RBAP, a Rhodamine B-Based Derivative: Synthesis, Crystal Structure Analysis, Molecular Simulation, and Its Application as a Selective Fluorescent Chemical Sensor for Sn2+

A new fluorescent chemosensor based on a Rhodamine B and a benzyl 3-aminopropanoate conjugate (RBAP) was designed, synthesized, and structurally characterized. Its single crystal structure was obtained and analyzed by X-ray analysis. In a MeOH/H2O (2:3, v/v, pH 5.95) solution RBAP exhibits a high selectivity and excellent sensitivity for Sn2+ ions in the presence of many other metal cations. The binding analysis using the Job’s plot suggested the RBAP formed a 1:1 complex with Sn2+.


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instrumentally intensive methods often also require extensive sample preparation prior to analysis and sophisticated experimental procedures [14].Thus, a simple and inexpensive method for detecting and quantifying metal ions is essential for real-time monitoring in biological samples.Ions of tin (Sn), a type of heavy metal, are usually found in the environment at low levels.Humans are usually exposed to organic tin complexes through packaged foods, soft drinks, biocides, and dentifrices [15].Little attention has been paid to the toxicity of Sn 2+ as an environmental pollutant in natural waters.Although tin is not a highly toxic element [16], at high concentrations of approximately 0.1-1.0g/L, Sn 2+ may affect water flavor and cause diarrhea.Recent literature reports have revealed that, in forms such as SnCl 2 , Sn 2+ can be readily taken up by human white blood cells and cause DNA damage [17,18].Thus, it is desirable to develop a reliable and sensitive analytical method to qualitatively and quantitatively evaluate the level of Sn(II) ions present in environmental and biological systems.Rhodamine B derivatives are extensively employed as molecular probes in the study of complex biological systems due to their high absorption coefficients, high fluorescence quantum yields, and long-wavelength absorptions and emissions [19,20].On the basis of the spirolactam/ring-opened amide equilibrium of rhodamine, several fluorescence-based sensing systems for metal ions have been developed.Most of the reported sensors based on rhodamine B derivatives are fluorescent chemosensors for the detection of Pb 2+ , Cd 2+ , Cu 2+ , Fe 3+ and Hg 2+ ions [21][22][23][24][25][26][27][28][29].Unfortunately, to the best of our knowledge, only a few rhodamine B-based chemosensors have been reported for tin ions [30].Therefore, a rhodamine B architecture was selected for the development of a new chemosensor that can selectively detect Sn 2+ .Recently, we reported rhodamine B-based sensor 1 (Figure 1), which consists of two rhodamine B moieties linked through the two amines of a 4,13-diaza-18-crown-6 ether as a highly sensitive fluorescent probe for monitoring Cr 3+ in a MeOH/H 2 O (3:2, v/v, pH 7.2) solution and in living cells [31].Unfortunately, single crystals of sensor 1 could not be grown in the presence or absence of Cr 3+ and were not analyzed by X-ray diffraction.In this paper, we report the design and synthesis of a rhodamine B-based derivative bearing a benzyl 3-aminopropanoate group (RBAP) that selectively displays a colorimetric response and a fluorescence "turn-on" response at 583 nm via a rhodamine ring-opening process with Sn 2+ in the presence of many other metal ions.We also report the single crystal X-ray analysis of RBAP, which further confirms its structure in the absence of Sn 2+ .Additionally, a DFT computational study was carried out for better understanding of the formation of a complex between RBAP and Sn 2+ .To the best of our knowledge, RBAP is the first rhodamine B-based sensor for the Sn 2+ ion utilizing the rhodamine ring-opening equilibrium approach.

Synthesis of RBAP
RBAP was synthesized according to the procedure published in the literature [31] (Scheme 1).Specifically, compound 2 was prepared in a 92% yield by treating 1 with 3-aminopropanoic acid and HCl gas at 120 °C for 12 h, which was followed by coupling with rhodamine B in the presence of DCC (1 eq.), HOBt (1 eq.), and TEA (3 eq.) in CH 2 Cl 2 at room temperature for 12 h to give RBAP in a 90% yield.Scheme 1. Synthesis of RBAP.

X-ray Crystallographic Analysis
A single crystal of RBAP suitable for X-ray diffraction studies was grown by vapor diffusion of a CH 2 Cl 2 solution of RBAP.The chemical structure of RBAP was further confirmed by X-ray analysis as shown in Figure 2, and the two-dimensional network structure of RBAP is shown in Figure 3.

pH Response of RBAP
To evaluate the pH response of RBAP, acid-base titration experiments were performed in a MeOH/H 2 O (2:3, v/v, pH 5.95) solution.The fluorescence intensities of RBAP at 583 nm in solutions at different pH levels were recorded.As shown in Figure 4, RBAP did not emit any obvious and characteristic fluorescence (excitation at 561 nm) in the pH range of 5.0 to 12.0.However, the fluorescence intensity at 583 nm was obviously enhanced at pH levels below 5.0 due to the ring-opening mechanism of the spirocyclic moiety of rhodamine B (Scheme 2).These results suggested that RBAP was insensitive to pH from 5.0 to 12.0 and may have been able to sense Sn 2+ under approximate physiological conditions with very low background fluorescence.Therefore, further UV-Vis and fluorescence studies were carried out in a MeOH/H 2 O (2:3, v/v, pH 5.95) solution.
The absorption spectra of RBAP upon titration with Sn 2+ in MeOH/H 2 O (2:3, v/v, pH 5.95) solution were recorded to gain further insight into the binding of RBAP and Sn 2+ .When no Sn 2+ ions were added to the solution of RBAP, the free RBAP remained colorless and did not exhibit any apparent absorption above 400 nm in MeOH/H 2 O (2:3, v/v, pH 5.95) solution, indicating that the spirolactam form of RBAP was the predominant species.Upon addition of the Sn 2+ ion, a new and strong absorption band centered at 561 nm was observed (Figure 6), resulting in the color change from colorless to pink.This indicated that the ring-opened form of RBAP exists in a significant concentration in the examined solution (Scheme 2).Next, a Job's plot was generated by continuously varying the mole fraction of Sn 2+ from 0 to 1 in a solution of [Sn 2+ ] + [RBAP] with a total concentration of 100 μM.The Job's plot analysis exhibited a maximum at approximately 0.5 mole fraction, indicating a 1:1 stoichiometry for the RBAP-Sn 2+ complex (Figure 7 inset).

Fluorescence Titration of RBAP with Metal Ions
To further evaluate the selectivity of RBAP for Sn 2+ , the change in fluorescence intensity upon the addition of various metal ions under the same conditions was also investigated.The fluorescence spectra of RBAP (10 μM) in MeOH/H 2 O (2:3, v/v, pH 5.95) displayed a very weak fluorescence at 583 nm (λ ex = 561 nm), indicating that the predominant form of RBAP was the spirolactam form.When Sn 2+ (50 μM, 5 equiv.)was added to the RBAP solution, a fluorescence enhancement of greater than 300-fold was observed (Figure 8), indicating that the Sn 2+ ion induced the formation of the ring-opened RBAP-Sn 2+ complex, which exhibited strong fluorescence (Scheme 3) The solution changed from colorless to pink.(Figure 8 inset) other various metal ions (Mg 2+ , Sn 2+ , Cr 3+ , Ag + , Ca 2+ , Na + , Pb 2+ , K + , Mn 2+ , Zn 2+ , Cu 2+ , Cd 2+ , Li + , Ba 2+ , Fe 2+ , Co 2+ , Fe 3+ , Hg 2+ , Al 3+ and Sn 4+ ) did not induce any apparent fluorescence enhancement after the addition of five equiv. of the metal ions.Over a 300-fold fluorescence enhancement was observed under saturation conditions (10 equiv.).The association constant was also calculated using the following formula: F − F 0 = △F = [Sn 2+ ] (F max − F 0 )/(1/K a + [Sn 2+ ]) based on a 1:1 stoichiometry, where F is the obtained fluorescence intensity, F 0 is the fluorescence intensity of free RBAP at the emission wavelength, and F max is the saturated fluorescence intensity for RBAP-Sn 2+ complex.When the reciprocal of △F was plotted as a function of Sn 2+ concentration, a linear relationship was obtained (y = A + Bx), and K a was calculated from A/B.Therefore, the binding association constant for Sn 2+ , K a , was estimated to be 2.65 × 10 4 M −1 in the MeOH/H 2 O (2:3, v/v, pH 5.95) solution, as inferred from the fluorescence titration curves of RBAP with the Sn 2+ ion.The detection limit of Sn 2+ by RBAP was determined to be 0.044 μM (Supporting Materials, S5).

NMR Study of RBAP with Sn 2+
To further elucidate the binding mode of RBAP with Sn 2+ , 1 H-NMR spectra of RBAP in the presence and absence of Sn 2+ were acquired.As shown in Figure 10, after the addition of 1 equiv. of Sn 2+ into a solution of RBAP in acetone-d 6 :D 2 O (5:1, v/v), The singlet peak of H f proton displayed a downfield shift from 4.15 ppm to 4.21 ppm (Δδ = 0.06 ppm), which originates from the coordination of the carbonyl oxygen of ester group on RBAP with Sn 2+ .In addition, the H g multiplet, (the signals of H g1 and H g2 overlapped) displayed an apparent downfield shift and signal splitting from one multiplet peak centered at 3.41 ppm to two multiplet peaks centered at 3.65 ppm (Δδ = 0.24 ppm) and 3.50 ppm (Δδ = 0.09 ppm), and the singlet peak of the symmetric H e proton also displayed a significant downfield shift and signal splitting, from a peak centered at 6.4 ppm to three doublets peaks centered at 6.78 ppm (Δδ = 0.38 ppm), 7.0 ppm (Δδ = 0.60 ppm) and 7.31 ppm (Δδ = 0.91 ppm), due to the Sn 2+ ion-induced ring-opening process of the rhodamine B spirocycle.(Supporting Materials Figure S3).The above results indicate an interaction mode of RBAP and Sn 2+ as proposed in Scheme 3, in which Sn 2+ is coordinated with the two carbonyl oxygen atoms of RBAP.

Density Functional Theory (DFT) Calculations
For better understanding of the nature of Sn 2+ coordination with RBAP, energy optimized structures of RBAP and RBAP-Sn 2+ (Figure 11) were obtained using DFT calculations with the B3LYP method using 6-31+G(d) as a basis set.The spatial distributions and orbital energies of the HOMO and LUMO of RBAP and RBAP-Sn 2+ were also generated using DFT calculations (Figure 12).The results indicated that, in RBAP the HOMO is spread out on the xanthenes of the rhodamine B moiety, while the LUMO is centered on the spirocycle of the rhodamine B moiety and more localized.As a result, the C-N bond on the spirocycle of the rhodamine B moiety breaks to facilitate the binding of Sn 2+ ions with the two RBAP carbonyl oxygen atoms.The π electrons in the HOMO of the RBAP-Sn 2+ complex are mainly located in rhodamine B, while the LUMO is mostly located on the guest Sn 2+ ion.The energy gap between the HOMO and LUMO was calculated to be 55.74 kcal/mol, an almost 50% decrease from that of RBAP, which was 102.38 kcal/mol (Supporting Materials).The results clearly suggest that the binding of Sn 2+ to RBAP stabilizes the system because the calculated orbital energies of both the HOMO and LUMO were lowered for the complex.In the RBAP-Sn 2+ complex, the distances of the two "Sn-O" bonds were calculated to be 2.01 and 2.21 Å, respectively (Figure 11).

Materials and General Methods
All reagents and organic solvents were ACS grade or higher and were used without further purification.Unless otherwise noted, all chemicals were purchased from J&K Scientific (Shanghai, China) and were used as received.All solvents were of analytical grade, and double distilled water was used in all experiments.The salts used to prepare metal ion stock solutions were Ba(NO 3 ) 2 , AgNO 3 , The reactions were performed under an argon atmosphere using standard Schlenk techniques.Thin-layer chromatography was performed on a HAIYANG silica gel F254 plate, and compounds were visualized under UV light (λ = 254 nm).Column chromatography was performed using HAIYANG silica gel (type: 200-300 mesh ZCX-2).
1 H (500 MHz) and 13 C-NMR (126 MHz) spectra were recorded on an Avance 500 spectrometer (Bruker; Billerica, MA, USA).The chemical shifts are reported in δ units (ppm) downfield relative to the chemical shift of tetramethylsilane.The abbreviations br, s, d, t, and m denote broad, singlet, doublet, triplet, and multiplet, respectively.Mass spectra were obtained with a Finnigan TSQ Quantum LC/MS Spectrometer (Thermo Fisher Scientific Corp., Waltham, MA, USA).High-resolution mass spectra (HRMS) were acquired under electron ionization conditions with a double-focusing high-resolution instrument (Thermo Fisher Scientific Corp., Waltham, MA, USA) The pH levels of stock solutions were measured using a PHS-25C Precision pH/mV Meter (Aolilong, Hangzhou, China).UV-Vis and fluorescence spectra were obtained on a UV-3600 UV-VIS-NIR spectrophotometer (Shimadzu, Kyoto, Japan) and an Edinburgh FLS920 fluorescence spectrophotometer (Livingston, UK), respectively at room temperature.

Experimental Procedure for X-ray Crystallographic Analysis
Crystallographic data of complexes were collected at 296 K on a Bruker APEX-II CCD system equipped with graphite-monochromated Mo-Kα radiation (λ = 0.071073 nm) using the ω-φ scan technique.Diffraction data were integrated using the SAINT program, which was also used for intensity corrections for Lorentz and polarization effects.A semi-empirical absorption correction was applied using SADABS.The structures were solved by direct methods, and all non-hydrogen atoms were refined anisotropically on F2 by full-matrix least-squares using the SHELXL-97 crystallographic software package.Summary of Data CCDC 969599, Compound Name: Formula: C 39 H 44 N 3 O 4 , Unit Cell Parameters: a 12.5467(15) b 22.338(3) c 12.0855(14) P21/c.

Computational Studies
Computational studies were carried out to investigate the nature of Sn 2+ coordination with RBAP using the Gaussian software package.All geometries for RBAP and RBAP-Sn 2+ were optimized using ab initio HF and density functional theory (DFT) calculations.The geometries were first optimized at the HF/3-21G level.The resulting structures were further optimized by DFT calculations using B3LYP with larger basis sets, i.e., the SDDAll basis set for Sn 2+ and 6-31+G(d) for all other atoms in the complex.

Conclusions
In conclusion, we report a rhodamine B derivative, RBAP, which is a selective and sensitive chemosensor that specifically recognizes the Sn 2+ ion in a MeOH/H 2 O (2:3, v/v, pH 5.95) solution by UV/vis and fluorescence spectroscopy.The chemical structure of RBAP was analyzed by 1 H-NMR, 13 C-NMR, and HRMS, and its structure was further confirmed by X-ray analysis.The 1:1 coordination mode was proposed on the basis of Job's plot.The Sn 2+ binding ability of RBAP was further demonstrated by DFT calculations, which suggested that both the HOMO and LUMO orbitals in the RBAP-Sn 2+ complex were stabilized and that the optical detection resulted from the significant decrease in the HOMO-LUMO energy gap.

Figure 9
Figure9displays the fluorescence spectrum of RBAP in the presence of different concentrations of Sn2+ .The free RBAP chemosensor (10 μM) exhibited a very weak fluorescence (λ ex = 561 nm) at 583 nm.The titration of Sn 2+ with RBAP led to a rapid increase in the emission intensity at 583 nm.Over a 300-fold fluorescence enhancement was observed under saturation conditions (10 equiv.).The association constant was also calculated using the following formula: F − F 0 = △F = [Sn 2+ ] (F max − F 0 )/(1/K a + [Sn 2+ ]) based on a 1:1 stoichiometry, where F is the obtained fluorescence intensity, F 0 is the fluorescence intensity of free RBAP at the emission wavelength, and F max is the saturated fluorescence intensity for RBAP-Sn 2+ complex.When the reciprocal of △F was plotted as a function of Sn 2+ concentration, a linear relationship was obtained (y = A + Bx), and K a was calculated from A/B.Therefore, the binding association constant for Sn 2+ , K a , was estimated to be 2.65 × 10 4 M −1 in the MeOH/H 2 O (2:3, v/v, pH 5.95) solution, as inferred from the fluorescence titration curves of RBAP with the Sn 2+ ion.The detection limit of Sn 2+ by RBAP was determined to be 0.044 μM (Supporting Materials, S5).