Chiral separation of phenyllactic acid by helical structure from spring dextrin

Article abstract of DOI:10.1007/s10847-015-0487-x

We performed the enzymatic synthesis of spring dextrin (SD) with a helical conformation and investigated its chiral-recognition properties in relation to the stereoselective phenyllactic acid (PLA), using reversed-phase high-performance liquid chromatography. The effects of column temperature, buffer pH and methanol content on enantioselective separation were investigated. Baseline chromatographic separation was achieved on an Inertsil ODS-SP column using 1 % SD as the chiral mobile-phase additive. Helical structure was necessary for chiral separation of PLA, according to visible spectroscopy. Molecular dynamic simulations to predict the interactions between SD and PLA showed that van der Waals attractions played an important role in enantiomer separation.

Full text of DOI:10.1007/s10847-015-0487-x

J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-015-0487-x
SHORT COMMUNICATION
Chiral separation of phenyllactic acid by helical structure
from spring dextrin
•
•
•
Jin Xu Xueming Xu Qiang Wang
Xuerong Fan
Received: 15 September 2014 / Accepted: 19 February 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract We performed the enzymatic synthesis of spring
dextrin (SD) with a helical conformation and investigated its
chiral-recognition properties in relation to the stereoselec-
tive phenyllactic acid (PLA), using reversed-phase high-
performance liquid chromatography. The effects of column
temperature, buffer pH and methanol content on enan-
tioselective separation were investigated. Baseline chro-
matographic separation was achieved on an Inertsil ODS-SP
column using 1 % SD as the chiral mobile-phase additive.
Helical structure was necessary for chiral separation of PLA,
according to visible spectroscopy. Molecular dynamic
simulations to predict the interactions between SD and PLA
showed that van der Waals attractions played an important
role in enantiomer separation.
living organisms often show different biological activities
towards different enantiomers [2], the detection and as-
signment of chirality at the molecular level continues to be
an important issue. Phenyllactic acid (PLA) is frequently
used as the component of pharmaceuticals and natural
antibiotic agents [3]. L-PLA is used for the synthesis of
antiviral and antitumour compounds (pederin, onnamides
and theopederins) [4]. However, D-PLA is an integral part
of a cyclic depsipeptide (a novel Gq/11 protein inhibitor)
[5]. Therefore, it’s important for chiral separation of the
PLA enantiomers to synthesize the pharmaceuticals.
Dextrins, such as cyclodextrin (CD) and maltodextrin
(MD) (Fig. 1a), are optically-active polymers that can be
readily exploited for the chiral resolution of racemic
compounds. Their torch-like structure with a hydrophobic
internal surface and hydrophilic external surface gives CDs
the ability to form complexes with a variety of chiral
compounds [6, 7]. CDs are therefore widely used for the
chiral separation of enantiomers in the pharmaceutical in-
dustry, as mobile-phase additives or as chiral stationary
phases [8–11]. MDs, which are mixtures of malto-oligo and
polysaccharides, can also be used as chiral selectors [12,
13]. The mechanism of chiral recognition has been shown
to be attributable to the fact that the helical structure of the
MD mimics the cavity responsible for chiral recognition in
CDs [14, 15]. Amylose forms a helical structure with small
molecules, such as iodine, which can be included in the
hydrophobic interior of MDs (iodine color reaction), as in
the cavity of CDs. The helix structure of amylose is
therefore important in resolving racemic mixtures [16–18].
Spring dextrin (SD) [19] is a spiral polymer composed
of repeating (1-4)-a-D-glucose units that can be produced
by the enzymatic hydrolysis of amylose (by a-amylase)
[20] or by enzymatic synthesis (with amylosucrase) [21].
Recent studies demonstrated effective single-walled carbon
Keywords Spring dextrin ꢀ Helical structure ꢀ Chiral
discrimination
Introduction
The chromatographic resolution of enantiomers is one of
the most difficult challenges in analytical chemistry [1]. As
J. Xu ꢀ Q. Wang ꢀ X. Fan (&)
Key Laboratory of Eco-textiles of Ministry of Education,
College of Textiles and Clothing, Jiangnan University, 1800
Lihu AVE, Wuxi 214122, People’s Republic of China
e-mail: wxfxr@163.com
X. Xu
State Key Laboratory of Food Science and Technology, Jiangnan
University, Wuxi 214122, People’s Republic of China
X. Xu
School of Food Science and Technology, Jiangnan University,
1800 Lihu AVE, Wuxi 214122, People’s Republic of China
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J Incl Phenom Macrocycl Chem
Fig. 1 a Chemical structure of the cyclodextrin (left) and maltodextrin (right); b HPAEC-PAD profiles of the SD (DP values are indicated above
the peaks.)
nanotube (SWNT) dispersion with SD, and reported that
SD wrapped around SWNTs in a helical manner [22].
Further, SD has been used to improve the stability of
polyunsaturated fatty acids by encapsulating them within
with helical SD [23]. Importantly, analogs of SD (MD or
amylose) have been used for the chiral separation of dozens
of enantiomers [24–26]. Therefore, the current study aimed
to clarify the molecular mechanisms responsible for SD,
with a view to the future development of chiral mobile-
phase additives capable of resolving enantiomeric com-
pounds such as the PLA enantiomers.
purchased from Hui Cheng Biological Technology Co.,
Ltd. (Shanghai, China). All other chemicals were analytical
reagents purchased from commercial sources.
Enzymatic synthesis of SD by phosphorylase
and chain-length distribution
SD was synthesized from glucose 1-phosphate (G-1-P)
using glucan phosphorylase. Briefly, an aqueous solution of
G-1-P (10 mL, 4 mg/mL) was adjusted to pH 6.5 with
2 mol/L acetic acid and mixed with 0.1 M citrate buffer pH
6.5 (4 mL). Twenty milliliters of the phosphorylase solu-
tion was mixed with an aqueous solution of maltotetraose
and made up to 100 mL with water, and incubated at 40 °C
for 24 h. After the reaction, the mixtures were centrifuged
at 5000g for 10 min, and the precipitate was washed five
times with distilled water and then freeze-dried. The chain-
length distribution of the SD was characterized by high-
performance anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) [20].
Experimental
Chemicals and reagents
D-(?)-3-Phenyllactic acid (D-PLA), L-PLA, DL-PLA, tri-
fluoroacetic acid (TFA), triethylamine (TEA), methanol,
phosphorylase and glucose 1-phosphate were purchased
from Sigma-Aldrich (Shanghai, China). Maltotetraose was
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J Incl Phenom Macrocycl Chem
High-performance liquid chromatography analysis
and optimization of mobile-phase parameters
Method development
We investigated the effects of column temperature, buffer
pH and methanol content on separation of the PLA enan-
tiomers. The effect of column temperature on enan-
tioselectivity was investigated using 0.05 % TFA buffer
(adjusted with TEA, pH 2.5) with 1 % SD containing 10 %
methanol as the mobile phase. Rs decreased with increas-
ing temperature (20–60 °C), and baseline separation was
observed at 30 °C (Fig. 2a). The temperature effects on
enantioselectivities usually depend on the thermodynamic
interactions between the analytes and chiral selectors, an
enthalpy-driven process or an entropy-driven process [28].
The enantioselectivity for this assay in the relative lower
temperature ranges can probably be attributed to the slower
rotation of the guest and host molecules, hence a steric fit is
possible [29]. We therefore fixed the temperature at 30 °C
for further optimization. Since PLA has one carboxylic
acid functionality (pKa 3.46) [30], the molecule dissociates
in aqueous solution by releasing a proton. The effect of
buffer pH on the Rs of the PLA enantiomers was investi-
gated using 0.05 % TFA buffer (adjusted with TEA) con-
taining 1 % SD and 10 % methanol as the mobile phase.
The Rs decreased with increasing pH from 2.0 to 4.0
(Fig. 2b), indicating better separation at lower pH values.
This phenomenon can be possibly attributed to the decrease
of the formation of inclusions caused by the increasing
dissociation of the solute [31]. A pH value of 2.5 was used
for further studies, given that higher pH reduces life-cycle
costs. The effect of methanol concentration on the Rs of the
enantiomers was also investigated using 0.05 % TFA
buffer (adjusted with TEA, pH 2.5) with 1 % SD con-
taining 10–30 % methanol as the mobile phase. The Rs of
PLA decreased with increasing methanol concentrations in
the mobile phase (Fig. 2c). This finding was consistent
with previous research [31] showing that the Rs of analytes
decreased with increasing methanol concentrations, which
could be probably attributed to the increase of SD-analytes
inclusion complexes caused by the organic modifier [32].
Mobile-phase buffer containing 10 % methanol was
therefore used in subsequent experiments. Based on the
above results, baseline separation of PLA (Fig. 3a) was
performed on an Inertsil ODS-SP column (150 9 4.6 mm,
particle size 5 lm) under the specified conditions (0.05 %
TFA buffer (1 % SD, 10 % methanol, pH 2.5, 30 °C).
Stock solutions of L-PLA, D-PLA and DL-PLA were pre-
pared (1 mg/mL) by dissolving in distilled water, and then
stored at 4 °C. The stock solutions were diluted with dis-
tilled water to the required concentrations before use.
Separation and detection of L-PLA and D-PLA were
performed on a Shimadzu liquid chromatography system
consisting of an LC-20AT pump, an SIL-10A injector,
Inertsil ODS-SP column (150 9 4.6 mm, particle size
5 lm) and an SPD-10A UV–Vis detector (254 nm) (Shi-
madzu Corp., Tokyo, Japan). Data were analyzed using LC
solution software (Shimadzu Corp.). The injection volume
was 20 lL. The mobile phase was a mixture of 0.05 %
TFA buffer (adjusted with TEA, pH 2.5) containing 1 %
SD and methanol (90:10, v/v) at 30 °C with a flow rate of
1.0 mL/min (unless otherwise specified). The effects of
column temperature, pH and methanol content on the
enantiomeric separation ability [resolution (Rs)] were
studied. The Rs was based on the average of at least three
independent determinations of each solute.
Molecular dynamics simulations
The interactions between the two enantiomers of PLA and
SD were simulated using the molecular dynamics module
in HYPERCHEM 8.0 software (Hypercube Inc., Waterloo,
Canada). Simulations were performed in a periodic box
˚
(30 9 30 9 50 A), according to the size and shape of PLA
and SD. All established models were energetically pre-
optimized and obtained using the AMBER force field. The
pre-optimized configurations were heated for 1 ps to
303.15 K (30 °C) and then simulated at 303.15 K for 2 ps
at a step size of 0.001 ps containing water molecules. In-
formation for the equilibrated conformations was then
computed and parameters such as non-bonded interactions,
including van der Waals attractions (VdWs), hydrogen
bonds (HBs), and electrostatic forces (EFs) were subse-
quently analyzed.
Results and discussion
SD chain-length distribution
Calibration, precision and accuracy
Synthetic SD was analyzed by HPAEC-PAD (Fig. 1b). The
individual SD homologs were baseline resolved on a Car-
boPAC PA200 column (Thermo Fisher Scientific, Inc.,
United States). Individual and sharp peaks were well-re-
solved, strongly suggesting that the SDs were linear [27].
Calibration curves for D-PLA and L-PLA were linear over
concentrations ranging from 0.5 to 15 lg/mL, according to
linear regression analysis of the peak area (y) of each
enantiomer versus the concentration (x). The regression
equations
of
the
calibration
curves
were
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J Incl Phenom Macrocycl Chem
Fig. 2 The influences of
column temperature (a), pH
(b) and methanol content (c) on
the HPLC separation. Vis
spectra of the aqueous solutions
of SD-iodine complexes at
different temperatures (d)
Fig. 3 a Chromatographic
separation of PLA optical
isomers; b Curves of total
energy, potential energy, and
simulation temperature during
the simulation performed at
293.15 K in 3 ps (left L-PLA;
right D-PLA)
y = 29,375x ? 512,473, with a regression coefficient (r2)
of 0.9984 for D-PLA, and y = 31,529x ? 513,016
(r2 = 0.9951) for L-PLA. The limits of detection (LOD)
were 0.4 and 0.8 lg/mL in receptor medium for D-PLA and
L-PLA, respectively, measured based on a signal-to-noise
ratio (S/N) C3. The limits of quantitation (LOQ) (S/
N C 10) were 0.9 and 1.3 lg/mL for D-PLA and L-PLA,
respectively, with a relative standard deviation (RSD)
\9.7 %. The intra- and inter-day precisions and accuracy
of the method are shown in Table 1. The RSD values for
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J Incl Phenom Macrocycl Chem
intra- and inter-day assays were \5 %, indicating the re-
producibilities of the chromatographic conditions and the
method (Table 1). The accuracy of the method was
demonstrated by a percentage recovery of 93.7–102.6 %,
determined by spiking D-PLA to 1.0 mg/mL L-PLA at 0.07,
0.2, and 0.5 % levels.
Table 2 Changes of single point energies of SD and PLA complex
SD complex
Single point energies (kcal/mol)a
DEVdWb
DEHBb
DEEFb
L-PLA
D-PLA
a
224.3 0.2
154.4 0.1
-5.8 0.1
-3.3 0.2
1368.9 0.2
1378.0 0.2
Values are means standard deviations from three experiments of
simulation
Conformational transition of SD with temperature
b
Van der Waals attractions, Hydrogen bonds, Electrostatic forces
The transition of SD conformation with temperature was
investigated using the visible spectrum, with iodine as a
probe for helical structure. The visible spectra profiles of
the SD-iodine complexes at 20, 30, 40, 50, 60 and 70 °C,
respectively, are shown in Fig. 2d. The absorption at
548 nm decreased with increasing temperature, indicating
that the helical structures were gradually destroyed at
higher temperatures.
1
k0
1
k1½SDꢁ
¼
þ
/k½Aꢁ /k½Aꢁ
where k0 is chromatographic definition of capacity factor, /
is the phase ratio, A is a stationary phase adsorption site,
SD stands for spring dextrin, and k, k1and k2 are the re-
spective equilibrium constants. Plots of 1/k versus SD
concentration for the enantiomer gave the straight lines
Evaluation of inclusion complex formation
0
(regression
equation 1/k1
= 5.3218[SD] ? 0.1012;
The organic molecules could undergo non-covalent inter-
actions with dextrin leading to the inclusion complex for-
mation. The chiral HPLC method with chiral mobile phase
additive is an efficient way to determine the binding con-
stant of a solute to a dextrin molecule [33]. For a 1:1
complex the following equation could be used for deter-
mination of formation constant and the stoichiometry for
the inclusion complex.
0
1/k2 = 3.7956[SD] ? 0.1061), and the correlation coeffi-
cients of plots were greater than 0.998 indicating that the
stoichiometry was 1:1 for the inclusion complex. The
binding constants were 54.428 and 37.674 L/g,
respectively.
Molecular dynamic simulations
Stable conformations were obtained at 303.15 K (30 °C)
after heating for 1 ps to 303.15 K followed by simulation
at 303.15 K for 2 ps, according to the stable levels of total
energy, potential energy, and simulation temperature fi-
nally achieved. The curves of these parameters during
simulation at 303.15 K for 3 ps are shown in Fig. 3b. The
optimized conformation was in equilibrium after about
1.1 ps simulation at 303.15 K. Table 2 summarizes the
single-point energies of SD with the L- and D- enantiomers
(the symbol ‘-’ in Table 2 represents the force orientation).
Electrostatic energy showed a maximum value, indicating
that electrostatic energy was the main driving force re-
sponsible for complex formation. However, the maximum
change between the L-PLA complex and D-PLA complex
was DEVdW, suggesting that VdW interactions played an
important role during enantiomer separation.
Table 1 Precision and recovery data of this assay
Spiking level of the D-PLA (%) Parameter
RSD (%)
D-PLA L-PLA
Precision data
Repeatability (n = 5)
0.3
Retention time 0.12
Peak area 3.6
Retention time 0.16
Peak area 4.7
0.15
2.9
0.07(LOD)
0.19
3.2
Inter-day precision (n = 3)
0.3
Retention time 0.14
Peak area 4.4
Retention time 0.23
0.21
3.5
0.07 (LOD)
0.19
3.4
Peak area
Recovered (lg/mL)
4.1
Added (lg/mL)
% Recovery
% RSD
Conclusions
Recovery data
0.696
SD as a chiral mobile-phase additive provides a flexible
alternative for the separation of enantiomers by allowing
separations to be performed on conventional columns. We
developed a simple, specific, precise and rugged reversed-
0.652
2.038
5.202
93.7
96.4
4.6
2.9
4.1
2.114
5.070
102.6
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Acknowledgments This work was supported by National Natural
Science Foundation of China (No. 31401647), Nature Science
Foundation of Jiangsu Province (No. BK20140150), Fundamental
Research Funds for the Central Universities (JUSRP11449), Program
for Changjiang Scholars and Innovative Research Team in University
(IRT1135) and Priority Academic Program Development of Jiangsu
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