Enzymatic Baeyer-Villiger oxidation of Benzo-Fused ketones: Formation of regiocomplementary lactones

Article abstract of DOI:10.1002/ejoc.200900084

Baeyer-Villiger monooxygenases (BVMOs) are enzymes that are known to catalyse the Baeyer-Villiger oxidation of ketones in aqueous media using O2 as oxidant. Herein, we describe the oxidation of a set of diverse benzo-fused ketones by three different BVMOs

Full text of DOI:10.1002/ejoc.200900084

FULL PAPER
DOI: 10.1002/ejoc.200900084
Enzymatic Baeyer–Villiger Oxidation of Benzo-Fused Ketones: Formation of
Regiocomplementary Lactones
Ana Rioz-Martínez,[a] Gonzalo de Gonzalo,[a] Daniel E. Torres Pazmiño,[b]
Marco W. Fraaije,[b] and Vicente Gotor*[a]
Keywords: Bioorganic chemistry / Oxidoreductases / Oxidation / Lactones / Regioselectivity
Baeyer–Villiger monooxygenases (BVMOs) are enzymes that
are known to catalyse the Baeyer–Villiger oxidation of
ketones in aqueous media using O2 as oxidant. Herein, we
describe the oxidation of a set of diverse benzo-fused ketones
by three different BVMOs in both aqueous and non-conven-
tional reaction media. Most of the tested ketones, for exam-
ple, 1-tetralone and 1- and 2-indanone, were converted by
one of the employed biocatalysts. The catalytic efficiency
could be improved by performing the oxidation reactions at
a relatively high pH and by adding organic cosolvents. One
striking observation is that absolute and complementary re-
gioselectivities were obtained when oxidizing a range of 1-
indanones using two different BVMOs. The conversion of 1-
indanone by 4-hydroxyacetophenone monooxygenase
(HAPMO) results in the formation of the expected lactone,
3,4-dihydrocoumarin. In contrast, by using a phenylacetone
monooxygenase mutein (M-PAMO), conversion of 1-in-
danone leads to the formation of only the unexpected lac-
tone, 1-isochromanone. This illustrates that by the appropri-
ate choice of BVMO as biocatalyst, the effective and regiose-
lective conversion of a wide range of benzo-fused ketones is
feasible.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
BVMOs, mild reaction conditions are applied, and the envi-
ronmental impact is minimized to a great extent when com-
pared with those used in chemical approaches to this reac-
tion. In addition, high regio- and/or enantioselectivities are
obtained in processes that are difficult to perform when
using other oxidizing reagents. The regioselectivity of the
Baeyer–Villiger oxidation is established by steric, conforma-
tional and electronic effects leading to the migration of the
higher substituted (the more nucleophilic) carbon centre. In
some rare cases, however, the use of Baeyer–Villiger
monooxygenases has led to the formation of unexpected
lactones with high regioselectivities,[4] increasing the syn-
thetic potential of this class of enzymes. It has been sug-
gested that in these cases the chiral environment of BVMOs
imposes restrictions that allow only a single conformation
of the Criegee intermediate formed during the oxidation.
Only the carbon centre located in a determined conforma-
tion will migrate, regardless of its nucleophilicity.
As biological catalysts, enzymes have evolved to perform
their catalytic activity in aqueous medium. Nevertheless, it
has also been demonstrated that some biocatalysts can act
in the presence of organic solvents. In fact, organic solvents
have been widely studied and applied in biocatalysis, with
the main advantages as follows: (1) catalysis of processes
unfavourable in aqueous medium, (2) the partial or total
suppression of water-induced side-reactions and (3) the sol-
ubilization of hydrophobic substrates.[5] Recently, it has
been shown that BVMOs can also be employed in non-con-
ventional media (mixtures of aqueous buffer with miscible
Introduction
Since its discovery more than 100 years ago, the Baeyer–
Villiger reaction is one of the key reactions in organic syn-
thesis.[1] In this reaction a carbon–carbon bond located ad-
jacent to a carbonyl group is oxidatively cleaved, and the
subsequent insertion of an oxygen atom into that position
results in the conversion of (cyclic) ketones into esters and
lactones. Baeyer–Villiger oxidations can be carried out by
using peroxy acids or by employing hydrogen peroxide or
oxygen as milder oxidants in the presence of Lewis acids or
organometallic compounds.[2] The products of this reaction
offer an attractive and simple entry to several classes of
biologically active compounds.
In the last few years, the development of enzymatic meth-
odologies using Baeyer–Villiger monooxygenases (BVMOs)
has allowed the preparation of several compounds that are
of high interest in organic synthesis.[3] These flavoproteins
are oxidoreductases that catalyse the Baeyer–Villiger oxi-
dation as well as other oxidative processes that employ at-
mospheric oxygen as the natural oxidant. When using
[a] Departamento de Química Orgánica e Inorgánica, Instituto de
Biotecnología de Asturias, Universidad de Oviedo,
c/ Julián Clavería 8, 33006 Oviedo, Spain
Fax: +34-985-103448
E-mail: vgs@fq.uniovi.es
[b] Laboratory of Biochemistry, Groningen Biomolecular Sciences
and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 2526–2532

Enzymatic Baeyer–Villiger Oxidation of Benzo-Fused Ketones
or immiscible organic solvents).[6] For all the cosolvents lactone to be achieved after 96 h. The use of the same or-
tested, two main effects were observed: (1) a decrease in ganic cosolvents was studied in the PAMO-catalysed oxi-
enzyme activity and stability and (2) an increase and in dations; however, the formation of 1b was not observed.
some cases a reversal in enantioselectivity. Furthermore, it
has been shown that phenylacetone monooxygenase
(PAMO) can be employed in the enantioselective Baeyer–
Villiger oxidation of ketones on a preparative scale when
working in biphasic aqueous buffer/organic cosolvent sys-
tems.[7]
Table 1. Effect of organic cosolvents on the HAPMO-biocatalysed
oxidation of 1-tetralone (1a).[a]
Benzo-fused lactones have received considerable atten-
tion as useful intermediates in organic synthesis and are the
key structural elements of a variety of biologically active
compounds. In a recent study it was shown that a set of
substituted 1-indanones could be oxidized with good to ex-
cellent yields by the wild-type bacterium Pseudomonas sp.
NCIMB 9872. In these reactions it was shown that the ki-
netic behaviour of the reactions was influenced by the posi-
tion and size of the substituents.[8] In this paper we report
the BVMO-mediated oxidation of several benzo-fused
ketones catalysed by three BVMOs employing non-conven-
tional reaction media. The three biocatalysts are phenylace-
tone monooxygenase (PAMO) from Thermobifida fusca,[9]
4-hydroxyacetophenone monooxygenase (HAPMO) from
Pseudomonas fluorescens ACB[10] and a recently created mu-
tant of PAMO, the M446G PAMO mutein (M-PAMO).[11]
All three BVMOs have previously been shown to be effec-
tive biocatalysts in the oxidation of racemic benzyl ketones
and aromatic sulfides.
Entry
Cosolvent
logP
Conv. [%][b]
1
2
3
4
5
6
7
8
9
none
–
5
5% MeOH
5% 1,4-dioxane
5% iPrOH
5% CH2Cl2
5% tBuOMe
5% iPr2O
5% toluene
5% 2-octanol
5% hexane
–0.76
–0.27
0.07
1.25
1.35
2.00
2.50
2.72
3.50
Յ3
Յ3
Յ3
Յ3
7
Յ3
26
25
15
10
[a] For the reaction conditions, see the Exp. Sect. [b] Determined
by GC.
2-Tetralone (2a) was also subjected to BVMO-catalysed
oxidation, but no reaction was observed with the three bio-
catalysts, even after modifying the reaction parameters (pH,
temperature, and organic cosolvents).
Results and Discussion
Enzymatic Oxidation of 2-Indanone Catalysed by BVMOs
Our experiments were dedicated to the biocatalysed oxi-
dation of different benzo-fused ketones (tetralones, in-
danones and benzocyclobutanone). All the reactions were
carried out by using purified enzymes. An auxiliary enzy-
matic system (glucose-6-phosphate with glucose-6-phos-
phate dehydrogenase) was included in the reaction mixture
to regenerate the NADPH coenzyme, which is required by
BVMOs.[12]
After analysing the oxidation reactions of tetralones, our
efforts were devoted to the study of the enzymatic Baeyer–
Villiger reactions of indanones. The oxidation reactions of
2-indanone (3a) carried out in buffer with HAPMO and
PAMO did not show the formation of 3-isochromanone
(3b). When modifying the reaction medium by adding dif-
ferent cosolvents, a slight improvement in the enzymatic ac-
tivity was observed only in the HAPMO-biocatalysed oxi-
dations. After 72 h, only 10% of 3b was recovered in the
media containing 5% hexane or CH2Cl2. No oxidation was
observed by changing the reaction media when PAMO was
BVMO-Catalysed Oxidation of Tetralones
We first analysed the enzymatic bio-oxidation of 1-tet- used. Oxidation of 3a catalysed by M-PAMO in 50 m Tris-
ralone (1a) by employing the three BVMOs. The reactions HCl buffer at pH 9.0 led to 3b with 50% conversion after
were performed in buffer (50 m Tris-HCl, pH 9.0) at 30 °C 72 h. Different water-miscible and -immiscible organic co-
for the two PAMO enzymes, whereas 20 °C was a more ap- solvents (5% v/v) were tested, as shown in Table 2. In all
propriate temperature for HAPMO.[9d] After 96 h, no reac- cases, this resulted in a more effective oxidation of 3a. The
tion was observed with PAMO and M-PAMO, whereas best results were obtained in both a hydrophilic (1,4-diox-
only 5% of the expected regioisomer 4,5-dihydro-1-benzo- ane, Entry 3) and a hydrophobic solvent (DIPE, Entry 7),
xepin-2(3H)-one (1b) was obtained when employing giving 3b with conversions close to 90% after 72 h. As a
HAPMO. As ketone 1a is not very soluble in water, its oxi- further attempt at medium engineering and to optimize the
dation catalysed by HAPMO in buffer containing 5% of reaction conditions, we decided to analyse the effect of pH
different organic cosolvents was studied. Table 1 shows that on the oxidation of 3a in buffer containing 5% 1,4-dioxane.
only cosolvents with a high hydrophobic character (logP Ն As shown for PAMO and HAPMO in other oxidative reac-
2.0)[13] led to a significant increase in the formation of 1b. tions,[9b,9d] there was a significant increase in M-PAMO ac-
Thus, the addition of 5% (v/v) of toluene or 2-octanol (En- tivity when working at relatively high pH values. No reac-
tries 8 and 9, respectively) allowed a 25% conversion to the tion was observed at pH 6.0, and low conversions were
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2527

V. Gotor et al.
FULL PAPER
measured below pH 9.0. At pH 9.0–10.5, the enzymatic ac- than 70% after 72 h (Table 3). The employment of hydro-
tivity remained high (conversion, c = 88–90% after 72 h, phobic cosolvents led to a deactivation of M-PAMO, giving
Entries 3, 14 and 15).
the unexpected lactones in lower yields than those obtained
for the oxidation performed in Tris-HCl buffer.
Table 2. M-PAMO-catalysed biooxidation of 2-indanone (3a) in
non-conventional reaction media.[a]
Entry
Cosolvent
logP
pH
Conv. [%][b]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
none
5% MeOH
5% 1,4-dioxane
5% iPrOH
5% CH2Cl2
5% tBuOMe
5% iPr2O
5% toluene
5% 2-octanol
5% hexane
5% 1,4-dioxane
5% 1,4-dioxane
5% 1,4-dioxane
5% 1,4-dioxane
5% 1,4-dioxane
–
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
6.0
7.0
8.0
10.0
10.5
50
83
90
72
69
69
89
71
55
77
Յ3
11
37
90
88
–0.76
–0.27
0.07
1.25
1.35
2.00
2.50
2.72
3.50
–0.27
–0.27
–0.27
–0.27
–0.27
[a] For the reaction conditions, see the Exp. Sect. [b] Determined
by GC.
Figure 1. Effect of different organic solvents in aqueous/organic
solvent (5% v/v) systems on the enzymatic oxidation reactions of
4a catalysed by HAPMO to synthesize lactone 4b or by M-PAMO
to give the unexpected lactone 4c (t = 72 h); logP (᭡) values have
been plotted to correlate enzyme activities with the hydrophobicity
of the organic solvents.
Biocatalysed Oxidation of 1-Indanone and Its Derivatives
The three Baeyer–Villiger monooxygenases were em-
ployed as biocatalysts in the enzymatic oxidation of 1-in-
danone (4a). The reaction performed in the presence of
HAPMO led to 3,4-dihydrocoumarin (4b) with a 26% con-
version after 72 h. When PAMO was employed, no oxi-
dation products were formed in the same reaction time. Sur-
prisingly, the oxidation catalysed by M-PAMO led to the
formation of an unexpected compound (67% conversion)
with the same molecular weight as 4b. This compound cor-
responded to the product obtained from the oxidation of
isochromane with NaClO2 and is the unexpected Baeyer–
Villiger product 1-isochromanone (4c). Thus, by the appro-
priate choice of the BVMO employed, two possible re-
gioisomers from the Baeyer–Villiger oxidation of 1-in-
danone can be obtained.
As shown in Figure 1, the effect of different organic co-
solvents (5% v/v) with HAPMO or M-PAMO as the biocat-
alyst was studied. The use of hydrophobic solvents in the
HAPMO-catalysed oxidation reactions led to an increase in
the enzymatic activity. Thus, the oxidation performed in 5%
2-octanol (logP = 2.70) or hexane (logP = 3.50) allowed
the preparation of lactone 4b with 39 and 44% conversions,
Table 3. Effect of pH on the oxidation of 1-indanone (4a) catalysed
by HAPMO or M-PAMO in different reaction media.[a]
Entry
BVMO
Cosolvent
pH
Conv. [%][b]
4b
4c
1
2
3
4
5
6
7
8
9
HAPMO
HAPMO
HAPMO
HAPMO
HAPMO
M-PAMO
M-PAMO
M-PAMO
M-PAMO
M-PAMO
5% MeOH
5% hexane
5% hexane
5% hexane
5% hexane
5% MeOH
5% MeOH
5% MeOH
5% MeOH
5% MeOH
7.0
8.0
9.0
10.0
10.5
7.0
8.0
9.0
10.0
10.5
Յ3
39
44
47
21
Յ3
Յ3
Յ3
Յ3
Յ3
Յ3
Յ3
Յ3
Յ3
Յ3
Յ3
17
79
72
72
10
[a] The reactions were performed at 30 °C for M-PAMO, whereas
20 °C was used for HAPMO. [b] Determined by GC.
The effect of pH on the enzymatic Baeyer–Villiger oxi-
respectively. On the other hand, the use of hydrophilic sol- dations of 4a catalysed by HAPMO in aqueous buffer with
vents led to a reduction of the enzymatic activity. M-PAMO 5% hexane and by M-PAMO in Tris-HCl with 5% meth-
showed the opposite behaviour in respect of the nature of anol were analysed in more detail. As described for pre-
the organic cosolvent. The use of hydrophilic solvents such vious oxidation reactions, low pH values led to no reaction
as methanol, 1,4-dioxane or 2-propanol, as well as the or to low conversion after long reaction times. For both
ethers tBuOMe or iPr2O, led to 4c with conversions higher enzymes, no oxidation was observed at pH 7.0. An increase
2528
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 2526–2532

Enzymatic Baeyer–Villiger Oxidation of Benzo-Fused Ketones
in the pH led to higher activities. M-PAMO was even able tive (6a) when using only buffer, but in the presence of 5%
to catalyse the formation of 4c with high conversions at hexane the opposite effect was observed: 5a was converted
pH 10.5 (Entry 10, c = 72%), whereas HAPMO seemed to more efficiently (c = 87%) than 6a (c = 56%) after 72 h
be deactivated at this pH: a moderate decrease in conver- (Table 4). A similar behaviour was also observed for M-
sion was observed when the pH was increased from pH 10.0 PAMO in the presence of 5% MeOH. HAPMO showed a
(Entry 4, c = 47%) to pH 10.5 (Entry 5), giving only 21% lower activity towards the oxidation of 5-bromo-1-in-
of 4b after 72 h.
danone (7a) than towards the chlorine derivative, whereas
The concentration of the organic cosolvent in both enzy- 6-bromoisochroman-1-one (7c) can be obtained with an ex-
matic systems was also studied. As shown in Figure 2, the cellent conversion (95%) in a process catalysed by M-
highest activity for HAPMO was found using 5% hexane. PAMO. The oxidation of 1-indanones bearing a methoxy
When this cosolvent concentration was increased to 30%, group in the aromatic ring occurred with lower activities
only a slight decrease in the conversion to 4b was observed. than the halogenated substrates. Thus, 4-methoxy-1-in-
Higher concentrations of hexane resulted in (partial) deacti- danone (8a) was not a substrate for the HAPMO-catalysed
vation of the enzyme, although HAPMO was still able to reaction, whereas the Baeyer–Villiger oxidations of 9a and
oxidize 4a in Tris-HCl buffer containing 70% hexane (c = 10a led to the expected lactones 9b and 10b with moderate
5% after 72 h). The enzymatic Baeyer–Villiger oxidation of conversions (32% after 72 h when employing 5% hexane).
4a catalysed by M-PAMO in the presence of methanol was On the other hand, the best results of M-PAMO were
found to be the highest at 5% (v/v), whereas higher cosol- achieved with ketone 8a (58% conversion in the presence
vent concentrations led to a progressive loss in enzymatic of 5% methanol). 6-Methoxyisochroman-1-one (9c) was
activity. The formation of lactone 4c after 72 h was not ob- obtained with a moderate conversion (c = 32% after 72 h),
served using mixtures containing 50% methanol.
whereas 6-methoxy-1-indanone (10a) was not oxidized by
this biocatalyst, which indicates that the position of the
methoxy group has a strong effect on the enzymatic activity.
Table 4. Enzymatic oxidation of substituted 1-indanones catalysed
by BVMOs in different reaction media.[a]
Entry
X
Cosolvent
BVMO
Conv. [%]
5–10b[b] 5–10c[b]
1
2
3
4
5
6
7
8
9
5-Cl
5-Cl
6-Cl
6-Cl
5-Br
5-Br
5% hexane
5% MeOH
5% hexane
5% MeOH
5% hexane
5% MeOH
HAPMO
M-PAMO
HAPMO
M-PAMO
HAPMO
M-PAMO
HAPMO
M-PAMO
HAPMO
M-PAMO
HAPMO
M-PAMO
87
Յ3
56
Յ3
52
Յ3
Յ3
Յ3
32
Յ3
32
Յ3
92
Յ3
79
Յ3
95
Յ3
58
Յ3
32
Յ3
Յ3
4-MeO 5% hexane
4-MeO 5% MeOH
5-MeO 5% hexane
5-MeO 5% MeOH
6-MeO 5% hexane
6-MeO 5% MeOH
10
11
12
Figure 2. Effects of the hexane concentration on the HAPMO oxi-
dation of 1-indanone (4a) and of the MeOH concentration on the
M-PAMO oxidation.
Յ3
[a] The reactions were performed at 30 °C for M-PAMO, whereas
20 °C was used for HAPMO. [b] Determined by GC.
To analyse the effect of different substituents on the aro-
matic moiety of 1-indanone, we studied the oxidation of
various 1-indanone derivatives 5a–10a catalysed by the
three BVMOs. As described for 1-indanone, HAPMO and
Baeyer–Villiger Oxidation of Benzocyclobutanone
Finally, the three enzymes were tested in the enzymatic
M-PAMO showed again a complete and opposite regiose- oxidation of benzocyclobutanone (11a). The Baeyer–
lectivity in the oxidation of the 1-indanone derivatives. Re- Villiger reaction of this compound led to a five-membered
actions performed with HAPMO led to the expected lac- lactone as the only regioisomer, 2-coumaranone (11b).
tones 5b–10b, whereas M-PAMO yielded the unexpected When employing HAPMO at 20 °C in aqueous buffer, this
lactones 5c–10c. In all the reactions tested, the use of 5% lactone was obtained in high yield after 72 h (Entry 1,
hexane with HAPMO and of 5% methanol with M-PAMO Table 5). The presence of 5% hexane increased the yield of
resulted in higher conversions. No reactions were observed 11b slightly (c = 93%). Good results were also obtained
for any of the substituted derivatives when wild-type PAMO when using PAMO. This biocatalyst showed low activity in
was employed. Halogenated derivatives 5a–7a were shown the oxidation of indanones and tetralones, but was able to
to be converted more efficiently by M-PAMO than by oxidize 11a with a 51% conversion at 30 °C in a solution
HAPMO, yielding high amounts of the unexpected lactones containing only buffer. By performing this reaction in the
5c–7c. The oxidation of 5-chloro-1-indanone (5a) by presence of 5% hexane, higher yields could be obtained
HAPMO was less effective than that of the 6-chloro deriva- (Entry 4). The use of 5% MeOH did not improve the results
Eur. J. Org. Chem. 2009, 2526–2532
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2529

V. Gotor et al.
FULL PAPER
recombinant 4-hydroxyacetophenone monooxygenase (HAPMO)[10a]
were overexpressed and purified as described previously. 1.0 unit of
BVMO will oxidize 1.0 µmol of phenylacetone to benzyl acetate per
minute at pH 9.0 and 25 °C in the presence of NADPH. Glucose
6-phosphate dehydrogenase from Leuconostoc mesenteroides was
obtained from Fluka-Biochemika. Starting ketones 1–6a and 8a
and lactone 11b were supplied by Sigma-Aldrich-Fluka, ketone 7a
was a product of TCI Europe and compounds 9a and 10a were
purchased from Acros Organics. All other reagents and solvents
were of the highest quality grade available and were obtained from
Sigma-Aldrich-Fluka and Acros Organics. Chemical reactions were
monitored by analytical TLC, performed on Merck silica gel 60
F254 plates and visualized by UV irradiation. Flash chromatog-
raphy was carried out with silica gel 60 (230–240 mesh, Merck).
Melting points were measured in open capillary tubes with a Gal-
lenkamp apparatus and are uncorrected. IR spectra were recorded
with a Perkin-Elmer 1720-X IR Fourier-transform spectrometer
obtained for this biocatalyst in buffer alone (c = 42% after
72 h, Entry 5). M-PAMO showed low activity towards 11a
(Entry 6), giving only a 14% yield of 11b after 72 h. The
yield of this lactone could be doubled by using 5% MeOH
as cosolvent.
Table 5. Enzymatic oxidation of 11a catalysed by BVMOs in non-
conventional reaction media.[a]
Entry
Cosolvent
BVMO
T [°C]
Conv. [%][b]
1
2
3
4
5
6
7
none
5% hexane
none
5% hexane
5% MeOH
none
HAPMO
HAPMO
PAMO
PAMO
PAMO
M-PAMO
M-PAMO
20
20
30
30
30
30
30
87
93
51
81
42
14
31
1
using KBr pellets. H and 13C NMR and DEPT spectra were re-
corded with tetramethylsilane (TMS) as the internal standard with
a Bruker AC-300 DPX spectrometer (1H: 300.13 MHz; 13C:
75.5 MHz). The chemical shifts (δ) are given in ppm. Mass spectra
were recorded in EI+ mode with a Hewlett-Packard 5973 mass
spectrometer. GC analyses were performed with a Hewlett-Packard
6890 Series II chromatograph. For all the analyses, the injector
temperature was 225 °C and the FID temperature was 250 °C. Lac-
tones 1b–10b were synthesized by Baeyer–Villiger oxidation of the
corresponding starting ketones (100 mg) with m-CPBA in CH2Cl2
at 0 °C (yields from 30 to 85%). 1-Isochromanone (4c) was pre-
pared according to the literature,[14] by oxidizing isochromane with
NaClO2 and N-hydroxyphthalimide in CH3CN/H2O (66% yield).
Compounds 1b,[15a] 2b,[15b] 3b,[15c] 4b,[15d] 5b,[8] 7b,[15e] 9b,[15f]
10b,[15g] 4c,[14] 6c[15h] and 9c[15i] exhibit physical and spectral proper-
ties in accord with those reported previously.
5% MeOH
[a] For the reaction conditions, see the Exp. Sect. [b] Determined
by GC.
Conclusions
The oxidation reactions of a set of benzo-fused ketones
biocatalysed by three BVMOs have been performed in order
to obtain benzo-fused lactones. Tetralones were poor sub-
strates for the three enzymes, whereas the bio-oxidation of
indanones produced the corresponding lactones in good
yields. The appropriate choice of reaction medium, for ex-
7-Chloro-3,4-dihydrochromen-2-one (6b): Chemical yield: 71.3 mg
ample, by the addition of different organic cosolvents (5%, (65%). Colourless solid, m.p. 53–54 °C. IR (KBr): ν˜ = 3086, 2918,
1744, 1487, 1450 cm–1. 1H NMR (300.13 MHz, CDCl3, 25 °C): δ =
v/v) to Tris-HCl buffer, led to higher conversions. This dem-
3
3
2.78 (t, JH,H = 6.8 Hz, 2 H), 2.98 (t, JH,H = 6.8 Hz, 2 H), 7.07–
7.14 (m, 3 H) ppm. 13C NMR (75.5 MHz, CDCl3, 25 °C): δ = 23.2
(CH2), 28.9 (CH2), 117.3 (CHar), 121.1 (CHar), 124.5 (CHar), 128.8
(CHar), 133.4 (Car), 152.3 (Car), 167.6 (C=O) ppm. MS (EI): m/z
(%) = 182 (100) [M]+, 154 (94), 147 (21), 119 (56), 77 (56). HRMS
(EI): calcd. for C9H7ClO2 [M]+ 182.01346; found 182.01217.
onstrates again that the addition of organic solvents can be
beneficial for enzymatic reactions. For HAPMO, the best
results were obtained when using hydrophobic organic co-
solvents, whereas M-PAMO seemed to prefer hydrophilic
solvents. Strikingly, these two enzymes also presented a di-
vergent regiospecificity in the oxidation of 1-indanone and
its derivatives. Employment of HAPMO as the biocatalyst
resulted in the formation of the expected lactones, whereas
the unexpected lactones were obtained with the PAMO mu-
tant. Wild-type PAMO was unable to convert most of the
1-indanones. This illustrates that the M446G mutation cre-
ates an active site in PAMO that is dedicated to accepting
1-indanones (see below) or indoles.[11] Thus, by choosing
carefully the reaction medium and the biocatalyst, both re-
gioisomeric Baeyer–Villiger products of 1-indanone deriva-
tives can be obtained in high yields. PAMO and HAPMO
are also able to catalyse the synthesis of 2-coumaranone
5-Methoxy-3,4-dihydrochromen-2-one (8b): Chemical yield: 23.1 mg
(42%). Pale-yellow solid, m.p. 43–45 °C. IR (KBr): ν = 3096, 2965,
˜
2360, 1770, 1612, 1469 cm–1. 1H NMR (300.13 MHz, CDCl3,
3
3
25 °C): δ = 2.71 (t, JH,H = 7.0 Hz, 2 H), 2.96 (t, JH,H = 7.0 Hz, 2
3
3
H), 3.85 (s, 3 H), 6.67 (t, JH,H = 7.6 Hz, 2 H), 7.19 (t, JH,H
=
8.2 Hz, 1 H) ppm. 13C NMR (75.5 MHz, CDCl3, 25 °C): δ = 17.4
(CH2), 28.5 (CH2), 55.7 (CH3), 106.1 (CHar), 109.3 (CHar), 111.2
(Car), 128.2 (CHar), 152.5 (Car) 156.6 (Car), 168.5 (C=O) ppm. MS
(EI): m/z (%) = 178 (100) [M]+, 150 (48), 136 (97), 77 (42). HRMS
(EI): calcd. for C10H10O3 [M]+ 178.06299; found 178.07031.
Synthesis of Benzocyclobutanone (11a): Ketone 11a was synthesized
according to the literature[16] by treating at reflux bromobenzene
with excellent conversions, which illustrates the synthetic with NaNH2 and 1,1-dimethoxyethylene in THF. The ketal ob-
tained was hydrolysed without further purification using a mixture
of THF/H2O containing traces of HCl. The final product 11a was
obtained in 83% yield.
potential of BVMO using benzo-fused ketones as sub-
strates.
General Method for the Enzymatic Baeyer–Villiger Oxidation of
Benzo-Fused Ketones: In a typical experiment, the starting ketones
1a–11a (15–30 mm) were dissolved in a 50 m Tris-HCl (pH from
Experimental Section
General Methods: Recombinant histidine-tagged phenylacetone
monooxygenase (PAMO),[9a] its M446G mutant (M-PAMO)[11] and 6.0 to 10.5)/organic cosolvent system (0.5 mL) containing glucose-
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Eur. J. Org. Chem. 2009, 2526–2532

Enzymatic Baeyer–Villiger Oxidation of Benzo-Fused Ketones
6-phosphate (2.0 equiv.), glucose-6-phosphate dehydrogenase (10.0
units), NADPH (0.2 mm) and the corresponding Baeyer–Villiger
monooxygenase (1.0 unit). The mixture was shaken at 250 r.p.m.
and the selected temperature in a rotatory shaker for the times indi-
cated. The reactions were then stopped, the mixtures worked up by
extraction with ethyl acetate (2ϫ0.5 mL), dried with Na2SO4 and
analysed directly by chiral GC to determine the conversion. For all
the reaction media tested, control experiments in the absence of
enzyme resulted in no conversion.
financed by the European Social Fund. G. d. G. (Juan de la Cierva
Program) thanks MICINN for personal funding. This work was
supported by the MICINN (Project CTQ2007-61126). M. W. F.
and D. E. T. P. received support from the EU-FP7 “Oxygreen” pro-
ject.
[1]
[2]
A. Baeyer, V. Villiger, Ber. Dtsch. Chem. Ges. 1899, 32, 3625–
3633.
a) S. Xu, Z. Wang, X. Zhang, X. Zhang, K. Ding, Angew.
Chem. Int. Ed. 2008, 47, 2840–2843; b) G. Peris, S. J. Miller,
Org. Lett. 2008, 10, 3049–3052; c) G.-J. Ten Brink, I. W. C. E.
Arends, R. A. Sheldon, Chem. Rev. 2004, 104, 4105–4123; d)
S.-I. Murahashi, S. Ono, Y. Imada, Angew. Chem. Int. Ed.
2002, 41, 2366–2368; e) M. Renz, B. Meunier, Eur. J. Org.
Chem. 1999, 4, 737–750.
General Procedure for the Enzymatic Synthesis of Lactones 5c, 7c
and 8c on the Multimilligram Scale: Reactions were performed in
duplicate. Ketones 5a, 7a and 8a (50 mg) were dissolved in 50 m
Tris-HCl buffer (pH 9.0, 10 mL) containing glucose-6-phosphate
(1.5 equiv.), glucose-6-phosphate dehydrogenase (20 units),
NADPH (0.02 mm) and M446G phenylacetone monooxygenase
(1.5 units). The mixtures were shaken at 30 °C and 250 r.p.m. for
72 h. Once finished, the reaction mixtures were extracted with
EtOAc (3ϫ25 mL), and the combined organic layers were dried
with Na2SO4, filtered and concentrated under reduced pressure.
The residues were purified by flash chromatography on silica gel
with hexane/ethyl acetate (8:2) to afford the corresponding lactones
5c and 7c and with hexane/ethyl acetate (7:3) to obtain compound
8c.
[3]
For some recent bibliographies, see: a) M. M. Kayser, Tetrahe-
dron 2009, 65, 947–974; b) D. E. Torres Pazmiño, M. W. Fraaije
in Future Directions in Biocatalysis (Ed.: T. Matsuda), Elsevier,
Dordrecht, 2007, pp. 107–127, and references cited therein; c)
M. W. Fraaije, D. B. Janssen in Modern Biooxidation, Enzymes,
Reactions and Application (Eds.: R. D. Schmid, V. D. Urlacher),
Wiley-VCH, Weinheim, 2007, pp. 77–94; d) M. D. Mihovilovic,
Curr. Org. Chem. 2006, 10, 1265–1287; e) N. M. Kamerbeerk,
D. B. Janssen, J. H. van Berkel, M. W. Fraaije, Adv. Synth. Ca-
tal. 2003, 345, 667–678; f) V. Alphand, G. Carrea, R. Wohlge-
muth, R. Furstoss, J. M. Woodley, Trends Biotechnol. 2003, 21,
318–323; g) M. D. Mihovilovic, B. Müller, P. Stanetty, Eur. J.
Org. Chem. 2002, 3711–3730.
6-Chloroisochroman-1-one (5c): Yield: 39.4 mg (72%). Colourless
solid, m.p. 58–59 °C. IR (KBr): ν = 3052, 2970, 1740, 1475 cm–1.
˜
1H NMR (300.13 MHz, CDCl3, 25 °C): δ = 3.08 (t, 3JH,H = 7.5 Hz,
3
3
2 H), 4.57 (t, JH,H = 7.5 Hz, 2 H), 7.40 (d, JH,H = 7.0 Hz, 1 H),
[4]
For some examples, see: a) M. D. Mihovilovic, P. Kapitan, P.
Kapitánová, ChemSusChem 2008, 1, 143–148; b) P. Cernu-
7.60 (s, 1 H), 7.94 (d, JH,H = 7.0 Hz, 1 H) ppm. 13C NMR
3
ˇ
(75.5 MHz, CDCl3, 25 °C): δ = 27.7 (CH2), 67.2 (CH2), 125.1
(CHar), 127.1 (CHar), 127.6 (Car), 130.2 (CHar), 133.5 (Car), 139.4
(Car), 164.9 (C=O) ppm. MS (EI): m/z (%) = 182 (90) [M]+, 154
(80), 138 (12), 126 (70), 77 (60). HRMS (EI): calcd. for C9H7ClO2
[M]+ 182.01346; found 182.01504.
chova, M. D. Mihovilovic, Org. Biomol. Chem. 2007, 5, 1715–
1719; c) R. Snajdrova, G. Grogan, M. D. Mihovilovic, Bioorg.
Med. Chem. Lett. 2006, 16, 4813–4817; d) F. Petit, R. Furstoss,
Tetrahedron: Asymmetry 1993, 4, 1341–1352; e) A. J. Carnell,
S. M. Roberts, V. Sik, A. J. Willets, J. Chem. Soc. Perkin Trans.
1 1991, 2385–2390.
a) G. Carrea, S. Riva in Asymmetric Organic Synthesis with
Enzymes (Eds.: V. Gotor, I. Alfonso, E. García-Urdiales),
Wiley-VCH, Weinheim, 2008, pp 3–20, and references cited
therein; b) A. M. Klibanov, Nature 2001, 409, 241–246.
a) G. de Gonzalo, G. Ottolina, F. Zambianchi, M. W. Fraaije,
G. Carrea, J. Mol. Catal. B 2006, 39, 91–97; b) C. Rodríguez,
G. de Gonzalo, D. E. Torres Pazmiño, M. W. Fraaije, V. Gotor,
Tetrahedron: Asymmetry 2008, 19, 197–203.
6-Bromoisochroman-1-one (7c): Yield: 34.9 mg (65%). Pale-yellow
[5]
[6]
solid, m.p. 63–65 °C IR (KBr): ν = 3052, 2980, 1736, 1470 cm–1.
˜
1H NMR (300.13 MHz, CDCl3, 25 °C): δ = 3.12 (t, 3JH,H = 6.8 Hz,
3
3
2 H), 4.60 (t, JHH = 6.8 Hz, 2 H), 7.50 (d, JH,H = 7.2 Hz, 1 H),
7.55 (s, 1 H), 7.91 (d, JH,H = 7.2 Hz, 1 H) ppm. 13C NMR
3
(75.5 MHz, CDCl3, 25 °C): δ = 26.5 (CH2), 65.6 (CH2), 127.3 (Car),
128.8 (CHar), 132.0 (CHar), 132.4 (Car), 132.6 (CHar), 144.1 (Car),
165.2 (C=O) ppm. MS (EI): m/z (%) = 226 (100) [M]+, 188 (70),
182 (10), 154 (60), 77 (80). HRMS (EI): calcd. for C9H7BrO2
[M]+ 225.96294; found 225.95931.
[7]
[8]
[9]
F. Schulz, F. Leca, F. Hollman, M. T. Reetz, Bels. J. Org. Chem.
2006, 1, 10.
M. C. Gutiérrez, V. Alphand, R. Furstoss, J. Mol. Catal. B
2003, 21, 231–238.
a) M. W. Fraaije, J. Wu, D. P. H. M. Heuts, E. W. van Helle-
mond, J. H. Lutje Spelberg, D. B. Janssen, Appl. Microbiol.
Biotechnol. 2005, 66, 393–400; b) G. de Gonzalo, D. E.
Torres Pazmiño, G. Ottolina, M. W. Fraaije, G. Carrea, Tetra-
hedron: Asymmetry 2005, 16, 3077–3083; c) M. Bocola, F.
Schulz, F. Leca, A. Vogel, M. W. Fraaije, M. T. Reetz, Adv.
Synth. Catal. 2005, 347, 979–986; d) C. Rodríguez, G. de Gon-
zalo, M. W. Fraaije, V. Gotor, Tetrahedron: Asymmetry 2007,
18, 1338–1344.
5-Methoxyisochroman-1-one (8c): Yield: 15.4 mg (28%). Pale-yel-
low solid, m.p. 44–46 °C. IR (KBr): ν = 3040, 2975, 1747,
˜
1482 cm–1. 1H NMR (300.13 MHz, CDCl3, 25 °C): δ = 3.13 (t,
3
3JH,H = 7.0 Hz, 2 H), 3.80 (s, 3 H), 4.61 (t, JH,H = 7.0 Hz, 2 H),
3
7.03–7.09 (m, 2 H), 7.67 (d, JH,H = 7.8 Hz, 1 H) ppm. 13C NMR
(75.5 MHz, CDCl3, 25 °C): δ = 28.5 (CH2), 55.7 (CH3), 65.9 (CH2),
106.2 (CHar), 109.4 (CHar), 123.2 (Car), 128.2 (CHar), 140.8 (Car),
152.6 (Car), 168.3 (C=O) ppm. MS (EI): m/z (%) = 178 (100)
[M]+, 150 (90), 134 (45), 106 (12), 77 (40). HRMS (EI): calcd. for
C10H10O3 [M]+ 178.06299; found 178.06547.
[10]
a) N. M. Kamerbeek, M. J. H. Moonen, J. G. M. van der Ven,
W. J. H. van Berkel, M. W. Fraaije, D. B. Janssen, Eur. J.
Biochem. 2001, 268, 2547–2557; b) N. M. Kamerbeek, A. J. J.
Osthorrn, M. W. Fraaije, D. B. Janssen, Appl. Environ. Micro-
biol. 2003, 69, 419–426; c) M. D. Mihovilovic, P. Kapitan, J.
Rydz, F. Rudroff, F. H. Ogink, M. W. Fraaije, J. Mol. Catal. B
2005, 32, 135–140.
Supporting Information (see footnote on the first page of this arti-
cle): Complete results of the enzymatic oxidation of 1-indanone
and its derivatives in all the reaction media, as well as the GC data
1
and H and 13C NMR spectra of lactones 5c, 6b, 7c, 8b and 8c.
Acknowledgments
[11]
[12]
D. E. Torres Pazmiño, R. Snajdrova, D. V. Rial, M. D. Mihovi-
lovic, M. W. Fraaije, Adv. Synth. Catal. 2007, 349, 1361–1368.
C.-H. Wong, G. M. Whitesides, J. Am. Chem. Soc. 1981, 103,
4890–4899.
A. R.-M. (FPU Program) thanks the Spanish Ministerio de Ciencia
e Innovación (MICINN) for her predoctoral fellowship which is
Eur. J. Org. Chem. 2009, 2526–2532
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www.eurjoc.org
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V. Gotor et al.
FULL PAPER
[13] Logarithm of the partition coefficient of a given compound in
a standard n-octanol/water two-phase system.
381–390; f) R. C. Cambie, L. H. Mitchell, P. S. Rutledge, Aust.
J. Chem. 1998, 51, 1167–1174; g) D. J. Collins, L. M. Downes,
A. G. Jhingran, S. B. Rutschmann, G. J. Sharp, Aust. J. Chem.
1989, 42, 1235–1248; h) G. Ordasso, G. Winters, Farmaco, Ed.
Sci. 1978, 33, 148–155; i) P. Mohr, M. Decker, C. Enzensperger,
J. Lehmann, J. Med. Chem. 2006, 49, 2110–2116.
[14] S. M. Silvestre, J. A. R. Salvador, Tetrahedron 2007, 63, 2439–
2445.
[15] a) P. Gorka, S. J. Miller, Org. Lett. 2008, 10, 3049–3052; b) E.
Metay, E. Leonel, C. Sulpice-Gaillet, J. Nedelec, Synthesis
2005, 10, 1682–1688; c) E. W. Lindsell, W. Edward, D. D.
Palmer, P. N. Preston, G. M. Rosair, R. V. Jones, A. J. Whitton,
Organometallics 2005, 24, 1119–1133; d) V. Kumar, A. Sharma,
A. K. Sinha, Helv. Chim. Acta 2006, 89, 483–495; e) S. G. Dav-
ies, D. Pyatt, C. Thompson, J. Organomet. Chem. 1990, 387,
[16]
R. V. Stevens, G. S. Bissacchi, J. Org. Chem. 1982, 47, 2393–
2396.
Received: January 26, 2009
Published Online: April 9, 2009
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Eur. J. Org. Chem. 2009, 2526–2532