Characterization of L-type Calcium Channel Binding-Site of a new class of Calcium modulators by a Multidisciplinary approach

Many potential diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transient and on recombinant L-VDCC. Twenty particular compounds were selected by the ligand-based virtual screening procedure (LBVS). From these compounds, five of them (5b, M2, M7, M8 and P1) showed a potent and selective inotropic activity on guinea-pig left atria driven 1 Hz. Further assays displayed an interesting negative inotropic effect of M2, M8, P1 and M7 on guinea pig isolated left papillary muscle driven at 1 Hz, a relevant vasorelaxant activity of 5b, M2, M7, M8 and P1 on K+-depolarized guinea-pig ileum longitudinal smooth muscle and a significant inhibition of contraction of 5b, M2, M8 and P1 on carbachol stimulated ileum longitudinal smooth muscle. Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus Leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding site for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this findings, the relative order of increased rates of contraction and relaxation at lower concentrations s(≤10-6M) in unpaced hearts was M7>M2>M8>P1. Similar increases in Ca2+ transient were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5b had no significant effect. Diltiazem blocks Ca2+current in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8, M2 showed ~ 10% tonic block; in addition, M8, M2 and P1 shifted the steady state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6 ± 11.5 ms, 226 ± 14.5 vs. 269 ± 12.9 vs. 199.28 ± 8.19 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. Only P1 showed a significantly decrease of the time for the recovery from inactivation. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8, M2 and P1 might directly decrease the binding affinity or allow rapid dissociation from the benzothiazepine binding site.

La comunicazione chimica in Blattaria: contributo al miglioramento della lotta

CHEMICAL COMMUNICATION IN BLATTARIA: CONTRIBUTION TO THE IMPROVEMENT OF THE CONTROL TECNIQUES The management of cockroach infestations in urban environment has undergone some changes in recent years by moving to the predominant use of baits, thanks to the awareness of the risks connected with the use of spray insecticides. The effectiveness of a bait is determined by the collective performance of its components, including active and inert ingredients, the food attractant and any other attractive odour. This research has focused on the behavioral responses of Italian sinanthropic cockroaches to semiochemicals and food attractants, for the purpose of evaluating a possible practical application and of contributing to the improvement of control techniques. Behavioral and morphological studies have been carried out on Supella longipalpa (F.), a small cockroach that is spreading in Italy. Behavioral assays showed that the fourth and fifth tergites of females are significantly more attractive than other region of the body. Cuticular pores and ducts ending in glandular structures (observed with a S.E.M. = Scanning Electron Microscope) are present in large number on these tergites, suggesting that they could be involved in the production and the release of sexual pheromone. Cockroaches produce an aggregation pheromone that is excreted along with their frass and that consists of volatile and non-volatile compounds, mainly amines and steroidal glycosides. The effectiveness of faecal extracts obtained from excrements of Blattella germanica (L.), Blatta orientalis L., Periplaneta americana (L.) and S. longipalpa was evaluated, both at intraspecific and interspecific level, using a "Y" tube olfactometer. Bioassays showed that faecal extracts obtained with methanol have a good intraspecific attractiveness and, in some cases, they showed also interspecific behavioral responses. A gel was prepared, having physical characteristics that could give a good resistance to dehydration, as a potential basis for a new bait; the gel was then added faecal extracts, obtained with methanol from B. germanica and S. longipalpa frass. Arena-tests showed that the new gel containing faecal extracts is more attractive than some commercial gel formulations used as comparison: it was the only product that could attract 100% of insects placed in the arenas in 4-5 days. In conclusion, the substances involved in chemical communication of Blattaria may be able to effectively increase the attractiveness of products for monitoring and controlling cockroaches.

Conformation and Stereodynamic of Hindered Aromatic System

The preparation of conformationally hindered molecules and their study by DNMR and computational methods are my thesis’s core. In the first chapter, the conformations and the stereodynamics of symmetrically ortho-disubstituted aryl carbinols and aryl ethers are described. In the second chapter, the structures of axially chiral atropisomers of hindered biphenyl carbinols are studied. In the third chapter, the steric barriers and the -barrier of 1,8-di-aylbiphenylenes are determined. Interesting atropisomers are found in the cases of arylanthrones, arylanthraquinones and arylanthracenes and are reported in the fourth chapter. By the combined use of dynamic NMR, ECD spectroscopy and DFT computations, the conformations and the absolute configurations of 2-Naphthylalkylsulfoxides are studied in the fifth chapter. In the last chapter, a new synthetic route to ,’-arylated secondary or tertiary alcohols by lithiated O-benzyl-carbamates carrying an N-aryl substituent and DFT calculations to determinate the cyclic intermediate are reported. This work was done in the research group of Prof. Jonathan Clayden, at the University of Manchester.

De Novo Design of secondary structures: Synthesis and conformational studies

Chemists have long sought to extrapolate the power of biological catalysis and recognition to synthetic systems. These efforts have focused largely on low molecular weight catalysts and receptors; however, biological systems themselves rely almost exclusively on polymers, proteins and RNA, to perform complex chemical functions. Proteins and RNA are unique in their ability to adopt compact, well-ordered conformations, and specific folding provides precise spatial orientation of the functional groups that comprise the “active site”. These features suggest that identification of new polymer backbones with discrete and predictable folding propensities (“foldamers”) will provide a basis for design of molecular machines with unique capabilities. The foldamer approach complements current efforts to design unnatural properties into polypeptides and polynucleotides. The aim of this thesis is the synthesis and conformational studies of new classes of foldamers, using a peptidomimetic approach. Moreover their attitude to be utilized as ionophores, catalysts, and nanobiomaterials were analyzed in solution and in the solid state. This thesis is divided in thematically chapters that are reported below. It begins with a very general introduction (page 4) which is useful, but not strictly necessary, to the expert reader. It is worth mentioning that paragraph I.3 (page 22) is the starting point of this work and paragraph I.5 (page 32) isrequired to better understand the results of chapters 4 and 5. In chapter 1 (page 39) is reported the synthesis and conformational analysis of a novel class of foldamers containing (S)-β3-homophenylglycine [(S)-β3-hPhg] and D- 4-carboxy-oxazolidin-2-one (D-Oxd) residues in alternate order is reported. The experimental conformational analysis performed in solution by IR, 1HNMR, and CD spectroscopy unambiguously proved that these oligomers fold into ordered structures with increasing sequence length. Theoretical calculations employing ab initio MO theory suggest a helix with 11-membered hydrogenbonded rings as the preferred secondary structure type. The novel structures enrich the field of peptidic foldamers and might be useful in the mimicry of native peptides. In chapter 2 cyclo-(L-Ala-D-Oxd)3 and cyclo-(L-Ala-DOxd) 4 were prepared in the liquid phase with good overall yields and were utilized for bivalent ions chelation (Ca2+, Mg2+, Cu2+, Zn2+ and Hg2+); their chelation skill was analyzed with ESI-MS, CD and 1HNMR techniques and the best results were obtained with cyclo-(L-Ala-D-Oxd)3 and Mg2+ or Ca2+. Chapter 3 describes an application of oligopeptides as catalysts for aldol reactions. Paragraph 3.1 concerns the use of prolinamides as catalysts of the cross aldol addition of hydroxyacetone to aromatic aldeydes, whereas paragraphs 3.2 and 3.3 are about the catalyzed aldol addition of acetone to isatins. By means of DFT and AIM calculations, the steric and stereoelectronic effects that control the enantioselectivity in the cross-aldol addition of acetone to isatin catalysed by L-proline have been studied, also in the presence of small quantities of water. In chapter 4 is reported the synthesis and the analysis of a new fiber-like material, obtained from the selfaggregation of the dipeptide Boc-L-Phe-D-Oxd-OBn, which spontaneously forms uniform fibers consisting of parallel infinite linear chains arising from singleintermolecular N-H···O=C hydrogen bonds. This is the absolute borderline case of a parallel β-sheet structure. Longer oligomers of the same series with general formula Boc-(L-Phe-D-Oxd)n-OBn (where n = 2-5), are described in chapter 5. Their properties in solution and in the solid state were analyzed, in correlation with their attitude to form intramolecular hydrogen bond. In chapter 6 is reported the synthesis of imidazolidin-2- one-4-carboxylate and (tetrahydro)-pyrimidin-2-one-5- carboxylate, via an efficient modification of the Hofmann rearrangement. The reaction affords the desired compounds from protected asparagine or glutamine in good to high yield, using PhI(OAc)2 as source of iodine(III).

Luminescent Systems for Genomics and Proteomics

The present PhD project was focused on the development of new tools and methods for luminescence-based techniques. In particular, the ultimate goal was to present substantial improvements to the currently available technologies for both research and diagnostic in the fields of biology, proteomics and genomics. Different aspects and problems were investigated, requiring different strategies and approaches. The whole work was thus divided into separate chapters, each based on the study of one specific aspect of luminescence: Chemiluminescence, Fluorescence and Electrochemiluminescence. CHAPTER 1, Chemiluminescence The work on luminol-enhancer solution lead to a new luminol solution formulation with 1 order of magnitude lower detection limit for HRP. This technology was patented with Cyanagen brand and is now sold worldwide for Western Blot and ELISA applications. CHAPTER 2, Fluorescescence The work on dyed-doped silica nanoparticles is marking a new milestone in the development of nanotechnologies for biological applications. While the project is still in progress, preliminary studies on model structures are leading to very promising results. The improved brightness of these nano-sized objects, their simple synthesis and handling, their low toxicity will soon turn them, we strongly believe, into a new generation of fluorescent labels for many applications. CHAPTER 3, Electrochemiluminescence The work on electrochemiluminescence produced interesting results that can potentially turn into great improvements from an analytical point of view. Ru(bpy)3 derivatives were employed both for on-chip microarray (Chapter 3.1) and for microscopic imaging applications (Chapter 3.2). The development of these new techniques is still under investigation, but the obtained results confirm the possibility to achieve the final goal. Furthermore the development of new ECL-active species (Chapter 3.3, 3.4, 3.5) and their use in these applications can significantly improve overall performances, thus helping to spread ECL as powerful analytical tool for routinary techniques. To conclude, the results obtained are of strong value to largely increase the sensitivity of luminescence techniques, thus fulfilling the expectation we had at the beginning of this research work.

Application of FTIR microscopy to cultural heritage materials

Research in art conservation has been developed from the early 1950s, giving a significant contribution to the conservation-restoration of cultural heritage artefacts. In fact, only through a profound knowledge about the nature and conditions of constituent materials, suitable decisions on the conservation and restoration measures can thus be adopted and preservation practices enhanced. The study of ancient artworks is particularly challenging as they can be considered as heterogeneous and multilayered systems where numerous interactions between the different components as well as degradation and ageing phenomena take place. However, difficulties to physically separate the different layers due to their thickness (1-200 µm) can result in the inaccurate attribution of the identified compounds to a specific layer. Therefore, details can only be analysed when the sample preparation method leaves the layer structure intact, as for example the preparation of embedding cross sections in synthetic resins. Hence, spatially resolved analytical techniques are required not only to exactly characterize the nature of the compounds but also to obtain precise chemical and physical information about ongoing changes. This thesis focuses on the application of FTIR microspectroscopic techniques for cultural heritage materials. The first section is aimed at introducing the use of FTIR microscopy in conservation science with a particular attention to the sampling criteria and sample preparation methods. The second section is aimed at evaluating and validating the use of different FTIR microscopic analytical methods applied to the study of different art conservation issues which may be encountered dealing with cultural heritage artefacts: the characterisation of the artistic execution technique (chapter II-1), the studies on degradation phenomena (chapter II-2) and finally the evaluation of protective treatments (chapter II-3). The third and last section is divided into three chapters which underline recent developments in FTIR spectroscopy for the characterisation of paint cross sections and in particular thin organic layers: a newly developed preparation method with embedding systems in infrared transparent salts (chapter III-1), the new opportunities offered by macro-ATR imaging spectroscopy (chapter III-2) and the possibilities achieved with the different FTIR microspectroscopic techniques nowadays available (chapter III-3). In chapter II-1, FTIR microspectroscopy as molecular analysis, is presented in an integrated approach with other analytical techniques. The proposed sequence is optimized in function of the limited quantity of sample available and this methodology permits to identify the painting materials and characterise the adopted execution technique and state of conservation. Chapter II-2 describes the characterisation of the degradation products with FTIR microscopy since the investigation on the ageing processes encountered in old artefacts represents one of the most important issues in conservation research. Metal carboxylates resulting from the interaction between pigments and binding media are characterized using synthesised metal palmitates and their production is detected on copper-, zinc-, manganese- and lead- (associated with lead carbonate) based pigments dispersed either in oil or egg tempera. Moreover, significant effects seem to be obtained with iron and cobalt (acceleration of the triglycerides hydrolysis). For the first time on sienna and umber paints, manganese carboxylates are also observed. Finally in chapter II-3, FTIR microscopy is combined with further elemental analyses to characterise and estimate the performances and stability of newly developed treatments, which should better fit conservation-restoration problems. In the second part, in chapter III-1, an innovative embedding system in potassium bromide is reported focusing on the characterisation and localisation of organic substances in cross sections. Not only the identification but also the distribution of proteinaceous, lipidic or resinaceous materials, are evidenced directly on different paint cross sections, especially in thin layers of the order of 10 µm. Chapter III-2 describes the use of a conventional diamond ATR accessory coupled with a focal plane array to obtain chemical images of multi-layered paint cross sections. A rapid and simple identification of the different compounds is achieved without the use of any infrared microscope objectives. Finally, the latest FTIR techniques available are highlighted in chapter III-3 in a comparative study for the characterisation of paint cross sections. Results in terms of spatial resolution, data quality and chemical information obtained are presented and in particular, a new FTIR microscope equipped with a linear array detector, which permits reducing the spatial resolution limit to approximately 5 µm, provides very promising results and may represent a good alternative to either mapping or imaging systems.

Biomimetic Materials for Biomedical Applications

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Innovative analytical methods for Central Nervous System Drug analysis in biological fluids

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

New routes to enantioenriched substances through small organic molecules

In this thesis we will disclose the results obtained from the diastereoisomeric salt formation (n salt, p salt and p1,n1 salt) between non-racemic trans-chrysanthemic acid (trans-ChA) and pure enantiomers of threo-2-dimethylamino-1-phenyl-1,3-propanediol (DMPP). The occurrence of p1,n1 salt formation can have profound effects on enantiomer separation of scalemic (non-racemic) mixtures. This phenomenon when accompanied by substrate self-association impedes the complete recovery of the major enantiomer through formation of an inescapable racemate cage. A synthetic sequence for the asymmetric synthesis of bicyclo[3.2.0]heptanones and bicyclo[3.2.0]hept-3-en-6-ones through a cycloaddition strategy is reported. The fundamental step is a [2+2]-cycloaddition of an enantiopure amide derived from the reaction between a set of acids and an oxazolidinone as the chiral auxiliary. The inter- and intramolecular cycloaddition of in situ-generated keteniminium salts gives bicycles with a good enantioselection. A key intermediate of Iloprost, a chemically stable and biologically active mimic of prostacyclin PGI2 is synthesized following a ‘green approach’. An example of simple optical resolution of this racemic intermediate involving the diastereoisomeric salt formation is described.