Selective Baeyer-Villiger oxidation of racemic ketones in aqueous-organic media catalyzed by phenylacetone monooxygenase

Article abstract of DOI:10.1016/j.tetasy.2007.12.008

The enantioselective kinetic resolution of a set of racemic substituted 3-phenylbutan-2-ones employing phenylacetone monooxygenase (PAMO) in non-conventional media was performed. The studies have revealed the effects of a range of solvents on the biocatalytic properties of the biocatalyst. Also, the enzymatic oxidation of α-acetylphenylacetonitrile was performed using organic cosolvents. This has resulted in a dynamic kinetic resolution of this cyanoketone yielding enantiopure (R)-2-acetoxyphenylacetonitrile with moderate yields depending on the reaction conditions employed.

Full text of DOI:10.1016/j.tetasy.2007.12.008

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 197–203
Selective Baeyer–Villiger oxidation of racemic ketones in
aqueous–organic media catalyzed by phenylacetone
monooxygenase
a
a
b
´
Cristina Rodrıguez, Gonzalo de Gonzalo, Daniel E. Torres Pazmino,
˜
Marco W. Fraaijeb and Vicente Gotora,*
aDepartamento de Quı´mica Orga´nica e Inorga´nica, Instituto Universitario de Biotecnologı´a de Asturias,
Universidad de Oviedo, 33006 Oviedo (Asturias), Spain
bLaboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Received 5 November 2007; accepted 18 December 2007
Available online 24 January 2008
Abstract—The enantioselective kinetic resolution of a set of racemic substituted 3-phenylbutan-2-ones employing phenylacetone mono-
oxygenase (PAMO) in non-conventional media was performed. The studies have revealed the effects of a range of solvents on the bio-
catalytic properties of the biocatalyst. Also, the enzymatic oxidation of a-acetylphenylacetonitrile was performed using organic
cosolvents. This has resulted in a dynamic kinetic resolution of this cyanoketone yielding enantiopure (R)-2-acetoxyphenylacetonitrile
with moderate yields depending on the reaction conditions employed.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
respectively, using molecular oxygen.5 Due to the mild
reaction conditions employed and the high regio- and/or
enantioselectivities obtained, BVMOs represent a valuable
alternative for the chemical Baeyer–Villiger oxidation to
synthesize chiral esters and lactones.
Chiral ketones as well as optically active esters and lactones
are organic molecules that are of interest for use in chem-
ical synthesis. Since its discovery more than 100 years
ago,1 the Baeyer–Villiger oxidation has represented a very
useful tool for the preparation of these high-added value
compounds.2 This reaction involves the oxidative cleavage
of a carbon–carbon bond adjacent to a carbonyl function
and is carried out chemically with peracids or with perox-
ides and a Lewis acid. Unfortunately, the use of such
strong oxidants presents several drawbacks. To overcome
these limitations, some ‘greener’ procedures have been
developed in recent years, for example, the use of organo-
catalytic compounds with mild oxidants3 or the employ-
ment of enzymes as chiral catalysts.4
Some years ago, phenylacetone monooxygenase (PAMO)
from Thermobifida fusca was first reported as a Baeyer–Vil-
liger monooxygenase.6 This biocatalyst is NADPH depen-
dent, monomeric, thermostable and has been successfully
employed with high selectivity in the preparation of chiral
esters and sulfoxides.7 It has also been shown that it can
oxidize amines and boron-containing compounds. PAMO
exhibits a narrow substrate specify but some mutants have
been prepared to overcome this drawback.8
Enzymes perform their function in aqueous buffer, but it
has been shown that some of them can act as catalysts in
organic solvents with low water contents.9 Recently, it
has been discovered that certain isolated BVMOs can per-
form oxidation when working in non-conventional media
(mixtures of aqueous buffer with miscible or immiscible
organic solvent).10 For all cosolvents tested, a decrease in
the enzymatic activity and stability was observed. The best
Baeyer–Villiger monooxygenases are oxidoreductases that
catalyze a set of oxidative reactions, including the oxida-
tion of aliphatic and cyclic ketones into esters and lactones,
*
Corresponding author. Tel./fax: +34 985 103448; e-mail: vgs@fq.
uniovi.es
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.12.008

198
C. Rodrı´guez et al. / Tetrahedron: Asymmetry 19 (2008) 197–203
results were obtained when working in mixtures of buffer
and 10% methanol. It was also discovered that the addition
of certain water-miscible cosolvents such methanol or
ethanol when employing PAMO or ethionamide monooxy-
genase (EtaA)11 led to a significant increase and in some
cases to a reversal in the enzyme enantioselectivity for the
enzymatic oxidation of phenyl sulfides to the correspond-
ing chiral sulfoxides. Wild type PAMO and some derived
mutant enzymes have been employed in the enantioselec-
tive Baeyer–Villiger oxidation of several ketones on a
preparative scale when working in biphasic (aqueous
buffer–organic cosolvent) systems.12
The
enzymatic
oxidation
of
( )-3-(4-methoxy-
phenyl)butan-2-one 1a in buffer alone led to (S)-2a
(c = 31% after 1 h) with a moderate enantioselectivity
(E = 32, Table 1, entry 1).14 When the Baeyer–Villiger
oxidation of ( )-1a was conducted in buffer with 30% of
cosolvents displaying a high hydrophilic character, for
example, DMSO and DMF, a loss in enzymatic activity
was observed, as shown in entries 2 and 3. Nevertheless,
the cosolvents only marginally affected the enantioselectiv-
ity. As previously found in the enzymatic oxidation of
organic sulfides,10 the presence of 30% methanol in the
reaction medium had an important effect on the enzymatic
selectivity. Again, a significant increase in the PAMO
enantioselectivity was achieved: ( )-1a was oxidized to
(S)-2a, thereby yielding the (R)-ketone with E = 49, in a
process slower than in buffer only (c = 13% after 3 h).
In the present study, we report on the enantioselective
Baeyer–Villiger oxidation of racemic benzylketones cata-
lyzed by PAMO when employing non-conventional media.
For the rest of the solvents tested in a 30% concentration
(entries 4–12), low PAMO activities were achieved. When
2. Results and discussion
Table 1. Effect of organic cosolvents and methanol concentration on
PAMO-catalyzed oxidation of ( )-3-(4-methoxyphenyl)butan-2-one, 1aa
2.1. PAMO-catalyzed oxidation of 3-phenylbutan-2-ones
in aqueous–organic media
Entry Cosolvent
t (h) eeb (%) eeb (%) cc (%) Ed
(R)-1a
(S)-2a
Our initial experiments were dedicated to investigate the
effect of a broad range of organic solvents, presenting
different physico-chemical properties, on the activity and
selectivity of PAMO when this enzyme catalyzed the Bae-
yer–Villiger oxidation of a racemic substituted 3-phenyl-
butan-2-one, as shown in Scheme 1. Purified PAMO is
effective in the oxidation of molecules with this structure,
as described in previous reports.7c Two different situations
were observed when the enzymatic reactions were carried
out in the solvents tested: the use of water-miscible organic
solvents resulted in a one-phase system, while the water-
immiscible solvents led to a biphasic system. In the latter
case, the enzyme and the hydrophilic components of the
mixture will be mainly found in the aqueous layer whereas
the substrate will be present mostly in the organic phase.
All the reactions were carried out using a 50 mM Tris/
HCl buffer at pH 8.0 as the aqueous phase and were con-
ducted using the isolated enzyme. As a consequence, a sec-
ond ancillary enzymatic system (glucose-6-phosphate with
glucose-6-phosphate dehydrogenase) was included in the
reaction mixture to regenerate the NADPH coenzyme.13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
None
1
3
3
3
6
3
3
3
4
4
4
10
4
1
8
8
8
44
23
7
14
n.d.
60
6
39
20
31
17
n.d.
54
21
15
n.d.
n.d.
91
90
93
95
n.d.
50
13
54
86
53
61
n.d.
79
94
95
n.d.
n.d.
31
20
7
13
61.0
54
31
42
19
39
22
61.0
40
18
13
61.0
61.0
32
24
30
49
n.d.
5.5
1.5
5.0
16
4.0
5.0
n.d.
14
30% DMSO
30% DMF
30% MeOH
30% AcCN
30% Dioxane
30% EtOH
30% i-PrOH
30% AcOEt
30% t-BuOMe
30% i-Pr2O
30% Toluene
30% Hexane
10% MeOH
50% MeOH
70% MeOH
90% MeOH
43
46
n.d.
n.d.
n.d. not determined.
a For reaction conditions see Section 4.
b Determined by GC.
c Conversion, c = ees/(ees + eep).
d Enantiomeric ratio, E = ln[1 ꢀ c(1 + eep)]/ln[1 ꢀ c(1 ꢀ eep)].
PAMO/ T (ºC)/ 250 rpm
Organic solvent
O
+
O
O
O
X
X
X
( )-1a-d
(R)-1a-d
(S)-2a-d
NADP+
NADPH
+ O2 + H+
+ H2O
D-Gluconate-6-P
G6PDH
D-Glucose-6-P
( )-1a; X=4-OMe
( )-1b; X=H
( )-1c; X=4-Br
( )-1d; X=3-CF3
Scheme 1. PAMO-catalyzed oxidation of substituted 3-phenylbutan-2-ones in aqueous–organic media.

C. Rodrı´guez et al. / Tetrahedron: Asymmetry 19 (2008) 197–203
199
employing 30% acetonitrile and toluene, no oxidation was
observed. Concerning the selectivity of the reaction, a
considerable loss in the E values was also observed. The
highest enantioselectivities when using water-immiscible
cosolvents were measured in 30% ethyl acetate and hexane
(entries 9 and 13, E = 16–14, respectively). PAMO almost
completely lost its selectivity when carrying out the oxida-
tions in any other solvent, both working in a monophasic
or a biphasic system.
E = 14 at 30% MeOH, the latter being almost five times
more enantioselective than in the absence of the organic
solvent.
The same effect was observed in the PAMO-catalyzed
Baeyer–Villiger oxidation of a meta-substituted benzyl-
ketone such ( )-3-(3-trifluoromethylphenyl)butan-2-one
(1d). When the reaction was performed at 30 °C in Tris/
HCl buffer pH 8.0, (S)-2d was obtained with a 52% conver-
sion in a process of low enantioselectivity (entry 7). Addition
of 10% or 30% methanol led to a slight loss in the PAMO
activity (entries 8 and 9) and a significant increase in the E
value (E = 10 at 30% MeOH), confirming the positive effect
of methanol on the enantioselectivity of this enzyme.
To study the effect of cosolvent concentration, the effect of
different amounts of methanol was analyzed. As shown in
Table 1 (entries 14–17), the addition of higher contents of
methanol produced a gradual inactivation of the enzyme.
No reaction was observed when working at methanol con-
centrations of 70% and 90%, but it is interesting to notice
that PAMO can still work at 50% MeOH contents, yielding
a conversion of 13% after 8 h. For all the MeOH concen-
trations in which the Baeyer–Villiger oxidation of ( )-1a
was observed, better E values were achieved than in buffer
alone, demonstrating that even at moderate concentra-
tions, methanol is able to improve the selectivity of PAMO.
2.2. Enzymatic oxidation of a-acetylphenylacetonitrile
catalyzed by PAMO
To probe the biocatalytic potential of PAMO for dynamic
kinetic resolutions, the conversion of a-acetylphenylaceto-
nitrile 3 was studied. For this oxidation reaction, it is
important to note that cyanoketone 3 presents a hydrogen
atom with a very acidic character. As a result, this com-
pound readily racemizes. For this reason, chiral 4 could
be obtained in high yields and high enantiomeric purity
in a dynamic kinetic resolution process. When the enzy-
matic Baeyer–Villiger reaction of a-acetylphenylacetonitri-
le was performed in 50 mM Tris/HCl at different pHs and
temperatures, no oxidation product 4 was observed even
after long reaction times. Only the side byproduct phenyl-
acetonitrile 5 was observed, as shown in Table 3. This com-
pound was also found in the blank reactions performed in
the absence of biocatalyst and corresponds to the hydroly-
sis product of a-acetylphenylacetonitrile in basic medium.
The formation of 5 was faster at higher pHs and tempera-
Once it was established that methanol was the best cosol-
vent for the enzymatic oxidation of ( )-1a in terms of
selectivity, the study was extended to other 3-phenyl-
butan-2-ones, as shown in Table 2. As previously reported,
the enzymatic oxidation of racemic 3-phenyl-2-butanone
1b catalyzed by PAMO was a process with a high enanti-
oselectivity (E = 188) leading to (R)-1b and (S)-2b with a
conversion of 27% after 1 h (entry 1). The addition of
10% or 30% methanol to the reaction medium did not alter
the selectivity of the resolution, yielding excellent E values
with a slight decrease in the enzymatic activity.
Table 2. Effect of methanol (10% and 30% v vꢀ1) on PAMO-catalyzed
oxidation of substituted 3-phenylbutan-2-onesa
Table 3. Effect of different cosolvents on PAMO-catalyzed oxidation of
a-acetylphenylacetonitrile 3a
Entry Ketone Cosolvent
eeb (%) eeb (%) cc (%)
(R)-ket (S)-est
E
CN
O
1
2
3
4
5
6
7
8
9
(
(
(
(
(
(
(
(
(
)-1b
)-1b
)-1b
)-1c
)-1c
)-1c
)-1d
)-1d
)-1d
None
10% MeOH 20
30% MeOH 24
None
10% MeOH 22
30% MeOH 24
None
10% MeOH 42
30% MeOH 60
36
98
99
99
44
73
83
43
52
69
27
17
20
24
23
22
52
45
46
188
174
179
3.0
7.9
14
4.5
5.0
10
O
CN
(R)-4
14
BVMO/Buffer pH 8.0/Cosolvent
+
O
G6P/G6PDH/NADPH
48 h/30 (ºC)/250 rpm
CN
3
47
5
a For reaction conditions see Section 4. Reactions were stopped after 1 h.
b Determined by GC.
c Conversion, c = ees/(ees + eep).
Entry Cosolvent
3b (%) (R)-4b (%) 5b (%) eeb (%) (R)-4
1
2
3
4
5
6
7
8
9
10% DMSO
10% MeOH
10% Dioxane
10% AcOEt
10% i-Pr2O
60
90
80
70
76
7.0
7.0
6.0
22
33
3.0
15
8.0
11
P98
96
97
P98
P98
P98
P98
P98
P98
When PAMO was employed as biocatalyst in the enzy-
matic oxidation of a 3-phenylbutanone presenting an elec-
tron-withdrawing group in the para-position of the
aromatic ring as ( )-1c, compounds (S)-2c and (R)-1c were
obtained in a resolution with a low E value (E = 3.0, Table
2, entry 4). Increasing amounts of methanol did not alter
the PAMO activity, but had a profound effect on the
enantioselectivity of the oxidation: it was possible to obtain
an E value of 7.9 when working at 10% methanol and
13
10% t-BuOMe 79
8.0
2.0
2.0
5.0
13
10% CH2Cl2
10% Toluene
10% Hexane
95
92
91
3.0
6.0
4.0
a [3] = 2.0 mg mLꢀ1. Reactions were carried out employing 0.5 units of
PAMO.
b Determined by GC.

200
C. Rodrı´guez et al. / Tetrahedron: Asymmetry 19 (2008) 197–203
tures. To confirm this, when 3 was dissolved in a 0.5 M
sodium hydroxide solution at room temperature, 5 was
the only product observed after a few minutes.
the Baeyer–Villiger oxidation was performed at pH 9.0, 4
was obtained with a slight decrease in enzyme selectivity
(ee = 93%) and good yield, but the side product was
formed to a much higher extent than at lower pH (entry
3). Ethyl acetate was a better cosolvent for this process.
When using 1.0 unit of PAMO at pH 8 and 30 °C, it was
possible to obtain 48% of enantiopure (R)-2-acetoxy-
phenylacetonitrile after 48 h and 58% after 72 h. These
data confirm that the reaction involves a dynamic kinetic
resolution. Longer reactions times did not increase the
amount of oxidation product to a great extent.
In view of these results, the oxidation was performed in a
one-phase system of aqueous buffer and methanol. Several
experiments were carried out at different concentrations,
temperatures and pHs. Under all the conditions, it was
possible to obtain some enantiopure oxidation product
(R)-4, but with low conversions after 2 days reaction time.
Also, the presence of methanol led to the formation of the
side product 5. Entry 2 of Table 3 shows the best oxidation
achieved with this cosolvent (10% methanol, pH 8.0 and
30 °C led to 7.0% of (R)-4 after 48 h).
The effects of temperature and pH of this enzymatic pro-
cess were also studied (Table 4). Performing the reaction
at 20 °C led to 4 in a high yield, and with only a minor
quantity of 5. On the contrary, oxidation of 3 at 40 °C
formed phenylacetonitrile in 62%, with only 4% of (R)-4.
Carrying out the conversion at a higher pH resulted in a
decrease in PAMO activity and selectivity (entry 9), while
at pH 7.5 almost all the starting material was recovered
after 48 h (entry 8). In addition, the ethyl acetate concen-
tration was also analyzed as a key parameter in the enzy-
matic oxidation of 3. When working in 5% AcOEt, 25%
of 4 was formed with ee = 94% and also phenylacetonitrile
was synthesized in the same amount. The highest PAMO
activity in the formation of enantiopure 4 was observed
at 10% AcOEt. An increase in the cosolvent concentration
led to a progressive decrease in the enzymatic activity (en-
tries 11–13) and in the formation of 5. When performing
the oxidation in 50% AcOEt, no conversion was observed.
Concerning the biocatalyst selectivity, enantiopure (R)-
2-acetoxyphenylacetonitrile was synthesized until 40%
cosolvent concentration. As we were working in a biphasic
buffer–ethyl acetate system, 2.0 equiv of a phase transfer
reagent as Bu4NHSO4 was added in the enzymatic oxida-
tion performed at pH 8.0 and 30 °C. The presence of this
compound led to a total loss in the enzymatic activity, as
shown in entry 14.
We decided to study the effect of other organic cosolvents
with different properties in the PAMO-catalyzed oxidation
of 3 (Table 3). For all the reaction media tested, some
phenylacetonitrile 5 was formed, even when performing
the reactions without biocatalyst. As shown in Table 3, oxi-
dations were carried out in buffer pH 8.0 containing 10%
organic solvent and stopped after 48 h. Compound (R)-4
was obtained with high enantiomeric excess (ee P 96%)
in all conditions. The presence of 10% cosolvent led, in
most cases to low yields of enantiopure 4 (lower than
10%), the formation of higher quantities of byproduct 5
(see entries 1 and 3, employing DMSO or 1,4-dioxane)
and the recovery of high amounts of the starting ketone.
Only when working with two water immiscible solvents,
that is, i-Pr2O or AcOEt, was the formation of 2-acetoxy-
phenylacetonitrile was higher than 10% after 48 h, espe-
cially in the case of AcOEt (22%, entry 4).
As i-Pr2O and AcOEt were the best cosolvents for the Bae-
yer–Villiger oxidation, certain reaction parameters were
modified to study their effect on PAMO properties. In these
reactions, the amount of biocatalyst was increased to
obtain higher conversions (Table 4). As shown in entry 1,
a 10% i-Pr2O at pH 8 and 30 °C allowed us to obtain enan-
tiopure (R)-4 in about 30% after 48 h. When increasing the
cosolvent amount to 20%, the oxidation became much
slower (only 6.0% of enzymatic product formed). When
The influence of 3 concentration on the activity and selec-
tivity of PAMO was studied when conducting the oxida-
tions in buffer with 10% AcOEt (Fig. 1). After 42 h, at all
Table 4. Enzymatic oxidation of a-acetylphenylacetonitrile 3 employing i-Pr2O and AcOEt as cosolventsa
Entry
Cosolvent
pH
T (°C)
t (h)
3b (%)
(R)-4b (%)
5b (%)
ee (R)-4b (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14c
10% i-Pr2O
20% i-Pr2O
10% i-Pr2O
10% AcOEt
10% AcOEt
10% AcOEt
10% AcOEt
10% AcOEt
10% AcOEt
5% AcOEt
20% AcOEt
30% AcOEt
40% AcOEt
10% AcOEt
8.0
8.0
9.0
8.0
8.0
8.0
8.0
7.5
9.0
8.0
8.0
8.0
8.0
8.0
30
30
30
30
30
20
40
30
30
30
30
30
30
30
48
48
48
48
72
48
48
48
48
48
48
48
48
48
66
87
63
40
28
41
33
93
59
52
62
83
83
89
30
6.0
26
48
58
4.0
7.0
11
12
14
3
62
4.0
24
23
3.0
2.0
2.0
10
P98
P98
93
P98
P98
P98
P98
P98
95
56
5.0
3.0
17
25
35
15
15
61.0
94
P98
P98
95
—
a [3] = 2.0 mg mLꢀ1. For reaction conditions see Section 4. 1 unit of PAMO was employed.
b Determined by GC.
c Reaction performed with 2 equiv of Bu4NHSO4 as phase transfer reagent.

C. Rodrı´guez et al. / Tetrahedron: Asymmetry 19 (2008) 197–203
201
pH, the type and concentration of organic cosolvent when
performing oxidations catalyzed by PAMO, it is possible to
obtain better results in terms of selectivity and conversion.
0.010
0.008
0.006
0.004
0.002
0.000
40
35
30
25
20
15
10
5
4. Experimental
4.1. General
Recombinant histidine-tagged phenylacetone monooxy-
genase (PAMO) was obtained as previously described.6
One unit of BVMO will oxidize 1.0 lmol of phenylacetone
to benzyl acetate per minute at pH 8 and 30 °C in the pres-
ence of NADPH. Glucose-6-phosphate dehydrogenase
(170 U/mg) from Leuconostoc mesenteroides was obtained
from Fluka-BioChemika. All reagents and solvents em-
ployed were of the highest quality grade available and were
from Sigma–Aldrich–Fluka and Acros Organics.
0
0
2
4
6
8
10 12 14 16 18 20
[Cyanoketone] (g L-1
)
Figure 1. Effect of a-acetylphenylacetonitrile concentration on the for-
mation of (R)-4 (green line), 5 (black line) and on the reaction rate (red
line) in PAMO-catalyzed oxidation of 3 in 50 mM Tris/HCl pH 8.0, 10%
AcOEt at 30 °C.
The racemic ketones ( )-1a–d were prepared according to
the literature, using methyl iodide and NaOH in a biphasic
medium water/dichloromethane (yields from 20% to 80%,
depending on the substrate).15 Acetate ( )-2a was prepared
by direct oxidation with m-CPBA/dichloromethane with
75% yield, while racemic esters ( )-2b–d and ( )-4 were
synthesized by chemical acetylation of the corresponding
alcohols (yields from 40% to 90%).
the concentrations tested (1–20 g Lꢀ1), enantiopure (R)-2-
acetoxyphenylacetonitrile was obtained. This shows that
substrate concentration does not affect the enzyme
selectivity.
As expected, when the concentrations were higher, enantio-
pure (R)-4 was obtained in lower quantities and also the
formation of the side product 5 was slower at high concen-
trations. It is interesting to notice that at substrate concen-
tration of 1 g Lꢀ1 4 was formed in larger quantities than 5
(ratio 2:1), while by increasing the substrate concentration
the side product was synthesized to a higher extent than the
oxidation one (at 16 g Lꢀ1 the ratio of 4:5 was 1:8), indicat-
ing a decrease in the PAMO activity. The reaction rates
(expressed as grams of 3 consumed per liter and per hour)
were also studied, yielding a maximum value at low con-
centrations (1–2 g Lꢀ1). Higher ketone amounts in the
medium decreased the reaction rate. When working at
20 g Lꢀ1 no oxidation was observed.
Chemical reactions were monitored by analytical TLC,
performed on Merck silica gel 60 F254 plates and visualized
by UV irradiation. Flash chromatography was carried out
with silica gel 60 (230–240 mesh, Merck). IR spectra were
recorded on a Perkin–Elmer 1720-X infrared Fourier
transform spectrophotometer using KBr pellets. Optical
rotations were measured using a Perkin–Elmer 241
polarimeter and are quoted in units of 10ꢀ1 deg cm2 gꢀ1
.
1H NMR, 13C NMR, and DEPT spectra were with TMS
(tetramethylsilane) as the internal standard with Bruker
AC-300 (1H, 300.13 MHz and 13C, 75.4 MHz) and Bruker
AC-300-DPX (1H, 300.13 MHz and 13C, 75.4 MHz) spec-
trometers. The chemical shift values (d) are given in ppm
and the coupling constants (J) in Hertz (Hz). APCI+ using
a Hewlett Packard 1100 chromatograph mass detector or
EI+ with a Hewlett Packard 5973 mass spectrometer was
used to record mass spectra (MS).
3. Conclusion
The addition of different amounts of organic cosolvents to
the reaction medium in enzymatic Baeyer–Villiger oxida-
tion of racemic ketones catalyzed by PAMO can improve
the biocatalytic properties of this biocatalyst. Herein, two
different effects can be discriminated: (1) an increase of
the enantioselectivity in the resolution of different substi-
tuted 3-phenylbutan-2-ones by different concentrations of
methanol, up to a 50% v vꢀ1 and, (2) higher conversions
in the PAMO-catalyzed oxidation of a-acetylphenylaceto-
nitrile due to an increase of the substrate solubility in the
reaction medium when performing the reactions. For the
latter substrate, it was possible to perform a dynamic
kinetic resolution, achieving enantiopure (R)-2-acetoxyphe-
nylacetonitrile with good conversions in 50 mM Tris/HCl
pH 8.0 at 20 °C containing 10% ethyl acetate. So, by choos-
ing the reaction parameters properly, such as temperature,
GC analyses were performed on a Hewlett Packard 6890
Series II chromatograph equipped with the following
columns: Restek RtbDEXse (30 m ꢁ 0.25 mm ꢁ 0.25 lm,
1.0 bar N2) and Krompex Cydex B (25 m ꢁ 0.25 mm ꢁ
0.25 lm, 1.0 bar N2). For all the analyses, the injector tem-
perature is 225 °C and the FID temperature is 250 °C.
The absolute configuration of ester 2a was determined by
comparison of the GC chromatograms with the patterns
described in previous experiments for the known configura-
tion.16 For esters 2b–d, the absolute configuration was
established by comparison with an authentic sample pre-
pared from chemical acylation of the corresponding com-
mercial chiral (S)-alcohols. Absolute configuration of 4
was determined in the same way, starting in this case from
the commercial alcohol of (R)-configuration.

202
C. Rodrı´guez et al. / Tetrahedron: Asymmetry 19 (2008) 197–203
4.2. General procedure for the PAMO-catalyzed oxidation
of racemic ketones ( )-1a–d and 3
the ee by GC analysis: Cydex B, 90 °C (30 min), 5 °C/
min, 120 °C (30 min), 10 °C/min, 200 °C; tR (R) 69.0 min;
tR (S) 69.5 min.
Unless otherwise stated, the corresponding racemic ketones
( )-1a–d and 3 (0.02 mmol, 1.0 equiv) were dissolved in
50 mM Tris/HCl at different pHs/organic cosolvent system
(1.0 mL), containing glucose-6-phosphate (0.04 mmol,
2.0 equiv), glucose-6-phosphate dehydrogenase (10.0
units), NADPH (0.2 mM) and the Baeyer–Villiger mono-
oxygenase (1.0 unit). The mixture was shaken at 250 rpm
and the selected temperature in a rotatory shaker for the
times indicated. The reaction was then stopped, worked
up by extraction with ethyl acetate (3 ꢁ 0.5 mL), dried over
Na2SO4 and analyzed directly by chiral GC to determine
the conversion and the enantiomeric excesses of the
remaining ketones (R)-1a–d, 3 and the esters 2a–d of (S)-
configuration and (R)-4. In the case of ketones ( )-1a–d,
for all the solvents tested, control experiments in the ab-
sence of enzyme resulted in no conversion. For 3, the reac-
tions performed without PAMO showed 5 as undesired
byproduct.
1
4.3.3. (R)-3-Phenylbutan-2-one, (R)-1b. Colorless oil. H
3
NMR (CDCl3, 300.13 MHz): d 1.39 (d, 3H, JHH 7.0 Hz),
3
2.12 (s, 3H), 3.75 (q, 1H, JHH 7.0 Hz), 7.19–7.39 (m,
5Har). 13C NMR (CDCl3, 75.5 MHz): d 17.1 (CH3), 28.3
(CH3), 53.6 (CH), 127.1 (CHar), 127.7 (2CHar), 128.9
(2CHar), 140.5 (Car), 208.8 (C@O). MS (APCI+, m/z):
149 [(M+H)+, 100%]. Determination of the ee by GC anal-
ysis: RtbDEXse, 70 °C (5 min), 1 °C/min, 120 °C (5 min);
tR (R) 44.3 min; tR (S) 46.1 min.
1
4.3.4. (S)-1-Phenyethyl acetate, (S)-2b. Colorless oil. H
3
NMR (CDCl3, 300.13 MHz): d 1.56 (d, 3H, JHH 6.6 Hz),
3
2.10 (s, 3H), 5.91 (q, 1H, JHH 6.6 Hz), 7.28–7.39 (m,
5Har). 13C NMR (CDCl3, 75.5 MHz): d 20.9 (CH3), 21.7
(CH3), 71.8 (CH), 125.6 (2CHar), 127.4 (CHar), 128.0
(2CHar), 141.2 (Car), 169.8 (C@O). MS (APCI+, m/z):
165 [(M+H)+, 100%]. Determination of the ee by GC anal-
ysis: RtbDEXse, 70 °C (5 min), 1 °C/min, 120 °C (5 min).
tR (S) 42.7 min; tR (R) 50.1 min.
4.3. Enzymatic oxidation catalyzed by PAMO of racemic
ketones ( )-1a, ( )-1c and ( )-1d at multimilligram scale in
buffer containing 30% MeOH
4.3.5. (R)-3-(4-Bromophenyl)butan-2-one, (R)-1c. Color-
1
less oil. H NMR (CDCl3, 300.13 MHz): d 1.37 (d, 3H,
3
Racemic ketones ( )-1a, ( )-1c and ( )-1d (0.5 mmol)
were dissolved in 20 mL of Tris/HCl buffer (50 mM, pH
8.0), containing a 30% MeOH for substrates 1c and 1d, glu-
cose-6-phosphate (1.0 mmol), glucose-6-phosphate dehy-
drogenase (40 units), NADPH (0.8 mM) and PAMO (4
units). The mixtures were shaken at 20 °C and 250 rpm
for 72 h and extracted with ethyl acetate (3 ꢁ 20 mL).
The combined organic layers were dried over Na2SO4
and the solvent was evaporated under reduced pressure.
The crude residues were purified by flash chromatography
on silica gel employing as eluent hexane/diethyl ether 8:2,
to afford (R)-1a (46.7 mg, 52% yield); (S)-2a (23.0 mg,
24% yield); (R)-1c (75.4 mg, 67% yield); (S)-2c (17.1 mg,
14% yield); (R)-1d (46.4 mg, 43% yield) and (S)-2d
(42.8 mg, 37% yield).
3JHH 6.9 Hz), 2.05 (s, 3H), 3.71 (q, 1H, JHH 6.9 Hz),
7.08 (d, 2H, JHH 8.6 Hz), 7.45 (d, 2H, JHH 8.6 Hz). 13C
NMR (CDCl3, 75.5 MHz): d 17.2 (CH3), 28.4 (CH3), 53.1
(CH), 121.2 (Car), 129.5 (2CHar), 132.1 (2CHar), 139.5
(Car), 208.2 (C@O). MS (EI+, m/z): 226 (M+, 21%), 183
(92%), 104 (100%). Determination of the ee by GC analy-
sis: RtbDEXse, 100 °C (5 min), 3 °C/min, 150 °C (5 min),
3
3
10 °C/min, 200 °C; tR (R) 56.6 min; tR (S) 58.2 min.
25
½aꢂD ¼ ꢀ17:1 (c 1.50, CHCl3), ee 24%.
4.3.6. (S)-1-(4-Bromophenyl)ethyl acetate, (S)-2c. Color-
1
less oil. H NMR (CDCl3, 300.13 MHz): d 1.51 (d, 3H,
3JHH 6.6 Hz), 2.06 (s, 3H), 5.82 (q, 1H, JHH 6.6 Hz),
3
7.23 (2Har, JHH 8.4 Hz), 7.47 (2Har, JHH 8.5 Hz). 13C
NMR (CDCl3, 75.5 MHz): d 21.2 (CH3), 22.0 (CH3), 71.5
(CH), 121.6 (Car), 127.7 (2CHar), 131.5 (2CHar), 140.6
(Car), 170.1 (C@O). MS (EI+, m/z): 242 (M+, 32%), 184
(79%), 104 (73%). Determination of the ee by GC analysis:
RtbDEXse, 100 °C (5 min), 3 °C/min, 150 °C (5 min),
10 °C/min, 200 °C; tR (S) 54.1 min; tR (R) 57.6 min.
3
3
4.3.1. (R)-3-(4-Methoxyphenyl)butan-2-one, (R)-1a. Col-
1
orless oil. H NMR (CDCl3, 300.13 MHz): d 1.36 (d, 3H,
3
3JHH 7.0 Hz), 2.03 (s, 3H), 3.68 (q, 1H, JHH 6.9 Hz),
3
3.79 (s, 3H), 6.87 (d, 2Har, JHH 6.7 Hz), 7.13 (d, 2Har,
3JHH 6.8 Hz). 13C NMR (CDCl3, 75.5 MHz): d 17.1
(CH3), 28.1 (CH3), 52.8 (CH), 55.2 (CH3), 114.3 (2CHar),
4.3.7. (R)-3-(3-Trifluoromethylphenyl)butan-2-one, (R)-1d.
1
128.7 (2CHar), 132.6 (Car), 158.7 (Car), 209.1 (C@O). MS . Colorless oil. H NMR (CDCl3, 300.13 MHz): d 1.43 (d,
(EI+, m/z): 178 (M+, 35%), 135 (100%). Determination of
the ee by GC analysis: Cydex B, 90 °C (30 min), 5 °C/
3H, JHH 7.1 Hz), 2.08 (s, 3H), 3.83 (q, 1H, JHH 7.0 Hz),
3
3
7.39–7.55 (m, 4Har). 13C NMR (CDCl3, 75.5 MHz): d
17.3 (CH3), 28.5 (CH3), 53.3 (CH), 123.9 (CF3, JCF
1
min, 120 °C (30 min), 10 °C/min, 200 °C; tR (R) 68.0 min;
25
3
3
tR (S) 68.5 min. ½aꢂD ¼ ꢀ22:7 (c 1.12, CHCl3), ee 44%.
270.6 Hz), 124.1 (CHar, JCF 3.7 Hz), 124.6 (CHar, JCF
2
3.7 Hz), 129.4 (CHar), 131.1 (CHar), 131.2 (Car, JCF
4.3.2. (R)-1-(4-Methoxyphenyl)ethyl acetate, (S)-2a. Col-
32.1 Hz), 141.4 (Car), 207.8 (C@O). MS (EI+, m/z): 216
(M+, 16%), 173 (40%). Determination of the ee by GC
analysis: RtbDEXse, 100 °C (20 min), 2 °C/min, 150 °C
1
orless oil. H NMR (CDCl3, 300.13 MHz): d 1.54 (d, 3H,
3JHH 6.6 Hz), 2.07 (s, 3H), 3.82 (s, 3H), 5.87 (q, 1H,
3
3JHH 6.6 Hz), 6.90 (d, 2Har, JHH 8.5 Hz), 7.32 (d, 2Har,
(5 min), 10 °C/min, 200 °C; tR (R) 21.7 min; tR (S)
25
3JHH 8.8 Hz). 13C NMR (CDCl3, 75.5 MHz): d 20.9
(CH3), 21.4 (CH3), 54.7 (CH3), 71.5 (CH), 113.3 (2CHar),
127.1 (2CHar), 133.2 (Car), 158.8 (Car), 169.9 (C@O). MS
(EI+, m/z): 194 (M+, 48%), 134 (100%). Determination of
23.7 min. ½aꢂD ¼ ꢀ38:7 (c 1.15, CHCl3), ee 60%.
4.3.8. (S)-1-(3-Trifluoromethylphenyl)ethyl acetate, (S)-
2d. Colorless oil. 1H NMR (CDCl3, 300.13 MHz): d

C. Rodrı´guez et al. / Tetrahedron: Asymmetry 19 (2008) 197–203
203
3
3
4. (a) Roberts, S. M. J. Mol. Catal. B: Enzym. 1998, 4, 111–136;
(b) Urlacher, V. D.; Schmid, R. D. Curr. Opin. Chem. Biol.
2006, 10, 156–161.
1.55 (d, 3H, JHH 6.8 Hz), 2.10 (s, 3H), 5.91 (q, 1H, JHH
6.6 Hz), 7.47–7.61 (m, 4Har). 13C NMR (CDCl3,
75.5 MHz): d 21.2 (CH3), 22.2 (CH3), 71.5 (CH), 122.7
(CHar, JCF 3.7 Hz), 124.0 (CF3, JCF 270.6 Hz), 124.6
(CHar, JCF 3.7 Hz), 128.9 (CHar), 129.5 (CHar), 130.9
3
1
5. (a) Mihovilovic, M. D.; Muller, B.; Stanetty, P. Eur. J. Org.
¨
Chem. 2002, 3711–3720; (b) Kamerbeek, N. M.; Janssen, D.
B.; Van Berkel, J. H.; Fraaije, M. W. Adv. Synth. Catal. 2003,
345, 667–678; (c) Alphand, V.; Carrea, G.; Wohlgemuth, R.;
Furstoss, R.; Woodley, J. M. Trends Biotechnol. 2003, 21,
318–323; (d) Mihovilovic, M. D. Curr. Org. Chem. 2006, 10,
1265–1287.
3
2
(Car, JCF = 32.1 Hz), 142.7 (Car), 170.1 (C@O). MS
(EI+, m/z): 232 (M+, 11%), 190 (90%). Determination of
the ee by GC analysis: RtbDEXse, 100 °C (20 min), 2 °C/
min, 150 °C (5 min), 10 °C/min, 200 °C; tR (S) 18.3 min;
25
tR (R) 22.8 min. ½aꢂD ¼ þ78:7 (c 0.95, CHCl3), ee 69%.
6. Malito, E.; Alfieri, A.; Fraaije, M. W.; Mattevi, A. Proc. Nat.
Acad. Sci. U.S.A. 2004, 101, 13157–13162.
7. (a) Fraaije, M. W.; Wu, J.; Heuts, D. P. H. M.; van
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4.3.9. (R)-2-Acetoxyphenylacetonitrile, (R)-4. Pale yellow
1
oil. H NMR (CDCl3, 300.13 MHz): d 2.22 (s, 3H), 6.46
(s, 1H), 7.49–7.58 (m, 5H). 13C NMR (CDCl3,
75.5 MHz): d 20.4 (CH3), 68.8 (CH), 116.0 (CN), 127.8
(2CHar), 129.2 (2CHar), 130.3 (CHar), 131.6 (Car), 168.9
(C). MS (EI+, m/z): 175 (M+, 35%), 133 (98%), 115
(97%). Determination of the ee by GC analysis: RtbDEX-
se, 130 °C (1 min), 2 °C/min, 200 °C (5 min). tR (S)
15.2 min; tR (R) 17.0 min.
Torres Pazmino, D. E.; Ottolina, G.; Fraaije, M. W.; Carrea,
˜
´
G. Tetrahedron: Asymmetry 2005, 16, 3077–3083; (c) Rodrı-
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Tetrahedron: Asymmetry 2007, 18, 1338–1344; (d) Zambian-
chi, F.; Fraaije, M. W.; Carrea, G.; de Gonzalo, G.;
´
Rodrıguez, C.; Gotor, V.; Ottolina, G. Adv. Synth. Catal
2007, 349, 1327–1331.
8. (a) Bocola, M.; Schulz, F.; Leca, F.; Vogel, A.; Fraaije, M.
W.; Reetz, M. T. Adv. Synth. Catal. 2005, 347, 979–986; (b)
Torres Pazmino, D. E.; Snajdrova, R.; Rial, D. V.; Miho-
˜
vilovic, M. D.; Fraaije, M. W. Adv. Synth. Catal. 2007, 349,
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Acknowledgments
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´
Cristina Rodrıguez thanks the Principado de Asturias for
her predoctoral fellowship. Dr. Gonzalo de Gonzalo (Juan
de la Cierva Program) thanks the Ministerio de Educacion
y Ciencia (MEC) de Espana for personal funding. This
˜
work was supported by the MEC (Project CTQ-04-04185).
´
12. Schulz, F.; Leca, F.; Hollman, F.; Reetz, M. T. Beilstein J.
Org. Chem. 2005, 1, 10.
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