Binding Stoichiometry Research Articles

A highly selective colorimetric chemosensor based on Salicylaldehyde Schiff base for Cu+2 and F− ions detection with a turn-off response for copper (II) ion: Experimental and theoretical studies

A salicylaldehyde-Schiff base ligand (SS) containing a pyridine-2-carboxamidine group was synthesized via a simple and convenient method to facilitate the highly selective colorimetric detection of Cu+2 and F− ions. Employing colorimetric, UV–visible, and fluorescence techniques, as well as 1H NMR titration experiments, we assessed the sensing capabilities of the synthesized sensor SS towards Cu+2 and F− ions. The developed sensor showed remarkable selectivity in the presence of competing ions, with a color transition from colorless to yellow and orange in the presence of Cu+2 and F− ions respectively. The detection limits for Cu+2 and F− ions were determined to be 1.7×10−5 M and 1.94×10−4 M, respectively, using UV-visible analysis. Notably, the fluorescence experiment displayed a significantly lower detection limit for Cu+2 ion, approximately 6.6 μM. Analysis of the spectroscopic data using Job's plot revealed a 1:2 binding stoichiometry between Cu+2 and SS, while 1:1 complex formation was observed between SS and F− ions. Density functional theory (DFT) calculations enabled us to explore the optimized structures, electronic properties, and binding mechanisms of the SS, SS-F− adduct, and SS-Cu+2 complexes. These computations revealed a reduction in the HOMO-LUMO energy gap for SS upon its interaction with fluoride and copper species, consistent with the experimental observations.

Unraveling the impact of tungsten disulfide quantum dots on human serum albumin conformational dynamics and fibrillation pathways: An integrated multi-spectroscopic, biochemical, and molecular docking investigation

Herein, the intricate molecular interplay between human serum albumin (HSA) and tungsten disulfide quantum dots (WS2 QDs) was probed using spectroscopic techniques and sophisticated molecular simulation methods. Fluorescence spectroscopy demonstrated that under physiological conditions, WS2 QDs forge a non-fluorescent ground-state complex with HSA, facilitated by hydrogen bonding and van der Waals forces, ultimately resulting in the static quenching of the protein's intrinsic fluorescence. Complementary site competition experiments and molecular docking simulations reinforced a precise 1: 1 binding stoichiometry, predominantly targeting HSA's Site I. Three-dimensional fluorescence spectroscopy revealed that WS2 QDs perturb the HSA polypeptide backbone, subtly modifying the microenvironment surrounding aromatic amino acid residues. This alteration was further corroborated by circular dichroism spectroscopy, marked by a decrease in helical content and a transition towards irregular peptide conformations. Thermal stability assays illuminated the reduced thermal resilience of the HSA − WS2 QD complex. Laser confocal microscopy coupled with thioflavin T staining yielded compelling evidence that WS2 QDs effectively inhibit amyloid fibril formation in both HSA and lysozyme, underscoring their potential as potent anti-amyloidogenic agents. This comprehensive study offers pivotal insights into multifaceted impact of WS2 QDs on protein structure and function, thereby expanding their horizon of potential applications within the burgeoning field of nanomedicine.

Colorimetric reversibility: Aqueous phase recognition of cyanide using smart phone-based device with real sample analysis

A novel colorimetric 2-hydrazinobenzothiazole based probe (BTHP) was successfully synthesized and employed as an optical cyanide sensor. The interaction of CN− with BTHP produced a distinct color variation (dark pink color) with a red shift (96 nm) in its absorption spectra. The probe was selective, sensitive and robust towards cyanide ion sensing even in the presence of other competing ions. It could detect cyanide ion upto 0.77 µM, even lower than the limit recommended by WHO (1.9 µM). The binding stoichiometry for BTHP-CN− complex (1:1) was confirmed via Job’s plot. A binding constant of 3.19*102 M−1 was calculated in the recognition process. The cyanide ion recognition mechanism was confirmed using 1H NMR titration and DFT calculation. The probe coated filter paper test strips displayed dark pink color upon cyanide exposure. BTHP can be used reversibly for the visible detection of cyanide in liquid (real water) as well as solid phase (soil). Further, the practical applications of the developed sensor include smartphone based sensing, logic gate fabrication, molecular keypad lock and write-read-erase-read devices.

A Click-generated chalcone allied triazole sensor for Co (II) with INHIBIT logic gate construction and its antioxidant properties

This article presents the synthesis of chalcone allied 1,2,3-triazole via an aldol-click reaction. The newly synthesized compound chalcogenyl based 1,2,3-triazole (7) selectively detects Co (II) ion over other metal cations by UV–visible photophysical study with the limit of detection value of 1.24 × 10-8 M. Job’s plot confirms that compound (7) and the Co (II) metal ion have 1:1 binding stoichiometry. The binding mechanism between Co (II) and sensor was confirmed by using 1H NMR spectroscopy, mass spectrometry and DFT studies. The reversible behaviour of 7 towards Co (II) was used to construct a molecular logic gate. The compound (7) demonstrated good antioxidant activity with an IC50 value of 3.04 µM in comparison to 1.31 µM of DPPḢ. Further, to explore the antioxidant effect of ligand 7, the molecular docking analysis was performed with xanthine oxidase protein and the obtained binding energy was −11.08 kcal/mol.

Naturally Occurring Flavonol, Quercetagetin 5,6,7,3',4'-Pentamethyl Ether (Marionol), as a Nontoxic Plant-Based Fluorescent Probe for Rapid, Sensitive, and Selective Detection of Cu2+ in Water.

Herein, we report a naturally occurring flavonol, quercetagetin 5,6,7,3',4'-pentamethyl ether (known as marionol), as a nontoxic fluorescent probe for rapid, sensitive, and selective detection of Cu2+ in water. The interaction between marionol and Cu2+ ions is studied by various state-of-the-art spectroscopic techniques such as 1H NMR, UV-vis, fluorescence, and high-resolution mass spectrometry (HRMS). Marionol shows strong fluorescence quenching in the presence of Cu2+, whereas other interfering cations such as Fe3+, Co2+, Al3+, Pb2+, Hg2+, Zn2+, Mg2+, Mn2+, Ca2+, Cu+, Ag+, Na+, and K+ produce negligible changes in fluorescence. The binding stoichiometry between marionol and Cu2+ is found to be 2:1, according to Job's plot and HRMS analyses. The binding constant (K a) of Cu2+ with marionol is calculated to be 2.89 × 109 M-2, and the limit of detection is found to be 0.15 μM. In addition, marionol is further used for the quantification of Cu2+ in natural spring water samples. The good recoveries (92-110% with RSD <5%) and its low toxicity to MCF-7 human breast cancer cells make marionol a unique plant-based fluorescent probe for the detection of Cu2+ ions.

Crown-Ether-Based Artificial K+ Selective Ionic Filter.

In this study, we have successfully synthesized bis (cholesterol-dibenzo-18-crown-6-ether)-pillar[5]arene compound 1 through a click reaction, which could spontaneously insert into lipid bilayers to form ion channel due to the membrane anchor cholesterol group and show significant transport activity of K+ superior to Na+, with a permeability ratio of K+/Na+ equal to 4.58. Compound 1 two crown ether modules act as selective filters similar to natural K+ channel, which are determined to 1 : 2 binding stoichiometry to K+ by Job's plot and NMR titration. This structurally unambiguously unimolecule artificial channel provides ideas for constructing highly K+/Na+ selective molecular filters.

Dual-mode strategy for glyphosate detection: Leveraging competitive interaction with iron and zinc-organic frameworks

The extensive use of glyphosate poses a potential threat to food safety, human health, and the ecological environment. Hence, detecting glyphosate residues in food is of great significance for food safety. In this work, the synthesis of zinc-organic frameworks (Zn-MOFs) was achieved through the coordination of Zn2+ with the 2,5-dihydroxyterephthalic acid (DOBDC) ligand. Upon excitation at 350 nm, these Zn-MOFs exhibited robust fluorescence emission at 533 nm. However, upon the addition of iron ions, the Zn-MOFs structure disintegrated, forming a blue Fe-DOBDC complex, resulting in fluorescence quenching. In the presence of glyphosate, the iron ions were chelated by glyphosate, causing the system to lighten in color and restore fluorescence. Further, the mechanism of this strategy was investigated, which was achieved by utilizing the competitive interaction of glyphosate and Zn-MOFs for iron ions. The binding stoichiometry and binding constant of glyphosate:Fe3+ are 1:1 and 7.59 × 103 M−1 using the Job's plot and Benesi-Hildebrand equation, respectively. To enhance glyphosate detection, this study introduced an ingenious dual-mode strategy. By analyzing the colorimetric and fluorescence changes before and after glyphosate addition, we can accurately determine its concentration in the system. The combined colorimetric and fluorescent modalities offer improved reliability and applicability, boasting simplicity, rapidity, and cost-effectiveness. This innovative method is anticipated to find widespread applications and offers novel perspectives on pesticide detection platforms based on ligand competition effects.

AIE active hydroxycoumarin-anthranilic acid coupled enamine: Sequential detection of Cu2+/S2− ions and live cell imaging application

In this contribution, we designed and developed 7-diethylamino-4-hydroxycoumarin coupled anthranilic acid-based enamine (DHCBA) and its Cu2+ complex (DHCBA-Cu)via simple synthetic strategy. DHCBA shows solvatochromic effect, aggregation induced emission feature at 80 % methanol–water mixture and colorimetric as well as fluorescence turn-off sensing features towards Cu2+ ions. The DHCBA-Cu2+ ensemble has been utilized as a turn-on fluorescence sensor for the detection of S2− through a decomplexation approach. The formation of a 1:1 DHCBA-Cu2+ ensemble was further supported by 1H NMR studies, HRMS analysis and Density Functional Theory (DFT) calculation. The detection limit for Cu2+ was found to be 6.5 nM and then the binding affinity and binding stoichiometry of the probe towards Cu2+ ion was calculated using the online BindFit v0.5 supramolecular software. Moreover, the 1:1 stoichiometric ratio between DHCBA and Cu2+ ions were confirmed via single crystal X-ray analysis. The structural analysis of DHCBA-Cu2+ indicates that it forms an interesting one-dimensional coordination polymeric chain-like structure, further transformed into a two-dimensional polymeric network through hydrogen bonding interactions. The reversible on–off–on emission character of the probe, DHCBA was successfully applied for the construction of the IMPLICATION logic gate as well as on-site detection applications using paper strip and cotton-swab tests. Furthermore, the sensing feature of the enamine against Cu2+/S2− ions were validated by intracellular imaging applications.

A Schiff-Base Molecular Probe for Selective Fluorescence Sensing of Maleic Acid with Recognition of Maleic Acid in Food Additives and Cell Imaging.

The 2,4-dinitrophenylhydrazine based Schiff base (L) acts as an effective fluorescence sensor for the selective detection of maleic acid. The detection limit of L towards maleic acid is observed to be 1.29 × 10-7 M. A 1:1 binding stoichiometry between L and maleic acid was obtained using Bensi-Hilderbrand method. The binding constant (Ka) was measured to be 5.17 × 106 M-1. The sensing behavior of L was confirmed through analysis using FT-IR, DLS and SEM analysis, alongside DFT calculations. Theoretical assessments clearly suggest that the L's mono-protonation and complexation in the solvent medium are the primary mechanisms in the sensing process. Additionally, L is used to imaging the maleic acid in living cells, demonstrating its potential biological uses. In addition, recognition of maleic acid in food additives was reported.

Stoichiometry of ligand binding and role of C-terminal lysines in Mycobacterium tuberculosis and human GAPDH multifunctionality.

Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; EC1.2.1.12) has several functions in Mycobacterium tuberculosis (Mtb) and the human host. Apart from its role in glycolysis, it serves both as a cell surface and a secreted receptor for plasmin(ogen) (Plg/Plm), transferrin (Tf), and lactoferrin (Lf). Plg sequestration by Mtb GAPDH facilitates bacterial adhesion and tissue invasion, while an equivalent interaction with host GAPDH regulates immune cell migration. In both, host and microbe, internalization of Tf/Lf-GAPDH complexes serves as a route for iron acquisition. To date, the structure of Mtb GAPDH or the residues involved in these moonlighting interactions have not been identified. This study provides the first known X-ray crystal structure of Mtb GAPDH. Through further mutagenesis and functional assays, we found that the C-terminal lysines of Mtb and human GAPDH affect enzyme activity and ligand binding. We also establish the stoichiometry of Plg, Tf and Lf interactions with the GAPDH tetramer. Lastly, molecular simulation studies reveal the interactions of the C-terminal lysine residues.

AIE active Schiff base derived Pd(II) complex as a ratiometric sensor for fluoride ion

A tripodal AIE active Schiff base (L4) was synthesized and utilized along with pyridine for the synthesis of Pd(II) metal complex (MH1) using a two-step process. X-ray diffraction studies were performed to establish the molecular geometry of the synthesized ligand (L4) and its metal complex (MH1), which revealed square planar geometry for the Pd(II) complex. DFT studies revealed a reduction in HOMO-LUMO gap upon complexation of the ligand with Pd(II) ion. The compound L4 displayed aggregation induced emission and viscosity induced emission enhancement was observed in MH1. The developed metal complex was utilized for the sensitive and selective recognition of F− ion through a ligand exchange reaction. A ratiometric “turn-off” response along with colorimetric change are the characteristic features of the Pd(II) complex (MH1). The probe revealed a binding constant of 1.4 * 107 M−1 as calculated using Benesi-Hildebrand equation. Moreover, the binding stoichiometry in the MH1-F− complex was found to be 1:1. The sensing mechanism was established using mass spectrometry as the presence of fluoride incorporated complex was noticed. The structure of the resulting complex was also established using DFT based studies.

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.

Selective Detection of Picric Acid by Benzodiazepine Containing Enaminone Core as Receptor and Its Application to Real Water Sample Analysis.

Nitroaromatic compounds are highly explosive and illegitimate substances. Over a decade, chemists have been affianced in extensive research on the selective and sensitive detection of these nitroaromatic explosives for homeland security and environmental protection. The benzodiazepine-based enaminone (BDE) receptor has been synthesized by aqueous extract of onion catalyzed three-component reaction between o-phenylenediamine, dimedone with an aldehyde. The BDE probe is well analyzed and applied to a sensor that selectively detects picric acid (PA). UV-Vis and fluorescence spectroscopy were used to investigate the photophysical responses of the receptor (BDE). From the observed results BDE found turn-off fluorescence with the addition of picric acid and the lowest limit of detection and limit of quantification was achieved about 24.6 nM and 73.8 nM. The fluorescence quantum yield was attained about 0.28. The BDE-PA adduct formation was confirmed by 1H NMR titration analysis. The Job's plot analysis was performed through 1H NMR titrations and established the binding stoichiometry ratio of the BDE-PA adduct as 1:1 ratio. Further, DFT calculations supported the observed photophysical responses of BDE-PA adduct to confirm the molecular level interactions. The receptor was effectively applied to approximate level of picric acid in real water sample analysis.

Versatile fluorenone sensor for swift identification of nerve agent mimic DCP and hazardous Cu2+/Hg2+ ions

A fluorenone and indole conjugated Schiff base (1) has been designed and synthesized through a one-pot reaction, and utilized as a multifunctional sensor for recognition of various analytes, DCP, Cu2+, Hg2+, and Cysteine. The UV–vis absorption spectra demonstrated the detection of the nerve agent mimic, DCP, indicated by a blue shift in the spectra in the solution of CH3CN. The sensor 1 showed high efficiency in detecting DCP among other phosphates and ions in absorption spectrum, with a detection limit of 1.16 µM. Additionally, when various anions and metal ions were introduced to the CH3CN solution of sensor 1 (λex 260 nm), the fluorescent probe was quenched completely in the presence of Cu2+ and Hg2+ ions. The association constants for Cu2+ and Hg2+ ions with sensor 1 were calculated to be 6.68 × 104 and 9.10 × 103 M−1, respectively, with a 1:1 binding stoichiometry. Moreover, the in-situ formed complex of sensor 1 and Cu2+ (1 + Cu2+) has been further utilized to synergistically recognize cysteine. The practical applications of sensor 1 were also investigated for detecting Cu2+ and Hg2+ ions in real water samples collected from different sources.

Multivalent Protein-Nucleic Acid Interactions Probed by Composition-Gradient Multiangle Light Scattering.

Many RNA-binding proteins, such as TDP-43 or CELF1, interact multivalently with nucleic acid repetitive elements. The molecular stoichiometry of protein to nucleic acid is often difficult to assess, particularly by standard electrophoretic mobility shift assays (EMSAs). Here, we investigate the use of composition-gradient multiangle light scattering (CG-MALS) for quantifying binding affinity and stoichiometry for two RNA-binding proteins with their nucleic acid partners of varied sequence and length: TDP43's N-terminal RNA recognition motifs with both TG/GU-repeat ssDNA and ssRNA, respectively, and CELF1's two N-terminal RNA recognition motifs with (TG/UGUU/GU) repeats and an experimentally defined cognate GU-rich element (GRE). Our CG-MALS data derived from each of these interactions is consistent with expected ranges of binding affinity and stoichiometry for proteins binding to shorter nucleic acid repeats. Furthermore, we conclude that CG-MALS can be an excellent method for obtaining quantitative estimates even for high (>2) protein-nucleic acid stoichiometric ratios.

A simple benzothiazolium-based sensor for cyanide detection: Applications in environmental analysis and bioimaging

A new sensor based on Ethylbenzothiazolium-2-hydroxynaphthaldehyde conjugate-based fluorescent sensor, (E)-3-ethyl-2-(2-(2-hydroxynaphthalen-1-yl) vinyl) benzo[d]thiazol-3-ium iodide (SU-1) was designed and synthesized. The structure of SU-1 was confirmed by 1H NMR, 13C NMR, HRMS, and single crystal XRD spectral analysis. SU-1 displayed a colorimetric and fluorometric response in a DMSO:H2O (1:1,v/v) matrix, changing color from pale yellow to colorless visible to the naked eye, accompanied by a ∼ 120 nm red-shift in the absorption spectra upon CN− addition. This shift, due to formation of deprotonation followed by the nucleophilic attack on the benzothiazolium ring’s double bond, disrupts π-conjugation, blocking intramolecular charge transfer within SU-1. However, competitive anions showed negligible interference while detecting CN−. The Limit of detection for CN− was determined to be 0.27 nM, significantly below the WHO’s permissible CN− concentration in drinking water (1.9 μM). Job’s plot analysis shows that the binding stoichiometry of SU-1 to CN− is a 1:1, with a stability constant (Ka) of 1.58 x 104 M−1. The sensor demonstrated practical applications in environmental water samples and fluorescence imaging of intracellular CN− in CAD cell line.

Exploiting cyanide anion detection in food samples and for constructing logic gates by turn-off fluorescent chemosensor

A high-performance turn-off fluorescent chemosensor MSAP was developed for the selective sensing of cyanide anion without any interferences from other competing anions. MSAP's spectral responses to cyanide anion have been investigated by UV–vis and fluorescence spectroscopy. The limit of detection of cyanide anion was about 2.8 μM, which is much less than the WHO acceptable threshold level. The binding stoichiometry between the MSAP and cyanide anion was about 1:1, which was well augmented by Job`s plot. The quenching constant between MSAP and CN− ion calculated from the Stern-Volmer equation was about 8.7351×104M−1. The proposed detection mechanism of MSAP for the sensing of cyanide anion was further demonstrated by 1H NMR titration, ESI-MS and by density functional theory (DFT) studies. At last, probe MSAP was fruitfully exploited to analyse the presence of cyanide ion in diverse food samples and a XOR molecular logic gate was fabricated.

Amide/urea-based simple fluorometric receptors for iodide and Hg2+ ions in aqueous medium: Aggregation induced emission and DFT studies

Herein, we report pyrene-tagged amide and urea-based sugar derivatives 1 and 2 in a simple synthetic pathway to recognize I− and Hg2+ ions. Both molecules showed absorbance and fluorescence selectivity towards iodide ions in THF/H2O (7/3, v/v) medium. The selectivity and sensitivity of 2 for iodide ions are superior to 1 due to more H-bond donors in 2. Interestingly, fluorometric receptor 2 exhibited aggregation-induced emission (AIE) at higher pH with a remarkable fluorometric color change. The AIE phenomenon might be explained by the self-association of 2 after forming imine functionality in the alkali medium. The Stern-Volmer plot showed the fluorescence quenching constant of each receptor with an iodide ion and indicated the quenching pathway. The LODs of 1 and 2 for iodide ions were evaluated as 0.84 and 0.17 µM, respectively. The 1:1 binding stoichiometry of 1 or 2 with iodide was found from the Job plot and verified by measuring the complex mass.Further, the complexes of each receptor with I− ions can detect Hg2+ ions selectively by fluorescence turn-on method with low sensitivities (LODs: 0.008 µM for 1 and 0.01 µM for 2). DFT results were used to understand the binding mode of receptors 1 and 2 with iodide ions and the quenching process in the aqueous THF medium. The real application of the receptors was established for the recovery of iodide and Hg2+ ions from natural water samples.

Dinitrophenyl and NBD platforms based fluorescent sensors for nanomolar detection of zinc ions: Synthesis, zinc ions sensing and DFT studies

The design and synthesis of two new fluorescent probes based on dinitrophenyl (1) and 7-nitrobenz-2-oxa-1,3-diazole (2) platforms is reported. Both probes, containing bis(pyridin-2-ylmethyl)amine as a receptor unit can efficiently detect zin ions (Zn2+) in aqueous solution, with limit of detections of 124 nM and 1.06 µM, respectively. Probe 1 and 2 led to 3-fold and 2.4-fold fluorescent enhancements, respectively, with the addition of Zn2+ which suggested that working mechanism of these probes is based on the inhibition of the photoinduced electron transfer from the electron donating receptor towards the excited fluorophore. Studying binding stoichiometry by job’s plot suggested 1:1 complexation between the probe and Zn2+. Moreover, the formation of probes-Zn2+ complexes was validated by both FT-IR and 1H NMR wherein marked changes in the absorption frequencies as well as distinct downfield shifts in the resonance peaks of pyridine function along with the CH2 group linked directly to the tertiary nitrogen of the DPA unit were observed. Density functional theory (DFT) calculations suggested that the electronic density of the HOMO in 1 was concentrated on the linker between DPA and dinitrophenyl, whereas the density of the LUMO was concentrated on dinitrophenyl moiety. Upon Zn2+ binding, the electronic density in HOMO and LUMO become more delocalized. However, in case of probe 2 the delocalization of electronic density does not appreciably occur. Based on DFT simulations, both sensors form thermodynamic stable complexes with Zn2+ ions. However, sensor 2 exhibited slightly better binding preference toward Zn2+ as compared to 1. The PET based probes exhibited good selectivity to detect Zn2+ in the presence of a variety of competing metal cations. These sensors have higher sensitivity and would be able to detect chronic level of Zn2+ in the freshwater (>1.84 μM) as permitted by the US EPA.

Amino acid – Squaramide conjugates as anion binding receptors

Squaramides have garnered significant attention as anion binding motifs and have become a popular supramolecular synthon owing to their unique structural features and propensity to form strong hydrogen bond interactions particularly with anionic species. However, their conjugation to biomaterials to form biomimetic receptors remains underexplored even though such species would combine the biocompatibility of amino acids with the strong hydrogen bonding propensity of squaramides and may prove useful across broad applications in the chemical and biological sciences. In this study, we present the design and synthesis of a series of amino acid – squaramide conjugates (AASqs) 1 – 5, as well as studying their anion recognition properties. 1–5 were prepared using facile synthetic methods and their anion binding properties were investigated via 1H NMR titrations with a range of anions in 0.5 % H2O in DMSO‑d6. Our findings constitute a cautionary tale, where it was shown that the presence of the C-terminal carboxylic acid of the amino acids complicate the binding behaviour of the receptors towards anions. We show that the C-terminal carboxylates compete with other anionic species for the squaramide binding site leading to complex binding equilibria in solution. However, upon esterification of the C-termini to afford compound 5, a simplification of the binding profile was observed and was easily fitted to a 1:1 (Receptor:Anion) binding stoichiometry. This work sheds light on the potential of AASqs as anion binding receptors whilst underlining the influence of the C-terminus on anion recognition. It also provides a direct solution to mitigate the complex binding behaviour of 1–4 through esterification and will prove useful in the design of more complex squaramide bioconjugates.