Efficient preparation of (R)-3-hydroxypentanenitrile with high enantiomeric excess by enzymatic reduction with subsequent enhancement of the optical purity by lipase-catalyzed ester hydrolysis

Article abstract of DOI:10.1271/bbb.120331

An efficient chemo-enzymatic procedure for the synthesis of (R)-3-hydroxypentanenitrile (1) with over 99% enantiomeric excess using two enzymatic reactions was successfully established. Initial enantioselective enzymatic reduction of 3-oxopentanenitrile with reductase S1 gave (R)-1 with an 81.5% ee which was then converted to (R)-1-(cyanomethyl) propyl n-butyrate (3b). Subsequent lipase-catalyzed enantioselective hydrolysis of 3b gave (R)-1 in a high yield with over 99% ee.

Full text of DOI:10.1271/bbb.120331

Biosci. Biotechnol. Biochem., 76 (9), 1796–1798, 2012
Note
Efficient Preparation of (R)-3-Hydroxypentanenitrile
with High Enantiomeric Excess by Enzymatic Reduction
with Subsequent Enhancement of the Optical Purity
by Lipase-Catalyzed Ester Hydrolysis
Shigeru KAWANO,y Junzo HASEGAWA, and Yoshihiko YASOHARA
Frontier Biochemical and Medical Research Laboratories, Kaneka Corporation,
1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
Received April 27, 2012; Accepted June 4, 2012; Online Publication, September 7, 2012
[doi:10.1271/bbb.120331]
An efficient chemo-enzymatic procedure for the syn-
thesis of (R)-3-hydroxypentanenitrile (1) with over 99%
enantiomeric excess using two enzymatic reactions was
successfully established. Initial enantioselective enzy-
matic reduction of 3-oxopentanenitrile with reductase
S1 gave (R)-1 with an 81.5% ee which was then
converted to (R)-1-(cyanomethyl) propyl n-butyrate
(3b). Subsequent lipase-catalyzed enantioselective
hydrolysis of 3b gave (R)-1 in a high yield with over
99% ee.
collection of various reductases and alcohol dehydro-
genases that could be coupled with a coenzyme-
regeneration system containing glucose dehydrogen-
ase,4,5) these tools being applied in the current study.
The yield, absolute configuration and optical purity of
1 obtained during this study were determined by gas
chromatography under the following conditions: A
Chiraldex G-TA (ASTEC, USA) column (0.25 mm
i.d. ꢁ 30 m) was used with He carrier gas at 150 kPa,
and respective injection and column temperatures of 200
and 115 ꢂC. An FID detector was used for visualization
at 200 ꢂC. The respective retention times (tR) for (R)-1
and (S)-1 were 7.5 and 8.3 min. Several enzymes
produced 1 with the (R) configuration, with carbonyl
reductase S17) showing the highest level of (R)-enantio-
selectivity, although the enantiomeric excess of (R)-1
obtained was only 81.5% ee. To enhance the enantio-
meric excess of (R)-1 obtained by enzymatic reduction
to greater than 99% ee, we investigated the hydrolysis of
acylated (R)-1 with a hydrolase (Fig. 1A).
We also simultaneously investigated the development
of a practical method for separating the remaining
substrate, acylated 1 (3), from product (R)-1 following
the hydrolysis reaction which did not involve column
chromatography. Focusing on the differences in the
lipophilicity of the two compounds, separation by
extraction with n-hexane was undertaken. A 10-mg
sample of racemic 3, (ꢀ)-1-(cyanomethyl)propyl acetate
(3a) or (ꢀ)-1-(cyanomethyl)propyl butyrate (3b) which
had been prepared from (ꢀ)-1 and acyl chloride
according to the reported procedures,2) 100 mg of (R)-
1 prepared as already described and 1.0 mL of a 0.5 M
potassium phosphate buffer (pH 7.0) were mixed and
extracted with n-hexane (3 ꢁ 0:3 mL). Extraction with
n-hexane recovered (ꢀ)-3a in a 45% yield, whereas (ꢀ)-
3b was recovered in an over 99% yield. More water-
soluble 1 was confined to the aqueous phase. Thus, 3b
could be removed in a high yield and separated from 1
by extracting with n-hexane, and butylated 1 (3b) was
selected as the substrate for the hydrolysis reaction.
With the exception of 3a,2) the enzymatic enantiose-
lective hydrolysis of acylated 1 (3) has not previously
been reported. Hence, fifteen commercially available
hydrolases (lipases and proteases) were tested for their
Key words: (R)-3-hydroxypentanenitrile; enantioselec-
tive enzymatic hydrolysis; enantioselective
enzymatic reduction
Chiral ꢀ-hydroxynitriles are very common intermedi-
ates in organic synthesis. (R)-3-Hydroxypentanenitrile
(1) is an important intermediate in the synthesis of an
immunosuppressive inosine 50-monophosphate dehydro-
genase inhibitor,1) in which (R)-1 with a high enantio-
meric excess (>99% ee) is required for the synthesis.
Only one preparation of (R)-1 with an ee in excess of
99% has been reported, involving the Pseudomonas
cepacia lipase-catalyzed kinetic resolution of (ꢀ)-1-
(cyanomethyl) propyl acetate (3a) in the presence of a
thiacrown ether.2) This method, however, is unsuitable
for the industrial-scale production of (R)-1, because of
the requirement for an expensive thiacrown ether
reagent and difficulties associated with the separation
of the (R)-1 product from the remaining substrate 3a
which can only be achieved by column chromatography.
Furthermore, the yield of (R)-1 from this process was
low (49%) because of the optical resolution. With this in
mind, we report here an efficient synthetic procedure for
(R)-1 which is amenable to industrial-scale production.
Many procedures have been developed for the
preparation of optically active alcohols.3) One of the
most convenient methods involves the enzyme-assisted
asymmetric reduction of carbonyl compounds.4,5) In
many cases, enzymatic reduction of carbonyl com-
pounds, under the appropriate conditions, affords a
chiral alcohol with high optical purity. It is convenient
that 3-oxopentanenitrile (2) could be easily prepared
from ethyl propionate and acetonitrile.6) We also had a
y
To whom correspondence should be addressed. Fax: +81-79-445-2668; E-mail: Shigeru Kawano@kn.kaneka.co.jp
Abbreviation: ee, enantiomeric excess

Chemo-Enzymatic Preparation of (R)-3-Hydroxypentanenitrile
1797
A
B
Fig. 1. Synthesis of (R)-3-Hydroxypentanenitrile.
A, Synthesis of (R)-3-hydroxypentanenitrile with over 99% ee. B, Synthesis of (R)-3-hydroxypentanenitrile by enantioselective hydrolysis of
racemic 1-(cyanomethyl) propyl n-butyrate.
Table 1. Hydrolysis of (ꢀ)-1-(Cyanomethyl) propyl Butyrate (3b) to 3-Hydroxypentanenitrile (1) with Different Hydrolases
Reaction
conditionsc
Reaction
time (h)
Yield
(%)c
Hydrolase (origin)a
Config.c
% eec
E value
Lipase SP398
Lipase M
(Candida sp.)
(Mucor javanicus)
A
A
A
A
A
20
20
20
20
20
27
36
52
0
R
R
R
82
94
82
14
53
27
Lipase SP388
Thermolysin
ꢁ-Chymotrypsin
(Rhizomucor miehei)
(Bacillus thermoproteolyticus)
(Bovine Pancreas)
42
S
35
0
Lipase AL
Lipase PL
(Achromobacter sp.)
(Alcaligenes sp.)
B
B
B
B
B
B
B
18
18
18
18
18
18
18
53
12
65
32
37
36
64
R
R
R
R
R
R
R
50
68
54
6
5
6
Novozyme 435
Lipase MY
Lipase AYS
Lipase AK
(Candida antarctica)
(Candida cylindracea)
(Candida rugosa)
18
1
5
1
(Pseudomonas fluorescence)
(Pseudomonas sp.)
91
49
35
8
TOYOBO LIP
Lipase AH
Lipase PS
Lipase SL
(Burkholderia cepacia)
(Burkholderia cepacia)
(Pseudomonas cepacia)
C
C
C
1.5
4
43
47
37
R
R
R
77
93
76
14
73
12
1.3
aLipase SP398, Lipase SP388 and Novozyme 435 were purchased from Novozymes, Denmark. Lipase M, Lipase AYS, Lipase AK, Lipase AH and Lipase PS were
purchased from Amano Enzyme, Japan. Lipase AL, Lipase PL, Lipase MY and Lipase SL were purchased from Meito Sangyo, Japan. TOYOBO LIP was purchased
from Toyobo, Japan. Thermolysin and ꢁ-Chymotrypsin were purchased from Nacalai Tesque, Japan.
bTen milligrams of (ꢀ)-3b, 5 mg of hydrolase and 0.5 mL of 0.5 M potassium phosphate buffer (pH 7.0) were mixed in a test tube and then stirred at 30 ꢂC (Reaction
conditions A). Fifty milligrams of (ꢀ)-3b, 0.25 mg of enzyme and 0.5 mL of 0.5 M potassium phosphate buffer (pH 7.0) were mixed in a test tube and then stirred at
30 ꢂC (Reaction conditions B). Fifty milligrams of (ꢀ)-3b, 1 mg of enzyme and 0.5 mL of 0.5 M potassium phosphate buffer (pH 7.0) were mixed in a test tube and
then stirred at 30 ꢂC (Reaction conditions C).
cYield, absolute configuration and ee value of 1 were determined by gas chromatography analysis under the following conditions. A Chiraldex G-TA (ASTEC, USA)
column (0.25 mm i.d. ꢁ 30 m) with He carrier gas at 150 kPa was used with injection and column temperatures of 200 and 115 ꢂC, respectively. Detection by FID was
conducted at 200 ꢂC. The retention times (tR) of (R)-1 and (S)-1 were 7.5 and 8.3 min, respectively.
ability to enantioselectivity hydrolyze (ꢀ)-3b (Fig. 1B).
Mixtures of (ꢀ)-3b, a hydrolase and a 0.5 M potassium
phosphate buffer (pH 7.0) were agitated at 30 ꢂC. The
yield, absolute configuration and optical purity of
obtained 1 were determined upon completion of the
reaction (Table 1). All of the lipases tested showed (R)-
selectivity, with Lipase PS showing the highest E value8)
of 73. Itoh et al. have reported that Lipase PS hydro-
lyzed (ꢀ)-3a to (R)-1 and (S)-3a with enantioselectivity
and an E value of 53.2) Hence, 3b was a more suitable
substrate than 3a for enantioselective hydrolysis when
using Lipase PS.
reactions. Two hundred grams of 2 was prepared from
ethyl propionate and acetonitrile (71.0% yield).6) Three
liters of a culture broth of E. coli HB101 carrying
pSNTS1G, co-overproducing the transformant of car-
bonyl reductase S1 and glucose dehydrogenase for
NADPH regeneration,9) 45 g of 2, 300 g of glucose and
100 mg of NADPþ were mixed and then stirred at 30 ꢂC
for 22 h. The pH value of the reaction mixture was
maintained at 6.5 by the automatic addition of 6 M
NaOH. A 45-g charge of 2 was given 2 h and 4 h after
the start of the reaction. The reaction mixture was
extracted twice with ethyl acetate upon completion of
the reaction. The organic layers were combined and the
solvent removed in vacuo to give (R)-1 with an 81.5% ee
(R)-1 of over 99% ee was prepared from inexpensive
materials by using two enantioselective enzymatic

1798
S. KAWANO et al.
obtained by the enzymatic reduction of 2); NMR ꢂH
(CDCl3): 1.00 (3H, t, J ¼ 7:6 Hz, –CH2–CH3), 1.64
(2H, dq, J ¼ 7:3, 7.3 Hz, –CH2–CH3), 2.38 (1H, bs,
–OH), 2.50 (1H, dd, J ¼ 16:6, 6.4 Hz, –CH2–CN), 2.58
(1H, dd, J ¼ 16:8, 4.6 Hz, –CH2–CN), 3.83–3.93 (1H,
m, –CHOH–). The total yield of (R)-1 from 2 was
68.6%.
In summary, we successfully prepared (R)-1 of high
enantiomeric purity (>99% ee) and good yield by using
two kinds of enantioselective enzymatic reactions. The
perspective of enantiomeric ratio (E value) for the
hydrolysis of 3 indicated 3b to be a better substrate for
Lipase PS than 3a. In addition, 3b was easily separated
from desired product (R)-1 by extracting with n-hexane
following the enzymatic hydrolysis. It is very important
for industrial-scale production to establish simple
methods for removing the impurities without using
column chromatography. Selecting 3b as a substrate for
the enzymatic hydrolysis enabled a chemo-enzymatic
procedure suitable for the industrial-scale production of
(R)-1 with over 99% ee according to Fig. 1A to be
successfully established.
Fig. 2. Enzymatic Hydrolysis of (R)-3b (81.5% ee) to (R)-1 by Using
Lipase PS.
Filled circles indicate the optical purity of (R)-1; filled triangles
indicate the reaction conversion.
(126.0 g, 91.4% yield). A mixture of (R)-1 (119.7 g),
114.6 g of pyridine, 150 mg of N,N-dimethyl-4-amino-
pyridine and 300 mL of tert-butyl methyl ether was
agitated at 0 ꢂC. n-Butyryl chloride (167.2 g) was then
added to the solution, and the resulting mixture was
stirred overnight at room temperature. The reaction
mixture was then extracted with ethyl acetate, and the
organic layer was collected, dried with anhydrous
Na2SO4 and the solvent removed in vacuo to give crude
(R)-3b. All of this (R)-3b and 1.25 g of Lipase PS were
added to 2.5 L of a 0.5 M potassium phosphate buffer
(pH 7.0), and the resulting mixture was stirred at 30 ꢂC
for 29 h. Time-course plots of the yield and optical
purity for (R)-1 are shown in Fig. 2. Following this
reaction, remaining 3b was removed by extracting with
n-hexane (4 ꢁ 3:0 L). The pH value of the remaining
aqueous phase was adjusted to 8.5 by using aqueous
NaOH, and (R)-1 was then extracted with ethyl acetate.
The combined organic layers were then washed with
aqueous NaHCO3 and dried with anhydrous Na2SO4.
The solvent was removed in vacuo, and the resulting
residue purified by distillation to give (R)-1 with 99.2%
ee as a colorless oil (89.8 g, 75.1% yield from (R)-1
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