Temperature-Sensitive Electrochemical Recognition of Tryptophan

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Temperature-Sensitive Electrochemical Recognition of Tryptophan Enantiomers Based on β‑Cyclodextrin Self-Assembled on Poly(L‑Glutamic Acid) Yongxin Tao, Jiangying Dai, Yong Kong,* and Yan Sha Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, School of Petrochemical Engineering, Changzhou University, Changzhou 213164, P. R. China ABSTRACT: A poly(L-glutamic acid)/β-cyclodextrin (P-L-Glu/β-CD)modified glassy carbon electrode (GCE) was prepared by electrochemical polymerization of L-glutamic acid on GCE and subsequent self-assembly of β-CD onto the obtained P-L-Glu. Electrochemical recognition of tryptophan (Trp) enantiomers with the P-L-Glu/β-CD was investigated by differential pulse voltammetry (DPV), and the results show that Trp enantiomers can be effectively recognized at the P-L-Glu/β-CD-modified GCE. It is also interesting to find that temperature plays a crucial role in the enantioselective recognition, which is evidenced by the variable-temperature UV spectra of the inclusion complexes of Trp enantiomers and β-CD. Under optimum conditions, the oxidation peak current ratio of L-Trp to D-Trp could reach 2.30, which is attributed to the stereoselectivity of β-CD to the enantiomeric pair of Trp.

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chemical point of view, which might be due to the little conductivity of β-CD. Herein, we first report on the electrochemical recognition of Trp enantiomers with β-CD self-assembled on poly(L-glutamic acid) (P-L-Glu) via its primary hydroxyl groups (−(HO)p). The molecular recognition ability of the P-L-Glu/β-CD toward Trp enantiomers is characterized by differential pulse voltammetry (DPV), indicating a stronger recognition ability for L-Trp. Though the hydrophobic indole ring of both L- and D-Trp can penetrate into the cavity of β-CD for complexation, P-L-Glu/βCD exhibits a higher affinity for L-Trp than D-Trp, which could be due to favorability of intermolecular hydrogen bonding formation between secondary hydroxyl groups (−(HO)s) of βCD and amino groups of L-enantiomers compared to those of D-enantiomers. The influence of temperature on the recognition ability was also discussed in detail, because the strength of intermolecular hydrogen bonding depends strongly on the temperature tested.

mino acids are molecular building blocks of life, which play a crucial role in life science and many other related fields. However, chiral discrimination has been a long-standing problem in the development, use, and action of amino acids. For example, one of the isomers is effective, while the other might be ineffective or even cause serious side-effects.1,2 Tryptophan (Trp) enantiomers, which have asymmetric carbon in their structure, have great impact on the biological system. As an essential amino acid, Trp has been determined to be a precursor of the neurotransmitter serotonin, and the level of Trp in plasma is closely related to the extent of hepatic disease.3 So, it becomes extraordinarily important to develop new methods for chiral identification of Trp enantiomers to meet both general and practical challenges, and there are several strategies concerning the recognition of enantiomeric pair of Trp.4−8 Among these developed methods, electrode column technology based on molecularly imprinted conducting polymers is most attractive, because it overcomes the shortcomings such as high cost and time consumption associated with high-performance liquid chromatography (HPLC) and capillary electrochromatography. Enantioselective recognition of enantiomers of Trp,4 aspartic acid (Asp),9 and glutamic acid (Glu)10 has been achieved by this technology in our group. Cyclodextrins (CDs) are cyclic oligosaccharides composed of α(1−4)-linked D-glucopyranose units featuring a hydrophobic central cavity suitable for the stable inclusion with various organic molecules.11−13 There was evidence that stable, highcoverage, β-CD-bonded media could be employed for stereoselective discriminations.14−17 However, as far as we are aware, little or no attention has been paid to the use of β-CD for selective recognition of Trp enantiomers from the electro© 2014 American Chemical Society

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EXPERIMENTAL SECTION Reagents and Apparatus. L-Trp (99%) and D-Trp (98%) were purchased from Aladdin Chemistry Co., Ltd. (China). βCD was obtained from Zibo Qianhui Biological Technology Co., Ltd. (China). L-Glu, potassium ferricyanide, and potassium ferrocyanide were purchased from Shanghai Lingfeng Chemical Co. Ltd. (China). All other chemicals not mentioned here were of analytical reagent grade and used as received. All solutions were prepared using doubly distilled water. Received: December 4, 2013 Accepted: February 3, 2014 Published: February 3, 2014 2633

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Scheme 1. Schematic Representation of Electropolymerization of L-Glu and Self-Assembly of β-CD on P-L-Glua

a

−(HO)p and −(OH)s represent the primary hydroxyl groups and secondary hydroxyl groups, respectively.

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RESULTS AND DISCUSSION Electropolymerization of L-Glu and Self-Assembly of β-CD on P-L-Glu. The overpotential for L-Glu oxidation in PBS of pH 7.0 is at ∼1.35 V,18 hence the upper potential in CV is set as 2.0 V. Since the acid dissociation constant (pKa) of Glu is 4.45, L-Glu exists in its anionic form (−COO−) at pH 7.0. During the electropolymerization process, L-Glu is first oxidized to its corresponding cation radicals, which in turn reacts with the L -Glu monomer −COO − group resulting in the propagation of polymer chains. The as-prepared P-L-Glumodified GCE was immersed into 0.01 M β-CD at room temperature overnight. β-CD can be self-assembled onto P-LGlu via hydrogen bonding between its −(HO)p and the carboxyl groups of P-L-Glu, and therefore, a functional surface for the electrochemical recognition of Trp enantiomers is obtained. The whole process can be illustrated as Scheme 1. The interaction mode between β-CD and P-L-Glu is deduced on the basis of the theory proposed by Ashton et al.,20 in which they ascribed the coupling of phenylalanine to β-CD to the hydrogen bonding between the backbone carboxylic acid functionality of phenylalanine and the primary face of the βCD torus. This conclusion would be applicable to any other amino acid, including L-Glu, which bears a less sterical demand. Electrochemical Characterization of β-CD Self-Assembled on P-L-Glu. The electrochemical property of the P-L-Glu/β-CD-modified GCE was investigated by CV in 0.1 M KCl using 5 mM Fe(CN)64‑/3‑ as the electroactive probe couple. As shown in Figure 1, a couple of well-defined redox peaks of the probe are observed at the bare GCE with ΔEp = 142 mV (curve a), whereas the response at the P-L-Glu is improved a little, and the ΔEp becomes relatively small (125 mV) (curve b). This indicates that P-L-Glu can act as a conducting bridge to facilitate the electron transfer of the probe and accelerate the whole redox process.19,21 The result agrees well with previously

Electrochemical experiments were carried out on a CHI 660D electrochemical workstation (CH Instruments) in a conventional three-electrode system. The variable-temperature UV spectra of the inclusion complexes of Trp enantiomers and β-CD were measured on a UV-1700 UV−vis spectrophotometer (Shimadzu, Japan). The temperatures tested in this work were controlled with an intelligent thermostatic bath (Scientz Biotechnology Co. Ltd., China). Preparation of β-CD Self-Assembled on P-L-Glu. According to the previous report with slight modification,18,19 electropolymerization of L-Glu was performed in 0.1 M phosphate buffer solution (PBS, pH 7.0) containing 0.05 M L-Glu in a three-electrode cell using a glassy carbon electrode (GCE) (3 mm in diameter) as working, a platinum foil as auxiliary, and a saturated calomel electrode (SCE) as reference electrode, respectively. Successive cyclic voltammograms (CV) were recorded between −0.6 and 2.0 V at the scan rate of 100 mV s−1 for 20 cycles. Afterward, the P-L-Glu-modified GCE was carefully washed with doubly distilled water and then immersed into a 0.1 M PBS (pH 7.0) containing 0.01 M β-CD overnight for the self-assembly of β-CD on P-L-Glu. Electrochemical Recognition of Trp Enantiomers. The prepared P-L-Glu/β-CD-modified GCE was immersed into a 25 mL solution containing 0.5 mM L-Trp or D-Trp. The host− guest inclusion complexation lasted for 80 s at different temperatures varying from −2 to 45 °C, during which a constant potential of −0.1 V was applied on the modifed electrode. Finally, the electrochemical responses of the two inclusion complexes were measured by differential pulse voltammetry (DPV) in 0.1 M PBS (pH 7.0) in the potential range from 0.4 to 1.0 V at 100 mV s−1, and the electrochemical recognition of the Trp enantiomers was based on the differences of the oxidation peak currents of L-Trp and D-Trp in the inclusion complexes. All DPV experiments were carried out three times. 2634

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reported findings,18 in which the authors revealed the P-L-Glu films on GCE by AFM to be a porous structure that undergoes extensive swelling in aqueous electrolytes. This feature accentuates the role of the probe dissolved in solution to occupy the empty spaces within the swollen films. After the self-assembly of β-CD onto the surface of P-L-Glu, an obvious decrease in peak current is observed together with a large ΔEp (218 mV) (curve c). The low electron transfer and suppressed electrochemical reversibility of Fe(CN)64‑/3‑ at the P-L-Glu/βCD is due to the introduction of β-CD with little conductivity. Enantioselective Recognition of L- and D-Trp. L-Trp or D-Trp is expected to be included with β-CD after the P-L-Glu/ β-CD-modified GCE was immersed into the solution containing L-Trp or D-Trp for 80 s with an applied potential of −0.1 V. After that, the electrochemical responses of Trp enantiomers bound to P-L-Glu/β-CD were investigated by DPV at 8 °C. For a better comparison, several other electrodes including bare GCE, P-L-Glu/GCE, and β-CD/GCE were used as the control electrodes, among which the β-CD/GCE was

Figure 1. Cyclic voltammograms of 5 mM Fe(CN)64‑/3‑ in 0.1 M KCl solution at bare GCE (a), P-L-Glu-modified GCE (b), and P-L-Glu/βCD-modified GCE (c).

Figure 2. Differential pulse voltammograms of (a) L-Trp and (b) D-Trp bound to bare GCE (A), P-L-Glu/GCE (B), β-CD/GCE (C), and P-L-Glu/ β-CD-modified GCE (D) in 0.1 M PBS (pH 7.0) at 8 °C, and the current ratio histogram obtained at different electrodes (E). 2635

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Figure 3. (A) Chemical structure of (a) Trp, (b) L-Trp, and (c) D-Trp. (B) Chemical structure (a) and functional structural scheme (b) of β-CD. (C) Schematic diagram showing the optimal orientation of L-Trp (a) and D-Trp (b) on the basis of the highest degree of hydrogen bonding and inclusion complexation.

Scheme 2. Models of the Inclusion Complexes of L-Trp (A) and D-Trp (B) in β-CDa

a

The red, gray, white, and blue spheres represent the oxygen, carbon, hydrogen, and nitrogen atoms, respectively; the dashed lines represent the hydrogen bonding between β-CD and the primary amino groups of D- and L-Trp.

prepared by immersing the GCE into 0.01 M β-CD at room temperature overnight. Figure 2A,B exhibit overlapped or almost overlapped differential pulse voltammograms of L-Trp and D-Trp bound to the bare GCE and the P-L-Glu/GCE, indicating no stereoselective recognition occurs on these two electrodes. A discernible peak current ratio of 1.42 (L-Trp to DTrp) was observed for the β-CD/GCE (Figure 2C), implying that Trp enantiomers can be recognized by the β-CD/GCE.

However, the identification efficiency is not satisfactory because of the small current ratio, and this might be due to the fact that the amount of β-CD adsorbed onto GCE is too small to recognize L- and D-Trp effectively. This problem can be overcome by modifying the GCE with P-L-Glu beforehand. As can be seen from Figure 2D, the peak current ratio of L-Trp to D-Trp is increased to 2.30 for the P-L-Glu/β-CD. The enantioselective ability of these electrodes toward the Trp 2636

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Figure 4. (A) Peak current ratio of L-Trp to D-Trp included with β-CD at different temperatures. Errors bars represent the standard deviation for three independent measurements. (B) Variable-temperature UV spectra of the inclusion complexes of 0.05 mM L-Trp and 0.01 M β-CD at different temperatures: (a) 4 °C; (b) 8 °C; (c) 25 °C; (d) 37 °C; and (e) 42 °C.

enantiomers is arranged in such an order: P-L-Glu/β-CD > βCD/GCE > P-L-Glu/GCE ≈ GCE (Figure 2E). The highest stereoselectivity of P-L-Glu/β-CD is attributed to the existence of P-L-Glu, which acts as an ideal substrate for the self-assembly of β-CD to enhance the loading amount of enantioselective βCD on the working electrode (Scheme 1). The loading amount of β-CD self-assembled onto the P-LGlu-modified GCE is calculated to be 1.5 × 10−5 mol, according to the calibration plot of the refractive index of β-CD solution to its concentration at room temperature (figure omitted), because the refractive index of β-CD solution is proportional to its concentration in a certain range. Mechanism of Stereoselective Discrimination of Trp Enantiomers by P-L-Glu/β-CD. Possessing a hydrophobic, tapered cavity suitable for the inclusion of various organic molecules, β-CD is regarded as an excellent receptor molecule for the inclusion of organic molecules. Good inclusion complexation might have occurred by insertion of the less polar part of the guest molecule (indole group of L- and D-Trp in the present work) into the cavity of β-CD, whereas the more polar group of the guest (amino group in this work) is exposed to the −(HO)s on β-CD rims. A large peak current ratio of LTrp to D-Trp in Figure 2D clearly indicates that the P-L-Glu/βCD preferably adsorbs L-Trp, which can be due to favorability of hydrogen bonding formation between the −(HO)s on the βCD rims and the amino group of L-Trp compared to that of DTrp. The structure of L-Trp, D-Trp, β-CD and the simplified mechanism of inclusion complexation between β-CD and the Trp enantiomers are illustrated in Figure 3. Important differences are observed between three-dimensional structures of L- and D-Trp with respect to the position of −NH2 at the chiral center of Trp. In this case, the −NH2 in the D-enantiomer is positioned less favorably for hydrogen bonding, leading to less inclusion complexation with β-CD. It is noteworthy that carboxyl group is also a more polar group of the guest molecule (Trp) compared to indole group, however, it plays little role in the inclusion complexation between β-CD and Trp. According to the theory proposed by Armstrong et al.,14 the chiral center of the guest molecule must be near to the mouth of the β-CD cavity to obtain a relatively tight fit between the host and guest. As can be seen from the models of the superimposed L- and D-Trp in β-CD (Scheme 2), the carboxyl group in both L-Trp and D-Trp is positioned less favorably for the formation of hydrogen bonding due to the relatively long bond distance. This result implies that in their

most stable configurations, L- and D-Trp can hydrogen bond to β-CD via their primary amino group but not carboxyl group. Temperature-Sensitive Characteristics of the Enantioselective Recognition. It is interesting to find that temperature has a close relationship with the electrochemical recognition of Trp enantiomers. Figure 4A shows the plots according to the results of the recorded DPVs, indicating that the highest discrimination efficiency of the P-L-Glu/β-CD is obtained at 8 °C. Because the enantioselective recognition of Land D-Trp is achieved via the intermolecular hydrogen bonding between β-CD and the Trp enantiomers, it is no doubt that temperature has a significant influence on the strength of the hydrogen bonding. To verify this assumption, the variabletemperature UV spectra of the inclusion complexes of 0.05 mM L-Trp and 0.01 M β-CD were recorded (Figure 4B). It is observed that the strongest absorbance of the complex (278 nm) appears at 8 °C, implying that the hydrogen bonding between β-CD and L-Trp has the biggest strength at 8 °C compared to other temperatures tested. The UV spectra of the inclusion complexes of D-Trp and β-CD present a similar trend (figure omitted). What causes this phenomenon? It can be explained by comparing different hydrogen bondings coexisting in this system. There are at least three types of hydrogen bondings in the aqueous solution containing β-CD and Trp, including hydrogen bonding between β-CD and H2O (HBβ‑CD‑H2O), hydrogen bonding between β-CD and Trp (HBβ‑CD‑Trp), and hydrogen bonding between Trp and H2O (HBTrp‑H2O). In the temperature range from −2 to 4 °C, HBβ‑CD‑H2O and HBTrp‑H2O predominate in this system due to the unique property of H2O molecules, and the suppressed HBβ‑CD‑Trp reduces the recognition ability of the P-L-Glu/β-CD. The strength of HBβ‑CD‑Trp is increased with the increase in temperature and reaches a maximum at 8 °C. The most stable HBβ‑CD‑Trp at 8 °C results in the most obvious discrimination efficiency of the P-LGlu/β-CD, because the hydrogen bonding between β-CD and the Trp isomers is the decisive factor in the enantioselective recognition. However, an excessively high temperature (from 10 to 45 °C) will cause the stable HBβ‑CD‑Trp to break, and this deteriorates the discrimination efficiency toward Trp enantiomers. In fact, the influence of temperature on the molecular recognition in β-CD complexes based on hydrogen bonding interactions is quite complicated, because changes in guest molecule conformations, positions, and orientations occur in 2637

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beneficial to the preparation of the modified GCE, which agrees well with the results of Figure 4A. Stoichiometry and Stability Constant for Inclusion Complexation. UV spectra of the inclusion complexes in aqueous solution were recorded, and the corresponding absorbance changes upon addition of different concentrations of β-CD to 0.05 mM L-Trp solution were shown in Figure 6A. The absorbance intensity of the inclusion complexes (β-CD-LTrp) increases gradually with increasing concentration of β-CD, and this might be attributed to the improved dissolution of the guest molecule, L-Trp, via the hydrophobic interaction between the guest molecule and the nonpolar cavity of β-CD. Shanmugam et al.23 have reported similar changes of the inclusion complexes of L-tyrosine and β-CD. A similar trend in the absorbance changes of 0.05 mM D-Trp upon adding different concentrations of β-CD was also observed (figure omitted), and the reason is the same as that of L-Trp. It is also important to determine the stoichiometric ratio of the inclusion complexation between β-CD and the Trp isomers, because it has a close relationship with the enantioselective recognition. Employing a classical method proposed by Benesi and Hildebrand,24 the stoichiometry ratio and stability constant for the combination of β-CD and the Trp isomers can be determined at various β-CD concentrations by the following equation:

the supramolecular host−guest inclusion at different temperatures.22 Here, it should be pointed out that temperature also influences the strength of hydrogen bonding between β-CD and P-L-Glu (HBβ‑CD‑P‑L‑Glu), although it is not the decisive factor for the chiral discrimination. Figure 5 shows the CVs of

Figure 5. Cyclic voltammograms of 5 mM Fe(CN)64‑/3‑ in 0.1 M KCl at the P-L-Glu-modified GCE (a) and the P-L-Glu/β-CD-modified GCE prepared at different temperatures for 4 h: (b) 0 °C; (c) 4 °C; (d) 8 °C; (e) 25 °C; and (f) 37 °C.

1 1 1 = + A − A0 Δε × [C Trp] (Δε × [C Trp] × K × [β ‐CD]n0 )

Fe(CN)64‑/3‑ at the P-L-Glu/β-CD-modified GCE prepared at different temperatures. The lowest peak current is obtained at 8 °C (curve d), indicating HBβ‑CD‑P‑L‑Glu is the strongest at 8 °C, because the introduction of β-CD with poor conductivity will hinder the electron transfer. So, the temperature of 8 °C is also

(1)

where A and A0 are the absorbance of Trp at each β-CD concentration and in the absence of β-CD, respectively. K is the stability constant for inclusion complexation. [β-CD]n0 and

Figure 6. (A) UV spectra of 0.05 mM L-Trp upon addition of β-CD of various concentrations at 8 °C: (a) 0 mM, (b) 0.5 mM, (c) 1 mM, (d) 2 mM, (e) 3 mM, (f) 5 mM, (g) 7 mM, and (h) 10 mM. Double reciprocal plots for L-Trp (B) and D-Trp (C) inclusion complexes with β-CD. 2638

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[CTrp] are the original concentrations of β-CD and Trp isomers, respectively, and n represents the stoichiometry ratio of the inclusion complex. Δε is the differential molar extinction coefficient of Trp in the absence and presence of β-CD. Different values of n were tested to give the plots of 1/ΔA versus 1/[β-CD]n0, and a straight line is obtained only when the value of n is 1 (Figure 6B,C), indicating formation of the 1:1 complex between β-CD and the Trp enantiomers. The values of the inclusion constant K could be calculated to be 254.6 (βCD-L-Trp) and 188.6 (β-CD-D-Trp), respectively, by dividing the intercept by the slope of the plots in Figure 6B,C. The larger value of K between β-CD and L-Trp indicates that β-CD exhibits higher affinity to L-Trp than D-Trp, which agrees well with the results of the DPVs of L- and D-Trp bound to the P-LGlu/β-CD-modified GCE (Figure 2D). The stability of the prepared P-L-Glu/β-CD-modified GCE was tested by successive recognition of Trp enantiomers. The current ratio of L-Trp to D-Trp decreases remarkably after three times of recognition (from the initial value of 2.30 to 1.46 on the fourth time), which is due to the weak interactions between β-CD and P-L-Glu, because the self-assembly of β-CD onto P-LGlu is achieved by hydrogen bonding. However, the recognition efficiency can be restored by immersing the modified GCE into the solution of 0.01 M β-CD overnight again for the reassembly of β-CD, and the current ratio is restored to 2.26, which is 98.3% of the initial value.

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CONCLUSIONS

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AUTHOR INFORMATION

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21275023), the Natural Science Foundation of Jiangsu Province (BK2012593), Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology (BM2012110) and PAPD.

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REFERENCES

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In summary, we have prepared a P-L-Glu/β-CD-modified GCE by electropolymerization of L-Glu as the substrate for selfassembly of β-CD via the hydrogen bonding between the −(HO)p of β-CD and the carboxyl groups of P-L-Glu. An interesting phenomenon of electrochemical recognition of Trp enantiomers is observed at the as-prepared electrode, which exhibits obvious peak current difference for the Trp enantiomers. More important, this chiral discrimination is temperature-dependent, because temperature significantly affects the strength of hydrogen bondings between H2O and β-CD and Trp, which compete with the hydrogen bonding between β-CD and Trp. The maximum peak current ratio could reach 2.30 at the temperature of 8 °C. The use of inclusion-complex formation with β-CD combined with simple electrochemical technology to recognize Trp enantiomers offers new avenues for isomers discrimination via electrochemical methods and would have great potentials in future studies. For example, the cavity size of the host molecule could be tuned by using different cyclodextrins (α-, β-, and γ-CD), and thus other chiral compounds containing aromatic ring would be potentially recognized by this method. Also, the possibility of increasing electrochemical signal differences between enantiomers are currently underway by means of a more comprehensive and in-depth investigation.

Corresponding Author

*E-mail: [email protected]. Fax: +86-519-86330167. Tel.: +86-519-86330256. Notes

The authors declare no competing financial interest. 2639

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