Dynamic kinetic resolution of α-aminonitriles to form chiral α-amino acids

Article abstract of DOI:10.1002/adsc.201100360

We have succeeded in the enzymatic synthesis of (R)-α-aminobutyric acid from racemic α-aminobutyronitrile. This has been demonstrated by the use of non-stereoselective nitrile hydratase (NHase) from Rhodococcus opacus 71D, D-aminopeptidase from Ochrobactrum anthropi C1-38 and α-amino-ε- caprolactam (ACL) racemase from Achromobacter obae. Racemic α- aminobutyronitrile was completely converted in 6 h at 30 °C to (R)-α-aminobutyric acid whose optical purity was more than 99%. (S)-α-Aminobutyric acid was also synthesized from α- aminobutyronitrile by NHase, ACL racemase and L-amino acid amidase from Brevundimonas diminuta TPU 5720. In a similar manner, other (R)- or (S)-α-amino acids with more than 97.5% ee could be synthesized from the corresponding α-aminonitriles. This is the first report on the dynamic kinetic resolution (DKR) of α-aminonitriles to form chiral α-amino acids. The key enzyme in this DKR is non-stereoselective NHase, which had been newly screened from soil samples, and its gene cloned. Copyright

Full text of DOI:10.1002/adsc.201100360

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DOI: 10.1002/adsc.201100360
Dynamic Kinetic Resolution of a-Aminonitriles to Form Chiral
a-Amino Acids
Kazuyuki Yasukawa,a Ryuji Hasemi,b and Yasuhisa Asanoa,*
a
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398, Japan
Fax : (+81)-766-562498; e-mail: asano@pu-toyama.ac.jp
Mitsubishi Gas Chemical Company, Inc., Niigata Research Laboratory, 182 Shinwari, Tayuhama, Kita-ku, Niigata
b
950-3112, Japan
Received: May 7, 2011; Published online: August 26, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100360.
Abstract: We have succeeded in the enzymatic syn-
thesis of (R)-a-aminobutyric acid from racemic a-
aminobutyronitrile. This has been demonstrated by
the use of non-stereoselective nitrile hydratase
(NHase) from Rhodococcus opacus 71D, d-amino-
peptidase from Ochrobactrum anthropi C1-38 and
a-amino-e-caprolactam (ACL) racemase from
Achromobacter obae. Racemic a-aminobutyronitrile
was completely converted in 6 h at 308C to (R)-a-
aminobutyric acid whose optical purity was more
than 99%. (S)-a-Aminobutyric acid was also syn-
thesized from a-aminobutyronitrile by NHase, ACL
racemase and l-amino acid amidase from Brevundi-
monas diminuta TPU 5720. In a similar manner,
other (R)- or (S)-a-amino acids with more than
97.5% ee could be synthesized from the correspond-
ing a-aminonitriles. This is the first report on the
dynamic kinetic resolution (DKR) of a-aminoni-
triles to form chiral a-amino acids. The key enzyme
in this DKR is non-stereoselective NHase, which
had been newly screened from soil samples, and its
gene cloned.
with the direct production of optical pure a-amino
acids via a nitrilase-catalyzed hydrolysis of a-aminoni-
triles, forming a-amino acids such as (S)-alanine,[1]
(S)-leucine,[2] and (S)-phenylglycine.[3] Only very little
work has been done on the conversion of mainly phe-
nylglycinonitrile to (S)- or (R)-phenylglycine by kinet-
ic resolution (KR) in from 25% up to 50% yield via
phenylglycinamide with a nitrile hydratase-amidase
system.[4–7] For example, Ewert et al.[4] reported that
nitrile hydratase (NHase) from Klebsiella oxytoca
preferentially converted the S-enantiomer to (S)-phe-
nylglycinamide with an E value of 7.8, and in the
presence of an S-stereoselective amidase, 1 mM (RS)-
phenylglycinonitrile was converted to (S)-phenylgly-
cine with >99% ee and (R)-phenylglycinamide with
>99% ee by KR. These KR of a-aminonitriles only
leave unreacted substrate or by-product in the reac-
tion mixture. We focused on a more efficient and
powerful method, dynamic kinetic resolution (DKR)
consisting of resolution and racemization steps of the
substrate in the reaction system. This DKR process
transforms a racemic starting material into only a
single chiral product in >99% theoretical yield. We
previously reported that a-amino-e-caprolactam
(ACL) racemase catalyzes the racemization of a-
amino acid amides as new substrates and is effective
for the DKR of a-amino acid amides, when used with
R- or S-stereoselective amino acid amide hydro-
lase.[8–10] Our purpose in this study is to obtain optical-
Keywords: amino acids; biotransformation; dynam-
ic kinetic resolution; nitrile hydratase
Optically active a-amino acids are important additives ly pure a-amino acids in theoretical yield with high
for animal feed and infusion as well as building enantiomeric excess by the DKR of racemic a-amino-
blocks for pharmaceuticals, agrochemicals, and artifi- nitriles. This DKR process consists of three steps: (i)
cial sweeteners. Racemic a-amino acids have been hydrolysis of both enantiomer of racemic a-aminoni-
synthesized by chemical hydrolysis of a-aminonitriles, trile by non-stereoselective NHase, (EC 4.2.1.84), (ii)
but biologically important chiral (R)- or (S)-a-amino stereoselective hydrolysis of the resulting racemic a-
acids cannot be produced in such a way.
amino acid amide by stereoselective amino acid
Most published works on stereoselective enzymatic amide hydrolase, shifting the equilibrium toward the
conversions of racemic a-aminonitriles have dealt product, and at the same time, (iii) racemization of
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Adv. Synth. Catal. 2011, 353, 2328 – 2332

Dynamic Kinetic Resolution of a-Aminonitriles to Form Chiral a-Amino Acids
Scheme 1. Dynamic kinetic resolution of a-aminonitriles to form chiral a-amino acids.
the remaining a-amino acid amide by ACL racemase nylglycinonitrile, they did not report E values and Km
(Scheme 1). A non-stereoselective NHase is a key values for the a-aminonitriles. We show that NHase
enzyme in this method, and most of the enzymes uti- has a wide substrate specificity toward aliphatic ni-
lized have been discovered and characterized during triles such as acrylonitrile (1,520 UmgÀ1), butyroni-
our original studies.
trile (3,770 UmgÀ1), alaninonitrile (770 UmgÀ1), a-
Asano et al. have isolated various nitrile-degrading aminobutyronitrile, valinonitrile (157 UmgÀ1) and leu-
microorganisms.[11,12] They first purified, characterized, cinonitrile (88.5 UmgÀ1). Moreover, aromatic nitrile
and named “nitrile hydratase” from Rhodococcus such as benzonitrile (758 UmgÀ1), phenylglycinonitrile
rhodochrous (formerly Arthrobacter sp.) J-1.[13,14] They (303 UmgÀ1), phenylalaninonitrile (446 UmgÀ1) and
also found that Pseudomonas chlororaphis B23 accu- manderonitrile (860 UmgÀ1) were also shown to be
mulates large quantities of amides from nitriles and is good substrates. Kinetic parameters for the several
suitable for the industrial production of acrylamide racemic a-aminonitriles were calculated by Hanes–
from acrylonitrile.[11–14] Moreover, nicotinamide and 5- Woolf plots. The Km values for alaninonitrile, a-ami-
cyanovaleramide are also industrially produced by nobutyronitrile, valinonitrile, leucinonitrile, tert-leuci-
NHase.[15,16] NHase has become one of the most im- nonitrile, phenylglycinonitrile, and phenylalninonitrile
portant enzymes in the chemical industry.
were calculated to be 17.3, 4.33, 0.20, 1.15, 1.59, 3.63,
In this report, we have isolated a new bacterial and 1.58 mM, respectively. Compared with Km values
strain of Rhodococcus opacus 71D from soil exhibit- for substrates with high affinities of known NHases
ing NHase activity with very low stereoselectivity such as P. chlororaphis B23,[17] Arthrobacter sp. J-1,[14]
toward a-aminobutyronitrile and purified and charac- and R. rhodochrous J-1,[23] these Km value for a-ami-
terized its NHase (see Supporting Information for de- nonitriles of NHase from R. opacus 71D revealed
taile). The specific activity of the purified enzyme was similar affinities.
1040 UmgÀ1 protein, when (RS)-a-aminobutyronitrile
Generally, NHases are rather not enantioselective
was used as a substrate. The amino acid sequence of with enantiomeric compounds. It has been shown that
this NHase showed that it would be a cobalt-contain- some NHases show moderate stereoselectivity, only
ing enzyme with the amino acids sequence of when synthetic substrates were specifically designed
CTLCSCY motif (see Supporting Information). One to fit the active sites. For instance, it has been report-
of the characteristics of Co-type NHase is that it is ca- ed that NHase from R. equi A4 show slight S-selectiv-
pable of hydrating aliphatic, aromatic, and heterocy- ity toward 2-(4-isobutylphenyl)-2-methylpropionitrile,
clic nitriles, whereas no Fe-type NHase has been re- 2-(4-chlorophenyl)-2-methylpropionitrile and 2-(4-
ported to have an activity toward aromatic and heter- methoxyphenyl)-2-methylpropionitrile, and NHase in
ocyclic substrates.[17–19] In the past, several NHase Rhodococcus sp. HT 40-6 has S-stereoselective activi-
have been purified and characterized from various ty toward mandelonitrile.[20] NHase from P. putida
microorganisms. However, none of these has been ex- 2D-11-5-1b and P. putida 5B-MNG-2P act on the R-
plored in detail for the catalytic activity to hydrolyze enantiomer of 2-(4-isobutylphenyl)-2-methylpropioni-
a-aminonitriles, probably because of the lack of avail- trile.[20] Thus, the stereoselectivity of NHase has been
ability of pure a-aminonitriles. While Bhalla et al. re- a very interesting topic, although there has been no
ported the substrate specificity of nitrilase from R. report on competent stereoselectivity of an NHase
rhodochrous PA-34[2] on a-aminonitriles such as alani- active toward a-aminonitrile. On the other hand, we
nonitrile, a-aminobutyronitrile, a-amino-n-valeroni- here demonstrate a successful utilization of non-ste-
trile, leucinonitrile, a-amino-n-capronitrile, and phe- reoselective NHase in DKR. The purified NHase hy-
Adv. Synth. Catal. 2011, 353, 2328 – 2332
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Kazuyuki Yasukawa et al.
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drated alaninonitrile, a-aminobutyronitrile, valinoni- while the remaining (S)-ABA-NH2 was racemized by
trile, leucinonitrile, phenylglycinonitrile and phenylal- ACL racemase. The concentration of (R)-ABA was
ninonitrile with E values[21] of 1.2 (S), 1.2 (S), 1.1 (S), the same as that of the ABN originally present in the
2.1 (S), 1.8 (S) and 1.0, respectively.
reaction mixture. All of (RS)-ABN was converted to
It is important that NHase should be resistant to (R)-ABA in 6 hours (conversion >99% with >99%
cyanide inhibition for biotransformation of a-amino- ee). (S)-ABA also could be obtained by DKR of race-
nitriles using NHase, because an a-aminonitrile is mic ABN by purified NHase, l-amino acid amidase
spontaneously decomposed into aldehyde and cyanide (LaaABd) from B. diminuta TPU 5720[26] and ACL
by a retro-Strecker reaction in aqueous solution, and racemase (see Supporting Information for details).
cyanide strongly inhibits NHase activity. Fe-type All of (RS)-ABN was converted to (S)-ABA in 12
NHase from P. chlororaphis B23[17] and Brevibacteri- hours (conversion >99% with >99% ee).
um sp. R312[22] were only slightly inhibited by cya-
As a proof of principle, the feasibility of achieving
nide, while Co-type NHase from Arthrobacter sp. J- the sequential conversion of racemic a-aminonitriles
1,[14] R. rhodochrous J-1,[23] and Rhodococcus sp. to chiral a-amino acids in a one-pot enzymatic reac-
YH3-3[24] were strongly inhibited by cyanide. NHase tion was demonstrated by using non-stereoselective
from R. opacus 71D showed a high performance NHase, ACL racemase and R- or S-stereoselective
toward a-aminonitrile hydrolysis more than the usual amidase.
Co-type NHases showing high resistanse toward cya-
We have reported the discoveries and properties of
nide even in 1 mM cyanide solution (data not shown). R-stereoselective amino acid amidases such as DAP,
This NHase has been proven to be very suitable for d-amino acid amidase (DaaA),[27] and alkaline d-pep-
conversion of several a-aminonitriles to a-amino acid tidase[30] and S-steroselective amino acidc amide hy-
amides. The time course of (R)-a-aminobutyric acid drolases such as LaaABd and l-amino acid amidase
(ABA) conversion from racemic (RS)-a-aminobutyro- (LaaAPa) from P. azotoformans IAM 1603.[29] By com-
nitrile sulfuric acid salt (ABN) by purified NHase, d- bination of non-stereoselective NHase, ACL race-
aminopeptidase (DAP) from O. anthropi C1-38[25] and mase and R- or S-stereoselective amidase, it is possi-
ACL racemase is shown in Figure 1.
ble to construct an efficient system to produce the
In the initial phase of the reaction, the NHase rap- corresponding (R)- or (S)-a-amino acids from racemic
idly converted ABN to racemic a-aminobutyramide a-aminonitriles. Actually, (S)-alanine with a yield of
(ABA-NH2) with an E value of 1.2. After 20 min, 99% and 99% ee was converted from 50 mM alanino-
ABN was completely converted to (R)- and (S)- nitrile using NHase (1.6 U), ACL racemase (0.5 U),
ABA-NH2. (R)-ABA-NH2 was hydrolyzed by DAP, and LaaAPa (2 U) in 3 hours. Moreover, using NHase,
ACL racemase, and LaaABd, 10 mM valinonitrile,
30 mM leucinonitrile, and 10 mM phenylalaninonitrile
were converted to the corresponding S-enantiomer of
the a-amino acids with yields of 99% (Table 1).
LaaABd acted on not only (S)-alaninamide and (S)-
phenylalaninamide, but also on R-form alaninamide
and slightly on phenylalaninamide.[26] (S)-Phenylala-
nine was produced from phenylalaninonitrile with a
little bit lower enantiomeric excess. (R)-Amino acids
also could be produced from several racemic a-ami-
nonitriles. DAP acted on (R)-amino acid amides with
small substituents, such as (R)-alaninamide and (R)-
a-aminobutyramide. On the other hand, DaaA
showed hydrolyzing activity toward bulky amino acid
amides like (R)-leucinamide and (R)-phenylalanin-
AHCTUNGTREGaNNUN mide. Alaninonitrile (50 mM), 30 mM leucinonitrile,
and 20 mM valinonitrile were converted to the corre-
sponding (R)-a-amino acids (Table 2).
Figure 1. The enzymatic conversion of (RS)-a-aminobutyro-
nitrile to (R)-a-aminobutyric acid. The reaction mixture
(total volume 1.0 mL) was composed of 50 mM potassium
phosphate buffer, pH 7.0, 20 mM (RS)-a-aminobutyronitrile,
500 nM pyridoxal phosphate, NHase (1.0 U), DAP (0.52 U),
and ACL racemase (1.6 U) at 308C. The NHase was added
to initiate the reaction. Symbols: (R)-a-aminobutyramide
Nitrilases have been used to produce only (S)-a-
amino acids from corresponding a-aminonitriles in
the kinetic resolution reaction, while a nitrilase-medi-
ated R-stereoselective hydrolysis of an a-aminonitrile
has not been reported. To the best of our knowledge,
this is the first report on the dynamic kinetic resolu-
tion of racemic a-aminonitriles to not only the corre-
sponding (R)-a-amino acids but also to (S)-a-amino
*
*
( ), (S)-a-aminobutyramide ( ), (R)-a-aminobutyric acid
&
( ), and (S)-a-aminobutyric acid (&).
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Dynamic Kinetic Resolution of a-Aminonitriles to Form Chiral a-Amino Acids
Table 1. Conversion of a-aminonitrile to (S)-a-amino acid by DKR. The reaction (1 mL) was carried out using 1.6 U NHase,
0.5 U ACL racemase, and 1.8 U LaaABd at 308C.
Substrate
Concentration [mM]
Reaction Time [h]
Yield [%]
Enantiomeric Excess [% ee]
alaninonitrile[a]
a-aminobutyronitrile
valinonitrile
50
50
10
30
10
3
3
6
9
12
>99
>99
99.0
>99
>99
>99
>99
>99
>99
97.5
leucinonitrile
phenylalininonitrile[b]
[a]
The reaction (1 mL) was carried out using 1.6 U NHase, 0.5 U ACL racemase, and 2 U LaaAPa.
The reaction (1 mL) was carried out using 1.6 U NHase, 1.0 U ACL racemase, and 1 U LaaABd. The yield and enantio-
meric excess were determined by HPLC (see Supporting Information).
[b]
Table 2. Conversion of a-aminonitrile to (R)-a-amino acid by DKR. The reaction (1 mL) was carried out using 1.6 U NHase,
0.5 U ACL racemase, and 1.9 U DaaA at 308C.
Substrate
Concentration [mM]
Reaction Time [h]
Yield [%]
Enantiomeric Excess [% ee]
alaninonitrile[a]
a-aminobutyronitrile[a]
valinonitrile
50
50
20
30
3
3
20
9
>99
>99
>99
>99
>99
>99
>99
>99
leucinonitrile
[a]
The reaction (1 mL) was carried out using 1.6 U NHase, 0.5 U ACL racemase, and 1.8 U DAP. The yield and enantiomer-
ic excess were determined by HPLC (see Supporting Information).
acids. This new method of dynamic kinetic resolution catalyzed the formation of 1 mmol of (S)-a-aminobutyric
acid per min at 308C.[26]
has the potential to be developed for the large-scale
production of optically active a-amino acids.
Analytical Procedures
(R)- or (S)-a-aminobutyramides were determined with an
HPLC equipped with a Crownpak CR (+) column at a flow
Experimental Section
rate of 0.3 mLminÀ1, using a solvent (60 mM HClO4). (R)-
and (S)-a-aminobutyric acids were determined with HPLC
Enzyme Reaction and Activity Assay
equipped with a SUMICHIRAL OA-5000 column at a flow
The standard reaction mixture (total volume 1.0 mL) for as-
saying the nitrile hydratase activity contained 50 mM KPB,
rate of 1.0 mLminÀ1, using a solvent system of 2 mM CuSO4
in 5% 2-propanol.
pH 7.0,
20 mM
(RS)-a-aminobutyronitrile,
0.1 mM
The identity of the (R)-a-aminobutyric acid formed by
NHase, ACL racemase, and DAP was confirmed by its isola-
tion. The reaction mixture (40 mL) contained 2.0 mmol
KPB (pH 8.0), 4.0 mmol (RS)-a-aminobutyronitrile sulfuric
acid salt (0.532 g), 80 nmol pyridoxal phosphate, 4.0 mmol
CoCl2·6H2O, 120 U of NHase, 39 U of ACL racemase, and
93 U of DAP. After the mixture was incubated at 308C for
10 h, (R)-a-amiobutyric acid formed was isolated by
Dowex-X8 column chromatography and recrystallized from
water-methanol-ether. The optical purity of the isolated (R)-
a-aminobutyric acid was more than 99% ee with an isolated
yield of 68% (0.28 g, 2.72 mmol); Optical rotations were
measured on a SEPA-300 (Horiba, Ltd., Kyoto, Japan) in-
CoCl2·6H2O and the appropriate amount of the enzyme.
One unit (U) of the enzyme activity was defined as the
amount of enzyme solution which catalyzed the formation
of 1 mmol of a-aminobutyramide from a-aminobutyronitrile
per min at 308C.
One unit (U) of ACL racemase activity was defined as
the amount of enzyme solution which catalyzed the forma-
tion of 1 mmol of (S)-a-aminobutyramide from (R)-a-amino-
butyramide per min at 308C.[8]
The d-aminopeptidase activity was assayed by the forma-
tion of (R)-a-aminobutyric acid from (R)-a-aminobutyr-
ACHTUNGTRENNUNGamide as follows. The reaction mixture contained 50 mM
KPB (pH 7.0), 50 mM (R)-a-aminobutyramide, and an ap-
propriate amount of the enzyme. One unit (U) of d-amino-
peptidase activity was defined as the amount of the enzyme
solution which catalyzed the formation of 1 mmol of (R)-a-
aminobutyric acid per min at 308C.[25]
l-Amino acid amidase activity was assayed by measuring
the production of (S)-a-aminobutyric acid from (S)-a-ami-
nobutyramide. One unit (U) of l-amino acid amidase activi-
ty was defined as the amount of the enzyme solution which
strument: [a]D20: À19.78 (c 1.00, 5 moldmÀ3 HCl) {ref.[30]
,
[a]D20: À20.58 (c 1.00, 5 moldmÀ3 HCl)}: 1H NMR (D2O,
400 MHz): d=3.60 (t, 1H, J=5.8 Hz), 1.79 (dq, 2H, J=5.8,
7.5 Hz), 0.87 (d, 3H, J=7.5 Hz); 13C NMR (400 MHz, D2O):
d=174.8, 55.8, 23.6, 8.5; MS (microTOF): m/z=104.0731,
calcd. for C4H10N1O2 [M+H]+: 104.0706. HPLC elution pro-
1
file and H NMR, 13C NMR, MS and IR spectra of the iso-
lated (R)-a-aminobutyric acid are described in the Support-
ing Information.
Adv. Synth. Catal. 2011, 353, 2328 – 2332
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Kazuyuki Yasukawa et al.
COMMUNICATIONS
[12] Y. Asano, T. Yasuda, Y. Tani, H. Yamada, Agric. Biol.
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[14] Y. Asano, K. Fujishiro, Y. Tani, H. Yamada, Agric.
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Supporting Information
Experimental details of the purification, characterization
and cloning of the enzyme are described in the Supporting
Information file.
[15] H. Yamada, M. Kobayashi, Biosci. Biotechnol. Bio-
chem. 1996, 60, 1391–1400.
[16] Y. Fukuda, M. Fukui, T. Harada, Y. Izumi, J. Ferment.
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Acknowledgements
This was supported in part by Grants-in-Aid for scientific re-
search A (23248015) and B (20380053) from Japan Society
for the Promotion of Science to Yasuhisa Asano.
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