A practical chemoenzymatic synthesis of (R)-isovaline based on the asymmetric hydrolysis of 2-ethyl-2-methyl-malonamide

Article abstract of DOI:10.1016/j.tetasy.2014.11.011

(R)-Isovaline has potential applications in drug development, and therefore the development of an efficient method for the production of (R)-isovaline is desired. Herein we have investigated the asymmetric hydrolysis of 2-ethyl-2-methyl-malonamide into (S)-2-ethyl-2-methyl-malonamic acid, a useful synthetic intermediate in the production of (R)-isovaline, using CsAM, which is a recombinant amidase originally derived from Cupriavidus sp. KNK-J915. The produced (S)-2-ethyl-2-methyl-malonamic acid (98.6% ee) could be easily converted into (R)-isovaline by the Hofmann rearrangement. Starting from diethyl 2-methylmalonate, we obtained (R)-isovaline (99.1% ee) in 58.6% yield over eight steps, including the CsAM-catalyzed asymmetric hydrolysis of 2-ethyl-2-methyl-malonamide.

Full text of DOI:10.1016/j.tetasy.2014.11.011

Tetrahedron: Asymmetry 26 (2015) 1–5
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A practical chemoenzymatic synthesis of (R)-isovaline based
on the asymmetric hydrolysis of 2-ethyl-2-methyl-malonamide
b
b
b
a
Masutoshi Nojiri a, , Fumi Yoshida , Yoshinori Hirai , Akira Nishiyama , Yoshihiko Yasohara
⇑
a Biotechnology Development Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
b Technology Management Department, QOL Division, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 October 2014
Accepted 12 November 2014
(R)-Isovaline has potential applications in drug development, and therefore the development of an effi-
cient method for the production of (R)-isovaline is desired. Herein we have investigated the asymmetric
hydrolysis of 2-ethyl-2-methyl-malonamide into (S)-2-ethyl-2-methyl-malonamic acid, a useful syn-
thetic intermediate in the production of (R)-isovaline, using CsAM, which is a recombinant amidase orig-
inally derived from Cupriavidus sp. KNK-J915. The produced (S)-2-ethyl-2-methyl-malonamic acid (98.6%
ee) could be easily converted into (R)-isovaline by the Hofmann rearrangement. Starting from diethyl 2-
methylmalonate, we obtained (R)-isovaline (99.1% ee) in 58.6% yield over eight steps, including the
CsAM-catalyzed asymmetric hydrolysis of 2-ethyl-2-methyl-malonamide.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
receptor and act as an analgesic amino acid,7 and it has potential
applications in drug development.
a
,
a
-Disubstituted amino acids have been reported to increase
Several synthetic methods for the production of (R)-isovaline
have been described in previous reports, including the use of chiral
auxiliaries,8 chiral phase-transfer catalysts,9,10 the Mitsunobu reac-
tion,11 and the resolution of racemic isovaline amide with ami-
dases.12 However, all of the available methods are unsatisfactory
in terms of reaction yield and enantiomeric excess, and therefore
the development of an efficient and inexpensive method for the
production of (R)-isovaline is desired.
the chemical stability, hydrophobicity, and metabolic stability of
their constituent peptides;1 they also represent a class of com-
pounds that are expected to be useful in peptide-based drug devel-
opment. Optically active
a,a-disubstituted amino acids are
structural components of some small molecule and peptide-based
drugs currently under development and constitute important chi-
ral building blocks in the bulk synthesis of such drugs.2–4 For
example, the
amino acid with a structure that is similar to that of the inhibitory
neurotransmitter
-aminobutyric acid (GABA),5 and is also a struc-
tural component of peptaibols, a type of microbial peptide antibi-
otic.6 In particular, (R)-isovaline, an optically active form of
isovaline, has been reported to activate the metabotropic GABAB
a
,
a
-disubstituted amino acid isovaline is a rare
Toward that end, enantiomerically pure (S)-2-ethyl-2-methyl-
malonamic acid, which is a useful synthetic intermediate in the
production of (R)-isovaline, was studied because it was expected
to be converted into (R)-isovaline by the Hofmann rearrangement
(Scheme 1). In addition, prochiral 2-ethyl-2-methyl-malonamide
was expected to be synthesized by using diethyl 2-methylmalo-
c
Hofmann
rearrangement
amidase
H2NOC
CONH2
H2NOC
CO2H
H2N
CO2H
+ H2O
(S)-2-ethyl-2-methyl-
(R)-isovaline
2-ethyl-2-methyl-malonamide
malonamic acid
Scheme 1. Chemoenzymatic production of (R)-isovaline.
⇑
Corresponding author. Tel.: +81 50 3133 7505.
E-mail address: Masutoshi.Nojiri@kaneka.co.jp (M. Nojiri).
http://dx.doi.org/10.1016/j.tetasy.2014.11.011
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

2
M. Nojiri et al. / Tetrahedron: Asymmetry 26 (2015) 1–5
Table 2
nate as a commercially available and inexpensive starting material.
An amidase derived from Rhodococcus rhodochrous NBRC15564 has
been reported to hydrolyze 2-ethyl-2-methyl-malonamide in an
(S)-selective manner, but the enantiomeric excess of the resulting
2-ethyl-2-methyl-malonamic acid was very low (12% ee).13,14 An
enantioselective hydrolysis of 2-ethyl-2-methyl-malonamide by a
chemical- or bio-catalyst that can differentiate between the methyl
group and ethyl group has not been reported on, with the excep-
tion of this example. Therefore we herein explored an amidase
capable of hydrolyzing 2-ethyl-2-methyl-malonamide with high
stereoselectivity toward the (S)-form, and attempted to develop a
biocatalytic process to prepare (S)-2-ethyl-2-methyl-malonamic
acid with high enantiomeric excess and yield using the identified
amidase. Furthermore, we also report the successful synthesis of
(R)-isovaline by the Hofmann rearrangement using highly enantio-
merically pure (S)-2-ethyl-2-methyl-malonamic acid, prepared by
this newly developed method.
Screening for recombinant amidases capable of asymmetric hydrolysis of 2-ethyl-2-
methyl-malonamide
Recombinant amidase
Conversion (%)
Produced EMA
Stereoselectivity (% ee)
Cs AM
Cn AM
99.9
93.5
99.0 (S)
99.0 (S)
2.2. CsAM-catalyzed asymmetric hydrolysis of 2-ethyl-2-meth-
yl-malonamide
The production of (S)-2-ethyl-2-methyl-malonamic acid from
2-ethyl-2-methyl-malonamide was investigated using cultured
cells of CsAM-producing recombinant Escherichia coli. The effect
of temperature and substrate concentration on the enantioselec-
tive hydrolysis of 2-ethyl-2-methyl-malonamide was studied. This
enzyme was stable at high temperatures, as reported previously,
since no decrease in activity was observed even after 30 min at
50 °C.15 As shown in Figure 1, under incubation at 30 °C, 208 mM
and 624 mM of 2-ethyl-2-methyl-malonamide were consumed
after 6 h and 19 h, respectively, while the enantiomeric excesses
of the produced (S)-2-ethyl-2-methyl-malonamic acid were 99.0%
ee and 98.6% ee, respectively. Under incubation at 40 °C, 208 mM
and 624 mM of 2-ethyl-2-methyl-malonamide were consumed
after 4 h and 10 h, respectively, and the enantiomeric excesses of
the produced (S)-2-ethyl-2-methyl-malonamic acid were 98.4%
ee and 98.0% ee, respectively. The reaction speed increased with
increasing temperature, from 30 °C to 40 °C. Higher reaction tem-
peratures and substrate concentrations led to lower enantiomeric
excesses of (S)-2-ethyl-2-methyl-malonamic acid (Table 3). The
2. Results and discussion
2.1. Screening for amidases capable of the asymmetric hydrol-
ysis of 2-ethyl-2-methyl-malonamide
We screened 21 microorganisms and
2
recombinant
amidases,15 CsAM and CnAM, for amidases that could catalyze
the stereoselective hydrolysis of 2-ethyl-2-methyl-malonamide.
Since we did not have authentic 2-ethyl-2-methyl-malonamic acid,
the analysis was first conducted based on the assumption that
2-ethyl-2-methyl-malonamic acid was represented by the new
product observed in the HPLC analysis of the reaction mixture of
2-ethyl-2-methyl-malonamide
treated
with
Rhodococcus
rhodochrous NBRC15564.13 When we successfully prepared (S)-2-
ethyl-2-methyl-malonamic acid as described in Section 4.8, the
new product was identified as 2-ethyl-2-methyl-malonamic acid.
Eight strains were found to produce 2-ethyl-2-methyl-malonamic
acid and six strains were found to produce the desired (S)-2-
ethyl-2-methyl-malonamic acid (Table 1). Two Cupriavidus strains
afforded the (S)-2-ethyl-2-methyl-malonamic acid product with
high enantiomeric excess (99.0% ee), but the conversion rate was
low. The enantiomeric excess of the product from Rhodococcus
rhodochrous NBRC15564 was 10.0% ee, which was comparable to
the reported value (12.0% ee). Comamonas sp. NITE BP-963 and
Pseudomonas sp. NITE BP-962 produced the undesired isomer (R)-
2-ethyl-2-methyl-malonamic acid with low enantiomeric excesses
(33.2% ee and 23.7% ee, respectively). The recombinant amidases
CsAM and CnAM produced the desired isomer (S)-2-ethyl-2-
methyl-malonamic acid, and the conversion rate and enantiomeric
excess were high (Table 2). CsAM and CnAM were derived from
Cupriavidus sp. KNK-J915 and Cupriavidus necator JMP134, respec-
tively. CsAM was chosen for the chemoenzymatic synthesis of
(R)-isovaline due to its superior activity and selectivity relative to
other test materials.
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10 12 14 16 18 20
Time (h)
Figure 1. Time courses of CsAM-catalyzed asymmetric hydrolysis of 2-ethyl-2-
methyl-malonamide. The reaction mixture consisted of EMM (30 mg, 208 mol, or
l
90 mg, 624
l
mol) and the cell suspensions (1Nm),L3).0T°hCe2b0i8ocmonMve(jrs)i,o4n0w°Cas62ca4rmrieMd
Table 1
out at 30 °C or 40 °C. Symbols: 40 °C 208 mM (
(4), 30 °C 624 mM (h).
Screening for microorganisms capable of asymmetric hydrolysis of 2-ethyl-2-methyl-
malonamide
Microorganisms
Conversion
(%)
EMA
Stereoselectivity (% ee)
Table 3
Effects of substrate concentration and temperature on the enantioselectivity of CsAM-
catalyzed asymmetric hydrolysis
Achromobacter denitrificans
NBRC15125
97.1
87.9 (S)
Brevibacterium iodinum NBRC3558
Cellulomonas fimi NBRC15513
Comamonas sp. NITE BP-963
Cupriavidus metallidurans NBRC101272 8.5
Cupriavidus necator NBRC102504
Pseudomonas sp. NITE BP-962
82.2
87.0
99.5
97.8 (S)
21.3 (S)
33.2 (R)
99.0 (S)
99.0 (S)
23.7 (R)
10.0 (S)
EMM
concentration (mM)
Temperature (°C)
Produced (S)-EMA
Stereoselectivity (% ee)
208
30
40
99.0
98.4
3.7
99.2
624
30
40
98.6
98.0
Rhodococcus rhodochrous NBRC15564 96.2

M. Nojiri et al. / Tetrahedron: Asymmetry 26 (2015) 1–5
3
amidase
HCONH2
C2H5Br
H2NOC
CONH2
H2NOC
CO2H
C2H5OOC
COOC2H5
NH3/MeOH
C2H5OOC
COOC2H5
MsO-
t-BuOK
MeONa/MeOH
TEA
MsOH
DCM
evaporate
Boc2O
NaOH
NaClO
NaOH
H2N
CO2H
+H3N
CO2H
BocHN
CO2H
acetone
Scheme 2. Preparative chemoenzymatic production of (R)-isovaline.
production of ethylmethylmalonic acid was not observed under
any conditions, thus suggesting that CsAM does not exert activity
on (S)-2-ethyl-2-methyl-malonamic acid, while (S)-2-ethyl-2-
methyl-malonamic acid is produced nearly quantitatively from 2-
ethyl-2-methyl-malonamide. Based on the aforementioned results,
30 °C and 624 mM were chosen as the optimal temperature and
substrate concentration for subsequent experiments.
D2O using a JNM-ECA500 FT-NMR spectrometer (500 MHz; Jeol,
Japan). Chemical shifts are expressed in parts/million (ppm), with
tetramethylsilane as the internal standard. 13C NMR spectra were
recorded in DMSO-d6 using a JNM-ECA500 FT-NMR spectrometer
(125 MHz; Jeol, Japan). Chemical shifts are expressed in parts/mil-
lion (ppm), with tetramethylsilane as the internal standard. Optical
rotations were measured with a SEPA-300 (Horiba, Japan). IR spec-
tra were measured with a FTIR-8400S (Shimadzu, Japan).
2.3. Chemoenzymatic production of (R)-isovaline
4.2. Screening of amidases for (S)-selective 2-ethyl-2-methyl-
Starting from diethyl 2-methylmalonate, a relatively inexpen-
sive raw material, we obtained (R)-isovaline in 58.6% yield over
eight steps, including the CsAM-catalyzed asymmetric hydrolysis
of 2-ethyl-2-methyl-malonamide (Scheme 2).
malonamide hydrolysis
Microorganisms were obtained from our laboratory collection.
The medium used for the screening consisted of 1% glycerol, 0.5%
peptone, 0.3% yeast extract, 0.3% malt extract, and 0.1% cyclohex-
anecarboxamide (pH 7.0). Each strain was inoculated into 8 mL
of the medium in a test tube, followed by incubation at 30 °C with
reciprocal shaking (at 300 rpm) for 2 d. Microorganisms were har-
vested by centrifugation. The cells from 8 mL of the culture broth
were suspended in 2 mL of 100 mM potassium phosphate (pH
7.0). E. coli HB101 was used as the host cell for CsAM or CnAM
expression.15 The transformed E. coli was cultured at 30 °C in a 2-
YT medium containing 1.6% BD Bacto™ Tryptone, 1.0% Bacto™
Yeast Extract, and 0.5% NaCl (pH 7.0). Each recombinant strain
was inoculated into 5 mL of the medium in a test tube, followed
by incubation at 30 °C with reciprocal shaking (at 300 rpm) for
20 h. The transformed E. coli cells were harvested by centrifuga-
tion. The cells from 5 mL of the culture broth were suspended in
5 mL of 100 mM potassium phosphate (pH 7.0). The resulting cell
suspensions (1 mL each) were mixed with 2 mg of 2-ethyl-2-
methyl-malonamide, and the reaction mixtures were stirred at
30 °C for 24 h. Aliquots of the reaction mixtures were then
withdrawn and centrifuged. The supernatants were analyzed by
high-performance liquid chromatography (HPLC) to determine
the conversion degree (%) of the substrate to the product and the
enantiomeric excess (% ee).
At first, 2-ethyl-2-methyl-malonamide (80 g, 0.55 mol) and the
cultured cell suspensions (860 mL) were stirred at 30 °C for 22 h.
The enantiomeric excess of the produced (S)-2-ethyl-2-methyl-
malonamic acid was 98.6% ee. Using the (S)-2-ethyl-2-methyl-malo-
namic acid solution, the preparation of (R)-isovaline (48.7 g,
0.42 mol) could be accomplished via the Hofmann rearrangement
and neutralization crystallization, in 74.9% yield. From acetone-
containing crude (R)-isovaline mesylate, (R)-isovaline was recrystal-
lized by the addition of triethylamine. The enantiomeric excess of
(R)-isovaline was increased to 99.1% ee due to the recrystallization.
Therefore the feasibility of a chemoenzymatic, large-scale produc-
tion of (R)-isovaline with high enantiomeric excess was confirmed.
3. Conclusion
An efficient chemoenzymatic process for the production of
(R)-isovaline was developed. Cultured cells of recombinant E. coli
producing CsAM served as an excellent catalyst in terms of reaction
efficiency, stereoselectivity, and the efficient preparation of (S)-2-
ethyl-2-methyl-malonamic acid. The concentration of 2-ethyl-2-
methyl-malonamide was optimized at 624 mM (90 g/L), which
yielded (S)-2-ethyl-2-methyl-malonamic acid with 98.6% ee. Using
the (S)-2-ethyl-2-methyl-malonamic acid solution, the preparative
scale production of (R)-isovaline with high enantiomeric excess
(99.1% ee) could be easily accomplished by the Hofmann rearrange-
ment, indicating that a stable biocatalytic process, which could be
applied on an industrial scale, has been established in this study.
To realize an even more efficient production of (R)-isovaline, further
studies regarding the enzymatic process are needed, including
improving the enantioselectivity of CsAM toward 2-ethyl-2-
methyl-malonamide under higher reaction temperatures and
substrate concentrations through optimization of the reaction pH,
protein engineering, preparing for practical immobilized CsAM,
and evaluation of the stability of the immobilized enzyme on
repeated batch reactions.
4.3. Analytical methods
The degree of conversion of 2-ethyl-2-methyl-malonamide to
2-ethyl-2-methyl-malonamic acid and the enantiomeric excess of
2-ethyl-2-methyl-malonamic acid were analyzed by reverse-phase
HPLC using a Shimadzu LC-VP system (Shimadzu, Kyoto, Japan)
equipped with two SUMICHIRAL OA-7000 columns (4.6 mm ꢀ
250 mm; Sumika Chemical Analysis Service Ltd, Osaka, Japan).
HPLC was conducted using acetonitrile/water (2:8 by vol, pH 2.5,
adjusted with phosphoric acid) as the mobile phase, a flow rate
of 1.0 mL/min, a column temperature of 30 °C, and ultraviolet
(UV) detection at 210 nm. The retention times of 2-ethyl-2-
methyl-malonamide, (S)-2-ethyl-2-methyl-malonamic acid, (R)-2-
ethyl-2-methyl-malonamic acid, and ethylmethylmalonic acid
were 9.7, 17.2, 18.9, and 80.7 min, respectively. The enantiomeric
excess of (R)-isovaline was analyzed by reverse-phase HPLC, as
described previously,11 using a Shimadzu LC-VP system (Shimadzu,
4. Experimental
4.1. Materials and instruments
All reagents and solvents were obtained from commercial
Kyoto, Japan) equipped with
(4.6 mm ꢀ 250 mm; Advanced Separation Technologies, Inc., New
a
Chirobiotic T™ column
sources. 1H NMR spectra were recorded in DMSO-d6, CDCl3, or

4
M. Nojiri et al. / Tetrahedron: Asymmetry 26 (2015) 1–5
Jersey, USA). HPLC was conducted using methanol/water (9:1 by
vol) as the mobile phase, a flow rate of 0.8 mL/min, a column tem-
perature of 25 °C, and ultraviolet (UV) detection at 204 nm. The
retention times of (R)-isovaline and (S)-isovaline were 4.0 and
8.3 min, respectively.
supernatant was adjusted to pH 12.0 with 30% sodium hydroxide
solution. The ammonia in the mixture was removed by evaporation
under reduced pressure and concentrated to give the crude mix-
ture (272.6 g), which contains (S)-2-ethyl-2-methyl-malonamic
acid.
A solution of 13% hypochlorous acid (481 g, 0.83 mol), 48%
sodium hydroxide solution (138 g, 1.65 mol), crude mixture
(272.6 g), which contains (S)-2-ethyl-2-methyl-malonamic acid,
and water (10 g) were stirred at 0 °C for 2 h. The mixture was
heated to 20 °C and stirred for 2.5 h. Subsequently, the pH of the
mixture was adjusted to 4.0 with concentrated hydrochloric acid
(238.3 g, 2.28 mol) and stirred at 20 °C for 25 h to give crude (R)-
isovaline hydrochloride. The resulting mixture, sodium hydroxide
(23.0 g, 0.56 mol), 2-propanol (168.8 g), and di-tert-butyl dicarbon-
ate (143.0 g, 0.65 mol) were stirred at 40 °C for 3.5 h, while the pH
was maintained at 11.0 by the addition of sodium hydroxide
(68.5 g, 1.66 mol). The resulting mixture was stirred at 40 °C for
2 h and then cooled to 0 °C and stirred for 15 h. Concentrated
hydrochloric acid (235.0 g, 2.26 mol) was added to the resulting
mixture and the mixture was heated to 18 °C. The organic layer
was separated and evaporated under reduced pressure. Toluene
(423.0 g) and water (212.0 g) were added to the concentrate and
the organic layer (560.0 g) was separated and evaporated under
reduced pressure to give the crude concentrate containing
(R)-N-Boc-isovaline (117.5 g, 0.54 mol). Crude (R)-N-Boc-isovaline
was dissolved in dichloromethane (1175 g), and methanesulfonic
acid (62.4 g, 0.65 mol) was added. The mixture was stirred at
24 °C for 5 h. The resulting mixture was evaporated under reduced
pressure to give the crude concentrate containing (R)-isovaline
mesylate. Crude (R)-isovaline mesylate was dissolved in acetone
(1175 g), and triethylamine (68.4 g, 0.68 mol) was added. The mix-
ture was stirred at 24 °C for 4 h. The solid matter was precipitated
and filtered, washed with acetone, and dried to give (R)-isovaline
(48.7 g, 0.42 mol) as a white solid in 74.9% yield. Characterization
of (R)-isovaline:11 1H NMR (500 MHz, D2O) d (ppm): 1.74 (m,
4.4. Bioconversion of 2-ethyl-2-methyl-malonamide to (S)-2-
ethyl-2-methyl-malonamic acid with cultured cells of
recombinant E. coli producing CsAM
The reaction mixture consisted of 2-ethyl-2-methyl-malona-
mide (30 mg, 208 lmol, or 90 mg, 624 lmol) and the cell suspen-
sions (1 mL) described in Section 4.2. The bioconversion was
carried out at 30 °C or 40 °C. Aliquots of the reaction mixtures were
withdrawn and centrifuged. The supernatants were analyzed by
HPLC to determine the conversion degree (%) of the substrate to
the product and the enantiomeric excess (% ee).
4.5. General procedure for the chemical synthesis of diethyl 2-
ethyl-2-methylmalonate
Diethyl 2-methylmalonate (231.7 g, 1.33 mol) was added drop-
wise to a solution of potassium tert-butoxide (177.5 g, 1.58 mol) in
THF (725 mL) at 0 °C. After stirring at 0 °C for 1 h, bromoethane
(202.5 g, 1.86 mol) was added dropwise and the mixture was
heated to 25 °C and stirred for 20 h. Water (500 mL) was then
added and the aqueous layer was removed. The organic layer
was removed under vacuum and concentrated to give diethyl 2-
ethyl-2-methylmalonate (244.7 g, 1.21 mol) as yellow oil in 91%
yield. Characterization of diethyl 2-ethyl-2-methylmalonate16 1H
:
NMR (500 MHz, CDCl3) d (ppm): 4.18 (4H, q, J = 7.5), 1.91 (2H, q,
J = 7.5), 1.39 (3H, s), 1.25 (6H, t, J = 7.0), 0.87 (3H, t, J = 7.5).
4.6. General procedure for the chemical synthesis of 2-ethyl-2-
methyl-malonamide
1H), 1.59 (m, 1H), 1.30 (s, 3H), 0.75 (t, 3H, J = 7.5 Hz). [
(c 1.0, H2O).
a]
25 = ꢁ8.2
D
Diethyl 2-ethyl-2-methylmalonate (244.7 g, 1.21 mol), a 20%
ammonia/methanol solution (447.7 g, 5.24 mol), formamide
(272.4 g, 6.05 mol), and 28% sodium methoxide/methanol solution
(152.2 g, 0.79 mol) were stirred at 30 °C for 20 h. Toluene (475 mL)
was then added dropwise to the mixture and stirred at 20 °C. After
stirring for 1 h, the mixture was cooled to 5 °C and stirred for 1 h.
The solid precipitate was collected by filtration and washed with
toluene. The filtrate was dried to give 2-ethyl-2-methyl-
malonamide (150 g, 1.04 mol) as a white solid in 86% yield. Char-
acterization of 2-ethyl-2-methyl-malonamide: 1H NMR (500 MHz,
DMSO-d6) d (ppm): 7.16 (s, 2H), 7.05 (s, 2H), 1.73 (q, 2H, J = 7.5 Hz),
1.20 (s, 3H), 0.76 (t, 3H, J = 7.5 Hz). 13C NMR (125 MHz, DMSO-d6) d
(ppm): 174.8, 53.3, 29.7, 19.4, 9.1. IR (KBr): 3379, 3296, 3227,
2997, 2968, 2945, 1691, 1630, 1452, 1408, 1366, 1281, 1188,
1105, 721, 679, 650, 627, 609. ESI–MS (m/z) = 145 [M+H]+.
4.8. (S)-2-Ethyl-2-methyl-malonamic acid
The crude mixture, which contains (S)-2-ethyl-2-methyl-malo-
namic acid, was prepared according to the method described in
Section 4.7. The pH was adjusted to 2.0 with concentrated hydro-
chloric acid. The mixture was evaporated and dried, and the dried
residue was dissolved in 2-propanol. The inorganic salts were
precipitated and then filtered. Hexane was added to the filtrate
to precipitate (S)-2-ethyl-2-methyl-malonamic acid, which was
subsequently filtered. Characterization of (S)-2-ethyl-2-methyl-
malonamic acid: 1H NMR (500 MHz, DMSO-d6) d (ppm): 7.18
(s, 1H), 7.10 (s, 1H), 1.75 (q, 2H, J = 8.0 Hz), 1.22 (s, 3H), 0.78 (t,
3H, J = 7.5 Hz). 13C NMR (125 MHz, DMSO-d6)
d
(ppm):
174.7, 174.0, 53.5, 28.7, 19.6, 9.0. IR (KBr): 3433, 3323, 3254,
2991, 2885, 1697, 1655, 1570, 1560, 1543, 1470, 1406, 1259,
4.7. General procedure for the chemoenzymatic synthesis of
(R)-isovaline
1171, 995. [a]
25 = +9.0 (c 0.1, MeOH). ESI–MS (m/z) = 146 [M+H]+.
D
At first, CsAM transformed E. coli was cultured at 30 °C for 10 h
in a test tube containing 5 mL of 2-YT medium. The seed culture
(1 mL) was then inoculated in a 500-mL Sakaguchi flask containing
50 mL of 2-YT medium. After 20 h of incubation at 30 °C with reci-
procal shaking (at 130 rpm), the cultured cells were collected by
centrifugation from 1.0 L of the cultured broth (from twenty-one
Sakaguchi flasks) and were suspended in 1.0 L of water. Next, 2-
ethyl-2-methyl-malonamide (80 g, 0.55 mol) and the cultured cell
suspensions (860 mL) were stirred at 30 °C for 22 h. The pH of the
reaction mixture was maintained at 7.0 with 30% sodium hydrox-
ide solution. The resulting mixture was centrifuged, and the
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