Selective recognition and determination of phenylalanine by a fluorescent probe based on cucurbit[8]uril and palmatine

Journal Pre-proof Selective recognition and determination of phenylalanine by a fluorescent probe based on cucurbit[8]uril and palmatine Pei-Hui Shan, Jie Zhao, Xin-Yu Deng, Rui-Lian Lin, Bing Bian, Zhu Tao, Xin Xiao, Jing-Xin Liu PII:

S0003-2670(20)30009-X

DOI:

https://doi.org/10.1016/j.aca.2020.01.007

Reference:

ACA 237366

To appear in:

Analytica Chimica Acta

Received Date: 21 October 2019 Revised Date:

23 December 2019

Accepted Date: 2 January 2020

Please cite this article as: P.-H. Shan, J. Zhao, X.-Y. Deng, R.-L. Lin, B. Bian, Z. Tao, X. Xiao, J.-X. Liu, Selective recognition and determination of phenylalanine by a fluorescent probe based on cucurbit[8]uril and palmatine, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Pei-Hui Shan: Investigation , Formal analysis, Validation, Methodology Jie Zhao: Formal analysis, Software Xin-Yu Deng: Validation Rui-Lian Lin: Data curation, Bing Bian: Software Zhu Tao: Resources, Visualization Xin Xiao: Conceptualization, Funding acquisition, Resources, Writing - Review & Editing, Supervision

Jing-Xin Liu: Funding acquisition, Writing- Original draft preparation

Selective recognition and determination of phenylalanine by a fluorescent probe based on cucurbit[8]uril and palmatine Pei-Hui Shan, Jie Zhao, Xin-Yu Deng, Rui-Lian Lin, Bing Bian, Zhu Tao, Xin Xiao and Jing-Xin Liu This paper demonstrated a simple and validated fluorescence enhancing method for the selective recognition and determination of the amino acid phenylalanine (Phe). Palmatine (PAL) can be encapsulated into the cucurbit[8]uril (Q[8]) in aqueous solution to form stable 1:2 host−guest inclusion complex. Interestingly, the addition of the Phe dramatically enhanced the fluorescence intensity of the inclusion complex. In contrast, the addition of any other natural amino acids gives no fluorescence variation. Accordingly, a new fluorescence enhancing method for the recognition and determination of the Phe was established.

Selective recognition and determination of phenylalanine by a fluorescent probe based on cucurbit[8]uril and palmatine Pei-Hui Shan,a,b Jie Zhao, b Xin-Yu Deng, b Rui-Lian Lin,c Bing Bian,d Zhu Tao,b Xin Xiao*a,b and Jing-Xin Liu*c a

State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key

Laboratory of Green Pesticide and Agricultural Bioengineering b

Key Laboratory of Macrocyclic and Supramolecular Chemistry of Guizhou Province, Guizhou

University, Guiyang 550025, China E-mail: [email protected] (X. Xiao) c

College of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002,

China. E-mail: [email protected] (J.-X. Liu) d

College of Chemical and Environmental Engineering, Shandong University of Science and

Technology, Qingdao 266590, China

ABSTRACT: This paper demonstrated a simple and validated fluorescence enhancing method to selectively recognize and discriminate the amino acid phenylalanine (Phe). 1H NMR spectroscopy reveal that the palmatine (PAL) can be encapsulated into the cucurbit[8]uril (Q[8]) in aqueous solution to form stable 1:2 host−guest inclusion complex PAL2@Q[8], which exhibits moderate intensity fluorescence property. Interestingly, the addition of the Phe into the inclusion complex PAL2@Q[8] leads to dramatically enhancing of the fluorescence intensity. In contrast, the addition of any other natural amino acids into the inclusion complex PAL2@Q[8] gives no fluorescence variation. Furthermore, it is easy to detect the concentration of Phe in target aqueous solution according to the linear relationship between fluorescence intensity and concentration of the Phe.

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Keywords: Cucurbit[8]uril Supramolecular assembly Selective recognition

Fluorescent probe

Host−guest chemisty

Introduction As the building blocks of proteins, enzymes and other biomolecules, twenty natural amino acids play crucial roles in many biological and physiological processes.1-3 The twenty natural amino acids are widely distributed in food, condiments, pharmaceutical preparations, and even metabolites of human body.4-7Therefore, developments of reliable methods for amino acid recognition and determination are of great significance in food testing, nutritional analysis, medical diagnostics, and many other fields.8-10 To date, many methods, including high performance liquid chromatography (HPLC),11 gas chromatography (GC),12 capillary electrophoresis (CE),13 and optical techniques,14-15 for the recognition and detection of the twenty natural amino acids have been established. Among the optical techniques, the fluorescence approach is particular attractive due to its advantages of excellent selectivity, fast response, low detection limits, and inexpensive instruments. Nevertheless, it is still a challenge to develop an effective fluorescent probe system for specific amino acids. As synthetic macrocyclic receptors, cucurbit[n]urils16-21 (n = 5–8, 10, abbreviated as Q[n]s Figure 1) comprise n glycoluril units bridged by 2n methylene groups, characterized hydrophobic cavities and carbonyl-laced portals. Over the past decade, Q[n]s and their derivatives have been reported to encapsulate a number of specific amino acids, peptides and proteins.22-38 It is well recognized that the encapsulation or release of guests by hosts usually brings about substantial alteration of chemical and physical properties of the guests.39-57 In particular, work by Chang and co-workers has demonstrated that the fluorescence of palmatine (PAL) in aqueous solution is enhanced greatly when encapsulated into the Q[7] cavity.58-61 The competitive reaction of some target organic compounds and the PAL for the occupancy of Q[7] cavity results in fluorescence quenching, which is utilized in recognition and determination of several different organic compounds. The studies of Chang prompted us to wonder

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whether the PAL probe can be bound within the larger Q[8] cavity to form inclusion complex, and whether the Q[8]·PAL can be used to sense the binding of any natural amino acids. In the present work, we studied the binding properties of Q[8] to PAL using 1H NMR spectroscopy, UV-vis and fluorescence spectroscopy, and isothermal titration calorimetry (ITC) techniques, and found that they form 1:2 host−guest inclusion complex, which displays moderate intensity fluorescence. Most importantly, when phenylalanine (Phe) was added to the 1:2 host−guest inclusion complex of Q[8] with PAL, the fluorescence intensity of the inclusion complex enhanced greatly. However, the addition of any other natural amino acids to the same inclusion complex does not lead to distinct fluorescence changes. Obviously, these observations can be used to selectively recognize and discriminate the Phe in aqueous solution.

Figure 1. Molecular structure of Q[8], PAL and amino acid Phe.

Results and Discussion Formation of inclusion 1:2 complexes in aqueous solution The binding interaction of Q[8] with PAL in aqueous solution was detected by 1H NMR titration experiments. Figure 2a shows the 1H NMR spectra of PAL in the absence and presence of different equivalent of Q[8] in neutral D2O solution. With the addition of more and more Q[8] host, the signals of the Hc, Hd, He, and Hf protons of the PAL shifted upfield, while those of the Hi, Hj protons of the PAL

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shifted downfield gradually. This behavior can be rationalized by the inclusion of the methoxy-isoquinoline of the PAL inside the cavity of the host, while the substituted benzene ring of the PAL remained outside. As can be seen in Figure 2b, the chemical shift of the He protons of PAL changes linearly with the Q[8] concentration up to 2.0 equiv, where it levels off, suggesting the formation of a 1:2 host–guest inclusion complex between the Q[8] host and the PAL guest. At each concentration of Q[8], only a single set of signals for the PAL was observed, indicating the guest exchange is fast compared to the NMR time scale.

Figure 2. (a) 1H NMR spectra of PAL (2.0 mmol/L) in the absence (A) and presence of 0.55 (B), 1.02 (C), 1.21 (D), 2.05 (E) 2.51 (F) equiv of Q[8] in D2O at 20 ºC; (G) 1H NMR spectrum of Q[8] (1.0 mmol/L) in D2O at 20 ºC. (b) 1H NMR chemical shift of the He proton of PAL as a function of the added concentration of Q[8]. UV-vis and fluorescence spectroscopy The binding interaction of Q[8] with PAL in aqueous solution was further detected by UV-vis (Figures 3 and 4) and fluorescence (Figure 5) spectroscopic titration experiments. As can be seen in Figure 3, PAL shows the characteristic absorption peaks at 225, 274, and 342 nm, respectively. We observed that the absorption peak of PAL at 274 nm was red shifted to 279 nm with the gradual addition of the Q[8]. Furthermore, the intensity of absorbance peak of the PAL decreased greatly, indicating the high binding affinity of the Q[8] with the PAL. The mol ratio titration method (Figure 3b) reveals that

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the binding interaction of Q[8] with PAL fitted well to a 1 : 2 (host : guest) binding model, which is in agreement with the results of 1H NMR titration. Based on the absorption spectrophotometric data, the binding constant for this PAL/Q[8] inclusion complex in aqueous solution was found to be 1.02×105 M−1. Continuous variation Job’s plot for Q[8] and PAL on the basis of UV-vis spectra (Figure 4) further confirmed that the PAL/Q[8] inclusion complex is formed with 1:2 stoichiometry. The fluorescence spectroscopy of PAL also showed large change upon addition of Q[8]. It is well known that PAL display no intrinsic fluorescence in aqueous solution.62 However, upon gradual addition of Q[8] the fluorescence intensity of PAL display a significant enhancement. The observed enhancement of fluorescence intensity for PAL in the presence of Q[8] is attributed to the formation of host−guest inclusion complex PAL2@Q[8], in which the Q[8] host provides a hydrophobic microenvironment for the PAL guest.

Figure 3. (a) UV-vis titration of PAL (2×10-5 mol·L-1) with increasing concentration of Q[8] and (b) absorbance (A) vs. the ratio of the number of mol of host and guest NQ[8]/NPAL.

Figure 4. Continuous variation Job’s plot for Q[8] and PAL on the basis of UV-vis spectra.

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Figure 5. Fluorescence titration of PAL (2×10-5 mol·L-1) with increasing concentration of Q[8]. Isothermal titration calorimetry (ITC) experiment To further understand the nature of host–guest complexation of Q[8] with PAL, were carried out isothermal titration calorimetry (ITC) experiments (Figure. 6). The obtained thermodynamic parameters reveal that the host–guest complexation is both enthalpically and entropically favorable (∆Hο = -21.29 kJ·mol-1, T∆Sο = 7.31 kJ·mol-1 , ∆Gο = -28.60 kJ·mol-1, T∆Sο = 24.51 kJ·mol-1). In addition to the ion−dipole interaction between the positively charged nitrogens on the PAL guest and the oxygen atoms on the portals of the Q[8] host, van der Waals interaction between the surfaces of the PAL guest and the inner wall of the Q[8] host contribute favorable enthalpy for the host–guest complexation. The removal of the water molecules from the Q[8] cavity and the Q[8] portals, and from the solvated shell of the PAL is likely responsible for this favorable entropic gain. According to the van’t Hoff equation (lnK = -∆Hο/ RT + ∆Sο/R) and the enthalpic and entropic values, a large binding constant (1.023 ± 0.2) × 105 of Q[8] with PAL were obtained, which is consistent with the UV-vis data.

Figure 6. ITC profile of host Q[8] with guest PAL at 298.15 K.

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Fluorescence enhancing of Q[8]2/PAL by Phe As mentioned above, PAL has no native fluorescence in aqueous solution. When the PAL was encapsulated into the Q[8] cavity, the formed inclusion complex PAL2@Q[8] emits moderate intensity fluorescence. To ascertain whether the inclusion complex PAL2@Q[8] can be used to recognize any natural amino acids, we carried out a series of fluorescence measurements by using 20 natural amino acids one by one. Interestingly, when Phe was added to the 1:2 inclusion complex of Q[8] with PAL, the fluorescence characteristic of the inclusion complex PAL2@Q[8] showed dramatic changes, namely, a slight blue shift and a deep in fluorescence intensity. However, the addition of any other 19 natural amino acids to the same inclusion complex PAL2@Q[8] does not lead to distinct fluorescence changes(Figure 7). These observations suggest that the inclusion complex PAL2@Q[8] can be used for selective recognition of the Phe in aqueous solution.

Figure 7. (a) The fluorescence spectra of PAL2@Q[8] with different amino acids in aqueous solution with λex = 343 nm. (b) fluorescence signal. Effect of Phe concentration on the fluorescence intensity of the inclusion complex PAL2@Q[8] The effect of varying Phe concentrations on the fluorescence intensity of the inclusion complex PAL2@Q[8] was also investigated. As can be seen in Figure 8a, the fluorescence intensity of the inclusion complex PAL2@Q[8] enhanced gradually as the Phe concentration increased. The concentrations of Phe varied from 0 to 6×10-5 mol·L-1. More importantly, the fluorescence intensity (F)

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exhibited a good linear relationship with the Phe concentration within a certain range of concentrations. The linear ranges was 0-2×10-5 mol·L-1. The linear regression equations were F = 25.4572C+18.8419 (C denotes the concentration (mol·L-1) of Phe) with correlation coefficients of 0.9917, indicating very good linearity.

Fig. 8 (a) The fluorescence spectra of Q[8]/PAL in different concentrations of Phe in aqueous solution

with λ = 343 nm. The concentrations of Phe (10-5 mol·L-1): (a) 0; (b) 0.2; (c) 0.4; (d) 0.6; (e) 0.8; (f) 1.0; (g) 1.2; (h) 1.4; (i) 1.6. CQ[8] = 1.0×10-5 mol L-1, CPAL = 2.0×10-5 mol L-1. (b) The corresponding plots of fluorescence intensity versus Phe concentration. The response mechanism of the fluorescent enhancing To understand the response mechanism of fluorescence enhancing of the inclusion complex PAL2@Q[8] upon the addition of Phe, we also recorded the 1H NMR spectra of Phe combined with the inclusion complex PAL2@Q[8]. The results show that all the peaks of the protons of the Phe are shifted to a higher field relative to those of the free Phe, indicating that the Phe is now located inside the cavity of Q[8] (Figure. 9). At the same time, the peaks of the protons of the PAL shifted to a lower field relative to those of the PAL in inclusion complex PAL2@Q[8] and broaden substantially. All these observations demonstrate that one included PAL molecule is replaced by the Phe molecule. As a result, PAL and Phe are bound simultaneously within the Q[8] cavity, forming a 1:1:1 ternary inclusion complex. In other words, a certain proportion of PAL molecule would be squeezed out of the Q[8] cavity and be replaced by the Phe molecule.

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Figure 9. 1H NMR spectra of Phe combined with Q[8] and PAL. It is documented that the fluorescence intensity of PAL greatly enhanced when it included in the hydrophobic cavity of Q[7].16 The structural analysis of PAL reveals that the isoquinoline ring and substituted benzene ring are not in a plane, and they cannot form a conjugated system, thereby resulting in a complete loss of fluorescence. When the PAL was encapsulated into the Q[7] cavity, the isoquinoline ring and the substituted benzene ring forms an effective conjugated system, which leads to a greater enhancement of the fluorescence intensity. If the PAL is squeezed out by other molecule, the only effect will be fluorescence quenching. In the present study, the larger Q[8] cavity can accommodate 2.0 equiv. of PAL, forming 1:2 host−guest inclusion complex PAL2@Q[8]. The protection of the Q[8] host leads to an enhancement of the fluorescence intensity of the PAL, which are remarkably similar to that of the inclusion complex PAL@Q[7]. When amino acid Phe was added to the host–guest system of PAL2@Q[8], the Phe competed to occupy the Q[8] cavity. As we observed in their 1H NMR spectra, the result is that the Q[8] cavity accommodated one PAL and one Phe simultaneously. On the basis of the 1H NMR spectra, a

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semiempirical computational model was constructed via the fast tight-binding quantum chemical method GFN-xTB, Figure 10, as implemented in the xtb 4.8 stand-alone program. 63 The model shows that the aromatic ring of the Phe forms π-π interaction with the isoquinoline ring of the PAL. This ternary inclusion complex PAL·Phe@Q[8] is likely more stable than the inclusion complex PAL2@Q[8]. The stable ternary inclusion complex leads to a greater enhancement of the fluorescence intensity. For the other 19 natural amino acids, they can’t replace the PAL and give no fluorescence variation.

Figure 10 Semiempirical model of the ternary inclusion complex PAL·Phe@Q[8]. Conclusions This paper has explored the binding properties of Q[8] to PAL in aqueous solution by a various of methods. The experimental results suggest that PAL can be encapsulated into the Q[8] cavity to form 1:2 host−guest inclusion complex. This inclusion complex PAL2@Q[8] display moderate intensity fluorescence property. Interestingly, the addition of the Phe into the inclusion complex PAL2@Q[8] dramatically enhanced its fluorescence intensity because of the formation of the ternary inclusion complex PAL·Phe@Q[8]. In contrast, the addition of any other natural amino acids gives no fluorescence variation. Accordingly, we developed fluorescence probe system based on Q[8] and PAL to recognize and determinate the amino acid Phe.

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Experimental Section Instruments: Absorption spectra of the host−guest complexes were recorded on an Aglient 8453 spectrophotometer at room temperature. Fluorescence spectra were recorded on a Varian RF-540 fluorescence spectrophotometer. All the NMR data were recorded on a Bruker DPX 400 spectrometer in D2O (pD = 2) at 293.15K.

Reagents and chemicals

Q[8] was prepared in our laboratory according to a literature method. 64 PAL and all the enantiomeric amino acids used in the experiment were commercially available and used as received without further purification. Stock standard solutions of PAL and all the enantiomeric amino acids (100 mg·mL-1) were prepared by dissolving them in double-distilled water. Stock solution of PAL was prepared by adding double-distilled water to a final concentration of 1.0×10-6 mol·mL-1. A Q[8] stock solution of 1.0×10-7 mol·mL-1 was prepared by dissolving Q[8] in double-distilled water. Stock standard solutions were stable for several weeks at room temperature. Standard working solutions were prepared by dilution of the stock standard solutions with double-distilled water before use. All chemicals were of analytical reagent grade, and double-distilled water was used throughout the procedure. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21861011, 21371004), the Innovation Program for High-level Talents of Guizhou Province (No. 2016-5657), the Major Program for Creative Research Groups of Guizhou Provincial Education Department (2017-028), the Science and Technology Fund of Guizhou Province (No. 2016-1030, 2018-5781) and the Natural Science Foundation of Anhui Province of China (1808085MB43).

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Synopsis

Selective recognition and determination of phenylalanine by a fluorescent probe based on cucurbit[8]uril and palmatine Pei-Hui Shan, Jie Zhao, Xin-Yu Deng, Rui-Lian Lin, Bing Bian, Zhu Tao, Xin Xiao and Jing-Xin Liu This study presents a simple and validated fluorescence enhancing method to selectively recognize and determinate the amino acid phenylalanine (Phe).

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This paper demonstrated a simple and validated fluorescence enhancing method for the selective recognition and determination of the amino acid phenylalanine (Phe). Palmatine (PAL) can be encapsulated into the cucurbit[8]uril (Q[8]) in aqueous solution to form stable 1:2 host−guest inclusion complex. Interestingly, the addition of the Phe dramatically enhanced the fluorescence intensity of the inclusion complex. In contrast, the addition of any other natural amino acids gives no fluorescence variation. Accordingly, a new fluorescence enhancing method for the recognition and determination of the Phe was established.

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: