A Tripodal Thiourea Receptor for Naked-Eye Detection of Sulfate via Fluoride Displacement Assay.
Breast cancer is the most common type of cancer in women in Mississippi. It has been shown that African American women in Mississippi have an age specific incidence in the 40-49 years higher than Caucasian women (329.73 vs 239.08 per 100,000) with a corresponding age specific mortality rate of 89.1 per 100,000 for African American women compared to 28.66 per 100,000 for Caucasian women (Mississippi Cancer Registry, 2012). Although breast cancer rates have declined nationally since 1990 (DeSantis et al., 2016), this improvement has not been distributed across all segments of the population. Disparities have been associated with race/ethnicity (DeSantis et al., 2016; Jacobellis and Cutter, 2002; Weir et al., 2003), geographic status (Liff et al., 1991; Higginbothan et al., 2001; Coughlin et al., 2002; McLafferty and Wang, 2009; Markossian et al., 2014), and socioeconomic status (Bradley et al., 2002; Barry and Breen, 2005; Nichols et al., 2014). Other factors that have been proposed to account for these disparities in breast cancer outcomes include more advanced stage at diagnosis, fewer physician recommendations for mammography, underutilization of cancer screening, higher prevalence of obesity, poorer patient physician relationship, and higher rates of hypertension among ethnic minorities, as well as differences in insurance coverage (Coleman and O'Sullivan, 2001; Harris et al., 2003; Li et al., 2003; Maloney et al., 2006; Siminoff et al., 2006; Braithwaite et al., 2009; Sail et al., 2012; Robbins et al., 2014).
The Mississippi River Delta region consists of 252 counties or parishes in eight states near the lower half of the Mississippi River. Disease burden and mortality rates from all causes, including cancer and heart disease, in these delta counties are 10% higher than other non-Delta counties in the same states and 20% higher than rates for the rest of the United States as a whole (Felix and Stewart, 2005; Cosby and Bowser, 2008). At a state level, the Delta is considered to be primarily Health District I and III. District V, which includes Hinds, Madison, and Rankin counties, is arguably the largest metropolitan area of the state and has less in common with the traditional concept of the Delta. District VII in the southwest corner of the state is therefore physically separated from the Delta and is not typically thought of when discussions of health disparities associated with Districts I and III. In an effort to better understand the different problems facing each of the health districts in Mississippi, this project sought to address questions of breast cancer rates in the state.
Keeton (2014) noted that geography had a significant impact on the stage of breast cancer at which the patient was diagnosed. Mayfield-Johnson et al. (2016) reported that in Mississippi, the relative burden of invasive breast cancer varies by age and by race/ethnicity. Although these studies were quite comprehensive and compared some data for each health district as well as surrounding states, questions remain. In particular, we wanted to focus on District VII and compare it to the remainder of the state.
While great progress has been made in research on the elimination of health disparities in the past few years, further work is necessary in translating research to practice (Scarinci, 2009). Community-Based Participatory Research is a promising methodology that not only fosters research and capacity building, but also promotes ownership and sustainability by mobilizing underserved communities as political and social actors in the elimination of cancer disparities. The World Health Organization (WHO) defines health promotion as the "process of enabling people and communities to take control over their health and its determinants". Thus, by definition health should be promoted through community involvement in which community members decide what, when, where, and how health will be promoted and disease will be prevented in their communities (Scarinci, 2009).
The ultimate goal of health disparity studies is to reduce breast, cervical, and colorectal cancer disparities between African-Americans and Caucasians in underserved counties in Mississippi. Research focused on developing and implementing a community action plan that should lead to reduced health disparities between African-Americans and Caucasians in these counties in Mississippi. These goals can only be met with careful analysis of the differences between communities of need. Anions play an important role in many environmental and biological systems, and the mechanistic understanding of selective anion recognition by synthetic receptors is critical in the field of supramolecular chemistry (Bowman-James and Bianchi, 2012). Although polyamine-based receptors are known to bind anions strongly, their binding occurs only at a certain pH, hampering their practical application under neutral conditions (Hossain, 2008). On the other hand, neutral receptors including amides, ureas, and thioureas are suitable for binding anions with their H-bond donor groups regardless of the solution pH (Bondy, 2003; Amendola et al, 2010; Bose et al, 2012). Recently, tren-based receptors bearing urea, or thiourea functional groups have been an area of focus for anion recognition, due to the directional conformation and enhanced chelation effect of the NH groups (Custelcean et al, 2005). The electron withdrawing nature of sulfur on thiourea functionalities increases the acidity of NH for H-bonding interactions with an anionic guest (Khansari et al, 2017). Furthermore, attaching chromophore groups to receptors often leads to a spectroscopic or color change, allowing them to serve as sensors for target analytes (Gale et al, 2016; Gale et al, 2017). Herein, we report a thiourea-based tripodal receptor L (Figure 1), showing strong selectivity for sulfate. The selectivity was further supported by competitive colorimetric studies, displaying a sharp visible color change upon the addition of sulfate to the fluoride complex of L.
General: All reagents and solvents were purchased as reagent grade and were used without further purification. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity INOVA 500 FT-NMR. Chemical shifts for samples were measured in DMSOd and calibrated against sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3d acid (TSP) as an external reference in a sealed capillary tube. NMR data were processed and analyzed with MestReNova Version 6.1.1-6384. The IR spectra were recorded on a Perkin Elmer-Spectrum One FT-IR spectrometer with KBr disks in the range of 4000-400 [cm.sup.-1]. The melting point was determined on a Mel-Temp (Electrothermal 120 VAC 50/60 Hz) melting point apparatus and was uncorrected. Mass spectral data were obtained at ESI-MS positive mode on a TSQ Quantum GC (Thermo Scientific). Elemental analysis was carried out using an ECS 4010 Analytical Platform (Costech Instrument) at Jackson State University.
Synthesis: The receptor L was synthesized following the literature procedure (Khansari et al, 2017).
NMR Binding Studies: Binding constants were obtained by [sup.1]H NMR titrations of L with the oxoanions (N[O.sub.3.sup.-], Cl[O.sub.4.sup.-], [H.sub.2]P[O.sub.4.sup.-], HS[O.sub.4.sup.-], and S[O.sub.4.sup.2-]) and halides ([F.sup.-], [Cl.sup.-], [Br.sup.-], and [I.sup.-]) using their tetrabutyl ammonium salts in DMSO-[d.sub.6]. Initial concentrations were [[L].sub.0] = 2 mM, and [[anion].sub.0] = 20 mM. Each titration was performed by 13 measurements at room temperature. The association constant K was calculated by fitting of several independent NMR signals with a 1:1 association model (Schneider et al, 1998).
UV-Vis Binding Studies: UV-Vis titration studies were performed by titrating L with anions in DMSO at 25 [degrees]C. In this case, initial concentrations of L and the anions were 1.5 x [10.sup.-4] M and 1.5 x [10.sup.-2] M, respectively. Each titration was performed by 15 measurements ([[[A.sup.-]].sub.0][[L].sub.0] = 0-35 equivalents), and the binding constant K was calculated by fitting the relative UV-Vis absorbance (I/[I.sub.0]) with a 1:1 association model (Schneider et al, 1998).
Cytotoxicity Assay: Primary human foreskin-derived fibroblasts (HF) and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum (SAFC, Lenexa, KS), 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/ml glucose, and 100 U/ml penicillin-streptomycin (Cellgro) at 37[degrees]C with 5% C[O.sub.2] (Freshney, 2005). Cells were seeded in 12-well plates and grown until they became confluent (approximately 24 hours). The media was removed, and fresh complete medium was added. A stock solution of L was made in 100% DMSO at 500 mM concentration. Cells were treated with L at a final concertation of 10 [micro]M to 500 [micro]M in different wells for 24 hours for cytotoxic assessment. In this experiment, 0.1% was the highest concentration of DMSO that the cells received. As a mock control, cells were treated with 0.1 % DMSO without L. At the end point, cells were observed under an inverted Evos-FL microscope (Thermo Fisher Scientific, Waltham, MA), and bright-field images of living cells were captured. After imaging, the viability of cells was determined using trypan blue exclusion assay as previously described (Strober, 2001; Archer et al, 2017; Freshney 2005).
NMR titration studies: 1H NMR titrations of L were performed to evaluate its binding affinity for a variety of anions ([F.sup.-], [Cl.sup.-], [Br.sup.-], [I.sup.-], Cl[O.sub.4.sup.-], N[O.sub.3.sup.-], [H.sub.2]P[O.sub.4.sup.-], HS[O.sub.4.sup.-], and S[O.sub.4.sup.2-]) using their tetrabutylammonium salts in DMSO-[d.sub.6]. Figure 1 shows the stacking of [sup.1]H NMR spectra as obtained from the titration of L with S[O.sub.4.sup.2-] (0-10 eq.). In the 1H NMR spectrum of L, one NH proton is observed at 10.01 (H1) ppm and the other one at 7.95 (H2) ppm. The addition of S[O.sub.4.sup.2-] to L resulted in a significant downfield shift of both NH signals ([DELTA][delta]= 1.49 ppm for H1 and [DELTA][delta]= 1.81 ppm for H2) with a sharp saturation at a 1:1 ratio (Figure 2), demonstrating strong interactions of the receptor and sulfate. Similar downfield shifts in the NH signals, but to a lesser extent, were also observed for HS[O.sub.4.sup.-] ([DELTA][delta]= 0.72 ppm for H1 and [DELTA][delta]= 0.71 ppm for H2), [Cl.sup.-] ([DELTA][delta]= 0.61 ppm for H1 and [DELTA][delta]= 0.38 ppm for H2) and [Br.sup.-] ([DELTA][delta]= 0.09 ppm for H1 and [DELTA][delta]= 0.06 ppm for H2) at the end of titrations. The NH signals of L were shown to be broadened and eventually disappeared upon the addition of [H.sub.2]P[O.sub.4.sup.-]. In this case, CH signals were used to calculate the binding constant. However, for [I.sup.-], N[O.sub.3.sup.-] and Cl[O.sub.4.sup.-], a negligible change in the NMR signals was observed. The binding constants of L for these anions were determined from a nonlinear regression analysis of the progressive changes of NH or CH signals with a 1:1 binding model (Schneider et al, 1988). The binding data are listed in Table 1, showing that the receptor binds strongly to S[O.sub.4.sup.2-], with an association constant larger than [10.sup.4] [M.sup.-1] (Table 1).
In contrast, upon the addition of fluoride to L, a new set of NMR signals appeared at downfield via a slow proton exchange between the free receptor and the complex. The signals of the free receptor disappeared completely upon the addition of one equivalent of fluoride (Figure 3). There is some evidence that highly basic anions can abstract acidic protons from NH of urea/thiourea-based compounds (Boiocchi et al, 2005). A detailed study on deprotonation and hydrogen bonding aspects between anions and urea/thiourea-based receptors reported by Perez-Casas and Yatsimirsky (2008) suggested that the deprotonation is accompanied by the disappearance of NMR signals of the abstracted protons, while the binding event results in the downfield shift of NMR signals of NH groups in a receptor. The distinct downfield shift of NH signals in our receptor is consistent with the formation of a hydrogen-bonded complex (instead of deprotonation). For further clarification, a control experiment was carried out using OH-, showing complete disappearance of NH signals due to the deprotonation of NH by highly basic hydroxide ions (E, Figure 3).
The binding constant for fluoride was calculated from the relative changes in the integrated intensity of NH signals for the free receptor and the complex, yielding a binding constant larger than [10.sup.4] [M.sup.-1] (Portis et al, 2017). To determine the selectivity of the receptor, competition experiments were performed in which sulfate was added to the receptor containing one equivalent of fluoride (C, Figure 3) or hydroxide (E, Figure 3) in DMSO-[d.sub.6]. As shown in Figure 3, the [sup.1]H NMR spectrum of L containing an equivalent amount of fluoride and sulfate (D), or hydroxide and sulfate (F), resembles the spectrum of L containing one equivalent of sulfate (B), thus demonstrating the selectivity of the receptor for sulfate. The receptor also exhibits good interactions for [Cl.sup.-], HS[O.sub.4.sup.-], and [H.sub.2]P[O.sub.4.sup.-] with association constants of 3.1, 2.9 and 3.0 (in log K), respectively. However, it does not show any appreciable affinity for [I.sup.-], N[O.sub.3.sup.-], or Cl[O.sub.4.sup.-].
Colorimetric studies: The receptor was further investigated by naked eye colorimetric studies for anions in DMSO. As shown in Figure 4, a visible color change from pale yellow to orange was observed after the addition of one equivalent of fluoride to L (2 mM), indicating a different optical absorption spectrum of the [[LF].sup.-] complex. However, the color remained almost unchanged for other anions. A similar color change was reported previously due to the addition of fluoride to related receptors (Khansari et al, 2014). To examine the visual selectivity, one equivalent of different anions was added separately to an orange solution of fluoride complex in DMSO. Interestingly, the color of [[LF].sup.-] was sharply changed to a pale yellow color (original color of the receptor) after the addition of sulfate. This observation suggests that sulfate can compete with fluoride for hydrogen bonding with NH groups, and displace the bound fluoride from the complex [[LF].sup.-] into solution, which agrees with NMR competition experiments (Figure 5). However, other anions are not strong enough to displace the bound fluoride, supporting the results of NMR and UV-Vis titrations. Thus, the fluoride-receptor complex serves as a colorimetric probe for visual identification of sulfate through fluoride displacement assay, a principle that is known as an indicator displacement assay widely used for optical sensing of analytes (Nguyen and Anslyn, 2006; Rhaman et al, 2014).
UV-Vis titration studies: UV-Vis titrations were also performed to investigate the interactions of the receptor with anions in DMSO. As shown in Figure 6, the addition of sulfate to a solution of L results in a systematic decrease in the absorbance with a red shift of the peak at 335 nm, suggesting the formation of a [[L(S[O.sub.4])].sup.2-] complex. The relative absorbance I/[I.sub.0] of L (where [I.sub.0] and I represent the absorbance of L before and after the addition of an anion, respectively) upon the gradual addition of S[O.sub.4.sup.2-] gave the best fit to a 1:1 binding mode yielding a binding constant of 6.40 (in log K). The host showed a similar spectral change when it was titrated with dihydrogen phosphate. The addition of fluoride anion to L also showed a decrease in the absorption at 335 nm, but no appreciable shift was observed as compared to that for sulfate or phosphate. However, the naked-eye colorimetric study shows an orange color after the addition of just one equivalent of fluoride to the receptor in which the concentration of L was different (2 mM) than that used in UV titrations (0.15 mM).
To confirm if the color originated from the binding with fluoride (instead of deprotonation), the receptor was deprotonated by adding one equivalent of hydroxide. The resulting intense red color of the deprotonated receptor is distinctly different than that developed for the fluoride complex, suggesting that the observed orange color (for the receptor containing fluoride) originated from the binding event (Figure 7). Further justification of this assumption is provided by control experiments from UV studies of the receptor containing one equivalent of hydroxide or fluoride in DMSO (Figure 8). In the UV spectrum, a new absorption band appeared at about 485 nm for the solution of L containing hydroxide anion, indicating an anion-induced deprotonation of L due to the removal of NH protons by highly basic O[H.sup.-]. However, such a band is absent for the solution of L containing fluoride. The addition of one equivalent sulfate to L mixed with fluoride (or hydroxide) shows a nearly similar spectrum to that obtained from the sulfate complex. This further supports the displacement of the bound fluoride by sulfate, which is in accordance with the NMR results discussed previously. On the other hand, the addition of other anions to L solution does not induce an appreciable change in the absorption spectrum. This observation is fully consistent with colorimetric observations, showing no visible color change for [Cl.sup.-], [Br.sup.-], [I.sup.-], Cl[O.sub.4.sup.-], N[O.sub.3.sup.-], and HS[O.sub.4.sup.-].
Cytotoxicity Assessment: The biocompatibility of L as a receptor was tested by analyzing the viability on HeLa cells. Each type of cells was treated with L at concentrations ranging from 10 [micro]M to 500 [micro]M for 24 hours, and the cell viability was quantified using a trypan blue exclusion assay. As a control, cells were treated with 0.1% DMSO. The results from the exclusion assay revealed that the cell viability of HeLa cells was almost unaffected up to 100 [micro]M concentration of the receptor (Figure 9). However, the cell cytotoxicity was observed at a higher concentration (500 [micro]M). Live cell imaging was also performed on HeLa cells at 24 hours post treatment, showing no cytotoxic effects up to 100 [micro]M (Figure 10). These results are in accord with the cell viability data, further demonstrating an excellent biocompatibility of the receptor on living cells.
In conclusion, we have synthesized and structurally characterized a thiourea-based tripodal receptor L, showing strong binding and selectivity for sulfate over other anions in DMSO. The selectivity of L for sulfate was further confirmed by the competitive colorimetric studies, displaying a sharp color change of [[LF].sup.-], while other anions showed no change in color. This observation suggests that the added sulfate displaces the bound fluoride in [[LF].sup.-], and this compound can be used as a colorimetric probe to detect sulfate in solution via a fluoride displacement assay. The strong selectivity of L for sulfate was further supported by UV-Vis titrations in DMSO. The receptor also shows an excellent biocompatibility in HeLa cells. The strong selectivity for sulfate and excellent biocompatibility towards living cells demonstrates that this receptor can be used as a potential sensing probe for the detection of sulfate anions for various biological and chemical applications.
The National Science Foundation is acknowledged for a CAREER award (CHE-1056927) to M.A.H. NMR core facility at Jackson State University was supported by the National Institutes of Health (G12RR013459). M.H.H. and R.T. are supported by American Heart Association (Award No. 14SDG20390009).
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Corey R. Johnson, (1) Mohammad H. Hasan, (2) Maryam Emami Khansari, (1) Ritesh Tandon (2) and Md. Alamgir Hossain * (1)
(1) Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS 39217
(2) Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, MS 39216
Corresponding Author: Alamgir Hossain Email: email@example.com
Caption: Figure 1. Receptor L.
Caption: Figure 2. Partial [sup.1]H NMR spectra of L (2 mM) showing changes in the NH chemical shifts with an increasing amount of S[O.sub.4.sup.2-] (20mM) in DMSO-[d.sub.6]. (H1 = CSNtfAr and H2 = C[H.sub.2]NHCS).
Caption: Figure 3. Partial [sup.1]H NMR spectra of L showing changes in the chemical shifts after the addition of one equivalent of different anions in DMSO-[d.sub.6].
Caption: Figure 4. Colorimetric studies of the receptor L (2 mM) with one equivalent of different anions in DMSO.
Caption: Figure 5. Colorimetric studies of [[LF].sup.-] after the addition of one equivalent of different anions in DMSO, showing a visual color change for sulfate.
Caption: Figure 6. UV-Vis titration spectra showing the changes in absorption spectra of L (1.5 x [10.sup.-4] M) with an increasing amount of S[O.sub.4.sup.2-] (1.5 x [10.sup.-2] M) in DMSO (Inset showing the titration plot).
Caption: Figure 7. Colorimetric studies of L (2 mM) after the addition of one equivalent of fluoride, hydroxide or a mixture of fluoride and hydroxide in DMSO, showing different color.
Caption: Figure 8. UV-vis spectra of L (1.5 x [10.sup.-4] M) with 5 equivalents of different anions in DMSO.
Caption: Figure 9. Effect of L on cell viability. Confluent HeLa cells were either mock treated (0.1 % DMSO-treated control) or treated with L (10 [micro]M to 500 [micro]M) for 24 hours. Triplicate samples were used, and error bars represent standard error of mean.
Caption: Figure 10. Bright-field images of HeLa cells. Cells were either mock treated (0.1% DMSO-treated control) or treated with L for 24 hours at the concentrations specified. Images are representative of three independent experiments.
Table 1. Binding constants (log K) and binding energies (E) of the anions complexes of L. Anion Log K (a) Log K (b) [F.sup.-] >4.0 (c) 5.1 [Cl.sup.-] 3.1 3.2 [Br.sup.-] 1.9 1.7 [I.sup.-] <1 (d) <1 (e) S[O.sub.4.sup.2-] > 4.0 6.4 HS[O.sub.4.sup.-] 2.9 2.8 [H.sub.2]P[O.sub.4.sup.-] 3.0 3.1 N[O.sub.3.sup.-] <1 (d) <1 (e) Cl[O.sub.4.sup.-] <1 (d) <1 (e) (a) Determined by [sup.1]H NMR titrations in DMSO-[d.sub.6]; (b) determined by UV titrations in DMSO; (c) slow proton exchange; (d) no appreciable change was observed in [sup.1]H NMR spectra; (e) no appreciable change was observed in UV spectra.