Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH)

Article abstract of DOI:10.1016/j.tetasy.2005.06.036

Alcohol dehydrogenase (LSADH) isolated from Leifsonia sp. S749 was used to produce (R)-chiral alcohols. The enzyme with a broad substrate range reduced various prochiral ketones and keto esters to yield optically active secondary alcohols with a high enantiomeric excess. LSADH transferred the pro-S hydrogen of NADH to the carbonyl moiety of phenyl trifluoromethyl ketone 13 through its re face to give (S)-1-phenyl-2,2,2-trifluoroethanol 40. LSADH was able to efficiently reproduce NADH when 2-propanol was used as a hydrogen donor in the reaction mixture.

Full text of DOI:10.1016/j.tetasy.2005.06.036

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 2539–2549
Production of (R)-chiral alcohols by a
hydrogen-transfer bioreduction with NADH-dependent
Leifsonia alcohol dehydrogenase (LSADH)
Kousuke Inoue, Yoshihide Makino and Nobuya Itoh*
Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan
Received 9 February 2005; revised 9 June 2005; accepted 10 June 2005
Available online 26 July 2005
Abstract—Alcohol dehydrogenase (LSADH) isolated from Leifsonia sp. S749 was used to produce (R)-chiral alcohols. The enzyme
with a broad substrate range reduced various prochiral ketones and keto esters to yield optically active secondary alcohols with a
high enantiomeric excess. LSADH transferred the pro-S hydrogen of NADH to the carbonyl moiety of phenyl trifluoromethyl
ketone 13 through its re face to give (S)-1-phenyl-2,2,2-trifluoroethanol 40. LSADH was able to efficiently reproduce NADH when
2
ꢀ
-propanol was used as a hydrogen donor in the reaction mixture.
2005 Elsevier Ltd. All rights reserved.
4
6,7
8
1
. Introduction
liver, Candida parapsilosis and Pseudomonas sp., and
+
NADP -dependent ADHs from Thermoanaerobium
brochii and Lactobacillus kefir,
9
10,11
In recent years, enantiometrically pure compounds
including amino acids, organic acids, amines, alcohols,
epoxides and so on, have been produced with the use
of biocatalysts, because such methods are superior to
general chemical methods in terms of enantioselectivity.
Among them, secondary alcohols are the most important
chiral synthons for pharmaceuticals and agrochemicals.
aldehyde reductase
1
2
from Sporobolomyces salmonicolor (EC 1.1.1.2), and
carbonyl reductase (EC 1.1.1.184) from Candida magno-
1
3
liae have been used for this purpose. However, in
many cases, they have the disadvantages of a narrow
substrate specificity, insufficient stereoselectivity or low
chemotolerancy to ketone substrates and organic sol-
vents. In addition, regenerating NAD(P)H is necessary
to efficiently continue the bioreduction process. There-
fore, such bioreduction processes are only economical
when the cofactor can be regenerated in situ in a second
catalytic cycle, for example, formate/formate dehydro-
genase (FDH) or glucose/glucose dehydrogenase
1
Chiral metal complexes such as BINAP-Ru have been
successfully used as catalysts in a number of cases of
asymmetric reduction processes, and industrially applied
for the synthesis of (ꢀ)-menthol and other terpenic sub-
2
stances. However, in the case of ketone reductions, dif-
1
4
ficulties remain in the operation, and in obtaining
sufficient enantiomeric excess and productivity.2,3
An alternative to the chemical asymmetric reduction
processes is a biocatalytic transformation system using
(GDH).
From the viewpoint of the regeneration of NAD(P)H,
2-propanol is another suitable hydrogen donor for bio-
reduction because of its chemical properties and low
4
enzymes or microorganisms.
6
–9,15–17
cost.
Recently, Itoh et al. reported that phenyl-
Until now, many examples of the reduction of ketones
with reductases and dehydrogenases have been
described. Oxidoreductases including NAD -depen-
dent alcohol dehydrogenases (ADHs) from yeast, horse
acetaldehyde reductase (PAR) from the styrene-assimi-
lating Rhodococcus (former Corynebacterium) sp. strain
ST-10 is a unique NADH-dependent alcohol dehydroge-
nase (ADH) with a broad substrate range and high
enantioselectivity to give (S)-alcohols from various car-
bonyl compounds without an additional coenzyme
regeneration system, because the enzyme itself is able
4
,5
+
*
Corresponding author. Tel.: +81 766 56 7500x560; fax: +81 766 56
498; e-mail: itoh@pu-toyama.ac.jp
1
7–21
2
to regenerate NADH in the presence of 2-propanol.
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.06.036

2540
K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
Table 1. Analysis of substrates and products of LSADH reaction
Substrate/product Analzying method/
conditions/retention time (min)
Determination method of (R) and
(S)-alcohols/conditions/retention time (min)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
/28
GC/DB-1, inj.: 250 ꢁC, det.: 250 ꢁC, col.: 40 ꢁC HPLC/Chiracel OB-H, hexane/2-propanol (49:1), 0.5 ml/min,
(
5 ꢁC/min) to 100 ꢁC/sub.: 3.9, pro.: 4.2
GC/DB-1, same as above/sub.: 6.3, pro.: 7.0
at 254 nm/, pro. (R)-form: 7.7, pro. (S)-form: 8.3
/29
HPLC/Chiracel OB-H, same above/pro.
(
R)-form: 7.5, pro. (S)-form: 8.0
HPLC/Chiracel OB-H, same above/pro.
R)-form: 7.2, pro. (S)-form: 7.9
/30
GC/DB-1, same as above/sub.: 9.9, pro.: 10.8
—
(
/31
GC/CP-cyclodextrin, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 80 ꢁC/sub.: 3.1, pro. (R)-form: 4.0, pro. (S)-form: 4.2
HPLC/Chiracel OD-H, hexane/2-propanol (49:1),
0.5 ml/min, 220 nm, 30 ꢁC/pro. (R)-form: 8.4, pro. (S)-form: 7.9
HPLC/Chiracel OB-H, hexane/2-propanol/TFA (9:1:0.1),
1.0 ml/min, 254 nm, 30 ꢁC/pro. (R)-form: 9.9, pro. (S)-form: 6.5
HPLC/Chiracel OB-H, same above/pro.
/32
GC/DB-1, inj.: 200 ꢁC, det.: 200 ꢁC,
col.: 160 ꢁC/sub.: 12.1, pro.: 12.9
GC/Thermon1000, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 160 ꢁC/sub.: 6.6, pro.: 11.2
/33
/34
Same as above/sub.: 7.4, pro.: 10.8
(
R)-form: 8.6, pro. (S)-form: 5.6
GC/DB-1, inj.: 200 ꢁC, det.: 200 ꢁC, col.: 70 ꢁC HPLC/Chiracel OB-H, same above/pro.
5 ꢁC/min raise) to 250 ꢁC/sub.: 24.4, pro.: 24.8 (R)-form: 7.3, pro. (S)-form: 5.6
/35
(
/36
GC/Thermon1000, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 70 ꢁC/sub.: 7.6, pro.: 10.1
HPLC/Chiracel OB-H, hexane/2-propanol (9:1),
1.0 ml/min, at 220 nm, 30 ꢁC/pro. (R)-form: 12.6, pro. (S)-form: 11.6
0/37
1/38
2/39
3/40
4/41
5/42
6/43
7/44
8/45
9/46
0/47
1/48
2/49
3/50
4/51
5/52
6/53
7/54
GC/DB-1, inj.: 200 ꢁC, det.: 200 ꢁC, col.: 70 ꢁC HPLC/Chiracel OB-H, same above/pro.
(
5 ꢁC/min raise) to 250 ꢁC/sub.: 11.2, pro.: 12.5 (R)-form: 18.8, pro. (S)-form: 18.2
GC/DB-1, same as above/sub.: 11.1, pro.: 12.3
HPLC/Chiracel OB-H, hexane/2-propanol (9:1),
1
.0 ml/min, 220 nm, 30 ꢁC/pro. (R)-form: 7.4, pro. (S)-form: 7.9
—
—
—
—
—
—
—
—
GC/CP-cyclodextrin, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 120 ꢁC/sub.: 4.3, pro. (R)-form: 6.0, pro. (S)-form: 6.5
GC/CP-cyclodextrin, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 130 ꢁC/sub.: 1.8, pro. (R)-form: 7.3, pro. (S)-form: 6.7
GC/CP-cyclodextrin, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 140 ꢁC/sub.: 5.1, pro. (R)-form: 8.7, pro. (S)-form: 9.1
GC/CP-cyclodextrin, same as above/sub.: 5.5, pro.
(
R)-form: 8.9, pro. (S)-form: 9.3
GC/CP-cyclodextrin, same as above/sub.: 5.5, pro.
R)-form: 8.9, pro. (S)-form: 9.3
GC/CP-cyclodextrin, same as above/sub.: 8.4, pro.
R)-form: 14.1, pro. (S)-form: 14.9
GC/CP-cyclodextrin, same as above/sub.: 8.2, pro.
R)-form: 10.4, pro. (S)-form: 10.0
(
(
(
GC/CP-cyclodextrin, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 160 ꢁC/sub.: 8.5, pro. (R)-form: 13.1, pro. (S)-form: 12.7
GC/DB-1, inj.: 200 ꢁC, det.: 200 ꢁC, col.: 70 ꢁC HPLC/Chiracel OB-H, hexane/2-propanol (9:1),
5 ꢁC/min raise) to 250 ꢁC/sub.: 18.7, pro.: 20.5 0.5 ml/min, 254 nm, 30 ꢁC/pro. (R)-form: 10.5, pro. (S)-form: 7.7
(
GC/DB-1, same as above/sub.: 17.4, pro.: 17.7
HPLC/Chiracel OB-H, hexane/2-propanol (9:1), 1.0 ml/min,
54 nm, 30 ꢁC/pro. (R)-form: 14.6, pro. (S)-form: 10.5
HPLC/Chiracel OB-H, same as above/pro.
R)-form: 13.8, pro. (S)-form: 15.1
2
GC/DB-1, same as above/sub.: 24.8, pro.: 25.2
(
—
—
GC/CP-cyclodextrin, inj.: 250 ꢁC, det.: 250 ꢁC,
col.: 110 ꢁC/sub.: 9.1, pro. (R)-form: 15.0, pro. (S)-form: 15.7
GC/CP-cyclodextrin, same as above/sub.: 15.8, pro.
(
R)-form: 22.9, pro. (S)-form: 23.6
GC/DB-1, inj.: 200 ꢁC, det.: 200 ꢁC, col.: 70 ꢁC HPLC/Chiracel OD-H, hexane/2-propanol (49:1), 1.0 ml/min,
5 ꢁC/min raise) to 250 ꢁC/sub.: 22.4, pro.: 22.7 285 nm, 30 ꢁC/pro. (R)-form: 17.1, pro. (S)-form: 16.0
(
GC/DB-1, same as above/sub.: 26.1, pro.: 26.8
HPLC/Chiracel OD-H, hexane/2-propanol (49:1), 1.0 ml/min,
54 nm, 40 ꢁC/pro. (R)-form: 31.0, pro. (S)-form: 23.6
HPLC/Chiracel OF, hexane/2-propanol (4:1), 0.5 ml/min,
2
GC/DB-1, same as above/sub.: 18.9, pro.: 21.1
2
20 nm, 40 ꢁC/pro. (R)-form: 11.5, pro. (S)-form: 10.3
Therefore, such a system is regarded as a superior asym-
metric hydrogen-transfer reduction process. Recently,
another chemotolerant ADH is reported in a R. ruber
DSM44541 strain. However, PAR can hardly trans-
form phenyl trifluoromethyl ketone (PTK) 13 to (R)-
or (S)-1-phenyl-2,2,2-trifluoroethanol (PTE) 40, which
would be a potential chiral synthon for liquid crystals.
In a previous study, we described the screening of a
microorganism (Leifsonia sp. strain S749), which can
reduce PTK 13 to (S)-PTE 40, the purification of the
corresponding ADH (LSADH: Leifsonia ADH), and
1
6
22
the characterization of the enzyme.
Herein, we report the application of LSADH for the
production of (R)-chiral alcohols from ketones includ-
ing acetophenone derivatives, 2-alkanones and keto

K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
2541
Table 2. Enantioselective reduction of alkanone and keto esters by LSADH
Substrate Relative activity (%)
Product
ee (%)
Conversion (%)
79
Yield (%)
O
OH
1
7
(R) >99
17
31
1
28
O
OH
1
2
4
04
29
88
(R) >99
(R) >99
(R) >99
83
87
2
29
O
OH
38
19
3
30
O
OH
O
O
100
O
O
4
31
O
OH
O
O
O
3
3
(R) >99
(R) 47
100
71
40
31
O
5
O
32
O
OH
O
O
1
O
O
6
33
O
OH
O
3
5
(R) 23
29
20
O
O
O
7
34
O
OH
O
O
(S) 85
95
58
47
O
8
35
O
O
O
OH
3
09
(R) >99
100
O
9
O
36
O
O
O
OH
1
8
64
09
(S) >99
(S) >99
100
100
35
54
Br
Br
O
10
O
37
O
O
O
OH
Cl
Cl
O
11
O
38
esters with a high enantioselectivity, and evaluate
LSADH as an asymmetric hydrogen-transfer biocatalyst
using 2-propanol.
and 2-alkanones, and cycloalkanones. With the LSADH
reactions, acetophenone 12, PTK 13 and 2-heptanone 3
are converted to (R)-1-phenylethanol 39 (99% ee), (S)-
PTE 40 (>99% ee) and (R)-2-heptanol 30 (>99% ee) in
the presence of 2-propanol. Therefore, LSADH was
thought to be a suitable biocatalyst for producing the
2
. Results and discussion
2
2
(
R)-form chiral alcohols. We further explored the sub-
2.1. Substrate spectrum and stereoselectivity of LSADH
for various ketones
strate specificity and stereoselectivity of LSADH for
various ketones. The results are summarized in Tables
2
and 3. It was found that LSADH was able to catalyze
As previously described, LSADH is a novel NADH-
the reduction of not only ketones but also a-keto and b-
keto esters. Until now, LSADH has been found to cat-
alyze the reduction of more than 40 ketones, indicating
a quite broad substrate spectrum. The data concerning
the stereoselectivities for the substrates clarified that
LSADH produced (R)-alcohols 39, 41–44, 47–52 from
2
2
dependent ADH with a broad substrate range. The en-
zyme is highly active toward medium-chain normal alde-
hydes and 2-alkanones between C and C , and various
5
8
aryl aldehydes and acetophenone derivatives, although
it barely acts on short-chain normal alkyl aldehydes

2542
K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
Table 3. Enantioselective reduction of arylketones by LSADH
Substrate
Relative activity (%)
Product
ee (%)
Conversion (%)
82
Yield (%)
40
O
O
O
OH
6
(R) 99
1
2
39
OH
CF3
100
CF
3
(S) >99
(R) >99
(R) >99
(R) >99
(R) >99
(S) >99
100
81
61
58
60
62
79
1
3
40
OH
7
6
0
0
90
Cl
14
Cl
41
O
OH
81
Cl
Cl
1
5
42
O
OH
1
51
95
Br
16
Br
43
O
OH
7
2
7
9
82
Br
17
Br
4
4
O
O
OH
Cl
Cl
Cl
Cl
100
1
8
45
OH
6
7
2
1
4
(S) >99
100
91
Cl
19
Cl
46
47
O
OH
(R) 79
12
8
Cl
Cl
Cl
OH
Cl
20
O
5
2
(R) 99
72
64
48
MeO
MeO
21
MeO
48
MeO
O
OH
(R) >99
88
MeO
22
MeO
4
9

K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
2543
Table 3 (continued)
Substrate
Relative activity (%)
Product
ee (%)
Conversion (%)
Yield (%)
O
OH
3
6
(
R) 99
8
2
O
3
50
OH
3
53
(R) 98
92
95
40
47
2
4
51
O
O
O
O
O
OH
8
6
(R) >99
(S) >99
62
29
2
5
52
O
OH
Cl
Cl
O
O
a
O
O
4
5
2
6
53
O
O
N
O
N
O
4
(R) >99
35
24
O
27
OH
5
4
a
The reductive activity for the substrate was determined by measuring the amount of produced alcohol by GC, because the substrate indicated strong
absorption at 340 nm and hindered the spectroscopic measurement of NADH formation.
O
OH
Reductive reaction
CF
3
CF
3
LSADH
Phenyl trifluoromethyl ketone
PTK) 13
(S)-1-Phenyl-2,2,2-tirfluoroethanol
((S)-PTE) 40
(
NAD+ [Conversion:99% (99mg/ml),
NADH
e.e.:>99% ]
O
OH
LSADH
Acetone
2-Propanol
Oxidative reaction
Figure 1. Principle of the LSADH-catalyzed reduction of ketones in combination with hydrogen-transfer via NADH from 2-propanol.
acetophenone or arylketone derivatives 12, 14–17, 20–25
and (R)-alkanols 28–30 from 2-alkanones 1–3, and (S)-
halohydrins 37, 38, 45, 46, 53 from 4-halo-3-oxobutano-
ate derivatives 10, 11, 2-chloroacetophenone (phenacyl
chloride) derivatives 18, 19 and chloromethyl 3,4-methyl-
enedioxyphenyl ketone 26 with an enantiomeric excess
of more than 98%, except from 2,4-dicholoroacetophe-
none 20 (R-form, 79% ee), methyl benzoylformate 6
6, 7 ethyl 2-oxo-4-phenylbutanoate 8 and 2,4-dicholoro-
acetophenone 20 gave low enantiomeric excesses for the
alcohols produced.
Although the reaction system was not optimized for each
substrate, conversions of 19 ketones out of a total of 27
tested reached more than 80%, and low conversion was
only limited to the substrates to which LSADH showed
a weak reactivity (less than 4% compared with PTK)
such as methyl or ethyl benzoylformate 6, 7 and 2,4-
dicholoroacetophenone 20, propiophenone 23 and 1-N-
Boc-3-pyrrolidinone 27 (Tables 2 and 3). The results
clearly indicated that the LSADH can function as a bio-
catalyst to transfer hydrogen from 2-propanol to various
[
(R)-form, 47% ee], ethyl benzoylformate 7 (R-form,
2
8
3% ee) and ethyl 2-oxo-4-phenylbutanoate 8 [(S)-form,
5% ee]. This suggested that the stereoselectivity of
2
3
LSADH strictly follows the anti-PrelogÕs rule. Interest-
ingly, it was observed that substrates with weak reactiv-
ity for LSADH such as methyl or ethyl benzoylformate

2544
K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
ketones through a smooth NADH-regeneration to give
enantiomerically pure (R)-alcohols with a practical level.
(a)
6
5
4
3
00
00
00
00
2
.2. Stereochemistry of LSADH
In order to elucidate the stereochemistry with respect to
NADH, the LSADH-catalyzing transfer of deutride (D)
or hydride from the D-labelled NADH at the pro-R po-
sition (NADD) was determined. NADD was obtained
with the reaction of yeast ADH and CH CD OH as
200
1
00
0
0
2.5
5
10 15
20
25
30
2-Propanol conc. (% (v/v))
3
2
1
described in the Experimental. H NMR and GC–MS
analyses of the PTE produced by LSADH with the
(b)
600
NADD gave the same profiles as those of authentic
5
4
00
00
1
PTE 40; H NMR (400 MHz, CDCl ) d (ppm) 4.99–
3
5
.04 (1H, m), 7.36–7.56 (5H, m, ArH), GC–EI-MS
+
300
00
m/z (M 176 for C H OF ) 137, 127, 107, 79, 51. These
8
7
3
2
results indicated that the C-2 hydrogen of PTE was not
replaced with D through LSADH reduction, in other
words, the pro-S hydrogen of NADD is transferred to
the re face of the carbonyl of PTK 13 (si face in the case
of acetophenone 12), as illustrated in Figure 2. The
result together with those of the section above indicated
that the stereochemistry of LSADH was the same as
that of Mucor javanicus ADH, and different from that
of the (R)-alcohol-producing ADH from Pseudomonas
100
0
0
0.2
0.4
0.6
0.8
1
2
+
NAD conc. (mM)
(c)
6
5
00
00
400
300
4
or Lactobacillus.
2
1
00
00
0
2.3. Optimized conditions for LSADH-catalyzed
production of (S)-PTE 40
4
5
6
7
8
9
10 11
To improve the LSADH reduction system using 2-pro-
panol as a hydrogen donor, reaction conditions were
optimized using PTK 13 as a representative substrate
pH
Figure 3. Optimization of (S)-PTE 40 production by LSADH. (a)
Effect of 2-propanol concentration on (S)-PTE 40 production. The
reaction mixture consisted of 10% (w/v) PTK 13, 1 lmol NAD ,
(
100 mg/ml: 574 mM, suspended in the reaction mix-
+
ture). With regard to the concentration of 2-propanol
in the reaction mixture, the optimal concentration was
between 5% (v/v) (0.65 M) and 10% (1.3 M), and pro-
duction of (S)-PTE 40 was inhibited at above 15% 2-
propanol concentration (Fig. 3a). This suggested that
more than 15% (v/v) 2-propanol in the reaction mixture
1
00 lmol KPB (pH 7.0), 0–30% (v/v) 2-propanol and 1 unit of
+
LSADH in a total volume of 1 ml. (b) Effect of NAD concentration
in the reaction mixture. The reaction mixture consisted of 10% (w/v)
PTK, 0–2.0 lmol NAD , 100 lmol KPB (pH 7.0), 5% (v/v) 2-
propanol and 1 unit of LSADH in a total volume of 1 ml. (c) Effect
of pH of the reaction mixture. The reaction mixture consisted of 10%
+
+
(
w/v) PTK, 1 lmol NAD , 100 lmol buffer [citrate–K
2
HPO
4
(pH 4.0
and 5.0), KPB (pH 6.0 and 7.0), Tris–HCl (pH 8.0 and 9.0) and
glycine–NaOH (pH 10.0 and 11.0)], 5% (v/v) 2-propanol and 1 unit of
LSADH in a total volume of 1 ml. The amount of (S)-PTE produced
after 24 h was drawn as a grey bar, and PTK remaining as a white bar
in all cases.
probably inhibited the LSADH reaction or denatured
the LSADH.
+
The optimal NAD concentration in the reaction mix-
ture was measured. As shown in Figure 3b, a sufficient
+
conversion was observed at an initial NAD concentra-
tion of more than 0.8 mM. Considering the K value of
m
+
22
NAD for 2-propanol oxidation (0.12 mM),
this
observation seemed reasonable. The effect of pH on
the reaction was also measured. As shown in Figure
3
c, the optimal pH was between 7 and 8.
2
.4. Time course of the production of (S)-PTE 40
Figure 2. Stereochemistry of the LSADH reaction compared with the
previously reported ADHs. In the case of PTK 13, this side is
designated as the re face.
Under the optimized conditions (10% 2-propanol, 1 mM
NAD and pH 7.0), the time course of (S)-PTE 40
+

K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
2545
1
1
1
400
200
000
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
LSADH gene, feeding of 2-propanol, elimination of
the acetone produced under reduced pressure, and adop-
tion of the solvent–water two-phase system and so on.
8
00
00
00
00
0
3. Conclusion
6
4
2
LSADH catalyzed the enantioselective reduction of
ketones with high conversion and enantiomeric
purity to give (R)-form alcohols. The stereochemistry
of LSADH has been found to use the pro-S hydrogen
of NADH. This bioreduction process would be industri-
ally applicable to the production of enantiomerically
pure alcohols because it can effectively regenerate
NADH by itself by transferring hydrogen from 2-propa-
nol to reduce the aimed ketones.
0
5
10
15
20
25
30
35
40
Reaction time (hr)
Figure 4. Time course of the production of (S)-PTE 40 and hydrogen
donor (2-propanol) concentration. The reaction mixture consisted of
0% (w/v) PTK 13, 1 lmol NAD , 100 lmol phosphate buffer (KPB)
+
1
(pH 7.0), 10% (v/v) 2-propanol and 1 unit of LSADH in a total volume
of 1 ml. Symbols: s, PTK remaining [initial concn of 10% (w/v)
4. Experimental
(
(
575 mM)]; d, (S)-PTE produced; m, 2-propanol [initial concn of 10%
v/v) (1306 mM)]; n, acetone; j, NAD ; h, NADH.
+
4.1. Preparation of LSADH and enzyme assay
LSADH was obtained from Leifsonia sp. S749 cells as
2
2
production was tested with a high concentration of PTK
3 (100 mg/ml: 574 mM) (Fig. 4). During the reaction,
reported previously.
A partially purified enzyme
1
preparation produced by ammonium sulfate precipita-
tion and sequential Butyl-Toyopearl and Bioassist Q
column chromatographies was used throughout the
experiments.
+
part of the NAD was rapidly reduced into NADH,
+
after which NAD and NADH concentrations were
maintained at 0.94–0.98 mM and 0.03–0.09 mM, respec-
+
tively. The concentration of NAD was eight times
+
higher than the K value for NAD (0.12 mM), whereas
LSADH activity was assayed at 25 ꢁC by measuring the
decrease in absorption at 340 nm of NADH as described
previously. One unit of enzyme was defined as the
amount that converted 1 lmol NADH and PTK 13 in
1 min. The substrate specificity of LSADH was mea-
sured spectrophotometrically by decreasing the absorp-
tion of NADH at 340 nm using the purified enzyme
and various ketones at 2 mM, unless otherwise indi-
cated, and their relative activity was compared with
PTK 13 as 100%.
m
that of NADH was almost equal to the K value for
m
2
2
22
NADH (0.048 mM). A similar phenomenon was re-
ported in the bioreduction of 2,3 -dichloroacetophenone
9 by PAR with 2-propanol. The result including the
0
1
7
1
case of PAR suggested that the reaction rate was kinet-
ically controlled by the enzyme itself in both cases.
Throughout the reaction, an almost equimolar produc-
tion of (S)-PTE 40 and acetone with a decrease in
PTK 13 and 2-propanol was observed in the range of
measurement error. The observed reaction rate constant
(
kcat) for the reduction of PTK 13 calculated from the
4.2. LSADH-catalyzed reduction coupled to NADH
regeneration with 2-propanol
ꢀ
1
initial velocity in Figure 4 was approximately 30 s
,
therefore, the overall reaction rate constant including
the oxidation of 2-propanol should double this
For determining the enantiomeric purity of the products
and assessing the coupling of NADH regeneration, we
constructed the LSADH and 2-propanol system (Fig.
1). The reaction mixture consisted of 0.5 mmol KPB
ꢀ1
(
60 s ). The value was comparable with that for PTK
ꢀ
1
ꢀ1
reduction (140 s ) and 2-propanol oxidation (47 s
)
2
2
reported for LSADH. Although, the 2-propanol used
for NADH regeneration seemed to compete for the ac-
tive site of the enzyme with the ketone substrate and
lower the turnover for the desired reaction, LSADH-cat-
alyzed hydrogen-transfer reduction proceeded efficiently
from the viewpoint of the regeneration and utilization of
NADH. After 35 h, (S)-PTE 40 was obtained at 79%
conversion (ca. 450 mM in the reaction mixture:
+
(pH 7.0), 50 mg of each substrate 1–27, 5 lmol NAD ,
5% (v/v) 2-propanol and 5 unit of purified LSADH in
a total volume of 5 ml, and was suspended in a 15-ml
polypropylene centrifuge tube with shaking (300 rpm)
for 24 h at 25 ꢁC. After the reaction, the mixture was
extracted twice with 7.5 ml of ethyl acetate. The com-
bined ethyl acetate extracts were dried over Na SO ,
2
4
7
9 mg/ml) with >99% ee When the amount of enzyme
added to the reaction mixture was increased by twice,
S)-PTE 40 was obtained at 99% conversion (ca.
70 mM in the reaction mixture: 99 mg/ml). The high
purified by silica gel (1.5 · 30 cm) chromatography
using n-hexane and ethyl acetate (3:1) as an eluent.
The fractions were condensed by evaporation, and ana-
(
5
lyzed by GC or HPLC. The purified product was also
1
accumulation of the product with a near perfect enantio-
meric purity showed the potential of the LSADH biore-
duction system for practical usage. The productivity
could be improved further by using a larger amount of
enzyme or recombinant E. coli cells expressing the
analyzed by H NMR (400 MHz, CDCl ) with tetra-
3
methylsilane (TMS) as a reference, and GC-electron ion-
ization (EI)-mass spectrometry (MS) (GC–EI-MS)
(QP-5000 GC–MS, Shimadzu): column DB-1
(0.25 mm · 60 m, J&W Scientific, CA, USA); flow rate

2546
K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
at a linear velocity of 35 cm/min of He; the split ratio of
4.4. Optimization of production of (S)-PTE 40
5
0; injection and detection temperature of 250 ꢁC; col-
umn temperature 40 ꢁC for 5 min then raised to 250 ꢁC
at 10 ꢁC/min and held at 250 ꢁC for 5 min.
In order to determine the 2-propanol and acetone con-
centrations in the reaction mixture, GC was performed
using a Shimadzu GC-14 A system equipped with a
coiled column (3 mm · 2 m) packed with Thermon
1000 (5% on Chromosorb W) with an FID, column tem-
perature of 30 ꢁC, injection temperature of 100 ꢁC,
detection temperature of 150 ꢁC and a flow rate of
General GC was performed using a Shimadzu GC-18A
system (Kyoto, Japan) equipped with a capillary column
(
DB-1, 0.25 mm · 30 m, J&W Scientific, CA, USA) with
an FID (flame ionization detector) with a flow rate of
ml/min of He and the split ratio of 60 under the con-
1
50 ml/min of N ; retention times (min): 2-propanol,
2
+
ditions described in Table 1. GC was also performed
using a Shimadzu GC-14 A system equipped with a
coiled column (3 mm · 2 m) packed with Thermon
4.4 min; acetone, 2.3 min. The NAD and NADH con-
centrations in the reaction mixture were also determined
1
7
by HPLC as previously described. The reaction mix-
tureÕs composition is mentioned in the legend of Figure
3, and the reaction was performed in a 2-ml polypropyl-
ene tube in a BioShakar MBR-022 (Taitec, Saitama,
Japan) with shaking (700 rpm) for 24 h at 25 ꢁC.
1
000 (5% on Chromosorb W) with an FID and a flow
rate of 50 ml/min of N . The conversions were deter-
2
mined on the basis of the peak areas of ketone substrates
and alcohol products from the GC.
To determine the absolute configuration and conver-
4.5. Chemicals
sions of some alcohols, the products 31, 39–46, 50 and
5
1, extracted with ethyl acetate from the reaction mix-
Ethyl 4-phenyl-2-oxobutanoate 8, methyl 4-bromo-3-
oxobutanoate 10, 2,3 -dichloroacetophenone 19, 2 ,4 -
0
0
0
ture, were analyzed using GC (HP 6890 GC system,
Hewlett Packard, USA) with a CP-cyclodextrin-b-236-
N19 chiral column (0.25 mm by 25 m, 0.25 lm film,
Chrompack, The Netherlands) and a FID. Helium gas
was used as a carrier at 15 psi (0.5 ml/min), and the split
ratio was 50. Details of the measuring conditions are
summarized in Table 1.
0
dichloroacetophenone 20, 3 -methoxyacetophenone 21,
0
0
3 ,4 -methoxyphenylacetone 22, piperonylacetone 25,
chloromethyl 3,4-methylenedioxyphenyl ketone 26,
1-N-Boc-3-pyrrolidinone 27, ethyl (RS)-4-phenyl-2-
hydroxybutanoate 35, methyl (RS)-4-bromo-3-hydroxy-
butanoate 37, (RS)-1-phenyl-2,2,2-trifluoroethanol
(PTE) 40, (RS)-2-chloro-1-(3-chlorophenyl)ethanol 46,
In order to determine the absolute configuration of the
(RS)-1-(2,4-dichlorophenyl)ethanol 47, (RS)-1-(3-meth-
oxyphenyl)ethanol 48, (RS)-1-(3,4-dimethoxyphenyl)-2-
propanol 49, (RS)-1-piperonyl-2-propanol 52, (RS)-1-
(3,4-methylenedioxyphenyl)-2-chloroethanol 53 and
(R)- and (RS)-1-N-Boc-3-pyrrolidinol 54 were kindly
supplied by Sumitomo Chemical Co. Ltd, Osaka, Japan.
2-Pentanone 1, 2-hexanone 2, 2-heptanone 3, ethyl 3-
oxobutanate 9, acetophenone 12, phenyl trifluoromethyl
2
-alkanols 28–30, they were converted into benzoyl
derivatives with benzoyl chloride and analyzed by
HPLC with a chiral column, as previously reported.19
Analytical HPLC was performed with a Shimadzu LC-
1
2
0AT system equipped with a Chiralcel OB-H (4.6 ·
50 mm, Daicel Chemical Industries, Tokyo, Japan).
The mobile phase was hexane/2-propanol (49:1) and
delivered at a flow rate of 0.5 ml/min (Table 1).
0
0
ketone (PTK) 13, 3 -chloroacetophenone 14, 4 -chloro-
0
0
acetophenone 15, 3 -bromoacetophenone 16, 4 -bromo-
acetophenone 17, propiophenone 23, benzylacetone 24,
(RS)-2-pentanol 28, (RS)-2-hexanol 29, (RS)-2-heptanol
30, ethyl DL-lactate 31, methyl (RS)-mandelate, methyl
(S)-mandelate 33, ethyl (RS)-mandelate, ethyl (S)-man-
delate 34, (RS)-1-phenylethanol 39, (RS)-1-(3-chloro-
phenyl)ethanol 41, (RS)-1-(4-chlorophenyl)ethanol 42,
(RS)-1-(3-bromophenyl)ethanol 43, (RS)-1-(4-bromo-
phenyl)ethanol 44, (RS)-1-phenyl-1-propanol 50 and
For determining the absolute configuration of the alco-
hols 32–38, 47–49 and 52–54, they were purified by silica
gel chromatography as described above, and analyzed
by HPLC with a chiral column. Analytical HPLC was
performed with a Shimadzu LC-10AT system using a
Chiralcel OB-H, OD-H or OF (4.6 · 250 mm, Daicel
Chemical Industries) under the conditions described in
Table 1.
(
RS)-4-phenyl-2-butanol 51 were purchased from Tokyo
4
.3. Stereochemistry of LSADH
Kasei Kogyo, Japan. Ethyl 3-methyl-2-oxobutyrate 5,
methyl benzoylformate 6, ethyl benzoylformate 7 and
other chiral alcohol standards including (R)-2-pentanol
28, (R)-2-hexanol 29, (R)-2-heptanol 30, ethyl (R)-lac-
tate 31, methyl (R)-mandelate 33, ethyl (R)-mandelate
34, ethyl (R)- 36 and (S)-3-hydroxybutanoates, ethyl
(R)- and (S)-4-chloro-3-hydroxybutanoates 38, (R)-1-
phenylethanol 39, (S)-PTE 40, (S)-2-chloro-1-phenyleth-
anol 45, (R)-1-phenyl-1-propanol 50 and (R)-4-phenyl-
2-butanol 51 were obtained from Sigma–Aldrich Chem-
icals, USA. Ethyl pyruvate 4 was purchased from Wako
Pure Chemicals, Japan, 2-chloroacetophenone (phen-
acyl chloride) 18, (RS)-2-chloro-1-phenylethanol 45
from Lancaster Synthesis UK, and ethyl 4-chloro-3-oxo-
butanoate 11 from Merck, Germany. Ethyl 2-hydroxy-
In order to determine the stereochemistry of LSADH,
deuterium (D)-containing NADH (NADD) at the pro-
R position in the nicotinamide moiety was synthesized
+
enzymatically from NAD and CH CD OH using yeast
3
2
1
9
ADH as previously reported. PTK 13 was reduced by
the purified LSADH using this cofactor as follows; the
reaction mixture consisted of 7 lmol PTK 13, 7 lmol
NADD, 5 units of LSADH and 0.1 mmol potassium
phosphate buffer (pH 6.0) in a total volume of 10 ml.
The reaction proceeded at 25 ꢁC for 48 h. The PTE 40
produced was purified by extraction with ethyl acetate
1
from the mixture and analyzed by H NMR and GC–
EI-MS.

K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
2547
1
3
-methylbutanoate 32 was synthesized from 5 by reduc-
Ethyl (R)-mandelate 34 (10 mg); H NMR (CDCl ) d
3
tion with sodium tetrahydroborate and confirmed by
GC–MS and H NMR as described above. All other
chemicals used herein were of analytical grade and were
available commercially.
(ppm) 1.23 (t, 3H, CH , 7.1 Hz), 3.46 (d, 1H, OH,
J = 5.6 Hz), 4.17 (dq, 1H, CH, J(d) = 10.7 Hz,
J(q) = 7.2 Hz), 4.27 (dq, 1H, CH, J(d) = 10.8 Hz,
3
1
J(q) = 7.1 Hz), 5.16 (d, 1H, CH, J = 5.4 Hz), 7.26–7.44
+
(
m, 5H, Ph), GC–EI-MS m/z (M 180 for C H O )
1
0
12
3
4
.6. Instrumental analyses of the purified products
163, 150, 135, 118, 107, 89, 79, 63, 51. The absolute con-
figuration was confirmed with the authentic standard.
The product after the reaction described in Section 4.2
was purified by silica gel chromatography. The products
obtained were analyzed by a H NMR and GC–EI-MS.
When the authentic chiral compound was available, our
products were compared with the authentic ones on GC
or HPLC equipped with a chiral column as described in
Table 1, and its absolute configuration was confirmed.
Optical rotation was measured with a JASCO P-1030
polarimeter.
Ethyl (S)-4-phenyl-2-hydroxybutanoate 35 (29 mg);
1
21
25
25
½aꢁ ¼ þ14.3 (c 0.5, CH Cl) {lit., ½aꢁ ¼ þ21.3 (c 1,
D
3
D
1
CH Cl)}, H NMR (CDCl ) d (ppm) 1.29 (t, 3H, CH ,
3 3 3
J = 7.1 Hz), 1.90–2.00 (m, 1H, CH ), 2.08–2.17 (m,
2
1H, CH ), 2.70–2.83 (m, 2H, CH ), 4.18 (dd, 1H, CH,
2
2
J = 3.9 Hz), 4.21 (q, 2H, CH , J = 7.2 Hz), 7.17–7.31
2
+
(m, 5H, Ph), GC–EI-MS m/z (M 208 for C H O )
1
2
16
3
190, 162, 144, 134, 117, 104, 91, 76, 65.
1
1
(
0
R)-2-Pentanol 28 (8.5 mg); H NMR (CDCl ) d (ppm)
Ethyl (R)-3-hydroxybutanoate 36 (23.5 mg); H NMR
(CDCl ) d (ppm) 1.23 (d, 3H, CH , J = 6.4 Hz), 1.28
3
.93 (t, 3H, CH , J = 7.1 Hz), 1.19 (d, 3H, CH ,
3
3
3
3
J = 6.4 Hz), 1.30–1.50 (m, 5H, CH , CH , OH), 3.76–
(t, 3H, CH3, J = 7.2 Hz), 2.42 (dd, 1H, CH,
J(d) = 8.7 Hz, J(d) = 16.5 Hz), 2.49 (dd, 1H, CH,
J(d) = 3.6 Hz, J(d) = 16.5 Hz), 3.05 (d, 1H, OH,
2
2
+
3
.87 (m, 1H, CH), GC–EI-MS m/z (M 87 for
+
C H O, M ꢀH 86) 69, 57, 45. The absolute configura-
5
12
tion was confirmed with the authentic standard.
J = 2.6 Hz), 4.18 (q, 2H, CH , J = 7.2 Hz), 4.12–4.23
2
+
(
m, 1H, CH), GC–EI-MS m/z (M 132 for C H OF ,
8
7
3
1
+
(
R)-2-Hexanol 29 (15.5 mg); H NMR (CDCl ) d (ppm)
M ꢀH 131) 117, 103, 87, 71, 60, 43. The absolute con-
3
0
.91 (t, 3H, CH , J = 7.1 Hz), 1.19 (d, 3H, CH ,
figuration was confirmed with the authentic standard.
3
3
J = 6.1 Hz), 1.25–1.52 (m, 7H, CH , CH , CH , OH),
2
2
2
+
3
.76–3.83 (m, 1H, CH), GC–EI-MS m/z (M 102 for
Methyl (S)-4-bromo-3-hydroxybutanoate 37 (17.5 mg);
+
22
D
26
25
D
C H O, M ꢀH 101) 87, 84, 69, 56, 45, 41. The absolute
½aꢁ ¼ ꢀ25.9 (c 0.5, CH Cl) {lit. ½aꢁ ¼ ꢀ16.2 (c 8,
6
14
3
1
configuration was confirmed with the authentic
standard.
CH Cl)}, H NMR (CDCl ) d (ppm) 2.65 (dd, 1H,
3 3
CH, J(d) = 7.5 Hz, J(d) = 16.5 Hz), 2.70 (dd, 1H, CH,
J(d) = 4.9 Hz, J(d) = 16.6 Hz), 3.09 (s, 1H, OH), 3.48
(dd, 1H, CH, J(d) = 5.6 Hz, J(d) = 10.5 Hz), 3.52 (dd,
1H, CH, J(d) = 5.1 Hz, J(d) = 10.5 Hz), 3.74 (s, 3H+,
1
(
0
R)-2-Heptanol 30 (19 mg); H NMR (CDCl ) d (ppm)
3
.89 (t, 3H, CH , J = 6.9 Hz), 1.19 (d, 3H, CH ,
3
3
J = 6.3 Hz), 1.26–1.51 (m, 9H, CH , CH , CH , CH ,
CH ), 4.22–4.28 (m, 1H, CH), GC–EI-MS m/z (M
2
2
2
2
3
+
OH), 3.75–3.84 (m, 1H, CH), GC–EI-MS m/z (M ꢀH
198, 196 for C H O Br) 167, 165, 125, 123, 103, 71,
5
9
3
1
15 for C H O) 101, 98, 83, 70, 55, 45. The absolute
61, 43.
7
16
configuration was confirmed with the authentic
standard.
1
Ethyl (S)-4-chloro-3-hydroxybutanoate 38 (27 mg); H
NMR (CDCl ) d (ppm) 1.29 (t, 3H, CH , J = 7.2 Hz),
3
3
1
Ethyl (R)-lactate 31 (9.5 mg); H NMR (CDCl ) d (ppm)
2.61 (dd, 1H, CH, J(d) = 7.6 Hz, J(d) = 16.6 Hz), 2.66
(dd, 1H, CH, J(d) = 4.8 Hz, J(d) = 16.5 Hz), 3.15
(d, 1H, OH, J(d) = 2.9 Hz), 3.60 (dd, 1H, CH,
J(d) = 5.6 Hz, J(d) = 11.2 Hz), 3.63 (dd, 1H, CH,
J(d) = 5.2 Hz, J(d) = 11.3 Hz), 4.19 (q, 1H, CH2,
J = 7.1 Hz), 4.22–4.30 (m, 1H, CH), GC–EI-MS m/z
3
1
.31 (t, 3H, CH , J = 7.2 Hz), 1.42 (d, 3H, CH ,
3
3
J = 7.1 Hz), 2.82 (s, 1H, OH), 4.25 (q, 2H, CH2,
J = 7.1Hz), 4.20–4.29 (m, 1H, CH), GC–EI-MS m/z
+
(
M 118 for C H O ) 103, 89, 75, 58, 45. The absolute
5
10
3
configuration was confirmed with the authentic
standard.
+
(M 168, 166 for C H O Cl) 141, 139, 123, 121, 117,
6
11
3
85, 71, 43. The absolute configuration was confirmed
Ethyl (R)-2-hydroxy-3-methylbutanoate 32 (20 mg);
with the authentic standard.
21
24
22
½
aꢁ ¼ ꢀ10.5 (c 0.5, CH Cl) {lit., ½aꢁ ¼ ꢀ7.9 (c 1,
D
3
D
1
1
acetic acid)}, H NMR (CDCl ) d (ppm) 0.84 (d, 3H,
(R)-1-Phenylethanol 39 (20 mg); H NMR (CDCl ) d
3
3
CH , J = 6.8 Hz), 1.03 (d, 3H, CH , J = 6.9 Hz), 1.31
(ppm) 1.50 (d, 3H, CH , J = 6.3 Hz), 1.88 (s, 1H,
3
3
3
(
(
t, 3H, CH , J = 7.2 Hz), 2.03–2.14 (m, 1H, CH), 2.72
OH), 4.90 (q, 1H, CH, J = 6.5 Hz), 7.25–7.39 (m, 5H,
3
+
s, 1H, OH), 4.03 (d, 1H, CH, J = 3.5Hz), GC–EI-MS
Ph), GC–EI-MS m/z (M 122 for C H O) 107, 91,
8
10
+
m/z (M 146 for C H O ) 128, 117, 104, 73, 55, 43.
79, 63, 51, 43. The absolute configuration was confirmed
with the authentic standard.
7
14
3
1
Methyl (R)-mandelate 33 (15.5 mg); H NMR (CDCl )
3
d (ppm) 3.44 (d, 1H, OH, J = 4.6 Hz), 3.77 (s, 3H,
(S)-1-Phenyl-2,2,2-trifluoroethanol (PTE) 40 (40.5 mg);
21
27
24
CH ), 5.18 (d, 1H, CH, J = 3.9 Hz), 7.26–7.46 (m, 5H,
½aꢁ ¼ þ23.5 (c 0.5, CH Cl), {lit. ½aꢁ ¼ þ25.1 (c
3
D
3
D
+
1
Ph), GC–EI-MS m/z (M 116 for C H O ) 150, 132,
0.81, CCl )}, H NMR (CDCl ) d (ppm) 2.81 (d, 1H,
9
10
3
4 3
1
18, 107, 79, 63, 51. The absolute configuration was con-
OH, J = 3.2 Hz), 5.03 (dq, 1H, CH, J(d) = 3.2 Hz,
firmed with the authentic standard.
J(q) = 6.6 Hz), 7.40–7.50 (m, 5H, Ph), GC–EI-MS m/z

2548
K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
+
22
D
(
M 176 for C H OF ) 137, 127, 107, 79, 51. The abso-
(R)-1-(3-Methoxyphenyl)ethanol 48 (32 mg); ½aꢁ
¼
8
7
3
3
4
28
D
lute configuration was confirmed with the authentic
standard.
þ44.4 (c 0.5, CH Cl) {lit.
½aꢁ ¼ þ39.0 (c 1.0,
3
1
CH Cl)}, H NMR (CDCl ) d (ppm) 1.49 (d, 3H,
3
3
CH , J = 6.6 Hz), 1.85 (s, 1H, OH), 3.82 (s, 3H, CH ),
3
3
2
D
1
(
R)-1-(3-Chlorophenyl)ethanol 41 (30.5 mg); ½aꢁ
¼
4.88 (dq, 1H, CH, J(d) = 1.8 Hz, J(q) = 6.1 Hz), 6.80–
2
8
21
D
+
þ42.7 (c 0.5, CH Cl) {lit. ½aꢁ ¼ þ44.0 (c 0.22,
7.29 (m, 4H, Ph), GC–EI-MS m/z (M 152 for
3
1
17
CH Cl)}, H NMR (CDCl ) d (ppm) 1.48 (d, 3H,
C H O ) 137, 121, 109, 94, 77, 65, 51, 43.
3
3
9
12
2
CH , J = 6.5 Hz), 1.93 (s, 1H, OH), 4.88 (q, 1H, CH,
3
J = 6.4 Hz), 7.22–7.38 (m, 4H, Ph) GC–EI-MS m/z
(R)-1-(3,4-Dimethoxyphenyl)-2-propanol 2549 (32 mg);
22
35
+
(
M
158, 156 for C H OCl) 143, 141, 121, 115, 113,
½aꢁ ¼ ꢀ21.7 (c 0.4, CH Cl) {lit. ½aꢁ ¼ ꢀ29.7 (c
3 3
8
9
D
3
D
1
9
1, 77, 51, 43.
1.01, CH Cl)}, H NMR (CDCl ) d (ppm) 1.25 (d,
3
H, CH , J = 6.1 Hz), 1.59 (s, 1H, OH), 2.61 (dd, 1H,
3
2
D
1
(
(
(
1
R)-1-(4-Chlorophenyl)ethanol 42 (29 mg); ½aꢁ ¼ þ53.9
CH, J(d) = 8.2 Hz, J(d) = 13.6 Hz), 2.75 (dd, 1H, CH,
J(d) = 4.6 Hz, J(d) = 13.6 Hz), 3.87 (s, 3H, CH ), 3.88
(s, 3H, CH ), 3.95–4.06 (m, 1H, CH), GC–EI-MS m/z
(M 196 for C H O ) 178, 163, 151, 137, 121, 107,
2
9
24
D
.
8
1
c 0.5, CH Cl) {lit. ½aꢁ
¼ þ44.9 (EtOH)}, H NMR
3
3
CDCl ) d (ppm) 1.47 (d, 3H, CH , J = 6.6 Hz), 1.89 (s,
3
3
3
+
H, OH), 4.88 (q, 1H, CH, J = 6.4 Hz),
1
1
16
3
+
7
.29–7.34 (m, 4H, Ph), GC–EI-MS m/z (M 158, 156
91, 77, 65, 44.
for C H OCl) 143, 141, 121, 115, 113, 103, 91, 77, 51, 43.
8
9
1
(
R)-1-Phenyl-1-propanol 50 (3 mg); H NMR (CDCl ) d
3
2
D
1
(
(
R)-1-(3-Bromophenyl)ethanol 43 (30 mg); ½aꢁ ¼ þ43.3
(ppm) 0.92 (t, 3H, CH , J = 7.4 Hz), 1.55 (s, 1H, OH),
1.71–1.89 (m, 2H, CH2), 4.61 (dt, 1H, CH,
J(d) = 2.8 Hz, J(t) = 6.6 Hz), 7.26–7.36 (m, 5H, Ph),
GC–EI-MS m/z (M 136 for C H O) 117, 107, 91,
79, 63, 51. The absolute configuration was confirmed
with the authentic standard.
3
3
0
25
D
1
c 0.5, CH Cl) {lit. ½aꢁ ¼ þ34.7 (c 1.35, CH Cl)}, H
3
3
NMR (CDCl ) d (ppm) 1.48 (d, 3H, CH , J = 6.6 Hz),
3
3
+
1
7
.87 (s, 1H, OH), 4.87 (q, 1H, CH, J = 6.4 Hz), 7.18–
.54 (m, 4H, Ph), GC–EI-MS m/z (M 202, 200 for
9
12
+
C H OBr) 187, 185, 121, 105, 103, 92, 77, 63, 51,
8
9
4
3.
1
(
R)-4-Phenyl-2-butanol 51 (23.5 mg); H NMR (CDCl )
3
2
D
1
(
(
R)-1-(4-Bromophenyl)ethanol 44 (31 mg); ½aꢁ þ 41.8
d (ppm) 1.23 (d, 3H, CH , J = 6.1 Hz), 1.43 (s, 1H, OH),
1.70–1.84 (m, 2H, CH ), 2.63–2.80 (m, 2H, CH ),
3.83 (tq, 2H, CH , J(t) = 6.1 Hz, J(q) = 6.1 Hz), 7.16–
7.30 (m, 5H, Ph), GC–EI-MS m/z (M 150 for
3
3
1
25
D
1
c 0.5, CH Cl) {lit. ½aꢁ ¼ þ32.1 (c 0.8, CH Cl)}, H
3
3
2
2
NMR (CDCl ) d (ppm) 1.47 (d, 3H, CH , J = 6.6 Hz),
3
3
2
+
1
.85 (d, 1H, OH, J = 2.7 Hz), 4.87 (dq, 1H, CH,
J(d) = 2.1 Hz, J(q) = 6.2 Hz), 7.23–7.49 (m, 4H, Ph),
C H O) 132, 117, 105, 91, 78, 65, 51, 45. The abso-
lute configuration was confirmed with the authentic
standard.
9
14
+
GC–EI-MS m/z (M 202, 200 for C H OBr) 187, 185,
8
9
1
59, 157, 121, 105, 103, 92, 77, 63, 51, 43.
1
23
D
(
(
1
S)-2-Chloro-1-phenylethanol 45 (39.5 mg); H NMR
(R)-1-Piperonyl-2-propanol 52 (31 mg); ½aꢁ ¼ ꢀ34.8 (c
3
6
20
D
1
CDCl ) d (ppm) 2.69 (d, 1H, OH, J = 3.0), 3.65 (ddd,
H, CH, J(d) = 1.0 Hz, J(d) = 8.8 Hz, J(d) = 11.2 Hz),
0.4, CH Cl) {lit. ½aꢁ ¼ ꢀ34.2 (c 1, CH Cl)},
H
3
3
3
NMR (CDCl ) d (ppm) 1.23 (d, 1H, CH , J = 6.3 Hz),
3
3
3
.75 (ddd, 1H, CH, J(d) = 0.7 Hz, J(d) = 3.4 Hz,
1.60 (s, 1H, OH), 2.60 (dd, 1H, CH, J(d) = 8.1 Hz, J(d) =
13.7 Hz), 2.71 (dd, 1H, CH, J(d) = 4.8 Hz, J(d) = 13.6
J(d) = 11.3 Hz), 4.91 (dt, 1H, CH, J(d) = 8.8 Hz,
J(t)=3.2 Hz), 7.31–7.40 (m, 5H, Ph), GC–EI-MS m/z
Hz), 3.92–4.00 (m, 1H, CH), 5.93 (s, 2H, CH ), 6.65–
6.78 (m, 3H, Ph), GC–EI-MS m/z (M 180 for
2
+
+
(
M
158, 156 for C H OCl) 138, 120, 107, 91, 79, 65,
8
9
5
. The absolute configuration was confirmed with the
C H O ) 135, 121, 106, 77, 51, 45.
1
0
12
3
authentic standard.
(
S)-1-(3,4-Methylenedioxyphenyl)-2-chloroethanol 53
22
1
(
S)-2-Chloro-1-(3-chlorophenyl)ethanol 46 (45.5 mg);
(14.5 mg); ½aꢁ ¼ þ45.6 (c 0.5, CH Cl), H NMR
D
3
21
32
20
½
aꢁ ¼ þ46.8 (c 0.5, CH Cl) {lit. ½aꢁ ¼ þ33.5 (c
(CDCl ) d (ppm) 2.69 (d, 1H, OH, J = 2.9 Hz), 3.60
D
3
D
3
1
1
1
.02, CH Cl)}, H NMR (CDCl ) d (ppm) 2.71 (d,
(dd, 1H, CH, J(d) = 8.8 Hz, J(d) = 11.2 Hz), 3.69 (dd,
1H, CH, J(d) = 3.4 Hz, J(d) = 11.2 Hz), 4.81 (dt, 1H,
3
3
H, OH, J = 3.1 Hz), 3.62 (dd, 1H, CH, J(d) = 8.7 Hz,
J(d) = 11.4 Hz), 3.74 (dd, 1H, CH, J(d) = 3.5 Hz,
J(d) = 11.3 Hz), 4.89 (dt, 1H, CH, J(d) = 8.5 Hz,
CH, J(d) = 8.8 Hz, J(t) = 3.2 Hz), 5.97 (s, 2H, CH ),
6.79–6.89 (m, 3H, Ph), GC–EI-MS m/z (M 202, 200
2
+
J(t)=3.2 Hz), 7.23–7.41 (m, 4H, Ph), GC–EI-MS m/z
for C H O Cl) 182, 164, 151, 135, 123, 105, 93, 77, 65,
51, 44. The absolute configuration was tentatively
9
9
3
+
(
M
194, 192, 190 for C H OCl ) 156, 154, 143, 141,
8
8
2
1
15, 113, 91, 77, 63, 51.
assigned to be S.
2
D
1
23
D
(
R)-1-(2,4-Dichlorophenyl)ethanol 47 (4 mg); ½aꢁ
¼
(R)-1-N-Boc-3-Pyrrolidinol 54 (12 mg); ½aꢁ ¼ ꢀ19.7 (c
3
3
20
D
37
23
D
1
þ44.0 (c 0.5, CH Cl) {lit. ½aꢁ ¼ þ4.5 (CH OH) 13%
0.3, CH Cl) {lit. ½aꢁ ¼ ꢀ22.7 (c 1.0, CH Cl)}, H
3 3 3 3
3
3
3
3
1
ee R}, H NMR (CDCl ) d (ppm) 1.47 (d, 3H, CH , J =
NMR (CDCl ) d (ppm) 1.46 (s, 9H, CH , CH , CH ),
3
3
6
.3 Hz), 1.93 (d, 1H, OH, J = 3.4 Hz), 5.25 (dq, 1H, CH,
1.92 (s, 1H, OH) 1.94–2.07 (m, 2H, CH ), 3.31–3.48
2
J(d) = 3.6 Hz, J(q) = 6.3 Hz), 7.26–7.56 (m, 3H, Ph),
(m, 4H, CH , CH ), 4.45 (s, 1H, CH), GC–EI-MS m/z
2
2
+
+
GC–EI-MS m/z (M 194, 192, 190 for C H OCl ) 179,
(M 187 for C H O N) 149, 132, 114, 103, 87, 74, 57.
8
8
2
9 17 3
1
5
77, 175, 147, 149, 151, 139, 114, 111, 102, 87, 75, 63,
0, 43.
The absolute configuration was confirmed with the
authentic standard.

K. Inoue et al. / Tetrahedron: Asymmetry 16 (2005) 2539–2549
2549
Acknowledgements
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1
8. Itoh, N.; Morihama, R.; Wang, J.-C.; Okada, K.;
We wish to thank Sumitomo Chemical Co. Ltd, Osaka,
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chiral alcohol standards, and for useful information
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