An efficient enzymatic aminolysis for kinetic resolution of aromatic α-hydroxyl acid in non-aqueous media

Article abstract of DOI:10.1016/j.tetlet.2016.10.054

A new and highly efficient enzymatic aminolysis approach for kinetic resolution of aromatic α-hydroxy acid in non-aqueous media has been developed. The corresponding α-hydroxyl acid ester was employed as the substrate, and commercially available Candida antarctica lipase B is used as the biocatalyst, anhydrous ammonia is the resolving agent. Reactions can be proceeded smoothly in organic solvent at ambient temperatures. High concentration of substrate is allowed due to the application of organic media and the products are obtained in yields of up to 49% with ee values of up to 99%, and with E value of >300, representing an appealing and promising protocol for large-scale preparations.

Full text of DOI:10.1016/j.tetlet.2016.10.054

Tetrahedron Letters 57 (2016) 5312–5314
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An efficient enzymatic aminolysis for kinetic resolution of aromatic
a-hydroxyl acid in non-aqueous media
Shan Chen a,b,y, Fuyan Liu a,y, Kuan Zhang a,y, Hansheng Huang a, Huani Wang a, Jiaying Zhou a, Jing Zhang a,
Yiwei Gong a, Dela Zhang a, Yiping Chen a, Chang Lin a, , Bo Wang a,
⇑
⇑
a Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, 58# Renmin Ave, Haikou 570228, PR China
b Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, Stockholm SE-106 91, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A new and highly efficient enzymatic aminolysis approach for kinetic resolution of aromatic a-hydroxy
acid in non-aqueous media has been developed. The corresponding a-hydroxyl acid ester was employed
as the substrate, and commercially available Candida antarctica lipase B is used as the biocatalyst, anhy-
drous ammonia is the resolving agent. Reactions can be proceeded smoothly in organic solvent at ambi-
ent temperatures. High concentration of substrate is allowed due to the application of organic media and
the products are obtained in yields of up to 49% with ee values of up to 99%, and with E value of >300,
representing an appealing and promising protocol for large-scale preparations.
Received 26 July 2016
Revised 14 October 2016
Accepted 17 October 2016
Available online 18 October 2016
Keywords:
Ammonia
Aminolysis
Kinetic resolution
Lipase
Ó 2016 Elsevier Ltd. All rights reserved.
The demand for enantiopure
a
-hydroxy acids keeps increasing
electivity and organic solvent tolerance by He and his co-
workers,1b with which, the desired product (R)-O-acetyl mandelic
acid was obtained with excellent ee value of 99% and yield of
49% using vinyl acetate as both solvent and the acyl donor
(Scheme 1a). Nevertheless, in his work, only one substrate
mandelic acid was tested and evaluated, no attempt was made
for extension of possible substrate spectrum for Pseudomonas
stutzeri LC2-8. And also, Yang and his co-workers6 have developed
a chemical hydroxylation approach using oppolzer’s sultam as chi-
ral auxiliary to obtain chiral amide derivatives in excellent
diastereoselectivities, the products could be yielded with drs of
up to >20:1.
Previously we have developed an enzymatic approach for
kinetic resolution of (R,S)-1-phenylethanol employing lipase/ester-
ase as the biocatalyst through aminolysis of the corresponding (R,
S)-1-phenylethyl acetate (Scheme 1b) to obtain both enantiomers,4
the desired product (R)-1-phenylethanol was afforded in yields of
up to >49% with ee values of up to 99%. The aminolysis reaction
itself proved to be a better and more reliable way to obtain high
enantiopure (R)-1-phenylethanol compared with conventional bio-
hydrolytic or esterification approaches. Due to high solubility of
substrate in organic media than in aqueous solution and excellent
enantioselectivities of lipase, we would like to extend the possible
substrate spectrum, for example, mandelic acid and its derivatives
in non-aqueous medium.
as they are valuable building blocks, auxiliaries and some are used
as resolving agents in chemical and pharmaceutical industry for
high value added substances.1 There has been a plethora of meth-
ods developed for the preparation of chiral enantiomer of a-hydro-
xyl acid, mandelic acid, and its related derivatives.2 Of them, the
employment of lipase for kinetic resolution (KR) or dynamic kinetic
resolution (DKR) of racemic a-hydroxyl acid has attracted consid-
erable attention due to high regioselectivities of lipases and usually
mild reaction conditions required for displaying their best activi-
ties.3 In most developed procedures with lipase as the biocatalyst,
substrate alkyl mandelic acid ester and its derivative are usually
involved, and till nowadays, most of the KR reactions are pro-
ceeded in aqueous solution via selective hydrolytic cleavage of an
ester bond (formed by a carboxylic group of an acid with hydroxyl
group of an mandelic acid) to achieve the purpose of resolution.
Nevertheless, these developed methods usually suffer from low
substrate solubility, and low ee values of the resulted product,
and some even suffer from treatment of serious downstream salt
water drainage.4,5 Recently, a high enantioselective lipase Pseu-
domonas stutzeri LC2-8 has been characterized with high enantios-
⇑
Corresponding authors. Tel./fax: +86 898 6627 9161 (C.L.), +86 898 6625 0736
(B.W.).
E-mail addresses: cl.changlin@yahoo.com (C. Lin), wangbo@hainu.edu.cn,
bo.wang@biotech.kth.se (B. Wang).
Herein, we present an efficient kinetic resolution of mandelic
acid and its derivatives catalyzed by lipase through the way of
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.tetlet.2016.10.054
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

S. Chen et al. / Tetrahedron Letters 57 (2016) 5312–5314
5313
and >200, respectively (Entry 2, 3, Table 1). Indicating that the
above two organic solvents are well compatible with biocatalyst
CALB when employed for aminolysis reactions using anhydrous
ammonia. And in future investigations, all reactions will be pro-
cessed in both isopropyl ether and isooctane.
Other reaction conditions such as temperatures and reaction
times were also investigated. When enzymatic aminolysis reac-
tions were proceeded in isopropyl ether at 25 °C, after 8 h, the
desired product could be afforded in the yield of 43% (Entry 2,
Table 1), and after 12 h, the product could be yielded in 49% (Entry
3, Table 1), indicating that a prolonged time will contribute posi-
tive effect on the conversion of substrate 2-acetoxy-2-phenylacetic
acid (R1 = H, and R2 = CH3) to the desired product (R)-2-hydroxy-2-
phenylacetic acid. And when reactions were performed at higher
temperatures, higher conversions of substrate could be achieved
(Entry 6, 7, Table 1). Nevertheless, it is not the same with the
resulted ee values, which decreased with increased reaction tem-
peratures (Entry 3, 4, 8, Table 1), and the same happened to E val-
ues. Therefore, in future investigations, all reactions will be carried
out at 25 °C, and reaction time 12 h.
Scheme 1. Developed methods relating to KR of mandelic acid and aminolysis: (a)
lipase Pseudomonas stutzeri LC2-8 catalyzed KR of mandelic acid; (b) lipase B from
Candida antarctica catalyzed aminolysis for KR of phenethyl alcohol.
O
O
O
OH
OH
OH
n
CALB/NH3
n
n
R
R
R
+
OAc
OH
OAc
Organic solvent
n = 0, 1
Scheme 2. Enzymatic aminolysis for kinetic resolution of mandelic acid ester and
its derivatives.
With all the optimized conditions in hand, a variety of
a-
hydroxy acid esters were then subjected to the enzymatic aminol-
ysis reactions, reactions were performed at 10 ml scale in isopropyl
ether and isooctane, respectively. And the results are summarized
in Table 2.
aminolysis, ammonia is used here as the resolving reagent, and
Lipase
B from Candida antarctica (CALB) as the biocatalyst
(Scheme 2). The reactions are proceeded in organic solvent, there-
fore, high concentrations of substrate are available as the sub-
strates are more soluble in organic solvent than in aqueous
solution, excellent ee values and high yields can be achieved.
And also no downstream salt water (usually derived from buffer
solution for biohydrolysis reactions) will be generated after reac-
tions. Therefore, it is considered to meet some principles of green
chemistry and therefore is more practical for large-scale
preparations.
Initial investigations for suitable organic solvents were per-
formed at a 5 mL scale using methyl 2-acet oxy-2-phenylacetic
acid (R1 = H, and R2 = CH3) as the model substrate, and CALB as
the biocatalyst. A variety of organic solvents such as ethyl ester,
n-hexane, n-octane, ethyl acetate, isopropyl ether, isooctane, and
cyclohexane were evaluated, and the results are summarized in
Table 1. It was indicated that when reactions were proceeded in
organic solvent isopropyl ether or isooctane, the desired product
(R)-2-hydroxy-2-phenylacetic acid could be obtained in yields of
up to 4% with ee values of up to 99%, and with E values of >300
All reactions were proceeded smoothly in both isopropyl ether
and isooctane. After reaction, the immobilized CALB can be easily
removed simply by filtration, and the yielded products were con-
densed and purified. Results indicated that most of the ee values
were obtained up to 99% with yields of up to 49% (Entry 1, 9,
Table 2). The substituted group on aromatic ring can cause positive
or negative effects on the conversions of substrates and ee values
of the desired products, when R is H and –OCH3, respectively, in
the reaction time of 12 h applying isopropyl ether as the solvent,
the conversions were 49% and 41% (Entry 1, 3, Table 2). Increased
temperatures will cause negative effects on the resulted ee values,
and also with E values, though conversions became higher (Entry 3,
4, 8, 9, Table 2). A higher concentration of racemic substrate was
also tested, for a complete conversion of substrate, more loading
of immobilized enzyme is needed, and the resulted product was
afforded in 42% yield (isolated) and ee value of 99%. Compared
with the chemical approach for chiral amides with good to excel-
lent yields of 89%, this enzymatic aminolysis methodology can
Table 1
Investigation of reaction parameters using 2-acetoxy-2-phenylacetic acid as the model substratea
Entry
Solvent
Time (h)
T (°C)
Yieldb (%)
eec (%)
Ed (%)
1
2
3
4
5
6
7
8
Ethyl acetate
Isopropyl ether
Isopropyl ether
Isopropyl ether
Isopropyl ether
Ethyl ester
Ethyl ester
Isooctane
Isooctane
Isooctane
15
8
12
6
12
12
12
6
12
15
10
15
25
25
25
35
15
25
15
35
25
15
25
15
32
43
49
41
42
45
23
47
48
29
39
26
93
99
99
97
99
98
99
97
99
99
97
98
12
41
>300
27
36
54
11
98
>200
14
9
10
11
12
n-Hexane
n-Hexane
22
11
a
Conditions: Reactions were carried out on a 2 mL scale, immobilized Candida antarctica lipase B, 2 mg, organic solvent, 2 mL, substrate concentration, 20 mM. Except
otherwise stated, ee values and conversions (conv) were determined by HPLC-analysis with a chiral OD-R column.
b,c
Yields and ee values of mandelic acid were determined by HPLC analysis equipped with a Chiralcel OD-3R column (150 ꢀ 2.1, Daicel Chemical Industries, Ltd) at a
wavelength of 254 nm at 30 °C.
d
E values were calculated using the formula: E = ln[(1 ꢁ c)(1 ꢁ eep)]/ln[(1 ꢁ c)(1 + eep)], c = ees/(ees + eep).

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S. Chen et al. / Tetrahedron Letters 57 (2016) 5312–5314
Table 2
Results of aminolysis reactionsa
O
O
O
OH
OH
OH
n
OAc
CALB/NH3
n
n
R
R
R
+
OH
OAc
Entry
Solvent
R
n
T (°C)
Yieldb (%)
eec (%)
Ed (%)
1
2
3
4
5
6
7
8
9
10
Isopropyl ether
Isopropyl ether
Isopropyl ether
Isopropyl ether
Isopropyl ether
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
H
o-Cl
p-OCH3
p-OCH3
H
H
o-Cl
p-OCH3
p-OCH3
H
0
0
0
0
1
0
0
0
0
1
25
25
25
35
25
25
25
25
35
25
49
43
41
47
40
48
47
44
49
45
99
99
99
97
99
99
98
99
98
99
>300
41
32
98
29
>200
99
48
>200
58
a
Conditions: Reactions were performed on a 10 mL scale, immobilized Candida antarctica lipase B, 2 mg, isopropyl ether/isooctane, 2 mL, substrate concentration, 20 mM.
Except otherwise stated, ee values and conversions (conv) were determined by HPLC-analysis with a chiral OD column.
b,c
Yields and ee values of aromatic
a-hydroxyl acid were determined by HPLC analysis equipped with a chiral OD column (150 ꢀ 2.1, Daicel Chemical Industries, Ltd) at a
wavelength of 254 nm at 30 °C.
d
E values were calculated by the formula: E = ln[(1 ꢁ c)(1 ꢁ eep)]/ln[(1 ꢁ c)(1 + eep)], c = ees/(ees + eep).
provide an alternative to obtain chiral amides with ee values of up
to 99%, though the yields cannot exceed 50%. Indicating that this
approach can be applied for large-scale preparations especially
for industrial purpose.
References and notes
1. (a) Mravik, A.; Bocskei, Zsolt; Katona, Z.; Markovits, I.; Pokol, G. Chem. Commun.
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Enzym. 2014, 99, 108–113.
In summary, we have developed an efficient enzymatic
2. (a) Yao, C. J.; Cao, Y.; Wu, S. S.; Li, S.; He, B. F. J. Mol. Catal. B: Enzym. 2013, 85–86,
105–110; (b) Ebert, Cynthia; Ferluga, G.; Gardassi, L.; Gianferrara, T.; Paolo, L.
Tetrahedron: Asymmetry 1992, 3, 903–912; (c) Martín, A.; Cocero, M. J. J.
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approach for kinetic resolution of
a-hydroxyl acid. The reaction
can proceed in both isopropyl ether and isooctane smoothly at
ambient temperatures, and anhydrous ammonia is employed as
the separation reagent. Due to the application of organic solvent,
the problems relating to low solubility of substrate are overcome
as the substrate a-hydroxyl acids are more soluble in organic sol-
vent than in aqueous solution. Simply by filtration, the biocatalyst
(immobilized) can be removed. A scale-up reaction was also per-
formed,7 and the prove that this protocol is more suitable for
large-scale preparations compared with conventional developed
methods, representing an appealing and promising protocol for
large-scale preparations.
7. Scale-up kinetic resolution of mandelic acid ester: a 250 ml round-bottom flask
was charged with mandelic acid ester of 75 mmol, 150 mL isopropyl ether and
immobilized lipase B from Candida antarctica 2 g, the flask was sealed and
anhydrous ammonia was added through the surface of solution for about 3 h.
The resulted mixture was stirred at 25 °C for another 9 h. Samples were taken at
Acknowledgments
Financial support from the National Natural Science Foundation
of China (No. 21502036), the Innovative Research team project of
Hainan Natural Science Foundation (No. 2016CXTD006), and Hai-
nan University (No. kyqd1408) is gratefully acknowledged.
1 h intervals, each time 20 ll of solution were used for analyzing. The separation
procedure is the same as described in Experimental section. And the resulted
crude residues were chromatographed on silica gel to afford the corresponding
pure a-hydroxyl acid in an isolated yield of 41%.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.10.
054.