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.

Metal coordination to solute binding proteins - exciting chemistry with potential biological meaning.

Zn(II) is essential for bacterial survival and virulence. In host cells, its abundance is extremely limited, thus, bacteria have evolved transport mechanisms that enable them to take up this essential metal nutrient. Paracoccus denitrificans encodes two solute binding proteins (SBPs) - ZnuA and AztC, which are responsible for zinc acquisition from the host cells. We focus on understanding the interactions of Zn(II) and Ni(II) (zinc's potential competitor, which is a biologically relevant metal ion essential for various bacterial enzymes) with the extracellular ZnuA and AztC's loops from P. denitrificans that are expected to be possible Zn(II) binding sites. In the case of Zn(II) complexes with ZnuA outercellular loop regions, the numerous histidines act as anchoring donors, forming complexes with up to four coordinated His residues, while in the AztC region, three imidazole nitrogens and one water molecule are involved in Zn(II) binding. In Zn(II) complexes with ZnuA His-rich loop regions, so-called polymorphic binding sites are observed. The large number of available imidazoles and carboxylic side chains also strongly affects the structure of Ni(II) complexes; the more histidines in the studied peptide, the higher the affinity to bind Ni(II) and the higher the pH value at which amide nitrogens start to participate in Ni(II) binding. Additionally, for Ni(II)-ZnuA complexes, a more rare octahedral geometry is observed and such complexes are more stable than the corresponding Zn(II) ones, in contrast to what was observed in the AztC region, suggesting that the numerous histidyl and glutamic acid side chains are more tempting for Ni(II) than for Zn(II).The general strong affinity of Zn(II)-zincophore complexes is also discussed.

Impact of C- and N-terminal protection on the stability, metal chelation and antimicrobial properties of calcitermin.

The main limitation to the use of antimicrobial peptides (AMPs) as regular drugs, against antibiotic and antifungal resistance, mainly relates to their rapid degradation by proteolytic enzymes. The introduction of suitable structural changes in the peptide chain can make the peptide less susceptible to the action of proteases, thus overcoming this problem. To improve the plasma stability of calcitermin, a metal-chelating AMP present in the human respiratory tract and investigated in the present study, C- and/or N- terminal modifications have been introduced in the native sequence. Evaluation of peptide stability has been performed to determine the half-life times in human plasma of both native calcitermin and its derivatives. However, the protection of the peptide termini can also affect its metal coordination behaviour. Thus, the characterization of Zn2+ and Cu2+ complexes has been performed by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV-Vis, circular dichroism and EPR. On the basis of the obtained results, it was possible to compare the biological activity of the studied systems, taking into account both the metal-binding ability and the peptide stability to search for a link among them. A significant result of this study is that the N-terminal protection increases the calcitermin half-life over seven times and the formation of metal complexes confers resistance towards degradation almost doubling its half-life.

Metal-protein solution interactions investigated using model systems: Thermodynamic and spectroscopic methods.

The first-row D-block metal ions are essential for the physiology of living organisms, functioning as cofactors in metalloproteins or structural components for enzymes: almost half of all proteins require metals to perform the biological function. Understanding metal-protein interactions is crucial to unravel the mysteries behind molecular biology, understanding the effects of metal imbalance and toxicity or the diseases due to disorders in metal homeostasis. Metal-protein interactions are dynamic: they are noncovalent and affected by the environment to which the system is exposed. To reach a complete comprehension of the system, different conditions must be considered for the experimental investigation, in order to get information on the species distribution, the ligand coordination modes, complex stoichiometry and geometry. Thinking about the whole environment where a protein acts, investigations are often challenging, and simplifications are required to study in detail the mechanisms of metal interaction. This chapter is intended to help researchers addressing the problem of the complexity of metal-protein interactions, with particular emphasis on the use of peptides as model systems for the metal coordination site. The thermodynamic and spectroscopic methods most widely employed to investigate the interaction between metal ions and peptides in solution are here covered. These include solid-phase peptide synthesis, potentiometric titrations, calorimetry, electrospray ionization mass spectrometry, UV-Vis spectrophotometry, circular dichroism (CD), nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Additional experimental methods, which can be employed to study metal complexes with peptides, are also briefly mentioned. A case-study is finally reported providing a practical example of the investigation of metal-protein interaction by means of thermodynamic and spectroscopic methods applied to peptide model systems.

Investigation of metal interactions with YrpE protein of Bacillus subtilis by a polyhistidine peptide model.

The use of model peptides that can simulate the behaviour of a protein domain is a very successful analytical method to study the metal coordination sites in biological systems. Here we study zinc and copper binding ability of the sequence HTHEHSHDHSHAH, which serves as model for the metal interactions with YrpE, a putative metal-binding protein of the ZinT family identified in Bacillus subtilis. Compared to other ZinT proteins secreted by Gram-negative bacteria, the metal-coordination properties of YrpE N-terminal histidine-rich domain have not been yet characterized. Different independent analytical methods, aimed at providing information on the stability and structure of the formed species, have been employed, including potentiometric titrations, electrospray ionization mass spectrometry, UV-Vis spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopy. The obtained speciation models and equilibrium constants allowed to compare the metal-binding ability of the investigated polyhistidine sequence with that of other well-known histidine-rich peptides. Our thermodynamic results revealed that the YrpE domain HTHEHSHDHSHAH forms more stable metal complexes than other His-rich domains of similar ZinT proteins. Moreover, the studied peptide, containing the alternated (-XH-)n motif, proved to be even more effective than the His6-tag (widely used in immobilized metal ion affinity chromatography) in binding zinc ions.

Calcitermin-Loaded Smart Gels Activity against Candida albicans: A Preliminary In Vitro Study.

Calcitermin is an antimicrobial peptide of 15 amino acids found in human nasal fluid characterized by antifungal and antibacterial properties. Candida albicans is the most common human fungal pathogen affecting many tissues, such as vaginal mucosa. In this study a formulation suitable for calcitermin administration on vaginal mucosa was developed for the treatment of fungal infections. To favor topical application, mucosal adhesion, and permanence, gels based on poloxamer 407 and xanthan gum were designed and compared with regard to their rheological behavior, erosion, and leakage. The selected gel was loaded with calcitermin, whose release kinetic was evaluated in vitro by Franz cells. An antifungal activity assay was conducted to assess the calcitermin anticandidal potential and the effect of its inclusion in the selected gel. The rheological study revealed the elastic and viscous moduli behavior as a function of poloxamer 407 and xanthan gum concentration. Xanthan gum presence decreased the transition temperature of the gel, while prolonging its erosion and leakage. Particularly, poloxamer 407, 18% and xanthan gum 0.4% were chosen. The calcitermin loading in the selected gel resulted in a transparent and homogeneous formulation and in a 4-fold decrease of the release rate with respect to the calcitermin solution, as evidenced by Franz cell study. The anticandidal activity tests demonstrated that calcitermin-loaded gel was more active against Candida albicans with respect to the peptide solution.

Lights and Shadows on the Therapeutic Use of Antimicrobial Peptides.

The emergence of antimicrobial-resistant infections is still a major concern for public health worldwide. The number of pathogenic microorganisms capable of resisting common therapeutic treatments are constantly increasing, highlighting the need of innovative and more effective drugs. This phenomenon is strictly connected to the rapid metabolism of microorganisms: due to the huge number of mutations that can occur in a relatively short time, a colony can "adapt" to the pharmacological treatment with the evolution of new resistant species. However, the shortage of available antimicrobial drugs in clinical use is also caused by the high costs involved in developing and marketing new drugs without an adequate guarantee of an economic return; therefore, the pharmaceutical companies have reduced their investments in this area. The use of antimicrobial peptides (AMPs) represents a promising strategy for the design of new therapeutic agents. AMPs act as immune defense mediators of the host organism and show a poor ability to induce antimicrobial resistance, coupled with other advantages such as a broad spectrum of activity, not excessive synthetic costs and low toxicity of both the peptide itself and its own metabolites. It is also important to underline that many antimicrobial peptides, due to their inclination to attack cell membranes, have additional biological activities, such as, for example, as anti-cancer drugs. Unfortunately, they usually undergo rapid degradation by proteolytic enzymes and are characterized by poor bioavailability, preventing their extensive clinical use and landing on the pharmaceutical market. This review is focused on the strength and weak points of antimicrobial peptides as therapeutic agents. We give an overview on the AMPs already employed in clinical practice, which are examples of successful strategies aimed at overcoming the main drawbacks of peptide-based drugs. The review deepens the most promising strategies to design modified antimicrobial peptides with higher proteolytic stability with the purpose of giving a comprehensive summary of the commonly employed approaches to evaluate and optimize the peptide potentialities.

Correction: Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica.

Correction for 'Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica' by Denise Bellotti et al., Dalton Trans., 2020, 49, 9393-9403, DOI: 10.1039/D0DT01626H.

Zn2+ and Cu2+ Binding to the Extramembrane Loop of Zrt2, a Zinc Transporter of Candida albicans.

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126-215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144-153), HxxHxxEHxD (181-193) and the Glu- and Asp- rich sequence DDEEEDxE (161-168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.

Thermodynamic Stability and Speciation of Ga(III) and Zr(IV) Complexes with High-Denticity Hydroxamate Chelators.

Increasing attention has been recently devoted to 89Zr(IV) and 68Ga(III) radionuclides, due to their favorable decay characteristics for positron emission tomography (PET). In the present paper, a deep investigation is presented on Ga(III) and Zr(IV) complexes with a series of tri-(H3L1, H3L3, H3L4 and desferrioxamine E, DFOE) and tetrahydroxamate (H4L2) ligands. Herein, we describe the rational design and synthesis of two cyclic complexing agents (H3L1 and H4L2) bearing three and four hydroxamate chelating groups, respectively. The ligand structures allow us to take advantage of the macrocyclic effect; the H4L2 chelator contains an additional side amino group available for a possible further conjugation with a biomolecule. The thermodynamic stability of Ga(III) and Zr(IV) complexes in solution has been measured using a combination of potentiometric and pH-dependent UV-vis titrations, on the basis of metal-metal competition. The Zr(IV)-H4L2 complex is characterized by one of the highest formation constants reported to date for a tetrahydroxamate zirconium chelate (log ß = 45.9, pZr = 37.0), although the complex-stability increase derived from the introduction of the fourth hydroxamate binding unit is lower than that predicted by theoretical calculations. Solution studies on Ga(III) complexes revealed that H3L1 and H4L2 are stronger chelators in comparison to DFOB. The complex stability obtained with the new ligands is also compared with that previously reported for other hydroxamate ligands. In addition to increasing the library of the thermodynamic stability data of Ga(III) and Zr(IV) complexes, the present work allows new insights into Ga(III) and Zr(IV) coordination chemistry and thermodynamics and broadens the selection of available chelators for 68Ga(III) and 89Zr(IV).

Deferoxamine B: A Natural, Excellent and Versatile Metal Chelator.

Deferoxamine B is an outstanding molecule which has been widely studied in the past decade for its ability to bind iron and many other metal ions. The versatility of this metal chelator makes it suitable for a number of medicinal and analytical applications, from the well-known iron chelation therapy to the most recent use in sensor devices. The three bidentate hydroxamic functional groups of deferoxamine B are the centerpiece of its metal binding ability, which allows the formation of stable complexes with many transition, lanthanoid and actinoid metal ions. In addition to the ferric ion, in fact, more than 20 different metal complexes of deferoxamine b have been characterized in terms of their chemical speciation in solution. In addition, the availability of a terminal amino group, most often not involved in complexation, opens the way to deferoxamine B modification and functionalization. This review aims to collect and summarize the available data concerning the complex-formation equilibria in solutions of deferoxamine B with different metal ions. A general overview of the progress of its applications over the past decade is also discussed, including the treatment of iron overload-associated diseases, its clinical use against cancer and neurodegenerative disorders and its role as a diagnostic tool.

How Zinc-Binding Systems, Expressed by Human Pathogens, Acquire Zinc from the Colonized Host Environment: A Critical Review on Zincophores.

Some transition metals, like manganese, iron, cobalt, nickel, copper and zinc, required for the biosynthesis of metalloenzymes and metalloproteins, are essential micronutrients for the growth and development of pathogenic microorganisms. Among the defenses put in place by the host organism, the so-called "nutritional immunity" consists of reducing the availability of micronutrients and thus "starving" the pathogen. In the case of metals, microorganisms can fight the nutritional immunity in different ways, i.e. by directly recruiting the metal ion or capturing an extracellular metalloprotein or also through the synthesis of specific metallophores which allow importing the metal in the form of a chelate complex. The best known and most studied metallophores are those directed to iron (siderophores), but analogous chelators are also expressed by microorganisms to capture other metals, such as zinc. An efficient zinc recruitment can also be achieved by means of specialized zinc-binding proteins. A deep knowledge of the properties, structure and action mechanisms of extracytoplasmic zinc chelators can be a powerful tool to find out new therapeutic strategies against the antibiotic and/or antifungal resistance. This review aims to collect the knowledge concerning zincophores (small molecules and proteins in charge of zinc acquisition) expressed by bacterial or fungal microorganisms that are pathogenic for the human body.

The N-terminal domain of Helicobacter pylori's Hpn protein: The role of multiple histidine residues.

Helicobacter pylori is a gram-negative bacterium with gastric localization that can cause many gastrointestinal disorders. Its survival in the host environment strictly requires an efficient regulation of its metal homeostasis, in particular of Ni(II) ions, crucial for the synthesis of some essential enzymes. Hpn is a protein of 60 amino acids, 47% of which are histidines, expressed by H. pylori and avid for nickel, characterized by the presence of an ATCUN (Amino Terminal Cu(II)- and Ni(II)-binding) motif and by two further histidine residues which can act as additional metal anchoring sites. We decided to deepen the following aspects: (i) understanding the role of each histidine in the coordination of metal ions; (ii) comparing the binding affinities for Cu(II), Ni(II) and Zn(II) ions, which are potentially competing metals in vivo; (iii) understanding the Hpn ability of forming ternary and poly-nuclear complexes. For these purposes, we synthesized the Hpn N-terminal "wild-type" sequence (MAHHEEQHG-Am) and the following peptide analogues: MAAHEEQHG-Am, MAHAEEQHG-Am, MAHHEEQAG-Am and MAHAEEQAG-Am. Our results highlight that the histidines in position 4 and 8 lead to the formation of Cu(II) binuclear complexes. The ATCUN motif is by far the most efficient binding site for Cu(II) and Ni(II), while macrochelate Zn(II) complexes are formed thanks to the presence of several suitable anchoring sites (His and Glu). The metal binding affinities follow the order Zn(II) < Ni(II) < < Cu(II). In solutions containing equimolar amount of wild-type ligand, Cu(II) and Ni(II), the major species above pH 5.5 are hetero-binuclear complexes.

Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica.

The ZinT mediated Zn(ii) uptake is one of the major differences in the metabolism of human and bacterial cells that can be challenged when looking for possible highly selective metal-based therapeutics. ZinT is a 216-amino acid periplasmic protein expressed by Gram-negative bacteria, which shuttles Zn(ii) ions to the ZnuABC transporter under zinc-limiting conditions. The suggested metal-binding sites of ZinT correspond to a domain containing three highly conserved histidine residues (His 167, 176 and 178) and to the N-terminal histidine-rich loop HGHHXH (residues 24-29). The coordination chemistry of the ZinT complexes with Zn(ii) and Cu(ii) has been investigated. The present work is focused on the protected peptides Ac-24HGHHSH29-NH2 and Ac-166DHIIAPRKSSHFH178-NH2 as models for the putative metal binding sites of ZinT from Escherichia coli (EcZinT), and Ac-24HGHHAH29-NH2 and Ac-166DHIIAPRKSAHFH178-NH2 from the ZinT protein expressed by Salmonella enterica sv. Typhimurium (SeZinT). The investigated peptides are able to form stable mono-nuclear complexes where the histidine residues represent the principal metal anchoring sites. The ZnuA (a periplasmic component of the ZnuABC transporter) metal binding site exhibits higher affinity for Zn(ii) than ZinT, suggesting that the interaction of the two proteins through the formation of a binary complex may involve the metal transfer from ZinT to ZnuA. In contrast, this would not occur in Cu(ii), since the ZinT complexes are more stable. Furthermore, at acidic pH, where the antimicrobial peptide calcitermin is biologically active, it also binds the metal ions with higher affinity than ZinT, representing a possible efficient competitor and antagonist of ZinT in the host human organism.

Cu(II) coordination to His-containing linear peptides and related branched ones: Equalities and diversities.

The two branched peptides (AAHAWG)4-PWT2 and (HAWG)4-PWT2 where synthesized by mounting linear peptides on a cyclam-based scaffold (PWT2), provided with four maleimide chains, through a thio-Michael reaction. The purpose of this study was primarily to verify if the two branched ligands had a Cu(II) coordination behavior reproducing that of the single-chain peptides, namely AAHAWG-NH2, which bears an Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) Motif, and HAWG-NH2, which presents a His residue as the N-terminal amino acid, in a wide pH range. The study of Cu(II) binding was performed by potentiometric, spectroscopic (UV-vis absorption, CD, fluorescence) and ESI-MS techniques. ATCUN-type ligands ((AAHAWG)4-PWT2 and AAHAWG-NH2) were confirmed to bind one Cu(II) per peptide fragment at both pH 7.4 and pH 9.0, with a [NH2, 2N-, NIm] coordination mode. On the other hand, the ligand HAWG-NH2 forms a [CuL2]2+ species at neutral pH, while, at pH 9, the formation of 1:2 Cu(II):ligand adducts is prevented by amidic nitrogen deprotonation and coordination, to give rise solely to 1:1 species. Conversely, Cu(II) binding to (HAWG)4-PWT2 resulted in the formation of 1:2 copper:peptide chain also at pH 9: hence, through the latter branched peptide we obtained, at alkaline pH, the stabilization of a specific Cu(II) coordination mode which results unachievable using the corresponding single-chain peptide. This behavior could be explained in terms of high local peptide concentration on the basis of the speciation of the Cu(II)/single-chain peptide systems.

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.

Thermodynamic and spectroscopic study of Cu(ii) and Zn(ii) complexes with the (148-156) peptide fragment of C4YJH2, a putative metal transporter of Candida albicans.

Candida albicans is a widespread human pathogen which can infect humans at different levels. Like the majority of microorganisms, it needs transition metals as micronutrients for its subsistence. In order to acquire these nutrients from the host, C. albicans employs various strategies, also involving chelating proteins specifically expressed to sequester metals from the environment. A histidine-rich protein sequence identified in the C. albicans genome, named C4YJH2, has been recently studied for its putative role in Zn(ii) transport. Two outer membrane major histidine-rich clusters of C4YJH2, namely the domains 131-148 (FHEHGHSHSHGSGGGGGG) and 157-165 (SHSHSHSHS), have been confirmed as strong binding sites for the Cu(ii) and Zn(ii) ions. Nevertheless, the 9-residue "linker" sequence 148-156 (GSDHSGDSK) between the two His-rich fragments of C4YJH2, containing an additional His residue, can also contribute to metal binding. In the present work, the protected peptide Ac-GSDHSGDSK-NH2 and some analogues (Ac-GSDHSGASK-NH2, Ac-GADHAGDAK-NH2, Ac-GSDH-NH2, and Ac-HSGD-NH2) have been synthesized and their metal binding properties have been studied in detail. The thermodynamics of complex-formation equilibria of the above reported ligands with Cu(ii) and Zn(ii) ions have been studied by potentiometry in a wide pH range and the stoichiometry of the formed species has been confirmed by mass spectrometry; the most likely solution structures of the metal complexes are also discussed on the basis of NMR, UV-vis, circular dichroism (CD) and EPR data. The results show the importance of Asp7 in the stabilization of Zn(ii) complexes and suggest a significant role of the (quite abundant) Ser residues in the task of metal uptake and regulation.

Bioinorganic chemistry of calcitermin - the picklock of its antimicrobial activity.

Calcitermin, an antimicrobial peptide from the fluid of the human airways, is a well-conserved, 15 amino acid C-terminal cleavage fragment of calgranulin C (VAIALKAAHYHTHKE), which is active under acidic pH conditions (pH 5.4). In an attempt to understand the impact of the coordination of Zn(ii) and Cu(ii) on the biological activity of calcitermin, we mutated each of the histidines with an alanine and studied the thermodynamics, binding mode and antimicrobial activity of wild type calcitermin and its H9A, H11A and H13A mutants and their Zn(ii) and Cu(ii) complexes. Both metals strongly enhance the antimicrobial activity of calcitermin-like peptides, although the link between the minimal inhibitory concentration (MIC) values and the stability, charge or structure of the complexes is not so obvious. As expected, the increase in the number of histidines makes the coordination of both metals more effective. There is no preferred Cu(ii) binding site in calcitermin: the stabilities of the Cu(ii)-H9A and Cu(ii)-H13A complexes are almost identical, while the Cu(ii)-H11A complex (in which two histidines are separated by three amino acids and only one His residue is involved in binding) is less stable. On the other hand, the higher stability of the Zn(ii)-H13A complex with respect to those formed by H9A and H11A suggests a pivotal role of His9 and His11 in Zn(ii) complexation. Impressive MIC breakpoints were obtained, similar and lower than those for commonly used antimicrobial agents that treat Candida albicans (Zn(ii) and Cu(ii) complexes of WT calcitermin and H9A, as well as H9A alone), Enterococcus faecalis (H11A, H13A and their metal complexes) and Staphylococcus aureus (H13A and its complexes).

Chiral Ligand-Exchange Chromatography of Pharmaceutical Compounds on Dynamically Coated (Home-Made) Stationary Phases.

It is well known for several decades that the two enantiomeric forms of a chiral compound can have very different effects on the human body. For this reason the synthesis or extraction from a natural source of a potential new drug, as well as its marketing, require a careful control of its optical purity. Chromatographic techniques can respond extremely well to this need, both in the analytical and in the preparative field. Among the several methods developed for this purpose, one of the first and of the most effective is the Chiral Ligand-Exchange Chromatography, which is based on the stability difference between the metallic diastereomeric complexes containing one or the other of the two enantiomers to be separated and a suitable chiral selector. This method has been effectively used for resolving racemic mixtures of products of biomedical and/or pharmacological interest, such as α - and ß-amino acids either proteinogenic or non-proteinogenic, oligopeptides, amino alcohols or ß-blockers. All these substances are linked together by their ability to bind metal ions, the most widely used of which is Cu(II). The chiral selector can be a component of either the mobile or the stationary phase, to which it can be either chemically bonded or dynamically adsorbed. The latter method has several advantages of convenience and, above all, cheapness. The preparation of dynamically-coated chiral stationary phases for Ligand-Exchange Chromatography has produced a large number of applications, the main of which, both in TLC and in HPLC, are reviewed below.

DOES hemopressin bind metal ions in vivo?

Hemopressin is a neuropeptide, derived from the degradation of the α(1)-chain of hemoglobin, and possesses several pharmacologic properties, such as the ability to block cannabinoid CB1 receptor activity, to cause dose-dependent hypotension and to inhibit food intake. Actually, human hemopressin (PVNFKLLSH) is only the precursor of a class of longer peptides, called "Pepcans", which bear additional residues at their amino-terminus and possess slightly different chemical and biological properties with respect to hemopressin. The presence of a histidyl residue and the free terminal amine imparts to hemopressin and its derivatives good binding properties towards transition metal ions. In this paper, we present a wide investigation on the complex-formation equilibria of human hemopressin and three analogues towards the Cu(ii) and Ni(ii) ions. The study showed that the main coordination site is always the amino terminus (if not protected), while the C-terminal histidine acts only as an anchoring site for the metal ions at acidic pH, with the formation of a macrochelate complex. The presence of additional residues in N-terminal position produces significant differences in the protonation and complex-formation behaviors of these peptides, which can be explained in terms of charge of the ligand and coordination environment. Although the participation of metal ions in the biological activity of hemopressin and Pepcans has not yet been demonstrated, the data reported here can help to shed light on the mechanisms governing the action of these neuropeptides in vivo.