Synthesis of Chiral 3-Alkyl-3,4-dihydroisocoumarins by dynamic kinetic resolutions catalyzed by a Baeyer-Villiger Monooxygenase

Article abstract of DOI:10.1021/jo902519j

"Chemical Equation Presented" Baeyer-Villiger monooxygenases have been tested, in the oxidation of racemic benzofused ketones. When employing a single mutant of phenylacetone monooxygenase (M.446G PAMO) under the proper reaction conditions, it was possible to achieve 3-substituted 3,4-dihydroisocoumarins with, high yields and optical purities through regioselective dynamic kinetic resolution processes.

Full text of DOI:10.1021/jo902519j

pubs.acs.org/joc
reported mainly following lateral and ortho-lithiations along
Synthesis of Chiral 3-Alkyl-3,4-dihydroisocoumarins
by Dynamic Kinetic Resolutions Catalyzed by a
Baeyer-Villiger Monooxygenase
with other strategies.2 Other recent syntheses include the
alkylation of 1,2-oxiranes followed by oxidative degrada-
tion,3 the reaction of benzocyclobutenoxides with alde-
hydes,4 domino [3 þ 3] cyclation/lactonization processes,5
the CuBr-catalyzed reaction of o-methoxycarbonylbenzene-
diazonium bromides with unsaturated compounds,6 and the
Ir-catalyzed cyclation of ketoaldehydes.7 All these methodo-
logies suffer from some drawbacks such as harsh reaction
conditions, multistep procedures, and low yields due to
functional group intolerance.
Today, enzymes are recognized as efficient catalysts for the
preparation of chiral compounds.8 A clear example of the
increasing interest in biocatalysis is the Baeyer-Villiger reaction,
a key process for the synthesis of esters and lactones.9 In the past
few years, enzymatic methodologies employing Baeyer-Villiger
monooxygenases (BVMOs) have been demonstrated to be a
very useful tool to perform selective Baeyer-Villiger oxida-
tions.10 BVMOs are flavoproteins that are able to catalyze the
oxidation of carbonylic and heteroatom-containing compounds
employing atmospheric oxygen as oxidant.11 Recent examples
of these biocatalysts are 4-hydroxyacetophenone monooxygen-
ase (HAPMO) from Pseudomonas fluorescens ACB12 and a
single mutant of the thermostable phenylacetone monooxygen-
ase (M446G PAMO) from Thermobifida fusca.13 This designed
mutant has been shown to have a different substrate profile and
selectivity when compared with the wild type (wt) enzyme.
Asymmetric BVMO-catalyzed oxidations have been deve-
loped by desymetrization of prochiral ketones or by kinetic
resolution of racemic ones. Strategies to increase the theoretical
yield of kinetic resolutions are of great importance. Thus, by
combining an in situ racemization of substrate with an enzy-
matic resolution, a dynamic kinetic resolution (DKR) can
Ana Rioz-Martınez,† Gonzalo de Gonzalo,†
‡
Daniel E. Torres Pazmino, Marco W. Fraaije, and
‡
~
Vicente Gotor*,†
†
´
ꢀ
ꢀ
Departamento de Quımica Organica e Inorganica,
´
Instituto de Biotecnologıa de Asturias, Universidad de Oviedo,
‡
ꢀ
´
Julian Claverıa 8, 33006, Oviedo, Spain, and Laboratory of
Biochemistry, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
vgs@fq.uniovi.es
Received November 26, 2009
Baeyer-Villiger monooxygenases have been tested in the
oxidation of racemic benzofused ketones. When employ-
ing a single mutant of phenylacetone monooxygenase
(M446G PAMO) under the proper reaction conditions,
it was possible to achieve 3-substituted 3,4-dihydroiso-
coumarins with high yields and optical purities through
regioselective dynamic kinetic resolution processes.
(3) Habel, A.; Boland, W. Org. Biomol. Chem. 2008, 6, 1601.
(4) Fitzgerald, J. J.; Pagano, A. R.; Sakoda, V. M.; Olofson, R. A. J. Org.
Chem. 1994, 59, 4117.
(5) Sher, M.; Ali, A.; Reinke, H.; Langer, P. Tetrahedron Lett. 2008, 49,
5400.
(6) Obushak, M. D.; Matiychuk, V. S.; Turytsya, V. V. Tetrahedron Lett.
2009, 50, 6112.
(7) Suzuki, T.; Yamada, T.; Watanabe, K.; Katoh, T. Bioorg. Med. Chem.
Lett. 2005, 15, 2583.
(8) (a) Matsuda, T.; Yamanaka, R.; Nakamura, K. Tetrahedron: Asym-
metry 2009, 20, 513. (b) Gotor, V.; Alfonso, I.; García-Urdiales, E. Asymmetric
Organic Synthesis with Enzymes, 1st ed.; Wiley-VCH: Weinheim, 2008. (c)
Woodley, J. M. Trends Biotechnol. 2008, 26, 321.
(9) (a) Punniyamurthy, T.; Velusamy, S.; Iqbal, J. Chem. Rev. 2005, 105,
2329. (b) Ten Brink, G. J.; Arends, I. W.; Sheldon, R. A. Chem. Rev. 2004,
104, 4105. (c) Renz, M.; Meunier, B. Eur. J. Org. Chem. 1999, 737.
(10) For recent reviews, see: (a) Kayser, M. M. Tetrahedron 2009, 65, 947.
3,4-Dihydroisocoumarins and their derivatives are com-
pounds that widely exist in nature as key intermediates in the
synthesis of biologically active molecules. As these com-
pounds are known to have interesting activities (e.g., anti-
fungal, antiallergenic, antiulcer, and antimalarial), they are
regarded as highly attractive molecules in organic chemis-
try.1 To date, several synthetic routes for these derivatives
have been described. A number of methods have been
~
(b) Torres Pazmino, D. E.; Fraaije, M. W. In Future Directions in Biocatalysis;
Matsuda, T., Ed.; Elsevier: Dordrecht, 2007; p 107. (c) Mihovilovic, M. D. Curr.
Org. Chem. 2006, 10, 1265.
(11) (a) Szolkowy, C.; Eltis, L. D.; Bruce, N. L.; Grogan, G. ChemBio-
Chem 2009, 10, 1208. (b) Snajdrova, R.; Braun, I.; Bach, T.; Mereiter, K.;
Mihovilovic, M. D. J. Org. Chem. 2007, 72, 9597. (c) Clouthier, C. M.;
Kayser, M. M.; Reetz, M. T. J. Org. Chem. 2006, 71, 8431. (d) Mihovilovic,
M. D.; Rudroff, F.; Winniger, A.; Schneider, T.; Schulz, F.; Reetz, M. T. Org.
Lett. 2006, 8, 1221.
(1) (a) Jiao, P.; Gloer, J. B.; Campbell, J.; Shearer, C. A. J. Nat. Prod.
2006, 69, 612. (b) Zidorn, C.; Lohwasser, U.; Pschorr, S.; Salvenmoser, D.;
€
Ongania, K.-H.; Ellmerer, E. P.; Borner, A.; Stuppner, H. Phytochemistry
~
2005, 66, 1691. (c) Umehara, K.; Matsumoto, M.; Nakamura, M.; Miyase,
T.; Kuroyanagi, M.; Noguchi, H. Chem. Pharm. Bull. 2000, 48, 566.
(2) (a) Mandal, S. M.; Roy, S. C. Tetrahedron Lett. 2007, 48, 4131. (b)
Uchida, K.; Fukuda, T.; Iwao, M. Tetrahedron 2007, 63, 7178. (c) Kurosaki,
Y.; Fukuda, T.; Iwao, M. Tetrahedron 2005, 61, 3289. (d) Superchi, S.;
Minutolo, F.; Pini, D.; Salvadori, P. J. Org. Chem. 1996, 61, 3183.
(12) (a) de Gonzalo, G.; Torres Pazmino, D. E.; Ottolina, G.; Fraaije, M.
W.; Carrea, G. Tetrahedron: Asymmetry 2006, 17, 130. (b) Kamerbeek, N.
M.; Olsthoorn, A. J. J.; Fraaije, M. W.; Janssen, D. B. Appl. Environ.
Microbiol. 2003, 69, 419.
(13) Torres Pazmino, D. E.; Snajdrova, R.; Rial, D. V.; Mihovilovic, M.
D.; Fraaije, M. D. Adv. Synth. Catal. 2007, 349, 1361.
~
DOI: 10.1021/jo902519j
r
Published on Web 02/18/2010
J. Org. Chem. 2010, 75, 2073–2076 2073
2010 American Chemical Society

Rioz-Martınez et al.
JOCNote
TABLE 1. Enzymatic Oxidation of 2-Methyl-1-indanone (()-2a Cata-
lyzed by BVMOs in Buffer Containing Hexane (5% v/v)a
time
(h)
temp
(°C)
ee of
2ab (%)
ee of
2b,cb (%)
cb
(%)
entry
BVMO
1c
2c
3
HAPMO
M446G
M446G
M446G
72
72
168
168
20
30
40
40
5
10
7
6
92
82
74
13
33
84
89
FIGURE 1. Time progress of the DKR of (()-2a catalyzed by
M446G PAMO in buffer pH 10 containing 5% hexane: 2, c (%),
9, ee (S)-2a (%), b, ee (R)-2c (%).
4d
5
aFor experimental details, see the Supporting Information. bDeter-
mined by GC. c[2a] = 4.0 mg mL-1. dpH = 10.5.
(R)-2b was obtained with low conversion and selectivity. Longer
reaction times did not change the results obtained. Biooxidations
catalyzed by M446G PAMO led to a different outcome. (R)-
3-Methyl-3,4-dihydroisocoumarin (R)-2c was obtained with
high optical purity (ee = 92%) and 33% conversion after 3
days (entry 2).19 The starting ketone, presenting (S)-configura-
tion, was recovered with only a 10% enantiomeric excess. This
process was carried out with a smaller amount of substrate at
40 °C and stopped after 168 h, obtaining (R)-2c with 84%
conversion and 82% enantiomeric excess. This indicates that a
DKR process has been performed on this substrate by using high
pHs and the appropriate biocatalyst. (S)-2a was achieved almost
racemic (ee = 7%). More drastic conditions for the substrate
racemization were achieved by using pH 10.5. After one week,
conversion was improved to 89% but the optical purity of (R)-2c
decreased to 74%.
The progress of the M446G PAMO-catalyzed oxidation
of (()-2a in buffer pH 10.0 containing 5% hexane was
analyzed in more detail. As shown in Figure 1, the reaction
led to 50% conversion after 6 h, presenting a classical kinetic
resolution behavior with optical purities of ee = 38% for (S)-
2a and ee = 92% for (R)-2c. After this first phase, racemiza-
tion of starting ketone became predominant, producing an
important loss in optical purity of 2a, while the ee of (R)-2c
remained almost constant. After 72 h, oxidation had reached
78% conversion and the chiral lactone was obtained with a
88% enantiomeric excess, while (S)-2a was almost racemic.
Longer reaction times only resulted in a slight increase in the
conversion (84%), but this was accompanied with a slight
loss in optical purity of (R)-2c due to a partial racemization
of this lactone.
be achieved,14 which allows the transformation of both enan-
tiomers of the starting material into a single enantiomeric
product in 100% theoretical yield. Recently, the first
BVMO-based DKRs have been described in which re-
combinant Escherichia coli cells containing cyclohexanone
monooxygenase were used at relatively high pH or combined
with ion-exchange resins.15
Previously, it has been shown than HAPMO was able to
convert 1-indanones into the normal lactones, whereas
M446G PAMO yielded the nonexpected ones.16 In view of
this opposite regioselectivity and attending to the structure
of substituted benzofused ketones (which possess an acidic
hydrogen and are therefore being able to racemize at high
pH), we report herein the enantioselective oxidation of these
substrates using isolated BVMOs in order to obtain the
corresponding chiral lactones.
We first analyzed the kinetic resolution of racemic 2-
methyl-1-tetralone (()-1a. No oxidation of this compound
was observed when using HAPMO. Resolutions catalyzed
by M446G PAMO allowed us to recover (S)-1a and lactone
(R)-3-methyl-4,5-dihydrobenzo[b]oxepin-2(3H)-one (1b), al-
beit with very low optical purities and conversions (data
shown in the Supporting Information).
Next, we focused on the enzymatic resolution of 2-alkyl-1-
indanones. Oxidation of rac-2-methyl-1-indanone (()-2a was
performed in a biphasic system, Tris-HCl buffer pH 10.0 con-
taining 5% hexane (Table 1),17 as these biocatalysts have been
shown to work in aqueous-organic solvent media.18 When
HAPMO was employed, (R)-3-methyl-3,4-dihydrocoumarin
Once it had been established that the best optical purity in
combination with a reasonable conversion of lactone (R)-2c
could be obtained after 48 h, several reaction parameters
were varied in order to improve the reaction. First, the
enzymatic resolution of (()-2a was performed in different
reaction media. As shown in Table 2, the oxidation catalyzed
by M446G PAMO in aqueous buffer led to 76% of the (R)-
lactone with a 79% enantiomeric excess. This is slightly
€
(14) See, for example: (a) Martín-Matute, V.; Backvall, J. E. In Asymmetric
Organic Synthesis with Enzymes; Gotor, V., Alfonso, I., García-Urdiales, E., Ed.;
Wiley-VCH: Weinheim, 2008; p 89. (b) Kim, M.-J.; Park, J.; Choi, Y. K. In Multi-
Step Enzyme Catalysis; García-Junceda, E., Ed.; Wiley-VCH: Weinheim, 2008;
p 1. (c) Pellissier, H. Tetrahedron 2008, 64, 1563. (d) Pamies, O.; Backvall, J.-E.
Chem. Rev. 2003, 103, 3247.
(15) (a) Berezina, N.; Alphand, V.; Furstoss, R. Tetrahedron: Asymmetry
ꢀ
2002, 13, 1953. (b) Gutierrez, M.-C.; Furstoss, R.; Alphand, V. Adv. Synth.
Catal. 2005, 347, 1051.
(16) Rioz-Martınez, A.; de Gonzalo, G.; Torres Pazmino, D. E.; Fraaije,
ꢁ
€
~
M. W.; Gotor, V. Eur. J. Org. Chem. 2009, 2526.
(17) Other buffers were tested for the enzymatic oxidation of (()-2a (see
the Supporting Information); the best results were achieved in Tris/HCl.
(19) There is a previous example of a BVMO from Pseudomonas sp. which
catalyzed the oxidationof2a toobtain 2c, but the reaction transcurred with low
selectivity. See: Iwaki, H.; Wang, S.; Grosse, S.; Bergeron, H.; Nagahashi, A.;
Lertvorachon, J.; Yang, J.; Konishi, Y.; Hasegawa, Y.; Lau, P. C. K. Appl.
Environ. Microbiol. 2006, 72, 2707.
~
(18) (a) Rodrıguez, C.; de Gonzalo, G.; Torres Pazmino, D. E.; Fraaije,
M. W.; Gotor, V. Tetrahedron: Asymmetry 2008, 19, 197. (b) de Gonzalo, G.;
Ottolina, G.; Zambianchi, F.; Fraaije, M. W.; Carrea, G. J. Mol. Catal. B:
Enzym. 2005, 39, 91.
2074 J. Org. Chem. Vol. 75, No. 6, 2010

Rioz-Martınez et al.
JOCNote
TABLE 2. Enzymatic Oxidation of (()-2a Catalyzed by M446G PAMO
TABLE 3. Enzymatic Resolution of Racemic 2-Alkyl-1-indanones Cata-
lyzed by M446G PAMOa
in Different Reaction Media after 48 ha
temp
(°C)
ee of
2ab (%)
ee of
2cb (%)
entry
solvent
none
pH
cb (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15c
10
10
10
10
10
10
10
10
10
10
9
40
40
40
40
40
40
30
20
30
20
40
40
40
40
40
e3
11
10
35
22
13
26
5
79
54
76
50
82
88
74
82
81
77
87
88
83
83
84
76
83
73
85
73
73
61
13
60
20
50
38
64
60
56
5% MeOH
5% dioxane
5% i-PrOH
5% i-Pr2O
5% hexane
5% MeOH
5% MeOH
5% hexane
5% hexane
5% MeOH
5% MeOH
5% hexane
5% hexane
5% hexane
ee of
3-5ab
(%)
ee of
3-5cb
(%)
28
6
time
(h)
cb
(%)
entry
R
solvent
none
5% hexane
5% MeOH
5% MeOH
none
5% hexane
5% MeOH
none
5% hexane
5% MeOH
e3
16
19
34
e3
1
2
Et
Et
Et
Et
i-Pr
i-Pr
i-Pr
n-Bu
n-Bu
n-Bu
96
96
96
120
104
104
104
144
96
6
11
10
16
17
17
16
17
11
20
91
91
60
85
g97
g97
g97
92
92
88
72
36
89
45
46
53
42
80
26
78
8
9
8
10
3
4c
5
aFor experimental details, see the Supporting Information. bDeter-
6
7
8
9
10
mined by GC. cReaction in the presence of 5 equiv of Lewatit MP-62.
lower when compared with the optical purity obtained in 5%
v/v hexane. Reaction rates were improved by employing 5%
of short chain alcohols, such as methanol or 2-propanol in
the reaction medium. The results are in accordance with
those described for M446G PAMO in the oxidation of
nonracemic indanones.16 However, these two solvents affec-
ted negatively the enzymatic selectivity. (R)-2c was obtained
only with 54% and 50% enantiomeric excess, as shown in
entries 2 and 4. The use of 1,4-dioxane or i-Pr2O allowed us
to obtain conversions and optical purities similar to those
obtained in aqueous buffer (entries 3 and 5).
As shown in entries 7-10, the effect of the temperature was
analyzed when 5% v/v hexane and methanol were used. As
expected, the decrease of the reaction temperature caused a loss
in the conversions, as previous studies have indicated that the wt
enzyme shows a higher activity by increasing the temperature.20
When methanol was employed at lower reaction temperatures
(entries 7 and 8), an important gain in the M446G PAMO
selectivity was achieved. By reducing the pH of the medium,
reactions became much slower and resulted in an increase in
enantioselectivity (ee = 87-88% at pH 8.0 or 9.0; entries 11 and
12) when buffer containing 5% methanol was used.
The effect of different anion-exchange resins in the
dynamic kinetic resolution of (()-2a using buffer with 5%
hexane was also studied. For all of the resins analyzed (see
the Supporting Information), racemization of the starting
ketone was complete. The enzymatic selectivity was un-
altered, but a partial inactivation of M446G PAMO was
observed. Strong anion-exchange resins were found to be
especially harmful for enzyme activity. The best result was
obtained using Lewatit MP-62, a weak anion-exchange
resin, which led to 56% of (R)-2c with 84% ee (entry 15).
Using cell free extract containing M446G PAMO, we were
also able to perform a conversion of 250 mg (()-2a, resulting
in 221 mg of isolated (R)-2c (80% yield). This illustrates that
no enzyme purification is needed to perform these reactions
on a multimilligram scale.
144
aFor experimental details, see the Supporting Information. bDeter-
mined by GC. cReaction performed at pH 8.0.
was observed when the corresponding ketones were incubated
in the presence of HAPMO, while M446G PAMO was able to
convert these ketones. Resolution of racemic 2-ethyl-1-inda-
none (()-3a led to (R)-3c with a 72% conversion after 96 h and
a high optical purity (ee = 91%) when employing buffer Tris-
HCl pH 10.0. The use of 5% hexane led to an important
decrease in enzymatic activity (entry 2), while the presence of
5% methanol improved the enzymatic activity (c= 89%) with a
loss in the enantiomeric excess of (R)-3c. As shown in entry 4,
oxidation of (()-3a was carried out using this cosolvent at pH
8.0 in order to increase the selectivity. This resulted in 45% of
(R)-3c with 85% optical purity. This oxidation was also per-
formed at 20 °C, but the enzyme selectivity was not increased,
while a lower conversion was observed after 120 h (data not
shown). (()-2-Isopropyl-1-indanone (()-4a was oxidized with
complete selectivity to (R)-4c (ee g 97%) in the presence of
M446G PAMO by employing Tris-HCl buffer as well as
mixtures of buffer containing 5% hexane or methanol. Oxida-
tion was slightly faster when 5% hexane (c = 53% after 104 h,
entry 6) was added than when other reaction media (conver-
sions lower than 50%) were used. Longer reaction times did not
improve the results obtained. (R)-3-Butyl-3,4-dihydroisocou-
marin (5c) can also be obtained with high enantiomeric excess
(optical purities around 90%) in all of the reaction media
analyzed (entries 8-10). The first set of experiments showed
that the DKR of (()-5a was much faster in buffer or in the
presence of 5% methanol. It was possible to achieve (R)-5c with
conversions close to 80%, as shown in entries 8 and 10. Results
obtained indicate that alkyl chains longer than ethyl have a
negative effect on enzyme activity, which is in accordance with
previous results obtained for this BVMO.20b
The biocatalyzed oxidations were extended to other race-
mic 1-indanones (Table 3). No formation of lactones 3-5b
Finally, we have focused on the oxidation of racemic
3-methyl-1-indanone, (()-6a. For this compound, the base-
induced racemization was not feasible. Therefore, a kinetic
resolution was performed. Depending on the biocatalyst
employed, opposite regioselectivities were again observed.
After the reaction conditions (see Supporting Information)
(20) See, for example: (a) Rodrıguez, C.; de Gonzalo, G.; Fraaije, M. W.;
Gotor, V. Tetrahedron: Asymmetry 2007, 18, 1338. (b) Rodrıguez, C.;
~
de Gonzalo, G.; Torres Pazmino, D. E.; Fraaije, M. W.; Gotor, V. Tetra-
hedron: Asymmetry 2009, 20, 1168.
J. Org. Chem. Vol. 75, No. 6, 2010 2075

Rioz-Martınez et al.
JOCNote
were optimized, the expected oxidation product (R)-4-
methyl-3,4-dihydrocoumarin (6b) was achieved with 23%
conversion when using HAPMO. The M446G PAMO-
biocatalyzed oxidation led to (R)-4-methyl-3,4-dihydro-
isocoumarin (6c) in a 12% conversion. For both enzymes,
high enantioselectivities (E g 200) were measured.
layer were separated by centrifugation, dried onto Na2SO4, and
analyzed directly by GC in order to determine the conversion
of the oxidations and the enantiomeric excesses of the ketones
(S)-1-6a and the lactones obtained (R)-1-6b or (R)-2-6c.
Enzymatic Preparation of Chiral 3-Alkyl-3,4-dihydroisocou-
marins (()-2-5c Catalyzed by M446G PAMO on a Multimilli-
gram Scale. Ketones (()-2-5a (60 mg, 0.41-0.32 mmol) were
dissolved in a Tris-HCl buffer (50 mM, pH 10.0, 30 mL)
containing 5% hexane for the oxidation of compound (()-2a
and (()-4a. Then, NADPH (0.2 mM), glucose 6-phosphate
(147-213 mg, 0.82-0.64 mmol), glucose 6-phosphate dehydro-
genase (300 units), and M446G PAMO (30 units) were added.
The mixture was shaken at 250 rpm at 40 °C during 120 h for
(()-2a, 96 h for (()-3a, 104 h for (()-4a, and 144 h for (()-5a.
The reaction was then stopped by extraction with ethyl acetate
(4 ꢀ 15 mL), the combined organic layers were dried over
Na2SO4, and the solvent was evaporated under reduced pres-
sure. Afterward, the crude residue were purified by flash chro-
matography using as eluent hexane/ethyl acetate 9:1 in order to
obtain (R)-2c (49.9 mg, 75%), (R)-3c (42.9 mg, 65%), (R)-4c
(32.8 mg, 50%), and (R)-5c (47.5 mg, 73%).
Enzymatic Preparation of Chiral 3-Methyl-3,4-dihydroisocou-
marin (()-2c Employing Cell Free Extract Containing M446G
PAMO on a 250 mg Scale. Compound (()-2a (250 mg, 1.71
mmol) was dissolved in a Tris-HCl buffer (50 mM, pH 10.0,
40 mL). Then, NADPH (0.2 mM), glucose 6-phosphate (890 mg,
3.4 mmol), glucose 6-phosphate dehydrogenase (1250 units),
and M446G PAMO cell free extract (46 mL) were added. The
mixture was shaken at 250 rpm at 40 °C and monitored by GC.
After 48 h, the reaction was stopped by extraction with EtOAc
(5 ꢀ 30 mL), and the organic layers were dried on Na2SO4 and
evaporated under reduced pressure. The crude residue were
purified by flash chromatography using as eluent hexane/ethyl
acetate 9:1 in order to obtain (R)-2c (221 mg, 80% yield).
The enzymatic preparation of different 3-alkyl-3,4-dihy-
droisocoumarins has been developed with high yields and
enantiomeric excesses by choosing properly the reaction
parameters for enzymatic Baeyer-Villiger oxidations. These
substrates were obtained from the corresponding 2-alkyl-
1-indanones due to the regioselectivity displayed by M446G
PAMO. The presence of an acidic hydrogen in the R-position
to the carbonylic moiety can induce substrate racemization
by working at high pHs (pH 10.0), allowing effective
dynamic kinetic resolution of the starting ketones. Addition
of 5% methanol increases the enzymatic activity with a loss
of selectivity, while the use of hexane leads to the best
selectivities. Higher conversions were obtained when ketones
presenting short alkyl chains were oxidzed. This study shows
that it is possible to obtain all targeted 3,4-dihydroiso-
coumarins with high optical purities. An enzymatic oxidation
has been scaled up to 250 mg scale using cell-free extract.
Experimental Section
General Methods. Recombinant histidine-tagged phenylace-
tone mononoxygenase mutant (M446G PAMO) and recombi-
nant 4-hydroxyacetophenone monooxygenase (HAPMO) were
overexpressed and purified as previously described.12b,13 A 1.0
unit of BVMO will oxidize 1.0 μmol of phenylacetone to benzyl
acetate per minute at pH 9.0 and room temperature in pre-
sence of NADPH. Glucose 6-phosphate dehydrogenase from
Leuconostoc mesenteroides, racemic ketones (()-1-3a and
(()-5-6a, and all other reagents and solvents were of the highest
quality grade available and were purchased from commercial
sources.
Acknowledgment. This work was supported by the MI-
CINN (Project CTQ2007-61126). A.R-M. (FPU program)
ꢀ
thanks the Spanish Ministerio de Ciencia e Innovacion
General Procedure for the Biooxidation of Racemic Ketones
(()-1-6a Catalyzed by BVMOs. The corresponding racemic
ketones (()-1-6a (14-28 mM) were dissolved in a 50 mM
Tris-HCl buffer at different pH’s containing 5% organic coso-
lvent if stated (1.0 mL), glucose 6-phosphate (28-56 mM),
glucose 6-phosphate dehydrogenase (10.0 units), NADPH (0.2
mM), and the corresponding Baeyer-Villiger monooxygenase
(1.0 unit). Reactions were shaken at 250 rpm, and the tempera-
tures and times were established. Once finished, the crude
reactions were extracted with EtOAc (2 ꢀ 500 μL). The organic
(MICINN) for her predoctoral fellowship, which is financed
by the European Social fond. G.d.G. thanks MICINN for
personal funding (Juan de la Cierva program). M.W.F. and
D.E.T.P. are supported by the EU-FP7 “Oxygreen” project.
Supporting Information Available: Complete results, experi-
mental data, as well as the achiral and chiral GC data and
product characterization. This material is available free of
charge via the Internet at http://pubs.acs.org.
2076 J. Org. Chem. Vol. 75, No. 6, 2010