Substrate screening of amino transaminase for the synthesis of a sitagliptin intermediate

Article abstract of DOI:10.1016/j.tet.2016.06.039

We reported herein a new enzymatic route to synthesize a sitagliptin intermediate using an aminotransferase. Substrate profile indicated that hydroxyethyl-3-oxo-4-(2,4,5-trifluorophenyl)butanoate, among 11 analogs, showed the best biocatalytic performance, partially due to its best solubility in the enzymatic system. The corresponding amino esters showing strong product inhibition on the reaction, were inclined to autohydrolyze, thus driving the reaction forward, which indicated the contribution of the rapid hydrolysis of hydroxyethyl ester to the biocatalytic performance. The reaction was performed at 100?mM with 82% conversion in 24?h. The amino ester product was further transformed to Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid, the key intermediate of sitagliptin.

Full text of DOI:10.1016/j.tet.2016.06.039

Accepted Manuscript
Substrate screening of amino transaminase for the synthesis of a sitagliptin
intermediate
Anwei Hou, Zixin Deng, Hongmin Ma, Tiangang Liu
PII:
S0040-4020(16)30552-X
10.1016/j.tet.2016.06.039
TET 27853
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 11 April 2016
Revised Date: 12 June 2016
Accepted Date: 13 June 2016
Please cite this article as: Hou A, Deng Z, Ma H, Liu T, Substrate screening of amino transaminase for
the synthesis of a sitagliptin intermediate, Tetrahedron (2016), doi: 10.1016/j.tet.2016.06.039.
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Substrate screening of amino transaminase
for the synthesis of a sitagliptin intermediate
Leave this area blank for abstract info.
Anwei Houa, Zixin Denga, b, Hongmin Maa, and Tiangang Liua, b,
aKey Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan
University School of Pharmaceutical Sciences, Wuhan 430071, China. bHubei Engineering Laboratory for
Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China.

1
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Tetrahedron
journal homepage: www.elsevier.com
Substrate screening of amino transaminase for the synthesis of a sitagliptin
intermediate
Anwei Houa, Zixin Denga, b, Hongmin Maa, and Tiangang Liua, b,
aKey Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences,
Wuhan 430071, China
bHubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China
ARTICLE INFO
ABSTRACT
Article history:
Received
We reported herein a new enzymatic route to synthesize sitagliptin intermediate using an
aminotransferase. Substrate profile indicated that hydroxyethyl-3-oxo-4-(2,4,5-
Received in revised form
Accepted
Available online
trifluorophenyl)butanoate, among 11 analogs, showed the best biocatalytic performance, partially
due to its best solubility in the enzymatic system. The corresponding amino esters showing
strong product inhibition on the reaction, were inclined to autohydrolyze, thus driving the
reaction forward, which indicated the contribution of the rapid hydrolysis of hydroxyethyl ester
to the biocatalytic performance. The reaction was performed at 100 mM with 82% conversion in
24 h. The amino ester product was further transformed to Boc-(R)-3-amino-4-(2,4,5-
trifluorophenyl)butyric acid, the key intermediate of sitagliptin.
Keywords:
Aminotransferase;
Enzymatic reaction;
Sitagliptin;
2016 Elsevier Ltd. All rights reserved
.
Product inhibition
acetyl-2-amino-3-(2,4,5-trifluorophenyl) propanoic acid with α-
chymotrypsin followed by Arndt-Eistert reaction,10 and
asymmetric synthesis of R-3-amino-4-(2,4,5-trifluorophenyl)
butyric ester with aminotransferase PYR-1 from Mycobacterium
vanbaalenii have also been adopted in some studies to obtain
4.11,12 Recently, enzymatic routes have been attracting increasing
attention because of their eco-friendliness, high stereoselectivity,
and catalytic efficiency;13-15 however, so far only a few studies
have focused on aminotransferase-based synthesis of 4.11,12 In
this study, we presented a chem-enzymatic route for the synthesis
of this intermediate, as described in Scheme 1. Our method
involves the transformation of the ketoester to amino ester by
using an aminotransferase, followed by hydrolysis and protection
to form 4.
1. Introduction
Sitagliptin, marketed as a phosphate salt under the trade name
Januvia, is an oral anti-diabetic drug of the dipeptidyl peptidase-4
(DPP-4) inhibitor class.1 It was developed by Merck and was
approved by the U.S. Food and Drug Administration (FDA) in
2006. Sitagliptin is the top-selling drug in its class and has
brought in about $6 billion in 2014 for Merck.2
Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid 4, the
key intermediate of sitagliptin, is mainly synthesized through the
hydrolysis and protection of 3-amino-4-(2,4,5-trifluoro-
phenyl)butyric ester 2. So far, many studies have focused on the
synthesis of the above-mentioned compound: some have used the
induction of the chiral amino group with a chiral pyrazine
compound followed by Arndt-Eistert Homologation to obtain
4,3,4 while others used the stereoselective reduction of methyl 3-
oxo-4-(2,4,5-trifluorophenyl)butanoate to obtain an S
configuration chiral alcohol, followed by the Mitsunobu reaction
to obtain R-3-benzyloxyamino-4-(2,4,5-trifluoruophenyl)butyric
acid.5,6 In addition to these methods, asymmetric resolution of
rac-3-amino-4-(2,4,5-trifluorophenyl)butyric ester with mandelic
acid and other organic acids,7,8 asymmetric hydrogenation of
enamines with [Rh(COD)Cl]2,9 enzymatic resolution of rac-N-
An
aminotransferase
variant,
ATA117-rd11,
from
Arthrobacter sp. was chosen owing to its broad substrate
profile,16-18 and the fact that it had been specifically developed for
the asymmetric catalysis of pro-sitagliptin.16 The enzymatic
binding pocket can be divided into a larger pocket and a smaller
16
one.
Considering the ester part of the substrates should be
accommodated in the larger one to ensure satisfactory
stereoselectivity,16,19 some large substituents of the ester, such as
benzyl, phenylethyl, and cyclohexyl groups, were chosen. Some
small substituents such as the methyl, ethyl, and propyl groups
were also chosen to fully map the substrate profile of the enzyme.
———
Corresponding author. Tel.: +86 27 68756713; fax: +86 27 68756713; e-mail: hongminma@whu.edu.cn(H, Ma)
Corresponding author. Tel.: +86 27 68755086; fax: +86 27 68755086; e-mail: liutg@whu.edu.cn(T, Liu)

2
Tetrahedron
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Scheme 1. The chem-enzymatic route for the synthesis of Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid 4
Further, 1 M isopropylamine was chosen as the amino donor to
accelerate the reaction and to increase the conversion.16
1b were even a little turbid. The reaction tendencies of the 11
substrates at this higher concentration were totally different from
those at low concentrations, and the products formed were found
to be prone to undergo autohydrolysis to form 3-amino-4-(2,4,5-
trifluorophenyl)butyric acid 3 at high substrate concentrations
(Scheme 1, Fig. 1). Therefore, the conversions of all the
substrates into the amino esters 2a-k and the amino acid 3 were
analyzed. Surprisingly, 1b (conversion 41.66%), and 1k
(conversion 37.73%) showed the highest conversion in the first 1
h, while 1g (conversion 24.98%) showed lower conversion. This
phenomenon may be attributed to the poor solubility of 1g and
the good solubility of 1b and 1k in the enzymatic system. In the
fourth hour, the conversion of 1b and 1k was 90.52% and
82.93%, respectively, and that of 1g was only 45.66%, indicating
that at high concentrations, 1b or 1k should be a better candidate
than 1g. In the eighth hour, the conversion of 1b reached 95.87%
and that of 1k was 97.36%, indicating that the conversion of 1b
increased only by 5% during the last four hours compared with
the 15% increase in the conversion of 1k. Upon comparing 1b
and 1k carefully, it was found that the amino product 2k was
easier to hydrolyze than 2b, because in 4 and 8 h, 2k hydrolyzed
33.21% and 58.48%, respectively, while 2b hydrolyzed 16.59%
and 34.48%. This autohydrolysis might reduce product inhibition
and thus promote the enzymatic reaction.
2. Results and discussion
2.1. Enzymatic reactions using different substrates
The enzymatic activities as well as the biocatalytic
performances of the eleven substrates including alkyl esters
(methyl 1a, ethyl 1b, n-propyl 1c, n-butyl 1d), branched alkyl
esters (iso-propyl 1e, tert-butyl 1f), aromatic esters (benzyl 1g,
phenylethyl 1h, phenylpropyl 1i), the cyclohexyl ester 1j, and the
hydroxyl ethyl ester 1k were tested (Table 1, Fig. 1). For the
alkyl esters, as the chain prolonged, the enzyme activity
increased almost linearly from (1.70 ± 0.05)×10-3 U/mL to (14.06
± 0.38)×10-3 U/mL (Table 1, 1a-d). With the hydroxyl group, the
activity of 1k became lower than that of 1b (Table 1, 1k). The
activity of 1e was almost same as that of 1d; when the iso-propyl
group was substituted with the tert-butyl group, the activity
decreased (Table 1, 1e-f). 1g had the highest activity among all
the 11 substrates; when the chain prolonged, the activity
decreased unlike what was observed for the alkyl esters (Table 1,
1g-i). It seemed that, among the aromatic esters, 1g containing
the benzyl group was more similar to pro-sitagliptin in structure
as they both have the methylene-aromatic ring motif, which
probably granted it the highest activity. Despite the highest
activity of 1g, it had poor solubility in the enzymatic system:
about 1 mM 1g was required to ensure that the reaction mixture
was homogeneous. However, high substrate concentrations are
necessary for the industrial application of enzymatic reactions. In
our enzymatic reaction mixture, DMSO was chosen as
the cosolvent to increase the solubility of the substrates as
described previously.16 Our attempts to find alternative solvents
were unsuccessful: methanol and ethanol were tested, but they
caused the transesterification of the substrates in the enzymatic
system, and other water-miscible organic solvents such as DMF
and THF did not show better behavior than DMSO. Moreover, it
was found that, among the 11 substrates, 1k and 1b had good
solubility in the enzymatic system. The substrates were then
tested at a raised concentration of 20 mM (Fig. 1). At this
concentration, the reaction mixture of all of the substrates were
heterogeneous with 1k as the only exception, and those of 1a and
Table 1 Enzyme activity of the different substrates
substrates
Enzyme activityb
Ua/mg
order
1a
1b
1c
1d
1e
1f
1g
1h
1i
(1.70±0.05)×10-3
11
8
4
3
2
5
1
7
6
(5.65±0.44)×10-3
(9.69±0.39)×10-3
(14.06±0.38)×10-3
(14.11±0.50)×10-3
(9.05±0.03)×10-3
(18.94±0.47)×10-3
(6.53±0.72)×10-3
(6.58±0.96)×10-3
(2.53±0.17)×10-3
(2.63±0.34)×10-3
1j
1k
10
9
a
One unit of ATA117-rd11 was defined as 1 mg crude enzyme producing
1 ꢀmol of the amino product per minute.
b The values are given as mean ± SD of triplicates.

3
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Fig. 1. Enzymatic reactions for different substrates at 1, 4, and 8 h. Red color represents the conversion of the substrates 1a-k into the corresponding products 2a-
k, and green color represents the conversion of the substrates into the hydrolyzed amino acid 3. Error bars represent standard deviation of triplicates.
DMSO. Thus, 100 mM of 1k and 20% DMSO were chosen as
the optimum conditions for further experiments.
2.2. The inhibition of amino acid 3 and amino ester 2b to the
aminotransferase
The hydroxyl substitution of 1k granted it the highest
solubility, which is pivotal for high reaction rate caused by the
increased concentration in the aqueous phase. The fact that the
amino ester 2k was easiest to hydrolyze indicated that this
behavior might reduce product inhibition on the enzyme. Hence,
the enzymatic activities at different concentrations of the amino
acid 3 and amino ester 2b were tested (Fig. 2). Considering the
poor solubility of the amino acid 3 in a solution containing
DMSO or not at neutral pH, pH 10 was adopted to ensure good
solubility of 3 in the reaction system. Experimentally, the activity
of the aminotransferase at the presence of 3 at 100 mM was the
same as that without the addition of the amino acid, which
proved that 3 has no inhibitory effect on the enzyme reaction at
concentrations of 0–100 mM. In the case of rac-amino ester 2b,
at 20 mM of the rac-amino ester 2b, the enzyme showed only
about 60% relative activity, and the relative activity ceased to
Fig. 2. Inhibition of enzyme activity by the amino ester 2b and the amino acid
3. Error bars represent standard deviation of triplicates.
reduce at higher concentrations. The inhibition of enzyme
activity with rac-amino ester 2b is shown in Fig 2. Thus, the
amino ester 2b exerted an obvious inhibitory effect on enzyme
activity. The reason why the inhibition caused by 2b did not
increase with increasing concentrations of 2b may be attributed
to the poor solubility of the rac-amino acid ester 2b in the
reaction mixture, which was indicated by the fact that the
reaction solution became turbid when 2b was used at a
concentration of 40 mM.
2.3. Optimization of the enzymatic reaction conditions
Because of the good solubility and apparent biocatalytic
behavior of 1k and the auto-hydrolysis ability of the
corresponding amino ester 2k, 1k was believed to be the best
substrate for use in industrial applications. The reaction
conditions, such as the concentrations of 1k and DMSO, were
then optimized, as shown in Fig. 3. At 50 mM of 1k, 85%
conversion was observed in 16 h and 95% conversion could be
achieved in 24 h in the presence of 10% or 20% DMSO. At 100
mM, the conversion of 1k reduced to 60% in 16 h and to about
80% in 24 h, which may possibly resulte from the slight
decomposition during the reaction. The conversion at the
presence of 10% DMSO was slightly less than that at 20%
Fig. 3. Time course of 1k at different concentrations of 1k and in the
presence of 20% or 10% DMSO. Error bars represent standard deviation of
triplicates.
2.4. Preparation
of
Boc-(R)-3-amino-4-(2,4,5-trifluoro-
phenyl)butyric acid 4
As the amino acid ester 2k tended to hydrolyze to form the
amino acid 3, which was hardly soluble in organic solvents such

4
Tetrahedron
ACCEPTED MANUSCRIPT
as ethyl acetate, it was difficult to use organic solvents to
of iso-propyl-β-D-thiogalactoside (IPTG) to obtain a final
extract all the products needed. However, Boc-(R)-3-amino-4-
(2,4,5-trifluorophenyl)butyric acid 4 shows good solubility in
organic solvents. Therefore, we developed an optimized method
to synthesize 4 after the enzymatic reaction. Because the
concentration of isopropyl amine (1 M) was much higher than
that of the products, it was necessary to remove as much
isopropyl amine as possible to avoid the consumption of Boc
anhydride during the chemical synthesis of 4. The pH of the
biocatalytic product was adjusted to 10 to remove isopropyl
amine as a volatile free base by rotary evaporation, and this
alkaline condition also allowed 2k to become completely
hydrolyzed to form amino acid 3. Using this strategy, only about
2 eq. Boc anhydride was added and all of the amino acid 3 was
transformed to 4. Then, using a 10 mL scale reaction with 100
mM 1k in 20% DMSO, about 200 mg of Boc acid 4 was obtained
(60.6% yield, 98.6% ee) as a white solid.
concentration of 1 mM. The cells were then cultivated for
another 16–20 h, after which they were harvested by
centrifugation (4500 rpm, 15 min, 4°C) and the supernatant was
discarded. Next, the cell pellets were resuspended in 100 mM
triethanolamine (chloride) buffer (pH 8.5) containing 1 mM PLP
and were then harvested by centrifugation using the same
conditions as described above. One gram of the wet cell pellets
were then resuspended in 10 mL cold triethanolamine (chloride)
buffer containing PLP, and the suspension was subjected to
ultrasonic disruption for 12 min. Ultrasonic cell disruption
followed by centrifugation (16000 rpm, 20 min, 4°C) finally
yielded the crude enzyme solution. The crude enzyme solution
could be used immediately or stored at 4°C for several days;
alternatively, the cell pellets can also be stored at -80°C until use.
Protein concentrations were determined using Bradford method.
4.3. Preparation of rac 2a-k compounds
3. Conclusion
The rac compounds were prepared using a previously
described method21 with minor modifications. The ketoester 1b
(198 mg, 0.76 mmol), ammonium acetate (586 mg, 7.6 mmol),
and sodium cyanoborohydride (50 mg, 79 mmol) were added in 4
mL methanol, and the solution was stirred overnight at room
temperature. Concentrated HCl was then added to the solution
until the final pH was 2, and then the methanol was removed by
rotary evaporation. Next, 5 mL water was added to the residue
and 10 M NaOH aqueous solution was used to adjust the pH to
10. Ethyl acetate (2× 5 mL) was added to this solution to extract
the product. The organic layers were combined and dried using
anhydrous sodium sulfate and concentrated to obtain the crude
product. The crude product was then purified by preparative thin
layer chromatography (PE/EA=5/3) to yield compound 2b (86
mg, 43.9%) as a colorless oil. 1H NMR (400 MHz, Chloroform-d)
δ 7.04 (ddd, J = 10.5, 8.7, 6.7 Hz, 1H), 6.90 (ddd, J = 10.1, 9.2,
6.6 Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 3.52 – 3.34 (m, 1H), 2.78 –
2.56 (m, 2H), 2.50 – 2.23 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, Chloroform-d) δ 172.00, 156.10 (ddd, J =
244.1, 9.2, 2.6 Hz), 148.74 (ddd, J = 250.0, 14.3, 12.4 Hz),
146.56 (ddd, J = 244.8, 12.4, 3.6 Hz), 121.84 (ddd, J = 18.3, 5.5,
4.2 Hz), 118.95 (ddd, J = 18.9, 6.1, 1.4 Hz), 105.48 (dd, J = 28.7,
20.8 Hz), 60.58, 48.57, 41.70, 36.37, 14.17. HRMS (ESI)
calculated for C12H15F3NO2 [M+H]+: m/z 262.1049; m/z found:
262.1051.The other rac compounds were prepared in a similar
way.
Here we developed a new enzymatic route to synthesize the
intermediate of sitagliptin using an aminotransferase. Through
substrate screening of 11 substrates, hydroxyethyl-3-oxo-4-
(2,4,5-trifluorophenyl)butanoate 1k was found to be the best
substrate for use in industrial applications. 1k had the best
biocatalytic performance owing to its high solubility in the
enzymatic system and its corresponding amino ester 2k was easy
to autohydrolyze to drive the reaction forward. The reaction
could be performed using 100 mM of 1k and showed 82%
conversion in 24 h. The amino product was then transformed to
Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl) butyric acid 4, the key
intermediate of sitagliptin, with 60.6% total yield and 98.6% ee.
4. Experimental section
4.1. General
2,4,5-Trifluorophenylacetic acid, Meldrum’s acid, and
ketoester 1a were purchased from Chemlin Chemical Industry
Co. (Nanjing, China). N,N-dimethylaminopyridine, N,N-
diisopropyl-ethylamine, and pyridoxal 5’-phosphate (PLP) were
purchased from Energy Chemicals (Shanghai, China). The
ketoesters 1b-k were synthesized as described previously.20 All
other organic substrates (AR or AG) were obtained from
1
commercial sources and used without further purification. H
NMR spectra and 13C NMR were recorded on an Agilent 400
MHz instrument (DirectDrive2)(California, USA). Chemical
shifts were recorded in ppm downfield from tetramethylsilane. J
4.4. Enzyme activity with different substrates
1
60 ꢀL crude enzyme solution at 0.5-1 mg/mL concentration,
100 ꢀL of 60 mM isopropyl amine (chloride) solution (pH 8.5),
and 40 ꢀL of a solution of 1a-k (5 mM) in DMSO were added
into a 2 mL Eppendorf tube. The mixture was then placed in a
shaking incubator at 45°C for 0.5 h, during which the
conversions were guaranteed to be less than 10 %. Then 0.8 mL
acetonitrile was then added to the reaction mixture. The
conversion was then analyzed by HPLC.
values were given in Hz. Abbreviations used in H NMR are s
(singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
High-resolution mass spectrometry (HRMS) experiments were
performed using
(Massachusetts, USA).
a
Thermo LTQ XL Orbitrap instrument
4.2. Expression of ATA117-rd11
4.5. Enzymatic reactions using different substrates
The ATA117-rd11 gene was codon-optimized and synthesized
using a C-terminal His-tag by Tianyi Huiyan Co. (Wuhan,
China). The gene was then inserted into a pRSFDuet-1
expression vector at the BamHI and XhoI restriction sites, and
the plasmid was transformed into Escherichia coli BL21 (DE3)
cells. The recombinant cells were cultivated in 500 mL LB broth
containing 50 mg/L kanamycin at 37°C. When the OD600
reached 0.6–0.8, enzyme expression was induced by the addition
A 125 ꢀL crude enzyme solution, 275 ꢀL of 1.83 M isopropyl
amine (chloride) solution (pH 8.5), and 100 ꢀL of a solution of
1a-k (100 mM) in DMSO were added into a 2 mL Eppendorf
tube. The mixture was then placed in a shaking incubator at 45°C
for 8 h. Twenty microliters of the sample was taken at 1, 4, and 8

5
h and diluted with 380 ꢀL methanol for use in HPCL to analyze
conversation.
Hz, 1H), 2.63 – 2.41 (m, 2H), 1.32 (s, 9H). 13C NMR (101
ACCEPTED MANUSCRIPT
MHz, Chloroform-d) δ 176.08, 174.84, 157.12, 155.12, 150.14,
147.79, 145.30, 121.04, 118.89, 105.36 (dd, J = 28.6, 20.8 Hz),
79.90, 47.56, 37.80, 32.97, 28.21. HRMS (ESI) calculated for
C15H17F3NO4 [M-H]-: m/z 332.1115; m/z found: 332.1123.
4.6. The inhibition of amino acid 3 to the aminotransferase
A 50 ꢀL crude enzyme solution, 110 ꢀL of 1.83 M isopropyl
amine (chloride) solution (pH 10) containing 0–183 mM amino
acid 3, and 40 ꢀL of a solution of 1b (100mM) in DMSO were
added into a 2 mL Eppendorf tube. The mixture was then placed
in a shaking incubator at 45°C for 30 min. Twenty microliters of
the sample was taken and diluted with 380 ꢀL methanol, and the
conversation was analyzed using HPLC. The relative activity
was determined by analysis of the consumption of the substrate
1b at the presence of amino acid 3, and the activity at the absence
of amino acid 3 was used as the control.
4.10. Analytical methods
The conversion rate was determined using an ultimate 3000
HPLC equipped with a Waters Xterra RP C18 column (3.9 × 150
mm, 5 ꢀm) using 10 mM NH4Ac/acetonitrile as the eluent. The
gradient was 1–10 min: 10%-90% acetonitrile, 10–11 min: 90%
acetonitrile, 11–12 min: 90%-10% acetonitrile, and 12–15 min
10% acetonitrile.
The enantiomeric excess of Boc-(R)-3-amino-4-(2,4,5-tri-
fluorophenyl)butyric acid 4 was determined using a Shimadzu
LC 20 with CHIRALPAK AD-H (4.6 × 250 mm, 5 ꢀm) using
Hexane/ IPA = 90/10 as the eluent at a flow rate of 1 mL/min.
4.7. The inhibition of amino ester 2b to the aminotransferase
A 50 ꢀL crude enzyme solution, 110 ꢀL of 1.83 M isopropyl
amine (chloride) solution (pH 8.5), 20 ꢀL of a solution of 1b
(200 mM) in DMSO, and 20 ꢀL of a solution of 2b (0–1000 mM)
in DMSO were added into a 2 mL Eppendorf tube. The mixture
was then placed in a shaking incubator at 45°C for 30 min.
Twenty microliters of the sample was taken and diluted with 380
ꢀL methanol, and the conversation was analyzed by HPLC. The
relative activity was determined by analysis of the consumption
of the substrate 1b at the presence of amino ester 2b, and the
activity at the absence of amino ester 2b was used as the control.
Legends
Scheme 1 The chem-enzymatic route for the synthesis of Boc-
(R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid 4.
Table 1. Enzyme activity of the different substrates.
Fig. 1. Enzymatic reactions for different substrates in 1, 4, and 8
h. Red color represents the conversion of the substrates 1a-k into
the corresponding products 2a-k, and green color represents the
conversion of the substrates into the hydrolyzed amino acid 3.
Error bars represent standard deviation of triplicates.
4.8. Enzymatic reaction using different concentrations of 1k
A 125 ꢀL crude enzyme solution, 275 ꢀL of 1.83 M isopropyl
amine (chloride) solution (pH 8.5), and 100 ꢀL of a solution of
1k (100–500 mM) in DMSO were added into a 2 mL Eppendorf
tube. The mixture was then placed in a shaking incubator at 45°C
for 24 h. Twenty microliters of the sample was taken at 1, 4, 8,
12, and 24 h, and diluted with 380 ꢀL methanol to analyze the
conversation using HPLC.
Fig. 2. Inhibition of enzyme activity by the amino ester 2b and
the amino acid 3. Error bars represent standard deviation of
triplicates.
Fig. 3. Time course of 1k at different concentrations of 1k and in
the presence of 20% or 10% DMSO. Error bars represent
standard deviation of triplicates.
4.9. Preparation
of
Boc-(R)-3-amino-4-(2,4,5-trifluoro-
phenyl)butyric acid 4
A 2.5 mL crude enzyme solution, 5.5 mL of 1.83 M isopropyl
amine (chloride), and a 2 mL solution of 1k (500 mM, 1 mmol)
in DMSO were added into a 50 mL sample bottle. The bottle was
then incubated at 45°C for 24 h, and the conversion was found to
be 80%. Anhydrous lithium hydroxide (192 mg, 8 mmol) was
added to this mixture and incubated for another 3 h to hydrolyze
all the products. The mixture was then centrifuged (6000 rpm, 10
min), and the residue was washed twice with 4 mL 0.4 M lithium
hydroxide aqueous solution. Next, the aqueous layer was mixed
and concentrated in high vacuum to remove the remaining
isopropyl amine. When almost all of the isopropyl amine was
removed, 3 mL tetrahydrofuran and di-tert-butyl pyrocarbonate
(427 mg, 1.96 mmol) was added to this solution. The mixture
was then stirred overnight at 35°C to complete the reaction. Ethyl
acetate (2× 15 mL) was used to extract the product. The organic
layer was combined and dried using anhydrous sodium sulfate,
and then concentrated by rotary evaporation. The crude product
was purified by silica column chromatography (PE/EA/acetic
acid=100/50/1) to yield Boc acid 4 (200 mg, 60.6% yield, 98.6%
Acknowledgements
Our research was supported by grants from the 973
(2012CB721000) Project and the Project supported by the
Science and Technology Department of Hubei Province and by
J1 Biotech Co. Ltd. (2014091610010595).
References and notes
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