Boosting Energy-Transfer Processes via Dispersion Interactions.

The generation of open-shell intermediates under mild conditions has opened broad synthetic opportunities during this century. However, these reactive species often require a case specific and tailored tuning of experimental parameters in order to efficiently convert substrates into products. We report a general approach that can overcome these ubiquitous limitations for several visible-light promoted energy-transfer processes. The use of either naphthalene (5-20 equiv.) or simple binaphthyl derivatives (10-30 mol %) greatly increases their efficiency, giving rise to a new strategy for catalysis. The trend is consistent among different media, photocatalysts, light sources and substrates, allowing one to improve existing methods, to more easily optimize conditions for new ones, and, moreover, to disclose otherwise inaccessible reaction pathways.

Insights on the Structure in Solution of Paramagnetic LnIII/GaIII 12-Metallacrown-4 Complexes Using 1D 1H NMR and Model Structures.

The solution structure of LnIIINaI(OBz)4[12-MCGaIII(N)Shi-4] complexes was studied through paramagnetic 1H NMR and DFT models. Although isostructural in the solid state, their 1H NMR spectra in DMSO-d6 are extremely different from one another due to the magnetic anisotropy of the lanthanide(III) ions. NMR data were analyzed by the "all-lanthanide" method that were compared to X-ray structures and model structures, allowing to establish the extent of the structural changes that occur from the solid state to the solution phase. Major structural changes involve the phenyl groups of the benzoate ions that, quite surprisingly, in solution present preferential orientations lowering the symmetry of the complex contrary to what observed in the solid state. Overall, DFT methods and 1D NMR data allowed us to clarify aspects related to molecular rearrangement processes in solution that could not be predicted by a simple look at the X-ray structures of these complexes.

Semirigid Ligands Enhance Different Coordination Behavior of Nd and Dy Relevant to Their Separation and Recovery in a Non-aqueous Environment.

Rare-earth elements are widely used in high-end technologies, the production of permanent magnets (PMs) being one of the sectors with the greatest current demand and likely greater future demand. The combination of Nd and Dy in NdFeB PMs enhances their magnetic properties but makes their recycling more challenging. Due to the similar chemical properties of Nd and Dy, their separation is expensive and currently limited to the small scale. It is therefore crucially important to devise efficient and selective methods that can recover and then reuse those critical metals. To address these issues, a series of heptadentate Trensal-based ligands were used for the complexation of Dy3+ and Nd3+ ions, with the goal of indicating the role of coordination and solubility equilibria in the selective precipitation of Ln3+-metal complexes from multimetal non-water solutions. Specifically, for a 1:1 Nd/Dy mixture, a selective and fast precipitation of the Dy complex occurred in acetone with the Trensalp-OMe ligand at room temperature, with a concomitant enrichment of Nd in the solution phase. In acetone, complexes of Nd and Dy with Trensalp-OMe were characterized by very similar formation constants of 7.0(2) and 7.3(2), respectively. From the structural analysis of an array of Dy and Nd complexes with TrensalR ligands, we showed that Dy invariably provided complexes with coordination number (cn) of 7, whereas the larger Nd experienced an expansion of the coordination sphere by recruiting additional solvent molecules and giving a cn of >7. The significant structural differences have been identified as the main premises upon which a suitable separation strategy can be devised with these kind of ligands, as well as other preorganized polydentate ligands that can exploit the small differences in Ln3+ coordination requirements.

Role of the Cysteine in R3 Tau Peptide in Copper Binding and Reactivity.

Tau is a widespread neuroprotein that regulates the cytoskeleton assembly. In some neurological disorders, known as tauopathies, tau is dissociated from the microtubule and forms insoluble neurofibrillary tangles. Tau comprises four pseudorepeats (R1-R4), containing one (R1, R2, R4) or two (R3) histidines, that potentially act as metal binding sites. Moreover, Cys291 and Cys322 in R2 and R3, respectively, might have an important role in protein aggregation, through possible disulfide bond formation, and/or affecting the binding and reactivity of redox-active metal ions, as copper. We, therefore, compare the interaction of copper with octadeca-R3-peptide (R3C) and with the mutant containing an alanine residue (R3A) to assess the role of thiol group. Spectrophotometric titrations allow to calculate the formation constant of the copper(I) complexes, showing a remarkable stronger interaction in the case of R3C (log Kf = 13.4 and 10.5 for copper(I)-R3C and copper(I)-R3A, respectively). We also evaluate the oxidative reactivity associated to these copper complexes in the presence of dopamine and ascorbate. Both R3A and R3C peptides increase the capability of copper to oxidize catechols, but copper-R3C displays a peculiar mechanism due to the presence of cysteine. HPLC-MS analysis shows that cysteine can form disulfide bonds and dopamine-Cys covalent adducts, with potential implication in tau aggregation process.

Lanthanide Identity Governs Guest-Induced Dimerization in LnIII [15-MC Cu II N(L-pheHA) -5])3+ Metallacrowns.

Series of lanthanide-containing metallic coordination complexes are frequently presented as structurally analogous, due to the similar chemical and coordinative properties of the lanthanides. In the case of chiral (LnIII [15-MC Cu II N(L-pheHA) -5])3+ metallacrowns (MCs), which are well established supramolecular hosts, the formation of dimers templated by a dicarboxylate guest (muconate) in solution of neutral pH is herein shown to have a unique dependence on the identity of the MC's central lanthanide. Calorimetric data and nuclear magnetic resonance diffusion studies demonstrate that MCs containing larger or smaller lanthanides as the central metal only form monomeric host-guest complexes whereas analogues with intermediate lanthanides (for example, Eu, Gd, Dy) participate in formation of dimeric host-guest-host compartments. The driving force for the dimerization event across the series is thought to be a competition between formation of highly stable MCs (larger lanthanides) and optimally linked bridging guests (smaller lanthanides).

Different Behavior of the Histidine Residue toward Cadmium and Zinc in a Cadmium-Specific Metallothionein from an Aquatic Fungus.

Metallothioneins (MTs) are a large superfamily of ubiquitous cysteine-rich metalloproteins with main functions in metal ion homeostasis and detoxification. Neclu_MT1 is a metallothionein from the aquatic fungus Heliscus lugdunensis and so far the only known MT that is solely induced by CdII but not by ZnII or copper ions. In addition to eight cysteine residues, Neclu_MT1 also contains a less common single C-terminal histidine residue. To better understand the role of this histidine residue in metal ion binding, for the first time, potentiometric pH titrations are applied, revealing insights into the protonation and metal ion binding processes. Additional studies with absorption and NMR spectroscopy complement the finding that while the histidine residue is not crucial for the overall metal binding capacity, it does serve as a ligand in the ZnII but not in the CdII form of the protein.

Binding and Reactivity of Copper to R1 and R3 Fragments of tau Protein.

Tau protein is present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble neurofibrillary tangles of tau are associated with several neurological disorders known as tauopathies, among which is Alzheimer's disease. In neurons, tau binds tubulin through its microtubule binding domain which comprises four imperfect repeats (R1-R4). The histidine residues contained in these fragments are potential binding sites for metal ions and are located close to the regions that drive the formation of amyloid aggregates of tau. In this study, we present a detailed characterization through potentiometric and spectroscopic methods of the binding of copper in both oxidation states to R1 and R3 peptides, which contain one and two histidine residues, respectively. We also evaluate how the redox cycling of copper bound to tau peptides can mediate oxidation that can potentially target exogenous substrates such as neuronal catecholamines. The resulting quinone oxidation products undergo oligomerization and can competitively give post-translational peptide modifications yielding catechol adducts at amino acid residues. The presence of His-His tandem in the R3 peptide strongly influences both the binding of copper and the reactivity of the resulting copper complex. In particular, the presence of the two adjacent histidines makes the copper(I) binding to R3 much stronger than in R1. The copper-R3 complex is also much more active than the copper-R1 complex in promoting oxidative reactions, indicating that the two neighboring histidines activate copper as a catalyst in molecular oxygen activation reactions.

Elucidation of 1H NMR Paramagnetic Features of Heterotrimetallic Lanthanide(III)/Manganese(III) 12-MC-4 Complexes.

The paramagnetic one-dimensional 1H NMR spectra of twelve LnIIINaI(OAc)4[12-MCMnIII(N)shi-4] complexes, where LnIII is PrIII-YbIII (except PmIII) and YIII, are reported. Their solid-state isostructural nature is confirmed in methanol-d4 solution, as a similar pattern in the 1H NMR spectra is observed along the series. Notably, a relatively well-resolved spectrum is reported for the GdIII complex. The chemical shift data are analyzed using the "all lanthanides" method, and the Fermi contact and pseudo-contact contributions are calculated for the lanthanide-induced shift (LIS). For the TbIII-YbIII complexes, the pseudo-contact contributions are typically 1 order of magnitude higher than the Fermi contact contributions; however, for the GdIII complex, the Fermi contact is the main contribution to the paramagnetic chemical shift. For the methyl protons of the axial acetate (-OAc) ligands, the LIS is opposite in sign, with respect to that of the aromatic salicylhydroximate (shi3-) protons, because of structural rearrangements that occur upon dissociation of the NaI cation in solution. The calculated crystal field parameters (BLn) for the TbIII (360 cm-1), DyIII (250 cm-1), HoIII (380 cm-1), ErIII (410 cm-1), TmIII (620 cm-1), and YbIII (380 cm-1) complexes are not constant, likely as a consequence of the inaccuracy of the Bleaney's constants and, to a smaller extent, the small structural changes that occur in solution. Overall, the metallacrown scaffold retains structural integrity and similarity in solution for the entire series; however, small structural features, which do not affect the overall similarity, do likely occur.

Design of 2D Porous Coordination Polymers Based on Metallacrown Units.

A 12-metallacrown-4 (MC) complex was designed and employed as the building block in the synthesis of coordination polymers, one of which is the first permanently porous MC architecture. The connection of the four-fold symmetric MC subunits by Cu(II) nodes led to the formation of 2D layers of metallacrowns. Channels are present in the crystalline architecture, which exhibits permanent porosity manifested in N2 and CO2 uptake capacity.

Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils.

One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu(T2) center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu(T2) site in the natural enzyme. The species Cu(II)(TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.

Tailoring the Use of 8-Hydroxyquinolines for the Facile Separation of Iron, Dysprosium and Neodymium.

Permanent magnets (PMs) containing rare earth elements (REEs) can generate energy in a sustainable manner. With an anticipated tenfold increase in REEs demand by 2050, one of the crucial strategies to meet the demand is developing of efficient recycling methods. NdFeB PMs are the most widely employed, however, the similar chemical properties of Nd (20-30 % wt.) and Dy (0-10 % wt.) make their recycling challenging, but possible using appropriate ligands. In this work, we investigated commercially available 8-hydroxyquinolines (HQs) as potential Fe/Nd/Dy complexing agents enabling metal separation by selective precipitation playing on specific structure/property (solubility) relationship. Specifically, test ethanolic solutions of nitrate salts, prepared to mimic the main components of a PM leachate, were treated with functionalized HQs. We demonstrated that Fe3+ can be separated as insoluble [Fe(QCl,I)3] from soluble [REE(QCl,I)4]- complexes (QCl,I -: 5-Cl-7-I-8-hydoxyquinolinate). Following that, QCl - (5-Cl-8-hydroxyquinolinate) formed insoluble [Nd3(QCl)9] and soluble (Bu4N)[Dy(QCl)4]. The process ultimately gave a solution phase containing Dy with only traces of Nd. In a preliminary attempt to assess the potentiality of a low environmental impact process, REEs were recovered as oxalates, while the ligands as well as Bu4N+ ions, were regenerated and internally reused, thus contributing to the sustainability of a possible metal recovery process.

Bifunctional octadentate pseudopeptides as Zirconium-89 chelators for immuno-PET applications.

BACKGROUND: Positron emission tomography (PET) is a highly sensitive method that provides fine resolution images, useful in the field of clinical diagnostics. In this context, Zirconium-89 (89Zr)-based imaging agents have represented a great challenge in molecular imaging with immuno-PET, which employs antibodies (mAbs) as biological vectors. Indeed, immuno-PET requires radionuclides that can be attached to the mAb to provide stable in vivo conjugates, and for this purpose, the radioactive element should have a decay half-life compatible with the time needed for the biodistribution of the immunoglobulin. In this regard, 89Zr is an ideal radioisotope for immuno-PET because its half-life perfectly matches the in vivo pharmacokinetics of mAbs. RESULTS: The main objective of this work was the design and synthesis of a series of bifunctional octadentate pseudopeptides able to generate stable 89Zr complexes. To achieve this, here we investigated hydroxamate, N-methylhydroxamate and catecholate chelating moieties in complexing radioactive zirconium. N-methylhydroxamate proved to be the most effective 89Zr-chelating group. Furthermore, the increased flexibility and hydrophilicity obtained by using polyoxyethylene groups spacing the hydroxamate units led to chelators capable of rapidly forming (15 min) stable and water-soluble complexes with 89Zr under mild reaction conditions (aqueous environment, room temperature, and physiological pH) that are mandatory for complexation reactions involving biomolecules. Additionally, we report challenge experiments with the competitor ligand EDTA and metal ions such as Fe3+, Zn2+ and Cu2+. In all examined conditions, the chelators demonstrated stability against transmetallation. Finally, a maleimide moiety was introduced to apply one of the most promising ligands in bioconjugation reactions through Thiol-Michael chemistry. CONCLUSION: Combining solid phase and solution synthesis techniques, we identified novel 89Zr-chelating molecules with a peptide scaffold. The adopted chemical design allowed modulation of molecular flexibility, hydrophilicity, as well as the decoration with different zirconium chelating groups. Best results in terms of 89Zr-chelating properties were achieved with the N-methyl hydroxamate moiety. The Zirconium complexes obtained with the most effective compounds were water-soluble, stable to transmetallation, and resistant to peptidases for at least 6 days. Further studies are needed to assess the potential of this novel class of molecules as Zirconium-chelating agents for in vivo applications.

Inhibitory interactions of the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold with Bunyavirales cap-snatching endonucleases expose relevant drug design features.

The World Health Organization (WHO) identifies several bunyaviruses as significant threats to global public health security. Developing effective therapies against these viruses is crucial to combat future outbreaks and mitigate their impact on patient outcomes. Here, we report the synthesis of some isoindol-1-one derivatives and explore their inhibitory properties over an indispensable metal-dependent cap-snatching endonuclease (Cap-ENDO) shared among evolutionary divergent bunyaviruses. The compounds suppressed RNA hydrolysis by Cap-ENDOs, with IC50 values predominantly in the lower µM range. Molecular docking studies revealed the interactions with metal ions to be essential for the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold activity. Calorimetric analysis uncovered Mn2+ ions to have the highest affinity for sites within the targets, irrespective of aminoacidic variations influencing metal cofactor preferences. Interestingly, spectrophotometric findings unveiled sole dinuclear species formation between the scaffold and Mn2+. Moreover, the complexation of two Mn2+ ions within the viral enzymes appears to be favourable, as indicated by the binding of compound 11 to TOSV Cap-ENDO (Kd = 28 ± 3 µM). Additionally, the tendency of compound 11 to stabilize His+ more than His- Cap-ENDOs suggests exploitable differences in their catalytic pockets relevant to improving specificity. Collectively, our results underscore the isoindolinone scaffold's potential as a strategic starting point for the design of pan-antibunyavirus drugs.

Clarifying the mechanism of cation exchange in Ca(II)[15-MC(Cu(II)ligand)-5] complexes.

The calcium metallacrown Ca(II)[15-MC(Cu(II)N(Trpha))-5](2+) was obtained by self-assembly of Ca(II), Cu(II), and tryptophanhydroxamic acid. Its X-ray structure shows that the core calcium ion is well-encapsulated in the five oxygen cavity of the metallacrown scaffold. The kinetics of Ca-Ln core metal substitution was studied by visible spectrophotometry by addition of Ln(III) nitrate to solutions of Ca(II)[15-MC(Cu(II)N(Trpha))-5](2+) in methanol solution at pH 6.2 (Ln(III) = La(III), Nd(III), Gd(III), Dy(III), Er(III)) to obtain the corresponding Ln(III)[15-MC(Cu(II)N(Trpha))-5](3+) complexes on the hours time scale. The reaction is first order in the two reactants (second order overall) with different rate constants across the lanthanide series. In particular, the rate for the Ca-Ln substitution decreases from La(III) to Gd(III) and then increases slightly from Gd(III) to Er(III). This substitution reaction occurs with second order rate constants ranging from 0.1543(3) M(-1) min(-1) for La(III) to 0.0720(6) M(-1) min(-1) for Gd(III). By means of the thermodynamic log K constants for the same reaction previously reported, the rate constants for the inverse Ln-Ca substitution were also determined. In this study, we demonstrated that the substitution reaction proceeds through a direct metal substitution and does not involve the disassembly of the MC scaffold. These observations in concert allow the proposition of a hypothesis that the dimension of the core metals play the major role in determining the rate constants of the substitution reaction. In particular, the largest lanthanides, which do not require complete encapsulation in the MC cavity, displace the Ca(II) ion faster, whereas in the back reaction Ca(II) displaces the smaller lanthanides faster as they interact relatively weakly with the metallacrown oxygen cavity.

Gallium(III)-pyridoxal thiosemicarbazone derivatives as nontoxic agents against Gram-negative bacteria.

Many bacterial strains are developing mechanism of resistance to antibiotics, rendering last-resort antibiotics inactive. Therefore, new drugs are needed and in particular metal-based compounds represent a valid starting point to explore new antibiotic classes. In this study, we have chosen to investigate gallium(III) complexes for their potential antimicrobial activity against different strains of Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa which have developed different type of resistance mechanism, including the expression of ß-lactamases (NDM-1, ESßL, or AmpC) or the production of biofilm. We studied a series of thiosemicarbabazones derived from pyridoxal, their related Ga(III) complexes, and the speciation in solution of the Ga(III)/ligand systems as a function of the pH. Proton dissociation constants and conditional stability constants of Ga(III) complexes were evaluated by UV/Vis spectroscopy, and the most relevant species at physiological pH were identified. The compounds are active against resistant Gram-negative strain with minimal inhibitory concentration in the µM range, while no cytotoxicity was detected in eukaryotic cells.

Copper binding agents acting as copper ionophores lead to caspase inhibition and paraptotic cell death in human cancer cells.

We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.

Toxic metal sequential sequestration in water using new amido-aminoacid ligand as a model for the interaction with polyamidoamines.

Polyamidoamines are low cost and easily synthesized materials that may find applications in cations sequestration and water treatment. In this paper a new amido-aminoacid ligand containing methionine has been designed as a monomeric model of the corresponding polyamidoamine. The amido-aminoacid ligand has been synthesized in high yield, by reacting acrylamide and methionine via aza-Michael addition in water and mild temperature conditions. The reaction has been monitored by NMR and Raman spectroscopies and the crystal structure has been determined by X-ray diffraction analysis. The coordination ability of the ligand towards Cu2+ cations in water, as well as its affinity for Ni2+ and Co2+ has been studied by potentiometric and spectrophotometric techniques. The divalent metal cations sequestration from water may occur with sequential selection by changing the pH of the solution. The copper complex with two coordinated ligands has been fully characterized in the solid state by single crystal X-ray diffraction. The results are discussed with a view to use these materials in the treatment of water contaminated by toxic transition metal ions.

The relationship between the electrospray ionization behaviour and biological activity of some phosphino Cu(I) complexes.

Electrospray ionization mass spectrometry was usefully employed for the characterization of three phosphino copper(I) complexes of medicinal interest. This technique revealed that the original [CuL(4)](+) pro-drugs (L = hydrophilic tertiary phosphine) underwent dissociation with production of coordinative unsaturated [CuL(3)](+) and [CuL(2)](+) species, which represented key intermediates for the activation of potential biological properties. The more favoured was the displacement of the ligands from the [CuL(4)](+) parent complex, the more favoured was in turn the possibility for the metal ion to directly interact with biological substrates, including pharmacological targets related to cancer proliferation. An inverse correlation between the stability and the cytotoxic activity of the three copper(I) complexes investigated has been clearly established.

Thermodynamics of self-assembly of copper(II) 15-metallacrown-5 of Eu(III) or Gd(III) with (S)-alpha-alaninehydroxamic acid in aqueous solution.

The equilibria of self-assembly of 15-metallacrown-5 (15-MC-5) complexes of Cu(2+) and (S)-alpha-alaninehydroxamic acid (alpha-Alaha, HL) with the lanthanide (Ln) ions Eu(3+) or Gd(3+) in aqueous solution are described. The binary Ln(3+)/alpha-Alaha systems were first studied by potentiometric and calorimetric in-cell titrations; the latter technique allowed us to define the most suitable speciation model. On the contrary, because the kinetics of formation of the Ln(3+) 15-MC-5 complexes is slow, their stability constants were determined by out-of-cell (batch) potentiometric titrations. Two 15-MC-5 complexes are formed with both Eu(3+) and Gd(3+), namely, {Ln[Cu(5)L(5)H(-5)]}(3+) and {Ln[Cu(5)L(5)H(-5)](OH)}(2+), with the latter being the hydroxo species of the former. The acidity of the former to give the hydroxo species is remarkably high (log K = 4.40-4.69). Moreover, our potentiometric and spectrophotometric investigations clearly indicate that the hydroxide ion is coordinated to the central Ln ion, as was reported for several 15-MC-5 in the solid state. The formation of {Ln[Cu(5)L(5)H(-5)]}(3+) starts at ca. pH 3.5, which converts at ca. pH 4.5 into the {Ln[Cu(5)L(5)H(-5)](OH)}(2+) species, which predominates up to pH 7, where a purple precipitate occurs. The coexistence of both 15-MC-5 species and the copper(II) 12-MC-4 species of alpha-Alaha ([Cu(5)L(4)H(-4)](2+)) was observed under appropriate experimental conditions (pH and ligand and metal concentrations). A complete ESI-MS investigation of the Ln(3+)/Cu(2+)/alpha-Alaha system at different pH's confirmed the formation of the two 15-MC-5 species. The 15-MC-5 stability constants were employed to quantitatively evaluate the solution behavior of Ln(III) MCs regarding their integrity, ligand substitution, and transmetalation processes. In particular, EDTA or DOTA, added in equimolar amounts, should not appreciably interfere with the MC integrity, as found in previous experimental investigations, although it is expected that at higher amounts of EDTA, the MC should be disrupted. Our results also demonstrate that an excess of alpha-aminohydroxamate does not interfere with the integrity of the MC, and the disappearance of the CD spectra upon addition of the R enantiomer to 15-MC-5 containing the S enantiomer is due to a very rapid ligand exchange with formation of all possible isomers with no selectivity. The stability of the 15-MC-5 complexes in the presence of transferrin, serum albumin, or an excess of Zn(2+) is also discussed. With regards to the latter metal ion, we found that the MCs are stable toward Gd(3+)/Zn(2+) transmetalation. although the presence of a phosphate buffer promotes the disruption of the MC scaffold by formation of stable Gd(3+)/phosphate species.