Enantioselective resolution of 4-chloromandelic acid by liquid–liquid extraction using 2-chloro-N-carbobenzyloxy-L-amino acid

Article abstract of DOI:10.1002/chir.22738

A liquid–liquid extraction resolution of 4-chloro-mandelic acid (4-ClMA) was studied by using 2-chloro-N-carbobenzyloxy-L-amino acid (2-Cl-Z-AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4-ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)-4-ClMA was much higher than that of (S)-4-ClMA in organic phase due to a higher stability of the complex formed between (R)-4-ClMA and 2-Cl-Z-AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2-Cl-Z-Valine dissolved in dichloromethane, pH of 2.0, concentration of 4-ClMA of 0.11 mmol/Land T of 296.7K.

Full text of DOI:10.1002/chir.22738

Received: 18 January 2017
DOI: 10.1002/chir.22738
Revised: 28 June 2017
Accepted: 30 June 2017
R E G U L A R A R T I C L E
Enantioselective resolution of 4‐chloromandelic acid by
liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐
L‐amino acid
Ruichen Lu1
| Quan He2
| Cai Feng1 | Yangfeng Peng1
1 School of Chemical Engineering, East
China University of Science and
Technology, Shanghai, China
2 Department of Engineering, Faculty of
Agriculture, Dalhousie University, Truro,
Nova Scotia, Canada
Abstract
A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was
studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a
chiral extractant. Important factors affecting the extraction efficiency were
investigated, including the type of chiral extractant, pH value of aqueous phase,
initial concentration of chiral extractant in organic phase, initial concentration
of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that
the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in
organic phase due to a higher stability of the complex formed between (R)‐4‐
ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02
mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration
of 4‐ClMA of 0.11 mmol/Land T of 296.7K.
Correspondence
Y‐F Peng, School of Chemical
Engineering, East China University of
Science and Technology, 130 Meilong
Road, Shanghai, China, 200237.
Email: yfpeng@ecust.edu.cn
KEYWORDS
2‐chloro‐N‐carbobenzyloxy‐L‐amino acid, 4‐chloromandelic acid, chiral separation, liquid–liquid
extraction, N‐(2‐chlorobenzyloxy)‐succinimide, valine
1 | INTRODUCTION
and metalloids,8,9 tartaric acid esters,10,11 dibenzoyl
tartaric acid (DBTA),12 mandelic acid ester, and cyclodex-
With a rapidly rising demand on single enantiomers of
chiral compounds, especially of chiral drugs, development
of resolution technology has received increasing attention.
Methods of enantioseparation include chromatographic
resolution, diastereomeric salts formation, electrophoretic
techniques, kinetic resolution, membrane‐assisted
separation, and enantioselective liquid–liquid extraction
(ELLE).1-6 Among these methods, ELLE stands out as a
promising option due to its low cost, readiness for scaling
up, and great potential for continuous operation.
ELLE is closely related to host–guest chemistry,
involving both chiral discrimination and solvent extrac-
tion.7 A good chiral extractant has to strike a balance
between its chiral recognition ability and its distribution
in two extraction phases. Currently, the most commonly
used resolving agents are crown ethers, metal complexes
trin as well as its derivative.13-15 Amino acids are a group
of chiral compounds abundantly available in nature,
which could potentially be used as chiral extractants.
However, amino acids generally have poor solubility in
organic solvents, which limits their application in
enantioselective extraction. A slight modification of amino
acids might not only improve their dissolving ability in
organic phase, but also change the chemical structure,
possibly beneficial to their chiral recognition ability. In
this study, a series of 2‐chloro‐N‐carbobenzyloxy‐L‐amino
acid (2‐Cl‐Z‐AA) were synthesized from L‐amino acids
and
N‐(2‐chlorobenzyloxy‐carbonyloxy)‐succinimide
(2‐Cl‐Z‐osu). These newly developed chiral extractants
were tested in the resolution of 4‐chloromandelic acid
(4‐ClMA), including 2‐Cl‐Z‐alanine, 2‐Cl‐Z‐phenylalanine,
2‐Cl‐Z‐proline, 2‐Cl‐Z ‐isoleucine, and 2‐Cl‐Z valine.
Chirality. 2017;1–8.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

2
LU ET AL.
4‐Chloromandelic acid (4‐ClMA) is currently
ric acid were provided by Shang‐hai LingFeng Chemical
Reagent (China) and were of analytical‐reagent‐grade
except for acetonitrile.
marketed in its racemic form. Enantiopure (R)‐4‐ClMA
is an important chiral intermediate to synthesize drugs
for diabetes and lipid disorder treatment.16 A number of
resolution methods have been reported, such as chro-
matographic methods and diastereomeric salts.17-20 How-
ever, there are some drawbacks, such as low yield and
high cost of the resolving agents. This work is an attempt
for enantioselective liquid–liquid extraction of 4‐ClMA
using amino acid derivatives. Chemical structures of
substrate and chiral extractants in this resolution are
shown in Figures 1 and 2.
2.2 | Instruments
The HPLC system (Hegong Instrument, Shanghai,
China) consists of a Vertex STI P5000 pump, an STI UV
detector (wavelength range of 190–700 nm), a 10 μL
sample loop (Rheodyne, Sigma Aldrich, St. Louis, MO),
and a 7725i sampler. A Leici pHS‐25 pH meter equipped
with an E‐201‐C combined glass electrode (Shanghai,
China) was used for pH measurement. DLSB‐5/10 low‐
temperature coolant (Yu Hua Instrument, China) was
used for temperature control during each extraction
process.
2 | MATERIALS AND METHODS
2.1 | Materials
4‐ClMA and 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid
were purchased from Dongtai Dongjin Medical and Tech-
nology (China), with purity of 98%. Hydroxypropyl‐β‐
Cyclodextrin (HP‐β‐CD) with purity of 99.5% was pur-
chased from Shandong Binzhou Zhiyuan Biotechnology
(China). Acetonitrile (high‐performance liquid chroma-
tography [HPLC]‐grade), five organic diluents like dichlo-
romethane, listed in Table 1, disodium hydrogen
phosphate, sodium dihydrogen phosphate, and phospho-
2.3 | Extraction experiments
Altogether, 0.02 g (0.107 mmol) 4‐ClMA and 0.01 g
(0.25 mmol) NaOH were added to 50 mL of distilled
water, giving 4‐chloromandelic acid sodium salt aqueous
phase containing 0.4 g/L (2.14 mmol/L) of 4‐ClMA. For
each extraction resolution, aqueous phases with a preset
pH were prepared by a certain amount volume of aqueous
phase described above, and a buffer solution containing
0.01 mol/L phosphate salt. 2‐Cl‐Z‐amino such as alanine,
phenylalanine, proline, isoleucine, and valine were dis-
solved respectively in organic diluents to prepare organic
phases. Equal volumes (each 5 mL) of the aqueous and
the organic phase were mixed in a 25‐mL glass bottle
and set in a water bath, maintaining a preset temperature
for 1 h. After settling and phase separation, the concentra-
tions of 4‐ClMA enantiomers in the aqueous phase were
analyzed by HPLC. Due to a low immiscibility between
the aqueous phase and organic phase, the change in
volume was negligible. The concentrations of 4‐ClMA
enantiomers in the organic phase can be calculated by
mass difference; the distribution coefficient K is defined
as follows:
FIGURE 1 Molecular structure of 4‐ClMA
FIGURE 2 Molecular structure of 2‐cl‐Z‐AA
KR ¼ CRO=CRW
KS ¼ CSO=CSW
(1)
(2)
TABLE 1 Effects of different organic solvents on K and α
Organic diluent
KS
KR
α
1,2‐dichloroethane
0.309
0.311
1.004
Dichloromethane
n‐octyl alcohol
Isoamyl alcohol
n‐butyl alcohol
0.474
18.1
21.0
23.6
0.510
18.8
22.3
25.8
1.08
1.04
1.06
1.09
The separation factor is defined as follows:
α ¼ KR=KS
(3)
where CRO and CRW represent the concentration of (R)‐
4‐ClMA in organic phase and aqueous phase, respectively;
Extraction conditions: concentration of 4‐ClMA = 0.11 mmol/L; concentra-
tion of 2‐Cl‐Z‐alanine = 0.01 mol/L; pH = 2.5; T = 276.7 K.

LU ET AL.
3
CSO and CSW represent the concentration of (S)‐4‐ClMA
in organic phase and aqueous phase, respectively.
2.4 | Determination of the dissociation
constant
The dissociation constant of 4‐ClMA was determined by
an acid–base potentiometric titration method using a
pHS‐25 pH meter equipped with an E‐201‐C combined
glass electrode (Leici).21 The pH electrode was calibrated
by buffer solutions with pH of 4.00 and pH of 6.86,
respectively, before use. 4‐ClMA (ab 1 mmol) added to
a beaker with magnetic stirring and temperature control
(T = 20 0.1 °C), was mixed with 20 mL absolute etha-
nol, 20 mL deionized water, and 5 mL potassium chlo-
ride (c = 1 mol/L). Potassium chloride served to
stabilize the ionic strength of the solution. An initial
pH value was measured, then 0.1 mol/L NaOH solution
was slowly dropped into the beaker; furthermore, the
volume of NaOH solution added and the pH value of 4‐
ClMA solution were recorded until the pH value reached
13. This procedure was repeated one time. The dissocia-
tion constant was calculated by the average value of
two experiments according to the method reported in
the literature.21
FIGURE 3 Chromatogram of 4‐ClMA enantiomers in the
aqueous phase
3 | RESULTS AND DISCUSSION
3.1 | Selection of organic diluents
In this study, organic solvents such as dichloromethane,
1,2‐dichloroethane, n‐octyl alcohol, isoamyl alcohol, and
n‐butyl alcohol were examined in extractive resolution
of 4‐ClMA using 2‐Cl‐Z‐amino as an extractant. The
effects of different organic solvents on K and α are listed
in Table 1. It indicates that 4‐ClMA is completely dis-
solved in solvents like n‐octyl alcohol, isoamyl alcohol,
and n‐butyl alcohol. Therefore, these solvents are not suit-
able to be used as organic phases in this study. Both
dichloromethane and 1,2‐dichloroethane gave proper Ks
and KR, indicating that 4‐ClMA was partially dissolved
in those two solvents. The measured Ks and KR of 4‐ClMA
in solvent 1,2‐dichloroethane were 0.309 and 0.311,
respectively, giving a selectivity of 1.004, while for dichlo-
romethane, the values of Ks and KR were 0.474 and 0.510,
respectively, giving a selectivity of 1.08. The concentration
of 4‐ClMA in this study was determined by an HPLC
method with an experimental error of ~2% as described
in the “Analytical methods.” Based on the error propaga-
tion rule, the relative error of K and α would be 4% and
8%, respectively. Taking the uncertainty into consider-
ation, there is no significant difference in separation fac-
tor between dichloromethane and 1,2‐dichloroethane.
Dichloromethane with a relatively high solubility of 4‐
ClMA was therefore selected as an organic solvent for
the following studies.
2.5 | Determination of solubility
To a solution of 0.02 mol/L of 2‐Cl‐Z‐Val in dichlorometh-
ane (5 mL), 0.01 g (R)‐4‐ClMA or (S)‐4‐ClMA was added
and stirred for 0.5 h at temperatures of 276.7 K, 281.7 K,
286.7 K, 291.7 K, 296.7 K, 303.2 K, 313.2 K, and 323.2 K,
giving a series of saturation solutions. The saturation solu-
tions were filtered and the concentrations of 4‐ClMA
enantiomers were determined by HPLC analysis against
a calibration curve.
2.6 | Analytical methods
The 4‐ClMA enantiomers were analyzed by HPLC
using a C18 column (Hedera ODS‐2 silica gel C18 col-
umn (250 mm × 4.6 mm × 5 μm, Hanbang, China)
with a UV detector at a wavelength of 220 nm at
293.2 K. The mobile phase consisted of a mixture of
aqueous buffer (pH of 2.0, 8 mM sodium dihydrogen
phosphate and 5 mM hydroxypropyl‐β‐cyclodextrin)
and acetonitrile with a volumetric ratio of 95:5 at a
flow rate of 1.0 mL/min. The retention time of (S)‐4‐
ClMA was less than that of (R)‐4‐ClMA, as shown in
Figure 3. The accuracy of the method was evaluated
by the standard additions method, in which the
recoveries were 100.2% and 102.0% for (S)‐4‐ClMA and
(R)‐4‐ClMA, respectively.
3.2 | Selection of chiral extractants
According to their structures, amino acids can be classi-
fied as aliphatic, aromatic, heterocyclic amino acids, and
heterocyclic imino acids. In this study, five amino acids
were selected for examining the resolution of 4‐ClMA in

4
LU ET AL.
the solvent dichloromethane, including alanine, phenylal-
anine, proline, isoleucine, and valine. These amino acids
can be easily modified to 2‐Cl‐Z‐AA through reacting with
2‐Cl‐Z‐osu, which have been widely applied in peptide
synthesis.22,23 After such modifications, the solubility of
amino acids in organic solvents were improved consider-
ably, and this group of amino acids could serve as promis-
ing chiral extractants in extractive resolution. Due to the
different structure of the parent amino acids, the chiral
recognition abilities of 2‐Cl‐Z‐AA with 4‐ClMA were dif-
ferent. The resolution performance is listed in Table 2.
Among the five 2‐Cl‐Z‐AA (alanine, phenylalanine, pro-
line, isoleucine, valine), the distribution coefficient K
was in the range of 0.253–0.526, and the separation factor
α was from 1.01–1.4. The separation factor of 2‐Cl‐Z‐
phenylalamine was the lowest, while 2‐Cl‐Z‐valine exhib-
ited the highest separation factor. From these results, it is
difficult to establish a clear correlation between the struc-
ture of amino acids and the extraction selectivity. For
example, comparing the three aliphatic amino acids of
interest, 2‐Cl‐Z‐alanine has the simplest structure, while
a low α; 2‐Cl‐Z‐valine's structure is bigger than 2‐Cl‐Z‐ala-
nine and it showed the highest α of 1.41; however, 2‐Cl‐Z‐
isoleucine, with one more methyl group than 2‐Cl‐Z‐
valine and two chiral carbons, exhibited a moderate α of
1.19. Hereby, a good interpretation of the experimental
results escaped our studies, and more research is needed
in terms of the effects of structure of amino acid on chiral
recognition ability.
dissociation constant of 4‐ClMA was determined to be
5.77 × 10−5. The varying distribution of ionic and molec-
ular form of 4‐ClMA with pH is displayed in Figure 4.
When the pH value was low, the enantiomers of 4‐ClMA
were mostly in their molecular forms. With an increase
in pH value, the fraction of free 4‐ClMA decreased and
the fraction of the ionic form increased. These directly
led to varied quantities of 4‐ClMA in organic and aque-
ous phases. In the process of extraction resolution, the
concentration of ionic and free form of 4‐ClMA influ-
enced the K and α significantly. As shown in Figures 5
and 6, with increasing pH, the distribution coefficients
of both R and S forms of 4‐ClMA decreased, indicating
a decrease in concentration of molecular form in aque-
ous phase, while α showed an increasing trend. When
the pH was greater than 4, a maximum separation factor
was observed; however, under such conditions the KR
was only 0.2, leading to a low separation efficiency. Con-
sidering a compromise between α and K, the pH value
was set at 2.
2‐Cl‐Z‐valine showed the highest α among the 2‐Cl‐Z‐
amino acids tested in this study, and was therefore
selected as the chiral extractant for the following studies.
3.3 | Effect of pH on chiral extraction
FIGURE 4 Species fraction as a function of pH: Free 4‐ClMA,
anion 4‐ClMA
For a substrate with a carboxyl or amino group, the pH
value has a great impact on its existing form in the aque-
ous phase.23 4‐ClMA is an organic acid, and can exist in
the ionic form, molecular form, or a mixture of two
forms depending on the value of pH. The acid
TABLE 2 Effects of resolving agents on K and α
Chiral extractant
KS
KR
α
2‐cl‐Z‐alanine
0.474
0.510
1.08
2‐cl‐Z‐phenylalanine
2‐cl‐Z‐proline
0.466
0.253
0.444
0.364
0.471
0.388
0.526
0.508
1.01
1.30
1.19
1.41
2‐cl‐Z‐isoleucine
2‐cl‐Z‐valine
Extraction conditions: concentration of 4‐ClMA = 0.11 mmol/L; concentra-
tion of 2‐Cl‐Z‐amino = 0.01 mol/L; organic diluents: dichloromethane;
pH = 2.5; T = 276.7 K.
FIGURE 5 Effect of pH on K

LU ET AL.
5
FIGURE
6
Effect of pH on α/Extraction conditions:
FIGURE 8 Effect of 2‐Cl‐Z‐Valine concentration on α/Extraction
conditions: concentration of 4‐ClMA = 0.11 mmol/L; organic
diluents: dichloromethane; pH = 2.0; T = 276.7 K
concentration of 4‐ClMA = 0.11 mmol/L; concentration of 2‐Cl‐Z‐
Valine =0.01 mol/L; organic diluents: dichloromethane; T = 276.7 K
Taking the cost of chiral extractants into consideration,
the 2‐Cl‐Z‐Valine concentration of 0.02 mol/L obviously
obtained the best separation performance when the con-
centration of the substrate, 4‐ClMA, was 0.11 mmol/L.
3.4 | Effect of 2‐cl‐Z‐valine concentration
on chiral extraction
The influence of 2‐Cl‐Z‐Valine concentration on K and α
is shown in Figures 7 and 8, respectively. With the con-
centration of 2‐Cl‐Z‐Valine increased from 0.01 to
0.03 mol/L, K increased accordingly and then stayed
almost unchanged when the concentration of 2‐Cl‐Z‐
Valine was beyond 0.02 mol/L. The value of α was
enhanced greatly, and then less significantly with the
increasing concentration of 2‐Cl‐Z‐Valine. This was due
to more and more complexes formed between 4‐ClMA
enantiomers and 2‐Cl‐Z‐Valine in the organic phase14
The stability of complexes formed between (R)‐4‐ClMA
and 2‐Cl‐Z‐Valine was much larger than the complexes
formed between (S)‐4‐ClMA and 2‐Cl‐Z‐Valine; as a
result, KR increased faster than KS, and thus α was
improved considerably. It was also observed that when
the concentration of 2‐Cl‐Z‐Valine was greater than
0.02 mol/L, the increase in both K and α was insignificant.
3.5 | Effect of 4‐ClMA concentration of
chiral extraction
The concentration of resolution substrate is also one of
the factors that affect chiral extraction. The effects of 4‐
ClMA concentration on K and α are shown in Figures 9
and 10. It was found that both K and α increased with
an increase in 4‐ClMA concentration, and α reached the
maximum value when the concentration of 4‐ClMA was
0.11 mmol/L. To obtain an efficient chiral separation,
the 4‐ClMA concentration was set at 0.11 mmol/L.
3.6 | Effect of temperature on chiral
extraction
The effect of temperature on the extraction process was
investigated as shown in Figures 11 and 12. In the
FIGURE 7 Effect of 2‐cl‐Z‐valine concentration on K
FIGURE 9 Effect of 4‐ClMA concentration on K

6
LU ET AL.
the observations reported in other studies, in which a
low temperature was favorable for extractive resolu-
tions.13,24,25 In the resolution of 4‐ClMA with 2‐Cl‐Z‐
Valine, the separation factor was 2.0 at a temperature of
276.7 K, and then increased to 3.05 when the temperature
reached 296.7 K. This could be attributed to the difference
in solubility change of (R)‐4‐ClMA and (S)‐4‐ClMA in
organic phase with temperature. The degrees in solubility
change with temperature of a pair of enantiomers, (R)‐4‐
ClMA and (S)‐4‐ClMA, in aqueous phase stayed the same
due to the absence of a chiral extractant. However, in the
organic phase, dichloromethane containing 2‐Cl‐Z‐
Valine, the solubility changes with T of (R)‐4‐ClMA and
(S)‐4‐ClMA showed a difference. The measured solubility
data are summarized in Table 3. Figure 13 plots ln(x) as a
FIGURE 10 Effect of 4‐ClMA concentration on α/Extraction
conditions: concentration of 2‐Cl‐Z‐Valine =0.02 mol/L; organic
diluents: dichloromethane; pH = 2.0; T = 276.7 K
TABLE 3 The solubility of (R)‐and (S)‐4‐ClMA in the organic
phase and water phase, respectively
4
4
4
T(K)
XRO(×10 )
XSO(×10 )
xw(×10 )
276.7
2.522
2.663
3.104
3.375
3.681
4.060
5.298
6.432
2.667
2.815
3.227
3.475
3.819
4.197
5.470
6.604
1.045
1.103
1.209
1.325
1.417
1.673
2.070
3.133
281.7
286.7
291.7
296.7
303.2
313.2
323.2
FIGURE 11 Effect of T on K
Organic phase: dichloromethane containing 0.02 mol/L 2‐Cl‐Z‐Valine; water
phase: 0.01 mol/L phosphate salt buffer solution with pH = 2.0; xRO and xSO
means the molar fraction of (R)‐and (S)‐4‐ClMA in the organic phase, respec-
tively; xw means the molar fraction of (R)‐or (S)‐4‐ClMA in the water phase.
FIGURE 12 Effect of T on α/Extraction conditions: concentration
of 4‐ClMA = 0.11 mmol/L; concentration of 2‐ Cl‐Z‐Valine
=0.02 mol/L; organic diluents: dichloromethane; pH = 2.0
FIGURE 13 The solubility of (R)‐and (S)‐4‐ClMA in the solution
of organic phase and water phase respectively/Organic phase:
dichloromethane containing 0.02 mol/L 2‐Cl‐Z‐Valine; water phase:
0.01 mol/L phosphate salt buffer solution with pH = 2.0
temperature range of 276.7 K to 296.7 K, the distribution
coefficients K decreased while α increased with the rise
of temperature. However, this result was different from

LU ET AL.
7
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lnxw ¼ −1:9494−2023:1=T; R ¼ ‐0:977
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lnxSO ¼ −1:9489−1749:5=T; R ¼ ‐0:995
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(6)
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Comparing the slopes of two lines for (R)‐4‐ClMA and
(S)‐4‐ClMA, it was found that the degree of solubility
change of (R)‐4‐ClMA in the organic phase was slightly
greater than that of (S)‐4‐ClMA, indicating that a higher
temperature favored formation of complexes between
(R)‐4‐ClMA and 2‐Cl‐Z‐Valine. Therefore, an increasing
temperature increased KR more significantly than KS,
and consequently the separation factor, α = KR/KS
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explained our observations in this study.
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In this study, 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐
Cl‐Z‐AA) for the first time was demonstrated to be a use-
ful extractant for enantioselective extraction of 4‐ClMA.
Among five amino acid derivatives tested in this work,
2‐Cl‐Z‐valine exhibited the best chiral discrimination abil-
ity, leading to an efficient resolution, with a separation
factor of 3.05. The optimal extraction conditions were
determined to be dichloromethane as a solvent, 2‐Cl‐Z‐
Valine concentration of 0.02 mol/L in, pH of 2.0, concen-
tration of 4‐ClMA of 0.11 mmol/L, and T of 296.7 K. 2‐
Chloro‐N‐carbobenzyloxy‐L‐amino acid holds promise in
the separation of 4‐ClMA, and the work broadened the
library of chiral selectors for enantioselective liquid–liq-
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