Pyrene Functionalized Luminescent Phenylalanine for Selective Detection of Copper (II) Ions in Aqueous Media

A novel pyrene-based fluorescent chemosensor 1 (pyren-1-ylmethyl)-L-phenylalanine was designed and synthesized by combining 1-pyrenecarboxyaldehyde and L-phenylalanine. 1 was characterized by several analytical methods and used as a fluorescent chemosensor for the selective and sensitive detection of Cu2+ ions through “turn-off” mechanism with a detection limit of 2 × 10–8 M. 1 can also be used to detect Cu2+ ions in a natural water sample and exhibits gelation properties with high thermal stability.


Introduction
In the last few decades, molecular recognition and host-guest inclusion have been promising in supramolecular chemistry, which detect many ionic and neutral species based on noncovalent interaction between host and guest moiety [1,2]. It provides a great tool for creating chemosensors to detect various biologically and environmentally crucial metal ions [3][4][5][6][7][8]. Due to their electronic transition, chemosensors binding with the analyte produce a measurable analytical signal [9]. In 1867, the first fluorescence-based chemosensor for the detection of Al [3] + ions was reported by F. Goppelsroder. Fluorescence-based chemosensors have attractive features, such as selectivity and sensitivity towards various metal ions such as Cu 2+ , Zn 2+ , and Ni 2+ [10][11][12]. The judicious design of the chemosensors is the crucial factor for tuning selectivity. Recently, various fluorescent chemosensors have been synthesized and utilized for sensing of different metal ions [13]. On comparing fluorescence with other competitive methods like ICP-MS, atomic absorbance spectroscopy (AAS) [14], Flame atomic absorption spectrometry(FAAS), their easy operation technique, detection of various metals even at their sublethal concentration, fast response, and cost efficiency give us the possibility for their application [15].
Fluorescence-based chemosensors are promising for the sensitive and selective testing of heavy metal ions, which act as biological and environmental hazards. In terms of natural abundance, copper is the third most abundant element in the human body and functions in many biological and physiological processes [16,17]. The toxic nature of copper causes dysfunctions in the cellular processes. It results in neurodegenerative diseases such as Wilson's [18,19] amyotrophic lateral cases of sclerosis [20,21], Alzheimer's [22], etc. Excess amount of copper also affects the plant in terms of victimization of physiological arrest, stunted development, and even death [23][24][25]. Moreover Copper is widely used in several agriculture and industrial applications, that causes copper as a major pollution source in the environment [26,27]. The acceptance level of copper in drinking water is 2mgL −1 (30 μM) recommends by World health organization (WHO) [28]. In human blood, normal concentrations of copper vary in the range of 100-150 g/dl (15.7-23.6 μM) [15]. Thus, the detection of copper in a highly effective and selective manner is huge importance for environmental and biological sciences.
However, in the field of supramolecular chemistry, selfassembly of low molecular weight species attract great interest due to their application in various fields such as drug delivery, construction of novel soft material, biosensor, gene therapy, etc [29][30][31][32]. The driving force for the formation of gel through self-assembly are non-covalent interactions such as electrostatic interaction, hydrophobic interaction, π-π interaction, hydrogen bonding interaction, and other 1 3 supramolecular forces [33,34]. Fluorescent supramolecular gels derived from biologically relevant molecules are currently attracting many researchers worldwide because of their great application potential in several fields.
Pyrene derivatives are widely helpful for developing fluorescence-based chemosensors due to their several properties such as strong fluorescence, long fluorescence lifetime, π-π * stacking, polarity sensitive vibronic emission, high charge carrier mobility, and chemical stability [35][36][37][38][39][40]. in recent years many study have been done using pyrene derivatives to sense various ions such as Al 3+ , Cu 2+ , Fe 3+ , Cr 3+ [41,42]. Yang et. al develop a pyrene based fluorescent sensor for the detection of Cu 2+ ions [43]. Here we have designed a fluorescent chemosensor 1 (pyren-1ylmethyl)-L-phenylalanine for selective and sensitive detection of Cu 2+ ions. 1 detects Cu 2+ through a fluorescence turn-off mechanism in the presence of various other metal ions. 1 also has the property to form a gel through noncovalent interaction. Moreover, 1 exhibits a promising tool for the detection of Cu 2+ ions in natural water samples.

Materials and Methods
FTIR and UV-visible spectra were recorded on a 8400S and Agilent Cary 60 single beam, UV-Visible spectrometer with serial no.-MY19329220, respectively, and fluorescence spectra were measured on a Fluoromax 4CP plus spectrofluorometer with a 10 mm quartz cell at 25°C. The melting point was measured using an EZ-Melt (automated melting point apparatus). 1 H and 13 C-NMR spectra were recorded on a JEOL AL 300 FT-NMR at an operating frequency of 500 MHz ( 1 H) and 126 MHz ( 13 C), respectively. Spectrometer operation at 500 MHz and 126 MHz in DMSO-d 6 solutions respectively are given in parts per million (ppm) related to Tetramethyl silane (TMS, δ = 0.00 ppm), and splitting pattern designated as s (singlet), d (doublet), t(triplet). High-resolution mass spectra (ESI-HRMS) were recorded on SCIEX X500R (TOF-MS) mass spectrometer. For atomic force microscopy NT-MDT, Model: Solvernext was used where samples were produced on tiny glass slides, which were then positioned beneath the device. On an MCR 72 Modular Compact Rheometer, rheological measurements were made utilizing a 25 mm diameter parallel plate geometry with a 1 mm gap. To determine the storage or elastic modulus, G′, and loss or viscous modulus, G′′, the gel sample was put on the lower plate and a stress amplitude sweep experiment was conducted at a constant frequency and 25 °C. The frequency sweep measurements were performed at constant stress in the linear viscoelastic range. From each set, vials containing 1 g of compound 1 for rheological experiments.
All chemicals are purchased from a commercial supplier and used without further purification. Lithium hydroxide and L-phenylalanine were purchased from spectrochem. 1-pyrenecarboxyaldehyde was purchased from Sigma-Aldrich, and sodium borohydride was purchased from S. d. fine. Metal salts Ni(NO 3 were purchased from Himedia.

General Methods for Measurements
Spectroscopy Measurement Alkali, alkaline earth, and transition metal ions were utilized for sensing to investigate variations in the emission and absorption spectra of 1. Nitrate salts of different metals were used to provide the metal ions: A stock solution of 1 (10 μM) was prepared in Water: DMSO (9:1, v/v at room temperature). While to make a stock solution of metal ions (Na + , K + , Pb 2+ , Ca +2 , Hg + , Ni +2 , Cu +2 , Co +2 , Zn +2 , Cd +2 , Ag + ) dissolve their nitrate salt in distilled water, (10 mM). Afterward, 3.0 ml of the compound 1 was taken in a quartz cuvette (4 ml, path length, 1 cm) for the titration experiment, and progressively metal ion solutions were added using a micropipette. The maximal excitation and emission wavelengths for fluorescence measurements were 342 nm and 375 nm, respectively, while the wavelength range for absorbance measurements was 200-800 nm.

Synthesis of (pyren-1-ylmethyl)-L-phenylalanine
L-phenylalanine (0.1651 g; 1 mmol) and LiOH (0.041 g; 1 mmol) were dissolved in methanol (10 ml), and previously dissolved 1-pyrenecarboxyaldehyde (0.230 g; 1 mmol) in hot methanol was added, drop-wise. The reaction mixture was refluxed for 5-6 h. at 65°C followed by cooled to room temperature, NaBH 4 (0.0378 g 1 mmol) was added portion wise and stirred at room temperature for 4-5 h. The solvent was removed under vacuum, and the crude product was dissolved in distilled water (10 ml), it was brought to a pH of 5-6 by adding 1 N dil. HCl. The resultant solid was filtered off and dried in a vacuum. Light yellow-colored solid compound 1 formed (Scheme 1

Synthesis and Characterization
The desired chemosensor 1 was synthesized by utilizing L-phenylalanine and 1-pyrenecarboxyaldehyde (PyCHO). The complete characterization has been done by FT-IR, UVvis, 1 HNMR, and 13 CNMR, HRMS. 1 shows excellent solubility in DMSO. The idea for designing this type of probe is based on fluorescent receptor moiety. Here we have aimed to design a molecule that possesses a free binding site in the form of -COOH group. This free-binding site can be applicable for a multifunctional application by providing a binding site for interaction with different cations, anions, and neutral species.
Interaction of 1 with metal ions does not show any remarkable change in the absorption spectrum of 1 except Cu 2+ ion. The addition of Cu 2+ ion with 1, shows a decrease in its absorbance (Fig. 1a). Further, to explore the association properties of 1 with Cu 2+ ion. UV-Visible titration experiment of 1 with the addition of several aliquots of Cu 2+ ion has been done. As a result, the formation of several isosbestic points (262, 269, 271, 279, 305,334, 348) gives a site for the possibility of the several binding modes between Cu 2+ ion and 1 (Fig. S5).
To gain insight into the selectivity of 1 towards various metal ions through fluorescence upon excitation at 342 nm. Neat 1 exhibited a characteristic emission band for pyrene monomer at 375 nm, 394 nm, and 416 nm [44,45] (Fig. S6).
While on the addition of metal ions (Na + , Pb 2+ , K + , Cd +2 , Ca +2 , Ag + , Co +2 , Ni +2 , Zn +2 , Hg + ) (100 mM) into a solution of 1 (10 μM) in Water: DMSO (9:1, v/v) doesn't exhibit any considerable change in the emission spectra. Only Cu 2+ ion was able to make a significant change in the emission spectra of 1 (Fig. 1b). Fluorescence lifetime measurement of 1 was measured at monomer emission i.e., 375 nm, exhibits a decay time is 1.43 ns (Fig. 2). Addition of 10 eq. of Cu 2+ ions show quenching in the emission intensity of 1 up to 85% in the view of the paramagnetic nature of Cu 2+ ions. These findings suggest that 1 shows excellent selectivity towards Cu 2+ ions. A fluorescence titration experiment is carried out to examine the effect on the intensity of the emission of 1 by adding several aliquots of Cu 2+ ions. On addition of several aliquots of Cu 2+ (1μL) to the solution of 1 immediately tends to "turn-off" fluorescence at 375 nm (29%), 394 nm (26%), and 416 nm (24%) (Fig. S7). Increasing Cu 2+ concentration (2-15 μL) lead to a continuous decrease in the emission intensity at 375 nm (85%), 394 nm (86%), and 416 nm (74%).
The total amount of Cu 2+ required to concentrate quench in the emission intensity of 1 is 15 μM (5 eq.) to the concentration of 1. The fluorescence quenching of 1 with Cu 2+ ion is explored by the classical Stern-Volmer equation.
where, I 0 and I are the emission intensity of neat 1 and 1 with Cu 2+ , respectively. [Q] represents the concentration of Cu 2+ ion, and K sv represents the Stern-Volmer quenching constant. The plot of I 0 /I vs varying concentration of

Scheme 1 Synthetic Scheme for 1
Cu 2+ ion represents good linearity (R 2 = 0.9725) with the calculated K sv value of 2.65 X 10 5 M −1 . The obtained K sv value strongly suggests the quenching behavior of 1 concerning Cu 2+ ions (Fig. S8). The quantum yield of 1 for the monomer emission was determined to be 0.23 and after the addition of Cu 2+ is 0.06 with quinine sulfate monohydrate (ϕ = 0.54 in 0.5 M sulfuric acid) being used as a standard (see ESI for details).
The selectivity of any probe towards a particular metal ion is the most desirable thing. Therefore, to investigate the selectivity of 1 towards Cu 2+ ions, we examine the emission characteristics of 1 with Cu 2+ ions in the presence of other competing metal ions. As a result, we found that none of the competitive metals can alter the emission quenching caused by the interaction of Cu 2+ with 1 (Fig. 3a). These results specify that 1 can be utilized as a selective sensor for Cu 2+ ions in presence of other competing metal ions. Now to get insight into the binding stoichiometry of 1 towards Cu 2+ for the formation of complex analyzed by jobs plot method. As a result, we found 2:1 stoichiometry between 1 and Cu 2+ ions complex (Fig. 3b). A proposed binding mechanism for the formation of a 1:2 complex of Cu 2+ ions with 1 (Fig. S13). Where Cu 2+ coordinates with the oxygen atom of carboxylic moiety.

Determination of Binding Constant and Sensitivity
The binding constant between 1 with Cu 2+ ion was calculated by using the Benesi-Hildebrand equation.
I represent the emission intensity of the 1-Cu 2+ complex, which varies with the concentration of Cu 2+ . I 0 represents the emission intensity of 1, and I max is the fluorescence intensity of the 1-Cu 2+ complex at the maximum concentration of Cu 2+ ions. Binding constant K a was evaluated by plot between 1/(I 0 -I) vs 1/[Cu 2+ ]. Plot between 1/(I 0 -I) vs 1/ [Cu 2+ ] shows good linearity (R 2 = 0.9822) with the value of binding constant K a = 5 × 10 4 M −1 (Fig. S9).
The sensitivity of 1 has been observed by adding varying concentrations of Cu 2+ (10 -11 to 10 -3 M) to 1 (10 µM) solution. A plot between I 0 /I vs. [Cu 2+ ] shows a significant change at 10 -8 M Cu 2+ concentration (Fig. S10). Therefore, for accuracy in the detection limit, further addition of varying concentrations of Cu 2+ ion (1 × 10 -8 to 9 × 10 -8 M) to the   (Fig. S11). The LOD value is lower than the concentration of Cu 2+ recommended by WHO 16 μM in the drinking water and from previously reported LOD value for Cu 2+ sensor (Table S1). Therefore, it exhibits that chemosensor 1 could be potentially used as a sensor for detecting Cu 2+ ions.

Reversibility of 1-Cu +2 with EDTA
The chemical reversibility phenomenon of the binding tendency of 1 with Cu 2+ ions was also examined by the addition of EDTA solution to the 1 + Cu 2+ system. As results indicate, the addition of EDTA solution replaces the Cu +2 ion from 1 + Cu +2 complex by regaining the emission intensity of 1 due to the great stability of EDTA-Cu 2+ complex, which confirms the regeneration of 1 from 1 + Cu 2+ complex, and hence 1 can be useful in the binding process. These findings suggest the chemical reversibility of the binding of 1 to Cu 2+ ions (Fig. S12).

Rheology Study
The gel has great relevance for application; therefore, we examine the strain recovery rheology studies for the viscoelastic characteristics (storage modulus G′ > loss modulus G′′) and mechanical strength of a gel. The mechanical characteristics of gel materials have been investigated using rheology. 1 g of 1 was measured for the storage modulus (G′) and loss modulus (G′′) about applied stress and frequency. Figure 3 represents the variation in G′ and G″ to the applied frequency on 1. This is observed that at any applied frequency, both G′ and G′′ are essentially frequency independent, and at any given point, G′ is bigger than G′′ over the entire frequency range, confirming the actual gellike nature of the material (Fig. 4a).
The shear-thinning tendency of gel was characterized by plotting a shear rate vs viscosity rheology plot. In the shear rate vs viscosity plot, the power-law equation was fit to the linear region as per the formula η is for viscosity, n and K are for shear-thinning coefficient and γ is for shear rate.
As we saw in the viscosity graph, it also confirms that the viscosity of 1 increases with increasing stress (Fig. 4b). This result indicates the formation of hard gel on increasing the stress on 1. Moreover, to explore the thermal stability and photoluminescence of synthesized 1 as a gel, to check the thermal stability of gel, heat up to 100 °C in an oil bath and no effect was observed, this indicates the high thermal stability of 1. Although 1 also exhibits blue emission on long-range wavelength (Fig. 5).

AFM Study
AFM studies were carried out to investigate the surface properties for 1. The 2D and 3D morphology of 1 exhibit uniformly distributed, with root mean square roughness (Rq) = 8.010 nm, average roughness (Ra) = 6.344 nm, and maximum profile peak height = 19.802 nm (Fig. 6a and  c). It is observed that particles of 1 are uniformly distributed and comprised of the sphere shape. The formation of = K n−1 Fig. 3 a Interference of cations on 1 and Cu 2+ ions selectivity. b Job's graph displays a 2:1 binding stoichiometry between 1 and Cu 2+ non-covalent interaction forms this sphere shape, i.e., Intermolecular hydrogen bonding between -COOH moiety in 1 (Fig. 6b). Here the interaction between -COOH moiety is more dominant than the π-π interaction between pyrene moiety. It was also observed by the fluorescence spectra of 1 where a significant pyrene excimer peak was not observed.

Water Samples Preparation for Cu 2+ Detection
To analyze the copper content in different water samples, tap water (TW), distilled water (DW), River water (RW), and Pond water (PW) were collected. The collected water was pretreated by boiling and filtration to avoid insoluble impurities present, before being used to detect copper content analysis by 1. Water samples were filtered to separate the insoluble substance from them. To remove dissolved salts like chlorine content from water samples, they were boiled for one hour and then cooled at room temperature.

Analysis of Cu 2+ in Various Natural Water Samples
To explore the applicative study of 1 in an environmental approach, we  The earlier process was used to well prepare the stock solution for analysis of the water samples. Afterward, fluorescence spectra of 1 with different water samples were recorded. Here we take 2.5 ml of water samples (Pond water (PW), Distilled water (DW), Lake Water (LW), and Tap water (TW)) in a quartz cuvette with the addition of 250 μL of 1 (100 mM; Water: DMSO; 9:1, v/v at room temperature). Impressively, we found quenching (̴ 43%) in the case of pond water to distilled water (Fig. 7a). Furthermore, the addition of 20 μL Cu 2+ ion to the solution of 1 and water sample (250 μL + 2.5 mL), gives further quenching in the emission spectra.
The linearity response of 1, to the addition of various natural water samples (100-500 μl) subsequently in 2.5 ml of 1 (10 μM), a pithy decrease in the emission of 1 exhibited good linearity (Fig. 7b). This result strongly suggests that 1 works as a fluorescence sensor detect Cu 2+ ions in biological and environmental contexts. Moreover, the quenching in the spectrum of 1 with the addition of pond water with 20 μL Cu 2+ ion (10 ppb), respectively. It suggests that the present concentration of Cu 2+ ion in pond water at that stage is < 10 ppb. This indicates that pond water contains a much higher amount of Cu 2+ as compared to tap water. Therefore, we can say that 1 feasibly works better to detect Cu 2+ ions in a real water sample.

Conclusion
In this article, we have reported the synthesis of a new (pyren-1-ylmethyl)-L-phenylalanine, fluorescent compound 1 for selective and sensitive sensing of Cu 2+ ions. The mechanism for Cu 2+ ions detection is based on a fluorescent "turn off" mechanism. The above study showed that 1 has a specific fluorescence quenching towards Cu 2+ ions in a Water-DMSO solution (9:1, v/v, r.t. 25˚C) in the existence of the several competitive metal ions (K + , Na + , Ca +2 , Co +2 , Fe +3 , Ni +2 , Zn +2 , Cu +2 , Hg + , Cd +2 , Ag +2 , Pb 2+ ). The LOD value of the chemosensor for specific detection of Cu 2+ ions reached 2 × 10 -8 M showing that 1 was highly applicable for quantitative analysis of Cu 2+ ions. In natural water analysis, 1 showed excellent results for the detection of Cu 2+ , exhibiting its potential as an ideal chemosensor. Moreover, 1 also exhibits properties of the gel, and even at high temperature and high-pressure this gel exhibits excellent stability, which increases its applicative potential in the environmental application.