Article pubs.acs.org/JPCB

Enantioselective Recognition Mechanism of Ofloxacin via Cu(II)Modulated DNA Wei Li,*,† Xiongfei Chen,† Yan Fu,‡ and Jinli Zhang‡ †

Key Laboratory for Green Chemical Technology MOE and ‡Key Laboratory of Systems Bioengineering MOE, School of Chemical Engineering and Technology, Tianjin University, Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Tianjin 300072, People’s Republic of China

Wei Li School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China S Supporting Information *

ABSTRACT: The specific interactions of Cu2+ with self-complementary DNA sequences involving d[G4C4(GC)2G4C4], d[(GC)10], and d[(AT)10], as well as the chiral recognition mechanism of ofloxacin enantiomers via the CuIImodulated DNAs, were investigated using characterizations of circular dichroism, gel electrophoresis, FT-IR spectroscopy, UV melting measurement, electron paramagnetic resonance, and HPLC. The CuII-coordinated GC-rich DNAs exhibit amplified enantioselectivity toward the S-enantiomer of ofloxacin. Especially in the case of d[G4C4(GC)2G4C4], ofloxacin enantiomers intercalate into the two adjacent guanine bases through the minor groove mediated by Cu2+, which leads to a more favorable binding between S-ofloxacin and DNA. The highest ee value of ofloxacin enantiomers in the permeate after being adsorbed by the CuII−DNA complex is obtained as 49.2% in the Renantiomer at the [Cu2+]/[base] molar ratio of 0.25, while at the [Cu2+]/ [base] molar ratio of 0.05 the highest ee value of ofloxacin enantiomers in the retentate reaches 26.3% in the S-enantiomer. This work illustrates a novel promising route to construct DNA-based chiral selectors toward certain drug enantiomers through the programmable enantioselective recognition on the basis of DNA chirality and the specific binding of transition metal ions.

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INTRODUCTION

so as to explore practical chiral selectors to produce drugs with optical purity. On the basis of the well-ordered nanostructure and highly programmable nature of DNA, conjugating DNA with small molecules, metal ions, or nanoparticles has attracted great interest in developing new DNA-based devices that exhibit versatile chemical and physical properties.11−13 In the fields of bioinorganic chemistry, specific interactions of transition metal ions including Cu2+, Zn2+, Ni2+, Co2+, Mn2+, and so on with DNA nucleobases and sequences have been extensively studied for many decades,14−16 providing the opportunity for the controllable synthesis of functional nanomaterials such as chiral catalysts,17 electrochemical sensors,18 and metal nanocrystals.19 In particular, Cu2+ exhibits highly specific recognitions toward DNA sequences, being considered a remarkable building block for constructing DNA nanomaterials. It has been reported that Cu2+ has higher binding affinity with GC-rich DNA than AT-

Deoxyribonucleic acid (DNA) is a natural chiral biomacromolecule characterized by a variety of well-established helical structures, ranging from canonical right-handed B form to the left-handed Z form.1 DNA with unique secondary structure often exhibits specific stereoselective recognition for chiral molecules such as antibiotics, amino acids, metallo-supramolecular complexes, etc. For example, A-form duplex DNA was reported to cooperatively bind to the Λ-enantiomer of tris(tetramethylphenanthroline)Ru(II).2 The Z-form DNA with left-handed double helix was found selectively bind to (−)-daunorubicin, whereas B-form DNA has the preferential affinity with its enantiomer (+)-daunorubicin.1 To date, several methods and applications using DNA as chiral selectors have been explored to discriminate chiral enantiomers including oligopeptide,3 adenosine,4 amino acids and their derivatives,5,6 chiral metal complexes7,8 and chiral drugs.9,10 However, the stereoselectivity of DNA is still ambiguous in some cases, and it needs extensive investigation on how to enhance the DNAbased chiral recognition toward one enantiomer of chiral drugs © 2014 American Chemical Society

Received: December 19, 2013 Revised: April 6, 2014 Published: May 5, 2014 5300

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rich DNA, mainly to the N7 and O6 of guanine as well as the O2 of cytosine.20,21 In recent years, a DNA-based metalloenzyme has been developed by assembling DNA with Cu2+, and could catalyze the asymmetric Friedel−Crafts reaction with high enantioselectivity.17 Therefore, it is intriguing to explore the highly base-specific DNA−Cu2+ binding in enantioslective recognition of target chiral compounds. Ofloxacin (Figure 1), a member of the quinolone antibiotics, which exhibits antibacterial activity by inhibiting the action of

Article

EXPERIMENTAL SECTION

Materials. Oligonucleotides d[G 4 C 4 (GC) 2 G 4 C 4 ], d[(GC)10], and d[(AT)10] (denoted as G4C4, GC, and AT, respectively) were purchased from Takara Biotechnology (Dalian) Co., Ltd., with a purity of higher than 98.0%. Racemic and S-ofloxacin were purchased from Jianglai Biological Technology Co., Ltd. (Shanghai, China) with a purity higher than 98.0%. CuCl2·2H2O was purchased from Alfa Aesar. Sodium chloride (NaCl), methyl green (MG), Hoechst 33258, and ethidium bromide (EB) were purchased from SigmaAldrich. Deuterium oxide (D2O) was purchased from Aladdin Industrial Corp. Deuterium chloride solution (DCl, 20 wt % in D2O) was purchased from J&K Scientific Ltd. Ultrafiltration centrifugal tubes (Amicon Ultra-0.5, Ultracel-3K, regenerated cellulose 3000 MW) were purchased from Millipore Co., Ltd. All DNA samples were dissolved in a Tris-HCl buffer (10 mM, pH7.2), annealed at 95 °C for 5 min, and cooled down to room temperature. The CuCl2 stock solution (10 or 100 mM) for each experiment was prepared by appropriate dilution of a 1 M CuCl2 aqueous solution in Tris-HCl buffer (10 mM, pH 7.2). The S- and racemic ofloxacin stock solutions were prepared by dissolving the solid materials in distilled water. All experiments were performed in Tris-HCl buffer (10 mM, pH 7.2) at 25 °C unless stated otherwise. Enantioselective Adsorption of the CuII−DNA Complexes. Appropriate volume (0, 1, 2, 5, and 10 μL) of 10 mM CuCl2 aqueous solution was added to 450 μL of annealed DNA sample solution. After an incubation of 15 min at 25 °C, the obtained CuII−DNA complex was mixed with 25 μL of 1 mM ofloxacin racemic solution. The total volume was made up to 500 μL by adding a Tris-HCl buffer (pH 7.2) so that the final concentrations of oligonucleotides and racemic ofloxacin were 20 and 50 μM, respectively. After 15 min the mixture was separated through ultrafiltration centrifugation at a speed of 8000 rpm for 20 min. The permeation solution is enriched with one enantiomer, while the retentate in the centrifugal tubes is composed of the CuII−DNA complexes with the adsorbed ofloxacin enriched with another enantiomer. In order to analyze the composition of the retentate, the desorption experiments were performed by adding an equivolume of Tris-HCl buffer solution (10 mM, pH 9.0) containing excessive EDTA into the centrifugal tubes, so as to make the preexisted ofloxacin enantiomer desorbed from the CuII−DNA complexes by the repulsive forces between the fully deprotonated ofloxacin and the DNA phosphate groups (Figure 1) as well as the CuIIEDTA chelation. To study the competitive adsorption of small ligands against the ofloxacin, the DNA samples were incubated in Tris-HCl buffer solution (10 mM, pH 7.2) containing dye molecules of MG, Hoechst 33258, or EB. Concentrated solution of MG, Hoechst 33258, or EB was added into 20 μM annealed DNA sample in Tris-HCl buffer solution (10 mM, pH 7.2) with the final [dye]/[base] molar ratio of 1:5, and the mixtures were incubated for 15 min. Then the following procedures were the same as the enantioselective adsorption experiments described above. Circular Dichroism Spectroscopy (CD). CD experiments were carried out using Jasco J-810 spectropolarimeter equipped with a Julabo temperature controller. CD spectra were recorded from 350 to 190 nm at 25 °C with a scanning speed of 100 nm/ min. A quartz glass cuvette of 0.1 cm path length was used and each CD spectrum was an average of three scans.

Figure 1. Molecular structure of ofloxacin (the asterisk denotes the chiral carbon atom).

Type II topoisomerase, is originally proposed to bind to DNA.22 The S-enantiomer has been reported to be 8−128 times more potent in inhibiting the multiplication of both Gram-positive and Gram-negative bacteria than the Renantiomer.23,24 Importantly, divalent and trivalent metal cations, such as Cu2+, Mg2+, Ni2+, Co2+, Fe3+, Cr3+, etc., were reported to affect the interactions of quinolone antibiotics with DNA targets and enhance the biological activities since the carboxylic and carbonyl groups of drug molecule can be ligated by cations.25,26 In the previous work, we found that in the presence of copper ions some double-stranded oligonucleotides with successive GC pairs, including RET, c-kit2, and VEGF that are enriched in transcription initiation sites of proto-oncogenes, can exhibit high enantioselectivity toward S-ofloxacin with the selectivity of 1.7−2.3.27 In order to disclose the enantioselective recognition mechanism of various DNA sequences in the presence of copper ions, in this study, we adopted three typical oligonucleotides with distinct structures, including d[G4C4(GC)2G4C4], d[(GC)10], and d[(AT)10], and studied the dependence relation between DNA sequences and the enantioselective recognition toward ofloxacin enantiomers, as well as the interactions among DNA, ofloxacin enantiomers, and copper ions, using characterizations of circular dichroism, native gel electrophoresis, FT-IR, electron paramagnetic resonance, HPLC, etc. It is indicated that for the GC-rich sequence Cu2+ probably interacts with the O6 and N7 atoms of guanine as well as the O2 of cytosine, while for the AT sequence Cu2+ interacts with the O2 and O4 atoms of thymine. The CuII-coordinated GC-rich DNAs exhibit amplified enantioselectivity toward the S-enantiomer of ofloxacin. Especially in the case of the G4C4 sequence including successive guanine bases, ofloxacin enantiomers intercalate into the two adjacent guanine bases through minor groove mediated by Cu2+, which leads to a more favorable binding between S-ofloxacin and DNA. The highest ee value of ofloxacin enantiomers in the permeate after being adsorbed by the CuII-G4C4 complex is obtained as 49.2% in the Renantiomer with the highest αp value of 2.93 at [Cu2+]/[base] ratio of 0.25, while at the [Cu2+]/[base] ratio of 0.05 the highest ee value of ofloxacin enantiomers in the retentate reaches 26.3% in the S-enantiomer. 5301

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Figure 2. (a) CD spectra of 20 μM G4C4 titrated with Cu2+ at the [Cu2+]/[base] molar ratio from 0 to 10 at 25 °C. Inset: the profile of CD intensity at 262 nm versus the [Cu2+]/[base] ratio. (b) Gel electrophoretic images of 20 μM G4C4 at different [Cu2+]/[base] ratios: lane 1, 20-bp DNA ladder; lane 2, DNA alone; lanes 3−7, DNA in the presence of Cu2+ with the [Cu2+]/[base] ratio of 0.05, 0.1, 0.25, 0.5, and 1, respectively. (c) FT-IR spectra of 2 mM G4C4 in the presence of Cu2+ with the [Cu2+]/[base] ratio of 0.25 and 0.5 in D2O at pH 7, together with the guanine− cytosine base pairing, of which the interactive sites with Cu2+ are marked in red.

Isothermal Titration Calorimetry (ITC). ITC measurements were performed using a VP-isothermal titration calorimeter (Microcal, Northampton, MA). Titration was carried out by injecting 10 μL aliquots per injection of a 500 μM S-ofloxacin solution into a 20 μM CuII−DNA complex solution under continuous stirring at 180 s intervals at 25 °C for a total of 28 injections. Titration curves were corrected for heat of dilution by injecting the ofloxacin solution into the buffer. The binding constant KA, the stoichiometry N, the enthalpy change ΔH0, and the entropy change ΔS0 were obtained by fitting the integrated heats of binding isotherm to the one-site binding model using MicroCal Origin 7.0 software. Electron Paramagnetic Resonance Spectroscopy (EPR). The X-band CW-EPR measurements were carried out on a Bruker A300 spectrometer under the experimental conditions of microwave frequency 9.50 GHz, microwave power 20 mW, and modulation amplitude 1 mT. Samples of the CuII−DNA complexes were prepared by adding 100 mM CuCl2 aqueous solution into the annealed G4C4 or AT buffer solution (10 mM, pH 7.2) with the [Cu2+]/[base] molar ratio of 0.05. And samples of ternary CuII−DNA−ofloxacin complexes were prepared by further mixing S-ofloxacin (10 mM in Tris-HCl buffer, pH 7.2) with the binary CuII−DNA complexes ([Cu2+]/[base] = 0.05) to obtain the [Cu2+]/ [ofloxacin] molar ratio of 1. The final concentration of Cu2+ in all EPR samples was 1 mM. Cu2+ alone and binary CuII− ofloxacin complex in the buffer solution were carried out as a control. Cooling of the samples was performed with a liquid nitrogen Dewar vessel at 110 K and a Bruker temperature control unit. HPLC Measurements. The compositions of ofloxacin enantiomers in the permeate and retentate solutions were analyzed by HPLC (Agilent 1200 Series), using a Kromasil C18

Native Gel Electrophoresis (PAGE). The PAGE experiments were carried out using 20% acrylamide (29:1 acrylamide/bis(acrylamide)) at 10 V/cm and 4 °C for 8 h in 1 × TB buffer. The gel was stained in GelRedTM 10000X solution for 30 min. Then the stained gel was imaged using a GDS8000 imaging system (UVP. Inc., USA). FT-IR Spectroscopy. FT-IR spectra of DNA and the CuII− DNA complex were measured by using a Thermoscientific Nicolet iS10 spectrophotometer. The DNA (G4C4 and AT) solutions were dissolved in D2O, with the pH value adjusted to 7.0 by addition of 0.1 M DCl. IR samples were prepared by addition of appropriate volumes of Cu2+ stock solution into the annealed G4C4 or AT solution to reach the [Cu2+]/[base] molar ratio of 0, 0.25, and 0.5 respectively. Deuteration experiments were performed by drying the sample solutions in vacuum and redissolving in equivalent volume of D2O (pH 7.0). Finally, the solutions were deposited between two ZnSe windows without spacer at a concentration of 2 mM oligonucleotide strand. All spectra were recorded over the region from 1800 to 800 cm−1 with a resolution of 4 cm−1 and 64 scans at 25 °C. UV Melting Measurment. The thermal denaturalization studies were performed on a Varian Cary 300 UV−vis spectrophotometer equipped with a Peltier temperature controller. A sample of 20 μM CuII−G4C4 or CuII−AT complex at the [Cu2+]/[base] molar ratio of 0.05 was respectively mixed with 50 μM S- and racemic ofloxacin and then diluted with Tris-HCl buffer (10 mM, pH 7.2) to the final DNA concentration of 10 μM. All the melting curves were recorded against Tris-HCl buffer at 260 nm in a 0.1 cm path length quartz cuvette. The programmed temperature was set from 20 to 95 °C with a heating rate of 0.6 °C/min. 5302

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Figure 3. (a) CD spectra of 20 μM AT titrated with Cu2+ at the [Cu2+]/[base] molar ratio from 0 to 10 at 25 °C. Inset: the profile of CD intensity at 267 nm versus the [Cu2+]/[base] ratio. (b) Gel electrophoretic images of 20 μM AT at different [Cu2+]/[base] ratios: lane 1, 20-bp DNA ladder; lane 2, DNA alone; lanes 3−7, DNA in the presence of Cu2+ with the [Cu2+]/[base] ratio of 0.05, 0.1, 0.25, 0.5, and 1, respectively. (c) FT-IR spectra of 2 mM AT and the complexes with Cu2+at the [Cu2+]/[base] ratio of 0.25 and 0.5 in D2O at pH 7, together with the adenine−thymine base paring, of which the interactive sites with Cu2+ are marked in red.

column (5 μm, 4.6 × 250 mm) and a UV detector at 293 nm with a mobile phase consisting of a mixture of methanol and water (20:80, v:v), 2.5 mM L-isoleucine, and 0.6 mM Cu2+ at a flow rate of 0.5 mL/min.28 The injected sample volume was 20 μL. The enantiomeric excess value (eep) and the separation factor (αp) in the permeate were calculated using the following equation (R-enantiomer excess) ee p(%) =

αp =

Cp,R − Cp,S Cp,R + Cp,S

AS(%) =

AR (%) =

αr =

Cf,R × V

× 100%

RESULTS AND DISCUSSION Sequence-Specific Interactions between Cu2+ and Oligonucleotides. Three typical DNA oligonucleotides with distinct structures, d[G4C4(GC)2G4C4], d[(GC)10], and d[(AT)10] (abbreviated as G4C4, GC and AT, respectively), were adopted to investigate the sequence-dependent interactions between DNA and copper ions. For the G4C4 sequence containing successive guanine bases, as shown in Figure 2a, CD spectrum exhibits a major positive band at 262 nm with a shoulder peak around 284 nm, as well as two negative bands at 233 and 213 nm, respectively, indicating that G4C4 mainly adopts the A-form duplex conformation.29 Upon titrating Cu2+ with the [Cu2+]/[base] molar ratio less than 1.0, the positive band at 262 nm shows a tiny fluctuation, while the shoulder peak at 284 nm obviously decreases, suggesting a structural transition induced by Cu2+. When the [Cu2+]/[base] molar ratio is increased up to 2, a significant decrease of the positive band at 262 nm is observed with an obvious inflection at the [Cu2+]/[base] ratio of 3.1 (see the inset of Figure 2a). As shown in Figure 2b, PAGE images show that the G4C4 sequence initially adopts the hairpin structure with a fast electrophoretic mobility, and then transfers into the doublestranded helical structure with a low mobility as the [Cu2+]/ [base] ratio is higher than 0.5.

where Cp,R and Cp,S represent the concentration of R- and Sofloxacin in the permeate solution, respectively. Cf,R and Cf,S represent the concentration of R- and S-ofloxacin in the feed solution, respectively. The enantiomeric excess value (eer) and the separation factor (αr) in the retentate were calculated using the following equations (S-enantiomer excess) Cr,S + Cr,R

Cr,R × V

× 100%

■

× 100%

Cf,R /Cf,S

Cr,S − Cr,R

Cf,S × V

where V represents the volume of the separation system.

Cp,R /Cp,S

eer(%) =

Cr,S × V

× 100%

Cr,S/Cr,R Cf,S/Cf,R

where Cr,S and Cr,R represent the concentration of S- and Rofloxacin in the retentate, respectively. The adsorption percentage (A) of S- and R-ofloxacin was calculated respectively using the following equations 5303

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Figure 4. CD titrations of 20 μM (a) G4C4 and (b) AT with S-ofloxacin in the presence of Cu2+ ([Cu2+]/[base] = 0.05). Inset: the changes of CD intensity with the concentrations of S- or racemic ofloxacin ranging from 0 to 50 μM; UV melting curves of the (c) CuII−G4C4 and (d) CuII−AT complexes with 50 μM S- or racemic ofloxacin, respectively, at the [Cu2+]/[base] ratio of 0.05 in Tris-HCl buffer solution (10 mM, pH 7.2).

FT-IR spectra of G4C4 in the presence of Cu2+ were recorded to discern the potential interactive sites with Cu2+. As shown in Figure 2c and Table S1 in the Supporting Information, when the [Cu2+]/[base] ratio rises from 0 to 0.25, the absorption bands at 1695 and 1652 cm−1, assigned to the C6O6 vibrations of guanine coupled with C2O2 vibrations of cytosine,30,31 shift to 1687 and 1650 cm−1, respectively. Meanwhile, the absorption band at 1491 cm−1, assigned to C8N7 vibrations of guanine bases,30 shifts to 1494 cm−1. It is indicated that Cu2+ probably interacts with the O6 and N7 atoms of guanine as well as the O2 atom of cytosine (Figure 2c).21 When the [Cu2+]/[base] ratio increases to 0.5, the absorption bands of the C6O6 vibrations of guanine coupled with C2O2 vibrations of cytosine become one strong peak at 1646 cm−1. Additionally, the in-plane ring vibration band of cytosine at 1578 cm−1 disappears, and the C8N7 vibration band of guanine shifts to 1481 cm−1, suggesting more Cu2+ ions interact with DNA bases. On the other hand, as the [Cu2+]/[base] ratio increases from 0 to 0.5, the antisymmetric stretching band of the phosphate groups at 1234 cm−1, indicative of the typical A-form conformation,30,32 gradually shifts to 1223 cm−1. The symmetric stretching band of the phosphate groups at 1085 cm−1 slightly shifts to 1084 cm−1 at the [Cu2+]/[base] ratio of 0.25, and then to a shoulder band at 1086 cm−1 at the [Cu2+]/[base] ratio of 0.5; meanwhile, the vibration band of C−O−P backbone at 1062 cm−1 shifts to 1065 cm−1 and then back to 1063 cm−1. It is known that the absorption bands of DNA deoxyribose and the phosphate groups (800 cm−1-1400 cm−1) are closely associated with the conformation of DNA. Therefore, it is reasonable to conclude that the specific interaction between Cu2+ and the guanine base results in a great conformational change of G4C4 sequences, which consequently induces the structural transition

from the hairpin to the double-stranded helix at high [Cu2+]/ [base] ratio,33,34 as reflected by the PAGE images in Figure 2b. For the GC sequence, the positive CD band at 284 nm decreases as titrating Cu2+, with two distinct inflections at the [Cu2+]/[base] ratio of 0.5 and 3.3, respectively (Figure S1a in the Supporting Information). Moreover, similar to the G4C4 sequence, a structural transfer from the hairpin to the doublestranded helix structure is also observed at the [Cu2+]/[base] ratio higher than 0.5 (Figure S1b in the Supporting Information). In the case of the AT sequence, the influence of Cu2+ on its conformation is different from that on GC-rich sequences. As shown in Figure 3a, CD spectrum of the AT sequence shows a positive band at 267 nm and a negative band at 245 nm, which is indicative of a B-form conformation.35 Upon titrations of Cu2+, the intensities of both CD bands gradually decrease accompanied by a slight red shift, with two inflections at the [Cu2+]/[base] ratio of 1.5 and 8.0, respectively (see the inset of Figure 3a). Unlike the G4C4 and GC sequences, there is no transfer from the hairpin to double-stranded helix even at the [Cu2+]/[base] ratio of 1.0 (Figure 3b). As shown in Figue 3c and Table S1, at the [Cu2+]/[base] ratio of 0.25, the absorption bands at 1698 and 1667 cm−1, assigned to the C2O2 and C4O4 stretching of thymine,30,31 shift to 1693 and 1663 cm−1, respectively, suggesting that Cu2+ probably interact with the O2 and O4 atoms of thymine (Figure 3c). When the [Cu2+]/[base] ratio increases to 0.5, the C4O4 stretching band of thymine shifts to 1660 cm−1, whereas the C2O2 stretching band of thymine does not change, indicating Cu2+ preferentially interact with the atom O4 of thymine rather than with O2 of thymine at high [Cu2+]/[base] ratio. Additionally, as the [Cu2+]/[base] ratio increases from 0 to 0.5, the antisymmetric stretching band of the phosphate groups at 1229 cm−1 gradually shifts to 1221 5304

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cm−1, indicative of the typical B-form conformation without great changes.30 The symmetric stretching band of the phosphate groups slightly shifts from 1093 to 1096 cm−1, the vibration band of C−O−P backbone slightly shifts from 1062 to 1065 cm−1, and there is no obvious change in the relative intensity between the two absorption bands. Therefore, it is suggested that the relative weak interaction between Cu2+ with the AT sequence cannot result in a structure transfer of DNA, which is consistent with one band of hairpin AT shown in the PAGE images (Figure 3b). In combination with CD titrations, PAGE and FT-IR experiments, copper ions exhibit distinct sequence-specific interactions with DNA oligonucleotides. That is, Cu2+ interacts strongly with N7 and O6 of the guanine base as well as the O2 of cytosine in GC-rich DNA so as to make the DNA conformation distorted along the molecular backbone, whereas Cu2+ can bind to O4 and O2 of the thymine base in AT-rich DNA but with no significant disruption on the DNA conformation. Enantioselective Recognition of Ofloxacin by the CuII−DNA Complexes. On the basis of sequence-specific interactions of Cu2+ with DNAs, the enantioselective recognition of ofloxacin enantiomers via the CuII-coordinated DNAs was investigated at low [Cu2+]/[base] molar ratio. In the CD spectra of the CuII−G4C4 complex ([Cu2+]/[base] = 0.05) titrated with S-ofloxacin (Figure 4a), the positive peak at 262 nm decreases while a small shoulder band at 284 nm gradually increases, suggesting a structural transition induced by ofloxacin binding. Importantly, the ratio of θ284 nm/θ262 nm representative of the relative variation is increased more significantly for the Senantiomer than that for the racemate (see the inset of Figure 4a), indicating the intriguing enantioselective recognition of the CuII-coordinated G4C4 toward S-ofloxacin against R-ofloxacin. However, as a control, in the absence of Cu2+ there is almost no change of the CD band at 284 nm upon S-ofloxacin addition (Figure S2a in the Supporting Information). In the case of GC sequence, the Cu2+-induced enantioselectivity of DNA toward ofloxacin enantiomers is also observed. As shown in Figure S3a in the Supporting Information, the S-ofloxacin binding results in a minor increase of the positive band at 284 nm and a slight decrease of the negative band at 252 nm. The plots of the CD intensity at 284 nm suggest the stereoselective discrimination of the CuII−GC complex toward S-ofloxacin against R-ofloxacin (see the inset of Figure S3a), compared with the control spectra without the CuII coordination (Figure S3b). In contrast, for the CuII-coordinated AT sequence, addition of S-ofloxacin causes only a subtle decrease of the positive band at 267 nm, but accompanied by the emergence of a positive band around 300 nm (Figure 4b). However, the plots of the intensity of CD band at 300 nm with ofloxacin concentration show little difference between S- and racemic ofloxacin binding (see the inset of Figure 4b), suggesting that the enhancement of enantioselective recognition induced by CuII coordination is quite weak for the AT sequence. The thermal denaturation behaviors of the CuII−DNA complexes bound with S- or racemic ofloxacin were further studied to understand deeply the enantioselective binding of ofloxacin enantiomers to the CuII-modulated DNAs. As shown in Figure 4c, the melting temperature (Tm) of G4C4 alone is 64.0 °C, while at the [Cu2+]/[base] ratio of 0.05 the Tm value increases up to 74.5 and 70.8 °C for the CuII−DNA complex with S- and racemic ofloxacin binding, respectively. The significant stabilization of ofloxacin on the CuII−G4C4 complex

suggests that in the presence of Cu2+ the ofloxacin enantiomers probably interact with DNA by an intercalative binding mode,36,37 rather than the groove binding that is mainly adopted by the DNA−ofloxacin interaction without Cu2+.24 Moreover, the higher Tm value of the CuII−G4C4 complex with S-ofloxacin indicates that the CuII−G4C4 complex preferentially binds to S-ofloxacin than R-enantiomer. ITC measurements were carried out for G4C4 with the titration of S-ofloxacin in the presence of Cu2+ (Figure S4 in the Supporting Information). The calculated thermodynamic parameters ΔH0 and TΔS0 are −27.51 and 4.09 kJ/mol for the formation of the CuII−G4C4−ofloxacin complex, suggesting a strong enthalpy-driven process accompanied by a favorable entropy contribution. As a control, in the absence of Cu2+, the ITC profile indicates weak interactions between G4C4 and S-ofloxacin. In addition, the binding stoichiometry of S-enantiomer to the CuII−G4C4 complex is calculated to be 1.1 S-ofloxacin per duplex, and the association constant KA is determined as 3.4 × 105 M−1 which is much higher than the reported KA (4.7 × 103 M−1) for the interaction of Dargininamide with its related DNA aptamer through electrostatic binding.38 It is illustrated that Cu2+ can facilitate the formation of the CuII−G4C4 complex with S-ofloxacin. In the case of AT, the melting temperature of AT alone is 40.8 °C, while at the [Cu2+]/[base] ratio of 0.05, the Tm value increase to 43.7 and 44.5 °C in the presence of racemic and Sofloxacin, respectively (Figure 4d). It is suggested that the interaction among AT, Cu2+ and ofloxacin is not strong enough to generate enantioselectivity toward ofloxacin enantiomers. The X-band EPR measurements were performed in a frozen buffer solution to study the coordination environments surrounding the CuII−DNA complexes upon binding to Sofloxacin. As shown in Figure 5, EPR spectra of the CuII−G4C4 and CuII−AT complexes in the presence of S-ofloxacin are typical of CuII complexes with a dx2−y2 ground state (g∥ > g⊥).39,40 As a control, EPR spectra of lonely Cu2+ and the CuII− ofloxacin binary complex in buffer solution were measured and are shown in Figure S5 in the Supporting Information. Table 1 lists the g values and the CuII hyperfine parameters, suggesting

Figure 5. X-band EPR spectra of (a) the CuII−G4C4 and (b) the CuII−AT complexes with S-ofloxacin ([Cu2+]/[base] = 0.05, [Cu2+]/ [ofloxacin] = 1) in Tris-HCl buffer (10 mM, pH 7.2) at 110 K. 5305

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Table 1. g Values and CuII Hyperfine Parameters of Different CuII Species in a Frozen Buffer Solution species

g⊥

g∥

A∥ (G)

CuII−Tris CuII−S-isomer CuII−G4C4 CuII−G4C4−S-isomer CuII−AT CuII−AT−S-isomer

2.079 2.091 2.041 2.046 2.063 2.051

2.382 2.315 2.245 2.275 2.291 2.277

135 150 177 173 157 167

are within the range of either 2O2N or 3O1N coordination mode.46 It has been reported that, in the DNA sequences containing successive guanines, Cu2+ prefers to chelate with N7 and O6 of the two adjacent guanine bases to form “G−CuII−G sandwich” complex.20 Therefore, in combination with the results of FT-IR spectra, it is reasonable to conclude that the CuII−G4C4 complex exhibits a 2O2N equatorial coordination mode. Upon addition of S-ofloxacin, the g∥ value increases to 2.275 but the A∥ value decreases to 173 G, respectively, which is attributed to the coordination of CuII with the carboxylic and the carbonyl groups of ofloxacin molecules. In the case of the CuII−AT complex, it shows a relatively high g∥ value and low A∥ value (Figure 5b), indicating the coordinated ligands around CuII are probably oxygen atoms involving the O2 and O4 of the thymine base of AT duplex as well as the hydroxyl groups of buffer or water molecules.47 Upon addition of ofloxacin, the solvent molecules coordinated to Cu2+ are replaced by the oxygen atoms from the carboxylic and carbonyl groups of ofloxacin. Besides, the relatively high g∥ value and low A∥ value probably result from a strong distortion of the coordination geometry in the CuII−AT complex from the planarity,48 and the distortion is decreased after addition of S-ofloxacin, causing a decrease of g∥ value but an increase of A∥ value.48 Chiral Separation of Ofloxacin Enantiomers Using the CuII−DNA Complexes. The separation of ofloxacin enantiomers using the CuII-coordinated DNAs as the chiral selectors was performed through centrifugal ultrafiltration. The compositions in both the permeate and retentate solutions were analyzed using HPLC (Figure S6 in the Supporting Information), from which the enantiomeric excess (ee) and the separation factor (α) were calculated. As shown in Figure 6, a and b, adopting the CuII−G4C4 complex at the [Cu2+]/ [base] ratio of 0.25, the eep value of the permeate solution reaches the highest of 49.1% in R-enantiomer with the highest

the coordination of four ligands in a square-planar or squarepyramidal geometry.41,42 In the case of lonely Cu2+ in the frozen buffer, Cu2+ prefers to interact with Tris molecules rather than water through coordinating the hydroxyl and amino groups of Tris.43 In the presence of S-ofloxacin with the [Cu2+]/[ofloxacin] molar ratio of 1, the g|| value decreases but the A∥ value increases, indicating that the Tris ligands coordinated to CuII are exchanged by the ofloxacin molecule so as to form a CuII−S-ofloxacin complex. As shown in Figure S5, both EPR spectra of CuII−Tris and CuII−S-ofloxacin binary systems exhibit a broad, intense, and unstructured resonance line accompanied by relatively weak hyperfine peaks, suggesting the presence of a spin−spin exchange interaction between paramagnetic centers, possibly CuII dimer.44 However, in the presence of DNA, the well-resolved hyperfine structures are visible in all the EPR spectra of the CuII−DNA binary and CuII−DNA−ofloxacin ternary systems (Figure 5a,b), and the EPR parameters exhibit relatively lower g∥ values and a higher A∥ value (Table 1), compared to the binary CuII−Tris and CuII−ofloxacin systems, indicating the formation of new CuII species in which Cu2+ coordinates the DNA bases so as to generate higher ligand field strength and more rigid bonds.45 In the case of G4C4 (Figure 5a), the g∥ and A∥ values for the CuII−G4C4 complex are 2.245 and 177 G, respectively, which

Figure 6. (a) eep and (b) αp value in the permeate solution after 50 μM racemic ofloxacin being adsorbed by 20 μM CuII−DNA complexes at different [Cu2+]/[DNA] molar ratio at pH 7.2. (c) eer and (d) αr value in the retentate at different [Cu2+]/[DNA] molar ratio at pH 9.0. 5306

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αp value of 2.93, while in the absence of Cu2+, the eep and αp value are only 7.5% and 1.16, respectively. At the [Cu2+]/[base] ratio either higher or lower than 0.25, both the eep and αp value are smaller than that obtained at the [Cu2+]/[base] ratio of 0.25 for the CuII−G4C4 complex adsorption separation. When another two sequences GC and AT are adopted, the highest eep and αp value of the permeate solution are also obtained at the [Cu2+]/[base] ratio of 0.25 (42.7% and 2.49 for the CuII−GC complex, and 13.7% and 1.32 for the CuII−AT complex, respectively), both of which are greatly larger than those without Cu2+ (4.6% and 1.10 for GC, and 2.2% and 1.05 for AT). It is suggested that the CuII coordination can significantly enhance the enantioselective recognition of the G4C4 and GC sequences toward ofloxacin; so does the AT sequence but to a much smaller extent. In order to analyze the composition of the retentate absorbed on the CuII−DNA complexes, a programmable desorption process was implemented by tuning pH levels and adding excessive EDTA. Figure 6, c and d, shows that the eer value of the retentate adsorbed on the CuII−G4C4 and CuII−GC complexes at the [Cu2+]/[base] ratio of 0.05 reaches the highest value of 26.3% and 13.4% in S-enantiomer with the highest αr value of 1.71 and 1.31, respectively. In contrast, for the AT sequence, the highest eer and αr value are obtained in the absence of Cu2+, while adding Cu2+ leads to the decrease of both eer and αr value (7.9% and 1.17). At the [Cu2+]/[base] ratio of 0.25, the highest adsorption percentage for S- and Renantiomers adopting all the three sequences are obtained (Table S2 in the Supporting Information). For example, S- and R-ofloxacin can be adsorbed on the CuII−G4C4 complexes at the percentage of 93.3% and 81.1%, respectively. It is suggested that the adsorption capability to ofloxacin enantiomers are amplified by addition of low concentration of Cu2+. In addition, it is interesting to find out that addition of EDTA results in the recovery of CD spectra of DNA (Figure S7 in the Supporting Information), which provides an opportunity for regeneration of the DNA-based selectors. The competitive binding assay was performed by using methyl green (MG), Hoechst 33258, and ethidium bromide (EB) as the probes, aiming to investigate the effects of these competitive binders on the enantioselectivity of the CuII−DNA complex. It is well-known that EB can intercalate into DNA base pairs through the minor groove, and Hoechst 33258 and MG are recognized as the classical minor groove and major groove binders, respectively.49,50 As shown in Table 2, addition of MG leads to a little decrease of the eep and αp value after ofloxacin is adsorbed by the CuII−G4C4 complexes at the [Cu2+]/[base] ratio of 0.05 and 0.25, respectively, which indicates that major groove-interacting probes exhibit little

effect on the chiral performance of the CuII−G4C4 complexes. In contrast, adopting the minor groove-binding ligands EB or Hoechst 33258, an apparent decrease of the eep and αp value is detected, indicating that either Hoechst 33258 or EB significantly weakens the enantioselectivity of the CuII−G4C4 complexes. The same trends are also obtained for the competitive assay involving the CuII−GC and CuII−AT complexes (Table S3 in the Supporting Information). Therefore, it is concluded that ofloxacin binds to the same site of DNA as EB and Hoechst 33258 with the aid of Cu II coordination. In combination with the results of CD titrations, UV melting, FT-IR, EPR, and competitive binding experiments, it is confirmed that the distinct coordination of CuII with GC-rich and AT-rich DNA is attributed to the different enantioselectivity of DNA toward ofloxacin enantiomers. It has been reported that ofloxacin at neutral pH value is favorable for enantioselective binding of S-ofloxacin to DNA,51 which can be described by the steric hindrance between the methyl group at the oxazine ring of R-enantiomer and the phosphate groups of DNA.52 In the case of G4C4, it is suggested that Cu2+ anchors inside the major groove through the G−Cu(II)−G coordination mode, while ofloxacin enantiomers interact with the CuII− DNA complex through the minor groove pathway of the DNA duplex. Therefore, S-ofloxacin partially intercalates into the adjacent GC pairs and Cu2+ acts as a bridge between the N7 and O6 atoms of guanine and the carboxylic and carbonyl groups of ofloxacin (Figure 7a). However, the protrusion of Rofloxacin into the minor groove is prohibited to some extent by the steric hindrance between the methyl group at the C-3 position of the oxazine ring of ofloxacin, which contains the chiral center, and the phosphate backbone of DNA (Figure S8a in the Supporting Information). However, for the AT sequence, Cu2+ mainly interacts with the O2 site of the thymine base located in the minor groove of AT DNA at low [Cu2+]/[DNA] ratio. Consequently, ofloxacin can easily interact with CuII through the groove binding model without intercalation, resulting in low enantioselectivity (Figure 7b and Figure S8b).

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CONCLUSIONS The specific interactions of Cu2+ with three typical oligonucleotide sequences involving G4C4, GC, and AT show that Cu2+ prefers to binding with the N7 and O6 atoms of guanine as well as the O2 of cytosine of GC-rich DNA, whereas with the O2 and O4 atoms of thymine of AT-rich DNA. At low [Cu2+]/ [base] ratio, the CuII-coordinated GC-rich DNAs exhibit amplified enantioselectivity toward S-enantiomer of ofloxacin, compared to AT-rich DNA. Especially for the G4C4 sequence including successive guanine bases, ofloxacin could intercalate into the two adjacent guanine bases sequence through the minor groove of DNA duplex with the aid of CuII coordination, which leads to a more favorable binding between S-ofloxacin and DNA. In the chiral separation of ofloxacin enantiomers, the highest ee value of ofloxacin enantiomers in the permeate after being adsorbed by the CuII−G4C4 complex is obtained as 49.2% in R-enantiomer at [Cu2+]/[base] molar ratio of 0.25, while at [Cu2+]/[base] molar ratio of 0.05 the highest ee value of the retentate reaches 26.3% in the S-enantiomer. This work provides deep insights into the enantioselective recognition based on chirality of DNA structures and the specific binding of transition metal ions, and is promising for the construction of DNA-based chiral selectors toward other chiral compounds.

Table 2. eep and αp Value of 50 μM Racemic Ofloxacin after Being Adsorbed by the G4C4-dye Complexes at the [Cu2+]/ [base] Molar Ratio of 0.05 and 0.25 at pH 7.2, Respectively (R-Enantiomer Excess) 0.05a

a

0.25a

species

eep (%)

αp

eep (%)

αp

no dye MGb Hoechst 33258b EBb

32.1 28.8 26.9 21.9

1.95 1.81 1.74 1.56

49.2 46.0 41.1 34.0

2.93 2.70 2.40 2.03

The [Cu2+]/[base] molar ratio. bThe [dye]/[base] molar ratio is 1:5. 5307

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Figure 7. Schematic illustration of the intercalative and groove binding of S-ofloxacin to (a) G4C4 and (b) AT in the presence of Cu2+, respectively. The purple dashed circles denote the methyl group at the C-3 position of the oxazine ring of S-ofloxacin and the phosphate backbone of G4C4, respectively, between which there is no apparent steric hindrance upon S-ofloxacin binding to G4C4. The green dashed lines indicate the coordination geometry of CuII in the CuII−DNA−ofloxacin complexes.

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ASSOCIATED CONTENT

* Supporting Information S

Additional CD spectroscopy, PAGE images, and ITC data for ofloxacin−DNA binding in the absence and presence of Cu2+. EPR spectra of the binary CuII−Tris and CuII−S-ofloxacin complexes. HPLC chromatogram of chiral separations of ofloxacin enantiomers by the CuII−DNAs. Tables for FT-IR bands assignments of DNA, adsorption percentage of S- and Rofloxacin, and the results of competitive binding assay. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-22-27890643. Fax: +8622-27890643. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (21076141, 21206107) and PCSIRT. The authors are grateful to Institute of Biomedical Engineering (Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China) for ITC measurements.

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REFERENCES

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