Asymmetric reduction and oxidation of aromatic ketones and alcohols using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus

Article abstract of DOI:10.1021/jo0616097

An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the corresponding optically active secondary alcohols was achieved with W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol (30%), v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement with Prelog's rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers its pro-R hydride to the re face of the ketone. A series of phenyl ring-containing ketones, such as 4-phenyl-2-butanone (1a) and 1-phenyl-1,3-butadione (2a), were reduced with good to excellent yields and high enantioselectivities. On the other hand, 1-phenyl-2-propanone (7a) was reduced with lower ee than 2-butanone derivatives. (R)-Alcohols, the anti-Prelog products, were obtained by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols using W110A TESADH in Tris buffer/acetone (90:10, v/v).

Full text of DOI:10.1021/jo0616097

Asymmetric Reduction and Oxidation of Aromatic Ketones and
Alcohols Using W110A Secondary Alcohol Dehydrogenase from
Thermoanaerobacter ethanolicus
Musa M. Musa,† Karla I. Ziegelmann-Fjeld,‡ Claire Vieille,‡ J. Gregory Zeikus,‡ and
Robert S. Phillips*,†,§,|
Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for
Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602, and Department of Biochemistry
and Molecular Biology, Michigan State UniVersity, East Lansing, Michigan 48824
rsphillips@chem.uga.edu
ReceiVed August 2, 2006
An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the
corresponding optically active secondary alcohols was achieved with W110A secondary alcohol
dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol
(30%, v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance
the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also
to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement
with Prelog’s rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers
its pro-R hydride to the re face of the ketone. A series of phenyl ring-containing ketones, such as 4-phenyl-
2-butanone (1a) and 1-phenyl-1,3-butadione (2a), were reduced with good to excellent yields and high
enantioselectivities. On the other hand, 1-phenyl-2-propanone (7a) was reduced with lower ee than
2-butanone derivatives. (R)-Alcohols, the anti-Prelog products, were obtained by enantiospecific oxidation
of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols using W110A TESADH in Tris
buffer/acetone (90:10, v/v).
Introduction
their racemic alcohols via enantiospecific kinetic resolution
(KR).3,4 Chiral metal complexes have been used as catalysts
for these purposes;5 however, these methods produce toxic
residual metals that create environmental problems. Enzymes
are recognized to be among the most effective catalysts for
producing optically active alcohols. Among their advantages
are their chemo-, regio-, and stereoselectivities due to the strict
recognition of a particular substrate by a given enzyme.
Tremendous efforts have been made in recent years to
establish enantioselective routes to enantiomerically pure com-
pounds, due to their importance in pharmaceutical, agricultural,
and food industries.1 Recent developments in medicine have
shown that a single enantiomer is biologically active in most
chiral drugs.2 Optically active alcohols are one of the most
important synthons. They can be produced from their corre-
sponding prochiral ketones via asymmetric reduction, or from
(1) (a) Garc´ıa-Urdiales, E.; Alfonso, I.; Gotor, V. Chem. ReV. 2005, 105,
313-354. (b) Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr. Opin.
Chem. Biol. 2004, 8, 120-126. (c) Nakamura, K.; Yamanaku, R.; Matsuda,
T.; Harada, T. Tetrahedron: Asymmetry 2003, 14, 2659-2681. (d)
Nakamura, K.; Matsuda, T.; Harada, T. Chirality 2002, 14, 703-708. (e)
Phillips, R. S. Can. J. Chem. 2002, 80, 680-685.
* Corresponding author. Phone: (706) 542-1996. Fax: (706) 542-9454.
† Department of Chemistry, University of Georgia.
‡ Michigan State University.
§ Department of Biochemistry and Molecular Biology, University of Georgia.
| Center for Metalloenzyme Studies, University of Georgia.
(2) Ariens, E. J. Med. Res. ReV. 1986, 6, 451-466.
10.1021/jo0616097 CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/10/2006
30
J. Org. Chem. 2007, 72, 30-34

Asymmetric Reduction and KR Using W110A TESADH
Biocatalytic processes also are less hazardous and energy
consuming than conventional chemistry methodologies. They
are normally carried out under mild conditions, which minimize
problems of product isomerization, racemization, or epimeriza-
tion. Biocatalysts are easily produced at low cost and with
minimum waste, and they can be decomposed in the environ-
ment after use. Unfortunately, they do have some disadvantages.
For example, many enzymes are thermally unstable. Another
disadvantage is the limited solubility of most organic substrates
in water; this leads to larger reaction volumes, a need for
cosolvents, and complicated product recovery.6
FIGURE 1. Prelog’s rule for predicting the stereochemistry of alcohols
formed from their corresponding ketones by asymmetric reduction with
ADHs.
Alcohol dehydrogenases (ADHs, EC 1.1.1.X, X ) 1 or 2)
are enzymes that catalyze the reversible reduction of ketones
and aldehydes to the corresponding alcohols. The asymmetric
reduction of ketones using the commercially available yeast
ADH and horse liver ADH is limited not only due to their
temperature sensitivity, but also due to their sensitivity toward
organic solvents and their loss of activity upon immobilization.
An additional disadvantage of horse liver ADH is its low affinity
for acyclic ketones.1b,7 Secondary ADH from Thermoanaero-
bacter ethanolicus (TESADH, EC 1.1.1.2), a highly thermo-
stable enzyme, has been isolated and characterized.8 NADPH
is required by this enzyme, from which the hydride is transferred
to the carbonyl carbon. Because NADPH is a costly cofactor,
alcohols like 2-propanol or ketones like acetone are used as
hydrogen source or hydrogen sink to regenerate the cofactor
and therefore make both processes catalytic. This enzyme is
stable at temperatures up to 80 °C, and it exhibits high activity
in the asymmetric reduction of ketones.9 Because of its
thermostability, resistance to organic solvents, and reactivity
for a wide variety of substrates, it is a useful biocatalyst for
synthetic applications.10
propargyl alcohols using wild-type TESADH.10a The behavior
of TESADH has been shown to be similar to results obtained
from reductions with a very highly homologous (99% identity),8b
NADPH-dependent,ThermoanaerobiumbrockiiADH(TBADH).11
For TBADH, Keinan et al. suggested that the two alkyl groups
of substrates occupy two hydrophobic sites, which differ from
one another in volume and also in their affinities toward the
alkyl groups (Figure 1).11 It was also shown that the small site,
which has higher affinity toward the alkyl groups of the ketone,
can accommodate up to three carbon substituents, like the
isopropyl group.10a,b,11
We have recently reported a new mutant of TESADH, where
tryptophan-110 was substituted by alanine (W110A TE-
SADH).12 This replacement makes the large pocket able to
accommodate phenyl ring-containing substrates that are not
substrates for wild-type TESADH.10b Its modified substrate
range makes this mutant enzyme useful for the enantioselective
reduction of phenyl ring-containing ketones such as 4-phenyl-
2-butanone (1a) and, in the reverse direction, for the enanti-
oselective oxidation via KR of racemic phenyl ring-containing
secondary alcohols.
A series of ethynyl ketones and ethynylketoesters were
reduced enantioselectively to the corresponding nonracemic
(3) (a) Zhu, D.; Yang, Y.; Hua, L. J. Org. Chem. 2006, 71, 4202-4205.
(b) Stampfer, W.; Kosjek, B.; Faber, K.; Kroutil, W. J. Org. Chem. 2003,
68, 402-406. (c) Yadav, J. S.; Nanda, S.; Reddy, P. T.; Rao, A. B. J. Org.
Chem. 2002, 67, 3900-3903. (d) Schubert, T.; Hummel, W.; Mu¨ller, M.
Angew. Chem., Int. Ed. 2002, 41, 634-636. (e) Stampher, W.; Kosjek, B.;
Moitzi, C.; Kroutil, W.; Faber, K. Angew. Chem., Int. Ed. 2002, 41, 1014-
1017. (f) Nakamura, K.; Inoue, Y.; Matsuda, T.; Misawa, I. J. Chem. Soc.,
Perkin Trans. 1 2000, 2397-2402. (g) Bortolini, O.; Fantin, G.; Fogagnolo,
M.; Giovannini, P. P.; Guerrini, A.; Medici, A. J. Org. Chem. 1997, 62,
1854-1856. (h) Grunwald, J.; Wirz, B.; Scollar, M. P.; Kilbanov, A. M.
J. Am. Chem. Soc. 1986, 108, 6732-6734.
(4) (a) Birman, V. B.; Jiang, H. Org. Lett. 2005, 7, 3445-3447. (b)
Ghanem, A.; Aboulenein, H. Y. Chirality 2005, 17, 1-15. (c) Martin-
Matute, B.; Edin, M.; Bogar, K.; Bachva¨ll, J. E. Angew. Chem., Int. Ed.
2004, 43, 6535-6539. (d) Birman, V. B.; Uffman, E. W.; Jiang, H.; Li,
X.; Kilbane, C. J. J. Am. Chem. Soc. 2004, 126, 12226-12227. (e)
Nakamura, K.; Matsuda, T.; Harada, T. Chirality 2002, 14, 703-708. (f)
Kim, K.; Song, B.; Choi, M.; Kim, M. Org. Lett. 2001, 3, 1507-1509.
(5) (a) Weinert, C. S.; Fanwick, P. E.; Rothwell, I. P. Organometallics
2005, 24, 5759-5766. (b) Noyori, R.; Okhuma, T. Angew. Chem., Int. Ed.
2001, 40, 40-73. (c) Blake, A. J.; Cunningham, A.; Ford, A.; Teat, S. J.;
Woodward, S. Chem.-Eur. J. 2000, 6, 3586-3594.
(6) (a) Gervais, T. R.; Carta, G.; Gianer, J. L. Biotechnol. Prog. 2003,
49, 389-395. (b) Cao, L.; Langen, L. V.; Sheldon, R. A. Curr. Opin.
Biotechnol. 2003, 14, 387-394. (c) Kilbanov, A. M. Nature 2001, 409,
241-246. (d) Koeller, M. K.; Wong, C. H. Nature 2001, 409, 232-240.
(e) Riva, S.; Carrea, G. Angew. Chem., Int. Ed. 2000, 39, 2226-2254.
(7) (a) Kovalenko, G. A.; Sokolovskii, V. D. Biotechnol. Bioeng. 1983,
25, 3177-3184. (b) Pollak, A.; Blumenfeld, H.; Wax, M.; Baughn, R. L.;
Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 6324-6336.
(8) (a) Burdette, D.; Zeikus, J. G. Biochem. J. 1994, 302, 163-170. (b)
Burdette, D. S.; Vieille, C.; Zeikus, J. G. Biochem. J. 1996, 316, 115-122.
(9) (a) Pham, V. T.; Phillips, R. S. J. Am. Chem. Soc. 1990, 112, 3629-
3632. (b) Pham, V. T.; Phillips, R. S.; Ljugdahl, L. G. J. Am. Chem. Soc.
1989, 111, 1935-1936.
Results and Discussion
A series of phenyl ring-containing ketones, which could not
be reduced by wild-type TESADH, were reduced by W110A
TESADH to produce the corresponding nonracemic alcohols
with good yields and high optical purities (Table 1). The
reductions were carried out in Tris buffer containing 30% (v/v)
2-propanol, which serves as both cosolvent and hydride source
to reduce the oxidized coenzyme. The use of such a high
percentage of 2-propanol was crucial not only to enhance the
solubility of the hydrophobic phenyl ring-containing ketone
substrates in aqueous media, but also to shift the equilibrium
into the reduction direction. The produced alcohols had S
configuration, in agreement with Prelog’s rule, in which the
NADPH cofactor transfers its pro-R hydride to the re face of
the ketone (Figure 1).1b,c,13
Phenyl ring-containing 2-butanone derivatives were reduced
to the corresponding (S)-alcohols with excellent stereoselec-
(10) (a) Heiss, C.; Phillips, R. S. J. Chem. Soc., Perkin Trans. 1 2000,
2821-2825. (b) Heiss, C.; Laivenieks, M.; Zeikus, J. G.; Phillips, R. S.
Bioorg. Med. Chem. 2001, 9, 1659-1666. (c) Heiss, C.; Laivenieks, M.;
Zeikus, G.; Phillips, R. S. J. Am. Chem. Soc. 2001, 123, 345-346. (d) Tripp,
A. E.; Burdette, D. S.; Zeikus, J. G.; Phillips, R. S. J. Am. Chem. Soc.
1998, 120, 5137-5141.
(11) Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J. Am. Chem.
Soc. 1986, 108, 162-169.
(12) Ziegelmann-Fjeld, K. I.; Musa, M. M.; Phillips, R. S.; Zeikus,
J. G.; Vieille, C. Protein Eng. Des., in press.
(13) Prelog, V. Pure Appl. Chem. 1964, 9, 119-130.
J. Org. Chem, Vol. 72, No. 1, 2007 31

Musa et al.
TABLE 1. Asymmetric Reduction of Phenyl Ring-Containing
Ketones Using W110A TESADH
FIGURE 2. GC chromatograms illustrating (a) the products of NaBH4
reduction of 6a, (b) the products of W110A TESADH reduction of
6a, and (c) (-)-(2S,3S)-6c produced from (+)-(2S,3R)-6b.
conv.
ee
substrate
R
producta
(%)b
(%)d
SCHEME 1. Conversion of (+)-(2S,3R)-6b into
(-)-(2S,3S)-6c
1a
2a
3a
4a
5a
6a
7a
8a
9a
PhCH2CH2
(S)-1b
(S)-2b
(S)-3b
(S)-4b
(S)-5b
(2S,3R)-6b
(S)-7b
(S)-8b
(S)-9b
99
98
64
>99
>99
>99
91
Ph(CdO)CH2
(E)-Ph-HCdCH
p-MeOC6H4(CH2)2
PhOCH2
p-ClC6H4CH2CHCl
PhCH2
87
>99
83c
95
97
>99
>99e
>99
37e
p-MeOC6H4CH2
>99e
71e
a The absolute configurations of the products were determined by
comparison of the signs of the optical rotation with those reported previously.
b % conversion was determined by GC. c Isolated yield. d Unless otherwise
mentioned, ee was determined by chiral stationary phase GC for the
produced alcohol. e The ee was determined for the corresponding acetate
derivative (see the Supporting Information).
mixture of syn- and anti-R-chlorohydrins), in which the syn-6b
had a different retention time than (+)-(2S,3R)-6b by injection
in a chiral column GC (Figure 2a,b). Reduction of 6a to almost
a single stereoisomer, (+)-(2S,3R)-6b, using W110A TESADH
indicated that the process involves a KR, and this should be
combined with isolation of (S)-6a as unreacted enantiomer and
a maximum yield of 50% of the produced R-chlorohydrin. We
have noticed that the yield is higher than 50%, and the isolated
unreacted 6a is a racemic mixture. This indicates that the
reduction of 6a with W110A TESADH proceedes by dynamic
kinetic resolution via a facile buffer-catalyzed enolization, which
enables the unreacted enantiomer (S)-6a to racemize after the
depletion of (R)-6a starts.16 The R-chlorohydrin (+)-(2S,3R)-
6b was then converted quantitatively to the corresponding
tivities and moderate to excellent yields (Table 1). 4-Phenyl-
2-butanone (1a) was reduced stereoselectively to produce (S)-
4-phenyl-2-butanol ((S)-1b) with excellent chemical and optical
yields. The â-diketone 1-phenyl-1,3-butadione (2a) was reduced
regio- and stereoselectively to furnish the monohydroxy ketone
(S)-3-hydroxy-1-phenyl-1-butanone ((S)-2b) with excellent yield
and ee, leaving the other keto group at C-1 intact. (E)-4-Phenyl-
3-butene-2-one (3a) was reduced with moderate yield and
excellent optical purity to produce the allylic alcohol (S)-4-
phenyl-3-butene-2-ol ((S)-3b). The presence of the methoxy
group at the para position of the phenyl ring in 4-(4-methox-
yphenyl)-2-butanone (4a) affected the ee of the produced (S)-
4-(4-methoxyphenyl)-2-butanol ((S)-4b) (91% ee), which is
lower than for (S)-1b. Phenoxy-2-propanone (5a) was reduced
with very high yield and optical purity to produce the corre-
sponding (S)-phenoxy-2-propanol ((S)-5b). When the R-chlo-
roketone, 3-chloro-4-(4-chlorophenyl)-2-butanone (6a), was
reduced with W110A TESADH, (+)-(2S,3R)-3-chloro-4-(4-
chlorophenyl)-2-butanol ((+)-(2S,3R)-6b) was produced with
high enantioselectivity (>99% ee) and diastereoselectivity (92:8
mixture of anti- and syn-R-chlorohydrins). The absolute con-
figuration of (+)-(2S,3R)-6b was confirmed by comparing the
sign of the optical rotation with that reported previously for
the very similar compound, (+)-(2S,3R)-4-phenyl-3-bromo-2-
butanol) ([R]20D +29.2, c 2.08, CHCl3; lit.14 [R]25J +37, c 0.06,
CHCl3, 95% ee). In a separate experiment, reduction of 6a with
NaBH4, which is expected to give mainly the syn product,15
afforded a mixture of four diastereomers ((()-6b) (88:12
epoxide,
(-)-(2S,3S)-4-(4-chlorophenyl)-2,3-epoxybutane
((-)-(2S,3S)-6c), without racemization using 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) (Scheme 1, Figure 2c).3d The absolute
configuration of (-)-(2S,3S)-6c was confirmed by the compari-
son of the sign of optical rotation with that reported for the
(15) (a) Usami, Y.; Ueda, Y. Chem. Lett. 2005, 34, 1062-1063. (b)
Hamamoto, H.; Mamedov, V. A.; Kitamoto, M.; Hayashi, N.; Tsuboi, S.
Tetrahedron: Asymmetry 2000, 11, 4485-4497. (c) Tsuboi, S.; Sakamoto,
J.; Yamashita, H.; Sakai, T.; Utaka, M. J. Org. Chem. 1998, 63, 1102-
1108. (d) Tsuboi, S.; Yamafuji, N.; Utaka, M. Tatrahedron: Asymmetry
1997, 8, 375-379. (e) Duhamel, P.; Leblond, B.; Biodois-Sery, L.; Poirier,
J. J. J. Chem. Soc., Perkin Trans. 1 1994, 2265-2271. (f) Tsuboi, S.;
Sakamoto, J.; Sakai, T.; Utaka, M. Chem. Lett. 1989, 8, 1427-1428. (g)
Satoh, T.; Sugimoto, A.; Yamakawi, K. Chem. Pharm. Bull. 1989, 37, 184-
186.
(16) (a) Feske, B. D.; Kaluzna, I. A.; Stewart, J. D. J. Org. Chem. 2005,
70, 9654-9657. (b) Poessl, T. M.; Kosjek, B.; Ellmer, U.; Grubber, C. C.;
Edegger, K.; Faber, K.; Hildebrandt, P.; Bornscheuer, U. T.; Kroutil, W.
AdV. Synth. Catal. 2005, 347, 1827-1834.
(14) Besse, P.; Fernard, M. F.; Veschambre, H. Tetrahedron: Asymmetry
1994, 5, 1249-1268.
32 J. Org. Chem., Vol. 72, No. 1, 2007

Asymmetric Reduction and KR Using W110A TESADH
TABLE 2. Enantiospecific Kinetic Resolution of Phenyl
to the corresponding ketones 1a and 3a, respectively, leaving
their (R)-alcohols as unreacted enantiomers with excellent
enantiomeric ratios (Table 2). The production of optically active
1b is important as it is a precursor for antihypertensive agents,
such as bufeniode and labetalol.3b,17 For rac-4b, it was resolved
by oxidative KR to furnish (R)-4b with moderate stereopref-
erence (77% ee at 75% conversion). Under the same conditions,
KR of rac-5b furnished (R)-5b with 25% ee at only 19%
conversion, indicating that the KR of this alcohol takes place
with high enantiomeric discrimination. Even with addition of
more enzyme and acetone, we were not able to push the reaction
to higher yield. The racemic 1-phenyl-2-propanol (rac-7a) was
resolved, as expected, with low enantiospecificity because it
was reduced with low ee. (S)-1-(4-Methoxyphenyl)-2-propanol
((S)-8b) was oxidized with excellent enantioselectivity to its
corresponding ketone 8a, leaving (R)-8b as enantiomerically
pure unreacted enantiomer. Although 9a was reduced with high
yield and moderate ee, rac-9b was not oxidized by W110A
TESADH. The same results for rac-9b were obtained by
Stampfer et al. using Rhodococcus ruber DSM 44541.3b
Resistance of TESADH to organic cosolvents allowed the
redox reactions in both directions to be carried out at relatively
high substrate concentration (35 mM in the reduction pathway
and 70 mM in the oxidation pathway). The design of new
TESADH mutants such as W110A TESADH in addition to
TESADH’s resistance to organic solvents and high concentra-
tions of substrates make this enzyme useful for synthetic
applications.
Ring-Containing rac-Alcohols Using W110A SADH
conv.
ee
substrate
R
producta
(%)b
(%)c
rac-1b
rac-3b
rac-4b
rac-5b
rac-7b
rac-8b
PhCH2CH2
(R)-1b
(R)-3b
(R)-4b
(R)-5b
(R)-7b
(R)-8b
50
50
75
19
49
48
>99
>99
77d
25
(E)-Ph-HCdCH
p-MeOC6H4(CH2)2
PhOCH2
PhCH2
p-MeOC6H4CH2
39d
92d
a The absolute configurations of the unreacted alcohols were confirmed
by co-injection in a chiral column GC with their S enantiomers prepared
by the asymmetric reduction of the corresponding ketones employing
W110A TESADH (Table 1). b % conversion was determined by GC.
c Unless otherwise mentioned, ee was determined by a chiral stationary phase
GC for the alcohols. d The ee was determined for the corresponding acetate
derivative (see the Supporting Information).
very similar compound (-)-(2S,3S)-4-phenyl-2,3-epoxybutane
([R]20D -26.2, c 2.32, CHCl3; lit.14 [R]25J -27, c 0.04, CHCl3,
>98% ee).
Conclusion
Unexpectedly, 1-phenyl-2-propanone (7a) was reduced to
produce (S)-1-phenyl-2-propanol ((S)-7b) with poor enantiose-
lectivity, indicating that 7a can fit in alternative modes in the
active site within the large pocket, allowing the NADPH cofactor
to deliver its pro-R hydride from either re or si faces. 1-(4-
Methoxyphenyl)-2-propanone (8a) was reduced to produce (S)-
1-(4-methoxyphenyl)-2-propanol ((S)-8b) with excellent chemi-
cal yield and ee, which means that the sterically bulky para
methoxy substituent in 8a restricts the substrate to only a single
binding mode within the active site. The cyclic ketone 2-tetra-
lone (9a) was reduced with high yield and moderate stereopref-
erence to produce (S)-2-tetralol ((S)-9b). Enzymatic asymmetric
reduction of substrates with sterically hindered groups on both
sides of the carbonyl, like 9a, is of great interest because these
substrates are typically either poor or non-substrates for ADHs;
therefore, very few ADHs are able to achieve such asymmetric
reductions.1b,3c
Oxidation via KR of phenyl ring-containing rac-alcohols was
used to produce their (R)-alcohols, the anti-Prelog configurated
alcohols, as unreacted enantiomers with moderate to high
enantiomeric ratios using W110A TESADH. The reactions were
carried out in Tris buffer containing 10% (v/v) acetone. The
amount of acetone needed was less than the amount of
2-propanol used in the reduction pathway simply because
alcohols are more soluble than their corresponding hydrophobic
ketones in aqueous media. As with all KRs, these reactions
suffer from the limitation that the maximum theoretical yield
with high enantiomeric ratio of a single enantiomer, (R) in this
case, is 50% (Table 2). As expected, the substrates reduced with
high ee showed high stereospecificities in the oxidation pathway
and vice versa.
We have been able to produce both enantiomers of a series
of phenyl ring-containing secondary alcohols by asymmetric
reduction and enantioselective oxidation via KR using W110A
TESADH. (S)-Alcohols were produced via asymmetric reduction
with high chemical and moderate to high optical yields using
2-propanol as a cosubstrate for coenzyme regeneration and as
a cosolvent. A number of racemic phenyl ring-containing
alcohols were resolved with W110A TESADH using acetone
as a hydrogen acceptor and a cosolvent. These reactions
produced a mixture of (R)-alcohols as unreacted enantiomer with
good enantiomeric ratios and the corresponding ketones, which
could be recycled. The use of 2-propanol (30%, v/v) and acetone
(10% v/v) in high concentration in the reduction and oxidation
pathways was crucial not only to enhance the solubility of
hydrophobic phenyl ring-containing substrates, but also to shift
the equilibrium to the desired direction. It is of great interest to
produce optically active alcohols of both enantiomers using the
same enzyme because the two enantiomers are often of equal
importance and only a few anti-Prelog enzymes are available.
W110A TESADH will be of great interest to organic chemists
for the preparation of optically active phenyl ring-containing
alcohols because of its thermal stability and high tolerance to
organic cosolvents.
Experimental Section
General Procedures. Capillary gas chromatographic measure-
ments were performed on a GC equipped with a flame ionization
detector and a Supelco â-Dex 120 chiral column (30 m, 0.25 mm
1
[i.d.], 0.25 µm film thickness) using helium as the carrier gas. H
The enantiospecific oxidation via KR using W110A TESADH
exclusively oxidized the S enantiomers of rac-1b and rac-3b
(17) Johnson, J. A.; Akers, W. S.; Herring, V. L.; Wolf, M. S.; Sullivan,
J. M. Pharmacotherapy 2000, 20, 622-628.
J. Org. Chem, Vol. 72, No. 1, 2007 33

Musa et al.
NMR and 13C NMR spectra were recorded on a 400 MHz
spectrometer at room temperature in CDCl3 using either solvent
peak or tetramethylsilane as internal standard. Column chromatog-
raphies were carried out on standard grade silica gel (60 Å, 32-63
µm) with ethyl acetate in hexane as eluent.
Materials. Commercial grade solvents were used without further
purification. NADP+, Novozyme 435, and NaBH4 were used as
purchased from commercial sources. Substrates 1a-6a, 9a, rac-
1b, rac-7b, (R)-7b, and (S)-7b were used as purchased from
commercial suppliers. 7a and 8a were prepared as described
previously.18 rac-3b, rac-4b, rac-5b, rac-8b, and rac-9b were
prepared by reducing the corresponding ketones with NaBH4.19
Gene Expression and Purification of W110A TESADH.
W110A TESADH was expressed in recombinant E. coli HB101-
(DE3) cells and purified as described.12
General Procedure for Asymmetric Reduction of Phenyl
Ring-Containing Ketones with W110A TESADH. Reactions were
conducted with 0.34 mmol of substrate, 2 mg of NADP+, and 0.75
mg of W110A TESADH in 10.0 mL of 50 mM Tris-buffer (pH
8.0)/2-propanol (70:30, v/v). The reaction mixture was stirred at
50 °C for 10 h, and then it was extracted with 3 × 5 mL of CH2-
Cl2. The combined organic layers were dried with Na2SO4 and
concentrated under vacuum. The remaining residue was analyzed
by chiral column GC to determine the percent conversion and ee
of the produced alcohols and then purified with silica gel using
hexane/ethyl acetate (85/15) (95/5 for 6b).
Hz, J ) 9.8 Hz), 3.10 (dd, 1H, J )14.6 Hz, J ) 4.2 Hz), 3.96 (qd,
1H, J ) 6.4, J ) 4.0), 4.14 (td, 1H, J ) 9.6, J ) 4.0), 7.17 (d, 2H,
J ) 8.0), 7.29 (d, 2H, J ) 8.0). 13C NMR, δ: 18.8, 39.2, 69.4,
70.3, 128.9, 130.8, 132.9, 136.3. HRMS calcd for C10H12OCl2 [M
+ H]+, 219.0343; found, 219.0347.
General Procedure for Kinetic Resolution of Phenyl Ring-
Containing Racemic Alcohols with W110A TESADH. Reactions
were conducted with 0.34 mmol of substrate, 1 mg of NADP+,
and 0.38 mg of W110A TESADH in 5.0 mL of 50 mM Tris-buffer/
acetone (90:10) (v/v). The reaction mixture was stirred at 50 °C
for 12 h, and then it was extracted with 3 × 5 mL of CH2Cl2. The
combined organic layers were dried with Na2SO4 and concentrated
under vacuum. The remaining residue was analyzed by chiral
stationary phase GC to determine the percent conversion to ketone
and ee of the unreacted (R)-alcohol.
Synthesis of (-)-(2S,3S)-4-(4-Chlorophenyl)-2,3-epoxybutane
((-)-(2S,3S)-6c). It was prepared from (2S,3R)-6b using a previ-
ously reported procedure for epoxidation.3d [R]20 -26.2 (c 2.32,
D
CHCl3) >99% ee, 84% de. 1H NMR, δ: 1.23 (d, 3H, J ) 5.2 Hz),
2.71-2.80 (m, 4H), 7.10 (d, 2H, J ) 8.4), 7.20 (d, 2H, J ) 8.8).
13C NMR, δ: 17.1, 37.9, 54.6, 59.6, 128.8, 130.5, 132.6, 136.1.
HRMS calcd for C10H11OCl [M + H]+, 183.0576; found, 183.0571.
Determination of Absolute Configuration. The absolute con-
figurations of the following compounds were determined by
comparing of the sign of the optical rotation with that reported in
the literature: (S)-1b,20 (S)-2b,21 (S)-3b,22 (S)-4b,23 (S)-5b,24 (S)-
7b,25 (S)-8b,26 and (S)-9b.3b The absolute configuration of (S)-7b
was also demonstrated by co-injection on a chiral column GC with
commercially available (R)-7b and (S)-7b. The absolute configu-
ration of (S)-1b was confirmed by co-injection on a chiral column
GC with (R)-1b, which was prepared by KR of rac-1b using
Novozyme 435.27 The absolute configurations of (R)-1b, (R)-3b,
(R)-4b, (R)-5b, (R)-7b, and (R)-8b were elucidated by co-injection
on GC using a chiral stationary phase with their S enantiomers
prepared from asymmetric reduction of the corresponding ketones
using W110A TESADH.
(+)-(2S,3R)-3-Chloro-4-(4-chlorophenyl)-2-butanol ((+)-(2S,3R)-
6b). [R]20D +29.2 (c 2.08, CHCl3) >99% ee, 84% de. 1H NMR, δ:
1.33 (d, 3H, J ) 6.4 Hz), 1.91 (brs, 1H), 2.91 (dd, 1H, J ) 14.6
(18) King, J. A.; McMillan, F. H. J. Am. Chem. Soc. 1951, 73, 4911-
4915.
(19) Yadav, V. K.; Babu, K. G. Tetrahedron 2003, 59, 9111-9116.
(20) Nakamura, K.; Inoue, Y.; Matsuda, T.; Misawa, I. J. Chem. Soc.,
Perkin Trans. 1 1999, 2397-2402.
(21) Ahmad, K.; Koul, S.; Taneja, S. C.; Singh, A. P.; Kapoor, M.; Riyaz-
ul-Hassan; Verma, V.; Qazi, G. N. Tetrahedron: Asymmetry 2004, 15,
1685-1692.
Acknowledgment. Financial support from the National
Science Foundation (grant number 0445511) to R.S.P., J.G.Z.,
and C.V. is gratefully acknowledged.
(22) Mastranzo, V. M.; Quintero, L.; Parrodi, C. A.; Juaristi, E.; Walsh,
P. J. Tatrahedron 2004, 60, 1781-1789.
(23) Donzelli, F.; Fuganti, C.; Mendozza, M.; Pedrocchi-Fantoni, G.;
Servi, S.; Zucchi, G. Tetrahedron: Asymmetry 1996, 7, 3129-3134.
(24) Nakamura, K.; Takenaka, K.; Fujii, M.; Ida, Y. Tetrahedron Lett.
2002, 43, 3629-3631.
Supporting Information Available: Chiral GC chromatogram
for both enantiomers of the acetate derivatives of alcohols 1b-5b
and 7b-9b. Optical rotation values for (S)-1b-5b and (S)-7b-9b.
1H NMR and 13C NMR spectra for compounds (S)-1b-5b, (S)-7b-
9b, (+)-(2S,3R)-6b, and (-)-(2S,3S)-6c. This material is available
free of charge via the Internet at http://pubs.acs.org.
(25) Erde´lyi, B.; Szabo´, A.; Seres, G.; Birincsik, L.; Ivanics, J.; Szatzker,
G.; Poppe, L. Tetrahedron: Asymmetry 2006, 17, 268-274.
(26) Ferrabochi, P.; Grisenti, P.; Manzocchi, A.; Santanielo, E. J. Chem.
Soc., Perkin Trans. 1 1990, 2469-2474.
(27) Choi, J. H.; Choi, Y. K.; Kim, Y. H.; Park, E. S.; Kim, E. J.; Kim,
M.; Park, J. J. Org. Chem. 2004, 69, 1972-1977.
JO0616097
34 J. Org. Chem., Vol. 72, No. 1, 2007