2° Convegno dell'Istituto di Chimica Biomolecolare del ... · Anna Maria Orlandoni ... Doemling...

2° Convegno dell'Istituto di Chimica Biomolecolare del CNR

Area della Ricerca di Sassari, 8-9 Ottobre 2015

2nd Convention of the CNR Institute of Biomolecular Chemistry

Sassari Reasearch Area, October 8-9, 2015


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Il comitato scientifico:

Nicola D’Antona Giovanna Delogu

Luciano De Petrocellis Corrada Geraci

Angela Patti Paolo Ruzza

Daniele Sanna Maurizio Solinas Antonio Trincone

Rosa Maria Vitale

Il comitato organizzatore

Emanuela Azara Antonella Dettori

Marina Pisano Gloria Rassu

Maria Serra


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“Biomolecular Chemistry” and the Italian Research Institute that has made a mission of it

When the CNR Institute of Biomolecular Chemistry (ICB) was founded in 2002, most of its

personnel hardly knew how to explain to the layman what this “new” discipline, Biomolecular

Chemistry, really was. Although obviously everybody knew what Chemistry is, and how it can be

elegantly applied to the understanding of the molecular mechanisms regulating biological systems

and processes (ICB was in fact founded starting from the National Coordination Institute for the

Chemistry of Biological Systems), the adjective “Biomolecular” seemed to define only a general,

and perhaps too vague, idea of what would have been the subjects of the studies of the new

institute. Yet, since from the first (and, until last year, only) general meeting of ICB, in 2002, it was

clear that researchers and technicians of the institute would have focused their research efforts on

the understanding of the multifaceted roles of biomolecules in all aspects of our life, and on their

technological application to respond to the ever increasing needs not only of the bio-medical field,

but also of the agricultural, environmental and renewable energy sectors. Those were also the

years of the rise of two new disciplines, Chemical Biology and Bioorganic Chemistry, which

seemed to be nicely tailored to what ICB researchers planned to work on.

In the meantime, the term “Biomolecular” has been increasingly used over the last 15 years.

Taking into account only the PubMed engine website, this word went from only 327 citations in

1996 to well above 5570 in 2014, thus indicating that its choice in 2002 had been both timely and

successful. “Biomolecular Sciences” is now a widespread discipline in Italian Universities, to the

point that, for example, doctorate courses on this specific subject have appeared in many faculties.

As a consequence, when I applied to become the Director of ICB, and since the beginning of my

mandate in June 2014, I thought that the mission of the Institute should continue to be the

“chemical study of biological systems and processes to understand their function at the molecular

level and explore their new technological applications” and “to operate in the fields of bioorganic

chemistry and chemical biology, with expertise in the disciplines of spectroscopy, structural and

synthetic chemistry, biochemistry, molecular modeling, microbiology, pharmacology, nutraceutics

and bioenergetics”. In other words, I strongly believed that ICB research activity should continue

to be centered on organic molecules and their biological properties.

Last October, researchers from the four ICB branches met again, in the Naples main branch of the

institute and for the first time in 12 years. During the course of two very intense days, they provided

an update of their current efforts towards fulfilling this mission, discussed their latest results, and

threw the bases for new intra-mural collaborations reflecting and exploiting the inter- and multi-

disciplinary nature of their expertise. This “experiment”, made necessary by the low degree (as

recognized by most of the personnel) of previous scientific contacts among researchers from the

different ICB branches, was very successful. It resulted in the forming of 5 “Inter-Section”

disciplinary groups and in the beginning of at least two new collaborations. The meeting also gave

momentum to the action of the 4 “Institute Services of Common Interest” (SICI), which are now

working very well to provide all sorts of information to ICB personnel on the subjects of their

competence, and to establish new contacts with the “outside world”.

From the scientific standpoint, the 2014 ICB meeting did not focus on any of the several

institutional topics of research in particular, as it aimed at providing ICB researchers with a general

view of the state of the art of all ICB experimental activities, “old” and “new”. For this year’s

meeting in Sassari, instead, the Scientific Committee has made a tremendous effort to select, out

of the many present in ICB, three main topics to focus the program on (Drug Discovery, Omics and

Green Chemistry), and have invited for each of these topics renowned scientists from outside the

institute and the CNR. This will increase the exchange of information between ICB and other

research institutions, and enhance further the visibility of our institute. At the same time, the

Committee have allowed for a wide participation of the institute’s research personnel to show the

results of their activities on all topics of interest of ICB in the form of posters, whose presentation

will be given equal importance as the oral presentations and over 3 hours for discussion.


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The result is what I believe to be an excellent and exciting program, for which I am very grateful to

the Scientific Committee, as it is emblematic of the great variety of the research interests and

activities of ICB, and their potential utility to help solving some of the unmet demands of our

society.

I hope we will enjoy this year’s annual meeting at least as much as we did last year, and I am

confident that new collaborations, ideas and strategies will come out of these two days together.

These, in turn, will help us coping with the public funding and, particularly, human resources

difficulties that affect our institute as well as research in Italy in general, and, at the same time, will

contribute at making ICB our home and a prestigious and renowned research institute in both our

country and abroad.

Vincenzo Di Marzo

Direttore Istituto di Chimica Biomolecolare


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Programma del 2° Convegno dell’Istituto CNR di Chimica Biomolecolare 8-9 Ottobre 2015, AULA MAGNA – Area di Ricerca CNR di Sassari

Giovedì 8 Ottobre 12:30–13:00 Messaggi di benvenuto del Direttore e del Responsabile UOS di Sassari – Ricordo dei colleghi Aniello Lopez ed Ugo Anselmi 13:00–14:30 Lunch di benvenuto PL1. 14:30–15:00 Moderatore: Giovanna Delogu Alba Canu – Centro di Restauro della Soprintendenza Archeologia della Sardegna, Ministero per i Beni e le Attività Culturali e il Turismo (MIBACT) – Sassari Restoration, Archaeometry and diagnostic: an essential collaboration OC1. 15:00–15:20 Pietro Spanu Potent non-covalent inhibitors of Caspase-1 therapeutically targeting neuroinflammation in neurodegenerative diseases OC2. 15:20–15:40 Davide Fabbri Sustainable coupling reactions of natural bioactive hydroxylated phenols 15:40–16:00 Coffee break 16:00–17:30 Poster session 17:30–19:00 Riunione Istituto di Chimica Biomolecolare Moderatore: Vincenzo Di Marzo

CENA SOCIALE

Venerdì 9 Ottobre PL2. 9:00–9:30 Moderatore: Giovanni Nicolosi Anna Maria Orlandoni – Matrìca S.p.A. – Porto Torres (SS) Third generation biorefineries: Matrìca case - Opportunity and new developments OC3. 9:30–9:50 Fabiana Piscitelli Endocannabinoidomics: “omics” approaches applied to endocannabinoids and endocannabinoid-like mediators OC4. 9:50–10:10 Paola Bureddu

Synthesis and characterization of aminoproline-based RGD semipeptides targeting V3 integrins and their utility in medicine OC5. 10:10–10:30 Maurizio Solinas Screening of N,N-bidentate pyridine-based ligands in copper and palladium catalysed reactions 10:30–10:50 Coffee break OC6. 10:50–11:10 Moderatore: Antonio Trincone Barbara Biondi Peptaibols in membrane-llike environments: use of nitroxyl-labeled peptides to get insight into mechanism of action drug discovery&recognition


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OC7. 11:10–11:30 Rosaria Villano Efficient and environmentally friendly methodologies for the synthesis of multifunctionalized scaffolds OC8. 11:30–11:50 Giuseppe Granata Assembly of a succinyl-calix[4]arene derivative in multilamellar vesicles 11:50–12:50 Poster session 12:50–14:00 Pranzo PL3. 14:00–14:30 Moderatore: Andrea Calderan Sebastiano Banni – Università degli Studi di Cagliari UNICA – Dipartimento di Biologia Sperimentale, Sezione di Fisiologia Generale Nutritional role of lipids and their potential use in functional foods and nutraceuticals OC9. 14:30–14:50 Adele Cutignano A 'sea' of bioactive natural products OC10. 14:50–15:10 Nicola Culeddu NMR metabolomic applications on olive oils: pedoclimatic and sensory differentiation. From single pulse NMR to adiabatic sequence PL4. 15:10–15:40 Moderatore: Mauro Marchetti Pier Andrea Serra – Università degli Studi di Sassari UNISS – Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia Electrochemical micro(bio)sensors as new analytical tools for Biomedical and Agri-Food applications 15:40–16:00 Coffee break 16:00–16:40 Poster session OC11. 16:40–17:00 Carmelo Drago One-pot microwave assisted catalytic transformation of waste frying oil into glycerol-free biodiesel OC12. 17:00–17:20 Margherita Gavagnin Carrying on the route of marine guanidines: isolation, synthesis and bioactivity of a guanidine terpene from the mollusk Actinocyclus papillatus 17:20 Conclusioni


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Relatori invitati – Invited speakers “Restoration, Archaeometry and diagnostic: an essential collaboration” Alba Canu – Centro di Restauro della Soprintendenza Archeologia della Sardegna, Ministero per i Beni e le Attività Culturali e il Turismo (MIBACT) – Sassari “Third generation biorefineries: Matrìca case - Opportunity and new developments” Anna Maria Orlandoni - Matrìca S.p.A. – Porto Torres (SS) “Nutritional role of lipids and their potential use in functional foods and nutraceuticals” Sebastiano Banni – Università degli Studi di Cagliari UNICA Dipartimento di Biologia Sperimentale, Sezione Fisiologia Generale “Electrochemical micro(bio)sensors as new analytical tools for Biomedical and Agri-Food applications” Pier Andrea Serra – Università degli Studi di Sassari UNISS Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia


Oral Presentations


Oral 1

POTENT NON-COVALENT INHIBITORS OF CASPASE-1 THERAPEUTICALLY TARGETING NEUROINFLAMMATION IN

NEURODEGENERATIVE DISEASES

Ulgheri F.1, Deligia F.1, Morra A.1, Fuggetta M.P.2, Chandgude A. L.3,

Doemling A.3, Spanu P.1

1Institute of Biomolecular Chemistry-CNR, Trav. La Crucca 3, 07100,Sassari,

Italy 2Institute of Translational Pharmacology-CNR, Via Fosso del Cavaliere,00133,

Rome, Italy 3Department of Drug Design, University of Groningen, Groningen, 9713 AV,

Netherlands

email: [email protected] Recent studies have shown the pivotal role of innate immune activation in the pathogenesis of major neurodegenerative diseases. It was discovered that microglia and other cell types components of the brain innate immune system, may be activated also by misfolded proteins or aberrant endogenous molecular patterns that are accumulated in the brain of patients affected by several neurodegenerative disorders. These stimuli are responsible for chronic neuroinflammation by triggering the inflammasome's formation and subsequent caspase-1 activation. Proinflammatory cytokines are processed by active caspase-1 into their mature form that leads to cytokines production and, ultimately, chronic inflammation. Furthermore, the extensive generation of proinflammatory cytokines (IL-1β, IL-18) mediated from activated caspase-1, leads to a new process of programmed cell death called pyroptosis that plays an important role in several neurological diseases. Moreover, recent findings indicate caspase-1 as modulator for the activation of caspase-6-mediated axonal degeneration in AD. These findings have changed the view of the role of chronic neuroinflammation in neurodegenerative diseases, showing that this could be the primary cause of the neurodegeneration and not a simple effect of the pathologies. Additionally, there is evidence of the key role of caspase-1 in neurodegeneration, and its inhibition might effectively interfere with the progression of neurological disorders. So, caspase-1 inhibitors are very promising candidates for the treatment of neurodegenerative diseases. For this purpose, we have designed and synthesized new potent and selective non-peptidic, non-covalent and cell permeable caspase-1 inhibitors, which have shown a very high activity in

suppressing the formation of IL-1 in LPS induced inflammation.

Acknowlegment: Research funded by Regione Autonoma della Sardegna- Legge Regionale 7 References: a) Heneka, M. T.; Kummer, M. P.; Latz, E. Nat. Rev. Immunol. 2014, 14, 463–477. b)

Agyemang, A. F.; Harrison, S. R.; Siegel, R. M.; McDermott, M. F. Semin. Immunopathol. 2015, 37,

335-347. c) Kaushal, V.; Dye, R.; Pakavathkumar, P.; Foveau, B.; Flores, J.; Hyman, B.; Ghetti, B.; Koller, B. H.; LeBlanc, a C. Cell Death Differ. 2015, 1–11.


Oral 2

SUSTAINABLE COUPLING REACTIONS OF NATURAL BIOACTIVE HYDROXYLATED PHENOLS

Fabbri D., Dettori M. A., Delogu G.

CNR - Institute of Biomolecular Chemistry, Traversa La Crucca 3, I-07100 Sassari, Italy email: [email protected]

Hydroxylated phenols like vanillin (4-hydroxy-3-methoxybenzaldehyde) 1, apocynin (1-(4-hydroxy-3-methoxyphenyl)-ethanone 2, iso-apocynin 1-(2-hydroxy-3-methoxyphenyl)ethanone), 3 creosol (2-methoxy-4-methylphenol) 4, zingerone (4-(4-hydroxy-3-methoxyphenyl)-2-butanone) 5 and eugenol (2-methoxy-4-(propen-2-yl)-phenol) 6, are naturally occurring compounds, present, as main components, in various plants like, Vanilla planifolia, Picrorhiza kurroa, Guaiacum officinale, Zingiber officinalis and Eugenia carofillata. These compounds show a broad spectrum of biological properties such as antiinflammatory, antiseptic, antioxidant and fungicide.a,b The corresponding C2-symmetric dimers, named hydroxylated biphenyls, generally show a higher bioactivity in comparison to the monomers.c Our interest in the field of hydroxylated biphenyls pushed us towards the optimization of oxidative coupling reactions of monomers 1-6 with particular attention to aspects of environmental sustainability of the synthesis. The reactions reported in the literature for C2 symmetric dimerization of phenols 1-6, contemplate reactions mediated by chemical inorganic oxidants,d,e typically iron sulphate and potassium persulfate, or enzymatic reactionsf (e.g. horseradish peroxidase). Surprisingly, to the best of our knowledge, there are not reported oxidative coupling reactions carried out with the aid of microwaves on natural phenols 1-6.

Dimers 7-12 are important precursors of most of bioactive hydroxylated biphenyls recently prepared by our group.g-l Our work is therefore aimed at optimizing, with the aid of microwaves, the synthetic process in terms of reduction of the amount of solvent, exclusive use of water as reaction medium and simplification of work-up operations. Acknowlegment: Research funded by: L.R. n.7 : "Promozione della ricerca scientifica e dell'innovazione

tecnologica in Sardegna”: crp-17136 “Salute & Trigu” 2012-1014, crp-25114 “Cosmesagro” 2012-1014 and

PRIN D.M. 1152/ric del 27/12/2011, “Cell wall determinants to improve durum wheat resistance to Fusarium

diseases”.

References: a) Kim, J-H. et al. Plos one, 2014, 9, 1-10. b) Lin, C-H. et al. Mol. Plant-Micr. Interact. 2009, 22, 942-952. c) Klees, R. et al. J. Biomed. Biotechnol. 2006, 2:87246. d) Forsythe, W. G. et al. Green Chem. 2013, 15, 3031-3038. e) Delomenede, M. et al. J. Med. Chem. 2008, 51, 3171-3181. f) Antoniotti, S. et al. Org. Lett. 2004, 6, 1975-1978. g) Dettori, M.A. et al. Lett. Drug Des. Discov. 2015, 12, 131-139; h) Marchiani, A. et al. Amino Acids, 2013, 45 (2), 327-338; i) Rozzo, C. et al. Mol. Cancer, 2013, 12:37. l) Pani, G. et al. J. Agr. Food. Chem. 2014, 62 (22), 4969-4977.

http://www.ncbi.nlm.nih.gov/pubmed/16883056


Oral 3

ENDOCANNABINOIDOMICS: “OMICS” APPROACHES APPLIED TO ENDOCANNABINOIDS AND ENDOCANNABINOID-LIKE

MEDIATORS

Piscitelli F. 1, Bisogno T.1, Giordano C.2, Luongo L.2, Maione S.2, Di

Marzo V.1

1 Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078, Pozzuoli (NA), Italy; 2 Department of Experimental

Medicine/Division of Pharmacology, Second University of Naples, via

Costantinopoli, 16 80138, Naples, Italy. email: [email protected]

Endocannabinoids (ECs) are defined as the endogenous ligands of cannabinoid receptors (CB1 and CB2) and a growing body of evidence has emerged on the role of the endocannabinoid system (ECS) in the regulation of several physiological conditions and numerous diseases. The development of analytical techniques for endocannabinoid measurement in several biological matrices has been helpful for the understanding of the physiological and pathological role of these mediators. Moreover, since ECs and EC-like molecules are all derivatives of fatty acids, several lipidomics approaches have been applied to date. The application of targeted lipidomics methods to the analysis of ECs and EC-related mediators is part of those methodologies used to investigate the “endocannabinoidome” and defined as “endocannabinoidomics” and increasing evidence has shown the importance of this strategy to identify new EC-like molecules and define their biological role and metabolic pathways (a). In particular, the application of “endocannabinoidomics” tecnique in a model of mouse traumatic brain injury (TBI) led to the identification of a poorly investigated EC-related molecule, the N-oleoylglycine (OlGly) in the injured brain. Therefore, to test the anti-hyperalgesic and neuroprotective effects of OlGly mice underwent TBI using the weight drop model and were divided into seven experimental groups: naïve, sham, sham+OlGly 50 and 100 mg/kg, TBI, TBI+OlGly 50 and 100 mg/kg. Animals were treated with OlGly (i.p.) for 14 days once a day, starting one day after injury and underwent, several behavioural tests to assess pain and depression. At the end of the treatment, the animals were decapitated and brains were dissected for endocannabinoid analysis. Interestingly, treatment with OlGly normalized motor impairment and reckless behavior; reduced thermal hyperalgesia and mechanical allodynia and, moreover, normalized aggressiveness and depression induced by TBI. On the other hand, the levels of endocannabinoids did not undergo any change 65 days after TBI. In conclusions, the application of a very targeted method for the discovery of new EC-like mediators is becoming necessary for the understanding of their biological role and the development of new diagnostic biomarkers and this study, described here, highlighted the neuroprotective role of a new bioactive lipid in a model of brain injury. References: a) Piscitelli F. In: Di Marzo & Wang (Eds.), Elsevier Academic Press, London, 2015, 137-149


Oral 4

SYNTHESIS AND CHARACTERIZATION OF AMINOPROLINE-

BASED RGD SEMIPEPTIDES TARGETING V3 INTEGRINS AND THEIR UTILITY IN MEDICINE

Burreddu P.1, Battistini L.2, Sartori A.2, Rassu G.1, Casiraghi G.2,

Zanardi F.2

1Istituto di Chimica Biomolecolare del CNR, Traversa La Crucca 3, 07100 Li Punti, Sassari, Italy. 2Dipartimento di Farmacia, Università degli Studi di Parma, V.le G.P.

Usberti 27A, 43124 Parma, Italy email: [email protected]

In recent past years, integrin receptors have been the focus of intense research directed to elucidate their structure, function and regulation.1 In the cancer-related field, the expression of particular integrins is correlated with disease progression and decreased patient survival in various tumor types, rendering these integrin

families appealing targets for cancer therapy. The V3 integrins, among others, have been identified as useful biomarkers of tumor angiogenesis and tumor progression, invasion and metastasis, being overexpressed on proliferating endothelial cells as well as various tumor-related cells. A number of specific, highly

potent V3-targeting small molecule ligands have been developed so far, which contain or mimic the essential RGD binding motif.2

Notable results from our laboratory in the design, synthesis and characterization of aminoproline-based integrin binders (AmpRGD) will be presented, which displayed

nanomolar binding affinity toward the isolated V3 integrin receptor.3 The preparation and evaluation of both covalent and nanostructured assemblies will also be discussed, wherein appropriate cytotoxic or imaging cargos are consigned to the AmpRGD semipeptide vectors, to be used as novel anti-angiogenic therapeutic/diagnostic tools. (1) Cox, D.; Brennan, M.; Moran, N. Nature Rev. Drug Discovery 2010, 9, 804-820. (2) Auzzas, L.; Zanardi, F.; Battistini, L.; Burreddu, P.; Carta, P.; Rassu, G.; Curti, C.; Casiraghi, G. Curr. Med.Chem. 2010 , 17, 1255-1299. (3) (a) Battistini, L.; Burreddu, P.; Sartori, A.; Arosio, D.; Manzoni, L.;Luigi Paduano, L.; D’Errico, G.; Sala, R.; Reia, L.; Bonomini, S.; Rassu, G.; and Zanardi, F. Mol Pharmaceutics, 2014, 11, 2280-2293. (b) Pilkington-Miksa, M.; Arosio, D.; Battistini, L.; Belvisi, L.; De Matteo, M.; Vasile, F.; Burreddu, P.; Carta, P.; Rassu, G.; Perego, P.;Carenini, N.; Zunino, F.; De Cesare, M.; Castiglioni, V.; Scanziani, E.; Scolastico, C.; Casiraghi, G.; Zanardi, F.; Manzoni, L. Bioconjugate Chem., 2012, 23, 1610-1622.


Oral 5

SCREENING OF N,N-BIDENTATE PYRIDINE-BASED LIGANDS IN COPPER AND PALLADIUM

CATALYSED REACTIONS

Solinas M.

Istituto di Chimica Biomolecolare, UOS di Sassari Traversa La Crucca, 3 – Regione Baldinca, Li Punti, 07100 Sassari

email: [email protected]

In the past few decades, a plethora of efficient chiral ligands has been developed in several asymmetric reactions. Some of these ligands are mixed bidentate donor ligands with P–P or P–N chelating mode, although several interesting examples of N,N-chelating ligands such as diamines, pyridine-based ligands (2,2’-bipyridines, 1,10-phenanthrolines, pyridyl amines, pyridyl oxazolines, etc.) and oxazoline-containing ligands have also proven their efficiency in several processes. Moreover, chiral N,N-ligands based on imino and pyridine moieties have become a large and important topic in asymmetric synthesis. In our ongoing research efforts aimed at the application of chiral ligands based on the pyridine framework to asymmetric catalysis, we present here the potential of a variety of chiral N,N-bidentate ligands in the asymmetric Cu(I)-catalysed allylic oxidation of cyclic alkene, in the Cu(II)-catalyzed Henry reaction and in the Pd-catalyzed allylic alkylation of 1,3-diphenylprop-2-enyl acetate with dimethyl malonate.

Acknowlegment: Research funded by Fondazione Banco di Sardegna References: a) Maurizio Solinas, Barbara Sechi, Salvatore Baldino, Giorgio Chelucci J. Mol. Catal. A Chem. 378 (2013) 206–212. b) Maurizio Solinas, Barbara Sechi, Giorgio Chelucci, Salvatore Baldino, José R. Pedro, Gonzalo Blay J. Mol. Catal. A Chem. 385 (2014) 73–77. c) Maurizio Solinas, Barbara Sechi, Giorgio Chelucci Appl. Organometal. Chem. 2014, 28, 831–834.


Oral 6

PEPTAIBOLS IN MEMBRANE-LIKE ENVIRONMENTS: USE OF NITROXYL-LABELED PEPTIDES TO GET INSIGHT INTO

MECHANISM OF ACTION

Biondi B.1, Bortolus M.2, Dalzini A.2, De Zotti M.2, Formaggio F.2, Peggion C.2

1Institute of Biomolecular Chemistry, Padova Unit, 35131, Padova

2Department of Chemical Sciences, University of Padova, 35131, Padova email: [email protected]

Peptaibols are a class of natural occurring antimicrobial peptides (AMPs), characterized by a high content of the non-coded Cα-tetrasubstituted amino acid Aib (α-aminoisobutyric acid), a C-terminal 1,2-amino alcohol, and acylated at the N-terminus. Peptaibols are particularly attractive for their resistance to enzymatic degradation and for a remarkable bioactivity, displayed even by short sequences, due to the presence of Aib that favors very stable secondary structures. Like all AMPs, peptaibols exert their antimicrobial action through a permeation of the bacterial membranesa. In this contribution, we report on the syntheses of various analogs of different peptaibols in which the Aib residue is replaced with the achiral tetrasubstituted α-amino acid 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine-4-carboxylic acid (TOAC). TOAC amino acid, which presents a stable nitroxyl radical fixed on a rigid heterocyclic structure, can be used as paramagnetic probe to study, by electron spin resonance (ESR), the mode in which peptides interact with a membraneb. In the last decade, a great attention has been devoted to the use of AMPs as anticancer agents. Despite differences in the sequences there are some common characteristics: they are often positively charged and adopt an amphiphilic helical structure in membrane environment. Most AMPs attack cancer cells by disrupting the cell membrane without binding to a specific receptor, thus by-passing the ability of cancer cells to adapt and developing treatment resistance. In this context, we synthesized various analogs of the natural peptaibol trichogin GA IVc, some of which bearing a nitroxyl probe at different positions, in order to evaluate the selectivity of trichogin analogs toward tumor cells and correlate the variance in bioactivity to different mechanism of peptide-membrane interaction.

Acknowlegment: CARIPARO Foundation (Progetto di Eccellenza 2011-12). References: a) Toniolo, C. and Brückner, H., Peptaibiotics, Wiley/VCH, Weinheim/ Zürich, 2009. b) Marsh, D., Jost, M., Peggion, C., Toniolo, C. Biophys. J., 2007, 92, 473-481.

c) De Zotti, M.; Biondi, B.; Peggion, C.; Formaggio, F.; Park, Y.; Hahm, K.-S.; Toniolo C. Org. Biomol. Chem., 2012, 10, 1285-1299.


Oral 7

EFFICIENT AND ENVIRONMENTALLY FRIENDLY METHODOLOGIES FOR THE SYNTHESIS OF

MULTIFUNCTIONALIZED SCAFFOLDS

Villano R.

ICB-CNR, trav. La Crucca 3, 07100 Sassari email: [email protected]

The vinylogous addition of Chan’s diene 11 to aldehydes is one of the most elegant and powerful methods for the synthesis of δ-hydroxy-β-ketoesters 2 (Scheme 1) which are useful key-intermediates in the synthesis of many natural products;2 so the development of innovative methodologies for this reaction represents an interesting research area.

Scheme 1 The reaction has been intensely investigated over the last decade and particular attention has recently focused on the use of low impact solvents and catalytic amounts of promoters, according to the growing demand for highly sustainable chemical procedures. In the light of the above, an overview of several convenient and efficient methodologies3 for the addition of Chan’s diene to aldehydes will be presented, with particular attention to experimental conditions and catalytic systems (including organometallic complexes and organocatalysts). Finally, some applications of these methodologies in the total synthesis of complex molecules will be discussed.

Acknowledgment: Research funded by Università degli Studi di Salerno References: 1) a) Yamamoto, K.; Suzuki, S.; Tsuji, J. Chemistry Letters 1978, 649. b) Chan, T.-H; Brownbridge, P. J. C. S. Chem. Comm. 1979, 578. c) Brownbridge, P.; Chan, T.-H; Brook, M. A.; Kang, G. J. Can. J. Chem. 1983, 61, 688. 2) a) Evans, D.A.; Hu, H.; Burch, J.D.; Jaeschke, G. J. Am. Chem. Soc. 2002, 124, 5654. b) Peterson, I.; Davies, R.D.M.; Marquez, R. Angew. Chem. Int. Ed. 2001, 40, 603. c) Evans, D.A.; Ripin, D.H.B.; Halstead, D.P.; Campos, K.R. J. Am. Chem. Soc. 1999, 121, 6816. d) Evans, D.A.; Carter, H.P.; Carreira, E.M.; Charette, A.B.; Prunet, J.A.; Lautens, M. J. Am. Chem. Soc. 1999, 121, 7540. 3) a) Soriente, A.; De Rosa, M.; Villano, R.; Scettri, A. Curr. Org. Chem. 2004, 8, 993. b) Villano, R.; Acocella, M. R.; Massa, A.; Palombi, L.; Scettri, A. Tetrahedron Asymmetry 2006, 17, 3332. c) Villano, R.; Acocella, M. R.; Massa, A.; Palombi, L.; Scettri, A. Tetrahedron 2009, 65, 5571. d) Villano,R.; Acocella, M. R.; De Sio, V.; Scettri, A. Cent. Eur. J. Chem. 2010, 8, 1172. e) Villano, R.; Acocella, M. R.; Scettri, A. Tetrahedron 2011, 67, 2768.

1

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mailto:[email protected]


Oral 8

ASSEMBLY OF A SUCCINYL-CALIX[4]ARENE DERIVATIVE IN MULTILAMELLAR VESICLES

Granata G.1, Consoli G. M. L.1, Geraci C.1, Lo Nigro R.2, Malandrino G.3

1 Istituto di Chimica Biomolecolare-C.N.R., Via P. Gaifami 18, 95126, Catania, Italy.

2 Istituto per la Microelettronica e Microsistemi-C.N.R., Strada Ottava 5, 95121, Catania, Italy.

3 Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125, Catania, Italy.


Self-assembled structures built from amphiphilic molecules find application in different fields. a The synthetic versatility of calixarene macrocycles, whose skeleton can be differently functionalized with a variety of hydrophilic and hydrophobic moieties, allows the development of amphiphilic derivatives able to aggregate in different higher order supramolecular architectures. b, c Here we describe the capability of an amphiphilic calix[4]arene derivative, bearing four carboxylic acid groups at the calixarene upper rim and four dodecyl chains at the lower rim, to form discrete and stable multilamellar vesicles in aqueous medium by using an emulsion/solvent evaporation method. Dynamic Light Scattering (DLS) measurements, Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) images provided information about size, size distribution and morphology of the nanostructured assemblies. The vesicular nature was confirmed by entrapment of a hydrophilic dye such as rodhamine B in aqueous lumen. A preliminary study showed that the self-assembled structures of the carboxy-calix[4]arene can entrap also a hydrophobic drug such as curcumin. The amphiphilic carboxy-calix[4]arene represents the first example of calix[4]arene derivative assembling in very stable multilamellar vesicles in aqueous medium with a narrow polydispersity. Suitable nanometer-size, very size narrow distribution, well-defined spherical shape, negatively charged hydrophilic multilamellar wall, high size stability (more than two years), and guest encapsulation ability make the vesicles of the carboxy-calix[4]arene derivative appealing in the research of drug delivery systems.

Acknowledgment: Research funded by PON R&C 02_00355_2964193 (MIUR, Rome). References: a) Hill, J. P.; Shrestha, L. K.; Ishihara, S.; Ji, Q.; Ariga, K. Molecules 2014, 19, 8589-8609. b) Helttunen, K.; Shahgaldian, P. New J. Chem. 2010, 34, 2704–2714. c) Consoli, G. M. L.; Granata, G.; Lo Nigro, R.; Malandrino, G.; Geraci, C. Langmuir 2008, 24, 6194–6200.


Oral 9

A ‘SEA’ OF BIOACTIVE NATURAL PRODUCTS

Cutignano A.

Institute of Biomolecular Chemistry – CNR, via Campi Flegrei 34 - 80078

Pozzuoli (NA) email: [email protected]

The marine environment is distinguished by unique groups of organisms that hide a treasure of fascinating and peculiar structures. The extraordinary chemical diversity of marine organisms often mirrors potent biological activities that represent the basis for the ecological function of marine natural products but also a promise for pharmacological applications. In our ongoing search for novel marine bioactive molecules we recently focused our attention on species from less explored habitats and on cultivable micro-organisms, either from commercial sources or isolated from the Mediterranean Area. Promising stories of bioactive natural or naturally inspired molecules, identified following traditional approaches will be discussed, highlighting the results obtained very recently through a multidisciplinary strategy that embraces expertise in the fields of the natural product chemistry, organic synthesis and pharmacological assays. Furthermore, in the frame of two PON projects aiming at discover new immunomodulant and anti-infective agents from marine sources, we validated and applied an automated chromatographic platform to the screening of a library of raw extracts from marine microalgae and sponges. The process is well suited for high throughput screening program and entails dramatic time saving along with effective desalting and reproducible fractionation of marine extracts. The potential of the developed protocol will be illustrated through key examples of bioactive molecules identified in few steps.

Acknowlegments: Research funded by PON Projects 01/00117 and 01/02093 and by PNRA Project 2009/A1.06


Oral 10

NMR METABOLOMIC APPLICATIONS ON OLIVE OILS:

PEDOCLIMATIC AND SENSORY DIFFERENTIATION. FROM

SINGLE PULSE NMR TO ADIABATIC SEQUENCES

Culeddu N.1, Chessa M.1, Molinu M.G.2, Deiana P.3, Dettori S.3 and

Santona M.3

1) CNR – Institute of Biomolecular Biochemistry UOS Sassari, via La crucca

3, I-7100 Sassari


2) CNR – ISPA Institute Of Sciences of Food Production - UOS Sassari via La

crucca 3, I-7100 Sassari

3) University of Sassari - Dept. of Sciences for Nature and Environmental

Resources, via De Nicola 9 I-7100 Sassari.

Olive oil is a fundamental constituent of the traditional Mediterranean diet, which is continuously attracting the interest of the scientific community for its health-protecting activities (1). The protective role of olive oil in fighting certain diseases has been attributed to its fatty acid composition and the presence of minor constituents, mainly phenolic compounds and squalene. Properly extracted from fresh ripe fruits, the oil is characteristically aromatic and slightly bitter. The sensory characteristic of virgin olive oil has been related with the presence of phenolic compounds derived from the hydrolysis of oleuropein, aldehydes and terpenes, (2)

We show some models obtained by metabolomic analysis of NMR data obtained from standard NMR sequences, multi suppression sequences and novel selective excitation scheme to analyze the aldehydes and polyphenols in olive oil without the need for extraction/concentration of these analytes.

These models take in account the relationship between NMR data vs pedoclimatic description, soil composition and sensory analysis.

We recognized also the instability of oleuropein when it react with methanol, leading to the artificial formation of isomers.

1) Frankel, E. N. J. Agric. Food Chem. 2011, 59, 785-792.

2) Boskou D., OLIVE OIL:Minor Constituents and Health CRC press 2009


Oral 11

ONE-POT MICROWAVE ASSISTED CATALYTIC TRANSFORMATION OF WASTE FRYING OIL INTO GLYCEROL-FREE BIODIESEL

Drago C., Nicolosi G.

Istituto di Chimica Biomolecolare, CNR, via Paolo Gaifami 18, I-95126 Catania; Italy, email: [email protected]

Biofuel production has increased in recent years and some commodities like corn, sugar cane or vegetable oil can be used either as food, feed, or to make biofuels. An alternative to overcome the "Food versus Fuel" dilemma is to use only non-food crops as feedstock for biofuel production.a However, the massive exploitation of land for biofuels production is often a consequence of its sterilization and desertification. So, long-term sustainability for biofuels can be accomplished by exploiting alternative feedstocks from non-arable land. A priceless and non-negligible feedstock would arise from waste recyclability and their reuse. In this contest our attention is focused to the WFOs (waste frying oils) from household and industrial sources.b Recently, We have reported a catalytic one-pot microwave assisted transformation of vegetable oils to a mixture of FAMEs (methyl esters of fatty acids) and glycerol ether derivatives, using the commercially available TBME (tert-butyl metyl ether) as single source both as transesterification and transetherification reagent.c,d The present study is an attempt to

adapt the already developed protocol to the transformation of WFO, which we received from a local restaurant, to a final potential biofuel. It is necessary to note that the high temperature of the frying processes and the water from the foods accelerate the hydrolysis of triglycerides, and it increases the FFAs (Free Fatty Acids) content in the oil.

Usually, vegetable oils with high content of FFA are not preferred for biodiesel production because they need a pretreatment to convert the FFA to the corresponding methyl esters.e Preliminary analysis on the received WFO showed a quantity of FFAs 10 times major to the same oil before its use. When a sample of WFO and TBME (1:10), in presence of 1% of the selected catalyst (w/w), was submitted to the microwave radiation, in 3 hours, we observed a complete conversion into the corresponding mixture of the corresponding FAMEs and a mixture of glycerol ethers derivatives. Interesting to highlight a complete conversion of FFAs to FAMEs and, as desired, a negligible traces of glycerol were detected.f There are several practical and cost-saving advantages making this process very attractive, among which the WFO could be directly transformed without any preliminary pretreatment, the obtained mixture, which is without glycerol, only needs to be separated from the catalyst, achieved by a very simple filtration, and it may be used directly in the energy chain.

Acknowledgements: The authors acknowledge the financial support provided by EFOR-CNR Project. References: a) Atabani, A. E.; Silitonga, A. S.; et al.; Renew. Sust. Energ. Rev.; 2013, 18, 211-245. b) Wermelinger, V. K.; Araujo; et al.; Bioresource Technology 2010, 101, 4415-4422. c) Drago, C.; Liotta, L. F.; La Parola, V.; Testa, M. L.; Nicolosi, G.; PCT N° PCT/IB2014/058783. d) Drago, C.; Liotta, L. F.; La Parola, V.; Testa, M. L.; Nicolosi, G.; Fuel, 2013, 113, 707. e) Melero, J. A.; Iglesias, J.; Morales, G.; Green Chem. 2009, 11,1285–1308. f) Drago, C.; Nicolosi, G.; et al.; manuscript in preparation.

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Oral 12

CARRYING ON THE ROUTE OF MARINE GUANIDINES: ISOLATION, SYNTHESIS AND BIOACTIVITY OF A GUANIDINE

TERPENE FROM THE MOLLUSK ACTINOCYCLUS PAPILLATUS

Gavagnin M.1, Carbone M.1, Ciavatta M. L.1, Kiss R.2,

Mathieu V. 2, Mollo E.1, Guo Y.-W.3

1Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078, Pozzuoli

2Laboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium,

3State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,

CAS, Zu Chong Zhi Road 555, 201203,Shanghai

email: [email protected] Guanidine is one of the most widely used scaffold in the design and synthesis of new bioactive compounds. Numerous guanidine compounds from natural sources as well as those obtained through synthetic strategies and virtual design have gained much interest over the last few decades due to their therapeutic potential.a-

c In nature the Y-shaped CN3 unit is mainly incorporated in peptide, polyketide, and aromatic structures whereas only a limited number of guanidine compounds possess a terpene framework. Despite their low occurrence, terpene guanidines have shown interesting biological activities, standing out as promising lead structures suitable for development of potential drugs. Prenylated guanidines from higher plants, for example, display antitumor, anti-angiogenic, antimicrobial, insulin-like and anti-lipolytic properties.c Continuing our studies on guanidines from marine mollusks that have led in recent years to the characterization of bioactive phidianidines,d-f we have now isolated actinofide (1), from the Chinese nudibranch mollusk Actinocyclus papillatus.

The structure exhibiting two terpenoid acyl residues linked to a guanidine unit has been determined by spectroscopic methods and confirmed by synthesis. Actinofide (1) has been tested for the inhibition of cellular growth on a panel of

cancer cell lines showing activity at a concentration less than 10 M against selected cell lines. Starting from the synthesis of 1, a small library of linear terpene acyl guanidines (with C5, C15 and C20 fragments) have been also prepared for the pharmacological screening. References: (a) Rauf, M.K.; Din, I.; Badshah A. Expert Opin. Drug. Discov. 2014, 9, 39-53. (b) Saczewski, F.; Balewski, L. Expert Opin. Ther. Patents 2013, 23, 965-995. (c) Berlinck, R.G.S.; Trindade-Silva, A.E.; Santos M.F.C. Nat. Prod. Rep. 2012, 29, 1382-1406. (d) Carbone, M.; Li, Y.; Irace, C.; Mollo, E.; Castelluccio, F.; Di Pascale, A.; Cimino, G.; Santamaria, R.; Guo, Y.-W.; Gavagnin, M. Org. Letters 2011, 13, 2516–2519. (e) Manzo, E.; Pagano, D.; Ciavatta, M.L.; Carbone, M.; Gavagnin M. 2013, PCT/IB2013/052,163; 2015, U.S. Patent 9.085.569. (f) Vitale, R.M.; Gatti, M.; Carbone, M.; Barbieri, F.; Felicità, V.; Gavagnin, M.; Florio, T.; Amodeo, P. ACS Chem. Biol. 2013, 8, 2762–2770.


Poster Presentations


Poster 1

INFLUENZA VIRUS PA ENDONUCLEASE: VIRTUAL SCREENING, DOCKING AND IN-VITRO VALIDATION OF NOVEL INHIBITORS

Pala N.1, Dallocchio R.2, Dessì A.2, Cadoni R.1, Carcelli M.3, Stevaert

A.4, Sechi M.1, Naesens L.4

1 di Sassari, Via Vienna 2, 07100

Sassari, Italy

2Istituto di Chimica Biomolecolare, CNR−Consiglio Nazionale delle Ricerche,

Sassari, 07100 Li Punti Italy, mail to [email protected], [email protected],

3 , Parco Area delle Scienze 17/A,

43124 Parma, Italy

4Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-

3000 Leuven, Belgium

Vaccination and antiviral agents are the primary measures against human influenza viruses. However, both interventions have shortcomings, and hence, there is an urgent need to develop new and more efficacious anti-influenza virus drugs. The influenza virus RNA-dependent RNA polymerase complex (RdRp), a heterotrimeric protein complex that is responsible for viral RNA transcription, represents an unexplored target for antiviral drug development. One particularly attractive approach is interference with the endonucleolytic “cap-snatching” reaction by the PA subunit of the RdRp, more precisely by inhibiting the metal-dependent catalytic activity which resides in the N-terminal part of PA (PA-Nter). In the last two decades, several small molecule PA inhibitors (PAIs) have been discovered. Among them, compounds belonging to the class of substituted 2,4-dioxobutanoic acids were identified as particularly potent and selective PAIs in both enzyme and cell-based assays. A few other classes of potential PAIs have been identified. All these diverse compounds bear distinct pharmacophoric fragments with chelating motifs able to bind the bivalent metal ions in the catalytic core of PA-Nter. More recently, the availability of crystallographic structures of PA-Nter has enabled rational design of PAIs with improved binding properties. Here, we present a new coupled pharmacophore/docking virtual screening approach that allowed us to identify novel PAIs having interesting inhibitory activity in a PA-Nter enzymatic assay, as well as antiviral activity in a cell-based influenza virus yield assay.

References: 1) Stevaert, A.; Dallocchio, R.; Dessì, A.; Pala, N.; Rogolino, D.; Sechi, M.; Naesens, L.; J. Virol. 2013, 87, 10524−10538. 2) Pala, N.; Dallocchio, R.; Dessì, A.; Brancale, A.; Carta, F.; Ihm, S.; Maresca, A.; Sechi, M.; Supuran, C. T.; Bioorg. Med. Chem. Lett. 2011, 21, 2515−2520. 3) Pala, N.; Stevaert, A.; Dallocchio, R.; Dessì, A.; Rogolino, D.; Carcelli,M.; Sanna, V.; Sechi, M.; Naesens, L.; ACS Med. Chem. Lett. 2015, Just Accepted Manuscript DOI: 10.1021/acsmedchemlett.5b00109.




Poster 2

THE HEMORPHINS: A NEW CLASS OF OPIOID PEPTIDES DERIVED FROM HEMOGLOBIN

Fenude E.1, Vite V.2

1ICB, Trav. La Crucca n.3, 07040, Sassari

2Corso di Laurea Specialistica in Chimica e Tecnologie Farmacautiche, Dipartimento di Chimica e Farmacia, Università di Sassari


Many biologically active peptides are generated by proteolytic processing of various higher molecular weight multifunctional precursor peptides and proteins. A series of ”nonclassical” endogenous opioid peptides (hemorphins) have been identified in the course of the study of proteolytic fragments of bovine blood hemoglobin. Depending on the N-terminal sequences the sub-families of LVV-emorphins, VV-hemorphins, and V-hemorphins can be distinguished. It was shown that the overall activity varied considerably among different hemorphins which points to the importance of the N-terminal hydrophobic residues on structure-function relationship. It is well accepted that the binding of the ligand to its receptor is mediated by ion-ion interactions, hydrogen bonding, dipole-dipole interactions, lipophilicity, and shape complementarity but the relative contributions of each of these interactions is still poor understood. We perused this goal by examination of the conformational and dynamic properties of synthetic analogues sequences carefully selected for their complementary biological properties. In this work it has been observed tetra- and penta-peptides fragments of N-terminal protected LVV-hemorphins which are well known to be opioid receptor ligands and immunoregulatory peptides in order to study the structural-functional peculiarity. Structural study in CHCl3 has been directed towards peptides with the follow aminoacidic sequence: Boc-Leu-Val-Val-OMe, Boc-Leu-Val-Val-Tyr-OMe, Boc-Leu-Val-Val-Phe-OMe, Boc-Leu-Val-Val-Tyr-Pro-OMe. These products have been synthesized, purified, and then analyzed by NMR spectroscopy, employing both mono- and bi- dimensional homo- and hetero- nuclear correlation 1H-1H, 1H-13C techniques through which it is possible to obtain structural and conformational informations. The result obtained are compared and discussed. References Fenude E., Dettori A., Demontis M.P., Alberico E., & al., Pharm. Res., 2003, 47, 53 Fenude E., Dedola S., Fais M., VII Convegno “Complex Systems: structure, properties, reactivity and dynamics, 2005, Alghero, 13-15 Giugno Fenude E., Roggio A.M., XXII Congresso Nazionale della Società Chimica Italiana, 2006,10-15 Settembre Firenze


Poster 3

POLYSACCHARIDE-BASED CHIRAL STATIONARY PHASES: EXPLOITING THE SIZE AND SHAPE OF THE MOLECULES FOR

CHIRAL RECOGNITION AND ENANTIOSEPARATIONS

Peluso P.



In keeping with the importance of chirality, chemistry invests resources to access pure enantiomers, reproducing homochirality in xenobiotic chemical contexts and understanding the mechanisms that govern the chiral discrimination processes. The real meaning of chirality lies in the relational and directional sense of the word. It means that two enantiomers have exactly the same properties in isotropic conditions. On the contrary, a chiral enantiopure molecule can recognize two enantiomers through stereoselective and anisotropic interactions. Nowadays, chemists largely use chromatography on chiral support for resolving racemic mixtures and a number of reasons make HPLC the preferred technique for enantioseparations. In the last decades, polysaccharide-based chiral stationary phases (CSPs) have been preferred for HPLC enantioseparations and, currently, the market makes different chiral columns available, which contain selectors based on cellulose and amylose derivatives. In this field, understanding the chiral recognition mechanisms is still a challenge.

The results obtained in this study a) prove that the anisotropic properties of the molecules as well as their size and shape deeply influence the enantioseparabilitya and b) explain why similar molecules produce very different enantioseparations (A) or why very diverse molecules are able to give analogous separations (B).b-d Acknowledgment: Research funded by Università Ca’ Foscari di Venezia (ADIR funds) and Regione Autonoma della Sardegna (L.R., August 7

th 2007, n. 7, call 2008).

References: a) Peluso, P.; Cossu, S. Chirality 2013, 25, 709-718. b) Peluso, P.; Mamane, V.; Aubert, E.; Cossu, S. J. Chromatogr. A 2012, 1251, 91-100. c) Peluso, P.; Mamane, V.; Aubert, E.; Cossu, S. J. Chromatogr. A 2014, 1345, 182-192. d) Peluso, P.; Mamane, V.; Cossu, S. Chirality 2015, DOI: 10.1002/chir.22485.


Poster 4

NOVOZYM-435 AS EFFICIENT CATALYST FOR THE SYNTHESIS OF BENZOIC AND (HETERO)AROMATIC CARBOXYLIC ACID

ESTERS

Sechi B., Giunta D., Marchetti M., Solinas M.



Hereby we report new reaction conditions to convert benzoic acids and more general (hetero)aromatic carboxylic acids into the corresponding n-heptyl esters by applying Novozym-435 as the biocatalyst in cyclohexane as the solvent. Very good yields are obtained in the esterification of a plethora of substituted acids with mild and straightforward reaction conditions. Direct esterification of the acid is favoured compared to transesterification of methyl benzoate under our reaction conditions in all cases studied. Recycling of the immobilised enzyme is feasible although with some minor limitations.

Acknowlegment: Research funded by Regione Autonoma della Sardegna (L.R., August 7

th 2007, n.

7, CRP_25257) References: a) Daniela Giunta, Maria Paola Masia, Mauro Marchetti, Raffaele Morrone, Maurizio Solinas Tetrahedron Letters 54 (2013) 5122–5125 b) Daniela Giunta, Barbara Sechi, Maurizio Solinas Tetrahedron 71 (2015) 2692-2697.


Poster 5

EFFICIENT BASE-FREE HYDROGENATION OF AMIDES TO ALCOHOLS AND AMINES CATALYSED BY WELL-DEFINED

PINCER IMIDAZOLYL-RUTHENIUM COMPLEXES

Alberico E.1, Cabrero-Antonino J. R.2, Drexler H.-J.2, Baumann W.2,

Junge K.2, Junge H.2, Beller M.2

1 Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Tr. La Crucca 3, 07100 Sassari (Italy); email: [email protected]

2 Leibniz-Institut für Katalyse e.V. Albert Einstein Str. 29a, 18059 Rostock (Germany).

Among the methods amenable to the synthesis of amines, catalytic hydrogenation of amides using molecular hydrogen1 is the most promising as to atom economy and waste prevention when compared to the use of LiAlH4 and B2H6 or metal promoted catalytic hydrosilylation. In order to overcome the low electrophilicity of their carbonyl groups the reduction of amides with molecular hydrogen is usually carried out at elevated pressures and temperatures, although recent improvement of heterogeneous catalysts has witnessed a mitigation of reaction conditions. The main drawback associated with the use of such catalysts is that they are incompatible with aromatic groups and double/triple CC bonds which are likewise reduced. This problem has been recently overcome by the development of a homogeneous ruthenium catalyst modified by 1,1,1tris(diphenylphosphinomethyl)ethane (Triphos) which operates in the presence of an acid cocatalyst. Yet it has a rather limited scope being best suited for 1° amides or substrates which bear a phenyl ring directly attached to the nitrogen atom and requires temperatures above 200 °C. Heterogeneous catalysts and the Ru/Triphos system promote the reduction of amides according to path a of Scheme 1. Homogeneous catalysts which rely on metal-ligand cooperation (bifunctional catalysis) instead pave the way to an alternative reactivity (Scheme 1, path b) in which the initial reduction of the carboxylic group is followed by collapse of the intermediate hemiaminal by cleavage of the C-N bond and reduction of the resulting aldehyde, overall affording the alcohol and amine. Herein we report the synthesis of two novel ruthenium pincer complexes bearing an imidazolylaminophosphino pincer ligand.2 The BH4-substituted one has demonstrated to be an efficient catalysts for the hydrogenation of different substituted amides to the corresponding alcohols and amines in excellent yields without the use of additional base. Scheme

Acknowlegment: Research funded by the ACS Pharmaceutical Roundtable References: 1) Smith A. M., Whyman R., Chem. Rev. 2014, 114, 5477; 2) Cabrero-Antonino J. R. ,Alberico E., Drexler H.-J., Baumann

W., Junge K., Junge H., Beller M. submitted to Chem.

Commun.



Poster 6

MOLECULAR DATABASE IMPLEMENTATION

Cosseddu A. Institute of Biomolecular Chemistry (ICB), Li Punti, 07100 Sassari


Implementation of Molecular Database ICB Support Units (UOS) located in Sassari has begun as activity in several projects to which the UOS of Sassari has participated. The database has been then extended and integrated with the largest and most comprehensive molecular database "StOrMoDB" generated by ICB (Amodeo P. et al). Over a span of time from 2000 to the present, new 915 molecules synthetized from five research groups of UOS of Sassari have been classified. The classification of individual molecules has been made through a common pattern that describes the main features such as structure, identification, purification, solubility, characterization (by NMR spectra, melting points, IR spectra, etc.), any biological activity. Each hit reports authors and scientific papers by which molecules has been described.

Structure Identification Spectra Bioattivity/Ecotox Reference

Chemical Name: (3E,3'E)-4,4'-(6,6'-dihydroxy-5,5'-dimethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one) Molecular Formula: C22H22O6 Molecular Weight: 382.41

Key Words: alfa-Synuclein, Biphenyl compounds, Curcumin-like molecules, Parkinson’s disease, Protein aggregation Label: VD040 Melting Point: 242-243 °C Source: synthetic compound Solubility: soluble in most common organic solvents Purification: filtration

1H, 13C NMR

Effect: -Synuclein interaction

-Synuclein aggregation

Test-System: circular dichroism and fluorescence spectroscopy Antioxidant activity Test-System: DPPH assays Test-System: TEAC assays

Neuroprotective activity Test-System: Cell viability Species: rat pheochromocytoma (PC12) cells

Amino Acids, 2013, 45:327–338 DOI: 10.1007/s00726-013-1503-3 CNR _ICB_UOS Padova [email protected] CNR _ICB_UOS Sassari [email protected] [email protected] [email protected] Food Chemistry, 2014, (157), 263–274 DOI: 10.1016/j.foodchem.2014.02.036 Institute of Organic Chemistry, Bulgarian Academy of Sciences [email protected] CNR _ICB_UOS Sassari [email protected] [email protected] [email protected]

In addition to entering into the most comprehensive Molecular Database StOrMoDB further application will be the possibility to carry out studies of the structure-activity against biological targets.

Acknowlegment: Research funded by BioTTasa - MISE- Dipartimento per lo Sviluppo e la Coesione Economica - Bando RIDITT Salude e Trigu – Regione Autonoma della Sardegna (L.R., August 7th 2007, n.7) Cluster Chimica Verde – MIUR - Avviso D.D. n. 257/Ric del 30 maggio 2012






Poster 7

ORGANOCATALYTIC, ASYMMETRIC ELIMINATIVE [4+2] CYCLOADDITION OF ALLYLIDENE MALONONITRILES WITH ENALS: RAPID ENTRY TO CYCLOHEXADIENE EMBEDDING

LINEAR AND ANGULAR POLYCYCLES

Zambrano V.1, Rassu G.1, Pinna L.2, Brindani N.3, Dell’Amico L.3,4, Curti C.3, Sartori A.3, Battistini L.3, Casiraghi G.3, Pelosi G.5, Greco D.3,

Zanardi F.3

1 Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Li Punti Sassari, Italy, 2 Dipartimento di Chimica e Farmacia Università degli Studi di

Sassari, Sassari, Italy, 3 Dipartimento di Farmacia,Università degli Studi di Parma, Parma, Italy. 4 Institute of Chemical Research of Catalonia, Spain Tarragona,

5 Dipartimento di Chimica, Università degli Studi di Parma, Parma, Italy email: [email protected]

Six-membered carbocycles, as well as five- and seven membered rings, are common motifs in nature and they can be found in many terpenoid, polyketide, and shikimate derived monocyclic and polycyclic molecular architectures.1 A direct aminocatalytic synthesis has been developed for the chemo-, regio-, diastereo-, and enantioselective construction of densely substituted polycyclic carbaldehydes containing fused cyclohexadiene rings2. The chemistry utilizes, for

the first time, remotely enolizable -extended allylidenemalononitriles as electron-rich 1,3-diene precursors in a direct eliminative [4+2] cycloaddition with both

aromatic and aliphatic -unsaturated aldehydes. The generality of the process is demonstrated by approaching 6,6-, 5,6-, 7,6-, 6,6,6-, and 6,5,6-fused ring systems, as well as biorelevant steroid-like 6,6,6,6,5- and 6,6,6,5,6-rings.

A stepwise reaction mechanism for the key [4+2] addition is proposed as a domino bisvinylogous Michael/Michael/retro-Michael reaction cascade. The utility of the malononitrile moiety as traceless activating group of the dicyano nucleophilic substrates is demonstrated. 1) P. M. Dewick in Medicinal Natural Products. A Biosynthetic Approach, 2nd ed., Wiley, Chichester, 2002. 2) N. Brindani, G. Rassu, L. Dell’Amico, V. Zambrano, L. Pinna, C. Curti, A. Sartori, L. Battistini, G. Casiraghi, G. Pelosi, D. Greco, F. Zanardi Angew. Chem. Int. Ed. 2015, 54, 7386 –7390. Financial support provided by the Università degli Studi di Parma


Poster 8

DETERMINATION OF FLAVOUR AND PIGMENT PROFILES IN PRE- AND POST-STABILIZATION FRUIT-BASED FOOD

FORMULATIONS

Sperlinga E., Napoli E., Siracusa L., Ruberto G.

ICB – CNR UOS CATANIA, Via Paolo Gaifami,18, 95126 Catania email: [email protected]

Shelf-life is defined as the maximum recommended time for which foods can be stored before becoming unsafe for consumption.a) In order to evaluate the shelf-life of a given food product there are many important parameters to consider, such as those related to its organoleptic features. The present research activity was carried out in collaboration with Dolfin s.p.a., a confectionery factory located in the province of Catania which provided the fruit-based formulation object of the study. Five aromatic commercial formulations (lemon, orange, berries, black cherry, strawberry) and three pigment commercial formulation (grape, shade lemon yellow, shade apple green) were used to proper set the analytical methods. The fruit-based formulation (apple mash, strawberry juice, water, strawberry flavour, natural red pigment) was subjected to accelerated shelf-life tests at different storage times (T0, T15, T30, T48, T62). Stabilization tests at room temperature were carried out after 90 and 180 days of storage. Finally, 3 different kind of secondary packaging were tested (984, NKME, cardboard) and they were compared to the one that is actually used. All the samples were subjected to the classical pasteurization process. Aroma compounds were extracted using the solid phase micro extraction (SPME) technique and the determination was carried out through GC-MS analysis. Pigments and phenolics were extracted using a solution of methanol/water/hydrochloric acid (80:19:1) and the extracts analyzed by HPLC-DAD, focusing on anthocyanins & cinnamic acids.

Results for accelerated tests samples showed that, after 15 days of storage, there was a significant (80%) decrement in anthocyanin content, which fell to more than 96% after 30 days. Chlorogenic acid (the dominant phenol in the formulation) was subjected to a slower degradation process; in fact, its content decreased of less than 50% after 62 days. Regarding flavours (mainly methyl cinnamate, ethyl

cinnamate and -decalactone) a quite stable trend all throughout the shelf-life tests was observed. Among the minor components, hexenyl acetate, ethyl eptanoate and α-terpineol were the ones that decreased more, while acetophenone and butyl isovalerate rose up. After 15 days, benzaldehyde, hexyl acetate and ethyl octanoate were formed. There were no significant differences among the other samples.

Acknowledgment: Research funded by “Programma Operativo Nazionale Ricerca e Competitività (R&C)”, 2007‐2013:SHELF‐LIFE PON02_00451_336190. References: a) Pasha, I.; Saeed, F.; Sultan, M.T.; Khan, M.R.; Rohi, M. Crit. Rev. Food Sci. Nutr. 2014, 54, 340-351.


Poster 9

EFFECTS OF TREHALOSE ON ALPHA-SYNUCLEIN STABILITY

REVEALED THROUGH SYNCHROTRON RADIATION CIRCULAR

DICHROISM

Ruzza P.1, Hussain R.2, Biondi B.1, Calderan A.1, Tessari I.3,

Bubacco L.3 and Siligardi G.2

1Institute of Biomolecular Chemistry of CNR, Padua Unit, Padua 35131, Italy;

2Diamond Light Source Ltd., Harwell Innovation Campus, Chilton, Didcot,

Oxfordshire OX11 0QX, UK; 3Department of Biology, University of Padua, Padua 35122, Italy.


Protein misfolding and aggregation characterized many neurodegenerative

diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases. Recently,

several research groups found that trehalose and other osmolytes are able to

interfere with protein misfolding and aggregation [a-c].

In the present work, we studied, by means of synchrotron radiation circular

dichroism (SRCD) spectroscopy, the dose-effect of trehalose on α-synuclein

conformation and/or stability to probe the capability of this osmolyte to interfere

with α-synuclein’s aggregation.

Our results indicated that a low trehalose concentration stabilizes α-synuclein

folding much better than at high concentration by blocking in vitro α-synuclein’s

polymerization [d].

These results suggested that trehalose could be associated with other drugs

leading to a new approach for treating Parkinson’s and other brain-related

diseases.

Acknowlegment.

We thank Diamond Light Source for access to beamline B23 (SM8034 and SM9079) that

contributed to the results presented here. The research leading to these results has received

funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under

grant agreement nº 226716.

References. a. Tanaka, M.; Machida, Y.; Niu, S.Y.; Ikeda, T.; Jana, N.R.; Doi, H.; Kurosawa, M.; Nekooki, M.; Nukina, N. Nature Medicine 2004, 10, 148-154. b. Liu, R.; Barkhordarian, H.; Emadi, S.; Park, C.B.; Sierks, M.R. Neurobiology of Disease 2005, 20, 74-81. c. Jiang, T.; Yu, W.B.; Yao, T.; Zhi, X.L.; Pan, L.F.; Wang, J.; Zhou, P. RSC Adv. 2013, 3, 9500-9508. d. Ruzza, P.; Hussain, R.; Biondi, B.; Calderan, A.; Tessari, I.; Bubacco, L.; Siligardi, G. Biomolecules 2015, 5, 724-734


Poster 10

IN SILICO IDENTIFICATION AND EXPERIMENTAL VALIDATION OF NOVEL MULTI-TARGETED ANTI-ALZHEIMER’S LIGANDS FROM MARINE

SOURCES

Vitale R.M.1, Desiderio D.4, Sgammato R.4, Thellung S.3, Carbone M.1, Ciavatta M.L.1, Mollo E.1, Gavagnin M.1, Felicità V.1,2, Rispoli V.2, Florio T.3,

Masullo M.4, Amodeo P1

1 Instituto di Chimica Biomolecolare (ICB)-CNR, Via Campi Flegrei 34, 80078 Pozzuoli. email: [email protected]

2.Dipartimento di Scienze della Salute, Università Magna Græcia of Catanzaro, Campus “Salvatore Venuta”, Viale Europa,I-88100 Catanzaro (CZ), Italy

3 Sezione di Farmacologia, Dipartimento di Medicina Interna (DiMI), e Centro di Eccellenza per la Ricerca Biomedica (CEBR) Università di Genova, viale Benedetto XV, 2,

16132 Genova 4 Dipartimento di Scienze Motorie e del Benessere Università degli Studi di Napoli

"Parthenope" Alzheimer’s disease (AD), the most common cause of dementia in elderly population, is a complex neurodegenerative disorder of the central nervous system, characterized by a progressive and irreversible degeneration of cholinergic neurons with the concomitant decrease of the hippocampal and cortical neurotransmitter acetylcholine (Ach) levels, and by the presence of amyloid plaques and neurofibrillary tangles. The current pharmacological therapy for AD is mainly based on acetylcholinesterase inhibitors (AChEI), aimed at restoring the Ach tone in the brain. However, due to the multifactorial aetiology of this pathology, compounds able to act simultaneously on two or more relevant biological targets is high desirable, as combining AChE inhibition with the impairment of β-amyloid peptide's aggregation and deposition by inhibition of β-secretase enzyme (BACE-1) and/or by directly hampering Aβ aggregation. The previous identification of a pseudozoanthoxanthin variant from the zoanthid crust coral Parazoanthus axinellae, as an acetylcholinesterase inhibitora prompted us to search other molecules sharing a similar scaffold in our collection of natural marine compounds, and to explore the potentiality of the newly-selected molecules as multiligand agents, by including as potential target BACE-1 and investigating the ability of this scaffold to inhibit β-amyloid aggregation. The most similar compounds among those available in suitable quantities for subsequent experimental validation within our collection were a pseudozoanthoxanthin from an unidentified caribbean zoanthid, differing from the analog reported by Turk and coll. for the number and position of methyl substituents on the azulene ring, and the bromo-pyrrole alkaloid stevensine. Docking and molecular dynamics studies were carried out on both AChE and BACE-1 enzymes as well as on β-amiloid fibrils. The positive results obtained in silico were then confirmed by biochemical assays that showed an inhibition for both enzymes in the low/sub-micromolar range as well as the ability of impairing β-amiloid aggregation. Acknowlegment: Research funded by POR Campania FESR 2007-2013 “FARMABIONET Rete integrata per le biotecnologie applicate a molecole ad attività farmacologica” References: a) Sepcić K, Turk T, Macek P. Toxicon. 1998, 36:937-40.


Poster 11

CALIX[8]ARENE BASED PH-RESPONSIVE HYDROGELATORS: USEFUL PLATFORM FOR DRUG DELIVERY

Cunsolo F., Mecca T.

CNR-Istituto di Chimica Biomolecolare, UOS of Catania, Via P. Gaifami 18, 95126, Catania email: [email protected]

Delivering drugs with improving safety and stability, controlled rate, slow delivery, targeted delivery is the aim that researchers of this area pursue with very different and attractive methods. Among the others, low molecular weight hydrogelators with stimuli responsive functionalities are considered to be of great interest as drug delivery systems.a Recently we have reported the first example of calix[8]arene polyelectrolyte derivatives as pH-responsive hydrogelators able to form gel phases in basic and acidic conditions.b In order to evaluate the utility of these hydrogels as delivery systems, two different set of hydrogels were prepared. The first one, obtained through basic pH-responsive calix[8]arene hydrogelators, has been studied for the loading and release capacity towards steroid and non-steroid anti-inflammatory drugs. Acidic pH-responsive calix[8]arene hydrogelators were instead used to check the improvement of aqueous solubility and stability of curcumin, a natural antioxidant that shows many pharmacological and protective activities, for which many nanoformulations have been proposed in recent years.c

Acknowlegment: Research funded by PON R&C 02_00355_2964193 (MIUR, Rome). References: a) Fleige, E.; Quadir, M. A.; Haag, R. Adv. Drug Delivery Rev. 2012, 64, 866-884. b) Mecca, T.; Messina, G. M. L.; Marletta, G.; Cunsolo, F. Chem. Commun. 2013, 49, 2530-2532. c) Naksuriya, O.; Okonogi, S.; Schiffelers, R. M.; Hennink, W. E. Biomaterials 2014, 35, 3365-3383.


Poster 12

PHOSPHOMANNOMUTASE 2: A NEW POTENTIAL TARGET FOR THE THERAPY WITH PHARMACOLOGICAL CHAPERONES

Andreotti G.1, Cimmaruta C.1,2, Citro V.2, Cubellis M.V.2

1 Istituto di Chimica Biomolecolare – CNR, via Campi Flegrei 34, 80078 Pozzuoli-Napoli email: [email protected]

2 Dipartimento di Biologia, Complesso Universitario di Monte S. Angelo, Università degli Studi di Napoli Federico II,

via Cinthia, 21, 80126 NAPOLI [email protected]

The most frequent glycosylation (CDG) disorder affecting the N-glycosylation pathway is caused by a deficiency of Phosphomannomutase (PMM2) the enzyme that isomerize Mannose-6-Phosphate. For this disorder, which is known as CDG-PMM2 (MIM: 212065) [a], there is no therapy at present, but, at least in principle patients could benefit from a therapy based on pharmacological chaperones (PC)[b]. PC are small molecules that preferentially bind the folded state of a protein and stabilize it. Disease mutations can lower the free energy difference between the folded and the unfolded protein shifting the equilibrium towards the latter one. Unstable proteins, although retaining the functional chemical groups needed for the biological activity, are sensitive to proteolysis and are cleared by the protein quality control systems in the cell. Hence for these mutations the reduction of the protein concentration in the cell is the primary effect and the reduction of total activity is only a secondary effect. Exploiting non-biased ligand migration and protein dynamics, we have produced the first accurate model of a ligand bound complex for PMM2 which can be used for high throughput screening to find PC for CDG-PMM2[c]. We have characterized in vitro the mutants that are most commonly found in European population, we have demonstrated that the genotype R141H/F119L is eligible for the therapy with PC. Moreover we have demonstrated that a small molecule, glucose-1,6 bisphosphate acts as PC for F119L stabilizing the mutant protein towards thermal induced denaturation and proteolysis. The same metabolite enhances the activity and promotes the correct quaternary structure of the mutant enzyme [d]. Acknowlegment: Research funded by Telethon (GGP12108) References: a) J. Jaeken, Congenital disorders of glycosylation, in: M.L. O. Dulac, and H.B. Sarnat, Editors (Ed.) Handbook of Clinical Neurology;Pediatric Neurology, vol. 113, Elsevier B.V, 2013, pp. 1737-1743. b) H.H. Freeze, Towards a therapy for phosphomannomutase 2 deficiency, the defect in CDG-Ia patients, Biochim Biophys Acta, 2009, 1792, 835-840. c) G. Andreotti, I. Cabeza de Vaca, A. Poziello, M.C. Monti, V. Guallar, M.V. Cubellis, Conformational Response to Ligand Binding in Phosphomannomutase2: INSIGHTS INTO INBORN GLYCOSYLATION DISORDER, J Biol Chem, 2014, 289, 34900-34910. d) G. Andreotti, E. Pedone, A. Giordano, M.V. Cubellis, Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation, Mol

Genet Genomic Med, 2013, 1, 32-44.


Poster 13

BIOTRANSFORMATION AND PREFERENTIAL CRYSTALLIZATION:

TWO PRACTICAL APPROACHES FOR THE RESOLUTION OF

MILNACIPRAN

Sanfilippo C. and Patti A.

1Istituto di Chimica Biomolecolar-CNR, via P. Gaifami 18, 95126, Catania


Enzymatic catalysis is a valuable approach used in organic synthesis to achieve chemical transformations in mild reaction conditions and with a high degree of selectivity. The use of enzymes, especially lipase, in organic solvent is an advantageous way to obtain single stereoisomers of a drug and biotransformations are today accepted as a powerful methodology for the industrial preparation of chiral pharmaceuticals. Enantiomerically pure molecules containing amino functions are important synthons for the preparation of drugs. Primary and secondary amines, where the amino group is directly located on a stereogenic carbon, have been obtained in good yields and optical purity by lipases-catalyzed kinetic resolution of the racemates through a transamination reaction in the presence of carboxylic esters as acyl donors.a However, aminomethyl compounds are challenging substrates for their high reactivity and distance from the chiral center. Milnacipran, Z-(±)-2-(aminomethyl)-N,N-diethyl-1-phenyilcyiclopropane, is an active antidepressant drug belonging to the class of inhibitors of the reuptake of serotonin and has recently attracted interest for its painkiller effects in the treatment of fibromyalgia.b Milnacipran is currently marketed in many countries, but not yet in Italy, in racemic form, however, recent pharmacokinetic studies on single enantiomers showed greater activity for (1S, 2R)-levomilnacipran. The aim of this study is the development of a simpler and more economical strategy alternative to the reported an enantioselective synthesis. The kinetic resolution of racemic milnacipran in the presence of lipase was then investigated and optimized by means of a careful choice of the reaction conditions (lipase source and form, temperature, solvent and acyl donor nature). The amide product and the unreacted substrate were obtained in satisfactory chemical yields and enantiomeric purities.c During this study we identified one of the amides obtained from the enzyme-catalyzed resolution as a conglomerate, whose nature was confirmed by the physical properties (melting points, solubility and X-ray diffraction). The properties of this specific amide of milnacipran were then exploited for its spontaneous resolution by conglomerate crystallization through a sequence of crystallization steps. Starting from a substrate with quite low enantiomeric excess, crystals of both enantiomers were obtained in enantiopure form.

References: a) Gotor-Fernández, V.; Busto, E.; Gotor, V.; Adv. Synth. Catal. 2006, 348, 797-812. b)

Ormseth, M. J.; Eyler, A. E.; Hammonds, C. L.; Boomershine, C. S.; J. Pain Res. 2010,3,15-24.c) Sanfilippo, C.; Nicolosi, G.; Patti, A. J. Mol. Catal. B: Enzym. 2014, 104, 82-86


Poster 14

ANTIOXIDANT ACTIVITY MEASUREMENTS BY ELECTRON SPIN RESONANCE SPECTROSCOPY

Sanna D.1, Fadda A.2, Serra M.1

1 Institute of Biomolecular Chemistry,

2 Institute of Sciences of Food Production, trav. La Crucca, 07040, Sassari email: [email protected]

Antioxidant activity measurements are currently used in many fields ranging from the cosmetic to the food industry. A variety of methods have been developed to assess antioxidant activity (AA), but the complexity of substrates like food or biological matrixes prevents from having a reliable method for antioxidant activity quantification.a The preferred methods for AA determinations are those which imply simple protocols, involve easy or none sample preparation, entail simple instrumentation, like a spectrophotometer. These simple experimental procedures allow for the determination of the AA of a lot of samples in a short time, but have some disadvantages. The more easily predictable ones are interferences and limitations of the method itself. When considering the outcome provided, in AA measurements, by simple spectrophotometric methods and when comparing these results with those obtained with complementary and alternative methods we reached the conclusion that those based on Electron Spin Resonance (ESR) spectroscopy are more reliable.b In fact these latter involve the determination of relatively stable radical species or the spin trapping of unstable radicals to form stable adducts. For this reason, since only species with unpaired electrons are detected, the interferences are scarce or missing. The comparison between the results obtained with spectrophotometric and ESR determination of DPPH scavenging activity is a clear example. The absorbance of the oxidation products of the antioxidants and aggregation phenomena which take place when using organic solvent/water mixtures can prevent the use of the spectrophotometric method but are of no account when using ESR spectroscopy. Another example is the spectrophotometric determination of the superoxide radical by using nitroblue tetrazolium. Attempts to generate this radical species, both with enzymatic or chemical methods, and spin trapping experiments with DMPO or DEMPO, were in most of the cases unsuccessful demonstrating that, in most of the cases, this species is invoked but not really involved in these systems.

References: a) Huang, D.; Ou B.; Pior, R.L. J Agric Food Chem 2005, 53,1841–1856 b) Sanna, D.; Delogu, G.; Mulas, M.; Schirra, M.; Fadda, A. Food Anal. Methods, 2012, 5, 759-766


Poster 15

BIOCATALYZED PROCEDURES TO RECOVER VALUABLE PRODUCTS FROM FISH WASTE

Morrone R., Baglieri A., Gambera G., Nicolosi G., D’Antona N.

CNR – Institute of Biomolecular Chemistry UOS Catania, Via P. Gaifami 18 - 95126 Catania, Italy


The waste derived from the fish processing represents approximately 45-50% of the weight of the raw material. Disposal of these wastes is expensive and produces environmental issues, therefore their treatment as “novel” raw material for the recovery of valuable products is certainly a valid alternative. Actually, the main derivatives obtained from fishing wastes are fish meal and oil. The oil from fish waste is commonly used to produce biodiesel but it represents too an excellent source of long chain polyunsaturated fatty acids (PUFAs) and especially of the precious omega-3 fatty acids, cis-5,8,11,14,17- eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA). The functional and biological properties of omega-3 fatty acids include among other: prevention of atherosclerosis, reduction in cardiovascular disease, protection against arrhythmias, and reduction of the blood pressure a. At least two meals of fish every week have been recommended by the American Heart Association to reduce the effect of cardiovascular diseases b. Since EPA and DHA are valuable products due to their beneficial properties on human health, the demand for fish oil is constantly growing and so any new sources are welcomed by the market. The main problem in the fish oil extraction procedures is the lability (mainly due to oxidative processes) of PUFAs when severe conditions, in terms of temperature and pH, are requested. To avoid such harsh conditions different “softer” enzymatic methods have been proposed c. The following work is focused on the evaluation, at a laboratory scale, of different proteases to recover oil from fish waste (head, bones, tail and gut). The adopted mild reaction conditions allowed avoiding any degradation of PUFAs. The fatty acid composition of the recovered oil was determined. Moreover, since omega-3 are commonly formulated and marketed as their corresponding ethyl esters too, an efficient irreversible biocatalyzed transesterification process in absence of organic solvents was used. Acknowlegment: Bio4Bio project, PON02_00451_3362376 References: a) Kim S.K., Mendis E. Food Res. Int. 2006, 39, 383-393. b) Kris-Etherton P.M., Harris W.S., Appel L.J. Circulation 2002, 106, 2747-2757. c) Ramakrishnan V.V., Ghaly A.E., Brooks M.S., Budge S.M. Enz. Eng. 2013, 2, 115.


Poster 16

SEARCH FOR MOLECULAR MARKERS OF THERAPEUTIC RESPONSE IN MALIGNANT MELANOMA THROUGH NGS: A

PROPOSAL OF ANTI-BRAF COMBINATION THERAPIES

Pisano M., Manca A., Palmieri G., Casula M., Cantara A., Dettori M. A.,

Fabbri D., Rozzo C.

Institute of Biomolecular Chemistry (ICB), National Research Council (CNR), Traversa la Crucca, 3, 07100 Sassari, Italy

email: [email protected]; [email protected]

Introduction: Recent development of new targeted agents is giving viable treatment options to melanoma therapies. Vemurafenib® specifically targets BRAF-V600E mutation, a highly common alteration found in malignant melanomas (MM). This targeted therapy is very effective on BRAF mutated MM patients but, most of them quickly develops an acquired drug resistance, limiting the therapeutic efficacy. We previously characterized the potential antitumor activity of several compounds on MM cells using a series of cell lines derived from MM patients showing different histopathological features (primary and/or metastatic, different tumor stage and site of onset). Using NGS approaches, we started the molecular characterization of Vemurafenib-resistant BRAF-mutated MM cell lines in comparison with their drug-sensitive counterparts, in order to identify genetic variants associated to the resistance. The evaluation of the antiproliferative activity of some new compounds - like the curcumin-related D6 molecule - on Vemurafenib-resistant MM cell lines is in progress. Materials and Methods: The NGS testing was performed on cell lines using the Ion-Torrent® technology. The validation of new markers will be carried out in vitro (same cell lines) and in vivo (MM paraffinated tissue samples) by Sanger

sequencing. BRAF-mutated MM cell lines were treated with Vemurafenib (20 M

to 5 M) to isolate resistant clones. These were treated with a combination of Vemurafenib and the curcumin-related D6 compound, before performing MTT proliferation assays. Results and discussion: A genetic heterogeneity was observed after characterization of several primitive and metastatic MM cell lines by cancer gene mutation profiling. Among them, four paired BRAF-mutated primitive and metastatic MM cell lines were selected to isolate Vemurafenib-resistant clones. These showed to be sensitive to D6 treatments, thus suggesting a combination therapy using Vemurafenib and D6. NGS results are being evaluated in order to identify alterations acting as markers for predicting the antiproliferative response following this treatment. Moreover, they will allow a better understanding of the molecular mechanisms underlying the development of resistance to Vemurafenib in MM. Conclusion: Our preliminary results suggest the feasibility of Vemurafenib-D6 combination treatment in drug-resistant MM. Our aim is to identify and validate new molecular markers associated to therapeutic response in order to obtain a better classification of the cases to be addressed to the treatment.




Poster 17

LIPASE CATALYSED RESOLUTION OF PROPAGYL ACRYLATES, AS SYNTHONS IN ENYNE METATHESIS

Lambusta D.1, Gambera G.1, D’Annibale A.2, Nicolosi G.1

1Institute CNR of Biomolecular Chemistry, via Paolo Gaifami 18, 95126 Catania

email: [email protected] 2Chemistry Department, University “La Sapienza”, P.le Aldo Moro 5, 00185 Roma

Five and six-membered lactones are widely present as structural subunits in a large number of natural compounds, showing important biological activities.1 Since the ring-closing enyne metathesis (RCEYM) reaction represents a powerful way to obtain cyclic structures, we were interested in exploring its potential in the synthesis of naturally occurring fragrances detectable in spirits featuring a chiral lactone ring system. Exploiting the enantiostereopreference of lipases when used in organic solvents, we developed an enzymatic resolution of propargyl acrylates racemic mixtures, in order to obtain enantiomerically pure esters subsequently undergoing a RCEYM reaction, in order to prepare enantiopure chiral lactones. In a first stage, we concentrated our attention on four propargyl alcohols (1a-d). The enzymatically catalyzed selective acetylation of each corresponding alcohol racemate was attempted, but unfortunately the use of lipases from Candida antarctica, Mucor miehei , Porcine Pancreas, and Pseudomonas fluorescens, was unsuccessful. We then moved our attention to the enzymatic alcoholysis of the corresponding racemic acrylates (2a-d) with n-butanol using lipase from C. Antarctica immobilized on support (Novozyme® 435) as the biocatalyst, in tert-butyl methyl ether.

In these conditions the alcoholysis occured for all the four esters with high enantioselectivity (E>200), and residual acrylates 2a-d were recovered with high enantiomeric excess. Both the alkyl-substituted derivative 2b and aryl derivative 2d, showed to react faster, leading in 24h to the total conversion of the recognised enantioform. According to our expectations, Lipase from C. antarctica brought to a marked R stereopreference. References:

1Kanchiswamy C.N.; Malnoy M.; Maffei M. E; Front. Plant Sci. 2015,6, 1-23


Poster 18

SYNTHESIS AND STUDY OF NEW HYDROXYLATED BIPHENOL DERIVATIVES AS POTENTIAL TYROSINASE INHIBITORS

Dettori M.A.1, Fabbri D.1, Fois X.2,

Rocchitta G.3, Dallocchio R.1, Dessì A.1, Serra P.A.3, Pantaleoni R.2,4 and Delogu G.1

1 CNR- Istituto di Chimica Biomolecolare, 2 CNR- Istituto per lo Studio degli

Ecosistemi,Traversa La Crucca 3- 07100 Sassari, Italy .3 UNISS- Dipartimento di Medicina Clinica e Sperimentale, V.le S. Pietro 43/b- 07100 Sassari, Italy.

4UNISS- Dipartimento di Agraria- Sezione di Entomologia,Via Enrico De Nicola 9- 07100 Sassari, Italy.


The role of melanin is to protect the skin from ultraviolet (UV) damage by absorbing UV sunlight Melanin is one of the most widely distributed pigments and is found in bacteria, fungi, plants and insects. Tyrosinase (polyphenol oxidase, E.C. 1.14.18.1) and laccase (phenol oxidase, E.C. 1.10.3.2) are, the keys enzymes in melanin biosynthesis, These inhibitors are also known to be useful in cosmetics as whitening agents.a The involvement of laccase in cuticle sclerotization or tanning is essential to insect survival.b

Hydroxylated biphenyls with a C2-symmetry axis have attracted considerable interest because of the biological activity exerted by a number of natural

occurring compounds containing this moiety.d It is known that 4,4’-

dihydroxybiphenyl 1 has been shown to be a potent inhibitor of tyrosinase.c

Our study is aimed to prepare new biphenol derivatives as potential inhibitors of melanin production starting from compound 1. Herein are reported some of the new synthesized biphenyls in which, with the aim to improve water-solubility, one hydroxyl is protected by aminoacid (2) gallic acid (3) and sugar (4 and 5) unit, respectively. Compounds 6-8 belong to the class of C-prenylated biphenyls.

Biological evaluation on PC12 cells, phytoiatric tests and docking studies of the new derivatives were carried on. Acknowledgments: This work has been supported by Sardinia Autonomous Region - L.R. 7 Augus2007, n. 7. References: a) Chang. T. S. Int. J. Mol. Sci. 2009, 10, 2440-2475. b) Dittmer, N. T.; Kanost, M. R. Insect Biochem. Mol. Biol. 2010, 40, 179-188. c) Kim Y. J.; No J. K.; Lee J. K. and Chung H. Y. Bio. Pharm. Bull. 2005, 28, 323-327. d) Nicolaou, K. C.; Christopher .C. N.; Brȁse B. S.; Nicholas N. Angew. Chem. Int. Ed., 1999, 38, 2096-2152.


Poster 19

PHYTOCHEMICAL STUDIES OF SOME

MARINE AND TERRESTRIAL MEDITERRANEAN PLANTS

Ciavatta M. L.1, Bitam F.2, Smadi A.3, Carbone M.1, Boumaraf M.4, Villani G.1,

Gavagnin M.1

1 Istituto di Chimica Biomolecolare-CNR, Via Campi Flegrei 34, I-80078 Pozzuoli (Naples)

2Département de Pharmacie, Faculté de Médecine, Université de Batna (05000), Algérie

3Laboratoire de Chimie et Chimie de l’Environnement (L.C.C.E), Département de Chimie, Faculté des Sciences, Université de Batna (05000), Algérie

4Department of Chemistry, Faculty of Exact Sciences, University of Mentouri-Constantine, 25000 Constantine, Algeria


Phytochemicals are biologically active, naturally occurring chemical compounds found in

plants, which are well-known to provide health benefits for humans.a In general, these

chemicals are produced to protect plant cells from environmental hazards such as

pollution, stress, drought, UV exposure and pathogenic attack.b They protect plants from

disease and damage and contribute to the plant’s colour, aroma and flavour.

Phytochemicals are usually accumulated in different parts of the plants, such as roots,

stems and leaves and belong to different structural types.c

Angiosperms are the largest group of plants on Earth (80% of the all known living plants)

and include aquatic and land species. They also are a rich source of new molecules with

interesting pharmacological properties that could be used as lead compounds for the

development of new drugs.d

In the frame of a scientific collaboration with researchers from Algerian universities we

have recently started a phytochemical screening on plants belonging to the Mediterranean

flora. Three angiosperms have been investigated: Cymodocea nodosa, a seagrass

collected in the Gulf of Naples, and Eryngium triquetrum and Pulicaria undulata, both

plants picked up from Algerian desert region.

A series of metabolites belonging to distinct structural classes have been isolated and

characterised by NMR and mass techniques. Phenolic and terpenoidic compounds have

been identified from C. nodosa, whereas polyacetylenes have been isolated for the first

time from E. triquetrum. Finally, novel terpenes belonging to the humulene class, have

been characterised from the lipophilic extract of P.undulata.

References:

a) Hasler C.M.; Blumberg J.B. J. Nutr.1999, 129: 756S-757S.

b) Piasecka A.; Jedrzejczak-Rey N.; Bednarel P. NewPhytologist2015, 206, 948-964..

c) Maag D.; ErbM.; Kollner T. G.; Gershenzon J. Bioessey2014, 37, 167-174.

d) Harvey A. L.; Edrada-Ebel R.; Quinn R. J. Nature Review Drug Discovery 2015, 14, 111-129.


Poster 20

DETERMINATION OF AVERMECTINES IN RAW WOOL BY LIQUID CROMATOGRAPHY COUPLED TO ORBITRAP MASS

SPECTROMETRY

Azara E.1, Bortolu S.2, Lo Curto P.3, Duce P.2

1Institute of Biomolecular Chemistry, National Research Council, Sassari, Italy 2Institute of Biometeorology, National Research Council, Sassari, Italy 3Clinical Epidemiology and Medical Statistics Unit, University of Sassari, Italy

email: [email protected]; [email protected]

The wool is a biodegradable renewable resource and, due to its complex chemical composition and physical structure, can find various value-added applications (e.g. thermal and acoustic insulation material, agricultural amendment, biomedical polymers, etc.). In order to investigate if wool fibre would be a significant route of human and environmental exposure, we developed a method to assess the presence of drugs residues, deriving from veterinary practices. Avermectins are macrocyclic lactones, which exhibit high anthelmintic, insecticide and acaricide activity. This class of compounds includes ivermectine (IVE), abamectine (ABA), emamectine (EMA), doramectine (DORA), eprinomectine (EPRI), moxidectina (MOXI). These molecules and their transformation products persist in the environment and can be toxic to no target organisms. The aim of this work was to identify and quantify Avermectins in Sarda sheep wool by High Liquid Chromatography coupled to Orbitrap mass spectrometry (LC-ESI-MS/MS). Microwave assisted extraction (MAE), an environmentally friendly method, was optimized with regard to the solvent type amount of solvent and duration of extraction time. Since wool is a complex matrix with high protein and fat content, which often interfere in analytical procedure, SPE Oasis hydrophilic lipophilic balance (HLB) cartridges and QuEChERS extraction method were compared as cleanup step. Chromatographic separation was achieved on a Kinetex C18 column using a linear gradient of methanol-Ammonium Formiate 1 mmol containing 0.1% formic acid. The Q Exactive was equipped with heated electrospray ionization source and operated in positive ionization mode. Orbitrap spectrometer operated in targeted-MS/MS mode with precursor ion fragmentation in HCD cell. The Full MS acquisition was performed with resolution power 70000 FWHM for parent ions and 17500 for the fragment ions with mass accuracy of 5 ppm. The calibration curves were linear with R2 always exceeding 0.99. The limit of detection ranged from 0.5 to 2 ng/g.

Acknowledgment: Research funded by Med-L@ine project, granted by the Cross Border Cooperation Programme Italy-Maritime 2013-2015 References: a) Rubies, A.; Antkowiiak, M.; Granados M.; Companyo R.; Centrich F. Food Chem 2015, 181, 57-63 b) Raich-Montiu, J.; Prat, M.D.; Granados M. Analytica Chimica Acta 2011, 697, 32-37


Presenting authors

Alberico E. ICB – SS Poster 5 Andreotti G. ICB – NA Poster 12 Azara E. ICB – SS Poster 20 Biondi B. ICB – PD Oral 6 Burreddu P. ICB – SS Oral 4 Ciavatta M. L. ICB – NA Poster 19 Cosseddu A. ICB – SS Poster 6 Culeddu N. ICB – SS Oral 10 Cutignano A. ICB – NA Oral 9 Dallocchio R. ICB – SS Poster 1 Dessì A. ICB – SS Poster 1 Dettori M.A. ICB – SS Poster 18 Drago C. ICB – CT Oral 11 Fabbri D. ICB – SS Oral 2 Fenude E. ICB – SS Poster 2 Gavagnin M. ICB – NA Oral 12 Granata G. ICB – CT Oral 8 Lambusta D. ICB – CT Poster 17 Mecca T. ICB – CT Poster 11 Morrone R. ICB – CT Poster 15 Peluso P. ICB – SS Poster 3 Pisano M. ICB – SS Poster 16 Piscitelli F. ICB – NA Oral 3 Rozzo C. ICB – SS Poster 16 Ruzza P. ICB – PD Poster 9 Sanfilippo C. ICB – CT Poster 13 Sanna D. ICB – SS Poster 14 Sechi B. ICB – SS Poster 4 Solinas M. ICB – SS Oral 5 Spanu P. ICB – SS Oral 1 Sperlinga E. ICB – CT Poster 8 Villano R. ICB – SS Oral 7 Vitale R.M. ICB – NA Poster 10 Zambrano V. ICB – SS Poster 7


Author Index Alberico E. (Poster 5) Amodeo P. (Poster 10) Andreotti G. (Poster 12) Azara E. (Poster 20) Baglieri A. (Poster 15) Battistini L. (Oral 4, Poster 7) Baumann W. (Poster 5) Beller M. (Poster 5) Biondi B. (Oral 6, Poster 9) Bisogno T. (Oral 3) Bitam F. (Poster 19) Bortolu S. (Poster 20) Bortolus M. (Oral 6) Boumaraf M. (Poster 19) Brindani N. (Poster 7) Bubacco L. (Poster 9) Burreddu P. (Oral 4) Cabrero-Antonino J. R. (Poster 5) Cadoni R. (Poster 1) Calderan A. (Poster 9) Cantara A. (Poster 16) Carbone M. (Oral 12) Carbone M. (Poster 10, Poster 19) Carcelli M. (Poster 1) Casiraghi G. (Oral 4, Poster 7) Casula M. (Poster 16) Chandgude A. L. (Oral 1) Chessa M. (Oral 10) Ciavatta M.L. (Oral 12, Poster 10, Poster 19) Cimmaruta C. (Poster 12) Citro V. (Poster 12) Consoli G.M.L. (Oral 8) Cosseddu A. (Poster 6) Cubellis M.V. (Poster 12) Culeddu N. (Oral 10) Cunsolo F. (Poster 11) Curti C. (Poster 7) Cutignano A. (Oral 9) D’Annibale A. (Poster 17) D’Antona N. (Poster 15) Dallocchio R. (Poster 1, Poster 18) Dalzini A. (Oral 6) De Zotti M. (Oral 6) Deiana P. (Oral 10) Deligia F. (Oral 1) Dell’Amico L. (Poster 7) Delogu G. (Oral 2, Poster 18) Desiderio D. (Poster 10) Dessì A. (Poster 1, Poster 18) Dettori M.A. (Oral 2, Poster 16, Poster 18) Dettori S. (Oral 10) Di Marzo V. (Oral 3) Doemling A. (Oral 1)

Drago C. (Oral 11) Drexler H.-J. (Poster 5) Duce P. (Poster 20) Fabbri D. (Oral 2, Poster 16, Poster 18) Fadda A. (Poster 14) Felicità V. (Poster 10) Fenude E. (Poster 2) Florio T. (Poster 10) Fois X. (Poster 18) Formaggio F. (Oral 6) Fuggetta M.P. (Oral 1) Gambera G. (Poster 15, Poster 17) Gavagnin M. (Oral 12, Poster 10, Poster 19) Geraci C. (Oral 8) Giordano C. (Oral 3) Giunta D. (Poster 4) Granata G. (Oral 8) Greco D. (Poster 7) Guo Y.-W. (Oral 12) Hussain R. (Poster 9) Junge H. (Poster 5) Junge K. (Poster 5) Kiss R. (Oral 12) Lambusta D. (Poster 17) Lo Curto P. (Poster 20) Lo Nigro R. (Oral 8) Luongo L. (Oral 3) Maione S. (Oral 3) Malandrino G. (Oral 8) Manca A. (Poster 16) Marchetti M. (Poster 4) Masullo M. (Poster 10) Mathieu V. (Oral 12) Mecca T.(Poster 11) Molinu M.G. (Oral 10) Mollo E. (Oral 12, Poster 10) Morra A. (Oral 1) Morrone R. (Poster 15) Naesens L. (Poster 1) Napoli E. (Poster 8) Nicolosi G. (Oral 11, Poster 15, Poster 17) Pala N. (Poster 1) Palmieri G. (Poster 16) Pantaleoni R. (Poster 18) Patti A. (Poster 13) Peggion C. (Oral 6) Pelosi G. (Poster 7) Peluso P. (Poster 3) Pinna L. (Poster 7) Pisano M. (Poster 16) Piscitelli F. (Oral 3) Rassu G. (Oral 4, Poster 7) Rispoli V. (Poster 10) Rocchitta G. (Poster 18) Rozzo C. (Poster 16)

2°Convegno dell'Istituto di Chimica Biomolecolare del CNR

Ruberto G. (Poster 8) Ruzza P. (Poster 9) Sanfilippo C. (Poster 13) Sanna D. (Poster 14) Santona M. (Oral 10) Sartori A. (Oral 4, Poster 7) Sechi B. (Poster 4) Sechi M. (Poster 1) Serra M. (Poster 14) Serra P.A. (Poster 18) Sgammato R. (Poster 10) Siligardi G. (Poster 9) Siracusa L. (Poster 8) Smadi A. (Poster 19)

Solinas M. (Oral 5, Poster 4) Spanu P. (Oral 1) Sperlinga E. (Poster 8) Stevaert A. (Poster 1) Tessari I. (Poster 9) Thellung S. (Poster 10) Ulgheri F. (Oral 1) Villani G. (Poster 19) Villano R. (Oral 7) Vitale R.M. (Poster 10) Vite V. (Poster 2) Zambrano V. (Poster 7) Zanardi F. (Oral 4, Poster 7)

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