Microbial deracemization of 1-Aryl and 1-heteroaryl secondary alcohols

Full text of DOI:10.1021/jo015770n

J . Org. Chem. 2001, 66, 6495-6497
6495
Sch em e 1. Secon d a r y Alcoh ols To Un d er go
Micr obia l Der a cem iza tion of 1-Ar yl a n d
1-Heter oa r yl Secon d a r y Alcoh ols
Der a cem isa tion
Graham R. Allan and Andrew J . Carnell*
Department of Chemistry, Robert Robinson Laboratories,
University of Liverpool, Liverpool L69 7ZD, U.K.
acarnell@liv.ac.uk
Received May 18, 2001
Chiral secondary alcohols are widely used as synthetic
intermediates, chiral auxiliaries, and analytical reagents
and can be obtained by resolution or asymmetric reduc-
tion processes. The deracemization of racemic alcohols
is an attractive approach since there is, in principle, no
need to discard or recycle an unwanted enantiomer.
Biocatalytic deracemization processes are an efficient
method by which chiral secondary alcohols can be ob-
tained.1 The combination of transition metal-catalyzed
racemization with an enantioselective lipase-catalyzed
esterification has been reported by several groups.2
Ba¨ckvall has used a ruthenium catalyst, 4-chlorophenyl
acetate, and Candida antarctica lipase (Novozyme 435)
to deracemize secondary alcohols and obtained, in most
cases, essentially enantiomerically pure (>99% ee) ac-
etates in up to 88% yield.3 Faber has used a lipase-
mandelate racemase two-enzyme system to achieve
deracemization of mandelic acid.4 We and other groups
have reported the deracemization of alcohols and diols
using microbial systems containing redox enzymes.5
Nakamura has reported the deracemization of 1-aryl
ethanols with Geotrichum Candidum IFO 5767.6 It is also
possible to use two isolated dehydrogenase enzymes with
matched selectivity to achieve similar transformations.
More attractive is the use of an oxidase, which does not
require the addition of nicotinamide cofactors, to catalyze
the initial enantioselective oxidation in combination with
a dehydrogenase for the second enantiocomplementary
reduction leading to overall deracemization. In fact the
second reduction step need not be selective and could be
performed with a chemical reagent compatible with the
oxidase enzyme as described by Faber.7
Ta ble 1. Resu lts of th e Der a cem iza tion of Secon d a r y
Alcoh ols
(R/S)
time
%
%
% ee
substratea
R1
R2
(days) ketone alcohol (R)10
1a
Ph
Ph
Ph
Ph
Ph
Ph
Me
Et
CHdCH2
CH2CH2Cl
nBu
6
5
5
5
5
5
5
5
5
5
4
4
5
5
4
5
16
8
9
21
11
0
21
17
43
0
19
21
14
15
27
71
84
90
90
79
89
100
79
81
56
100
81
78
85
85
73
29
969
993
4515
9911
6716
0
1b
1c
1d b
1e
1f17
1g
1h
1i
cyclohexyl
Me
p-FC6H4
p-NO2C6H4 Me
p-MeOC6H4 Me
2-pyridyl
C4H3O
C4H3S
C3H2NS
C10H7
9918
8919
993
0
1j9
1k
1l
1m
1n
1o
1p
Me
Me
Me
Me
Me
Me
-
999
999
979
993
983
993
C6H11
indan-1ol
a
Reactions were performed using a substrate concentration of
b
1 mg/mL in the growth media (ref 13). Substrate concentration
0.5 mg/mL. Yields and ee’s were determined by HPLC using a
Chiracel OB-H column using a quantitated method for determi-
nation of yield.
In connection with our search for enantioselective
alcohol oxidase enzymes we surveyed the literature for
microorganisms displaying high enantioselectivity for the
oxidation of secondary alcohols. The bacterium Sphin-
gomonas paucimobilis has been successfully used by
Fogagnolo for the resolution of 1-heteroaryl and 1-aryl-
2-propanols where the (S)-alcohol is oxidized to the
ketone with good selectivity.8 Bacillus stereothermophilus
was used by the same group to enantioselectively oxidize
aryl and heteroaryl ethanols with excellent selectivity.9
On screening aryl and heteroaryl alcohols 1a -p with
Sphingomonas paucimobilis NCIMB 8195, we made the
fortuitous discovery that this organism catalyzes the
efficient deracemization of a wide range of this class of
substrates leading to up to 90% yield of the (R)-alcohol
(Scheme 1, Table 1). The corresponding ketones 2a -p
were formed at various levels during each of the biotrans-
formations, indicating that stereoinversion of the (S)-
alcohol proceeds by sequential oxidation and reduction.
Substrates 1a -e, with a steady increase in steric demand
(1) Stecher, H.; Faber, K. Synthesis 1997, 1.
(2) (a) Allen, J . V.; Williams, J . M. J . Tetrahedron Lett. 1996, 37,
1859. (b) Dinh, P. M.; Howarth, J . A.; Hudnott, A. R.; Williams, J . M.
J .; Harris, W. Tertrahedron Lett. 1996, 37, 7623. (c) Larsson, A. L. E.;
Persson, B. A.; Ba¨ckvall, J . E. Angew. Chem., Int. Ed. Engl. 1997, 36,
1211. (d) Reetz, M. T.; Schimossek, K. Chimia 1996, 50, 668.
(3) Persson, B. A.; Larsson, A. L. E.; Le Ray, M.; Ba¨ckvall, J . E. J .
Am. Chem. Soc. 1999, 121, 1645.
(4) Strauss, U. T.; Faber, K. Tetrahedron: Asymmetry 1999, 10,
4079.
(5) Carnell, A. J . Adv. Biochem. Eng. Biotechnol./Biotransform. 1998,
57.
(6) Nakamura, K.; Inoue, Y.; Matsuda, T.; Ohno, A. Tertrahedron
Lett. 1995, 36, 6263.
(8) Fogagnolo, M.; Giovannini, P. P.; Guerrini, A.; Medici, A.;
Pedrini, P.; Colombi, N. Tetrahedron: Asymmetry 1998, 9, 2317.
(9) Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P.; Poli, S.
Tetrahedron: Asymmetry 1993, 4, 1607.
(7) Kroutil, W.; Faber, K. Tetrahedron: Asymmetry 1998, 9, 2901.
10.1021/jo015770n CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/23/2001

6496 J . Org. Chem., Vol. 66, No. 19, 2001
Notes
Ta ble 2. Resu lts of th e Der a cem iza tion of 1-Ar yl
P r op a n -2-ols
%
%
% ee
eemax
substratea
R1
C6H5
C4H3S
C4H3O
R2 ketone alcohol alcohol (E)
3a
3b
3c
3d
CH3
CH3
CH3
40
0
22
74
60
100
78
82
0
31
99
66%
-
22%
(9)
F igu r e 1. 1-Aryl propan-2-ols studied.
m-CF3C6H4 CH3
26
Of the three substrates that reacted, substrates 3a and
3c reacted more slowly than the corresponding aryl
ethanols 1a and 1k but still gave ee’s higher than the
theoretical maximum at the degrees of conversion shown.
Substrate 3d reacted at a faster rate although more
slowly than with the P. paucimobilis strain used by
Fogagnolo and with much lower selectivity (E value of 9
compared with around 200). These results suggest that
the Sphingomonas paucimobilis NCIMB 8195 strain we
have used is indeed a different strain to that used by
Fogagnolo. Our strain gives efficient deracemization of
eleven of the secondary aryl and heteroaryl ethanols
tested and partial slow deracemization of two (hetero)-
aryl propan-2-ols and resolution of another with low
enantioselectivity. The deracemization of 1-phenyl etha-
nol 1a was scaled up starting from 220 mg to provide
179 mg of the (R)-1a in 81% yield, 97% ee with 14%
residual ketone 2a after flash column chromatography.
Similarly from 250 mg of thiophenyl ethanol 1l we
obtained the (R)-1h with a 99% ee in 70% isolated yield
along with the ketone (24%).13
There is currently no evidence that Sphingomonas
paucimobilis NCIMB 8195 contains an alcohol oxidase
enzyme. This has been established from negative ABTS/
horseradish peroxidase plate tests which give a purple
coloration in response to hydrogen peroxide production
by sugar and alcohol oxidases.14 The likelihood is there-
fore that the deracemization is driven by dehydrogenase
enzymes within the cells which may be compartmental-
ized and have different cofactor requirements (i.e., NADH
and NADPH), thus providing the thermodynamic driving
force necessary for deracemization to occur. If this is the
case, then the reaction is unlikely to be viable in vitro
since satisfying these conditions in the absence of the cell
is likely to be difficult. We are therefore currently
focusing our efforts on the identification and isolation of
novel alcohol oxidases which can catalyze the irreversible
oxidation of secondary alcohols and do not require
nicotinamide cofactors. These enzymes will be coupled
with compatible chemical reductants for deracemization
reactions.
a
Reactions were run over 5 days at substrate concentration of
0.5 mg/mL in growth media (ref 13).
of the R2 group, show that deracemization of substrates
with a saturated chain proceeds well after 5-6 days.10
The allylic alcohol 1c undergoes a slower deracemiza-
tion, perhaps reflecting the lower reactivity of the inter-
mediate R,â-unsaturated ketone 2c. The alcohol 1d is of
particular interest as an intermediate for the synthesis
of Prozac.11 This substrate was toxic to the cells at 1 mg/
mL concentration so the biotransformation was carried
out at half the normal concentration. Substrate 1e
contains the nBu group which is obviously too sterically
demanding for high selectivity. With two large cyclic
groups flanking the carbinol center in 1f there was no
deracemization. Introduction of para substituents on the
aromatic ring showed a marked difference in outcome for
the substrate 1i, possessing the electron-donating meth-
oxy group, compared to the substrates 1g and 1h with
electron-withdrawing substituents. Substrate 1i gave a
much higher level of residual ketone 2i, perhaps a
reflection of the higher reduction potential of this alco-
hol.12
In the heterocyclic series tested, the pyridyl alcohol 1j
was not transformed whereas the 2-furanyl, 2-thiophenyl
and 2-thiazyl alcohols 1k -m were deracemized almost
equally efficiently. The naphthyl alcohol 1n shows that
larger aromatic groups can be accepted. Substrate 1o was
the only substrate tested with no aromatic ring meaning
that the alcohol is significantly less activated. However
this substrate was deracemized with reasonable ef-
ficiency, giving the alcohol with 98% e.e. The last susb-
trate, indan-1-ol 1p underwent kinetic resolution giving
1p in 99% ee after 71% conversion (E ) 10). Incubation
of (R)-1a as the substrate led to no conversion to ketone.
Incubation of pure (S)-1a resulted in a slower conversion
compared to the racemic substrate giving 70% ee for (R)-
1a and 16% residual ketone 2a after 7 days.
Fogagnolo used an unspecified noncommercially avail-
able strain of Pseudomonas paucimobilis for the kinetic
resolution of 1-aryl and 1-heteroaryl propan-2-ols.8 Since
we had used a strain listed as Sphingomonas (Pseudomo-
nas) paucimobilis NCIMB 8195, we were conscious that
we may well have used a related strain containing
different enzymes so we tested a small range of 1-aryl
propan-2-ols 3a -d , including 1-m-trifluoromethylphenyl
propan-2-ol 3d , tested by Fogagnolo, to determine if
they underwent kinetic resolution or deracemization
(Table 2).
Exp er im en ta l Section
Racemic alcohols 1a and 1n were purchased from Aldrich
Chemical Co. All other 2-substituted ethanols and alcohol 3d
were synthesized by sodium borohydride reduction of the com-
mercially available ketones and spectroscopic data were consis-
tent with those reported in the literature.
Gen er a l P r oced u r e for th e Syn th esis of 1-Ar yl-2-p r o-
p a n ols 3a -c. To a stirred solution of aryl bromide (20 mmol)
in dry THF (100 mL) at -30 °C was added n-butyllithium (22
mmol). The solution was stirred for 30 min at -30 °C whereupon
(10) The absolute configurations of alcohols 1a , 1b, and 1l were
proved by chiral GC (Lipodex A) correlation with standards prepared
by resolution with Lipase SAM-2, Laumen, K.; Schneider, M. P. J .
Chem. Soc., Chem Commun. 1988, 598. The absolute configuration of
alcohols 1e, 1i, 1n , 1o, and 1p were proved by chiral HPLC (Chiralcel
OB-H) correlation with standards prepared by resolution with No-
vozyme 435 and vinyl acetate as reported by Ba¨ckvall and co-workers
(ref 3).
(13) Sphingomonas paucimobilis was obtained from the National
Collections of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen,
Scotland, and was maintained on malt extract agar slopes. Sphin-
gomonas paucimobilis was grown in liquid culture on a nutrient media
composed of glucose (20 g/L), yeast extract (5 g/L), malt extract (5 g/L),
nutrient broth (10 g/L), and K2HPO4 (0.4 g/L). All media components
were purchased from Sigma-Aldrich Co. Ltd.
(11) Corey, E. J .; Reichard, G. A. Tetrahedron Lett. 1989, 30, 5207.
(12) Adkins, H.; Elofson, R. M.; Robinson, A. G. J . Am. Chem. Soc.
1949, 71, 3622.
(14) Danneel, H.-J .; Ullrich, M.; Giffhorn, F. Enzyme Microb. Tech-
nol. 1992, 14, 898.

Notes
J . Org. Chem., Vol. 66, No. 19, 2001 6497
propylene oxide (20 mmol) was added and the solution allowed
to warm to room temperature and stirred for a further 1 h. The
reaction was quenched by the addition of saturated ammonium
chloride (50 mL) and diethyl ether (100 mL) added. The organic
extract was washed with water and dried (MgSO4) and the
solvent removed under reduced pressure to yield the crude
products which were purified by flash column chromatography
on silica (eluant petroleum ether:ethyl acetate 2:1) to yield the
pure 1-aryl propan-2-ols 3a -c.
1-P h en yl p r op a n -2-ol (3a ) was isolated as a colorless oil:
yield 68%. Spectral data was consistent with that reported
previously.20
1-Th ien yl p r op a n -2-ol (3b) was isolated as a colorless oil:
yield 61%. Spectral data was consistent with that reported
previously.21
1-F u r yl p r op a n -2-ol (3c) was isolated as a pale yellow oil:
yield 77%. Spectral data was consistent with that reported
previously.22
Rep r esen ta tive Biotr a n sfor m a tion P r oced u r e. Bacteria
were grown in 250 mL of sterile liquid media,13 after inoculation
with an aseptic loop (30 µL), for 2 days at 32 °C with vigorous
aeration (200 rpm, New Brunswick orbital incubator). The
alcohol 1l (250 mg) was added in DMF (1 mL) and the reaction
shaken for 4 days. The cells were removed by centrifugation
(3000 rpm for 5 min), and the supernatant was extracted with
ethyl acetate (3 × 200 mL) and dried over magnesium sulfate
to afford, after purification by flash column chromatography
(ethyl acetate:petroleum ether; 2:8), the ketone 2l (60 mg, 24%)
and the (R)-alcohol 1l (175 mg, 70%); [R]25D ) +25.0 (c 5.0 CHCl3)
(lit.9 +24.3 c 5.0 CHCl3); ee > 99% by chiral HPLC (Chiralcel
OB-H).
(15) Mallavadhani, U. V.; Rao, Y. R. Tetrahedron: Asymmetry 1994,
5, 23.
(16) Shen, Z.-X.; Lu, J .; Zhang, Q.; Zhang. Y.-W. Tetrahedron:
Asymmetry 1997, 8, 2287.
Ack n ow led gm en t. We thank the BBSRC for a
PDRA to G.R.A.
(17) Davies, K. M.; Hickinbottom, W. J . J . Chem. Soc. 1963, 373.
(18) Nakamura, K.; Fujii, M.; Ida, Y. J . Chem. Soc., Perkin Trans.
1 2000, 3205.
(19) Naemura, K.; Murata, M.; Tanaka, R.; Yano, M.; Hirose, K.;
Tobe, Y. Tetrahedron: Asymmetry 1996, 7, 3285.
(20) Zushi, S.; Kodama, Y.; Fukuda, Y.; Nishihata, K.; Nishio, M.;
Hirota, M.; Uzawa, J . Bull. Chem. Soc. J pn. 1981, 54, 2113.
J O015770N
(21) Kumamoto, T.; Hosoya, K.; Kanzaki, S.; Masuko, K.; Watanabe,
M.; Shirai, K. Bull. Chem. Soc. J pn. 1986, 59, 3097.
(22) Arco, M. J .; Trammel, M. H.; White J . D. J . Org. Chem. 1976,
41, 2075.