Valeria Zito

External and Internal Guest Binding of a Highly Charged Supramolecular Host in Water: Deconvoluting the Very Different Thermodynamics

NMR, UV-vis, and isothermal titration calorimetry (ITC) measurements probe different aspects of competing host-guest equilibria as simple alkylammonium guest molecules interact with both the exterior (ion-association) and interior (encapsulation) of the [Ga(4)L(6)](12-) supramolecular assembly in water. Data obtained by each independent technique measure different components of the host-guest equilibria and only when analyzed together does a complete picture of the solution thermodynamics emerge. Striking differences between the internal and external guest binding are found. External binding is enthalpy driven and mainly due to attractive interactions between the guests and the exterior surface of the assembly while encapsulation is entropy driven as a result of desolvation and release of solvent molecules from the host cavity.

Conditions for calibration of an isothermal titration calorimeter using chemical reactions.

The reaction of protonation of 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) is a suitable one for the calibration of isothermal titration calorimeter (ITC), providing that experimental conditions are appropriately chosen. The conditions and methods for handling experimental data from a nanowatt-ITC are discussed. Also, the binding of Ba2+ to 18-Crown-6 is successfully used to check the accuracy and precision of the chemical calibration performed with TRIS. This latter reaction has the additional advantage that the data can also be used for a check on the determination of the value of a binding constant. The anomaly of the first injection in ITC is analyzed and, by combining calorimetric and spectroscopic measurements, it is shown that it mainly results from a backlash effect of the syringe plunger rather than from a diffusion effect.

A gemini guest triggers the self-assembly of a calixarene capsule in water at neutral pH

A gemini guest having both aromatic units and negative charges induces the self-assembling of a homodimeric capsule when interacting with a positively charged calixarene at pH 7.

Self-assembling, patterning and SPR imaging of a 1,3 alternate bis(dipyridyl)calix[4]arene derivative-Cu2+ complex immobilized on to Au(111) surfaces.

The electrochemically switchable Cu(2+) complex of a 1,3 alternate bis(dipyridyl)calix[4]arene derivative forms self-assembled monolayers on Au(111) surfaces. The receptor is patterned on the surface by using microcontact printing procedures and the resulting surface is imaged via SPR.

Inorganic stressors of ubiquitin.

Many neurodegenerative proteinopathies are characterized by ubiquitin (Ub)-containing intraneuronal inclusion bodies. Recent reports have shown that Ub is able to bind Cu(II) and Zn(II), the dyshomeostasis of which is a hallmark of neurodegeneration. Here we use complementary techniques like potentiometry, circular dichroism-visible, and electron spin resonance to unveil the Ub/metal species that form, at neutral pH, their binding constants and structural features. Next, we show that both Zn(II) and Cu(II) ions hinder the interactions between Ub and Ub-conjugating E2 enzymes and inhibit significantly both Lys48 and Lys63 self-polyubiquitination reactions in a cell-free medium. The effects of Zn(II) and Cu(II) on Lys63 and Lys48 polyUb chain synthesis are compatible with the hypothesis that metal binding to His68 modifies the Ile44 hydrophobic patch of Ub and makes the protein less available for polyUb. These findings contribute to further arguments for a close relationship between metal dyshomeostasis and abnormal protein degradative pathways in the upstream events, triggering neurodegeneration.

Stereochemistry and thermodynamics of the inclusion of aliphatic and aromatic anionic guests in a tetracationic calix[4]arene in acidic and neutral aqueous solutions

A cationic calix[4]arene derivative binds both aliphatic and aromatic anions in aqueous solution thanks to concerted electrostatic and hydrophobic interactions. 1H NMR and potentiometric data show that the host flexibility changes as a function of pH. 1H NMR and isothermal titration calorimetry (ITC) studies, also supported by ESI-MS data, show that inclusion yields supramolecular complexes of the guest⊂host type only. Binding becomes weaker as the pH increases due to rigidification of the host in neutral aqueous solutions, and is enthalpically favoured for all the supramolecular complexes, regardless of pH.

Tunable Zn2+ and Cu2+ calixarene complexes as polytopic building blocks for guest recognition

Two calix[4]arene receptors containing two or four 3-pyridylmethyl pendants at the lower rim and fixed in the cone conformation have been synthesised. Their complexes with Cu2+ and Zn2+ have been studied via UV–vis absorption spectroscopy in acetonitrile; 1H NMR spectra provide further insights into the binding sites and show that the macrocycles suitably arrange their structure to meet the coordination features of the metal ion. Both ligands form with both Cu2+ and Zn2+ multiple complex species with large stability. The large binding constant values allow to drive the system to the formation of the desired species by appropriately selecting the metal/ligand ratio. The host–guest binding properties of N-methylpyridinium (NMPy) with both the free ligands and their metal complexes have been investigated by 1H NMR spectroscopy in CDCl3/CD3CN (9:1, v/v). The metal ions do not negatively affect the affinity of the calixarenes for NMPy and also allow for the formation of systems with multiple cavities available for guest inclusion, thus paving the way to new heteroditopic receptors for molecular recognition in solution.

Anion binding to a tetracopper resorcinarene-based complex

UV–vis and electron paramagnetic resonance (EPR) spectroscopic studies have been carried out on the multinuclear copper(II) complex Cu4BpaRes. The copper atoms are in a tetragonal distorted geometry with nitrogens from bispicolylamine (Bpa) coordinating in the equatorial plane and water molecules or anions completing four coordination sites. The interaction of anions with this polynuclear copper complex in aqueous solution supports the formation of different complex species, which depend on the type and the concentration of the anions. In the presence of excess anions, frozen solution EPR parameters show the formation of species in which the in-plane coordination is characterised by the presence of three nitrogen atoms coming from the ligand and a donor atom from the specific anion. For the bidentate anion ligands and especially for malonate, UV–vis titrations indicate the formation of a 1:4 (Cu4BpaRes:anion) species. EPR experiments support the formation of such a species and indicate that the four copper centres are equivalent and reach penta-coordination via the coordination of both oxygens from the bidentate ligand.

Different and Often Opposing Forces Drive the Encapsulation and Multiple Exterior Binding of Charged Guests to a M4L6 Supramolecular Vessel in Water

The supramolecular assembly [Ga4 L6 ]12- acts as a nanoscale flask to mediate the reactivity of encapsulated reactive guests and also functions as a catalyst to carry out enzyme-like chemical transformations. The guest binding to the interior cavity and exterior of this host is difficult to untangle because multiple equilibria occur in solution, and only when refining simultaneously data obtained from different techniques, such as NMR, UV/Vis, and calorimetry, can the accurate solution thermodynamics of these host-guest systems be determined. This study reports the driving forces for the inclusion and stepwise exterior guest binding of different aliphatic quaternary ammonium guests to the [Ga4 L6 ]12- assembly. Encapsulation into the host cavity was found to be an entropy-driven process, whereas exterior ion association is driven either by enthalpically favorable attractive forces or by the entropy gain due to desolvation, depending on guest size and character. The analysis of the energetics of reaction may help predicting and understanding the intimate role and contribution of the transition state in those rate-accelerated reactions involving this supramolecular assembly as an enzyme-like molecular flask.

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5  m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.