Biocatalytic asymmetric hydrogen transfer employing Rhodococcus ruber DSM 44541

Article abstract of DOI:10.1021/jo026216w

Nonracemic sec-alcohols of opposite absolute configuration were obtained either by asymmetric reduction of the corresponding ketone using 2-propanol as hydrogen donor or by enantioselective oxidation through kinetic resolution of the rac-alcohol using acetone as hydrogen acceptor employing whole lyophilized cells of Rhodococcus ruber DSM 44541. The microbial oxidation/reduction system exhibits not only excellent stereo- and enantioselectivity but also a broad substrate spectrum. Due to the exceptional tolerance of the biocatalyst toward elevated concentrations of organic materials (solvents, substrates and cosubstrates), the process is highly efficient. The simple preparation of the biocatalyst and its ease of handling turns this system into a versatile tool for organic synthesis.

Full text of DOI:10.1021/jo026216w

Bioca ta lytic Asym m etr ic Hyd r ogen Tr a n sfer Em p loyin g
Rh od ococcu s r u ber DSM 44541
Wolfgang Stampfer, Birgit Kosjek, Kurt Faber, and Wolfgang Kroutil*
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz,
Heinrichstrasse 28, A-8010 Graz, Austria
Wolfgang.Kroutil@uni-graz.at
Received J uly 19, 2002
Nonracemic sec-alcohols of opposite absolute configuration were obtained either by asymmetric
reduction of the corresponding ketone using 2-propanol as hydrogen donor or by enantioselective
oxidation through kinetic resolution of the rac-alcohol using acetone as hydrogen acceptor employing
whole lyophilized cells of Rhodococcus ruber DSM 44541. The microbial oxidation/reduction system
exhibits not only excellent stereo- and enantioselectivity but also a broad substrate spectrum. Due
to the exceptional tolerance of the biocatalyst toward elevated concentrations of organic materials
(solvents, substrates and cosubstrates), the process is highly efficient. The simple preparation of
the biocatalyst and its ease of handling turns this system into a versatile tool for organic synthesis.
In tr od u ction
tions needed, and the preference for an aqueous medium.
However, their large-scale application has been impeded
by (i) their requirement for cofactor-recycling, (ii) their
Catalytic asymmetric hydrogen transfer based on
transition) metals and chiral organic ligands constitutes
3
(
instability toward elevated concentrations of organic
materials (such as substrates and acetone or 2-propanol
a general approach to synthesize nonracemic sec-alcohols
by asymmetric reduction of the corresponding ketone or
1
4
used as cosubstrates), and (iii) inhibition phenomena.
by kinetic resolution of rac-sec-alcohols via enantioselec-
As a consequence, biochemical reductions/oxidations
2
tive oxidation. Recently, the use of asymmetric hydrogen
employing sec-alcohol dehydrogenases on a large scale
transfer catalysts compatible with aqueous solutionsa
tremendous opportunity for the practice of “green
chemistry”shas garnered considerable interest and re-
5
are restricted to the use of fermenting cells and are
6
impeded by low (co)substrate concentration(s).
We have recently presented a highly enantioselective
sulted in the publication of the first water-compatible or
7
sec-alcohol dehydrogenase from Rhodococcus ruber DSM
soluble catalyst analogues.1
c,e,f,i
-
4
4541 that is exceptionally stable toward organic sol-
All biocatalytic methods for the asymmetric hydrogen
transfer are based on alcohol dehydrogenases requiring
nicotinamide-cofactors. They show several advantages
over the chemical methods, such as their intrinsic asym-
metry, the absence of side reactions (in particular the
aldol condensation), the essentially mild reaction condi-
(3) (a) Devaux-Basseguy, R.; Bergel, A.; Comtat, M. Enzyme Microb.
Technol. 1997, 20, 248-258. (b) Hummel, W. Adv. Biochem. Eng. 1997,
8, 145-184.
4) Faber, K. Biotransformations in Organic Chemistry, 4th ed.; pp
178-183, Springer: Heidelberg, 2000.
5) Active metabolism is required for cofactor regeneration: (a)
P e´ rez, H. I.; Luna, H.; Manjarrez, N.; Sol ı´ s, A. Tetrahedron: Asymmetry
001, 12, 1709-1712. (b) Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini,
5
(
(
2
*
To whom correspondence should be addressed. Tel: +43-316-380-
350. Fax: +43-316-380-9840.
1) (a) Noyori, R.; Ohkuma, T. Angew. Chem., Int. Ed. Engl. 2001,
P.; Fontana, S. Tetrahedron: Asymmetry 2000, 11, 2367-2373. (c)
P e´ rez, H. I.; Luna, H.; Manjarrez, N.; Sol ´ı s, A.; Nu n˜ ez, M. A. Biotechnol.
Lett. 1999, 21, 855-858. (d) Fogagnolo, M.; Giovannini, P. P.; Guerrini,
A.; Medici, A.; Pedrini, P.; Colombi, N. Tetrahedron: Asymmetry 1998,
9, 2317-2327. (e) Ohta, H.; Fujiwara, H.; Tsuchihashi, G. Agric. Biol.
Chem. 1984, 48, 317-322.
5
(
4
0, 40-73. (b) Haack, K.-J .; Hashiguchi, S.; Fujii, A.; Ikariya, T.;
Noyori, R. Angew. Chem., Int. Ed. Engl. 1997, 36, 285-288 (c) Bubert,
C.; Blacker, J .; Brown, S. M.; Crosby, j.; Fitzjohn, S.; Muxworthy, J .
P.; Thorpe, T.; Williams, J . M. J . Tetrahedron Lett. 2001, 42, 4037-
(6) In general, the substrate concentration was below 0.15 mol L-
and cosubstrate concentration was below 3% (v/v); see: (a) Nakamura,
K.; Inoue, Y.; Matsuda, T.; Misawa, I. J . Chem. Soc., Perkin Trans 1
1999, 2397-2402. (b) Wolberg, M.; Hummel, W.; Wandrey, C.; M u¨ ller,
M. Angew. Chem., Int. Ed. Eng. 2000, 39, 4306-4308. (c) Goswami,
A.; Bezbaruah, R. L.; Goswami, J .; Borthakur, N.; Dey, D.; Hazarika,
A. K. Tetrahedron: Asymmetry 2000, 11, 3701-3709. (d) Griffin, D.
R.; Yang, F.; Carta, G.; Gainer, J . L. Biotechnol. Prog. 1998, 14, 588-
593. For higher (co)substrate concentrations, see: (e) Heiss, C.; Phillips,
R. S. J . Chem. Soc., Perkin Trans. 1 2000, 2821-2825 [15% (v/v)
cosubstrate 2-propanol, 0.12 mol L- substrate]. (f) Matsuyama, A.;
Yamamoto, H.; Kawada, N.; Kobayashi, Y. J . Mol. Catal. B: Enzymatic
1
4
039. (d) Cross, D. J .; Kenny, J . a.; Houson, I.; Campbell, L.; Walsgrove,
T.; Wills, M. Tetrahedron: Asymmetry 2001, 12, 1801-1806. (e)
Thorpe, T.; Blacker, J .; Brown, S. M.; Bubert, C.; Crosby, J .; Fitzjohn,
S.; Muxworthy, J . P.; Williams, J . M. J . Tetrahedron Lett. 2001, 42,
4
041-4043. (f) Rhyoo, H. Y.; Park, H.-J .; Suh, W. H., Chung, Y. K.;
Tetrahedron Lett. 2002, 32, 269-272. (g) Murata, K.; Ikariya, T. J .
Org. Chem. 1999, 64, 2186-2187. (h) Mao, J .; Baker, D. C. Org. Lett.
1
999, 1, 841-843. (i) Rhyoo, H. Y.; Park, H.-J .; Chung, Y. K. Chem.
Commun. 2001, 2064-2065. (j) Noyori, R.; Yamakawa, M.; Hashiguchi,
S. J . Org. Chem. 2001, 66, 7931-7944. (k) Campbell, E. J .; Zhou, H.;
Nguyen S. T. Angew. Chem., Int. Ed. Engl. 2002, 41, 1020-1022.
1
-
1
+
(2) (a) Hashiguchi, S.; Fujii, A.; Haack, K.-J ., Matsumura, K.;
2001, 11, 513-521 (0.3 mol L substrate, no details on the NAD /
NADH recycling system). (g) Simpson, H. D.; Cowan, D. A. Protein
Pept. Lett. 1997, 4, 25-31 [11% (v/v) cosubstrate 2-propanol, 0.44 mol
Ikariya, T.; Noyori, R. Angew. Chem., Int. Ed. Engl. 1997, 36, 288-
89. (b) Faller, J . W.; Lavoie, A. R. Org. Lett. 2001, 3, 3703-3706. (c)
Iura, Y.; Sugahara, T.; Ogasawara, K. Tetrahedron Lett. 1999, 40,
735-5738. (d) Persson, B. A.; Larsson, A. L. E.; Le Ray, M.; Backvall,
J .-E. J . Am. Chem. Soc. 1999, 121, 1645-1650.
2
-
1
L
substrate]. (h) Hildebrandt, P.; Riermeier, T.; Altenbuchner, J .;
5
Bornscheuer, U. T. Tetrahedron: Asymmetry 2001, 12, 1207-1210,
2825 [20% (v/v) cosubstrate 2-propanol, 0.05 mol L- substrate].
1
1
0.1021/jo026216w CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/05/2002
4
02
J . Org. Chem. 2003, 68, 402-406

Biocatalytic Asymmetric Hydrogen Transfer
F IGURE 1. Asymmetric reduction of ketones employing
lyophilized whole cells.
F IGURE 2. Biocatalytic kinetic resolution of sec-alcohols by
enantioselective oxidation.
TABLE 1. Bioca ta lytic Asym m etr ic Red u ction of
Keton es Em p loyin g R. r u ber DSM 44541 (see F igu r e 1)
CHART 1
activity
(
µmol
%
%
-
1
g
-1) product conva ee
substr R′
R′′
h
1
2
3
4
5
6
7
8
9
a
a
a
a
a
a
a
a
a
CH3 n-C8H17
CH3 n-C7H15
CH3 n-C6H13
CH3 n-C5H11
CH3 (CH3)2CdCH(CH2)2
C2H5 n-C5H11
CH3 Ph
CH3 p-MeOC6H4
CH3 c-C6H11
69
84
97
93
95
74
103
nd
90
(S)-1b
(S)-2b
(S)-3b
(S)-4b
(S)-5b
(S)-6b
(S)-7b
(S)-8b
(S)-9b
(S)-10b
(S)-11b
94 >99
94 >99
92 >99
92 >99
93 >99
79
97
TABLE 2. Bioca ta lytic Red u ction of Cyclic Keton es (see
F igu r e 1 a n d Ch a r t 1)
81 >99
61 >99
87 >99
82 >99
52 >99
%
conversionb
substr
product
% ee
1
1
0a CH3 2-naphthyl
1a CH3 (E)-Ph-HCdCH
64
nd
b
12ba
ndc
7.8
83.2
12a
<0.2
0.2
19.8
1
1
3a
4a
13ba
a
2% v/v 2-propanol, 22 h reaction time. b nd, not determined.
2
(S)-14b
a
Absolute configuration was not determined due to low conver-
sion. b 22 h reaction time. c nd, not determined due to low conver-
sion.
vents. The enzyme’s activity remains high at concentra-
tions of up to 20% (v/v) acetone and 50% (v/v) 2-propanol,
allowing one to perform asymmetric hydrogen transfer
in aqueous systems at neutral pH. For preparative-scale
applications, whole lyophilized or resting cells can be
used as a readily available alternative.
transfer protocols,2a can be transformed to their corre-
sponding (S)-alcohols with exceptional prochiral recog-
nition. Enantiopure 4-phenyl-but-3-en-2-ol 11b can be
obtained either by kinetic resolution of the rac-alcohol
employing Sharpless epoxidation8 or via asymmetric
Resu lts a n d Discu ssion
9
hydrogenation of ketone 11a in 97% ee; however, metal-
Red u ction . Hydrogen-transfer reactions are generally
reversible and preparative-scale reactions are usually
driven into the desired reduction or oxidation direction
by employing the corresponding cosubstrate as hydrogen
acceptor/donor in excess to shift the system out of
equilibrium.
catalyzed hydrogen-transfer protocols failed due to insuf-
ficient chemodifferentiation between the CdC and CdO
double bond. Based on the intrinsic excellent chemo-
selectivity of alcohol dehydrogenases, the (S)-alcohol 11b
was obtained from ketone 11a employing whole cells of
R. ruber DSM 44541 in >99% ee.
Metal-catalyzed hydrogen-transfer reductions described
so far are often limited by the reversibility of the reaction
due to the low cosubstrate concentration used (0.1 M
However, in contrast to metal-catalyzed hydrogen-
transfer methods, 1-tetralone 12a and 1-indanone 13a
(Chart 1) could not be reduced by employing the microbial
2
a
2
-propanol ). In contrast, whole lyophilized cells of R.
method. Nevertheless, 2-tetralone 14a was reduced with
reduced activity and moderate stereopreference; thus,
alcohol (S)-14b was obtained in 83% ee.
The stereopreference of the dehydrogenase(s) involved
follows Prelog’s rule;10 thus, the attack of the (formal)
hydride preferentially occurs from the Re-side of the
ketone by assuming that the smaller group possesses the
lower CIP-priority.
ruber DSM 44541 perform the asymmetric hydrogen
transfer reaction at 2-propanol concentrations up to 50%
7
v/v ≈7.9 M , thus avoiding the problem of reversibility
due to the excess of cosubstrate (Figure 1).
n-Alkan-2-ones 1a -4a were reduced to the correspond-
ing (S)-alcohols 1b-4b with excellent stereoselectivity
(Table 1) using 22% (v/v) 2-propanol as cosubstrate.
Branching and the presence of a CdC double bond in the
alkyl chain at the ω-position of the alkyl chain (substrate
Oxid a tion . The microbial alcohol dehydrogenase sys-
tem not only tolerates 2-propanol but also acetone in
concentrations of up to 20% (v/v) to effect the reverse
5
a ) does not influence the ee of the product nor the
activity. A slight decrease in stereoselectivity was ob-
served when the carbonyl moiety was moved to the more
hindered 3-position (substrate 6a ). Acetophenone 7a and
even p-methoxyacetophenone 8a , which can be reduced
only with difficulties using metal-catalyzed hydrogen-
(8) (a) Martin, V. S.; Woodard, S. S.; Katsuki, T.; Yamada, Y.; Ikeda,
M.; Sharpless, K. B. J . Am. Chem. Soc. 1981, 103, 6237-6240. (b)
Kitano, Y.; Matsumoto, T.; Sato, F. J . Chem. Soc. Chem. Commun.
1
986, 1323-1325.
9) Ohkuma, T.; Koizumi, M.; Doucet, H.; Pham, T.; Kozawa, M.;
(
Murata, K.; Katayama, E.; Yokozawa, T.; Ikariya, T.; Noyori, R. J . Am.
Chem. Soc. 1998, 120, 13529-13530.
(10) Prelog, V. Pure Appl. Chem. 1964, 9, 119-130.
(
7) Stampfer, W.; Kosjek, B.; Moitzi, C.; Kroutil, W.; Faber, K.
Angew. Chem. Int. Ed. Engl. 2002, 41, 1014-1017.
J . Org. Chem, Vol. 68, No. 2, 2003 403

Stampfer et al.
TABLE 3. Bioca ta lytic Kin etic Resolu tion by Oxid a tion of r a c-Alk a n ols Em p loyin g Lyop h ilized Cells of R. r u ber DSM
4
4541 (see F igu r e 2)
activity
(µmol h g-1
)
-
1
time (h)
% conv
alcohol
% ee
E value
substr.
R′
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
CH(CH3)2
n-C3H7
R′′
1
5b
n-C10H21
140
420
840
1260
1400
1120
560
1540
1550
5
24
24
24
24
24
24
24
24
24
22
24
24
8.3
55
56
50
52
49
56
96
50
15ba
10.2
96.5
99.0
>99
>99
>99
97.5
34
ndb
34
43
>100
>100
>100
33
1
2
3
4
b
b
b
b
n-C8H17
n-C7H15
n-C6H13
n-C5H11
n-C4H9
(R)-1b
a
2b
(R)-3b
(R)-4b
(R)-16b
(R)-17b
(S)-18b
(R)-5b
(R)-6a
1
1
1
6b
7b
8b
n-C3H7
C2H5
1.3
5
6
b
b
(CH3)2CdCH(CH2)2
n-C5H11
n-C5H11
n-C4H9
>99
33.0
nd
>100
49.5
0.7
36.9
2.7
nd
a
1
2
9b
0b
nd
nd
19b
20ba
0
∼1
a
Absolute configuration of remaining alcohol not determined due to low conversion;. b nd, not determined due to low conversion.
reaction, namely, the kinetic oxidative resolution of rac-
sec-alcohols via biocatalytic hydrogen transfer (Figure 2).
The biocatalyst not only displays excellent stereoselec-
tivity for most substrates investigated but is also able to
differentiate between primary and secondary alcohols.
Only sec-alcohols are oxidized and primary alcohols (e.g.
CHART 2
1
-octanol, 2-phenylethanol) remain essentially untouched.
Since the catalyst always acts on the same enantiomers
the ketone is reduced to the (S)-alcoholsthe (S)-enanti-
omer is oxidized from a racemate in the oxidation mode,
by leaving the (R)-enantiomer untouched. By these
means, both enantiomers are accessible in nonracemic
TABLE 4. Bioca ta lytic Oxid a tion (for Kin etic
Resolu tion ) of Cyclic Alk a n ols (see F igu r e 2 a n d Ch a r t 2)
substrate
time (h)
% convb
alcohol
% ee
E value
rac-12b
rac-13b
rac-14b
24
24
24
24
24
4.9
11.3
0.2
8.7
98.0
(S)-12b
13ba
4.0
7.8
0.3
na
na
8.3
4.5
2
b
14ba
c
form using the same catalyst.
nd
c
2
2
1b
2b
na
na
Kinetic resolution of rac-n-2-alkanols of varying chain
length (C3-C10, substrate 1b-4b and 15b-17b) re-
vealed that the oxidative activity peaks at a maximum
of C7, as (S)-2-heptanol (S)-4b was oxidized fastest (Table
a
Absolute configuration of remaining alcohol not determined
due to low conversion. b 24 h reaction time. c na, not applicable;
nd, not determined due to low conversion.
3
). The activity for 2-dodecanol 15b was only one tenth
of the rate for 2-heptanol 4b. For 2-alkanols exceeding
0 carbon atoms (C10, C12) but also for shorter 2-al-
1
although â-tetralol rac-14b was not oxidized, the corre-
sponding ketone 14a was reduced with moderate stere-
opreference (ee ) 83%, Table 2). Interestingly, cyclohex-
anol 21b was oxidized at rather low rate (8.7% conversion
within 24 h), while cyclopentanol 22b was an excellent
substrate (98% conversion within 24h).
kanols (C5), both the activity and also the enantioselec-
tivity was reduced. Thus, for 2-pentanol 17b the enan-
tioselectivity decreased to E ) 33. However, upon reducing
the length of the carbon chain from C5 to C4 (substrate
1
8b), an unexpected phenomenon was observed: The
activity almost tripled but the enantioselectivity was very
low (E ) 1.3), going in hand with opposite “anti-Prelog”
stereopreference. Thus, the (R)-enantiomer was prefer-
entially oxidized instead of the (S)-enantiomer. By com-
paring the data for the oxidation with that of the
reduction, it becomes clear that stereoselectivities match
for 6-methyl-hept-5-en-2ol 5b/6-methyl-hept-5-en-2one 5a
but mismatch for 3-octanol 6b/3-octanone 6a . Although
the enantioselectivity for the oxidation of rac-3-octanol
Racemic 1-phenyl ethanol rac-7b was resolved by
kinetic oxidative resolution with excellent enantioselec-
tivity (E > 100) (Table 5). However its p-amino 23b
derivative was not accepted; similarly, p-hydroxy-1-
phenylethanol 24b exhibited moderate selectivity (E ∼
7
). All other 1-phenylethanol derivatives possessing a
substituent in the para-position, such as p-MeO 8b, p-F
5b, p-Cl 26b, p-Br 27b, and p-methyl 28b, gave excel-
2
lent results in kinetic resolution. Steric hindrance may
be the reason that ortho-substituted 1-phenylethanol
derivatives, e.g., 30b (o-Me-), 32b (o-Cl), or 34b (o-
methoxy-1-phenylethanol), were not accepted. However,
no difference in enantioselectivity was observed for the
para- and meta-position for a Me (29b), Cl (31b), or
methoxy (33b) derivative, and relative activities of meta-
derivatives were even higher than that of the para-
analogues. Interestingly, whereas the saturated system
1-cyclohexylethanol 9b was resolved with low enantio-
selectivity, the corresponding reduction of 1-cyclohexy-
lethanone 9a proceeded in excellent stereoselectivity (ee
>99%) and yield (Table 1). Expanding the aromatic
6
b was low (E ∼ 5, Table 3)sthe (S)-enantiomer was
oxidized only 5 times faster than (R)-6bsthe correspond-
ing reduction of ketone 6a furnished alcohol (S)-6b in
9
2
7% ee at 79% conversion (Table 1). The same is true for
-decanol 1b/2-decanone 1a . Nevertheless, more steri-
cally hindered alcohols, such as 6b and 19-20b, were
transformed much more slowly.
Sterically encumbered cyclic ketones R-tetralone 12a
and R-indanone 13a could not be reduced by this method
(see Table 2), but their corresponding rac-alcohols (R-
tetralol rac-12b) and R-indanol (rac-13b) (Chart 2) were
resolved through oxidation, albeit at low rates and
insufficient enantioselectivities (Table 4). In contrast,
4
04 J . Org. Chem., Vol. 68, No. 2, 2003

Biocatalytic Asymmetric Hydrogen Transfer
TABLE 5. Bioca ta lytic Kin etic Resolu tion of
r a c-1-P h en yleth a n ol Der iva tives by Oxid a tion Em p loyin g
R. r u ber DSM 44541 (see F igu r e 2)
versatile tool for organic synthesis for the following
reasons: (1) A broad spectrum of ketones can be reduced
in an asymmetric fashion giving (S)-configured alcohols
in high yields and excellent ee using 2-propanol as a
hydrogen donor at 22% v/v. (2) Likewise, the system can
tolerate acetone as a hydrogen acceptor for oxidation
reactions at a concentration of 20% v/v, and a large
variety of rac-alcohols can be resolved by kinetic resolu-
tion to furnish nonreacted (R)-alcohols and the corre-
sponding ketone, which may be recycled. (5) High sub-
strate concentrations make these systems applicable to
large-scale applications.13
activity
(
µmol
%
b
%
ee
E
-1
-1
substr
R′
R′′
h
g
)
conv alcohol
value
7
3b
4b
b
Ph
p-NH
p-OHC
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
133
44.4 (R)-7b
77.8 >100
c
23ba
2
2
2
-C
6
H
4
4
<1
nc
36
c
6
H
nd
(R)-24b
37
6.6
8
b
p-MeOC
p-FC
p-ClC
p-BrC
p-Me-C
m-Me-C
o-Me-C
m-ClC
o-ClC
m-MeOC
o-MeOC
cyclohexyl
2-naphthyl
6
H
4
456
241
257
208
432
573
<1
299
<1
nd
49.8 (R)-8b
43.5 (R)-25b
41.3 (R)-26b
43.8 (R)-27b
47.6 (R)-28b
>99
73.5
>100
96
2
2
2
2
2
3
3
3
3
3
5b
6b
7b
8b
9b
0b
1b
2b
3b
4b
6
H
4
6
H
4
78.2 >100
82.5 >100
97.3 >100
>100
76.1 >100
85.7 >100
6
H
4
6
H
4
6
H
4
50.1 (R)-29b >99
6
H
4
nc
42.7 (R)-31b
nc
32ba
45.4 (R)-33b
nc
34ba
32.0 (R)-9b
48.9 (R)-10b >99
30ba
6
H
4
4
H
Exp er im en ta l Section
6
6
H
4
6
H
4
<1
nd
nd
Cells were cultivated and prepared according to literature
methods.14
The following substrates and reference compounds were
commercially available:
9
0b
b
26
31
1
>100
a
Absolute configuration of remaining alcohol not determined
due to low conversion. b 24 h reaction time. nc, no conversion;
c
1
a -40a , 1b-7b, 12b, 13b, 15b-23b, 28b-31b, 35b, 37b,
nd, not determined due to low conversion.
3
9b, and 40b.
The following rac-alcohols were synthesized by reduction
NaBH , EtOH) of the corresponding ketone as previously
4
TABLE 6. Resu lts of th e Bioca ta lytic Kin etic
Resolu tion by Oxid a tion of r a c-1-P h en yleth a n ol
Der iva tives Em p loyin g Lyop h ilized Wh ole Cells of R.
r u ber DSM 44541 (see F igu r e 2)
(
reported, and products were identified by comparison of NMR
data:
1
5
16
16
16
18
8
b, 9b, 10b, 11b, 14b,17 24b, 25b,15 26b,15 27b,15
substr
R′
R′′
% convb alcohol % ee E value
3
2b, 33b, 34b, 36b, and 38b.
15
15
15
19
19
3
3
3
3
3
4
5b
6b
7b
8b
9b
0b
Ph
Ph
Ph
Ph
C2H5
C3H7
C4H9
C5H11
CH3
5.4
3.2
7.4
6.4
22.8
49.5
(R)-35b
36b
2.0
nd
5.6
nd
ndc
nd
nd
Micr obia l Tr a n sfor m a tion s. In a typical oxidation pro-
a
cedure, lyophilized cells (40 mg) were rehydrated in sodium
phosphate buffer (0.50 mL, pH 8.0, 50 mM) for 0.5 h before
substrate (200 µmol) and acetone (125 µL) were added, and
the reaction mixture was shaken for 24 h at 130 rpm (24 °C).
In a typical reduction procedure, lyophilized cells (40 mg)
were rehydrated in sodium phosphate buffer (0.50 mL, pH 7.5,
50 mM) for 0.5 h before substrate (100 µmol) and 2-propanol
(140 µL) were added, and the reaction mixture was shaken
for 22 h at 130 rpm (24 °C).
(R)-37b
a
38b
nd
Ph-CH2
Ph-(CH2)2 CH3
(R)-39b 34.0
(R)-40b 96.1
>100
>100
a
Absolute configuration of remaining alcohol not determined
due to low conversion. b 24 h reaction time. nd, not determined
due to low conversion.
c
Biotransformations were stopped by addition of ethyl acetate
0.7 mL), mixing, and centrifugation (5 min, 13 000 rpm). The
system from a phenyl to a naphthyl moiety (substrate
(
1
0b) resulted in an excellent kinetic resolution.
Substitution of the CH moiety in 1-phenylethanol by
organic phase was dried (Na SO ) and analyzed by GC on a
chiral stationary phase (see below).
2
4
3
Deter m in a tion of Absolu te Con figu r a tion . The absolute
configuration of the followings compounds was elucidated by
coinjection on GC using a chiral stationary phase and com-
mercially available reference samples: (S)-3b, (S)-4b, (S)-7b,
a longer alkyl group resulted in a decrease of activity (see
substrates 35b-38b, Table 6). In contrast, by keeping
3
the CH moiety and moving the phenyl moiety further
away from the alcohol group through -CH2- spacer
groups (substrates 39b and 40b), the activity did not
decrease and no negative influence on the enantioselec-
tivity was observed (E > 100). Substrate 40b is of
practical interest, since it constitutes a precursor for the
(S)-10b, (S)-12b, (R)-16b, (R)-17b, (S)-18b, and (R)-39b. The
absolute configuration of (S)-8b, (R)-35b, and (R)-37b was
proven by comparison of GC data with that of the literature.20
The absolute configuration of (S)-11b was determined by
hydrogenation of the CdC double bond (5% Pd/C, EtOH, H 1
2
bar) and comparison of the resulting product 40b with (S)-
40b by coinjection on GC using a chiral stationary phase. The
absolute configuration of the remaining compounds was
determined by comparison of optical rotation values with
literature data (see the Supporting Information).
synthesis of antihypertensive agents, such as bufeniode
or labetalol,11 and spasmolytics or antileptics, such as
1
2
emepronium bromide. Both enantiomers were obtained
either by oxidative kinetic resolution of rac-40b to give
(
(
R)-40b (see Table 6) or reduction of ketone 40a to give
S)-40b, with >99% ee at 98% conversion within 4.5 h
(
13) Stampfer, W.; Kosjek, B.; Kroutil, W.; Faber, K. Biotechnol.
Bioeng. in press.
14) Kroutil, W.; Mischitz, M.; Faber, K. J . Chem. Soc., Perkin Trans.
at 50% v/v 2-propanol.
(
1
1997, 3629-3636.
Con clu sion s
(
15) Nakamura, K.; Matsuda, T. J . Org. Chem. 1998, 63, 8957-8964.
(16) Blake, A. J .; Cunningham, A.; Ford, A.; Teat, S. J .; Woodward,
We have shown that the whole-cell microbial oxidation/
reduction system of R. ruber DSM 44541 represents a
S. Chem. Eur. J . 2000, 6, 3586-3594.
17) Palmer, M.; Walsgrove, T.; Wills, M. J . Org. Chem. 1997, 62,
226-5228.
18) Everhart, E. T.; Craig, J . C. J . Chem Soc. Perkin Trans 1 1991,
(
5
(
(
11) J ohnson, J . A.; Akers, W. S.; Herring, V. L.; Wolfe, M. S.;
Sullivan, J . M. Pharmacotherapy 2000, 20, 622-628.
12) Aasen, A. J .; Schjelderup, L. Acta Chem. Scand., Ser. B 1988,
2, 206-210.
1701-1707.
(19) Salvi, N. A.; Subrata, C. Tetrahedron 2001, 57, 2833-2839.
(20) Burk, M. J .; Hems, W.; Herzberg, D.; Malan, C.; Zanotti-Gerosa,
A. Org. Lett. 2000, 2, 4173-4176.
(
4
J . Org. Chem, Vol. 68, No. 2, 2003 405

Stampfer et al.
Ackn owledgm en t. This study was performed within
the Spezialforschungsbereich “Biokatalyse”, and finan-
cial support by the Fonds zur F o¨ rderung der wissen-
schaftlichen Forschung (Vienna, project no. F115) is
gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Achiral and chiral
GC data as well as the [R]20
values. This material is available
D
free of charge via the Internet at http://pubs.acs.org.
J O026216W
4
06 J . Org. Chem., Vol. 68, No. 2, 2003