Preparative separation of enantiomers based on functional nucleic acids modified gold nanoparticles

Article abstract of DOI:10.1002/chir.22208

The preparative-scale separation of chiral compounds is vitally important for the pharmaceutical industry and related fields. Herein we report a simple approach for rapid preparative separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs). The separation of _DL-tryptophan (_DL-Trp) is demonstrated as an example to show the feasibility of the approach. AuNPs modified with enantioselective aptamers were added into a racemic mixture of _DL-Trp. The aptamer-specific enantiomer (_L-Trp) binds to the AuNPs surface through aptamer-_L-Trp interaction. The separation of _DL-Trp is then simply accomplished by centrifugation: the precipitate containing _L-Trp bounded AuNPs is separated from the solution, while the _D-Trp remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured under 95 °C and a second centrifugation is then performed, resulting in the separation of AuNPs and _L-Trp. The supernatant is finally collected to obtain pure _L-Trp in water. The results show that the racemic mixture of _DL-Trp is completely separated into _D-Trp and _L-Trp, respectively, after 5 rounds of repeated addition of fresh aptamer-modified AuNPs to the _DL-Trp mixture solution. Additionally, the aptamer-modified AuNPs can be repeatedly used for at least eight times without significant loss of its binding ability because the aptamer can be easily denatured and renatured in relatively mild conditions. The proposed approach could be scaled up and extended to the separation of other enantiomers by the adoption of other enantioselective aptamers. Chirality 25:751-756, 2013. 2013 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22208

CHIRALITY (2013)
Preparative Separation of Enantiomers Based on Functional Nucleic
Acids Modified Gold Nanoparticles
*
*
RONG HUANG, DAIFANG WANG, SHUZHEN LIU, LONGHUA GUO, FANGFANG WANG, ZHENYU LIN, BIN QIU, AND GUONAN CHEN
Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety (Fuzhou University), and Department of Chemistry,
Fuzhou University, Fuzhou, Fujian, China
ABSTRACT
The preparative-scale separation of chiral compounds is vitally important for the
pharmaceutical industry and related fields. Herein we report a simple approach for rapid prepara-
tive separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs).
The separation of DL-tryptophan (DL-Trp) is demonstrated as an example to show the feasibility
of the approach. AuNPs modified with enantioselective aptamers were added into a racemic
mixture of DL-Trp. The aptamer-specific enantiomer (L-Trp) binds to the AuNPs surface through
aptamer-L-Trp interaction. The separation of DL-Trp is then simply accomplished by centrifugation:
the precipitate containing -Trp bounded AuNPs is separated from the solution, while the D-Trp
L
remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured
under 95°C and a second centrifugation is then performed, resulting in the separation of AuNPs
and L-Trp. The supernatant is finally collected to obtain pure L-Trp in water. The results show that
the racemic mixture of DL-Trp is completely separated into D-Trp and -Trp, respectively, after 5
L
rounds of repeated addition of fresh aptamer-modified AuNPs to the DL-Trp mixture solution.
Additionally, the aptamer-modified AuNPs can be repeatedly used for at least eight times
without significant loss of its binding ability because the aptamer can be easily denatured and
renatured in relatively mild conditions. The proposed approach could be scaled up and
extended to the separation of other enantiomers by the adoption of other enantioselective
aptamers. Chirality 00:000–000, 2013. © 2013 Wiley Periodicals, Inc.
KEY WORDS: aptamer; chiral compounds; DL-tryptophan; gold nanoparticles; preparative-scale
enantioseparation
INTRODUCTION
multiple of chiral selector(s) as stationary phase, chiral mobile
additives, or chiral counterions. Previously, crown ethers,21
cyclodextrins,22 cellulose and amylase,23 polysaccharides,24
maltodextrins,25 and macrocyclic antibiotic classes26 have been
extensively described for enantioseparation. Recently, a new
group of chiral selectors, nucleic acid aptamers, is emerging
in the enantioseparation field.27,28 Aptamers are synthetic
single-strand nucleic acids isolated by the systematic evolution
of ligands by exponential enrichment (SELEX) procedure and
characterized by very impressive affinity and selectivity proper-
ties to some given targets ranging from molecules to proteins
and even cells.29 Aptamers possess the advantages of chemical
stability, readily availability, simple modifiability, reusability,
and high flexibility.30–32 To date, aptamers have been used as
chiral additives in CE for separation of arginine33 and
tryptophan34 in CEC for the chiral separation of a series of
chiral herbicides,35 and as chiral stationary phase to resolve
Chirality is a fundamental property of nature. It is well
known that the metabolic and regulatory processes mediated
by biological systems are sensitive to stereochemistry and
different responses can be often observed when comparing
the activities of a pair of enantiomers.1,2 Therefore, chirality
and its consequences have to be considered when studying
xenobiotics, such as drugs and agrochemicals. In the past
few decades the increasing demand of pure chiral compounds
in the pharmaceutical industry has favored the rapid
developments in separation techniques. Although some
stereoselective syntheses have been developed to produce
single-enantiomeric substances, relatively few are selected
for large-scale preparations.3 Additionally, at the early stages
of the evaluation of a therapeutic drug, the development of
an asymmetric synthesis would be both expensive and time-
consuming.4 Consequently, approaches for preparative
separations of enantiomers from their racemic mixtures are
in great demand.
Hitherto, a number of strategies have been described and
applied for the separation of the enantiomeric composition
of chiral compounds.5–8 Among the diverse technologies,
chromatographic-based chiral resolution is the most widely
used techniques,9 e.g., high-performance liquid chromatogra-
phy (HPLC),10,11, capillary electrophoresis (CE),12,13 capillary
electrochromatography (CEC),14,15 gas chromatography
(GC),16,17 supercritical fluid chromatography (SFC),18 and
simulated moving bed chromatography (SMB)19,20 have been
successfully utilized for enantioseparation. One of the
common features of these techniques is the use of one or a
Contract grant sponsor: National Science Foundation for Fostering Talents in
Basic Research of China; Contract grant number: J1103303.
Contract grant sponsor: National Natural Science Foundation of China; Contract
grant number: 21277025, 21205017, 21275031.
Contract grant sponsor: National Science n of Fujian Province; Contract grant
number: 2012 J01036, 2011J01035.
Contract grant sponsor: Foundation of Fujian Educational Committee; Con-
tract grant number: JA12039.
*Correspondence to: Longhua Guo or Guonan Chen, Ministry of Education
Key Laboratory of Analysis Detection Technology for Food Safety (Fuzhou
University), and Department of Chemistry, Fuzhou University, Fuzhou,
Fujian, 350108, China. E-mail: guolh@fzu.edu.cn or gnchen@fzu.edu.cn
Received for publication 26 April 2013; Accepted 5 June 2013
DOI: 10.1002/chir.22208
Published online in Wiley Online Library
(wileyonlinelibrary.com).
© 2013 Wiley Periodicals, Inc.

HUANG ET AL.
racemates for HPLC36. It should be noted that most of the
above-mentioned approaches are utilized for enantioselective
determination of chiral compounds. In contrast, few of them
are developed for preparative-scale separation of enantiomers.
In this contribution, aptamers functionalized AuNPs were
utilized as chiral selectors for preparative-scale separation of
enantiomers. The nature of aptamers, which are easy to be
denatured and renatured, enables the reusability of the chiral
selectors, while the large specific surface area of the AuNPs
ensures the maximum amount of binding sites. Additionally,
the separation of the target enantiomer from its enantiomer
counterpart can be simply accomplished by centrifugation—
after centrifugation, the aptamer-specific entiomers were
coprecipitated with the AuNPs, while their entiomer counter-
parts remain in the supernatant. In view of the fact that
hitherto a number of aptamers targeted on different chiral
compounds have been isolated, the proposed approach has
the potential to be extended to the preparative separation of
a series of chiral compounds.
at room temperature for at least 16 h. Ten μL of 500 mM Tris acetate
(pH 8.2) buffer and 100 μL of 1 M NaCl were then added to the solution
dropwise with gentle hand shaking. The solution was allowed to stand
for another 24 h, followed by centrifugation for 15 min at 5000 rpm. As
much supernatant as possible was gently pipetted off to remove free
DNA. After discarding the supernatant, the pellets were washed,
recentrifuged, and redispersed in 1 mL of an aqueous solution containing
20 mM Tris acetate, pH 8.2.
Enantioseparation With Aptamer-AuNPs
The prepared aptamer-AuNPs was incubated with 0.5 mM DL-Trp for
30 min at room temperature in binding buffer. Then it was centrifuged
for 15 min at a proper rate. The supernatant was collected for future
separation. The pellets were dispersed in 100 μL double-distilled water
and denatured at 95 °C for 5 min, then immediately centrifuge for
15 min at a proper rate. The supernatant was collected for evaluating
the resolution effect.
RESULTS AND DISCUSSION
Principle of Enantioseparation Based on Functional
Nucleic Acids Modified AuNPs
Scheme 1 shows the proposed enantioseparation strategy.
The aptamer-modified AuNPs were used as probes to recog-
nize the corresponding target enantiomers with high affinity
and specificity. After interacting with the racemic trp, the
L-Trp bounded AuNPs were separated from the solution by
centrifugation. The L-Trp-bounded AuNPs were redispersed
in distilled water. The aptamers were denatured at 95 °C for
5 min, and immediately centrifuged at an appropriate speed
for 15 min. The supernatant was collected and the aptamer-
modified AuNPs were separated for the next round of separa-
tion. After several rounds of separation, the racemic trp solution
could be completely separated, as shown in Scheme 1.
Figure 1A shows a typical electropherogram of a racemic
mixture of DL-Trp separated in a buffer containing α-CD as
chiral selector. It can be clearly seen that under this
condition, the two enantiomers of Trp can be baseline-
separated. Figure 1B shows the electropherogram of a
sample containing the supernatant collected from a denatured
AuNP-aptamer solution after the proposed enantioseparation
procedures. The results show that only L-Trp was found in this
solution. This indicates that the proposed approach can
efficiently extract L-Trp from the racemic mixture of DL-Trp. In
order to confirm that the peak represented L-Trp, the other
two experiments were conducted. Figure 1C is the electrophe-
rogram after addition of the 10μL racemic mixture of DL-Trp
(1 mM) into B. Two distinct peaks were observed in this
electropherogram, and the first peak (corresponding to L-Trp)
was higher than the second one. This result strongly indicates
EXPERIMENTAL
Reagents and Materials
HAuCl4 ·4H2O and sodium citrate were purchased from Shanghai
Sinopharm Chemical Reagent Co. Ltd (China). DL-Trp, L-Trp, and α-cyclodextrin
(α-CD) were obtained from Sigma (St. Louis, MO). The sequence of DNA
aptamer against L-Trp was as follows: 5'SH-AGC ACG TTG GTT AGG
TCA GGT TTG GGT TTC GTG C-3’,37 which was synthesized by Shanghai
Sangon Biological Engineering Technology & Services Co. (Shanghai,
China) and purified by HPLC.
The DL-Trp was dissolved in binding buffer (137 mmol/L NaCl,
0.5 mmol/L MgCl2, 2.7 mmol/L KCl, 2 mmol/L KH2PO4, 10 mmol/L
Na2HPO4, pH 7.4) for further experiments.
The resolution effect was evaluated under mature CE method using
α-CD a chiral selector reported previously,38 and all CE separations were
conducted on a Beckman P/ACE MDQ instrument (Beckman Instruments,
Fullerton, CA) equipped with a UV system, and the data were captured by
P/ACE System MDQ Software. An uncoated fused-silica capillary of 60cm
(50cm effective length) with 75μM ID and 375 μM OD (Hebei Yongnian
Optic Fiber Factory, Hebei, China) was used for enantioseparation. The
cassette holding the capillary column was thermostatted at 25°C. The
wavelength of the UV detector was set at 214 nm.
Au Nanoparticles Synthesis
AuNPs used in this system are stabilized by citrate and synthesized
according to the method reported by Frens and Kolloid.39 All glassware
used in this preparation was thoroughly cleaned in aqua regia (three parts
HCl [37.5%], one part HNO3 [65%]), rinsed with double-distilled water,
and oven-dried prior to use. A 50-mL aqueous solution of 0.01% HAuCl4
was heated to boiling and vigorously stirred in a 100-mL round-bottom
flask, then 0.3 mL of 1% trisodium citrate was added quickly to this
solution. The color of the solution turned deep blue within 35 s, and then
the final color changed to dark-red after 120 s, indicating the formation of
the AuNPs. Boiling was continued for an additional 10 min. The solution
was cooled to room temperature with a continuous stirring for another
15 min. The AuNPs were stored in thedark at 4 °C.
that the enantiomer in solution B was -Trp. Another solution
L
containing 50 μL 1 mM pure D-Trp was added to solution C
and the electropherogram is shown in Figure 1D. Only the
second peak was significant enhanced, revealing that the
second peak corresponded to D-Trp.
Preparation of Aptamer -AuNP Conjugates
Optimization of the Experimental Conditions
The conjugation of AuNPs with aptamer was performed via a thiol
function according to the procedures in the literature.40 In this protocol,
the thiolated aptamer (primary aptamer) was used for conjugation with
AuNPs. Before conjugation, the thiolated aptamer was activated following
this procedure: 9 μL of thiolated aptamer (1 mM) was mixed with 1.5 μL of
10 mM Trichloroethyl phosphate (TCEP) and 1 μL of 500 mM acetate
buffer (pH 5.2) to incubate for 1 h at room temperature. One mL of 5-fold
concentrated AuNPs solution was added to the TCEP-treated thiol
aptamer solution with gentle shaking. The mixture was stored in a drawer
Multiple experiments were conducted in order to obtain
the best separation efficiency. First, we investigated the effect
of the AuNP size to the separation (Fig. 2). Three different
AuNPs with the diameters of 77 7.56 nm (Fig. 2A),
55 5.56 nm (Fig. 2B), and 20 2.30 nm (Fig. 2C) were inves-
tigated. In general, the smaller sized particles have a larger
surface area to volume ratio, so that more aptamers can be
modified on the surface. However, our investigation revealed
Chirality DOI 10.1002/chir

SEPARATION OF ENANTIOMERS BASED ON AUNP
Scheme 1. The schematic of the enantioseparation of DL-Trp based on functional nucleic acids modified Au nanoparticles.
of the separation efficiency. Therefore, AuNPs with an
average diameter of 55 nm were used for the experiments
afterwards.
Next, we investigated the effect of the centrifugal speed on
the separation efficiency. Figure 3 shows the electrophero-
grams of the D-Trp obtained after the aptamer-AuNP
separation under centrifugal speeds of 6000 rpm (Fig. 3A),
5000 rpm (Fig. 3B), 4500 rpm (Fig. 3C), and 4000 rpm
(Fig. 3D) using the same size of AuNPs (55 nm). The
separation efficiency was evaluated by the peak area of -Trp
L
under the mature CE conditions as described above. When
the centrifugal speed was higher than 5000 rpm, the peak area
of L-Trp was much lower. The result confirmed that a high
centrifugal speed would decrease the binding ability of aptamer
with the target molecules. When the centrifugal rate was lower
than 5000rpm, a longer centrifugal time was necessary. This
may also inhibit the binding of target molecules to the aptamers
because lower separation efficiencies were observed. There-
fore, 5000rpm is recommended as the optimal centrifugal
speed and was used for the subsequent experiments.
Fig. 1. The electropherograms of (A) the racemic mixture of DL-Trp; (B)
L-Trp obtained by the aptamer functionalized AuNPs; (C) solution after addition
of 10μl 1 mM DL-Trp into (B); and (D) solution after addition of 50μl 1 mM D-Trp
into (C).
Reusability of the Aptamer Functionalized AuNPs
One of the overwhelming advantages of aptamers that
compete with antibodies for biomolecule recognition is their
reusability.41 Aptamers can be denatured and renatured for
several rounds without losing their binding abilities, while
antibodies cannot. In our experiments, the aptamers were
denatured by heating up at 95°C for 5 min, followed by imme-
diate centrifuging for 15 min at 5000 rpm. The precipitate was
then redispersed in binding buffer to recover its binding
ability. Figure 4 shows the reusability of the aptamer function-
alized AuNPs evaluated by the CE peak area of L-Trp. Only a
Chirality DOI 10.1002/chir
that a smaller AuNP size is not better. The best separation
efficiency was observed when 55 nm AuNPs were used.
Further decreasing the AuNP size induced a serious
decrease of the separation efficiency. As is known, smaller
AuNPs require a higher centrifugation speed to be precipi-
tated. We inferred that the high centrifugation speed may
partially denature the aptamers, which led to the decrease

HUANG ET AL.
Fig. 2. The effect of AuNP size to the enantioseparation efficiency evaluated by the peak area of the electropherograms. The electropherograms of (A,B,C)
correspond to the use of AuNPs with average diameters of 20 2.30 nm (left top),55 5.56 nm (left middle), and 77 7.56 nm (left bottom), respectively. Scale
bars = 200 nm.
Fig. 4. The variation of separation efficiencies (evaluated by the
corresponding CE peak area) of the aptamer functionalized AuNPs for
10 repeated uses.
slight decrease of the binding abilities was observed during
the first eight rounds of reuse. This kind of slight decrease
in binding ability was most probably due to the reason that
a small portion of the aptamers have been irreversible
damaged during the denaturing and renaturing process.
Anyway, our results revealed that the aptamer functionalized
AuNPs can be reused at least eight times without significant
loss of the corresponding binding ability.
Fig. 3. The effect of centrifugal speed to the enantioseparation efficiency
evaluated by the peak area of the electropherograms. The centrifugal speeds
were 6000 rpm in (A), 5000 rpm in (B), 4500 rpm in (C), and 4000 rpm in (D).
Chirality DOI 10.1002/chir

SEPARATION OF ENANTIOMERS BASED ON AUNP
Two factors that affect the enantioseparation efficiency were
investigated. The results showed that maximum separation
efficiency was achieved with 50 nm-diameter AuNPs under a
centrifugation speed of 5000 rpm. Additionally, our investiga-
tion also revealed that the aptamer-functionalized AuNPs
can be reused for at least eight times without a significant
decrease of the binding ability. Finally, it was demonstrated
that complete enantioseparation was achieved after five
repeated additions of aptamer-functionalized AuNPs to a
0.5 mM racemic mixture of DL-Trp solution. The method
could be expanded to the preparative separation of other
chiral compounds after adaptation of the corresponding chiral
selective aptamers.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support of the
National Science Foundation for Fostering Talents in Basic
Research of China (No. J1103303), National Natural Science
Foundation of China (21277025, 21205017, 21275031), National
Science Foundation of Fujian Province (2012 J01036,
2011J01035), and the Foundation of Fujian Educational Com-
mittee (JA12039).
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Chirality DOI 10.1002/chir