Water Soluble Host–Guest Chemistry Involving Aromatic N-Oxides and Sulfonateresorcinarene

Resorcinarenes decorated with sulfonate groups are anionic in nature and water soluble with a hydrophobic electron-rich interior cavity. These receptors are shown to bind zwitterionic aromatic mono-N-oxides and cationic di-N-oxide salts with varying spacer lengths. Titration data fit a 1:1 binding isotherm for the mono-N-oxides and 2:1 binding isotherm for the di-N-oxides. The first binding constants for the di-N-oxides (K1: 104 M−1) are higher compared to the neutral mono-N-oxide (K: 103 M−1) due to enhanced electrostatic attraction from a receptor with an electron-rich internal cavity and cationic and electron deficient N-oxides. The interaction parameter α reveals positive cooperativity for the di-N-oxide with a four-carbon spacer and negative cooperativity for the di-N-oxides that have spacers with more four carbons. This is attributed to shape complementarity between the host and the guest.

Optimization of a Noncanonical Anti-infective: Interrogation of the Target Binding Pocket for a Small-Molecule Inhibitor of Escherichia coli Polysaccharide Capsule Expression

ABSTRACTWe previously identified a small-molecule inhibitor of capsule biogenesis (designated DU011) and identified its target as MprA, a MarR family transcriptional repressor of multidrug efflux pumps. Unlike other proposed MprA ligands, such as salicylate and 2,4-dinitrophenol (DNP), DU011 does not alter Escherichia coli antibiotic resistance and has significantly enhanced inhibition of capsule expression. We hypothesized that the potency and the unique action of DU011 are due to novel interactions with the MprA binding pocket and the conformation assumed by MprA upon binding DU011 relative to other ligands. To understand the dynamics of MprA-DU011 interaction, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS); this suggested that four peptide regions undergo conformational changes upon binding DU011. We conducted isothermal calorimetric titration (ITC) to quantitatively characterize MprA binding to DU011 and canonical ligands and observed a distinct two-site binding isotherm associated with the binding reaction of MprA to DU011; however, salicylate and DNP showed a one-site binding isotherm with lower affinity. To elucidate the binding pocket(s) of MprA, we selected single point mutants of MprA that included mutated residues predicted to be within the putative binding pocket (Q51A, F58A, and E65D) as well as on or near the DNA-binding domain (L81A, S83T, and T86A). Our ITC studies suggest that two of the tested MprA mutants had lower affinity for DU011: Q51A and F58A. In addition to elucidating the MprA binding pocket for DU011, we studied the binding of these mutants to salicylate and DNP to reveal the binding pockets of these canonical ligands.

Titration Simulations for Understanding of the Binding Equilibrium

<p>The reliability evaluation of the predicted binding constants in numerous models is a challenge for supramolecular host-guest chemistry. Here, I briefly formulate binding isotherm with the derivation of the multivalent equilibrium model for the chemist who wants to determine the binding constants of their compounds. This article gives an in-depth understanding of the stoichiometry of binding equilibrium to take divalent binding equilibria bearing two structurally identical binding sites as an example. The stoichiometry of binding equilibrium is affected by (1) the cooperativity of complex, (2) the concentration of titration media, and (3) the equivalents of guests. The simulations were conducted with simple Python codes.</p>

Titration Simulations for Understanding of the Binding Equilibrium

<p>The reliability evaluation of the predicted binding constants in numerous models is a challenge for supramolecular host-guest chemistry. Here, I briefly formulate binding isotherm with the derivation of the multivalent equilibrium model for the chemist who wants to determine the binding constants of their compounds. This article gives an in-depth understanding of the stoichiometry of binding equilibrium to take divalent binding equilibria bearing two structurally identical binding sites as an example. The stoichiometry of binding equilibrium is affected by (1) the cooperativity of complex, (2) the concentration of titration media, and (3) the equivalents of guests. The simulations were conducted with simple Python codes.</p>

Titration Simulations for Understanding of the Binding Equilibrium

<p>The reliability evaluation of the predicted binding constants in numerous models is also a challenge for supramolecular host-guest chemistry. Here, I briefly formulate binding isotherm with the derivation of the multivalent equilibrium model for the chemist who wants to determine the binding constants of their compounds. This article gives an in-depth understanding of the stoichiometry of binding equilibrium to take divalent binding equilibria bearing two structurally identical binding sites as an example. The stoichiometry of binding equilibrium is affected by (1) the cooperativity of complex, (2) the concentration of titration media, and (3) the equivalents of guests. The simulations were conducted with simple Python codes.</p>

Accurate Kd via Transient Incomplete Separation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i>_d, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i>_d via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate <i>K</i>_d value. <br></div><div><br></div><div>Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding <i>K</i>_d of protein-small molecule complexes.</div>

Accurate Kd via Transient Incomplete Separation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i>_d, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i>_d via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate <i>K</i>_d value. <br></div><div><br></div><div>Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding <i>K</i>_d of protein-small molecule complexes.</div>

Accurate Kd via Transient Incomplete Separation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i>_d, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i>_d via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate <i>K</i>_d value. <br></div><div><br></div><div>Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding <i>K</i>_d of protein-small molecule complexes.</div>

Competitive DNA binding of Ru(bpy)2dppz2+ enantiomers studied with isothermal titration calorimetry (ITC) using a direct and general binding isotherm algorithm

A simple algorithm allowing for binding isotherm calculations of almost any level of complexity is demonstrated here in a competitive ITC setting with enantiopure Ru-bpy intercalating into AT-DNA.