An Evidence for the Chiral Discrimination of Naproxen Enantiomers: A

Publication Date (Web): February 21, 2011. Copyright ... *E-mail: [email protected] (C.D.); [email protected] (S.M.S.); [email protected] (M.M.F.C.)...
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An Evidence for the Chiral Discrimination of Naproxen Enantiomers: A Combined Experimental and Theoretical Study Yanli Wei,† Sufang Wang,† Jianbin Chao,† Songbai Wang,† Chuan Dong,*,† Shaomin Shuang,*,‡ Man Chin Paau,§ and Martin M. F. Choi*,§ †

Research Center of Environmental Science and Engineering, and ‡School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China § Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, China

bS Supporting Information ABSTRACT: Naproxen enantiomers possess strong room temperature phosphorescence (RTP) in β-cyclodextrin (β-CD) system with a small amount of 1,2dibromoethane (1,2-DBE) under ambient conditions. The effects of pH, concentration of β-CD, and 1,2-DBE on the RTP of naproxen enantiomers have been investigated in detail. Time-resolved RTP spectroscopy shows that both naproxen enantiomers exhibit biexponential decay pattern with lifetimes of τ1 = 4.79 ( 0.13 and τ2 = 1.51 ( 0.096 ms for R-naproxen and τ1 = 6.67 ( 0.15 and τ2 = 2.13 ( 0.061 ms for S-naproxen. The lifetime differences between these enantiomers are Δτ1 = 1.88 and Δτ2 = 0.62 ms, indicating that chiral discrimination of naproxen enantiomers can be achieved in β-CD/1,2-DBE system. Naproxen enantiomers can form stable complexes with β-CD and 1,2-DBE in stoichiometric ratios of 1:1:2 and 1:1:1 (naproxen:β-CD:1,2-DBE), and the association constants are 3.20  103 M-4 and 2.43  103 M-3 for the S- and R-enantiomers, respectively. The chiral discrimination of R-naproxen and S-naproxen is realized via their difference in interaction with the chiral cavity of β-CD due to their difference in stereochemical structure. Finally, molecular modeling is performed to determine the chiral recognition on a molecular level, and the results are in good agreement with the experimental data.

1. INTRODUCTION The chiral discrimination is of great importance in many fields such as biology, chemistry, material science, and pharmaceutical since enantiomers often show different physiological activities depending on their absolute configurations. Chirality is a central factor in biological phenomenon, and it is common that the enantiomers of a chiral drug show striking differences in terms of biological activity, potency, toxicity, and transport mechanisms. An effective chiral selector is often required to interact with the enantiomers to form different diastereomeric complexes to achieve chiral discrimination. Among the chiral selectors, cyclodextrins (CDs) are the most commonly used supramolecular receptors for the chiral molecules. CDs are cyclic oligosaccharides composed of 6(R), 7(β), or 8(γ) D-glucose units that are joined by R-1,4-glucosidic linkage, and their three-dimensional structure can be considered as a truncated cone. For instance, β-CD has bottom and top diameters of 6.0 and 6.5 Å.1 The hydroxyl groups are located at the outer surface of the CD molecule, which makes them water-soluble but simultaneously generates an inner hydrophobic cavity. As a result, they can either partially or entirely accommodate suitably sized and shaped lipophilic molecules.2 Furthermore, these macrocycles are inherently dissymmetric and thus able to form different r 2011 American Chemical Society

diastereomeric host-guest complexes by interacting differently with each enantiomer.3 β-CD has been applied frequently in chiral separations and analyses attributing to its capability to form inclusion complexes, low price, and good availability.4 With the development of modern analytical instruments, various techniques including chromatography, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance, circular dichroism, and fluorescence have been used for chiral separation and analysis.5 Among these methods, fluorescence spectroscopy has received much attention due to its simplicity, speediness, and high sensitivity.6 As a sister technology to fluorescence spectroscopy, room temperature phosphorescence (RTP) is also a good choice for chiral discrimination since it possesses some distinctive advantages such as fewer spectral interferences, longer emission wavelength, larger Stokes shift, and longer and easily measurable triplet state lifetimes. It is wellknown that the unique chiral microenvironments of CDs or chiral surfactants allow chiral discrimination to occur in triplet excited states. In our previous study, the chiral discrimination Received: September 5, 2010 Revised: January 26, 2011 Published: February 21, 2011 4033

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Figure 1. Chemical structure of (A) R-naproxen and (B) S-naproxen.

between quinine and quinidine has been successfully performed based on their difference in phosphorescence lifetime in various chiral surfactant systems including digitonin and glycyrrhizic acid.7 Naproxen, a chiral compound, is often used as a nonsteroidal anti-inflammatory analgesic drug. Figure 1 displays the chiral Rcarbon atom of R-naproxen and S-naproxen. As only the (þ)isomer possesses the desirable properties and therapy, it is important that the naproxen enantiomers have to be distinguished. Both the protonated and deprotonated forms of naproxen can be included into β-CD and emit RTP in β-CD system in the presence of cyclohexane and thallium nitrate under deoxygenated conditions.8 However, to our knowledge, the exploitation of chiral discrimination of naproxen enantiomers by RTP lifetime measurement as well as the study of binary complexes and dynamics of naproxen in the supramolecular system of β-CD at ambient conditions have not been reported. In most previous studies,9 CD-induced RTP was stronger only in bromoalkanes or cyclohexane systems where turbidity or precipitates occurred. Herein, we report for the first time the RTP emission of naproxen in the system of β-CD/1,2-dibromoethane (1,2-DBE). Naproxen can show stronger RTP in a transparent solution containing βCD as the host molecule and 1,2-DBE presenting the heavy atom effect on naproxen. In addition, the chiral discrimination of naproxen enantiomers can be achieved by recording their timeresolved RTP spectra since their difference in RTP lifetimes is palpable. The interaction mechanisms between R-naproxen or Snaproxen with β-CD and 1,2-DBE were also explored by 1H NMR, 2D NMR, and molecular modeling. Our proposed work provides a better insight into the interactions between naproxen enantiomers with β-CD and 1,2-DBE as well as a convenient and simple method to distinguish between R-naproxen and Snaproxen.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. 1,2-Dibromoethane, Rnaproxen, and S-naproxen were purchased from Acros Organics (Geel, Belgium) and used without further purification. Stock solutions of 1.0 mM R-naproxen and S-naproxen were prepared by dissolving the solid R-naproxen and S-naproxen in 2.0 mM sodium hydroxide. β-CD and methyl-β-CD were obtained from

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Fluka Chemicals (Buchs, Switzerland) and used as received. Disodium hydrogen phosphate and monosodium dihydrogen phosphate were from Farco Beijing Supplies (Beijing, China). All other reagents of analytical grade or above were used as received. Purified water from a Milli-Q water purification system (Millipore, Bedford, MA) with a resistivity higher than 18 MΩ 3 cm was used to prepare all solutions. 2.2. Instrumentation. Phosphorescence spectra were conducted on a Cary Eclipse fluorescence spectrophotometer (Varian, Forest Hill, Victoria, Australia) equipped with a pulsed xenon lamp and a 10  10 mm standard quartz cell. The fluorescence and time-resolved spectra were performed on an Edinburgh FLS920 fluorescence lifetime spectrometer (Edinburgh, UK) operating in the time-correlated single photon counting mode. A xenon arc lamp as the excitation light source was used to record fluorescence spectra and a hydrogen laser operating at an excitation wavelength of 274 nm was used for fluorescence lifetime measurement. A microsecond pulsed xenon flash lamp was used as the excitation light source in the determination of RTP lifetime. The lifetimes were analyzed with the instrumentspecific F900 (Edinburgh, UK) software using the intensity decay equation n

IðtÞ ¼ A þ

∑ Bi expð- t=τi Þ i¼1

where I(t) is the intensity with time, A is the instrumental background (dc offset), Bi is the pre-exponential factor, and τi is the luminescence lifetime associated with the ith component of the sample. All our RTP decay curves show biexponential nature. Fitting results were judged on the basis of a reduced χ2 around 1.0 and a random distribution of the weighted residuals around zero. All one-dimensional and two-dimensional (2D) 1H NMR spectra were obtained on a Bruker Avance DRX 300 MHz nuclear magnetic resonance spectrometer (F€allenden, Switzerland) in D2O, and the chemical shifts (δ) are expressed in ppm with D2O as the reference. 2.3. Procedure. Typically, an appropriate amount of stock solution of R-naproxen or S-naproxen was transferred into a 10 mL comparison tube, and then appropriate volumes of 1,2-DBE and β-CD or methyl-β-CD in phosphate buffer solution (0.20 M, pH 3.0) were added. The mixture was diluted to the final 5.0 mL with a phosphate buffer solution (0.20 M, pH 3.0) and shaken thoroughly. The working solution was left to equilibrate for 2 h at room temperature and then transferred into a 1.0 cm standard quartz cell with a cover to record its phosphorescence spectrum and lifetime. The sample was excited at 274 nm, and the phosphorescence signal was monitored at 520 nm. The excitation and emission slit widths were both set at 20 nm. A delay time of 0.10 ms was used in order to remove interfering fluorescence and scattered light from the phosphorescence spectrum. The gate time was selected as 5.0 ms. 2.4. Molecular Modeling. The molecular mechanistic and dynamic calculations were performed with the Insight II program (version 2000, Molecular Simulations Inc., San Diego, CA) using the consistent valence force field (CVFF) method. The β-CD structure was obtained by energy minimization of a crystallographic geometry.10 The conformational search of naproxen enantiomers were performed by simulated annealing molecular dynamics-full energy minimization strategy. The lowest energy conformation of naproxen enantiomer was selected for further 4034

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Table 1. 1H Chemical Shifts Corresponding to β-CD in the Presence of Different Componentsa Δδ1 proton in β-CD

δ0

Δδ2

R-naproxen S-naproxen R-naproxen S-naproxen

H-1

4.970

-0.094

-0.096

-0.070

-0.081

H-2

3.531

-0.105

-0.096

-0.042

-0.081

H-3 H-4

3.874 3.491

-0.125 -0.099

-0.113 -0.090

-0.171 -0.002

-0.186 -0.081

H-5

3.758

-0.159

-0.154

-0.150

-0.167

H-6

3.824

-0.169

-0.170

-0.216

-0.233

a δ0 indicates the chemical shift of proton in β-CD. Δδi = δi - δ0 (i = 1, 2), where 1 and 2 correspond to the solutions of β-CD and naproxen, and β-CD, naproxen, and 1,2-DBE, respectively.

simulations. During the simulation, the structures of naproxen, CD and 1,2-DBE was allowed to fully optimize. In the calculation process, the complex of naproxen/β-CD was placed in the center of the cubic cell, which is 30 Å, and the cell is then filled with water using the PBC method.11

3. RESULTS AND DISCUSSION 3.1. 1H NMR and 2D NMR Studies. Nuclear magnetic reso-

nance (NMR) is an important technique for determining the interaction mechanism between chiral molecular and β-CD. To better understand the function model of the inclusion complex of naproxen/β-CD, 1H NMR spectra in the absence and presence of 1,2-DBE in D2O were measured and are displayed in Figures S1S8. The change of chemical shifts (Δδ) of various relevant protons in β-CD and naproxen are summarized in Table 1. The Δδ of the protons in β-CD with naproxen, and naproxen and 1,2-DBE are different. Upon addition of naproxen to β-CD, significant changes of H-3, H-5, and H-6 in β-CD were observed, and the Δδ are in the order H-6 > H-5 > H-3 > H-2 > H-1 ≈ H-4. Furthermore, the Δδ of the naphthyl ring protons were significant, inferring an obvious upfield shift of the aromatic protons in naproxen due to the interaction of naproxen with β-CD. The naphthyl moiety of naproxen could enter into the hydrophobic cavity of β-CD since CD has a toroidal/hollow truncated cone structure (vide supra). The Δδ of H-5 in β-CD is larger than that of H-3, suggesting that naproxen gets into the hydrophobic cavity from the narrower rim of the β-CD. In the tricomponent system of naproxen/βCD/1,2-DBE, the Δδ of H-3 is larger than that of H-5, indicating that 1,2-DBE enters into the cavity of β-CD through the wider rim of the β-CD. Table 1 displays the chemical shift of protons (δ0) of β-CD and Δδi = δi - δ0 (i = 1, 2) where 1 and 2 correspond to the solutions of β-CD and naproxen, and β-CD, naproxen, and 1,2DBE, respectively. It is noted that the differences of Δδ2 between R-naproxen and S-naproxen are larger than that of Δδ1, and the changes of H-6 in both the binary and tricomponent systems are the largest as compared to other protons, indicating that the addition of 1,2-DBE as a third component plays an important role in the chiral recognition of β-CD to the naproxen enantiomers; i.e., a R- or S-naproxen molecule can interact differently with the H-6 of β-CD molecule, leading to the chiral discrimination of R-naproxen and S-naproxen. 2D NMR spectroscopy has also been used to study the inclusion complexation of β-CD with naproxen in order to understand the binding model. Figure 2 displays spatial contour

Figure 2. Partial ROESY spectra of (A) 0.50 mM R-narproxen and (B) S-narproxen and β-CD in D2O at 20 °C.

plots of the ROESY spectra for S-naproxen and the S-naproxen/ β-CD system. The cross-peaks between naphthyl ring and β-CD protons were observed, indicating that the naphthyl ring of the naproxen molecule penetrates into the hydrophobic cavity from the narrower side of β-CD. The carboxylic acid moiety of the naproxen molecule is located at the outer rim of β-CD via the hydrogen bond between the carboxylic proton of naproxen and the external hydroxyl group of β-CD. 1,2-DBE acting as a spacefilling component and competitor inserts into the β-CD cavity from the wider opening of β-CD to form a stable tricomponent complex of naproxen/β-CD/1,2-DBE. 3.2. Fluorescence and Phosphorescence Spectra. Figure 3a, b depicts the fluorescence spectra of R-naproxen and S-naproxen in aqueous solution. Both naproxens show an emission peak maximum at ca. 360 nm. The fluorescence intensity of R-naproxen or S-naproxen decreases with a concomitant increase of RTP at ca. 520 nm when 1,2-DBE and β-CD are added to the naproxen sample solution as shown in Figure 3c, d, inferring that a rigid 4035

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Figure 3. Fluorescence spectra of 20 μM (a) S-naproxen and (b) Rnaproxen. RTP spectra of 20 μM (c) S-naproxen and (d) R-naproxen in the presence of 2.0 mM β-CD and 23 mM 1,2-DBE.

tricomponent inclusion complex is formed from naproxen, β-CD, and 1,2-DBE. The R-naproxen/β-CD/1,2-DBE and S-naproxen/ β-CD/1,2-DBE complexes exhibit very similar spectral characteristics with maximum emission peaks at 520 nm and excitation peaks at 274 and 320 nm (not shown). Since the RTP intensity is stronger when excited at 274 nm, this excitation wavelength was chosen for all the RTP lifetime and intensity measurements. 3.3. Optimization of Phosphorescence Conditions. The RTP of the naproxen enantiomers could be affected by pH and concentration of β-CD and 1,2-DBE. As such, these factors were investigated in detail. 3.3.1. Effect of pH. Naproxen is a weak organic acid in aqueous solution. It has been reported that the pKa of naproxen is 4.4112a or 5.55.12b As such, the existence of the protonated and deprotonated forms of naproxen depends on the pH of the solution and the RTP intensity of naproxen may vary with pH. The RTP emission intensity of naproxen gradually decreases with the increase in pH from 3.0 to 8.0 and almost disappears when the pH is higher than 8.0 (not shown), implying that the RTP of naproxen mainly origins from its protonated species due to the coexistence of hydrogen bonding, hydrophobic interaction, and dipole-dipole interactions of the naproxen/β-CD/1,2-DBE complex. Thus, phosphate buffer solution at pH 3.0 was chosen for most RTP determinations. 3.3.2. Effect of β-CD Concentration. The cavity of β-CD could provide a hydrophobic microenvironment and avoid quenching of the excited triplet states of naproxen from collision with oxygen. The effects of β-CD concentration on the RTP intensity of R-naproxen and S-naproxen in the presence of 1,2DBE were investigated. The results show that the RTP intensity increases with the increase in β-CD concentration and then decreases with further increase in β-CD at 2.0 mM. The maximum RTP was obtained at 2.0 mM β-CD, indicating that the formation of tricomponent complex completes at this concentration. When the concentration of β-CD is higher than 2.0 mM, the solution of naproxen/β-CD/1,2-DBE becomes slightly turbid which might be due to self-aggregation of β-CD,13 thus reducing its ability to interact with guest molecules14 with a concomitant decrease in RTP emission. As such, 2.0 mM β-CD was chosen for this work. 3.3.3. Effect of 1,2-DBE Concentration. 1,2-DBE is a wellknown external heavy atom perturber for CD-induced RTP. It

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Figure 4. Effect of concentration of 1,2-DBE on the phosphorescence intensity of 20 μM R-naproxen and S-naproxen. The inset displays the average lifetimes of R-naproxen and S-naproxen against concentration of 1,2-DBE. The concentration of β-CD is 2.0 mM.

not only enhances the spin-orbit coupling interaction but also increases the probability of intersystem crossing from the excited singlet (S1) to the triplet (T1) states. As mentioned above, 1,2-DBE can enter easily into the cavity of CD because of its small molecular volume and low polarity.15 The fluorescence intensity of naproxen is quenched by 1,2-DBE but at the same time, the intersystem crossing efficiency is remarkably promoted, leading to the increase in triplet state emission. Figure 4 displays the effect of 1,2-DBE on the RTP intensity of naproxen enantiomers. It is obvious that the RTP increases with the increase in concentration of 1,2-DBE and reaches the maximum intensity at 23 mM. Further increase in 1,2-DBE concentration causes a drop in RTP which is possibly due to the fact that excess 1,2-DBE can quench the RTP of naproxen/βCD/1,2-DBE complex. As such, the Stern-Volmer plots were used to determine the quenching constants of R- and S-naproxen: P0 ¼ 1 þ KSV ½1; 2-DBE P

ð1Þ

where P0 is the RTP intensity of naproxen in the presence of 23 mM 1,2-DBE and P is the RTP intensity at excess 1,2-DBE (>23 mM). [1,2-DBE] is the excess concentration of 1,2-DBE, and KSV is the Stern-Volmer quenching constant which is determined from eq 1 as 15.4 and 13.8 M-1 for R- and Snaproxen, respectively. The inset of Figure 4 depicts the average RTP lifetimes of naproxen against concentration of 1,2-DBE. SNaproxen displays a longer RTP lifetime than R-naproxen. Both S-naproxen and R-naproxen have the longest lifetimes at 23 mM of 1,2-DBE. Excess 1,2-DBE lead to the decrease in their lifetimes, possibly attributing to the dynamic quenching of 1,2-DBE on naproxen/β-CD/1,2-DBE complex. Furthermore, the inclusion constants (K) and stoichiometric ratios of the inclusion complex of naproxen and β-CD were determined by the nonlinear least-squares fitting of the experimental data obtained from the fluorescence measurement:16 1 ΔF ¼ fRð½H0 þ ½G0 þ 1=KÞ 2 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ( R2 ð½H0 þ ½G0 þ 1=KÞ2 - 4R2 ½H0 ½G0 g 4036

ð2Þ

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where [H]0 and [G]0 are the initial concentrations of host β-CD and guest naproxen, respectively. ΔF denotes the change of the fluorescence intensity of naproxen with the addition of β-CD. R is a sensitive factor of the structural change of the complex comprising naproxen and β-CD at the interactive course. The inclusion constants between naproxen enantiomers and β-CD in the absence of 1,2-DBE are determined as 566 and 502 M-1 for S- and R-naproxen, respectively. The ratio of KS-naproxen/ KR-naproxen is 1.13, inferring that native β-CD shows very little chiral discrimination without 1,2-DBE toward naproxen enantiomers, and this is consistent with a previous report.17 By contrast, in the presence of 23 mM 1,2-DBE, the inclusion constants between naproxens and β-CD are 265 and 198 M-1 for S- and R-naproxen, respectively, lower than that of without 1,2-DBE. It is plausible that 1,2-DBE can compete with naproxen for β-CD. But the ratio of KS-naproxen/KR-naproxen now turns to 1.34, inferring that the chiral discrimination ability of β-CD toward naproxen enantiomers is improved with 1,2-DBE and that 1,2-DBE not only acts as a heavy atom perturber but also as a space regulator. As a heavy atom perturber, 1,2-DBE could enhance the S1 f T1 intersystem crossing and thus induce stronger phosphorescence. On the other hand, as a space regulator, 1,2-DBE inserts into the cavity of β-CD and forms a tricomponent complex (naproxen/β-CD/1,2-DBE). The inclusion constants between naproxen and β-CD decrease to 10.8% and 25.1% for S- and R-naproxen, respectively, when the concentration of 1,2-DBE increases from 23 to 58 mM, inferring that excessive 1,2-DBE may interact with naproxen preventing its complexation with β-CD. For comparison, methyl-β-CD was also used as the chiral selector in this study. The inclusion constants of naproxen enantiomers and methyl-β-CD determined by the fluorescence measurements are 713 and 627 M-1 for R- and S-naproxen, respectively, in the absence of 1,2-DBE and are 276 and 260 M-1 for R- and S-naproxen, respectively, with 23 mM 1,2-DBE. So, the ratios of KS-naproxen/KR-naproxen are 1.14 and 1.06 in the absence and presence of 1,2-DBE, respectively, indicating that methyl-βCD is not an ideal chiral selector for naproxen enanotimers as compared to β-CD. This is probably related to the methyl moieties of methyl-β-CD which reduce the specific hydrogen bonding of the carboxylic acid moiety of the naproxen enantiomers. In addition, 1,2-DBE is a quencher for naproxen fluorescence. The Stern-Volmer plot is thus employed to determine its quenching effect on naproxen F0 ¼ 1 þ kq τ0 ½Q  ¼ 1 þ KSV ½Q  F

ð3Þ

where F0 and F are the fluorescence intensities of naproxen in the absence and presence of 1,2-DBE, respectively. kq is the bimolecular quenching constant, and τ0 is the lifetime of naproxen in the absence of quencher (1,2-DBE). For R- and S-naproxen, the fluorescence lifetimes (τ0) are 11.17 and 11.19 ns, respectively. Equation 3 is applied to determine KSV by plotting the linear regression of F0/F against concentration of 1,2-DBE. The bimolecular quenching constants (kq) are 1.37  109 and 1.52  109 M-1 s-1 for R- and S-naproxen, respectively. Figure S9 displays the fluorescence lifetime of naproxen enantiomers at different concentrations of 1,2-DBE. The fluorescence lifetimes decrease with the increase in concentration of 1,2-DBE. For instance, the lifetimes decrease to 9.18 and 9.34 ns for R- and Snaproxen when the concentration of 1,2-DBE is 32.5 mM.

Obviously, the fluorescence quenching effect of 1,2-DBE on Rand S-naproxen is more or less the same. 3.4. Stoichiometric Inclusion Constant. The formation of a tricomponent complex can be described by the following equilibria:19 K1

naproxen þ β-CD sF Rs naproxen 3 β-CD

ð4Þ

K2

naproxen 3 β-CD þ nð1; 2-DBEÞ sF Rs naproxen 3 β-CD 3 ð1; 2-DBEÞn ð5Þ K3

nð1; 2-DBEÞ þ β-CD sF Rs ð1; 2-DBEÞn 3 β-CD

ð6Þ

K4

ð1; 2-DBEÞn 3 β-CD þ naproxen sF Rs ð1; 2-DBEÞn 3 β-CD 3 naproxen

ð7Þ Ki is the corresponding equilibrium constant. The formation, stoichiometric ratio, and inclusion constant of the inclusion complex are determined by fluorescence measurements. The equilibrium described by K1 is measured by monitoring the fluorescence intensity of naproxen at 360 nm as a function of βCD concentration based on eq 2 and calculated as 502 and 566 M-1 for R- and S-naproxen, respectively, with 1:1 stoichiometric ratio. The equilibrium constant K2 can be expressed in terms of K1 as K2 ¼

½naproxen 3 β-CD 3 ð1; 2-DBEÞn  K1 ½naproxen½β-CD½1; 2-DBEn

ð8Þ

When the concentration of 1,2-DBE is much higher than the complex, which is one of our experimental conditions, the following equation can be applied to the tricomponent complex: 1 1 1 ¼ þ IF - I0 K2 k½1; 2-DBEn ½β-CD0 k½β-CD0

ð9Þ

where IF and I0 are the fluorescence intensities of naproxen in the presence and absence of 1,2-DBE. k is the combined quantum yield and instrumental factor, and the subscript refers to the initial concentration of β-CD. A double-reciprocal plot of 1/(IF - I0) against 1/[1,2-DBE]n yields K2 from the ratio of the intercept to slope. Figure S10 depicts the double-reciprocal plots of 1/(IF I0) against 1/[1,2-DBE]n for the tricomponent system when n = 1 and 2. The plot bends upward when n = 1 whereas it is linear when n = 2 for S-naproxen, indicating that a quaternary complex of S-naproxen/β-CD/(1,2-DBE)2 with a stoichiometric ratio of 1:1:2 is formed. For R-naproxen, the plot is linear when n = 1 and bends downward when n = 2, implying that a ternary complex of R-naproxen/β-CD/1,2-DBE with a stoichiometric ratio of 1:1:1 is resulted. On the basis of eq 8, the equilibrium constants K2 are 3.20  103 M-2 and 2.43  103 M-1 for S-naproxen quaternary and R-naproxen ternary complex, respectively. As such, a chiral diastereoisomer selectivity of 1.32:1 is achieved. The equilibrium constant K3 can be obtained by using phenolphthalein as the chromophoric reagent (Supporting Information), and the result is K3 = 752 M-1 for the 1:1 binary complex of 1,2-DBE/β-CD, which is in the same order of magnitude to K1. Since K2 . K1 ≈ K3, both naproxen enantiomers and 1,2-DBE could enter into the cavity of β-CD to form more stable tricomponent complex. The ratio of K3(S-naproxen)/ K3(R-naproxen) is 1.32, which is larger than that of K1(S-naproxen)/ K1(R-naproxen) = 1.13. Hence, it can be concluded that 1,2-DBE acts as not only a competitor but also a space regulator. 4037

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Figure 6. (top) Phosphorescence decay curves of 20 μM R-naproxen and S-naproxen. (bottom) Residual analysis of phosphorescence decay fitting of R-naproxen and S-naproxen. The concentrations of β-CD and 1,2-DBE are 2.0 mM and 23 mM, respectively.

Figure 5. Effect of concentration of β-CD on the fluorescence spectra of 10 μM (A) R- and (B) S-naproxen in the presence of 23 mM 1,2-DBE. The insets display the plot of change in fluorescence intensity against concentration of β-CD.

The inclusion constant K4 can be determined by monitoring the change in fluorescence intensity of naproxen upon addition of β-CD using the nonlinear least-squares fitting method based on eq 2. Figure 5 shows the effect of concentration of β-CD on the fluorescence spectra of R- and S-naproxen, with constant concentrations of naproxen (10 μM) and 1,2-DBE (23 mM). The fluorescence intensity increases with increasing β-CD concentration. The insets of Figure 5 display the plots of ΔF against concentration of β-CD using eq 2. The inclusion constants K4 of naproxen enantiomers and β-CD with 1:1 stoichiometric ratio are 198 and 265 M-1 for R- and S-naproxen, respectively, which are obviously lower than that of without 1,2-DBE (vide supra). The ratio of the inclusion constants is 1.34, inferring that the different stereochemical structures of R- and S-naproxen could affect their ability to interact with the chiral cavity of β-CD in the presence of 1,2-DBE. 3.5. Time-Resolved Spectroscopy. Phosphorescence lifetime is an important molecular luminescence parameter for characterizing luminescence compound. The chiral microenvironment of the CD cavity makes selective recognition of the analyte possible. By comparing the lifetimes of the diastereoisomeric complexes, it is possible to distinguish between the R- and S-enantiomers.3 The RTP lifetimes of R- and S-naproxen are taken in the presence

of 2.0 mM β-CD and 23 mM 1,2-DBE. Figure 6 depicts the phosphorescence decay curves and the residual analysis of R- and S-naproxen. Both of them exhibit a biexponential decay pattern, and the RTP lifetimes are τ1 = 4.79 ( 0.13 and τ2 = 1.51 ( 0.096 ms for R-naproxen and τ1 = 6.67 ( 0.15 and τ2 = 2.13 ( 0.061 ms for S-naproxen. The RTP lifetime of the quaternary complex of Snaproxen/β-CD/(1,2-DBE)2 is longer than that of the ternary complex of R-naproxen/β-CD/1,2-DBE. The RTP lifetimes are in the millisecond regime, indicating the π and π* triplet states character of the inclusion complexes.18 The differences in RTP lifetime (Δτ) and the percent differences (Δτ/τ) are Δτ1 = 1.88 and Δτ2 = 0.62 ms and Δτ1/τ1 = 32.9 and Δτ2/τ2 = 34.4%, respectively, indicating that the chiral discrimination of naproxen enantiomer is possible in the β-CD/1,2-DBE system. In essence, time-resolved RTP spectroscopy can be applied to distinguish the naproxen enantiomers without deoxygenation. 3.6. Molecular Modeling. Besides the experimental studies of naproxen enantiomers in the β-CD/1,2-DBE system, molecular modeling simulation is also performed with β-CD and naproxen by gradient energy minimization of the initial configuration for 200 iterations. The results of the 200 iterations are consistent with 1000 steps. In the trial for new configurations, βCD is regarded as the coordinate center. Then S-naproxen takes translational movement of 0.1 Å to x, y, and z axes and rotation of 60° around x, y, and z axes, whereas R-naproxen makes translational movement of 1.0 Å to x, y, and z axes and rotation of 15° around x, y, and z axes. The lowest energy structures of the naproxen enantiomer and β-CD complexes are determined and displayed in Figure 7. The molecular modeling calculation shows that S-naproxen could fit almost completely inside the cavity of β-CD, which is different from R-naproxen that is only partially inserted in the cavity of β-CD. The distances between the carboxylic hydrogen of S-naproxen and H-6, H-2, and H-3 of β-CD are 5.92, 10.35, and 9.45 Å and 4.54, 8.51, and 9.68 Å for R-naproxen, respectively. The corresponding binding energies are -64.5 and -68.7 kJ/mol for R- and S-naproxen, respectively. The energy difference between S- and R-naproxen inclusion complexes is 4.2 kJ/mol, suggesting that S-naproxen has stronger interaction with β-CD than R-naproxen. It can be concluded that 4038

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complexes. Enantio-discrimination could be successfully achieved based on the different interactions such as hydrogen bonds and dipole-dipole interactions between naproxen and β-CD molecules. A stable inclusion complex, namely, β-CD/naproxen, could be formed in the presence of 1,2-DBE and the complexes exhibit enantiomeric differentiation by their differences in RTP intensity and lifetime. This enables easy discrimination between R- and S-naproxen enantiomers based on time-resolved spectroscopy only. It is anticipated that such new information would lead to a better understanding of the relationship between chirality and conformational changes of chiral molecules. The use of β-CD/1,2-DBE for rapid chiral analysis of other enantiomeric pairs could also be explored.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed experimental procedures and calculation method of the equilibrium constant of K3, 1 H NMR, UV-vis spectra, and double-reciprocal plot of 1/(IF I0) against 1/[1,2-DBE]n. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (C.D.); [email protected] (S.M. S.); [email protected] (M.M.F.C.).

’ ACKNOWLEDGMENT Financial support from the National Natural Science Foundation of China (20875059), Natural Science Foundation of Shanxi Province (2010021010-2), and Research Project Supported by Shanxi Scholarship Council of China (200809) is gratefully acknowledged. ’ REFERENCES

Figure 7. The most stable conformation of the inclusion complex of (A) R-naproxen and (B) S-naproxen with β-CD.

the chiral discrimination of naproxen enantiomers by β-CD is governed by the different interactions of the naproxen enantiomers with H-6 of β-CD. The results of molecular modeling simulation are consistent with the experimental behavior of the respective inclusion complexes (vide supra).

4. CONCLUSION In this work, the use of the β-CD/1,2-DBE system to obtain strong and stable RTP of naproxen, to discriminate naproxen enantiomers, and the findings of the difference in phosphorescence lifetimes of these enantiomers are reported for the first time. The hydrophobic cavity of β-CD could provide a chiral microenvironment for naproxen enantiomers to form diastereomeric host-guest

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