Competitive Titration Research Articles

A reference scale of cucurbit[7]uril binding affinities.

The accurate determination of ultra-high binding affinities in supramolecular host-guest chemistry is a challenging endeavour because direct binding titrations are generally limited to affinities <106 M-1 due to sensitivity constraints of common titration methods. To determine higher affinities, competitive titrations are usually performed, in which one compound with a well established binding affinity serves as a reference. Herein, we propose a reference scale for such competitive titrations with the host cucurbit[7]uril (CB7) comprising binding affinities in the range from 103 to 1015 M-1. The suggested reference compounds are commercially available and will aid in the future determination of CB7 binding affinities for stimuli-responsive host-guest systems.

An Eco-Friendly Extraction and Purification Method of Nuciferine From Folium Nelumbinis Assisted With P-Sulfonatocalix[6]Arenes

Folium nelumbinis was used as vegetable, functional food and herbal medicine in Asia. In this study, p-sulfonatocalix[6]arenes (SC6A) assisted extraction of nuciferine from the Folium nelumbinis has been carried out to develop an eco-friendly extraction process with high extraction efficacy and facilitated operation. A single factor test was adopted to obtain the optimal extraction conditions, involved the use of 0.4:1:20 (g/g/mL) SC6A / Folium nelumbinis / solution ratio, below 180 μm granulometric fraction and 1 h extraction time with soaking method. Nuciferine and SC6A were separated easily by alkalization, the purity and recovery of nuciferine extracted with SC6A were increased 29.24 and 35.73 times compared with aqueous solution. Moreover, a good reusability of SC6A in the extraction of nuciferine was demonstrated. Competitive fluorescence titration, DSC and FT-IR characterization indicated that SC6A could form host-guest complexes with nuciferine at 1:1 ratio with a binding constant value of 5.55 × 105 M-1. The study provided an eco-friendly, safe and effective method for further development of nutrition supplements contains nuciferine

Binding of calcium and magnesium to human cardiac troponin C

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.

Deuterium equilibrium isotope effects in a supramolecular receptor for the hydrochalcogenide and halide anions.

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in an arylethynyl bisurea supramolecular anion receptor and use the Perrin method of competitive titrations to study the deuterium equilibrium isotope effects (DEIE) of anion binding for HS−, Cl−, and Br−. This work highlights the utility and also challenges in using this method to determine EIE with highly reactive and/or weakly binding anions.

Hydroxypyridinone-based stabilization of Np(IV) enabling efficient U/Np/Pu separations in the Adapted PUREX process

The classical process to recover uranium (U) and plutonium (Pu) from used nuclear fuel using tributyl phosphate (TBP), namely the Plutonium Uranium Redox EXtraction (PUREX) process, is complicated by the persistent presence of neptunium (Np) and thus requires extra purification steps. The concept of Adapted PUREX seeks to achieve Np recovery by adjusting the valence of the metal more effectively, thereby controlling its behavior more precisely. This study introduces the use of an aqueous hydroxypyridinone chelator, 3,4,3-LI(1,2-HOPO) (abbreviated as HOPO), to dictate the behavior of Np for recovery and meanwhile simplify cumbersome reprocessing steps. The interactions between Np and HOPO were probed mechanistically by way of absorption spectrophotometry, in conjunction with cyclic voltammetry. UV–Vis-NIR spectra illustrated the reduction of NpO22+ to Np4+, with a fast reaction rate. Cyclic voltammetry revealed quasi-reversible processes between the oxidized and reduced forms of the ligand and its Np complexes. The corresponding heterogeneous rate constants (k0) were estimated from the peak-to-peak separation potentials (ΔEp), at ~ 4 – 35 μm/s for both HOPO and NpHOPO, with scan rates of 0.01 – 0.4 V/s. Meanwhile, the electromotive force (EMF) as well as the change of Gibbs free energy (ΔG) were assessed from the half-wave potential (E1/2), demonstrating the completeness of NpO22+ reduction to Np4+ by HOPO. The cumulative formation constant of the resulting NpHOPO complex (logβ101) was determined by metal competition titration to be 42.0 ± 0.6, corroborating the extraordinarily high affinity of HOPO to tetravalent metal ions. The prowess of valence control by HOPO and the high stability of the formed complex resulted in enhanced separations of Np from U and of Pu from U, with a maximum separation factor of ~7000 for both, nearly 90- and 10300-fold higher, respectively, than the values obtained using conventional PUREX formulae.

Proposed Allosteric Inhibitors Bind to the ATP Site of CK2α.

CK2α is a ubiquitous, well-studied kinase that is a target for small-molecule inhibition, for treatment of cancers. While many different classes of adenosine 5'-triphosphate (ATP)-competitive inhibitors have been described for CK2α, they tend to suffer from significant off-target activity and new approaches are needed. A series of inhibitors of CK2α has recently been described as allosteric, acting at a previously unidentified binding site. Given the similarity of these inhibitors to known ATP-competitive inhibitors, we have investigated them further. In our thorough structural and biophysical analyses, we have found no evidence that these inhibitors bind to the proposed allosteric site. Rather, we report crystal structures, competitive isothermal titration calorimetry (ITC) and NMR, hydrogen-deuterium exchange (HDX) mass spectrometry, and chemoinformatic analyses that all point to these compounds binding in the ATP pocket. Comparisons of our results and experimental approach with the data presented in the original report suggest that the primary reason for the disparity is nonspecific inhibition by aggregation.

Transcription Factor Binding Affinities and DNA Shape Readout.

SummaryAn essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. Here, we use a binding assay we recently established to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, we find that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates our approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.

Rapid Thermodynamically Stable Complex Formation of [nat/111In]In3+, [nat/90Y]Y3+, and [nat/177Lu]Lu3+ with H6dappa.

A phosphinate-bearing picolinic acid-based chelating ligand (H6dappa) was synthesized and characterized to assess its potential as a bifunctional chelator (BFC) for inorganic radiopharmaceuticals. Nuclear magnetic resonance (NMR) spectroscopy was employed to investigate the chelator coordination chemistry with a variety of nonradioactive trivalent metal ions (In3+, Lu3+, Y3+, Sc3+, La3+, Bi3+). Density functional theory (DFT) calculations explored the coordination environments of aforementioned metal complexes. The thermodynamic stability of H6dappa with four metal ions (In3+, Lu3+, Y3+, Sc3+) was deeply investigated via potentiometric and spectrophotometric (UV-vis) titrations, employing a combination of acidic in-batch, joint potentiometric/spectrophotometric, and ligand-ligand competition titrations; high stability constants and pM values were calculated for all four metal complexes. Radiolabeling conditions for three clinically relevant radiometal ions were optimized ([111In]In3+, [177Lu]Lu3+, [90Y]Y3+), and the serum stability of [111In][In(dappa)]3- was studied. Through concentration-, time-, temperature-, and pH-dependent labeling experiments, it was determined that H6dappa radiolabels most effectively at near-physiological pH for all radiometal ions. Furthermore, very rapid radiolabeling at ambient temperature was observed, as maximal radiolabeling was achieved in less than 1 min. Molar activities of 29.8 GBq/μmol and 28.2 GBq/μmol were achieved for [111In]In3+ and [177Lu]Lu3+, respectively. For H6dappa, high thermodynamic stability did not correlate with kinetic inertness-lability was observed in serum stability studies, suggesting that its metal complexes might not be suitable as a BFC in radiopharmaceuticals.

Synthesis, characterization, X-ray structure and DNA binding study of palladium(II) complex with new thioether containing ONS donor ligand

A palladium(II) complex, [Pd(L)Cl] (1) with a new ONS donor azo–thioether ligand (L) (where L = 2,4-dihydroxy-5-((2-(methylthio)phenyl)diazenyl)(phenyl)(phenyl)methanone) is successfully synthesized. The ligand and complex are thoroughly characterized by several spectroscopic techniques. The geometry of the complex is confirmed by single-crystal X-ray analysis. Electronic structure and spectral properties are interpreted by DFT and TDDFT calculations. The interaction of the complex with CT DNA was investigated by UV-vis method and binding constant is found to be 5.42 × 104 M−1. Competitive binding titration with ethidium bromide (EB) by fluorescence titration method was carried out to understand the efficiency of the complex to displace EB from EB-DNA complex. From fluorescence titration Stern-Volmer dynamic quenching constants, Ksv was calculated and is found to be 4.15 × 104 M−1. Cyclic voltammogram of the complex exhibits significant shifting of the reduction couple to the negative potential region and decrease in current height in the presence of CT DNA. SYNOPSIS A palladium(II) complex, [Pd(L)Cl] (1) with a new ONS donor azo–thioether ligand (L) (where L = 2,4-dihydroxy-5-((2-(methylthio)phenyl)diazenyl)(phenyl)(phenyl)methanone) is successfully synthesized and characterized. The geometry of the complex is confirmed by single-crystal X-ray analysis. Electronic structure and spectral properties are interpreted by DFT and TDDFT calculations. The interaction of the complex with CT DNA is investigated by UV-vis, fluorescence and cyclic voltammetric method.

The Binuclear Transport Center of ZupT: A Zinc Transporter from E. coli

ZupT is a member of the ZIP family of transporters which are responsible for increasing the intracellular concentration of zinc from E. coli to humans. Defects in these transporters are linked to zinc deficiency, cancer, and cardiovascular disease. The mechanism of transport in this family is still not well‐understood, partly due to the bottleneck in the expression and purification of these integral membrane proteins. Recently a structure was solved of a ZIP transporter from B. bronchiseptica. The structure revealed a cadmium binuclear metal transport site, which is relatively uncommon for metal transporters. Using the crystal structure, a homology model was built for ZupT which shares 32% identity and a conserved binuclear center. In the ZupT model the metal ions are separated by roughly 4.2 A and coordinated by a bidentate Glu123 (orange). His148 and Glu152 round out site 1 (red) and Asn120, Asn149, and Glu181 site 2 (yellow). In this work, solution studies including competitive metal titrations were carried out with purified ZupT. Our results show that ZupT binds one equivalent of zinc, its primary substrate, at physiological concentrations. In contrast, it binds two equivalents of iron, a secondary substrate. Site directed mutagenesis was carried out to determine which residues are important in coordinating zinc and iron. Zinc and iron both bind to a ‘primary’ transport site (site 1‐red), while iron also binds to a secondary site (site 2‐yellow). Activity assays were used to determine the relationship between binding and function. ZupT showed transport activity with both iron and zinc; additionally, it was observed that iron positively regulated zinc activity. Our results also indicated that the highly conserved residue His119 (blue) is not essential for binding of either zinc or iron, but is important for overall transport activity, probably by facilitating metal release. Overall these results show that ZIP transporters have a single zinc transport site which can be regulated by iron binding to an adjacent secondary site.Binuclear metal center of ZupT.Utilizing the crystal structure of ZIPB from B. bronchiseptica a homology model was made with the SWISS‐MODEL server. Visualization of the binculear center was carried out with UCSF CHIMERA.Figure 1

Influence of DMβCD on the Interaction of Copper(II) Complex of 6-Hydroxychromone-3-carbaldehyde-3-hydroxybenzoylhydrazine with ctDNA.

The interaction mechanism between a scarcely soluble copper(II) complex of Cu(II)-6-hydroxychromone-3-carbaldehyde-(3′-hydroxy)benzoylhydrazone (CuCHz) in aqueous solution and its DMβCD complex was studied in the presence of ctDNA through spectroscopy and thermodynamic methods. The thermodynamic results indicate that the binding process of the CuCHz–DMβCD inclusion complex is a spontaneous process and the inclusion is enthalpy-driven. The binding constants of CuCHz and CuCHz–DMβCD with ctDNA are 2.69 × 103 and 14.7 × 103 L mol–1, respectively. The stoichiometry of the complex is 1:1, and the determined thermodynamic indicates that the process of binding is spontaneous and entropy-driven. A competitive binding titration with ethidium bromide revealed that CuCHz efficiently displaces EB from the EB–DNA system. In addition to the thermal denaturation experiments and docking studies, we can confirm that the mode of binding of this complex to ctDNA is intercalation mode. The presence of DMβCD enhances the aqueous solubility of CuCHz; nevertheless, the cyclodextrin did not affect the interaction of CuCHz with ctDNA because the inclusion complex breaks down when it binds with ctDNA.

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, 1H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440 nm upon excitation around 380 nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by 1H NMR study. The binding constants are calculated to be 5.3 × 107 and 6.8 × 106 M−1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC50 in the order of 0.023 mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.Communicated by Ramaswamy H. Sarma

PH‐Selective Fluorescent Enhancement with Glyphosate in Aqueous Media

AbstractIn this work the host–guest interactions between calixpyridinium and glyphosate were studied in aqueous media at pH 2.0 and 6.0 by using a calixpyridinium–dye complex. A pH‐selective fluorescent enhancement with glyphosate at pH 6.0 was found. The results of competitive binding titration experiments showed that calixpyridinium formed 1:1 host–guest complexes with glyphosate at pH 6.0. The association constant of calixpyridinium with glyphosate, extracted from the competitive binding titration, was (4.83±1.42)×102 M−1. The binding mode between calixpyridinium and glyphosate in D2O at pD 6.0 was further deduced by using 1H NMR spectroscopy. The result showed that the anionic carboxyl group, rather than the anionic phosphate group, was the main binding site of glyphosate to calixpyridinium accompanied by electrostatic interaction. pH 6.0 is in the pH range suitable for plant growth. Therefore, it is anticipated that these results are extremely important in the detection of glyphosate. These studies were further performed in some real aqueous samples, and the results were similar to those obtained in simple pure water. More importantly, this method indeed could be applied in the detection of glyphosate in the different real soil samples, and some potential background interferences could not affect the detection. All these studies in the real samples can greatly increase the usefulness of this work.

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Chemical and photo-induced nuclease activity of a novel minor groove DNA binder Cu(II) complex

A new type of copper(II) metal complex containing 1,10-phenanthroline (phen) and 8-(difluoromethoxy)-3,4-dihydro-2H-[1,3]thiazino[3,2-a]benzimidazole (dtb) ligands was prepared and characterized. The ds-DNA interaction of the complex was studied by UV?Vis spectrophotometry, competitive fluorometric titration with ethidium bromide (EB) and 4?,6-diamidino-2-phenylindole (DAPI), viscosity measurements and agarose gel electrophoresis. The results show that the complex can bind to ds-DNA in the minor groove by displacing DAPI molecules. DNA cleavage mechanism studies revealed that hydrogen peroxide radicals are responsible for DNA oxidative cleavage.

Interaction of Nucleotides with a Trinuclear Terbium(III)-Dizinc(II) Complex: Efficient Sensitization of Terbium Luminescence by Guanosine Monophosphate and Application to Real-Time Monitoring of Phosphodiesterase Activity.

An in-depth study of the interaction of a trinuclear terbium(III)-dizinc(II) complex with an array of nucleotides differing in the type of nucleobase and number of phosphate groups, as well as cyclic versus acyclic variants, is presented. The study examined the nature of the interaction and the efficiency at which guanine was able to sensitize terbium(III) luminescence. Competitive binding and titration studies were performed to help establish the nature/mode of the interactions. These established that (1) interaction occurs by the coordination of phosphate groups to zinc(II) (in addition to uridine in the case of uridine monophosphate), (2) acyclic nucleotides bind more strongly than cyclic counterparts because of their higher negative charge, (3) guanine-containing nucleotides are able to sensitize terbium(III) luminescence with the efficiency of sensitization following the order guanosine monophosphate (GMP) > guanosine diphosphate > guanosine triphosphate because of the mode of binding, and (4) nucleoside monophosphates bind to a single zinc(II) ion, whereas di- and triphosphates appear to bind in a bridging mode between two host molecules. Furthermore, it has been shown that guanine is a sensitizer of terbium(III) luminescence. On the basis of the ability of GMP to effectively sensitize terbium(III)-based luminescence while cyclic GMP (cGMP) does not, the complex has been utilized to monitor the catalytic conversion of cGMP to GMP by a phosphodiesterase enzyme in real time using time-gated luminescence on a benchtop fluorimeter. The complex has the potential to find broad application in monitoring the activity of enzymes that process nucleotides (co)substrates, including high-throughput drug-screening programs.

Strong binding and fluorescence sensing of bisphosphonates by guanidinium-modified calix[5]arene.

Based on the indicator displacement assay (IDA) approach, we herein report the fluorescence “switch-on” sensing and quantitative detection of bisphosphonates (BPs), a class of drugs extensively used in the treatment of patients with various skeletal diseases. Guanidinium-modified calix[5]arene (GC5A) affords strong binding on the micromolar to nanomolar level towards BPs dominantly via multiple salt bridge interactions, which was evaluated by fluorescence competitive titrations. Fluorescent IDA enables the highly sensitive and label-free detection of BPs in buffer solution, and more importantly, in artificial urine. Calibration lines were therefore set up in untreated artificial urine, allowing for quantifying the concentrations of BPs in the biologically relevant low range.

A small, steady, rapid and selective TICT based fluorescent HSA sensor for pre-clinical diagnosis

Quantitative detection of HSA in biological fluids is essential for pre-clinical diagnosis. Herein a TICT based HSA sensor NJUP1 was introduced for detection in healthy and hypertension blood samples. It interacted with domain IIA of HSA to generate a turn-on response. The detection mechanism was convinced by HSA destruction, site competition, molecular docking and isothermal titration calorimetry. NJUP1 suggested advantages such as rapid (response time 5 s), steady (more than 24 h), sensitive (LOD 18.1 nM, up to 200-fold enhancement), selective (∼6 fold over 10 times BSA in mass), wide detection range (covering blood samples requirements) and low toxicity, among which several were top class. Most importantly, it possessed only ∼300 molecular weight and one connecting double bond. It could be applied in urine system and blood samples. Thus, NJUP1 provided a potential approach for further applications.

Palladium(II) complexes with thioether containing azophenol ligands: Synthesis, characterization, X-ray structure and DNA binding study

The new palladium(II) complexes [Pd(L1/L2)Cl] (1/2), with ONS donor azo-thioether ligands (HL1/HL2), have been successfully synthesized. The complexes were thoroughly characterized by several spectroscopic techniques. The distorted square planar geometries of the complexes were confirmed by single crystal X-ray analysis. The electronic structures, redox and spectral properties are interpreted by DFT and TDDFT calculations. The interaction of the complexes with CT DNA was investigated by the UV–Vis method and the binding constant was found to be 3.56 × 104 M−1 for 1 and 5.72 × 104 M−1 for 2. A competitive binding titration with ethidium bromide (EB) by the fluorescence titration method revealed that the complexes efficiently displace EB from the EB–DNA system and the Stern–Volmer dynamic quenching constant, Ksv, was found to be 1.28 × 104 M−1 and 2.04 × 104 M−1 for 1 and 2 respectively.

Synthesis of a zinc(II) complex with hexadentate N4S2 donor thioether ligand: X-ray structure, DNA binding study and DFT computation

A new zinc(II) complex, [Zn(L)](ClO4) with hexadentate N4S2 donor azo-thioether ligand (HL) was synthesized and characterized by several spectroscopic techniques. The structure was confirmed by single crystal X-ray analysis. The interaction of the complex with CT DNA was investigated by UV–vis method and binding constant is found to be 6.6 × 104 M−1. Competitive binding titration with ethidium bromide (EB) by fluorescence titration method reveals that the complex efficiently displaces EB from EB-DNA system and the Stern-Volmer dynamic quenching constant, Ksv is found to be 2.6 × 104 M−1. DFT and TDDFT calculations were carried out to interpret the electronic structure and electronic spectra of the complex.