'Green' synthesis of important pharmaceutical building blocks: Enzymatic access to enantiomerically pure α-chloroalcohols

Article abstract of DOI:10.1016/j.tetasy.2005.08.037

Thirty one recombinant ketoreductase enzymes were screened for the reduction of six α-chloroketones, the precursors of pharmaceutically valuable α-chloroalcohols. Several highly active and enantioselective ketoreductases were found and their applications

Full text of DOI:10.1016/j.tetasy.2005.08.037

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3275–3278
‘Green’ synthesis of important pharmaceutical building
blocks: enzymatic access to enantiomerically pure a-chloroalcohols
Dunming Zhu, Chandrani Mukherjee and Ling Hua*
Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA
Received 9 August 2005; accepted 19 August 2005
Available online 28 September 2005
Abstract—Thirty one recombinant ketoreductase enzymes were screened for the reduction of six a-chloroketones, the precursors of
pharmaceutically valuable a-chloroalcohols. Several highly active and enantioselective ketoreductases were found and their appli-
cations to the synthesis of both enantiomers of these a-chloroalcohols were demonstrated on a preparative scale. This offers a con-
venient, ÔgreenÕ access to this type of important pharmaceutical building blocks.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
preparation of a large group of anti-depressants and
a- and b-adrenergic drugs.1–6 For example, 2-chloro-1-
phenylethanol 1 is the precursor for the synthesis of flu-
oxetine, tomoxetine, and nisoxetine,1 while 2-chloro-1-
(30-chlorophenyl)ethanol 2 has been used to synthesize
AJ-9677, a potent and selective b3 adrenergic receptor
agonist (Fig. 1).2a
Chiral halohydrins are valuable synthetic intermediates
for the preparation of a wide range of biologically
interesting compounds. 2-Chloro-1-phenylethanol 1,
2-chloro-1-(30-chlorophenyl)-ethanol 2, 2-chloro-1-(40-
chlorophenyl)ethanol 3, 2-chloro-1-(30,40-dichlorophen-
yl)ethanol 4, 2-chloro-1-(40-nitrophenyl)ethanol 5, and
2-chloro-1-(40-methanesulfonamidophenyl)ethanol
(Fig. 1) are particularly interesting as synthons for the
6
Amongst the various approaches to chiral chlorohyd-
rins, a straightforward process is the asymmetric reduc-
tion of prochiral halomethyl ketones, which can be
achieved chemically and biocatalytically.7,8 With ever-
increasing environmental concerns, the development of
Ôgreen methodsÕ to produce fine chemicals are highly
desirable. Biocatalysis accommodates several of the 12
principles of green chemistry defined by Anastas and
Warner,9 hence studies on the biocatalytic reduction of
prochiral ketones to the corresponding enantiomerically
pure alcohols have been developed rapidly over the last
decade.10
OH
R
Cl
X
O
X =
H
3'-Cl
4'-Cl
NHCH3
1
2
3
4
5
6
R = 4-CF3 Fluoxetine
R = 2-CH3 Tomoxetine
R = 2-OCH3 Nisoxetine
3',4'-Cl2
4'-NO2
4'-CH3SO2NH
Biocatalytic reductions can be carried out using either
whole cell systems11 or isolated ketoreductases.12 For
the biocatalytic transformation of a-chloroacetophe-
nones to the corresponding chiral a-chloroalcohols,
most of the reported methods involve whole cell reac-
tions.8,10c However, the use of isolated enzymes is
advantageous because undesirable enantiomer forma-
tion mediated by contaminating ketoreductases is mini-
mized,13 the enzymes can be stored and used as normal
chemical reagents and no microbiological knowledge is
required. In this regard, we have recently developed a
OH
H
Cl
N
OCH2CO2H
NH
AJ-9677
Figure 1. Structures of a-chloroalcohols 1–6 and four drugs.
*
Corresponding author. Tel.: +1 214 768 1609; fax: +1 214 768
4089; e-mail: lhua@smu.edu
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.08.037

3276
D. Zhu et al. / Tetrahedron: Asymmetry 16 (2005) 3275–3278
O
reductases KRED101, 111, 112, 113, 114, 115, and 118
were less active towards the reductions of 30,40-dichloro-
and 40-nitro-a-chloroacetophenones 10 and 11 than the
unsubstituted counterpart 7, and the other enzymes
showed a reverse tendency.
Cl
X
X =
7
8
H
3'-Cl
9
4'-Cl
3',4'-Cl2
4'-NO2
The enantioselectivity of ketoreductases KRED101, 107,
111, 112, 113, 114, 115, 118, 121, 123, 130, and 131 for
the reduction of a-chloroketones 7–12 were evaluated
using the NADPH recycle system of D-glucose dehydro-
genase and D-glucose. The results are presented in Table
2. As shown in Table 2, KRED101, 107, 112, 113, 130,
and 131 showed excellent enantioselectivity for the
reduction of all the tested a-chloroketones 7–12, and
the substituents on the benzene ring of the ketones
exerted minimal effect on their enantioselectivity. The
reductions catalyzed by KRED101, 107, 112, or 113 gave
(S)-chloroalcohols, while (R)-configuration products
were obtained using KRED 130 or 131 as catalyst. The
enantioselectivities of the other ketoreductase enzymes
were greatly affected by the substituents of substrates,
and the substituent even reverted the absolute configura-
tion of the major product alcohol. This was exemplified
by the KRED118-catalyzed reduction of 7 and 8, in
which the (R)-enantiomer was produced as the major
product with modest enantioselectivity, while the (S)-
enantiomers were obtained for other a-chloroketones
with upto 98% ee.
10
11
12
4'-CH3SO2NH
Figure 2. Structures of a-chloroketones 7–12.
ketoreductase tool-box of 31 recombinant ketoreductase
enzymes by genome mining and protein engineering,
and have shown that these isolated recombinant
enzymes efficiently catalyze the enantioselective reduction
of substituted aryl ketones and b-ketoesters.14 To
explore further the application of our isolated recombi-
nant ketoreductase collection, six a-chloroketones 7–12
were screened with our enzyme collection (Fig. 2). The
enantioselectivities of 12 selected ketoreductases with
high activities were then studied. It was found that both
enantiomers of the above-mentioned important pharma-
ceutical building blocks 1–6 could be obtained in enan-
tiomerically pure form and good to excellent isolated
yields.
2. Results and discussion
Good preparative applicability requires high activity
and enantioselectivity. Tables 1 and 2 show that, for
all the tested a-chloroketones, both enantiomers of the
product alcohols could be obtained in greater than
99% enantiomeric excess via the reduction catalyzed
by at least one enzyme in the ketoreductase tool box.
This was then further tested on a 1 mmol scale with
selected enzymes, allowing ready isolation and charac-
terization of the products. The isolated yield and enan-
tiomeric excess (ee) for the preparative scale reactions
are summarized in Table 3. The (R)-enantiomers of a-
chloroalcohols 1–6 were prepared with KRED130 in
good to excellent yields (76–99%) and essentially enantio-
merically pure form, while the enantiomerically pure
(S)-enantiomers were obtained in high isolated yields
(69–97%) using KRED112, 107, or 113. It is clearly dem-
onstrated that our ketoreductase collection is useful and
provides a ÔgreenÕ alternative access to enantiomerically
pure a-chloro alcohols of pharmaceutical importance.
The initial reaction rates of a-chloroketones 7–11 with
the ketoreductase enzymes KRED101–131 were deter-
mined by spectrophotometrically measuring the oxida-
tion of NADPH at 340 nm at room temperature. For
4-chloroacetylphenylmethanesulfonamide 12, the strong
absorbance at 340 nm prevented the measurement of
reaction rate by this method. As shown in Table 1,
KRED101, 107, 111, 112, 113, 114, 115, 118, 121, 123,
130, and 131 were efficient catalysts in the reduction of
all the tested a-chloroketones (the data for the less active
enzymes are not presented). For most of these 12 keto-
reductases, meta-chlorinated a-chloroketone 8 showed
a higher activity than the unsubstituted one 7, while
para-chloro substituent 9 decreased the activity. An
exception was KRED107. Seven out of twelve keto-
Table 1. Specific activity of ketoreductase-catalyzed reduction of
a-chloroketones 7–11a
KRED 7 (40-H) 8 (30-Cl) 9 (40-Cl) 10 (30,40-Cl2) 11 (40-NO2)
3. Experimental
101
107
111
112
113
114
115
118
121
123
130
131
1147
25.0
484
1424
1628
390
1095
327
95.7
1359
20.9
503
1580
1684
392
1466
365
97.6
523
91.1
128
388
331
61.8
715
61
15.7
73.4
163
7.5
273
75.3
262
396
513
674
106
142
1015
1397
271
531
276
104
162
161
The chiral GC analysis was performed on a Hewlett
Packard 5890 series II plus gas chromatograph equipped
with autosampler, EPC, split/splitless injector, FID
detector and 25 m · 0.25 mm CP-Chirasil-Dex CB chiral
capillary column. The chiral HPLC analysis was per-
formed on a Water prep4000 with UV detector and
(S,S)-Whelk-O1 (4.6 · 25 cm) chiral column from Regis
Technologies, Inc. All the ketoreductases were purified
recombinant enzymes, which were developed by genome
mining and protein engineering, and are commercially
available from BioCatalytics, Inc. a-Chloroketones were
88.6
348
211
116
170
299
104
148
17.2
167
257
94.6
82.7
90.4
a The specific activity was defined as nmol minÀ1 mgÀ1
.

D. Zhu et al. / Tetrahedron: Asymmetry 16 (2005) 3275–3278
3277
at 340 nm (e = 6.22 mMÀ1 cmÀ1) using a SpectraMax
M2 microplate reader (Molecular Devices). The activity
was measured at room temperature in a 96-well plate,
in which each well contained a-chloroketone (6.25
mM), and NADPH (0.25 mM) in a potassium phos-
phate buffer (100 mM, pH 7.0, 180 ll). The reaction
was started by the addition of the ketoreductase (20 ll
solution containing 2.5–100 lg of enzyme). The activ-
ity was defined as the number of nmol of NADPH
converted in one minute by 1 mg of enzyme (nmol
minÀ1 mgÀ1).
Table 2. Enantioselectivity of ketoreductase-catalyzed reduction of
a-chloroketones 7–12a
D-Glucose
D-Gluconic acid
GDH
NADP+
NADPH
KRED
OH
O
OH
Cl
Cl
Cl
+
X
X
X
3.2. Evaluation of the enantioselectivity
R
S
7-12
The enantioselectivity of the enzymatic reduction of
a-chloroketones was studied using an NADPH recycle
system. The general procedure was as follows: D-glucose
(4 mg), D-glucose dehydrogenase (0.5 mg), NADPH
(0.5 mg), ketoreductase (0.5 mg) and a-chloroketone
solution in DMSO (50 ll, 0.25 M) were mixed in a
potassium phosphate buffer (1 ml, 100 mM, pH 7.0)
and the mixture shaken at 25 ꢁC overnight. The mixture
was extracted with methyl tert-butyl ether (1 ml). The
organic extract was dried over anhydrous sodium sulfate
and subjected to chiral GC or HPLC analysis.
KRED
7
8
9
10
>99
>99
24
>99
>99
À10
30
11
12
b
101
107
111
112
113
114
115
118
121
123
130
131
98
98
2
>99
98
98
90
68
>99
98
6
66
À34
À30
48
98
98
–
>99
À24
>99
>99
À36
À24
96
>99
58
>99
>99
54
98
78
>99
>99
62
À42
0
54
98
14
70
90
60
À40
À62
26
88
À42
À62
À34
18
À40
À30
>À99
À96
>À99
>À99
>À99
À96
>À99
>À99
>À99
>À99
À88
>À99
3.3. Preparation of the a-chloroalcohols
a The absolute configuration was determined by comparing the sign of
the specific rotation with the literature data, the ee values were
measured by chiral GC or HPLC analysis, a positive ee means the
major product was (S)-configuration while a negative ee value means
(R)-enantiomer was the major product.
The preparative synthesis was carried out as follows:
D-glucose (400 mg), D-glucose dehydrogenase (5 mg),
NADPH (5 mg) and ketoreductase (5 mg) were mixed
in a potassium phosphate buffer (50 ml, 100 mM,
pH 7.0). To the mixture was added an a-chloroketone
solution (200 mg in 2.5 ml DMSO). The mixture was
stirred at room temperature and the pH controlled at
7.0 with the addition of a 0.5 M NaOH solution until
conversion was complete (usually overnight). The
mixture was extracted with methyl tert-butyl ether.
The organic extract was dried over anhydrous sodium
sulfate and removal of the solvent gave the product
b Conversion was less than 20% after 24 h.
Table 3. Isolated yield and enantiomeric excess of both enantiomers of
a-chloroalcohols in preparative experiments
22
c
a-Chloro alcohols
KRED
Yield (%)a
ee (%)b
½aꢀD
(S)-1
112
130
72
94
69
99
97
81
79
88
87
76
96
89
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
53.8
À53.1
43.5
(R)-1
(S)-2112
(R)-2130
(S)-3
(R)-3
(S)-4
(R)-4
(S)-5
(R)-5
(S)-6
1
alcohol, which was identified by the comparison of H
NMR with the literature data.16–19 The absolute config-
urations of the product alcohols were determined by
comparison of the optical rotation data with those in
the literature.16–19
À43.1
112
130
112
130
107
130
113
130
45.2
À46.0
34.2
À35.1
40.5
À40.4
32.0
À31.8
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