Biocatalytic cascade reactions for asymmetric synthesis of aliphatic amino acids in a biphasic reaction system

Article abstract of DOI:10.1016/j.molcatb.2015.07.011

Abstract Enantiopure aliphatic amino acids, including l-3-hydroxyadamantylglycine (l-Hag), l-tert-leucine (l-Tle) and l-norvaline, are essential chiral building blocks for a number of pharmaceutical drugs. Here, we developed cascade enzyme reactions in an extractive biphasic system using a branched-chain amino acid transaminase (BCTA) and an (S)-selective ω-transaminase (ω-TA) for asymmetric synthesis of the aliphatic amino acids from achiral α-keto acid precursors. The extractive cascade reactions enabled equilibrium shift of the BCTA reaction by recycling an amino acid cosubstrate as well as acceleration of the ω-TA reaction by removing an inhibitory ketone product from an aqueous phase. Starting with 20 mM α-keto acid, 4 mM rac-homoalanine and 50 mM rac-α-methylbenzylamine (rac-α-MBA), the biphasic cascade reactions afforded synthesis of four unnatural amino acids (i.e., l-Tle, l-Hag, l-norvaline and l-norleucine) and two natural amino acids (i.e., l-valine and l-Leucine) with >92% conversion yield and >99.9% ee. To demonstrate the industrial feasibility of the extractive cascade reaction, preparative-scale synthesis of l-Hag was performed in a reaction mixture consisting of 300 mL hexane and 50 mL aqueous solution (50 mM phosphate buffer, pH 7.0) charged with 50 mM keto acid substrate, 5 mM l-homoalanine, 120 mM rac-α-MBA, 2 U/mL BCTA and 16 U/mL ω-TA. Conversion yield of l-Hag reached 92% with >99.9% ee at 70 h. Product isolation led to 0.32 g white solid of l-Hag (62 % isolation yield).

Full text of DOI:10.1016/j.molcatb.2015.07.011

Accepted Manuscript
Title: Biocatalytic cascade reactions for asymmetric synthesis
of aliphatic amino acids in a biphasic reaction system
Author: Eul-Soo Park Jong-Shik Shin
PII:
S1381-1177(15)30033-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.07.011
MOLCAB 3203
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
26-5-2015
15-7-2015
16-7-2015
Please cite this article as: Eul-Soo Park, Jong-Shik Shin, Biocatalytic cascade reactions
for asymmetric synthesis of aliphatic amino acids in a biphasic reaction system, Journal
of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.07.011
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Biocatalytic cascade reactions for asymmetric synthesis of aliphatic amino
acids in a biphasic reaction system
Eul-Soo Park and Jong-Shik Shin*
Department of Biotechnology, Yonsei University, Seoul 120-749, South Korea
*To whom correspondence should be addressed.
Engineering Building 2 (Rm 507), Yonsei University, Shinchon-Dong 134, Seodaemun-Gu,
Seoul 120-749, South Korea (E-mail: enzymo@yonsei.ac.kr, Tel: (+82) 2-2123-5884, Fax:
(+82) 2-362-7265)
Keywords: amino acids; biocatalysis; asymmetric synthesis; product inhibition; cascade
reaction; transaminase
Highlights
• Extractive enzyme reaction for asymmetric synthesis of amino acids
• Cascade reactions by coupling two transaminases.
• Equilibrium shift by recycling a cosubstrate
• In situ selective extraction of an inhibitory ketone
• The biphasic reaction afforded synthesis of six amino acids with > 92 % yield and > 99.9 %
ee.

Abstract
Enantiopure aliphatic amino acids, including L-3-hydroxyadamantylglycine (L-Hag), L-tert-
leucine (L-Tle) and L-norvaline, are essential chiral building blocks for a number of
pharmaceutical drugs. Here, we developed cascade enzyme reactions in an extractive biphasic
system using a branched-chain amino acid transaminase (BCTA) and an (S)-selective ω-
transaminase (ω-TA) for asymmetric synthesis of the aliphatic amino acids from achiral α-keto
acid precursors. The extractive cascade reactions enabled equilibrium shift of the BCTA
reaction by recycling an amino acid cosubstrate as well as acceleration of the ω-TA reaction by
removing an inhibitory ketone product from an aqueous phase. Starting with 20 mM α-keto
acid, 4 mM rac-homoalanine and 50 mM rac-α-methylbenzylamine (rac-α-MBA), the biphasic
cascade reactions afforded synthesis of four unnatural amino acids (i.e.
norvaline and -norleucine) and two natural amino acids (i.e. -valine and
92 % conversion yield and > 99.9 % ee. To demonstrate the industrial feasibility of the
extractive cascade reaction, preparative-scale synthesis of -Hag was performed in a reaction
mixture consisting of 300 mL hexane and 50 mL aqueous solution (50 mM phosphate buffer,
L-Tle,
L-Hag, L-
L
L
L
-leucine) with >
L
pH 7.0) charged with 50 mM keto acid substrate, 5 mM
L
-homoalanine, 120 mM rac-α-MBA,
-Hag reached 92 % with > 99.9 % ee
L-Hag (62 % isolation yield).
2 U/mL BCTA and 16 U/mL ω-TA. Conversion yield of
L
at 70 h. Product isolation led to 0.32 g white solid of
1. Introduction
Optically pure amino acids are increasingly employed as important chiral motifs in various
pharmaceutical drugs [1, 2]. For example, -3-hydroxyadamantylglycine ( -Hag) and -tert-
leucine ( -Tle), which are unnatural amino acids carrying branched-chain aliphatic groups, are
L
L
L
L

essential components of a type-2 diabetes drug (i.e. saxagliptin) [3] and a HIV-protease
inhibitor [4], respectively. In addition, -norvaline carrying a linear aliphatic side chain is a key
L
intermediate of Perindopril (i.e. an ACE inhibitor) [5]. Growing industrial demands for the
aliphatic amino acids have spurred massive research efforts to develop preparative methods via
biocatalysis owing to the lack of the fermentative methods yet to access the unnatural amino
acids. Several biocatalytic strategies have been developed by kinetic resolution of racemic
amino acid derivatives using acylase, lipase and protease [6-8] or asymmetric amination of keto
acids using transaminase and dehydrogenase [9-11]. Because of a two-fold higher maximum
yield, the asymmetric synthesis is often favored over the kinetic resolution [12, 13].
Branched-chain amino acid transaminase (BCTA) is a major metabolic producer of the
natural branched-chain amino acids such as
displays a perfect match in the substrate specificity for a broad range of aliphatic α-keto acids,
which enables production of the unnatural amino acids such as -Tle and -Hag [11]. Moreover,
-valine, -leucine and -isoleucine [14]. BCTA
L L L
L
L
compared to the amino acid dehydrogenases [9, 10], no requirement of an expensive external
cofactor such as NADH renders BCTA promising for industrial applications in the asymmetric
synthesis of the aliphatic amino acids. However, a crucial setback in the scale-up of the BCTA
reaction is a neutral equilibrium position of the most BCTA reactions (i.e. equilibrium constant
≈ 1) [14]. To overcome the equilibrium problem, it was demonstrated that removal of a keto
acid coproduct by spontaneous decarboxylation or enzymatic conversion using dehydrogenases
could facilitate an equilibrium shift [11].
In the previous report, we demonstrated that the thermodynamically neutral BCTA reactions
can be driven to completion by coupling a ω-transaminase (ω-TA) reaction as an equilibrium
shifter [15]. Note that ω-TA is capable of employing primary amines as an amino donor and
thereby the resulting ω-TA reaction between the primary amine and the keto acid is

thermodynamically favorable [16]. To couple the BCTA and ω-TA reactions, -homoalanine
L
was used as a shuttling substrate. Isopropylamine was used as an amino donor for the ω-TA
from Ochrobactrum anthropi because isopropylamine was regarded as an ideal amino donor
due to easy removal of the volatile deamination product (i.e. acetone) [17]. However, this
strategy cannot be generalized to other ω-TAs because isopropylamine is rather a poor substrate
for most ω-TAs. For example, two typical ω-TAs, i.e. an (S)-selective ω-TA from Vibrio
fluvialis JS17 and an (R)-selective ω-TA form Arthrobacter sp., are known to show negligible
activities for isopropylamine [18, 19]. Note that the ω-TA from O. anthropi used in the
previous study shows exceptionally high activity for isopropylamine (i.e. 43 % activity relative
to that for (S)-α-methylbenzylamine ((S)-α-MBA)) [20]. In this study, we seek to develop a
generally applicable strategy in which an amine substrate of choice is reactive toward most ω-
TAs (i.e. benzylamine and α-MBA) and thereby any ω-TA displaying desirable enzymatic
properties for a manufacturing setting, including high operational stability and low product
inhibition, can be used for a process scale-up.
2. Materials and methods
2.1. Chemicals
Pyridoxal 5′-phosphate (PLP) was purchased from Sigma Aldrich Co. (St. Louis, USA).
Pyruvic acid was obtained from Kanto Chemical Co. (Tokyo, Japan). Trimethylpyruvate was
purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). 2-(3-Hydroxy-1-adamantyl)-2-
oxoethanoic acid was purchased from Hi-Tech Chemical Co. (Chongqing, China). n-Hexane
was purchased from Duksan Chemical Co. (Ansan, South Korea). Materials used for culture
media including yeast extract, tryptone and agar were purchased from Difco (Spark, USA). All
other chemicals were purchased from Sigma Aldrich Co. (St. Louis, USA) and of the highest

grade available.
2.2. Expression and purification of TAs
Overexpression of His6-tagged TAs was carried out as described previously with minor
modifications [21]. Escherichia coli BL21(DE3) cells carrying the expression vectors (i.e.
pET28a(+) harboring the TA gene) were cultivated in LB medium (typically 1 L) containing 50
µg/mL kanamycin. Protein expression was induced by IPTG at 0.4 OD600 and the cells were
allowed to grow for 10 h. The culture broth was centrifuged and the resulting cell suspension
was subjected to ultrasonic disruption. Protein purification was carried out as described
previously [21]. Molar concentrations of the purified TAs were determined by measuring UV
absorbance at 280 nm.
2.3. Enzyme assay
Typical enzyme assays were carried out at 37 °C in 50 mM phosphate buffer (pH 7). One unit
of BCTA is defined as the enzyme amount catalyzing formation of 1 μmole 2-oxobutyrate in 1
min at 20 mM trimethylpyruvate and 20 mM
L
-homoalanine. One unit of ω-TA from
Paracoccus denitrificans (PDTA) is defined as the enzyme amount catalyzing formation of 1
μmole acetophenone in 1 min at 20 mM pyruvate and 20 mM (S)-α-MBA. After 10 min
reaction, the enzyme reaction (100 µL reaction volume) was stopped by adding 600 µL
acetonitrile. For the initial rate measurements, 2-oxobutyrate and acetophenone for BCTA and
PDTA, respectively, were analyzed by HPLC.
2.4. Cascade enzyme reactions to produce aliphatic amino acids
Unless otherwise specified, the cascade reactions were performed in 50 mM phosphate
buffer (pH 7) at 37 oC. In the biphasic reactions, 3 or 6 mL of hexane was carefully overlaid on

the aqueous reaction mixture (1 mL) and the reactions were carried out without agitation.
Aliquots of the aqueous and organic phases (30 µL each) were taken at predetermined reaction
times and mixed with 180 µL acetonitrile. The reaction progresses were monitored by HPLC
analysis of the keto acid substrate or the amino acid product.
2.5. Preparative-scale synthesis and isolation of
Preparative-scale extractive cascade reaction for asymmetric synthesis of
out at 37 °C under magnetic stirring in an oven-dried beaker charged with 50 mL reaction
mixture containing 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (2.5 mmol), -homoalanine
L-Hag
L
-Hag was carried
L
(0.25 mmol), rac-α-MBA (6 mmol), PLP (5 µmol), BCTA (0.18 µmol), PDTA (0.7 µmol) and
potassium phosphate (50 mM, pH 7.0). Hexane (300 mL) was added to the aqueous reaction
mixture. When the conversion exceeded 90 %, the aqueous reaction mixture was subjected to
product isolation.
The aqueous reaction mixture (50 mL) was separated from the organic phase using a
separation funnel and the pH was adjusted to 1.0 by adding 5 N HCl (4.5 mL). Protein
precipitate was removed by filtration through a glass-fritted filter funnel. The pH of the filtrate
solution was adjusted to 7.0 by adding 5 N NaOH solution (6 mL). The resulting solution was
evaporated at 30 °C and 0.1 bar until the final volume reached around 2 mL. The precipitate
formed during the evaporation was obtained by filtration, washed by 10 mL EtOH and then
dried overnight in a drying oven (75 °C). The resulting white solid of -Hag was subjected to
L
mass spectrometric analysis and elemental analysis as described in the Supplementary Data.
2.6. HPLC analysis
Analysis of acetophenone was performed on a Waters HPLC system (Milford, USA) using
a Symmetry C18 column (Waters Co.) with isocratic elution of 60 % methanol/40 % water,

both containing 0.1 % trifluoroacetic acid, at 1 mL/min. Detection was done with a UV detector
tuned at 254 nm. Retention time of acetophenone was 3.8 min.
α-Keto acids were analyzed using an Aminex HPX-87H column (Bio-Rad, Hercules, USA)
with isocratic elution of 5 mM H2SO4 solution at 0.5 mL/min. UV detection was carried out at
210 nm. Column oven temperature was set to 40 °C. Retention times of the keto acids were
12.0 (2-oxobutyrate), 14.2 (2-oxopentanoic acid), 11.9 (3-methyl-2-oxobutyrate), 17.7 (2-
oxohexanoic acid), 15.3 (4-methyl-2-oxopentanoic acid), 9.7 (trimethylpyruvate) and 11.7 min
(2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid).
For determination of amino acid concentration and enantiomeric excess, 2,3,4,6-tetra-O-
acetyl-β-
The GITC-derivatives of the amino acids were resolved on the Symmetry column at a flow rate
of 1 mL/min with UV detection tuned at 254 nm. Retention time of -enantiomers of
homoalanine was 26.9/28.7 min at isocratic elution of 40 % methanol (A)/60 % water (B), both
containing 0.1 % trifluoroacetic acid,. Retention time of -enantiomers of Hag was 21.1/34.4
min at isocratic elution of 45 % A/55 % B. Retention time of -enantiomers of norvaline,
valine, leucine, Tle were 10.1/14.9, 8.8/13.3, 15.2/23.4 and 11.6/19.0 min, respectively, at
D-glucopyranosyl isothiocyanate (GITC) was used for amino acid derivatization [22].
L/D
/
L D
/
L D
isocratic elution of 50 % A/50 % B. Retention time of
6.0/7.9 min at isocratic elution of 60 % A/40 % B.
/ -enantiomers of norleucine was
L D
3. Results and discussion
3.1. Extractive cascade reaction
To construct the extractive cascade reaction setup (Figure 1), we chose a BCTA cloned
from Escherichia coli [15] and a (S)-selective ω-TA from Paracoccus denitrificans (PDTA)
[23]. -Homoalanine (i.e. a shuttling substrate) was used as an amino donor for the BCTA
L

reaction and its deamination product (i.e. 2-oxobutyrate) was converted back to L-homoalanine
by the ω-TA reaction in which the amino group to be transferred was supplied from a primary
amine, leading to recycling of the shuttling substrate and a consequent thermodynamic shift of
the BCTA reaction driven by the highly exergonic ω-TA reaction. Owing to the recycling of the
shuttling substrate, the cascade reaction requires -homoalanine in a much lower amount than
L
the keto acid substrate. Benzylamine and α-MBA, i.e. typical reactive amine substrates for all
known ω-TAs, were employed as the amino donor for PDTA in the cascade reactions. For
example, PDTA showed a 14-fold higher activity for (S)-α-MBA relative to that for
isopropylamine [20]. This renders (S)-α-MBA much more efficient as an amino donor for
PDTA like other ω-TAs. The ketone product from the ω-TA reaction was known to exert a
severe inhibition on the ω-TA activity [16]. For example, the product inhibition constant of
PDTA for acetophenone was measured to 2.4 mM in the reaction between (S)-α-MBA and
pyruvate [24]. Therefore, an organic solvent was overlaid on the aqueous phase where the
cascade reaction was occurring, so the inhibitory ketone could be selectively extracted to reduce
the product inhibition [16]. Note that all the substrates and products, except the ketone, are not
extractable because of their ionic characters. Hexane was used as the extractant of
benzaldehyde and acetophenone of which the partition coefficients were measured to be 11.1
[25] and 16.4 [26], respectively, in the hexane/water biphasic system.
3.2. Effect of ketone removal
To examine whether the extractive ketone removal improves the cascade reaction rate, we
compared reaction progresses of the synthesis of
L-Tle in a monophasic and a biphasic system
using benzylamine as the amine substrate (Figure 2). Note that PDTA possesses a high activity

for benzylamine (i.e. 96 % activity relative to (S)-α-MBA) [23]. Owing to a cheap price,
racemic homoalanine rather than an enantiopure -form was used as the shuttling substrate (0.1
molar equivalent of kinetically effective -homoalanine to trimethylpyruvate). As expected, the
L
L
biphasic reaction led to a much higher conversion yield than that of the monophasic reaction
devoid of extraction of the inhibitory benzaldehyde. In both reactions, enantiomeric excesses of
the produced L-Tle were higher than 99.9 % owing to the stringent stereoselectivity of BCTA.
3.3. Asymmetric synthesis of
L-Tle
The yield of -Tle in the biphasic system reached 81 % at 53 h but did not increase upon
L
further incubation until 107 h (Figure 2). We presumed that the leveling off of the yield resulted
from high amino acceptor reactivity of benzaldehyde. PDTA displayed 91 % amino acceptor
activity for benzaldehyde relative to 2-oxobutyrate [22]. Therefore, despite the extractive
removal, a low concentration of benzaldehyde in the aqueous phase would strongly compete
with 2-oxobutyrate for the amination step catalyzed by PDTA. This led us to change the amine
substrate from benzylamine to (S)-α-MBA because the amino acceptor activity of PDTA for
arylalkyl ketones such as propiophenone was negligible [23] and thereby the competition with
2-oxobutyrate for PDTA would be minimal. Indeed, substitution of benzylamine with α-MBA
significantly improved the asymmetric synthesis of L-Tle (i.e. 94 % yield and > 99.9 % ee of L-
Tle at 61 h) (Figure 3A). By the same reason with homoalanine, rac-α-MBA rather than (S)-α-
MBA was used as the amine substrate for PDTA (i.e. 1.25 molar equivalent of kinetically
effective (S)-α-MBA to trimethylpyruvate). This led to enantioenrichment of the racemic amine
substrate during the cascade reaction (i.e. 89 % ee of (R)-α-MBA at 61 h). This could benefit
the process economics when the partially enriched (R)-α-MBA was to be isolated and further

processed to enantiopure (R)-α-MBA.
To examine how well the shuttling substrate was recycled during the cascade reaction, we
monitored concentrations of
oxobutyrate was kept below 0.12 mM throughout the reaction, indicative of the efficient
regeneration of -homoalanine driven by the PDTA reaction. Only 0.03 mM 2-oxobutyrate was
L-homoalanine and 2-oxobutyrate (Figure 3B). The level of 2-
L
detectable at the end of the reaction, supporting that the thermodynamically favorable PDTA
reaction served well as an equilibrium shifter. The strong thermodynamic coupling of the two
TA reactions was attributed to the efficient removal of an inhibitory ketone and the ensuing
maintenance of the high PDTA activity. We monitored acetophenone levels in both phases,
showing that acetophenone was initially built up in the aqueous phase and then the aqueous
acetophenone was kept constant around 0.4 mM after 21 h (Figure 3C). The concentration ratio
of acetophenone in the aqueous to organic phase was 10.4 at the end of the extractive reaction
(i.e. below the partition coefficient), indicating that the hexane layer was capable of further
extraction of acetophenone.
3.4. Asymmetric synthesis of various amino acids
The cascade reaction performed with BCTA and PDTA employed
than -alanine, as the shuttling substrate. This choice was based on a much lower activity of
BCTA for -alanine than that for -homoalanine because the active site of BCTA has been
L-homoalanine, rather
L
L
L
optimized for branched-chain amino acids [15]. However, PDTA shows much higher activity
for pyruvate than 2-oxobutyrate, i.e. a 19-fold higher specificity constant for pyruvate than that
for 2-oxobutyrate [27]. Moreover,
whether -homoalanine is a shuttling substrate of choice better than
reaction progresses of the cascade reaction to synthesize -Tle in a monophasic reaction system
(Figure 4). The cascade reaction performed with -alanine led to much lower conversion than
L-alanine is cheaper than
L-homoalanine. To examine
L
L-alanine, we compared
L
L

that with L-homoalanine did, indicating that the BCTA reaction, rather than the PDTA reaction,
is rate-determining under the given reaction conditions. Therefore, we kept using
homoalanine for further reactions.
L
-
To apply the extractive cascade reaction to the synthesis of various aliphatic amino acids,
we performed asymmetric amination of five additional keto acids (Table 1). All the reactions
led to > 95 % yields of the amino acid products (i.e. three unnatural and two natural amino
acids) within 76 h. Owing to the high stereoselectivity of BCTA, enantiopurities of all the
amino acids produced were > 99.9 % ee. As observed during the asymmetric synthesis of L-Tle
in Fig. 3, all the reactions in Table 1 led to partial enantioenrichment of α-MBA substrate (i.e.
63 - 72 % ee of the resulting (R)-α-MBA).
To demonstrate industrial applicability of the extractive cascade reaction, we performed
preparative-scale synthesis of
mixture charged with 0.56 g 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (50 mM), 0.026 g
-homoalanine (0.1 molar eq. to the keto acid substrate), 0.773 mL rac-α-MBA (2.4 molar eq.),
L
-Hag under magnetic stirring in a 50 mL aqueous reaction
L
34 mg BCTA (2 U/mL) and 74 mg PDTA (16 U/mL). For in situ removal of acetophenone, 300
mL hexane (i.e. 6 vol. to the aqueous phase) was overlaid over the aqueous reaction mixture.
After 70 h, conversion yield of
phase was processed to product isolation, leading to recovery of 0.32 g white solid of
(62 % isolation yield, > 99.9 % ee). The isolated product was subjected to mass spectrometric
L
-Hag reached 92 % with > 99.9 % ee. The resulting aqueous
L
-Hag
analysis and elemental analysis, which corresponded to pure
Data).
L-Hag (see the Supplementary
4. Conclusions
In this study, we demonstrate that the extractive cascade reaction composed of BCTA and

ω-TA can afford asymmetric synthesis of various aliphatic amino acids by shifting the
equilibrium limit of the BCTA reaction and removing the product inhibition of the ω-TA
reaction. Because the amine substrate used in this study (i.e. α-MBA) is one of the most
reactive amino donors for ω-TAs, the present strategy is expected to be applicable to all the ω-
TAs. To the best of our knowledge, this is the first example of the TA-based asymmetric
synthesis of amino acids using extractive cascade reactions. However, feasibility of scaling up
this approach should be addressed by minimizing interfacial enzyme inactivation that might
result from a direct contact of the enzyme to an organic solvent (e.g. use of an enzyme
membrane reactor combined with an extraction module [28]).
Acknowledgements
This work was supported by the Advanced Biomass R&D Center (ABC-2011-0031358)
through the National Research Foundation of Korea and the R&D grant (S2173394) funded by
the Small and Medium Business Administration of Korea.
References
[1] M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Keßeler, R. Stürmer, T. Zelinski,
Industrial methods for the production of optically active intermediates, Angew. Chem. Int.
Ed., 43 (7) (2004) 788-824.
[2] J.S. Ma, Unnatural amino acids in drug discovery, Chimica Oggi, 21(6) (2003) 65-68.
[3] S.A. Savage, G.S. Jones, S. Kolotuchin, S.A. Ramrattan, T. Vu, R.E. Waltermire,
Preparation of saxagliptin, a novel DPP-IV inhibitor, Org. Process Res. Dev., 13 (6) (2009)
1169-1176.
[4] P. Lehr, A. Billich, B. Charpiot, P. Ettmayer, D. Scholz, B. Rosenwirth, H. Gstach,
Inhibitors of human immunodeficiency virus type 1 protease containing 2-aminobenzyl-

substituted 4-amino-3-hydroxy-5-phenylpentanoic acid:ꢀ Synthesis, activity, and oral
bioavailability, J. Med. Chem., 39 (10) (1996) 2060-2067.
[5] V. Michel, B. Jean, M Bernard, R. Georges, US Patent No. 4,902,817 (1990)
[6] S.L. Liu, Q.X. Song, D.Z. Wei, Y.W. Zhang, X.D. Wang, Preparation of optically pure tert-
leucine by penicillin G acylase-catalyzed resolution, Prep. Biochem. Biotech., 36 (3)
(2006) 235-241.
[7] N.J. Turner, J.R. Winterman, R. Mccague, J.S. Parratt, S.J.C. Taylor, Synthesis of
homochiral L-tert-leucine via a lipase-catalyzed dynamic resolution process, Tetrahedron
Lett., 36 (7) (1995) 1113-1116.
[8] E. Agosta, A. Caligiuri, P. D'Arrigo, S. Servi, D. Tessaro, R. Canevotti, Enzymatic
approach to both enantiomers of N-Boc hydrophobic amino acids, Tetrahedron Asymmetry,
17 (13) (2006) 1995-1999.
[9] R. Wichmann, C. Wandrey, A.F. Bückmann, M.R. Kula, Continuous enzymatic
transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration,
Biotechnol. Bioeng., 23 (1981) 2789-2802.
[10] R.L. Hanson, S.L. Goldberg, D.B. Brzozowski, T.P. Tully, D. Cazzulino, W.L. Parker,
O.K. Lyngberg, T.C. Vu, M.K. Wong, R.N. Patel, Preparation of an amino acid
intermediate for the dipeptidyl peptidase IV inhibitor, saxagliptin, using a modified
phenylalanine dehydrogenase, Adv. Synth. Catal., 349 (8) (2007) 1369-1378.
[11] E.Y. Hong, M.H. Cha, H.D. Yun, B.G. Kim, Asymmetric synthesis of L-tert-leucine and
L-3-hydroxyadamantylglycine using branched chain aminotransferase, J. Mol. Catal. B-
Enzym., 66 (2) (2010) 228-233.

[12] S. Servi, D. Tessaro, G. Pedrocchi-Fantoni, Chemo-enzymatic deracemization methods for
the preparation of enantiopure non-natural α-amino acids, Coordin. Chem. Rev., 252 (7)
(2008) 715-726.
[13] C.C. Gruber, I. Lavandera, K. Faber, W. Kroutil, From a racemate to a single enantiomer:
Deracemization by stereoinversion, Adv. Synth. Catal., 348 (14) (2006) 1789-1805.
[14] K. Hirotsu, M. Goto, A. Okamoto, I. Miyahara, Dual substrate recognition of
aminotransferases, Chem. Rec., 5 (3) (2005) 160-172.
[15] E.S. Park, J.Y. Dong, J.S. Shin, Biocatalytic asymmetric synthesis of unnatural amino
acids through the cascade transfer of amino groups from primary amines onto keto acids,
ChemCatChem, 5 (12) (2013) 3538-3542.
[16] M.S. Malik, E.S. Park, J.S. Shin, Features and technical applications of ω-transaminases,
Appl. Microbiol. Biotechnol., 94 (5) (2012) 1163-1171.
[17] C.K. Savile, J.M. Janey, E.C. Mundorff, J.C. Moore, S. Tam, W.R. Jarvis, J.C. Colbeck, A.
Krebber, F.J. Fleitz, J. Brands, P.N. Devine, G.W. Huisman, G.J. Hughes, Biocatalytic
asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture,
Science, 329 (2010) 305-309.
[18] J.S. Shin, B.G. Kim, Exploring the active site of amine:pyruvate aminotransferase on the
basis of the substrate structure-reactivity relationship: How the enzyme controls substrate
specificity and stereoselectivity, J. Org. Chem., 67 (9) (2002) 2848-2853.
[19] A. Iwasaki, K. Matsumoto, J. Hasegawa, Y. Yasohara, A novel transaminase, (R)-
amine:pyruvate aminotransferase, from Arthrobacter sp. KNK168 (FERM BP-5228):
Purification, characterization, and gene cloning, Appl. Microbiol. Biotechnol., 93 (4)
(2012) 1563-1573.

[20] E.S. Park, J.Y. Dong, J.S. Shin, ω-Transaminase-catalyzed asymmetric synthesis of
unnatural amino acids using isopropylamine as an amino donor, Org. Biomol. Chem., 11
(40) (2013) 6929-6933.
[21] E.S. Park, J.S. Shin, Deracemization of amino acids by coupling transaminases of opposite
stereoselectivity, Adv. Synth. Catal., 356 (17) (2014) 3505-3509.
[22] E. Park, M. Kim, J.S. Shin, One-pot conversion of L-threonine into L-homoalanine:
Biocatalytic production of an unnatural amino acid from a natural one, Adv. Synth. Catal.,
352 (18) (2010) 3391-3398.
[23] E.-S. Park, M. Kim, J.-S. Shin, Molecular determinants for substrate selectivity of ω-
transaminases, Appl. Microbiol. Biotechnol., 93 (6) (2012) 2425-2435.
[24] E.S. Park, J.S. Shin, ω-Transaminase from Ochrobactrum anthropi is devoid of substrate
and product inhibitions, Appl. Environ. Microbiol., 79 (13) (2013) 4141-4144.
[25] J.S. Shin, B.G. Kim, Transaminase-catalyzed asymmetric synthesis of L-2-aminobutyric
acid from achiral reactants, Biotechnol Lett., 31 (10) (2009) 1595-1599.
[26] J.S. Shin, B.G. Kim, Kinetic resolution of α-methylbenzylamine with ω-transaminase
screened from soil microorganisms: Application of a biphasic system to overcome product
inhibition, Biotechnol. Bioeng., 55 (2) (1997) 348-358.
[27] E.S. Park, J.S. Shin, Free energy analysis of ω-transaminase reactions to dissect how the
enzyme controls the substrate selectivity, Enzyme Microb. Tech., 49 (2011) 380-387.
[28] J.S. Shin, B.G. Kim, A. Liese, C. Wandrey, Kinetic resolution of chiral amines with ω-
transaminase using an enzyme-membrane reactor, Biotechnol. Bioeng., 73 (3) (2001) 179-
187.
Table 1. Asymmetric synthesis of aliphatic amino acids in the extractive cascade reaction.a

Side chain of the keto
acid substrate
Reaction
time (h)
Product
Yield b (%)
95.6
ee b (%)
> 99.9
61
L
-Hag
-norvaline
-norleucine
-valine
HO
L
> 99.9
> 99.9
> 99
97.8
76
40
L
L
> 99
95.2
> 99.9
> 99.9
40
40
L
-leucine
a
Reaction conditions were 6 mL hexane combined with 1 mL aqueous reaction mixture
containing keto acid (20 mM), rac-α-MBA (50 mM), rac-homoalanine (4 mM), BCTA (0.6
U/mL), PDTA (4.8 U/mL) and PLP (0.1 mM) in phosphate buffer (50 mM, pH 7). b Yield and
ee of the amino acid products were measured by performing quantitative chiral HPLC analysis.
Figure legends
Fig. 1 Schematics of the extractive cascade reaction. Chemicals enclosed by ovals represent
substrates that are initially fed in the reaction mixture. The desired amino acid product
is shown in a box.
Fig. 2 Comparison of the cascade reactions in a monophasic and a biphasic system for
asymmetric synthesis of
L-Tle. Aqueous reaction mixture (1 mL) contained
trimethylpyruvate (20 mM), benzylamine (30 mM), rac-homoalanine (4 mM), BCTA
(0.6 U/mL), PDTA (4.8 U/mL) and PLP (0.1 mM) in phosphate buffer (50 mM, pH 7).

To construct the biphasic system, hexane (3 mL) was overlaid on the aqueous reaction
mixture.
Fig. 3 Extractive cascade reaction for asymmetric synthesis of -Tle using α-MBA as the
L
amine substrate. Reaction conditions in the 1 mL aqueous phase were
trimethylpyruvate (20 mM), rac-α-MBA (50 mM), rac-homoalanine (4 mM), BCTA
(0.6 U/mL), PDTA (4.8 U/mL) and PLP (0.1 mM) in phosphate buffer (50 mM, pH 7).
For the ketone removal, 6 mL of hexane added to the aqueous reaction mixture. Time-
course monitoring of the reaction progress was done by analyzing (A)
trimethylpyruvate, (B) -homoalanine and 2-oxobutyrate, and (C) acetophenone in
both aqueous and organic phases.
L
-Tle and
L
Fig. 4 Comparison of
reactions were carried out under the same reaction conditions as those in Figure 3,
except that 4 mM rac-homoalanine was replaced by 2 mM -homoalanine or -alanine.
Organic solvent was not added over the aqueous reaction mixture.
-homoalanine and -alanine as a shuttling substrate. The cascade
L L
L
L

O
inhibitory ketone
R2
Organic phase
Aqueous phase
O
O
R2
R1
COO-
+
NH3
COO-
PDTA
BCTA
(Keq >> 1)
(Keq ≈ 1)
O
COO-
+
NH3
+
NH3
R2
R1
COO-
Fig. 1

100
80
60
40
20
0
monophasic
biphasic
0
20
40
60
80
100
120
reaction time (h)
Fig. 2

A
L
-Tle
20
15
10
5
trimethylpyruvate
0
0
10
20
30
40
50
60
reaction time (h)
B
2.5
2.0
1.5
1.0
0.5
0.0
L
-homoalanine
2-oxobutyrate
0
10
20
30
40
50
60
reaction time (h)
C
4
3
2
1
0
organic phase
aqueous phase
0
10
20
30
40
50
60
reaction time (h)
Fig. 3

60
50
40
30
20
10
0
L
-homoalanine
-alanine
L
0
10
20
30
40
50
reaction time (h)
Fig. 4