close

Вход

Забыли?

вход по аккаунту

?

Kinetic Resolution of 4-Hydroxy-2-ketones Catalyzed by a BaeyerЦVilliger Monooxygenase.

код для вставкиСкачать
Communications
Enantioselective Reactions
DOI: 10.1002/anie.200602986
Kinetic Resolution of 4-Hydroxy-2-ketones
Catalyzed by a Baeyer–Villiger
Monooxygenase**
Smith and Levenberg)[12] gave hydroxyalkyl acetates 2 a–c
with good enantioselectivity (Scheme 1, Figure 1, Table 1).
The oxidation product was always obtained in greater than
90 % ee and S configuration. E values[18] of around 50 should
also enable the isolation of the optically active starting
Anett Kirschner and
Uwe T. Bornscheuer*
Baeyer–Villiger monooxygenases (BVMOs)
belong to the class of oxidoreductases and
convert aliphatic, aryl aliphatic, and cyclic
ketones into esters and lactones, respectively, using molecular oxygen.[1, 2] Thus
they mimic the chemical Baeyer–Villiger
oxidation,[3] which is usually peracid catalyzed and proceeds through a two-step
Scheme 1. Enzymatic Baeyer–Villiger oxidation of racemic 4-hydroxy-2-ketones by BVMO
from P. fluorescens DSM 50106 expressed in E. coli. NAD = nicotinamide adenine dinucleomechanism.[4] Additionally, BVMOs are
tide.
capable of oxidizing heteroatom-containing
functions (including S, N, and P groups).[5]
In particular, for enantioselective Baeyer–
Villiger oxidations,[6] BVMOs represent a valuable alternative
to metal-based chiral catalysts.[7] While stereoselective
Baeyer–Villiger oxidations using enzymes have been described for prochiral or racemic mono- and bicyclic ketones,[8]
as well as for racemic aromatic ketones,[2, 9] so far no example
of an enantioselective BVMO-catalyzed conversion of racemic aliphatic acyclic ketones has been reported. Only the use
of 3-chloro-2-butanone as the substrate for a 4-hydroxyacetophenone monooxygenase has been reported but the conversion was poor, and the potential enantioselectivity of the
reaction was not addressed.[2] The chemical Baeyer–Villiger
oxidation of b-hydroxyketones using peracids gave acylated
Figure 1. Time-dependent conversion of 1 a into 2 a at 30 8C using
diols, but the reactions were not enantioselective.[10]
resting cells of E. coli, which afforded the BVMO from P. fluorescens
Herein we report the first BMVO-catalyzed kinetic
DSM 50106. &: (R)-1 a, ^: (S)-1 a, ~: (S)-2 a, *: (R)-2 a).
resolution of aliphatic acyclic ketones with racemic 4hydroxy-2-ketones serving as model substrates. We were
Table 1: Results of the kinetic resolution of 1 a–c.[a]
prompted to investigate these compounds as we recently
discovered that a BVMO from Pseudomonas fluorescens
Substr.
t [h]
Conv. [%][b]
Enantiomeric excess[c]
E[b]
DSM 50106, which we could recombinantly express in E. coli,
[% eeS]
[% eeP]
converts aliphatic 2-ketones into the corresponding acetates
1a
8
40
61
93
54
but shows little activity towards cyclic ketones.[11] Indeed, we
1b
4
48
84
91
55
were pleased to find that the racemic 4-hydroxy-2-ketones
1c
2
45
74
90
41
1 a–c (synthesized according to the method described by
[*] Dipl.-Biochem. A. Kirschner, Prof. Dr. U. T. Bornscheuer
Institute of Biochemistry
Dept. of Biotechnology & Enzyme Catalysis
Ernst Moritz Arndt University of Greifswald
Friedrich-Ludwig-Jahn-Strasse 18c, 17487 Greifswald (Germany)
Fax: (+ 49) 3834-86-80066
E-mail: uwe.bornscheuer@uni-greifswald.de
Homepage: www.chemie.uni-greifswald.de/ ~ biotech
[**] The authors thank the Fonds der Chemischen Industrie (Germany)
and the Studienstiftung des Deutschen Volkes (Germany) for
stipends (A.K.). The assistance of A. Gollin for the chemical
synthesis of b-hydroxyketones is acknowledged.
7004
[a] Using resting cells of E. coli to produce the BVMO from P. fluorescens
DSM 50106 at 30 8C. [b] Calculated according to Chen et al.[18] [c] Determined by GC analysis on a chiral stationary phase.
hydroxyketones if the reaction proceeds with approximately
55 % conversion. Although it is not relevant to the optical
purity of the product, we observed migration of the acetyl
group from the primary to the secondary hydroxy group,
which gave a 4:1 mixture of the acetylated 1,2-diols 2 a–c and
3 a–c as determined by GC-MS analysis. A similar observation
was reported by Park and Kozikowski, although their ratio of
7:3 possibly results from the different reaction conditions.[10]
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 7004 –7006
Angewandte
Chemie
Minor amounts ( 5 %) of the other possible product of a
Baeyer–Villiger oxidation, hydroxy acid methyl esters 4 a–c,
were also detected.
Although the BVMO from P. fluorescens DSM 50106
prefers short-chain aliphatic acyclic ketones, 1 b and 1 c were
converted much faster than 1 a, with close to 50 % conversion
after 4 h when resting cells were used. Since BVMOs are
flavin-dependent (mainly flavin adenine dinucleotide) and
require NAD(P)H to catalyze the reaction, the biotransformations were performed using E. coli recombinant whole
cells, either growing or resting, and thereby the Baeyer–
Villiger monooxygenase was expressed from P. fluorescens
DSM 50106.[11] Thus cofactor regeneration, and in our case
also enzyme stability, were not limiting factors. When growing
cells were used, the substrate was added at the same time as
induction of BVMO expression, whereas for resting cells the
enzyme was first expressed and then the harvested cells were
resuspended in phosphate buffer prior to the biotransformations.[13] In the latter case, glucose was added along with the
racemic substrate to facilitate efficient cofactor recycling.[14]
The major advantage of using resting cells rather than
growing cells was that reaction times were considerably
shortened while higher conversions were observed. Additionally, these resting cells could be stored in the refrigerator
for up to three weeks with only modest loss of activity (20–
30 %, data not shown). This is in contrast to the observations
made using recombinant cyclohexanone monooxygenase,
which is actively degraded during biocatalysis using E. coli
resting cells.[15]
Interestingly, this kinetic resolution can be used to
generate two different chemical species, which additionally
differ in their configuration. Besides the residual optically
active hydroxyketone (1 a–c), an optically active acetate of a
1,2-diol is formed. This feature makes this biotransformation
especially useful, as the regio- and enantioselective reduction
of a 2,4-diketone to afford (R)-1 would require a highly
specific ketoreductase since four diastereomers may be
formed.[16] The alternative kinetic resolution of a 1,2-diol
using a lipase or esterase proceeds with low enantioselectivity,[17] as these hydrolases preferentially cleave (or acylate) the
primary alcohol functionality. Thus, the reaction described
here not only broadens the synthetic applicability of Baeyer–
Villiger monooxygenases but also provides a synthetically
useful alternative to established enzymes such as hydrolases
and ketoreductases in organic synthesis.
Experimental Section
Unless otherwise specified, all chemicals were purchased from SigmaAldrich, Fisher Scientific, VWR, ABCR, and Roth GmbH and were
used without further purification. All solvents were distilled prior to
use. NMR spectra were recorded in CDCl3 on a 300-MHz (Bruker)
instrument.
The products from the BVMO-catalyzed reaction were synthesized separately for comparison purposes. Racemic 4-hydroxy-2ketones 1 a–c were synthesized by aldol condensation according to the
method described by Smith and Levenberg.[12] Corresponding
hydroxyalkyl acetates 2 a–c were synthesized enzymatically using
lipase B from Candida antarctica. Thus 5 mg of the immobilized
enzyme (Chirazyme L-2, C-2, Roche) was mixed with 300 mL of
Angew. Chem. Int. Ed. 2006, 45, 7004 –7006
isooctane and 300 mL of vinyl acetate in a 2-mL glass vial. The
reaction was started by addition of the 1,2-diol (10 mg), and the
mixture was incubated at 25 8C in a thermoshaker (Eppendorf) for
approximately 2 h until the conversion was complete. Samples were
used directly for GC analysis. NMR data were consistent with
previously published data.[19] Hydroxy acid methyl esters 4 a–b were
synthesized from the corresponding b-ketoacid methyl esters through
a reduction catalyzed by alcohol dehydrogenase, using the recombinant enzyme from P. fluorescens DSM 50106.[20] Thus, 5 mg of the
enzyme lyophilisate was mixed with 800 mL of phosphate buffer
(50 mm, pH 7.5), 200 mL of 2-propanol and 2 mL of b-ketoacid methyl
ester. Reaction mixtures were incubated at 20 8C for 24 h in a
thermoshaker. The reaction mixture was then extracted twice with
500 mL ethyl acetate. The combined organic phases were dried over
anhydrous sodium sulfate and concentrated by passing N2 through the
flask. The resulting products were analyzed by using GC. The bketoacid methyl esters that were not commercially available were
synthesized according to the method described by Oikawa et al.[21]
Absolute configurations of the hydroxyalkyl acetates 2 a–c were
determined by comparison with (R)-2-hydroxydecyl acetate ((R)-2 c),
which was synthesized from (R)-1,2-decanediol by using lipasecatalyzed transesterification. This result revealed a preferential
oxidation of the S enantiomers of 1 a–c by the BVMO.
Biocatalysis using resting cells: The expression of the BVMO in
E. coli JM109 pGro7 pJOE4072.6 + HT[11] was performed in 200 mL
of LBcm+amp containing 0.5 mg mL 1 l-arabinose at 30 8C. Cells were
grown up to an optical density of 0.5–0.6 at 600 nm, and BVMO
expression was induced by the addition of l-rhamnose (0.2 % (w/v)
final concentration). After further growth for 4 h, cells were
harvested by centrifugation and washed once with sterile phosphate
buffer (50 mm, pH 7.5). For biocatalysis reactions in small volume,
cells were resuspended in the same buffer to an OD of around 40, and
1-mL aliquots of this cell suspension were mixed in 2 mL Eppendorf
reaction vials with 5 mmol of 4-hydroxy-2-ketone 1 a–c and 10 mL of a
sterile 1m glucose solution. The vials were closed with air-permeable
caps (LidBac, Eppendorf) and incubated at 30 8C in a thermoshaker
(Eppendorf) at 1400 rpm. After set intervals, samples (300 mL) were
taken, extracted twice with ethyl acetate, and dried over anhydrous
sodium sulfate. Excess solvent was removed under nitrogen, and
samples were analyzed by using GC.
GC analyses on a chiral stationary phase (Table 2) were carried
out on a Shimadzu GC-14A gas chromatograph with a chiral bcyclodextrin column (Hydrodex-b-3P, Macherey–Nagel). Injection
and detection temperature were set at 220 8C. GC-MS analysis was
performed using a Shimadzu QP-2010 device equipped with the same
chiral column.
Table 2: GC analyses on a chiral stationary phase of 1 a–c and 2 a–c.[a]
Cmpd.
Heating Program (Column)
tR [min]
S/R[b]
1a
1b
1c
2a
2b
2c
20 min, 90 8C//20 8C min 1//110 8C, 15 min
15 min,120 8C//20 8C min 1//130 8C, 15 min
20 min, 135 8C//20 8C min 1//145 8C, 15 min
20 min, 90 8C//20 8C min 1//110 8C, 15 min
15 min,120 8C//20 8C min 1//130 8C, 15 min
20 min, 135 8C//20 8C min 1//145 8C, 15 min
17.8/18.6
12.7/13.3
18.3/19.1
23.3/24.1
23.3/24.1
22.4/23.2
[a] Using a chiral b-cyclodextrin column (Hydrodex-b-3P). [b] Elution
order of enantiomers is S before R as determined by using a standard of
(R)-2 c.
Received: July 25, 2006
Published online: October 2, 2006
.
Keywords: b-hydroxyketones · Baeyer–Villiger monooxygenases ·
enantioselectivity · enzyme catalysis · kinetic resolution
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7005
Communications
[1] M. D. Mihovilovic, B. MHller, P. Stanetty, Eur. J. Org. Chem.
2002, 3711.
[2] N. M. Kamerbeek, J. J. Olsthoorn, M. W. Fraaije, D. B. Janssen,
Appl. Environ. Microbiol. 2003, 69, 419.
[3] a) C. Walsh, Y. C. J. Chen, Angew. Chem. 1988, 100, 342; Angew.
Chem. Int. Ed. Engl. 1988, 27, 333; ; b) A. Baeyer, V. Villiger,
Ber. Dtsch. Chem. 1899, 32, 3625.
[4] R. Criegee, Justus Liebigs Ann. Chem. 1948, 560, 127.
[5] a) G. Carrea, B. Redigolo, S. Riva, S. Colonna, N. Gaggero, E.
Battistel, D. Bianchi, Tetrahedron: Asymmetry 1992, 3, 1063;
b) G. de Gonzalo, D. E. Torres Pazmino, G. Ottolina, M. W.
Fraaije, G. Carrea, Tetrahedron: Asymmetry 2006, 17, 130.
[6] M. D. Mihovilovic, F. Rudroff, B. GrKtzl, Curr. Org. Chem. 2004,
8, 1057.
[7] G. Strukul, Angew. Chem. 1998, 110, 1256; Angew. Chem. Int.
Ed. 1998, 37, 1199; .
[8] a) M. D. Mihovilovic, F. Rudroff, B. GrKtzl, P. Kapitan, R.
Snajdrova, J. Rydz, R. Mach, Angew. Chem. 2005, 117, 3675;
Angew. Chem. Int. Ed. 2005, 44, 3609; ; b) M. J. Taschner, D. J.
Black, Q. Z. Chen, Tetrahedron: Asymmetry 1993, 4, 1387;
c) M. J. Taschner, L. Peddada, J. Chem. Soc. Chem. Commun.
1992, 1384; d) M. J. Taschner, D. J. Black, J. Am. Chem. Soc.
1988, 110, 6892; e) A. J. Carnell, S. M. Roberts, V. Sik, A. J.
Willetts, J. Chem. Soc. Perkin Trans. 1 1991, 2385; f) V. Alphand,
R. Furstoss, J. Org. Chem. 1992, 57, 1306; g) A. Carnell, A.
Willets, Biotechnol. Lett. 1992, 14, 17; h) R. Gagnon, G. Grogan,
M. S. Levitt, S. M. Robets, P. W. H. Wan, A. J. Willetts, J. Chem.
Soc. Perkin Trans. 1 1994, 2537; i) M. T. Bes, R. Villa, S. M.
Roberts, P. W. H. Wan, A. Willets, J. Mol. Catal. B 1996, 1, 127;
j) J. D. Stewart, K. W. Reed, C. A. Martinez, J. Zhu, G. Chen,
M. M. Kayser, J. Am. Chem. Soc. 1998, 120, 3541; k) M. D.
Mihovilovic, P. Kapitan, J. Rydz, F. Rudroff, F. H. Ogink, M. W.
Fraaije, J. Mol. Catal. B 2005, 32, 135; l) M. D. Mihovilovic, F.
Rudroff, B. GrKtzl, P. Stanetty, Eur. J. Org. Chem. 2005, 809.
[9] V. Alphand, R. Furstoss, J. Mol. Catal. B 2000, 9, 209.
[10] P. Park, A. P. Kozikowski, Tetrahedron Lett. 1988, 29, 6703.
[11] A. Kirschner, J. Altenbuchner, U. T. Bornscheuer, Appl. Microbiol. Biotechnol. 2006, DOI: 10.1007/s00253-006-0556-6.
[12] A. B. Smith, P. A. Levenberg, Synthesis 1981, 567.
[13] A. Z. Walton, J. D. Stewart, Biotechnol. Prog. 2002, 18, 262.
[14] T. Endo, S. Koizumi, Adv. Synth. Catal. 2001, 343, 521.
[15] A. Z. Walton, J. D. Stewart, Biotechnol. Prog. 2004, 20, 403.
[16] a) G.-J. Shen, Y.-F. Wang, C. Bradshaw, C.-H. Wong, J. Chem.
Soc. Chem. Commun. 1990, 677; b) A. Fauve, H. Veschambre,
Biocatalysis 1990, 3, 95.
[17] a) F. Theil, J. Weidner, S. Ballschuh, A. Kunath, H. Schick,
Tetrahedron Lett. 1993, 34, 305; b) F. Theil, S. Ballschuh, A.
Kunath, H. Schick, Tetrahedron: Asymmetry 1991, 2, 1031;
c) A. J. M. Janssen, A. J. H. Klunder, B. Zwanenburg, Tetrahedron 1991, 47, 7409.
[18] C.-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem.
Soc. 1982, 104, 7294.
[19] a) A. Lethbridge, R. O. C. Norman, C. B. Thomas, J. Chem. Soc.
Perkin Trans. 1 1974, 1929; b) S. Uemura, K. Ohe, S.-I.
Fukuzawa, S. R. Patil, N. Sugita, J. Organomet. Chem. 1986,
316, 67.
[20] P. Hildebrandt, A. Musidlowska, U. T. Bornscheuer, Appl.
Microbiol. Biotechnol. 2002, 59, 483.
[21] Y. Oikawa, K. Sugano, O. Yonemitsu, J. Org. Chem. 1978, 43,
2087.
7006
www.angewandte.org
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 7004 –7006
Документ
Категория
Без категории
Просмотров
1
Размер файла
92 Кб
Теги
resolution, ketone, kinetics, baeyerцvilliger, monooxygenase, hydroxy, catalyzed
1/--страниц
Пожаловаться на содержимое документа