Enantioselective separation of chiral ofloxacin using functional Cu(ii)-coordinated G-rich oligonucleotides

Article abstract of DOI:10.1039/c3ra43251c

The DNA-based selector for discriminating chiral ofloxacin with high enantioselectivity and affinity is constructed through Cu(ii)-coordination with G-rich duplex containing successive guanines. Using this chiral selector, R- and S-ofloxacin can be direct

Full text of DOI:10.1039/c3ra43251c

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PAPER
Enantioselective separation of chiral ofloxacin
using functional Cu(II)-coordinated G-rich
oligonucleotides†
Cite this: RSC Adv., 2014, 4, 1329
Yan Fu, Xiaoli Duan, Xiongfei Chen, Jinli Zhang and Wei Li*
Received 27th June 2013
Accepted 30th September 2013
The DNA-based selector for discriminating chiral ofloxacin with high enantioselectivity and affinity is
constructed through Cu(II)-coordination with G-rich duplex containing successive guanines. Using this
chiral selector, R- and S-ofloxacin can be directly enriched from the racemate, with the enantiomeric
excess of 85% (R) and 78% (S) individually by three operational stages.
DOI: 10.1039/c3ra43251c
www.rsc.org/advances
crystallization. Herein we present a novel concept to construct a
Cu(II)-coordinated double-stranded DNA-based selector with
Introduction
Along with the increasing demand of enantiopure drugs in the augmented enantioseparation efficiency, of which the core is
pharmaceutical industry, more attention has been paid to the amplication of chiral recognition via the unique metal
construct highly enantioselective selectors to discriminate le- ion-coordinated DNA helix as well as the ligation between chiral
and right-handed enantiomers. Chiral diversity is an intriguing drug and metal ion. Using this DNA-based selector, R- and
nature of DNA macromolecules, which can serve as stereo- S-enantiomer can be easily enriched individually from racemic
selective selectors for discriminating metallo-supramolecular ooxacin, with the enantiomeric excess of 85% (R) and 78% (S),
complexes. The human telomeric antiparallel G-quadruplex can respectively, through three operational stages.
selectively interact with the P-Ni
junction preferentially binds to the M-Fe
hand, metals are in many respects the carriers of functions in
2 3
L , whereas the DNA three-way
1
2 3
L . On the other
Experimental section
biological systems, particularly, desired in the action of organic Materials
2
drugs targeting biomacromolecules. Transition metals such as
DNA oligonucleotides were purchased from the Japanese
Takara Bio. (Dalian) with purity higher than 98% measured by
HPLC. All DNA samples were annealed by heating the sample
2
+
2+
2+
Cu , Ni or Pt , preferentially coordinate at nitrogen N7 sites
of guanines, consequently, they can anchor inside three-
dimensional DNA structures to construct chiral microenviron-
ꢀ
cuvette to 95 C for 5 min in 10 mM Tris–HCl buffer (pH 7.0),
3
ments. Previously using SELEX procedure the single-stranded
followed by slowly cooling down to room temperature and
DNA oligonucleotide with high enantioselectivity has been
selected against a target enantiomer including oligopeptide,
adenosin, tyrosinamide, amino acid derivatives, ibuprofen, and
thalidomide derivative, showing attractive applications of DNA
ꢀ
storing at 4 C. Polydeoxyguanylic–polydeoxycytidylic acid
sodium salt (GC-DNA), polycytidylic acid–polyguanylic acid
sodium salt (GC-RNA), calf thymus (ct-DNA, %GC ¼ 42),
micrococcus lysodeikticus (ml-DNA, %GC ¼ 72), sh sperm (fs-
DNA, %GC ¼ 42) were purchased from Sigma-Aldrich (USA)
with the purities higher than 98%. Bovine serum albumin (BSA,
4
molecules for chiral resolution.
The chiral drug ooxacin, one of quinolone antibiotics
which exhibit antibacterial activity by inhibiting the action of
>97% purity) was also purchased from Sigma-Aldrich (USA).
5
topoisomerase II, is originally proposed to bind to DNA. The
Racemate and S-enantiomer of ooxacin with the purity of the
mass fraction higher than 99% were purchased from Shanghai
Jianglai Co., China. A Nanosep omega spin column (polyether
salfone, 3k molecular weight cut off, PALL Life Sciences) was
used to separate DNA–drug complex.
antibacterial activity of its S-enantiomer is 8–128 times higher
6
than that of the R-enantiomer. Since ooxacin racemates do
not exhibit enantiomeric phase separation in their crystals, it is
a prerequisite to separate partially one enantiomer from the
ooxacin racemates through chiral resolving agents before
Enantioenrichment of R- and S-ooxacin
Key Laboratory of Systems Bioengineering MOE, Key Laboratory for Green Chemical
Technology MOE, Tianjin University, Tianjin 300072, People’s Republic of China.
E-mail: liwei@tju.edu.cn
For the G-rich oligonucleotides, concentrated racemic ooxacin
aqueous solution was added into the obtained 20 mM annealed
DNA solution (strand concentration) in the absence or presence
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3ra43251c
2
+
2+
1
of Cu ([Cu ]/[base] ratio of 0.1) at pH 7.0, to a nal
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concentration of 0.02–0.2 mM. Aer 15 min incubation, the stage was utilized as the feed solution in the next stage.
mixture was transferred to the spin column. Aer centrifugation Subsequent operations of adsorption or separation were similar
at the speed of 8000 rpm for 20 min, the mixture solution was as the procedures used in the single-stage operation. Aer
separated using the spin column to provide the permeate, and three-stage adsorption, the enantiomeric excess in the permeate
the DNA–Cu(II) complex with adsorbed ooxacin enantiomers was denoted as e.e.
P
calculated by eqn (4):
were trapped on the membrane. The concentration of indi-
vidual ooxacin enantiomer in the permeate was measured by
high performance liquid chromatography (HPLC).
ðAR;P ꢂ AS;P
ðAR;P þ AS;P
Þ
Þ
e:e:
P
ð%Þ ¼
ꢁ 100ð%Þ
(4)
In order to remove the adsorbed ooxacin enantiomers from where AS,P and AR,P were the peak area of S- and R-enantiomer,
the DNA sequences, an equivalent volume of Tris–HCl–EDTA respectively, in the permeate.
buffer (pH 9.0) was added into the spin column to wash the
In the enantiomeric enrichment of S-ooxacin from the
trapped DNA on the membrane, and the mixture was incubated racemate, the adsorbed ooxacin enantiomers on the ltered
for 15 min. Aer centrifugation at the speed of 8000 rpm for 20 residue in the rst operational stage were desorbed using Tris–
min, the mixture solution was separated using the spin column HCl–EDTA buffer (pH 9.0). Then the S-enriched desorption
to provide the permeate, of which the individual concentration solution at the rst stage was adjusted to pH 7.0 and utilized as
of the desorbed ooxacin enantiomer from the ltered residue the feed solution in the next stage. Aer three cycles of
was measured by HPLC.
For the polynucleotides including sh sperm (fs-DNA), calf residue was denoted as e.e.
adsorption and desorption, the enantiomeric excess in the
R
calculated by eqn (5), and the
thymus (ct-DNA), micrococcus lysodeikticus (ml-DNA), polydG– adsorption ratio of S-enantiomer was denoted as A
polydC (GC-DNA), and polyG–polyC (GC-RNA), concentrated by eqn (6):
calculated
S
racemic ooxacin aqueous solution was added into the
ðAS;R ꢂ AR;R
ðAS;R þ AR;R
Þ
Þ
e:e:
R
ð%Þ ¼
ꢁ 100ð%Þ
(5)
(6)
obtained 800 mM DNA/RNA solution (base concentration) in the
2
+
2+
absence or presence of Cu ([Cu ]/[base] ratio of 0.1) at pH 7.0,
to a nal concentration of 0.1 mM. The following operations on
adsorption, desorption as well as separation were similar as the
procedures used for oligonucleotides.
A
S;R
S;F
A
S
¼
ꢁ 100ð%Þ
A
where AS,R and AR,R were the peak area of S- and R-enantiomer,
respectively, in the residue, AS,F was the peak area of S-enan-
tiomer in the racemic feed solution.
The adsorbed ooxacin enantiomers were able to dissociate
from DNA by using a desorption approach through both tuning
pH and adding EDTA, therefore, the adsorption amount of
individual R- or S-ooxacin was calculated by eqn (1) and (2):
C
S;R ꢁ V
Characterizations
q
S
¼ C
(1)
(2)
DNA ꢁ V ꢁ MDNA
High performance liquid chromatography (HPLC). The
enantiomeric excess of mixtures of ooxacin enantiomers was
analyzed by HPLC (Agilent 1200 Series). Chromatographic
separations were performed using a Kromasil C18 5 mm (4.6 ꢁ
C
R;R ꢁ V
q
R
¼ C
DNA ꢁ V ꢁ MDNA
where CS,R and CR,R were the concentrations of S- and R-enan-
tiomer in the ltered residue, respectively, CDNA was the
concentration of DNA, MDNA was the molecular weight of DNA,
and V was the volume of the separation system.
The enantioselectivity of DNA towards S-ooxacin was
denoted as a, calculated by eqn (3):
250 mm) column, and detected by a UV detector at 293 nm
using a mobile phase consisting of a mixture of methanol and
water (20 : 80, v/v), 2.5 mM L-isoleucine, 0.6 mM Cu at a ow
rate of 0.5 mL min . The injected sample volume was 20 mL.
Circular dichroism spectroscopy (CD). CD spectroscopy were
carried out on a Jasco J-810 spectropolarimeter at 20 C using a
2+
ꢂ1
ꢀ
ðCS;R=CR;R
Þ
Þ
quartz glass cuvette with 0.1 cm path length. All the CD spectra
were measured from 350 nm to 190 nm with a scanning speed
of 100 nm min . The cell holding chamber was ushed with a
constant stream of dry nitrogen gas to avoid water condensation
on the cell exterior.
Isothermal titration calorimetry (ITC). ITC measurements
were performed using a VP-isothermal titration calorimeter
a ¼
(3)
ðCS;F=CR;F
ꢂ1
where CS,F and CR,F were the concentration of S- and R-enan-
tiomer in the feed solution.
For bovine serum albumin (BSA), concentrated racemic
ooxacin aqueous solution was added into 0.3 mM BSA solution
at pH 9.0, to a nal concentration of 0.1 mM. The ooxacin
enantiomers in the ltered residue were desorbed by adjusting
the pH level to 3.0.
(Microcal, Northampton, MA). Titration was carried out by
injecting 10 mL aliquots per injection of the concentrated S- or
racemic ooxacin solution into 5 mM DNA solution at 210 s
ꢀ
intervals at 20 C for a total of 28 injections. Titration curves
Multi-stage operation for enantioenrichment of individual R-
or S-ooxacin using G-rich oligonucleotides
were corrected for heat of dilution by injecting the ooxacin
solution into the buffer. The binding constant KA and the
0
In the enantiomeric enrichment of R-ooxacin from the race- enthalpy change DH were obtained from the sigmoidal curve
mate, the R-enriched permeate obtained in the rst operational tting.
1330 | RSC Adv., 2014, 4, 1329–1333
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Native polyacrylamide gel electrophoresis (PAGE). PAGE
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measurements were carried out to characterize the electropho-
resis mobility of DNA structures using 20% acrylamide at 10 V
ꢂ1
ꢀ
ꢂ1
cm and 4 C. The gel was stained in a 3 mg mL Gelred
solution for 30 min. PAGE images were obtained using a
GDS8000 system (UVP, Inc., USA).
Results and discussion
In this study, double-stranded oligonucleotides with four tracts
of successive GC pairs, including RET, c-kit2, and VEGF
enriched in transcription initiation sites of proto-oncogenes,
7
are selected as the DNA scaffolds (Table S1†). CD spectra of all
the double-stranded oligonucleotides show a positive peak at
2
+
Scheme 1 (a) Chemical structure of S-ofloxacin; (b) Cu coordinates
at N7 sites of two successive guanines in the G-rich strand; (c) sche-
matic illustration of stereoselective recognition on G-rich double helix
2
65 nm and a negative band around 240 nm at pH 7.0, which is
8
an indication of duplex structure (Fig. S1a†). Upon addition of
for discriminating ofloxacin enantiomers (DNA model is constructed
2
+
2+
2+
Cu ([Cu ]/[base] ¼ 0.1), CD spectrum of double-stranded RET based on the crystal structure of d[G C ] , PDB 2ANA), Cu acts as a
4
4 2
at pH 7.0 shows a little decrease in the positive peak at 265 nm, bridge between the adjacent guanines of nucleic acids and the
carboxylic and carbonyl groups of ofloxacin.
and PAGE image suggests that RET–Cu(II) complex adopts a
2
+
duplex structure (Fig. S1b and S2†). Cu has been reported to
bind preferentially with the duplex containing successive
guanines on the same strand through the interaction between
the phosphate backbone of nucleic acids. Therefore, stereo-
selective recognition is achieved via Cu -coordinated G-rich
II
2
+
Cu with N7 of guanines. This binding model occurs at the low
2
+
9
double helix so as to discriminate ooxacin enantiomers.
The ability to tune the chiral recognition of DNA–Cu(II)
complex with external stimulus is considered to be a dominant
driving force to implement the chiral resolution through a
programmable adsorption–desorption process. For the
[
Cu ]/[base] ratio and stabilizes the G-rich duplex. The char-
acteristic band of RET–Cu(II) exhibits an obvious decrease at
65 nm upon addition of S-ooxacin, suggesting that the
2
binding of ooxacin occurs via an adaptive conformational
change of DNA–Cu(II) complex. The induced CD band at 287 nm
corresponds well to the UV absorption band of ooxacin, indi-
cating that the bound ooxacin molecules are arranged along
the helical DNA scaffold. Interestingly, CD spectrum of
RET–Cu(II)–S-ooxacin exhibits great difference to that of the
RET–Cu(II)–racemate, indicating the unique enantioselective
recognition towards S-ooxacin against R-ooxacin.
The stronger exothermicity is detected in the complexation
of RET–Cu(II) with S-ooxacin compared to that with the race-
mate, suggesting that RET–Cu(II) preferentially binds to the
S-enantiomer (Fig. S3a and S3b†). The association constant (K )
A
5
ꢂ1
of RET–Cu(II) is determined as 9.8 ꢁ 10 M for S-ooxacin,
which is comparable to the binding affinity of the DNA aptamer
4
e
(
IBA4) towards S-ibuprofen (K
d
¼ 1.5 mM). However, in the
2
+
absence of Cu , no detectable exothermicity occurs in the
binding of S-ooxacin to RET (Fig. S3c†). For the poly-
nucleotides involving natural calf thymus DNA, synthetic poly
[d(G-C)
2 2
] and poly[d(A-T) ], the association constant for the
formation of the S-ooxacin–DNA complex was determined as
3
ꢂ1
3
ꢂ1
3
ꢂ1
10
1
.4 ꢁ 10 M , 1.4 ꢁ 10 M and 1.0 ꢁ 10 M , respectively.
It is suggested that the Cu(II)-anchored DNA double helix
signicantly enhances the binding affinity of ooxacin mole-
cule owing to the coordination of carboxylic and carbonyl
II 11
groups of ooxacin with Cu . As illustrated in Scheme 1c, for
S-ooxacin, drug molecule partially intercalates into the
2
+
adjacent GC pairs and Cu acts as a bridge between the adja-
cent N7 sites of nucleic acids and the carboxylic and carbonyl
groups of ooxacin. In the case of R-ooxacin, its protrusion
into the minor groove of DNA is prohibited to some extent by
2
+
Fig. 1 (a) CD spectra of RET–Cu(II)–ofloxacin ([Cu ]/[base] ¼ 0.1, 50
2
+
mM S-ofloxacin, pH 7.0) adding equiv. EDTA to Cu alternately; (b) CD
2+
spectra of RET–Cu(II)–ofloxacin ([Cu ]/[base] ¼ 0.1, 50 mM S-oflox-
acin) at pH 7.0 and 9.0 reversibly by adjusting the pH through adding
+
ꢂ
the steric hindrance between the methyl group of ooxacin and H /OH , respectively.
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RET–Cu(II)–S-ooxacin at pH 7.0, as shown in Fig. 1a, upon
2
+
addition of equimolar EDTA to Cu , the CD band shis from
2
+
287 nm to 265 nm, suggesting that the strong chelation of Cu
induces the release of ooxacin molecules from DNA. Moreover,
2
+
the reversible CD spectra are recorded by adding EDTA and Cu
2
+
alternately. Aer removal of the bound Cu and ooxacin, RET
restores its conformation of duplex (Fig. S4†).
As shown in Fig. 1b, the CD band shis reversibly from 287
nm to 265 nm due to the pH level changing from 7.0 to 9.0 or
vice versa, suggesting the pH-responsive recognition for drug
2
+
enantiomers in the presence of Cu . The weaker affinities
2
+
between Cu ions and DNA bases at basic conditions mainly
contribute to the pH-dependent switches. Spectral reversibil-
2
+
ities triggered by Cu and pH value suggest that the supra-
molecular assembly of DNA–Cu(II)–ooxacin is highly
a
dynamic structure controlled by reversible coordination
bonding and electrostatic interactions, which are promising for
the enantiomeric enrichment of either R- or S-enantiomer.
Cu(II)-coordinated DNA exhibits different adsorption
2
+
behavior compared to that of DNA alone. Addition of Cu ions
obviously enhances the adsorption capacities of these three
sequences for both S- and R-ooxacin. Adopting the c-kit2-
Cu(II), the adsorbed amount for S- and R-ooxacin reaches to
II
0
.21 and 0.092 mmol(ooxacin)/g(DNA–Cu ) respectively with
the highest enantioselectivity (a) of 2.27 (Fig. 2a). Such
adsorption capacities of DNA–Cu(II) are more than 100-fold
higher compared to those of BSA molecules, the common
ꢂ4
ꢂ4
selector for chiral ooxacin (7.3 ꢁ 10 and 9.4 ꢁ 10 mmo-
l(ooxacin)/g(BSA) with an enantioselectivity of 1.28 towards
12
R-enantiomer). Meanwhile, the enantioselectivities (a) of
either RET–Cu(II) or c-kit2–Cu(II) increase signicantly in the
range of 20–100 mM racemic ooxacin in the feed solution, and
the saturation can be reached at 150 mM racemate (Fig. 2b). The
Fig. 2 (a) The adsorption amount of S- and R-ofloxacin individually as
VEGF–Cu(II) complex shows larger adsorption capacity which is well as the enantioselectivities (a) towards S-enantiomer at 100 mM
2
+
saturated with 200 mM racemate.
racemic feed solution, using 20 mM DNA–Cu(II) complexes ([Cu ]/
[
base] ¼ 0.1) or 20 mM each oligonucleotide alone; (b) the enantio-
Highly efficient enantioseparation is usually performed in an
operational mode of multi-stage adsorption. In the enantio-
meric enrichment of R-ooxacin from the racemate, aer three-
selectivities (a) towards S-enantiomer at different concentrations of
racemic feed solution; (c) the adsorption amount of S- and R-oflox-
acin individually as well as the enantioselectivities (a) towards
stage adsorption, the e.e.
5.0% for RET–Cu(II), c-kit2–Cu(II) and VEGF–Cu(II), respectively complexes ([Cu ]/[base] ¼ 0.1) or each polynucleotide alone.
Table 1). It is suggested that c-kit2–Cu(II) complex is the most
P
values can reach 74.8%, 85.1% and S-enantiomer at 100 mM racemic feed solution, using DNA–Cu(II)
2+
7
(
efficient chiral selector for R-ooxacin enrichment owing to the
highest enantioselectivity. In contrast, adopting G-rich DNAs
2
+
without addition of Cu , the highest e.e. is only 22.0% for
P
Table 1 The e.e.
enantiomer and the e.e.
enantiomer through three-stage adsorption using G-rich DNAs in the
presence or absence of Cu
p
in the permeate in the enrichment process of R-
in the residue in the enrichment process of S-
c-kit2 owing to lower adsorption capacity compared to that of
the Cu(II)-coordinated DNA (Tables S2 and S3†). Although the
similar enantioselectivities are detected for c-kit2 alone and
c-kit2–Cu(II), signicantly different efficiencies in the enrich-
ment of R-enantiomer are obvious for these two adsorbents,
which is attributed to the enhanced binding affinities of oox-
acin molecules to DNA through Cu(II)-mediated coordination.
In the enantiomeric enrichment of S-ooxacin from the
R
2+a
2
+
2+b
[Cu ]/base ¼ 0.1
e.e. (%)
Without Cu
Sequence
RET
P
e.e.
R
(%)
e.e. (%)
P
74.8
85.1
75.0
49.5
78.1
57.6
21.0
22.0
18.9
racemate, the DNA–Cu(II)-bound ooxacin enantiomers on the c-kit2
ltered residue in the rst operational stage are desorbed using VEGF
Tris–HCl–EDTA buffer (pH 9.0). Then the S-enriched desorption
solution is adjusted to pH 7.0 and then utilized as the feed pH 7.0. The e.e.
results in too low concentration.

a
Experimental conditions: 100 mM racemic feed solution, 20 mM DNA,
b
R
can not be determined since three-stage operation
solution in the next stage. Aer three cycles of adsorption and
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desorption, the enantiomeric excess in the residue can reach
Notes and references
7
3
8.1% (S) using c-kit2–Cu(II) as the selector, with the yield of
0.3%. However, S-enantiomer can be hardly enriched through
1 (a) L. Feng, C. Zhao, Y. Xiao, L. Wu, J. Ren and X. Qu, Chem.
Commun., 2012, 48, 6900; (b) L. Feng, B. Xu, J. Ren, C. Zhao
and X. Qu, Chem. Commun., 2012, 48, 9068.
2 Z. Guo and P. J. Sadler, Angew. Chem., Int. Ed., 1999, 38,
1512.
multi-stage operation using DNA without Cu(II)-coordination
due to the poor adsorption capacity at low concentration of
ooxacin in the feed solution (Tables 1, S4 and S5†). It is indi-
cated that amplication of both enantioselectivity and binding
affinity of DNA selector is critical to reduce operational stages to
obtain optically pure enantiomer. Therefore, Cu(II)-coordinated
G-rich DNAs are promising chiral selectors to produce both R-
and S-enantiomer from racemic ooxacin with highly optical
purity. Importantly, aer three repetitious recycling, double-
stranded c-kit2 maintains the adsorption capacity of S-enan-
tiomer (only reduced by 8%) as well as the enantioselectivity
3 C. Wang, G. Jia, J. Zhou, Y. Li, Y. Liu, S. Lu and C. Li, Angew.
Chem., Int. Ed., 2012, 51, 9352.
4 (a) M. Michaud, E. Jourdan, A. Villet, A. Ravel, C. Grosset and
E. Peyrin, J. Am. Chem. Soc., 2003, 125, 8672; (b) M. Michaud,
E. Jourdan, C. Ravelet, A. Villet, A. Ravel, C. Grosset and
E. Peyrin, Anal. Chem., 2004, 76, 1015; (c) C. Ravelet,
R. Boulkedid, A. Ravel, C. Grosset, A. Villet, J. Fize and
E. Peyrin, J. Chromatogr., A, 2005, 1076, 62; (d) P.-H. Lin,
S.-J. Tong, S. R. Louis, Y. Chang and W.-Y. Chen, Phys.
Chem. Chem. Phys., 2009, 11, 9744; (e) Y. S. Kim,
C. J. Hyun, I. A. Kim and M. B. Gu, Bioorg. Med. Chem.,
2010, 18, 3467; (f) A. Shoji, M. Kuwahara, H. Ozaki and
H. Sawai, J. Am. Chem. Soc., 2007, 129, 1456.
(2.15), showing high efficiency in regeneration and reusability.
For comparison, several polynucleotides including natural
sh sperm (fs-DNA), calf thymus (ct-DNA), micrococcus lyso-
deikticus (ml-DNA), and synthetic polydG–polydC (GC-DNA),
polyG–polyC (GC-RNA), are also selected to perform the chiral
resolution of ooxacin enantiomers. As a result, adopting ct-,
ml-, and GC-DNA, the a towards S-enantiomer is 1.92, 2.07 and
5 (a) L. L. Shen and A. G. Pernet, Proc. Natl. Acad. Sci. U. S. A.,
1985, 82, 307; (b) L. A. Mitscher, Chem. Rev., 2005, 105,
559.
1.56, respectively, whereas no stereoselectivity is detectable for
either fs-DNA or GC-RNA, indicating that the chiral recognition
is greatly associated with DNA conformation (Fig. S1c†). Inter-
6 (a) I. Hayakawa, S. Atarashi, S. Yokohama, M. Imamura,
K. Sakano and M. Furukawa, Antimicrob. Agents Chemother.,
1986, 29, 163; (b) R. H. Drew and H. A. Gallis,
Pharmacotherapy, 1988, 8, 35.
2
+
estingly, addition of Cu into ct-, ml-, or GC-DNA decreases the
enantioselectivity signicantly since polynucleotides are
susceptible to undergo compaction process in the presence of
13
transition metal ions. For example, the a decreases from 2.07
to 1.26 while the adsorption capacities of both S- and R-ooxacin
exhibit obvious increments (Fig. 2c). Compared to other nucleic
acid molecules, it is conrmed that the specic stereoselective
selector with high enantioselectivity and affinity is constructed
7 (a) K. Guo, A. Pourpak, K. Beetz-Rogers, V. Gokhale, D. Sun
and L. H. Hurley, J. Am. Chem. Soc., 2007, 129, 10220; (b)
K. Guo, V. Gokhale, L. H. Hurley and D. Sun, Nucleic Acids
Res., 2008, 36, 4598; (c) S.-T. Danny Hsu, P. Varnai,
A. Bugaut, A. P. Reszka, S. Neidle and S. Balasubramanian,
J. Am. Chem. Soc., 2009, 131, 13399.
2
+
through unique Cu coordination in the G-rich oligonucleo-
tides for efficient enrichment of either R- or S-enantiomer.
8 (a) W. Li, D. Miyoshi, S.-I. Nakano and N. Sugimoto,
Biochemistry, 2003, 42, 11736; (b) J. Zhang, Y. Fu, L. Zheng,
W. Li, H. Li, Q. Sun, Y. Xiao and F. Geng, Nucleic Acids
Res., 2009, 37, 2471.
Conclusions
The DNA-based selector for discriminating chiral ooxacin with
high enantioselectivity and affinity is constructed through
Cu(II)-coordination with G-rich duplex containing successive
9 (a) I. Sissoeff, J. Grisvard and E. Guille, Prog. Biophys. Mol.
Biol., 1978, 31, 165; (b) V. Andrushchenko, J. H. van de
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guanines. Furthermore, Cu(II)-coordinated DNAs exhibit 10 (a) E.-J. Lee, J.-A. Yeo, K. Jung, H. J. Hwangbo, G.-J. Lee and
stimuli-responsive recognition towards ooxacin enantiomers,
providing a programmable adsorption and desorption process
for the enantiomeric enrichment of either R- or S-enantiomer.
S. K. Kim, Arch. Biochem. Biophys., 2001, 395, 21; (b)
H. J. Hwangbo, B. H. Yun, J. S. Cha, D. Y. Kwon and
S. K. Kima, Eur. J. Pharm. Sci., 2003, 18, 197.
Using this chiral selector, R- and S-ooxacin can be directly 11 (a) C. Y. Chen, K. Chen, Q. Long, M. H. Ma and F. Ding,
enriched from the racemate, with the enantiomeric excess of
5% (R) and 78% (S) individually by three operational stages.
Compared to other biomacromolecules, Cu(II)-coordinated G-
Spectroscopy, 2009, 23, 103; (b) C. Y. Chen, Q. Z. Chen,
X. F. Wang, M. S. Liu and Y. F. Chen, Transition Met.
Chem., 2009, 34, 757.
8
rich DNAs are promising selectors for the enantioseparation of 12 (a) J. Haginaka, J. Chromatogr., A, 2000, 875, 235; (b) X. Zhu,
chiral ooxacin.
Y. Ding, B. Lin, A. Jakob and B. Koppenhoefer,
Electrophoresis, 1999, 20, 1869; (c) Y. Fu, T. Huang,
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Acknowledgements
This work was supported by NSFC (20836005, 21076141,
2
1206107), and the Special Funds for Major State Basic 13 J. C. Sitko, E. M. Mateescu and H. G. Hansma, Biophys. J.,
Research Program of China (2012CB720300).
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