Design and synthesis of trans-A2B2 and trans-A2BC dipyridylporphyrins: new building blocks for the self-assembly of metallacyclic supramolecular structures

2013/2014Artificial molecular structures forming stable pores in biological membranes may have important applications in the biomedical field and in the field of biotechnology, in particular as sensors. These structures have to meet specific characteristics of size, shape and solubility. In particular, they have to enter the membrane engaging hydrophobic interactions with the phospholipid bilayer and, at the same time, forming a polar conduit for the transport of the ions across the membrane. A molecular structure which meets these features is an amphipathic, rigid and tube-shaped one and, mostly important, long enough to span the entire membrane. The final goal of this thesis work is the design and preparation of structures that would reflect these characteristics obtained by the metal-mediated self-assembly of pyridylporphyrins. In particular to obtain structures long enough to span the membrane the focus was on the design of pyridylporphyrins equipped with complementary hydrogen bonding donor/acceptor moieties and of a polar subunit to increase membrane compatibility. Using transition metal complexes with an adequate geometry these “molecular panels” should self-assemble in metallasquares which, upon hydrogen-bonding driven dimerization in membrane, should form tubular empty structures long enough to span the phospholipid bilayer forming large pores. In the first part of the Thesis work, a versatile and straightforward synthetic strategy for the preparation of a library of amphiphilic trans-A2B2 and trans-A2BC dipyridylporphyrins directly from 5-(4-pyridyl)dipyrromethane has been developed and optimized. The major part of the porphyrins synthesized in this way are new compounds.The library members have been functionalized through different metal catalysed coupling reactions, showing their great potential and versatility towards the different employment which could be addressed, to obtain amphiphilic and dimeric derivatives, in some cases with very good and satisfying yields. The derivatization reactions have been performed on the free base porphyrins and, therefore, it has been necessary to carefully optimize the conditions of the metal catalysed reactions in order to avoid the insertion of the catalyst, or of the co-catalyst, in the porphyrin macrocycle. The functionalities that have been inserted into the dipyridylporphyrins scaffold are hydrogen-bonding complementary donor/acceptor moieties, like uracil and diacylaminopyridine, and an amphiphilic polyether chains. Starting from the porphyrin library and exploiting metal catalysed coupling reaction also three dipyridylporphyrins dimers have been prepared. The target amphiphilic dipyridylporphyrins have been principally utilized in self-assembly reactions exploiting the pyridyl groups present, in particular through the coordination-driven self-assembly approach, with cis-coordinating metal complexes like Re(CO)5Br and trans,cis,cis-[RuCl2(CO)2(DMSO-O)2], leading to the formation of molecular squares together with other kind of metallacyclic species. At the best of our knowledge, this is the first time that the Ru(II) complex have been employed for the self-assembly with trans-dipyridylporphyrins. The porphyrins, the dimers and supramolecules synthesized have been mainly characterized by mean of NMR spectroscopy, in particular through 1H, 13C, 1H-1H COSY, 1H-13C HSQC, 1H-DOSY. The latter technique, being more and more important and utilized in supramolecular chemistry either in the characterization either in the sample purity proof of the compounds, has been in fact thoroughly utilized both to confirm the dimensions in solution of all the molecules synthesized and to give an evidence of their purity. This last feature has been one of the more challenging to face because the sample purity was not so evident just analysing the 1H-NMR spectra due to the possible presence of isomers and conformers. In absence of X-ray spectroscopic and MS spectrometric data, PFG-NMR has been a powerful, helpful and straightforward way to rationalize the high complexity of the resonating signals pattern in these spectra and to confirm the higher molecular dimensions reached as relative to the parent porphyrins. Confirmation of the pyridyl-metal bond formation with the right configuration has come also from IR, UV-Vis and fluorescence emission spectra acquired both for the porphyrins and for the supramolecular metallacycles. Putting together all the data and although in some cases we were not able to unambiguously define the nuclearity of the metallacycle, the supramolecules synthesised have all cyclic and symmetric structure and retain the symmetry of their parent porphyrins.The most representative porphyrins, together with the supramolecular metallacycles have been then tested as transmembrane ion channels utilizing liposomes as model of biological membranes. Preliminary studies on the H+ transport assays have been reported.Le strutture molecolari artificiali capaci di formare nanopori stabili all’interno di una membrane biologica sono sempre più di ampio interesse, grazie alla possibilità di essere impiegate in campo biomedico e biotecnologico, soprattutto come sensori. Per poter formare nanopori questi sistemi devono soddisfare dei requisiti minimi in termini di forma, dimensioni e solubilità. Essi devono essere in grado di inserirsi facilmente in membrana tramite interazioni idrofobiche e al contempo formare condotti polari che consentano il passaggio degli ioni; quindi una struttura che presenti tali caratteristiche dovrà essere anfifilica, avere una forma allungata, essere abbastanza rigida e, soprattutto, essere sufficientemente lunga da attraversare completamente la membrana. Lo scopo di questo lavoro di tesi è quello di realizzare sistemi che soddisfino queste esigenze sfruttando il metal-mediated self-assembly di piridilporfirine su centri metallici. In particolare, per ottenere strutture sufficientemente lunghe da attraversare completamente il doppio strato fosfolipidico, si è focalizzata l’attenzione sulla realizzazione di piridilporfirine equipaggiate con gruppi accettori e donatori di legami ad idrogeno e con un catena anfifilica che ne aumenti la compatibilità con la membrana. Utilizzando complessi di metalli di transizione con una geometria adeguata questi “pannelli molecolari” dovrebbero assemblarsi a dare metallacicli di forma approssimativamente cubica in grado di dimerizzare in membrana, grazie alla formazione di legami ad idrogeno, formando così strutture tubulari cave sufficientemente lunghe da attraversare completamente la membrana. Nella prima parte della Tesi è stata messa a punto ed ottimizzata una strategia sintetica per ottenere una libreria di derivati anfifilici di trans-A2B2 e trans-A2BC dipiridilporfirine direttamente a partire dal 5-(4-piridil)dipirrometano. La maggior parte delle porfirine sintetizzate in questo modo sono composti nuovi. I membri della libreria sono stati quindi funzionalizzati attraverso reazioni di coupling metallo-catalizzate per ottenere sia derivati anfifilici che dimerici, dimostrando così il loro potenziale e la loro versatilità verso l’utilizzo per diverse applicazioni. Le reazioni di funzionalizzazione sono state condotte sulle porfirine free-base ed è stato dunque necessario ottimizzare accuratamente le condizioni delle reazioni metallo-catalizzate in modo tale da evitare che il catalizzatore, o l’eventuale co-catalizzatore, si potesse inserire nel macrociclo porfirinico. Le funzionalità che sono state inserite nelle dipiridilporfirine sono molecole con gruppi donatori/accettori di legame idrogeno tra di loro complementari , in particolare, derivati dell’uracile e della diacilamminopiridina, e residui anfifilici come catene polieteree. Partendo dalla libreria di porfirine sono anche stati sintetizzati dei dimeri di dipiridilporfirine. Le dipiridilporfirine target sono state principalmente utilizzate in reazioni di auto-assemblaggio sfruttando i gruppi piridinici presenti, in particolare attraverso il metodo coordination-driven self-assembly con complessi metallici cis-coordinanti come Re(CO)5Br e trans,cis,cis-[RuCl2(CO)2(DMSO-O)2], ottenendo la formazione di quadrati molecolari insieme con altre specie metallacicliche. Al meglio delle nostre conoscenze, il complesso di Ru(II) è stato utilizzato per la prima volta per l’auto-assemblaggio con trans-dipiridilporfirine. Le porfirine, i dimeri e gli addotti supramolecolari ottenuti sono stati caratterizzati principalmente tramite spettroscopia NMR, in particolare attraverso 1H, 13C, 1H-1H COSY, 1H-13C HSQC, 1H-DOSY. Quest’ultima tecnica, essendo divenuta sempre più importante in chimica supramolecolare sia per la caratterizzazione sia per provare la purezza dei composti, è stata utilizzata a fondo per confermare le dimensioni in soluzione delle molecole sintetizzate e per avere una prova della purezza dei campioni. Quest’ultimo aspetto è stato uno dei più difficili da affrontare perché non era certo evidente analizzando i soli spettri 1H-NMR acquisiti per la possibile presenza di isomeri e confomeri. In assenza di dati spettroscopici a raggi X e di spettrometria di massa, la tecnica PFG-NMR è stata uno strumento potente, utile e diretto per razionalizzare l’elevata complessità del pattern dei segnali osservato in questi spettri e per confermare le più elevate dimensioni raggiunte da queste molecole relativamente alle porfirine di partenza. Conferma dell’avvenuta formazione dei legami metallo-piridina con la giusta configurazione è venuta anche dai dati spettroscopici IR, UV-Vis e di emissione di fluorescenza, acquisiti per le porfirine così come per i metallacicli supramolecolari. Anche se non è stato possibile in alcuni casi assegnare in maniera non ambigua la nuclearità di queste supramolecole, poiché la geometria molecolare sia di specie a nuclearità [3+3] che [4+4] può essere approssimata dalla medesima sfera, le supramolecole sintetizzate sono specie cicliche e simmetriche e preservano la simmetria molecolare delle specie di partenza.Le porfirine più rappresentative e gli addotti metallaciclici sono stati testati per la loro capacità di formare canali ionici transmembrana utilizzando liposomi come modelli delle membrane biologiche. Gli studi preliminari sull’attività di trasporto di ioni H+ sono riportati nella Tesi.XXVII Ciclo198

Synthesis and characterization of trans-di-(4-pyridyl)porphyrin dimers

Preparation and characterization of a small library of symmetric trans-di(4-pyridyl) porphyrin dimers, obtained by either Glaser\u2013Hay or Sonogashira coupling reactions from appropriately prepared trans-di-4-pyridylporphyrin precursors, is presented. The porphyrin dimers are differentiated by a phenyl-alkynyl bridge of increasing length at one meso-position, while for all the derivatives the two remaining opposite meso-positions are tailored with a phenyl moiety bearing a short polyether chain. Coordination of the four pyridyl groups with appropriate metal fragments may be exploited to construct tubular hollow structures, with varied internal sizes, depending on the choice of the porphyrin dimer component

Guanine-based amphiphiles: synthesis, ion transport properties and biological activity

Novel amphiphilic guanine derivatives, here named Gua1 and Gua2, have been prepared through few, simple and efficient synthetic steps. In ion transport experiments through phospholipid bilayers, carried out to evaluate their ability to mediate H(+) transport, Gua2 showed high activity. When this compound was investigated for ion-selective transport activities, no major differences were observed in the behaviour with cations while, in the case of anions, selective activity was observed in the series I(-)>Br(-)>Cl(-)>F(-). The bioactivity of these guanine analogues has been evaluated on a panel of human tumour and non-tumour cell lines in preliminary in vitro cytotoxicity assays, showing a relevant antiproliferative profile for Gua2

Synthesis and characterization of a hydrophilic conjugated 4+4 Re(I)-porphyrin metallacycle

The preparation and the full characterization, including the X-ray structure determination, of a polar trans-dipyridylporphyrin functionalized with two short polyoxyethylene chains is reported. Reaction of the porphyrin with a Re(I) complex yielded a 4+4 metallacycle showing an improved solubility and a lower tendency to aggregate with respect to analogous porphyrin cyclic derivatives. These properties allowed a full NMR characterization of the metallacycle including VT 1H DOSY-NMR experiments and, for the first time, the recording of a 13C-NMR spectrum giving further insight into the structural definition of these type of metallacycles. Spray deposition of the metallacycle on a heated mica substrate shows the formation of regular ring-like nano-structures which are not formed by the parent porphyrin

O-annulation to polycyclic aromatic hydrocarbons: a tale of optoelectronic properties from five- to seven-membered rings

We take advantage of the Pummerer oxidative annulation reaction to extend PAHs through the formation of an intramolecular C–O bond with a suitable phenol substituent. Depending on the peripheral topology of the PAH precursor (e.g., pyrene, boron–dipyrromethene, or perylene diimide) five-, six-, and seven-membered O-containing rings could be obtained. The effect of the O-annulations on the optoelectronic properties were studied by various methods with the pyrano-annulated pyrene and BODIPY derivatives depicting quantitative emission quantum yields

Closed Mitral Valvotomy: Celebrating 100 Years of Surgical History

The year 2023 marks the 100th anniversary of the first successful valvotomy for mitral valve stenosis by Elliott C. Cutler in 1923. Closed-chest mitral valve commissurotomy developed further before being replaced by an open procedure after the advent of the heart-lung machine. Currently, because of the almost complete disappearance of rheumatic disease in the Western World, mitral commissurotomies are infrequently performed in those countries, although the procedure-either closed or open-is still performed in developing countries and select patients. This review retraces the 100-year journey from a historic operation to the current era-a milestone in the treatment of patients with mitral stenosis

New bone formation using antibiotic-loaded calcium sulfate beads in bone transports for the treatment of long-bone osteomyelitis

Purpose Bone transport is one of the most frequently used techniques for critical-sized bone defects due to trauma or infection. To fill the defect area and avoid the collapse of soft tissues during transport, some authors have described the use of polymethylmethacrylate or absorbable antibiotic carriers in the form of cylindrical blocks. Methods In this article, we present our experience in the treatment of post-traumatic osteomyelitis of the lower and upper limbs, using a bone transport technique with antibiotic-loaded calcium sulfate in the form of beads. Results With the progressive absorption of calcium sulfate, we observed the formation of a bone-like tissue envelope at the periphery of the defect area. Histological analysis and direct visualization during open revision surgery of the docking site in all patients confirmed the presence of newly formed bone tissue with a high presence of osteoblasts and few osteoclasts; no areas of necrosis or signs of infection were observed. This bone envelope maintained the mechanical protective function of the transport path and docking site, and also provided a biological stimulus to avoid the development of necrotic areas and optimize the consolidation phase. ConclusionBone transport with calcium sulfate beads improves biological and mechanical support and reduces the number of surgeries required

One health adoption within prevention, preparedness and response to health threats. Highlights from a scoping review

Introduction: As the COVID-19 pandemic has demonstrated, the complexity of factors involved in the emergence of health threats requires a holistic One Health (OH) approach to enhance the effectiveness of prevention, preparedness, and response (PPR) strategies. Therefore, we conducted a scoping review to explore how the OH approach has been adopted in the context of PPR strategies to health threats, and the challenges and benefits deriving from its integration. Methods: We defined the research questions and a strategy to guide the peer-reviewed and grey literature search to identify relevant articles and documents (identification). We assessed them for eligibility according to predefined criteria (screening) and finally included the ones that answered the research questions (inclusion). We performed a descriptive and thematic analysis of the results. Results: A total of 138 records were included in the review (57 from the peer-reviewed literature and 81 from the grey literature). The OH approach was mainly adopted in prevention strategies, particularly within the governance area. Human and animal health were the most integrated disciplines in the OH approach, while environmental and social sciences were the less integrated. The most targeted threats were antimicrobial resistance and zoonoses, with the African region being the most represented. Conducive factors for the adoption of OH PPR strategies were identified in resolutions and guidance emanating from international organisations. Discussion: The global governance of OH should utilise conducive factors, such as international resolutions and guidance, to enhance the adoption of multisectoral and multi-actor PPR strategies, that focus on national and international priorities and neglected threats, such as environmental hazards and pandemic risk. Integrated frameworks and metrics for the implementation and evaluation of OH PPR strategies need to be consolidated to contribute to the growing body of evidence supporting the adoption of the OH approach

Cytokine serum profile in a group of Sicilian Nonagenarians

The aim of our study was to evaluate the possibility of using multiplex analysis of the cytokine profile as a marker for successful aging by comparing cytokine plasmatic levels of a group of Sicilian nonagenarians with those of young controls. We analyzed a panel of 17 cytokines, comprehensive of haematopoietic factors T helper 1 (Th1), Th2, inflammation regulatory cytokines, and chemokines. The assay was carried out using the Luminex system. Interleukin (IL)-6 levels (p = 0.01) were increased in nonagenarians, whereas no modifications of other proinflammatory cytokines and chemokines were observed. Interferon-gamma (IFN-gamma) and IL-2 levels are unmodified, suggesting a substantial maintenance of relevant T cell functions. In addition, a significant increase of IL-12 serum levels in nonagenarians versus young controls that might be related to the increase of natural killer (NK) cell functions characterizing aging processes was observed. The analysis of Th2 cytokines show an increase of IL-13 and a reduction of IL-4 levels mirroring the maintenance of some effector's mechanisms of the immunoresponse in advanced ages. Our results suggest that the multiplex analysis of cytokine levels might be useful in defining a successful aging profile

Relevance of gamma interferon, tumor necrosis factor alpha, and interleukin-10 gene polymorphisms to susceptibility to Mediterranean spotted fever.

The acute phase of Mediterranean spotted fever (MSF) is characterized by dramatic changes in cytokine production patterns, clearly indicating their role in the immunomodulation of the response against the microorganism, and the differences in cytokine production seem to influence the extent and severity of the disease. In this study, the single nucleotide polymorphisms (SNPs) of tumor necrosis factor alpha (TNF-α) -308G/A (rs1800629) and interleukin-10 (IL-10) -1087G/A (rs1800896), -824C/T (rs1800871), and -597C/A (rs1800872) and the gamma interferon (IFN-γ) T/A SNP at position +874 (rs2430561) were typed in 80 Sicilian patients affected by MSF and in 288 control subjects matched for age, gender, and geographic origin. No significant differences in TNF-α -308G/A genotype frequencies were observed. The +874TT genotype, associated with an increased production of IFN-γ, was found to be significantly less frequent in MSF patients than in the control group (odds ratio [OR], 0.18; 95% confidence interval [95% CI], 0.06 to 0.51; P corrected for the number of genotypes [Pc], 0.0021). In addition, when evaluating the IFN-γ and IL-10 genotype interaction, a significant increase of +874AA/-597CA (OR, 5.31; 95% CI, 2.37 to 11.88; Pc, 0.0027) combined genotypes was observed. In conclusion, our data strongly suggest that finely genetically tuned cytokine production may play a crucial role in the regulation of the immune response against rickettsial infection, therefore influencing the disease outcomes, ranging from nonapparent or subclinical condition to overt or fatal disease