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Organic-inorganic (O-I) nanomaterials are versatile platforms for an incredible high number of applications, ranging from heterogeneous catalysis, molecular sensing, cell targeting, imaging, cancer diagnosis and therapy, just to name a few. Much of their potential stems from the unique control of organic environments around inorganic sites within a single O-I nanomaterial, which allows for new properties inaccessible using purely organic or inorganic materials. Structural and mechanistic characterization plays a key role in understanding and rationally designing such hybrid nanoconstructs. Here, we introduce a general methodology to identify and classify local (supra)molecular environments in an archetypal class of O-I nanomaterials, i.e. self-assembled monolayer-protected gold nanoparticles (SAM-AuNPs). By using an atomistic machine-learning guided workflow based on the Smooth Overlap of Atomic Positions (SOAP) descriptor, we analyze a collection of chemically different SAM-AuNPs, and detect and compare local environments in a way that is agnostic and automated, i.e. with no need of a-priori information and minimal user intervention. In addition, the computational results coupled with experimental electron spin resonance measurements prove that is possible to have more than one local environment inside SAMs, being thickness of the organic shell and solvation primary factors in determining number and nature of multiple co-existing environments. These indications are extended to complex mixed hydrophilic-hydrophobic SAMs. This work demonstrates that it is possible to spot out and compare local molecular environments in SAM-AuNPs exploiting atomistic machine-learning approaches, establishes ground rules to control them, and holds the potential for rational design of O-I nanomaterials instructed from data.

The highly basic and poorly nucleophilic phosphazene base P-1-t-Bu promotes the Darzens condensation of alpha-halo esters with aromatic aldehydes affording alpha,beta-epoxy esters in nearly quantitative yields under mild conditions and in short reaction times. The more basic P-4-t-Bu phosphazene was found useful with low reactivity aldehydes. These reactions can be performed in aprotic organic solvents of low polarity, thus minimizing the hydrolysis of alpha,beta-epoxy esters which often accompanies the base-promoted Darzens condensations.

The ability to control the properties of monolayer protected gold nanoparticles (MPNPs) discloses unrevealed features stemming from collective properties of the ligands forming the monolayer and presents opportunities to design new materials. To date, the influence of ligand end-group size and capacity to form hydrogen bonds on structure and hydration of small MPNPs (

Herein, we report a facile and flexible synthesis of porous and highly faceted platinum nanoparticles (NPs) performed in the liquid phase. The synthesis is performed by reduction of platinum 2,4-pentantedionate in the presence of oleylamine and oleic acid in dibenzyl ether at 200 °C. The growth process was monitored by time-course transmission electron microscopy (TEM), revealing a peculiar progressive evolution that, in comparison with previous methodologies, is quite unusual. In fact, the morphology evolves first through nanocubes, nanostars, and dendrites to arrive at porous multifaceted NPs. This offers the possibility to selectively obtain materials with different degrees of complexity at a different time of reaction with one synthetic approach. Moreover, fine tuning of the reaction conditions was achieved by assessing, in dedicated experiments, the effects of temperature, surfactant concentration, and surfactant ratio, allowing control on NPs' dispersity and shape reproducibility. The dimensionally monodispersed NPs have a mean diameter of 52 ± 2 nm and display small regular crystallites with uniform facets exposed on the surface as evinced by high-resolution-TEM analysis. The as-prepared multifaceted platinum NPs were tested for oxygen reduction and methanol oxidation reactions exhibiting improved catalytic activity with respect to conventional Pt-based nanomaterials.

Unprotected dipeptides are attractive building blocks for environmentally friendly hydrogel biomaterials by virtue of their low-cost and ease of preparation. This work investigates the self-assembling behaviour of the distinct stereoisomers of Ile-Phe and Phe-Ile in phosphate buffered saline (PBS) to form hydrogels, using transmission electron microscopy (TEM), attenuated total reflectance infrared spectroscopy (ATR-IR), circular dichroism (CD), and oscillatory rheometry. Each peptide purity and identity was also confirmed by 1 H- and 13 C-NMR spectroscopy and HPLC-MS. Finally, single-crystal XRD data allowed the key interactions responsible for the supramolecular packing into amphipathic layers or water-channels to be revealed. The presence of the latter in the crystal structure is a distinctive feature of the only gelator of this work that self-organizes into stable hydrogels, with fast kinetics and the highest elastic modulus amongst its structural isomers and stereoisomers.

Monoclonal antibody-based immunotherapy is one of the pillars of cancer treatment. However, for an efficient and personalized approach to the therapy, a quantitative evaluation of the right dose for each patient is required. In this study, we developed a simple, label-free, and rapid approach to quantify Trastuzumab, a humanized IgG1 monoclonal antibody used against human epidermal growth factor receptor 2 (HER2), overexpressed in breast cancer patients, based on localized surface plasmon resonance (LSPR). The central idea of this work was to use gold nanoparticles (AuNPs) as plasmonic scaffolds, decorated with HER2 binders mixed with oligo-ethylene glycol (OEG) molecules, to tune the surface density of the attached macromolecules and to minimize nonspecific binding events. Specifically, we characterized and optimized a self-assembled monolayer of mixed alkylthiols terminated with nitrilotriacetic acid (NTA), and OEG3 as a spacing ligand to achieve both excellent dispersibility and high reliability in protein immobilization. The successful immobilization of histidine-tagged HER2 (His-tagged HER2) on NTA via cobalt (II) chelates was demonstrated, confirming the fully functional attachment of the proteins to the AuNP surface. The proposed design demonstrates the capability of producing a clear readout that enables the transduction of a Trastuzumab/HER2 binding event into optical signals based on the wavelength shifts in LSPR, which allowed for detecting clinically relevant concentrations of Trastuzumab down to 300 ng/mL in the buffer and 2 µg/mL in the diluted serum. This strategy was found to be fast and highly specific to Trastuzumab. These findings make the present platform an auspicious tool for developing affordable bio-nanosensors.

Natural and synthetic anionophores promote the trans-membrane transport of anions such as chloride and bicarbonate. This process may alter cellular homeostasis with possible effects on internal ions concentration and pH levels triggering several and diverse biological effects. In this article, an overview of the recent results on the study of aniontransporters, mainly acting with a carrier-type mechanism, is given with emphasis on the structure/activity relationship and on their biological activity as antibiotic and anticancer agents and in the development of new drugs for treating conditions derived from dysregulation of natural anion channels.

Mixtures of fluorinated and hydrogenated compounds display peculiar properties arising from their mutual phobicity and the same applies to semifluorinated species in which a reciprocal phobicity pattern exists within the same molecule. The interest in assemblies comprising these species stems from the ease in which self-sorting can take place, allowing the preparation of compartmentalised molecular aggregates with unique hydrophobic patterns or surface features. Most importantly, this is the result of molecular properties rather than specifically designed fabrication techniques. This brief overview describes some examples that are instrumental to present the features of fluorinated/hydrogenated supramolecular assemblies in which self-sorting of the dislike units takes place at different length scales. This review is focussed on flat assemblies such as self-assembled monolayers on gold surfaces, Langmuir Blodgett films and on three dimensional assemblies such as micelles, vesicles and nanoparticles.

Two structurally different water-soluble homoligand gold nanoparticle systems, one featuring a rigid fluorous monolayer in the proximity of the gold core and the other featuring a flexible fluorinated region in a distal position, were studied as putative hosting systems by determining their binding constants for a series of fluorinated and non-fluorinated radical probes by means of ESR spectroscopy. Comparison of the binding constants obtained with hydrogenated homoligand nanoparticles of similar structure used as the reference evidenced that the binding of both hydrogenated and fluorinated guests is favoured in the presence of fluorinated nanoparticles. In addition, a flexible fluorinated monolayer acts as a better hosting system than the more rigid counterpart. In the latter case decreasing the size of the nanoparticles causes a small decrease of the binding affinities for both hydrogenated and fluorinated guests. The same nanoparticle systems were analysed for their ability to retard the phase transfer of a fluorescent dye from an aqueous solution to a toluene layer. All of the nanoparticles studied produced a significant decrease of the phase transfer rate of the dye because of the efficient interaction with the monolayer. These data support the introduction of fluorinated moieties in the monolayer of gold nanoparticles as a novel design tool for the development of drug delivery systems.

Low intrinsic toxicity, high solubility, and stability are important and necessary features of gold nanoparticles to be used in the biomedical field. In this context, charged nanoparticles proved to be very versatile, and among them charged mixed-monolayer gold nanoparticles, displaying monolayers with well-defined morphologies, represent a paradigm. By using mixtures of hydrogenated and fluorinated thiols, the formation of monolayer domains may be brought to an extreme because of the immiscibility of fluorinated and hydrogenated chains. Following this rationale, mixed monolayer gold nanoparticles featuring ammonium, sulfonate, or carboxylic groups on their surface were prepared by using amphiphilic hydrogenated thiols and 1H,1H,2H,2H-perfluoro-alkanethiols. The toxicity of these systems was assessed in HeLa cells and was found to be, in general, low even for the cationic nanoparticles which usually show a high cytotoxicity and is comparable to that of homoligand gold nanoparticles displaying amphiphilic-charge neutral-hydrogenated or fluorinated thiolates in their monolayer. These properties make the mixed ligand monolayer gold nanoparticles an interesting new candidate for medical application.

Gold nanoparticles carrying fluorinated ligands in their monolayer are, by themselves, contrast agents for 19F magnetic resonance imaging displaying high sensitivity because of the high density of fluorine nuclei achievable by grafting suitable ligands on the gold core surface. Functionalization of these nanoparticles with Gd(III) chelates allows adding a further functional activity to these systems, developing materials also acting as contrast agents for proton magnetic resonance imaging. These dual mode contrast agents may allow capitalizing on the benefits of 1H and 19F magnetic resonance imaging in a single diagnostic session. In this work, we describe a proof of principle of this approach by studying these nanoparticles in a high field preclinical scanner. The Gd(III) centers within the nanoparticles monolayer shorten considerably the 19F T1 of the ligands but, nevertheless, these systems display strong and sharp NMR signals which allow recording good quality 19F MRI phantom images at nanoparticle concentration of 20 mg/mL after proper adjustment of the imaging sequence. The Gd(III) centers also influence the T1 relaxation time of the water protons and high quality 1H MRI images could be obtained. Gold nanoparticles protected by hydrogenated ligands and decorated with Gd(III) chelates are reported for comparison as 1H MRI contrast agents.

Gold nanoparticles (AuNPs) covered with mixtures of immiscible ligands present potentially anisotropic surfaces that can modulate their interactions at complex nano–bio interfaces. Mixed, self-assembled, monolayer (SAM)-protected AuNPs, prepared with incompatible hydrocarbon and fluorocarbon amphiphilic ligands, are used here to probe the molecular basis of surface phase separation and disclose the role of fluorinated ligands on the interaction with lipid model membranes and cells, by integrating in silico and experimental approaches. These results indicate that the presence of fluorinated amphiphilic ligands enhances the membrane binding ability and cellular uptake of gold nanoparticles with respect to those coated only with hydrogenated amphiphilic ligands. For mixed monolayers, computational results suggest that ligand phase separation occurs on the gold surface, and the resulting anisotropy affects the number of contacts and adhesion energies with a membrane bilayer. This reflects in a diverse membrane interaction for NPs with different surface morphologies, as determined by surface plasmon resonance, as well as differential effects on cells, as observed by flow cytometry and confocal microscopy. Overall, limited changes in monolayer features can significantly affect NP surface interfacial properties, which, in turn, affect the interaction of SAM-AuNPs with cellular membranes and subsequent effects on cells.

Harnessing the reciprocal phobicity of hydrogenated and fluorinated thiolates proved to be a valuable strategy in preparing gold nanoparticles displaying mixed monolayers with a well-defined and pre-determined morphology. Our studies display that the organisation of the fluorinated ligands in phase-separated domains takes place even when these represent a small fraction of the ligands grafted on the gold surface. Using simple model ligands and by combining 19F NMR or ESR spectroscopies, and multiscale molecular simulations, we could demonstrate how the monolayer morphology responds in a predictable manner to structural differences between the thiolates. This enables a straightforward preparation of gold nanoparticles with monolayers displaying stripe-like, Janus, patchy, and random morphologies. Additionally, solubility properties may be tuned as function of the nature of the ligands and of the monolayer morphology obtaining gold nanoparticles soluble in organic solvents or in aqueous solutions. Most importantly, this rich diversity can be achieved not by resorting to ad hoc developed fabrication techniques, but rather relying on the spontaneous self-sorting of the ligands upon assembly on the nanoparticle surface. Besides enabling control over the monolayer morphology, fluorinated ligands endow the nanoparticles with several properties that can be exploited in the development of novel materials with applications, for instance in drug delivery and diagnostic imaging.

The development of fluorinated gold nanoparticles is presently arising and increasing attention across several fields of nanotechnology. The synthetic approaches evolved over time from the use of almost perfluorinated alkanethiols and perfluorinated arylthiols to amphiphilic fluorinated thiols capable of ensuring solubility in conventional organic solvents and water. The interest in these systems stems from the unique properties of both nanosized and fluorous compounds. In perspective, the development of our understanding of the fluorophilic interactions at the nanoscale will allow to devise novel strategies for self-assembly, molecular recognition and new materials for biomedical applications. In this paper we present, through selected examples, the potential of fluorous ligands in the synthesis of gold nanoparticles and the relevance of mastering the properties of these systems in the development of new materials.

The kinetic analysis of the place exchange reaction on the neutral Au25(SCH2CH2Ph)18 cluster by using 4-fluorobenzylthiol and a series of substituted arylthiols allowed us to establish, for the first time, that the selectivity for the inner and outer positions of the dimeric staples of the cluster can be modulated by using incoming thiols with different structures.

Molecular self-assembly is a topic attracting intense scientific interest. Various strategies have been developed for construction of molecular aggregates with rationally designed properties, geometries, and dimensions that promise to provide solutions to both theoretical and practical problems in areas such as drug delivery, medical diagnostics, and biosensors, to name but a few. In this respect, gold nanoparticles covered with self-assembled monolayers presenting nanoscale surface patterns—typically patched, striped or Janus-like domains—represent an emerging field. These systems are particularly intriguing for use in bio-nanotechnology applications, as presence of such monolayers with three-dimensional (3D) morphology provides nanoparticles with surface-dependent properties that, in turn, affect their biological behavior. Comprehensive understanding of the physicochemical interactions occurring at the interface between these versatile nanomaterials and biological systems is therefore crucial to fully exploit their potential. This review aims to explore the current state of development of such patterned, self-assembled monolayer-protected gold nanoparticles, through step-by-step analysis of their conceptual design, synthetic procedures, predicted and determined surface characteristics, interactions with and performance in biological environments, and experimental and computational methods currently employed for their investigation.

The spontaneous self-organization of dissimilar ligands on the surface of metal nanoparticles is a very appealing approach to obtain anisotropic "spherical" systems. In addition to differences in ligand length and end groups, a further thermodynamic driving force to control the self-assembled monolayer organization may become available if the ligands are inherently immiscible, as is the case of hydrogenated (H-) and fluorinated (F-) species. Here, we validate the viability of this approach by combining (19)F NMR experiments and multiscale molecular simulations on large sets of mixed-monolayer-protected gold nanoparticles (NPs). The phase segregation of blends of F- and H-thiolates grafted on the surface of gold NPs allows a straightforward approach to patterned mixed monolayers, with the shapes of the monolayer domains being encoded in the structure of the F/H-thiolate ligands. The results obtained from this comprehensive study offer molecular design rules to achieve a precise control of inorganic nanoparticles protected by specifically patterned monolayers.

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31–PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC50 of 14.6 ± 3.0 nM (oseltamivir 25 ± 4 nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC50 of 0.1 ± 0.03 nM (oseltamivir 0.2 ± 0.02 nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure–activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.

A novel dendrimeric peptide ligand dubbed D-PAM-Φ was designed to achieve a high capacity for human IgG through the decoration of the D-PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D-PAM structure were supported by the recently published solid-state structure of D-PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl-D-PAM (D-PAM-Φ). D-PAM-Φ was immobilised on an activated solid support and compared with the parent D-PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10-fold enhancement with respect to the D-PAM-functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory-scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D-PAM-Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy.

Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors.Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay.Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor.The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments.The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.

formed by immunoglobulins of the M class (IgM) and tumor-associated antigens was recently found in sera of individuals affected by neoplastic diseases such as hepatocellular carcinoma 1-3 and colorectal 4 and prostate cancer 5 in their early stages. The quantitation of these tumor marker–IgM immune complexes by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) was reported to be a useful approach for cancer early diagnosis. 6 As all immunometric methodologies, the obtainment of quantitative data by ELISA relies solely on the availability of suitable standards represented by samples of the analytes of known concentration. In principle, these reference calibrators could be of either human or synthetic origin; however, the very nature of immune complexes as species of ill-defined stoichiometry hinders any rational approach to the synthesis of artificial analogs. Unfortunately, this limits the preparation of reference calibrators to the processing of material of human origin, posing serious industrial limitations because the extraction of proteins from biological samples of human origin always requires strict care and costly procedures because of possible causes of infection. The development of effective processes for the profitable large-scale preparation of synthetic substitutes of the immune complexes to be used for calibration purposes in ELISA procedures represents an urgent industrial need. Such a methodology could allow significant cost reductions and enhance the safety standards associated with the production of diagnostic tools aimed to detect the onset of neoplastic diseases. Here we report a cost-effective semisynthetic methodology circumventing the synthetic issues correlated to the limited knowledge of the immune complexes’ structure. The method exploits a combinatorial bioconjugation approach, allowing the tethering of tumor antigens to human naive IgM after a quick and reproducible screening of a large number of reaction conditions. The conjugation chemistry exploited is based on the biotin-avidin technology because of the mild conditions it requires, although giving fast reactions and quantitative yields. 7-11 The selection of the best synthetic conditions is headed by a maximization process considering the immunoreactivity and solubility of the constructs. This combinatorial method represents a novel approach to the synthesis of protein conjugates, preserving the immunoreactivity of each partner involved. This issue is not a primary concern in the usual methodologies exploited for the protein-protein conjugation, 12,13 and to the best of our

Background: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation. Methods: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody. Results: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p2000 kDa) with both tests. Conclusions: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis. Clin Chem Lab Med 2010;48:217–23.

The use of binary blends of hydrogenated and fluorinated alkanethiolates represents an interesting approach to the construction of anisotropic hybrid organic–inorganic nanoparticles since the fluorinated and hydrogenated components are expected to self-sort on the nanoparticle surface because of their reciprocal phobicity. These mixed monolayers are therefore strongly non-ideal binary systems. The synthetic routes we explored to achieve mixed monolayer gold nanoparticles displaying hydrogenated and fluorinated ligands clearly show that the final monolayer composition is a non-linear function of the initial reaction mixture. Our data suggest that, under certain geometrical constraints, nucleation and growth of fluorinated domains could be the initial event in the formation of these mixed monolayers. The onset of domain formation depends on the structure of the fluorinated and hydrogenated species. The solubility of the mixed monolayer nanoparticles displayed a marked discontinuity as a function of the monolayer composition. When the fluorinated component content is small, the nanoparticle systems are fully soluble in chloroform, at intermediate content the nanoparticles become soluble in hexane and eventually they become soluble in fluorinated solvents only. The ranges of monolayer compositions in which the solubility transitions are observed depend on the nature of the thiols composing the monolayer.

The two peptides, rich in C(alpha)-tetrasubstituted amino acids, Ac-[Aib-L-(alphaMe)Val-Aib](2)-L-His-NH(2) (1) and Ac-[Aib-L-(alphaMe)Val-Aib](2)-O-tBu (2 a) are prevalently helical. They present the unique property of changing their conformation from the alpha- to the 3(10)-helix as a function of the polarity of the solvent: alpha in more polar solvents, 3(10) in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3(10)-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3(10)-helix and the use of the CD technique for its assessment.

Microwave dielectric heating proved to be an efficient method for the one-pot and stepwise syntheses of symmetrical and unsymmetrical naphthalenediimide derivatives of alpha-amino acids. Acid-labile side chain protecting groups are stable under the reaction conditions; protection of the alpha-carboxylic group is not required. The stepwise condensation of different amino acids resulted in high yields of unsymmetrical naphthalenediimides. The reaction proceeds without racemization and is essentially quantitative.

Two new synthetic ionophores in which the hydrophobic portion is represented by a short helical Aib-peptide (Aib=α-amino-isobutyric acid) and the hydrophilic one is a poly-amino (1a) or a polyether (1b) chain have been prepared. The two conjugates show a high ionophoric activity in phospholipid membranes being able to efficiently dissipate a pH gradient and, in the case of 1b, to transport Na(+) across the membrane. Bioactivity evaluation of the two conjugates shows that 1a has a moderate antimicrobial activity against a broad spectrum of microorganisms and it is able to permeabilize the inner and the outer membrane of Escherichia coli cells.

In this chapter, we discuss the relevant physical and chemical properties of gold nanoparticles that are exploited for analytical applications in a variety of different fields. This chapter was conceived to collect in a single place an overview of the properties of gold nanoparticles that are important for technological applications, with prevalent reference to the scientific literature that has appeared in the last six years. Given the extremely broad expertise needed for an in-depth discussion, the chapter is co-authored with experts in the specific field dealing with optical properties, ab initio calculations of electronic properties, magnetic, electrochemical and chemical properties.

The ionophoric activity of a calix[4]arene guanidinium conjugate (4G4Oct) has been investigated in bulk chloroform membrane and mainly in liposomes. In both membrane models 4G4Oct shows high activity in the transport of chloride. The mechanistic evidence suggests a carrier mechanism in which the calixarene binds the anions through electrostatic interactions and hydrogen bonds with the guanidinium moieties. Studies on anion transport in liposomes indicate a peculiar selectivity sequence inverse to the most commonly observed lyotropic series with chloride transported faster than bromide and iodide virtually non-transported. This selectivity sequence together with competitive inhibition of chloride transport by the non-transported ions suggests a strong anion binding to the guanidinium moieties in the membrane environment. The complex formed is lipophilic and is able to shuttle the ion across the membrane. The origin of the observed anion selectivity is suggested to be related to a less favourable ion exchange at the membrane interface of iodide with respect to chloride with OH2, which is the antiported ion in the transport process in liposome. These studies complement the reported ability of 4G4Oct to condensate plasmid DNA and to transfect cells, showing that the calixarene derivative is also able to efficiently transport chloride across a phospholipid bilayer.

A novel dendrimeric peptide ligand dubbed D-PAM-Φ was designed to achieve a high capacity for human IgG through the decoration of the D-PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D-PAM structure were supported by the recently published solid-state structure of D-PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl-D-PAM (D-PAM-Φ). D-PAM-Φ was immobilised on an activated solid support and compared with the parent D-PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10-fold enhancement with respect to the D-PAM-functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory-scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D-PAM-Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy.

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

SERPINB3 has been found in chronic liver damage and hepatocellular carcinoma, but not in normal liver. By direct mRNA sequencing, a new SERPINB3 polymorphism (SCCA-PD) has been identified, presenting the substitution Gly351Ala in the reactive center loop of the protein. The prevalence of the SCCA-PD isoform has been found to be significantly higher in patients with cirrhosis than in patients with chronic liver disease and in normal subjects. The aim of this study was to investigate the biological and functional activity of SERPINB3 isoforms using in vitro models. HepG2 and Huh7 cells lines were transfected with plasmid vectors containing wild-type SERPINB3 or its polymorphic variant SCCA-PD and their expression at transcriptional and protein level was determined. To assess the functional activity, both recombinant proteins were produced and kinetic analysis was carried out using papain and cathepsin-L as target proteases. In addition, the inhibition of JNK kinase activity by SERPINB3 isoforms was assessed. The crystal structure of wild-type SERPINB3 at 2.7 Å resolution was used for preparation of refined 3D models of the two isoforms. The results showed that transcriptional activity and protein expression of the two isoforms were similar in both transfected cell lines. Both SERPINB3 preparations exerted a dose-dependent protease inhibitory activity, but the effect of SCCA-PD was higher than that of the wild-type isoform. This result was supported by 3D modelling, where increased hydrophobic profile of the SCCA-PD isoform, introduced by the G351A mutation, was detected. In addition, at high protein concentration, SCCA-PD revealed a 16% higher inhibitory effect on c-Jun phosphorylation by JNK1, compared with wild-type SERPINB3. In conclusion, the single amino acid substitution in the SERPINB3 reactive site loop improves the functional activity of SCCA-PD isoform. This different antiprotease activity might favor disease progression in patients carrying this polymorphism.

New donor-acceptor hybrids of Zn(II)-metallated 5,15-diaryl porphyrins have been designed and synthesised via the porphyrin interactions with an electron acceptor molecule, di-n-hexyl N-substituted 1,2,4,8-naphthalenetetracarboxylic diimide (NDI). Binding interactions within these supramolecular complexes were investigated in the solid state by synchrotron X-ray diffraction and probed in solution by (1)H NMR spectroscopy. The systematic modulation of the porphyrin π-density was achieved, for the first time as multiple methoxy and fluorine groups were introduced as substituents to the 5,15-diaryls of the porphyrin. For these, the variation of the porphyrin-NDI binding strengths determined by (1)H NMR titrations was shown, using the Swain's type dual parameter approach, to be closely linked with the peripheral substitution pattern of the diaryl porphyrins validated by crystallography. The new 1:1 donor-acceptor complexes formed display characteristic features of the aromatic-stacked systems, i.e. the parallel arrangement and short interplanar separation between the substituted porphyrin and NDI. Synthetic modification of electron-density on the porphyrin surface by introducing substituents at peripheral sites of functionalised porphyrins represent a general solution towards electronically tunable aromatic surfaces: an understanding of their solution and solid state behaviour will significantly improve the rational design of new functional donor-acceptor supramolecular materials with potential applications ranging from new energy materials to dye-sensitised solar cells, photovoltaics and future drug delivery devices.

Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies.

Background and aim An increasing number of clinical data suggests the importance of the assessment of serum levels of the squamous cell carcinoma antigen (SCCA)-lgM immune complex for the diagnosis of hepatocellular carcinoma (HCC). In addition, monitoring of SCCA-IgM immune complexes has been described as a useful prognostic approach in patients with cirrhosis since the progressive increase of SCCA-IgM over time is associated with a higher risk of HCC development. Because other assays in patients with cirrhosis have been affected by interfering IgMs, the aim of the present study was to assess the specificity of SCCA-IgM reactivity in cirrhotic patients by evaluating SCCA-IgM detection dependence on different capturing phases and by measuring the recovery of SCCA-IgM reactivity after serum fractionation. Patients and methods Serum samples from 82 patients with cirrhosis (M/F ratio 3/1; mean age ± SD: 56 ± 9 years) were collected at the Liver Unit of the Department of Clinical and Experimental Medicine, University of Padua, according to the approved institutional procedures. Serum levels of SCCA-IgM were measured using two different ELISA tests: the reference assay based on a rabbit oligoclonal anti-human SCCA antibody (Hepa IC, Xeptagen, Italy) and a dedicated ELISA with a mouse monoclonal anti-SCCA as capture antibody. Results Serum levels of SCCA-IgM measured with the reference assay (median value 87 AU/mL) were higher than levels measured with the mouse monoclonal test (median value 78 AU/mL), but the differences were not statistically significant (Mann-Whitney U test, p>0.05). When SCCA-IgM levels measured with both tests were compared, a linear correlation was found (r = 0.77, p < 0.001). An in silico analysis using available structural data of SCCA showed the presence of up to three putative antigenic sites localized on the SCCA surface, thus providing evidence that the test with the mouse monoclonal anti-SCCA antibody could underestimate the total circulating immune complexes due to the steric hindrance of the enormous mass of IgM (900 kDa) that could mask on the SCCA (45 kDa) surface the binding site recognized by the monoclonal antibody. To show that the SCCA-IgM assay was not affected by interfering IgMs, we measured the recovery of SCCA-IgM reactivity after serum fractionation of ten of the most reactive samples in both assays. Total recovery of SCCA-IgM reactivity was obtained with both assays in the fractions corresponding to components with higher molecular weight than IgM and SCCA (>2000 kDa). Conclusions The results of this study indicate that the reactivity measured in cirrhotic patients is related only to SCCA-IgM immune complexes and is not affected by other serum components, supporting the analytical specificity of the SCCA-IgM assay and validating the importance of SCCA-IgM as a risk biomarker for HCC development.

We report a dynamic combinatorial library that, upon binding of an electronically-complementary guest, produces in high yield a tetrameric receptor with flat hydrophobic, electron-deficient surfaces and flexible, water-soluble disulfide-containing linkers; analysis of the dependence of library composition on template concentration gives insight into the binding behaviours of the species involved.

Understanding the chemical physical properties of protein binding sites is at the basis of the rational design of protein ligands. The hinge region of the Fc fragment of immunoglobulin G is an important and well characterized protein binding site, known to interact with several natural proteins and synthetic ligands. Here, we report structural evidence that a Staphylococcus aureus Protein A mimetic peptide dendrimer, deduced by a combinatorial approach, binds close to the Cgamma2/Cgamma3 interface of the constant fragment of a human IgG1 molecule, partially hindering the Protein A binding site. The X-ray analysis evidenced a primary binding site located between a terminal Arg residue of the ligand peptidic arm and a hydrophobic protein site consisting of Val308, Leu309, and His310. A molecular dynamic analysis of the model derived from the X-ray structure showed that in water at room temperature the complex is further stabilized by the formation of at least one more contact between a terminal Arg residue of the second arm of the peptide and the carboxylic group of a protein amino acid, such as Glu318, Asp312, or Asp280. It appears thus that stability of the Fc-dendrimer complex is determined by the synergetic formation of multiple bonds of different nature between the dendrimer arms and the protein accessible sites. The electrostatic and van der Waals energies of the complex were monitored during the MD simulations and confirmed the energetic stability of the two interactions.

Nanozymes with a heart of gold: A functional artificial protein has been prepared by grafting a dodecapeptide onto the surface of gold nanoparticles (see picture). The system catalyzes the hydrolysis of carboxylate esters and features enzyme-like properties. (Figure Presented).

ESR spectroscopy has been used to study the interaction of para-pentylbenzyl hydroxyalkyl nitroxide with the monolayer of water-soluble protected gold clusters having a core diameter ranging from 1.6 to 5.3 nm. The solubilization of the nitroxide probe in the more hydrophobic environment of the monolayer strongly depends on the size of the gold core. In particular, the partition equilibrium constant increases as the nanoparticle diameter decreases. These results have been attributed to the different packing of the chains in the monolayer resulting from the different radius of curvature of the investigated nanoparticles. This represents, to the best of our knowledge, the first report demonstrating that the core size of metallic nanoparticles affects the solvating properties of the protective organic monolayer.

This short review highlights recent results on the use of nanoclusters of gold protected by a monolayer of functional ligands in both molecular recognition and catalysis. Particular emphasis is given to the multivalent properties of these systems and, in the case of catalysis, to the cooperativity between functional groups present in the ligands of the monolayer. The outstanding catalytic efficiency of some of the functional nanoparticles synthesized led us to call them nanozymes by analogy with the activity of catalytic polymers (synzymes).

We report here the first example of peptide-functionalized gold nanoparticles hydrolytically active against carboxylate esters. The active units are constituted by His-Phe-OH terminating thiols. The confinement of the catalytic units in the monolayer covering the nanoparticles triggers a cooperative hydrolytic mechanism operative at pH < 7 in which a carboxylate and an imidazolium ion act as general base and general acid, respectively. Such a mechanism is absent with an analogous monomeric dipeptide, and this results in a more than 300-fold rate acceleration of the hydrolytic process at low pH in the presence of the functional nanoparticles.

EPR spectroscopy has been used to study the interaction of para-substituted benzyl hydroxyalkyl nitroxides with the monolayer of water-soluble protected gold cluster made by a short alkyl chain and a triethylene glycol monomethyl ether unit. The inclusion of nitroxide probes in the more hydrophobic environment of the monolayer gave rise to a reduction of the value of both nitrogen and beta-proton hyperfine splittings. The spectra also showed selective line broadening attributed to modulation of the spectroscopic parameters as the result of exchange between free and complexed nitroxide. The rate constants were obtained by analyzing the EPR line shape variations as functions of nanoparticle concentration and temperature. This represents, to the best of our knowledge, the first determination of rate constants for the solubilization of organic substrates in a monolayer-protected cluster.