show 97% ee, retention time on
GC/FID: 24.71 min for (2S,3S) and 24.85 min for (2R,3R). Retention
time on GC/MS(B): 8.22 min EI-MS m/z (relative intensity): 51 (11),
65 (14), 78 (67), 91 (100), 105 (37), 131 (59), 148 (67), 184 (M+, 5),
186 (2); IR (film): 3399, 3025, 2978, 2930, 1603, 1494, 1447, 1365,
1260, 1131, 750, 693; 1H NMR (250 MHz, CDCl3): ı 1.30 (d, 3H,
J = 6 Hz), 3.05 (dd, 1H, J1 = 14 Hz, J2 = 8 Hz), 3.23 (dd, 1H, J1 = 14 Hz,
J2 = 6.5 Hz), 3.89–3.96 (m, 1H), 4.05 (ddd, 1H, J1 = 8.3 Hz, J2 = 6.5 Hz,
J3 = 3.3 Hz), 7.22–7.35 (m, 5H); 13C NMR (62.5 MHz, CDCl3): ı 20.87,
41.14, 68.46, 69.87, 126.83, 128.50, 129.33, 137.62.
2.14. (2S, 3S)-4-phenyl-3-bromo-2-butanol (4b)
Following the general procedure for bioreduction of haloenones
mediated by P. stipitis with addition of DNB, after 24 h at 30 ◦C
compound 4b was obtained in 87% yield as pale yellow oil; [˛]D25
−21 (c 1.0, CHCl3) [lit. −28◦, c 0.03, CHCl3 for (2S,3S)-isomer, >95%
ee] [19]; chiral GC/FID analyses show 98% ee, retention time on
GC/FID: 23.10 min for (2S,3S) and 23.28 min for (2R,3R). Reten-
tion time on GC/MS(A): 7.82 min GC/MS(B): 7.94 min EI-MS m/z
(relative intensity): 51 (8), 65 (9), 78 (26), 91 (86), 105 (56), 131
(100), 148 (34), 228 (M+, 2), 230 (1); IR (film): 3402, 3026, 2978,
2929, 1603, 1492, 1445, 725, 694, 672; 1H NMR (250 MHz, CDCl3):
ı 1.29 (d, 3H), 3.17 (dd, 1H, J1 = 14.3 Hz, J2 = 8 Hz), 3.37 (dd, 1H,
J1 = 14 Hz, J2 = 7 Hz), 3.69–3.79 (m, 1H), 4.21 (ddd, 1H, J1 = 8 Hz,
J2 = 7 Hz, J3 = 3 Hz), 7.22–7.35 (m, 5H); 13C NMR (62.5 MHz, CDCl3):
ı 22.24, 42.09, 65.56, 68.13, 126.91, 128.57, 129.26, 138.29.
2.15. (2S, 3R)-4-Phenyl-2,3-epoxybutane (5)
Following the general procedure for epoxy ring closure from 4a,
compound 5 was obtained as colorless oil showing [˛]2D5 −25 (c 1.0,
CHCl3) [lit. −21◦, c 0.04, CHCl3 for (2S,3R)-isomer, >99% ee] [19].
Following the same procedure from 4b, compound 5 was obtained
as a colorless oil showing [∞]2D5 −22 (c 1.0, CHCl3). Both products

D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 289–293
293
radical reaction was obtained with 1.0 mg of DNB according to
Acknowledgments
GC/MS analyses of the reaction products. Interestingly, the corre-
sponding halohydrins 4a and 4b were formed in 38 and 87% yield in
high diastereoisomer ratio syn:anti (>99:1) and in high ee, 97 and
98%, respectively, together with some quantity of haloketones 3a
and 3b after 24 h at 30 ◦C. No ketone 2 was produced after 24 h of
reaction at 30 ◦C. The halohydrins 4a and 4b were isolated and the
configuration was assigned as (2S,3S) based on 13C NMR shifts of C2
and C3 and optical rotation of the corresponding epoxide derivative
5 [19].
The authors thank CNPq and FAPESP for financial support.
References
[1] S. Koul, D.H.G. Crout, W. Errington, J. Tax, J. Chem. Soc., Perkin Trans. 1 (1995)
2969–2988.
[2] Y. Kawai, K. Saitou, K. Hida, D.H. Dao, A. Ohno, Bull. Chem. Soc. Jpn. 69 (1996)
2633–2638.
[3] R. Stuermer, B. Hauer, M. Hall, K. Faber, Curr. Opin. Chem. Biol. 11 (2007)
203–213.
Summarizing the results, a general competition reaction system
may be proposed for the P. stipitis reduction of ␣-haloenones 1a and
1b (Scheme 2) through the intermediates 3a and 3b respectively.
From those intermediates there is a competition reaction between
the electron transfer process and the hydride transfer process. In
both cases, the electron transfer process prevails over the hydride
transfer process. However, the electron transfer process is inhib-
ited with addition of DNB and thus the C O bond of intermediates
3a and 3b are reduced mediated by an oxidoreductase producing
the corresponding halohydrins (2S,3S)-4a and (2S,3S)-4b in high
diastereoselectivity and high ee.
[4] T. Sakai, S. Matsumoto, S. Hidaka, N. Imajo, S. Tsuboi, M. Utaka, Bull. Chem. Soc.
Jpn. 64 (1991) 3473–3475.
[5] C. Fuganti, J. Minut, G.P. Fantoni, S. Servi, J. Mol. Catal. B: Enzym. 4 (1998) 47–52.
[6] J. Aleu, G. Fronza, C. Fuganti, V. Perozzo, S. Serra, Tetrahedron: Asymmetry 9
(1998) 1589–1596.
[7] E.P.S. Filho, J.A.R. Rodrigues, P.J.S. Moran, Tetrahedron: Asymmetry 12 (2001)
847–852.
[8] E.P.S. Filho, J.A.R. Rodrigues, P.J.S. Moran, J. Mol. Catal. B: Enzym. 15 (2001)
23–28.
[9] Y. Chu, B.L. Zhang, V. Silvestre, J.P. Cheng, Biorg. Chem. 34 (2006) 158–166.
[10] A. Kergomard, M.F. Renard, H. Veschambre, J. Org. Chem. 47 (1982)
792–798.
[11] G.J.A. Conceic¸ ão, P.J.S. Moran, J.A.R. Rodrigues, Tetrahedron: Asymmetry 14
(2003) 43–45.
In order to validate the proposed inhibition of electron trans-
fer process, other inhibitors of radical reaction were used. Thus,
the procedure stated in 2.6 of the experimental section was
followed with addition of 1.0 mg of Irganox® 1010 (pentaery-
thritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate)
or 1.0 mg of Irganox® 1076 (octadecyl 3,5-di-(tert-butyl)-4-
hydroxyhydrocinnamate) dissolved in 0.5 mL of ethanol, in place
of the addition of DNB, in parallel experiments. In both cases, halo-
hydrins 4a (60–77%) and 4b (42–69%) were formed jointly with 3a
(20–30%) and 3b (15–28%) and no formation of ketone 2 from 1a
and only 2% of 2 was formed from 1b, after 24 h of reaction at 30 ◦C.
Also, we found the Irganox 1010 to be slightly better than Irganox
1076 as inhibitor of radical process in this media.
[12] V.E.C.D. Ban, H.M. Willemen, H. Wassink, C. Laane, H. Haaker, Enzyme Microb.
Technol. 25 (1999) 251–257.
[13] M. Zagozda, J. Plenkiewicz, Tetrahedron: Asymmetry 17 (2006) 1958–1962.
[14] D. Horton, J.D. Wander, in: W. Pigman, D. Horton (Eds.), The Carbohydrates, vol.
1B, Academic Press, NY, 1980, p. 643.
[15] T. Kitazume, N. Ishikawa, J. Fluorine Chem. 29 (1985) 431–444.
[16] M. Utaka, S. Konishi, A. Takeda, Tetrahedron Lett. 27 (1986) 4737–4740.
[17] G. Fronza, C. Fuganti, S. Serra, Eur. J. Org. Chem. (2009) 6160–6171.
[18] G. Fronza, C. Fuganti, P. Grasselli, A. Mele, J. Org. Chem. 56 (1991)
6019–6023.
[19] P. Besse, M.F. Renard, H. Veschambre, Tetrahedron: Asymmetry 5 (1994)
1249–1268.
[20] L.M. Aleixo, M. de Carvalho, P.J.S. Moran, J.A.R. Rodrigues, Biorg. Med. Chem.
Lett. 3 (1993) 1637–1642.
[21] M. Hamdani, B. De Jeso, H. Deleuze, A. Saux, B. Maillard, Tetrahedron: Asym-
metry 4 (1993) 1233–1236.
[22] G. Jorg, M. Bertau, ChemBioChem 4 (2004) 87–92.
[23] O. Cabon, M. Larchevêque, D. Buisson, R. Azerad, Tetrahedron Lett. 33 (1992)
7337–7340.
4. Conclusion
[24] O. Cabon, D. Buisson, M. Larchevêque, R. Azerad, Tetrahedron: Asymmetry 6
(1995) 2199–2210.
Reductions of (Z)-3-chloro-4-phenyl-3-buten-2-one and (Z)-
3-bromo-4-phenyl-3-buten-2-one mediated by P. stipitis gave
4-phenylbutan-2-one through dehalogenation of the transient
3-halo-4-phenylbutan-2-one. The addition of 1,3-dinitrobenzene
avoids the dehalogenation and thus the corresponding (2S,3S)-
halohydrins were obtained in excellent enantiomeric excesses.
A general competition reaction system is proposed between a
hydride transfer and an electron transfer mechanisms for reaction
of the transient 3-halo-4-phenylbutan-2-one. The electron transfer
mechanism was efficiently inhibited by DNB, and thus, the hydride
transfer mechanism mediated by an oxidoreductase gave halohy-
drin in high ee. The electron transfer process was also efficiently
inhibited by Irganox® 1010 and 1076.
[25] (a) M. Bertau, Tetrahedron Lett. 42 (2001) 1267–1268;
(b) M. Bertau, Biocatal. Biotransform. 20 (2002) 363–367.
[26] D.D. Tanner, A.R. Stein, J. Org. Chem. 53 (1988) 1642–1646.
[27] D.D. Tanner, H.K. Singh, A. Kharrat, A.R. Stein, J. Org. Chem. 52 (1987)
2142–2146.
[28] D.D. Tanner, A. Kharrat, J. Org. Chem. 53 (1988) 1646–1650.
[29] W. Adam, M. Heil, R. Hutterer, J. Org. Chem. 57 (1992) 4491–4495.
[30] J.M. Smolen, E.J. Weber, P.G. Tratnyek, Sci. Technol. 33 (1999) 440–445.
[31] A. Ohno, N. Kito, Chem. Lett. (1972) 369–372.
[32] A. Ohno, T. Shio, H. Yamamoto, S. Oka, J. Am. Chem. Soc. 103 (1981) 2045–2048.
[33] J. Yuasa, S. Fukuzumi, J. Phys. Org. Chem. 21 (2008) 886–896.
[34] N. Kornblum, Angew. Chem. Int. Ed. Engl. 14 (1975) 734–745.
[35] Fundac¸ ão André Tosello Pesquisa e Tecnologia, Rua Latino Coelho, 1301, 13087-
010 Campinas-SP, Brazil, http://www.fat.org.br/.
[36] J.C. Banks, D. van Mele, C.G. Frost, Tetrahedron Lett. 47 (2006) 2863–2866.
[37] K.M. Kim, I.H. Park, Synthesis (2004) 2641–2644.