curriculum vitae prof. vincenzo pavone
Transcription
curriculum vitae prof. vincenzo pavone
Prof. Vincenzo Pavone CURRICULUM VITAE PROF. VINCENZO PAVONE Nome: Vincenzo Cognome: Pavone Luogo di nascita: Napoli, Italia Data di nascita: 29 gennaio 1952 Cittadinanza: Italiana Indirizzo di lavoro: Dipartimento di Chimica “Paolo Corradini” Università di Napoli “Federico II” Complesso Universitario Monte S. Angelo Via Cynthia, 46 80126 Napoli, Italia Telefono: +39-081674399 Fax: +39-081674090 e-mail: [email protected] 1 Prof. Vincenzo Pavone CARRIERA UNIVERSITARIA Settembre 1975 – dicembre 1975 Stage pre-laurea presso l’Istituto “G. Donegani” della Montedison, Novara, Italia Maggio 1976 Laurea in chimica (110/110 e lode) Settembre 1977 – agosto 1978 Borsa di studio del CNR presso l’Istituto Chimico dell’Università di Napoli 1978 – 1980 Visiting Fellow alla “Section of Macromolecular Biology - Laboratory of Chemical Biology - National Institute of Arthritis, Metabolism and Digestive Diseases (NIAMDD) - National Institutes of Health (NIH)”, Bethesda, MD, USA Luglio 1980 – novembre 1987 Ricercatore universitario presso la Facoltà di Scienze MM.FF.NN. dell’Università di Napoli “Federico II” 1987 Visiting Fellow alla Mitsubishi–Kasei, Institute of Life Science, Tokyo, Japan – borsa di studio CNR-NATO Novembre 1987 – novembre 1994 Professore Associato di Chimica Generale presso la Facoltà di Scienze MM.FF.NN. dell’Università di Napoli “Federico II” 2 Prof. Vincenzo Pavone Novembre 1994 Professore Ordinario di Chimica Generale presso la Facoltà di Scienze MM.FF.NN. dell’Università di Napoli “Federico II” 1997 Visiting Professor al “National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology”, Tsukuba, Japan 3 Prof. Vincenzo Pavone ATTIVITA’ MANAGERIALI PIU’ RECENTI 2000-2002 Membro del Consiglio amministrazione) del (con Polo funzioni delle di Scienze consiglio e di Tecnologie dell’Università di Napoli Federico II Il Polo delle Scienze e Tecnologie è una divisione dell’Università di Napoli “Federico II” a cui afferiscono i 38 Dipartimenti delle Facoltà di Scienze MM.FF.NN., Ingegneria e Architettura. 1997-2002 Vice direttore del Dipartimento di Chimica “Paolo Corradini” dell’Università di Napoli Federico II (Bilancio 2002: € 1.342.000,00) 2002-2008 Direttore del Dipartimento di Chimica “Paolo Corradini” dell’Università di Napoli Federico II (Bilancio 2007: € 2.867.000,00) Al Dipartimento di Chimica afferiscono 21 professori ordinari, 32 professori associati, 13 ricercatori, 9 assegnisti, 10 borsisti, 17 dottorandi di ricerca, 2 unità di personale di biblioteca, 6 unità di personale tecnico, 8 unità di personale amministrativo. La nuova sede del Complesso Universitario di Monte S.Angelo si estende su una superficie di circa 12.000 mq e consta di circa 30 laboratori di ricerca, 10 laboratori didattici e di un laboratorio di didattica informatizzata. 2008- Vice Presidente e membro del Consiglio di Amministrazione della Società Consortile per Azioni BioTekNet (Capitale Sociale: € 700.000,00) 4 Prof. Vincenzo Pavone La Società Consortile per Azioni BioTekNet di recente istituzione deriva dal Centro Regionale di Competenza in Biotecnologie Industriali BioTekNet che è nato con l'obiettivo di mettere a disposizione del mondo della produzione, con una logica di integrazione e potenziamento, il rilevante patrimonio di competenze biotecnologiche esistente in regione Campania. L'attività di BioTekNet è particolarmente focalizzata sullo sviluppo di processi industriali e di tecnologie che utilizzano sistemi biologici o loro componenti, con particolare riferimento a: nuove tecnologie fermentative; biosensori innovativi; biosistemi per la depurazione delle acque; applicazioni biologiche avanzate per il settore alimentare; enzimologia industriale. L’obiettivo strategico di BioTekNet è la creazione di una rete integrata di competenze scientifiche, tecnologiche, strumentali ed economico-gestionali, allo scopo di assicurare un efficace trasferimento del sapere al sistema industriale. 2007 a tutt’oggi Membro del Consiglio di Amministrazione del Consorzio Technapoli – Parco Scientifico e Tecnologico dell’Area Metropolitana di Napoli e Caserta (Fondo consortile: € 1.625.855,00) Il Consorzio Technapoli è il Parco Scientifico e Tecnologico (PST) dell’area metropolitana di Napoli e di Caserta, il cui obiettivo strategico ed istituzionale è quello di incrementare la competitività del sistema economico territoriale attraverso interventi volti a favorire la ricerca e l’innovazione tecnologica, l’internazionalizzazione delle imprese e l’attrazione di investimenti esteri. Technapoli, per il raggiungimento del suo obiettivo strategico, coopera sia con i soggetti economici locali (pubblici e privati) sia con network nazionali ed internazionali, sviluppando attività di collegamento e di integrazione operativa tra Imprese, Università, Pubblica Amministrazione e Centri di Ricerca pubblici e privati per la realizzazione di progetti congiunti su tematiche connesse all’innovazione tecnologica. 5 Prof. Vincenzo Pavone Il Consorzio svolge un’ampia gamma di attività volte a sostenere e diffondere l’importanza dell’innovazione tecnologica quale leva per acquisire vantaggi competitivi sul mercato globale, sia in termini di sistema delle imprese che in termini più generali di “territorio”. In particolare, Technapoli si è specializzato nella: erogazione di servizi telematici; promozione e valorizzazione della tutela della proprietà intellettuale - Marchi e Brevetti; redazione e gestione di progetti di ricerca, innovazione, formazione e trasferimento tecnologico, nonché di piani di sviluppo industriale; promozione dell'aggregazione di imprese che operano nello stesso settore industriale e/o in settori complementari. 1996-1998 Membro della comitato tecnico-scientifico del Consorzio ARPA L’ARPA, Agenzia Ricerca e Produzione Avanzata dell'Università degli Studi di Napoli Federico II, è una struttura di interfaccia tra domanda e offerta di servizi innovativi, in particolare nell’ambito della ricerca, della consulenza e dell'alta formazione. 2004 a tutt’oggi Membro della commissione spin-off dell’Università di Napoli “Federico II” 2001 a tutt’oggi Presidente della commissione di valutazione dei programmi di scambi internazionali tra l’Università di Napoli “Federico II” ed Università ed Istituti stranieri per la mobilità di breve durata di docenti, studiosi e ricercatori 6 Prof. Vincenzo Pavone COORDINAMENTO E PARTECIPAZIONE A PROGETTI DI RICERCA PIU’ RECENTI 1999-2000 PRIN-MIUR Anno 1998 - Prot. 9803184222; ∈ 145,000 Titolo: Metalloproteine e sistemi miniaturizzati: Struttura, attività e applicazioni. Abstract: Obiettivi principale di questo progetto è lo sviluppo di nuove metalloproteine miniaturizzate, basate sulla autoagreggazione di peptidi con ioni metallici, quali modello di ferritine e rubredossine. Ruolo: Coordinatore nazionale, responsabile unità locale. 1999-2000 CNR - Biotechnology and Molecular Biology Program; ∈ 40,000 Titolo: Nuove esterasi/lipasi di interesse industriale e loro inibitori. Abstract: Il progetto è rivolto ad uno studio delle relazioni strutturafunzione in nuove esterasi e/o lipasi, e di loro inibitori. Ruolo: Co-investigatore 1999-2000 European Community FP4 Programme; ∈ 215,000 Titolo: Novel compounds that inhibit the activation of CD4-positive T cells as immunosuppressive agents. Abstract: Il progetto è rivolto alla sintesi di peptidi e peptido-mimetici, corrispondenti ad un dominio discreto del complesso di maggiore istocompatibilità (MHC) di classe II. Sulla base di calcoli teorici e di preliminari risultati sperimentali, queste molecole potrebbero interferire con l’attivazione di cellule T – CD4 positive, attraverso un 7 Prof. Vincenzo Pavone nuovo meccanismo d’azione. Ruolo: Responsabile unità locale 1999-2000 CNR – Progetto Coordinato Agenda 2000, codice -CNRC00781B_002; ∈ 10,000 Titolo: Metalloproteine, interazioni tra metalli e proteine, e loro modelli. Abstract: Il progetto di ricerca ha come obiettivo lo studio di nuove metallo-proteine, dalla purificazione alla caratterizzazione strutturale e funzionale. Parte integrante del progetto è lo sviluppo di metalloproteine in miniatura come mimetici di basso peso molecolare di macromolecole complesse. Queste nuove molecole rappresentano valide alternative per applicazioni industriali dei prodotti biotecnologici di espressione. Ruolo: PI 2001-2002 PRIN-MIUR Anno 2000 - prot. MM03185591; ∈ 120,000 Titolo: Metalloproteine e sistemi miniaturizzati: Struttura, attività e applicazioni. Abstract: Obiettivi principale di questo progetto è lo sviluppo di nuove metalloproteine miniaturizzate basate sulla autoagreggazione di peptidi con ioni metallici, che possono trovare svariate applicazioni, quali ad esempio, catalizzatori, sensori o agenti detossificanti. Ruolo: Coordinatore nazionale, responsabile unità locale 2002-2006 HPMD-CT-2001-00113 Marie Curie Fellowships; € 262,000 Titolo: Miniaturized metalloproteins: new biocatalysts by design. 8 Prof. Vincenzo Pavone Abstract: Il progetto è dedicato al reclutamento di ricercatori stranieri, la cui attività è rivolta allo sviluppo di test catalitici, per la caratterizzazione funzionale di metalloproteine in miniatura sviluppate nel gruppo di ricerca ospitante. Ruolo: PI 2004-2005 PRIN-MIUR anno 2003 - prot. 2003037580; Paolo Maria Scrimin (PI); € 94,000 Titolo: Approccio supramolecolare a metalloproteine minimali. Abstract: Scopo del progetto è lo sviluppo di sistemi catalitici artificiali, attraverso un approccio supramolecolare. Nuove architetture molecolari complesse, in grado di coordinare ioni metallici, rappresentano fattori chiavi nella realizzazione di processi catalitici. Ruolo: Responsabile Unità locale 2003-2007 FIRB-MIUR RBNE01RB9B; John Guardiola (PI); € 134,000 Titolo: Analisi comparata in vitro ed in vivo di vaccini ricombinanti innovativi basati su sistemi procariotici di display, proteine di fusione e peptidi bioattivi. Abstract: Il progetto è rivolto allo sviluppo di nuovi vaccini, basati su differenti sistemi, e ad una analisi comparativa delle loro proprietà strutturali e funzionali. Ruolo: Responsabile Unità locale 2003-2007 FIRB-MIUR RBNE01LCKB; Giuseppe Scala (PI); € 200,000 Titolo: Identificazioni di peptidi e dominii proteici in grado di inibire 9 Prof. Vincenzo Pavone l'attività di geni coinvolti nella patogenesi di neoplasie delle cellule B e del carcinoma del colon-retto. Abstract: Il progetto è rivolto ad una migliore comprensione dei meccanismi molecolari, che sono alla base delle trasformazioni neoplastiche di cellule B e del carcinoma del colon. Ruolo dell’unità è lo sviluppo di peptido-mimetici, disegnati ad hoc. Ruolo: Responsabile Unità locale 2005-2006 PRIN-MIUR anno 2004 - prot. 2004055484; Roberto Santucci (PI); € 65,000 Titolo: Ingegnerizzazione e caratterizzazione di metalloproteine in soluzione ed immobilizzate su superfici. Abstract: Il progetto è finalizzato a produrre varianti di citocromi c e del citocromo P450, nonché di sistemi modello, allo scopo di ottenere (i) una migliore comprensione dei meccanismi che controllano il folding di metalloproteine, e (ii) una ottimizzazione del processo di immobilizzazione di questi biosistemi su superfici solide o sol-gel, mostrando i citocromi interessanti potenzialità d'impiego in area biosensoristica. Ruolo: Partecipante 2005 Legge Regionale N.5 - Annualità 2003; € 17,000 Titolo:. Modelli di metalloproteine contenenti siti ferro-ossigeno. Abstract: Obiettivo principale del progetto è lo sviluppo di nuovi composti (denominati DF: Due Ferri), basati su peptidi artificiali, capaci di auto-assemblare in presenza di ioni metallici, per costituire modelli di metalloproteine. In particolare, viene presa in esame la classe di proteine ferro-ossigeno, allo scopo di costruire molecole in 10 Prof. Vincenzo Pavone grado di catalizzare reazioni di ossidazione, usando ossidanti "puliti", quali ossigeno ed acqua ossigenata. Ruolo: PI 2005 – 2006 FP6-2003-LIFESCIHEALTH-3 (STReP); € 200,000 Titolo: Identification of novel epitopes as HIV-1 vaccine candidates. Abstract: Obiettivo finale del progetto è la selezione di epitopi peptidici, in grado di mimare i domini dell’”envelope” del virus dell’HIV-1. Essi sono interessanti candidati per lo sviluppo di vaccini. Ruolo: Co-investigatore 2003-2008 Regione Campania – POR 2000-2006 - misura 3.16; € 750,000 Titolo: Biosensori Innovativi. Abstract: Obiettivo del progetto è di sviluppare nuovi tipi di biosensori elettrochimici e/o di fluorescenza, che consentano, con tecnologie innovative, la determinazione di analiti di elevato interesse in vari comparti, quali quello clinico, chimico- farmaceutico, ambientale ed agro-alimentare. Ciò allo scopo di consentire una integrazione sinergica tra ricerca sperimentale e richiesta industriale. Ruolo: PI 2006-2009 MIUR 297/99; € 900,000 Titolo: Micro-sistema di diagnosi basato su biosensori elettrochimici innovativi (MicroDiaSym). Abstract: Obiettivo del presente progetto è la costruzione di un nuovo sistema di diagnosi (in seguito indicato come MicroDiaSym), basato 11 Prof. Vincenzo Pavone su biosensori elettrochimici, in grado di rilevare analiti (quali ad esempio sequenze di DNA, anticorpi, recettori) di elevato interesse clinico, ambientale ed agro-alimentare. Ruolo: PI 2007-2008 PRIN-MIUR Anno 2006- Prot. 2006039071; Paolo Maria Scrimin (PI); € 95,000 Titolo: Nanoproteine artificiali autoassemblate. Abstract: Obiettivo del progetto è la preparazione e lo studio di "nanoproteine artificiali" ottenute mediante un processo di autoassemblaggio di tioli, funzionalizzati con peptidi, sulla superficie di nanoparticelle di oro. Ruolo: Responsabile Unità locale 12 Prof. Vincenzo Pavone PRINCIPALI COLLABORAZIONI CON AZIENDE ITALIANE E STRANIERE 1. Carlo Gavazzi Space – Sviluppo di un biosensore elettrochimico per uso nelle navicelle spaziali. La Carlo Gavazzi Space è una delle principali imprese europee di medie dimensioni operante nella “space systems integration”, ed è parte di un cluster di imprese europee. La Carlo Gavazzi Space è stata fondata nel 1981 ed il suo quartier generale ha sede a Milano. La Società occupa fisici e ingegneri di alta qualificazione. 2. Sigma-Tau Research Switzerland – Sviluppo di nuovi agenti antitumorali. La Sigma-Tau Research Switzerland si occupa della promozione, sviluppo, coordinamento e prestazione di servizi attinenti le attività scientifiche, di ricerca di base, ricerca clinica, sperimentazione, sviluppo e produzione, per conto proprio o di terzi, di processi fisici, chimici, biologici e biotecnologici, di metodiche produttive e diagnostiche implicanti anche l'ingegneria genetica, di prodotti biotecnologici, di molecole, di preparati per uso cosmetico o farmaceutico, di prodotti intermedi o finiti destinati all'industria farmaceutica, biotecnologica, chimica e cosmetica al consumatore finale. 3. Menarini Industrie Farmaceutiche Riunite – Sviluppo di nuovi antagonisti della contrazione della muscolatura liscia. E’ il primo gruppo farmaceutico italiano in Europa. L'attività di Ricerca del Gruppo Menarini si svolge attraverso Menarini Ricerche, che si occupa di tutte le attività di Ricerca e Sviluppo dalla fase di ideazione dei nuovi progetti fino alla fase di registrazione del farmaco. Dall'acquisto del Centro di Ricerca Farmaceutica di Pomezia (Roma), nei primi anni '80, nascono un modernissimo centro di biotecnologie, Menarini Biotech, e un 13 Prof. Vincenzo Pavone Centro per la Ricerca e Sviluppo in grado di offrire a Menarini ed alle sue collegate un'organizzazione di ricerca razionale ed avanzata. Tutte queste iniziative contribuiranno ad elevare il livello della qualità della ricerca Menarini, portandola al 9° posto nel mondo secondo un'indagine indipendente dell' “Institute for Scientific Information” di Philadelfia - nel periodo che va dal 1981 al 1992. I programmi di ricerca, suddivisi nei centri di Firenze, Pomezia, Lomagna, Pisa, Barcellona e Berlino, riguardano essenzialmente le patologie cardiovascolari, l'oncologia e l'area dolore, infiammazione, asma con lo studio di antagonisti recettoriali. 4. PRIMM - Membro del “Scientifc Advisory Board”. La PRIMM è una SMI che offre servizi biotecnologici con sede e laboratori al San Raffaele Biomedical Science Park di Milano. La Primm possiede anche una consolidata esperienza in progetti R&D portati avanti in stretta collaborazione con importanti istituzioni italiane e straniere. Uno dei principali focus aziendali è il disegno, la sintesi e lo svilluppo di nuovi peptidi bioattivi per applicazioni terapeutiche. Le aree principali di applicazione sono l’infiammazione, l’asma e le malattie infettive. 5. AXXAM – Consulente scientifico sul progetto “Fotoproteine”. Axxam è una società privata con sede e laboratori al San Raffaele Biomedical Science Park di Milano ed è uno spin-off della Bayer HealthCare, Research and Development Organization. Nel 2006 ha inaugurato i laboratori al Polaris, il parco Scientifico e Tecnologico della Sardegna. Axxam ha un team di più di 60 unità di personale altamente qualificato. Il programma interno delle ricerche si sviluppa su due filoni principali: 1) Nuove piattaforme tecnologiche basate sulla Photina®, una nuova fotoproteina attivata dal Ca2+; 2) sviluppo di piattaforme per l’identificazione di nuovi target in particolari settori terapeutici come il dolore e l’osteoporosi. 14 Prof. Vincenzo Pavone 6. Norsk Hydro - Consulente scientifico del progetto “Cancer Vaccine”. La Norsk Hydro è una compagnia con una forte presenza dello Stato norvegese prevalentemente incentrata sulla produzione petrolifera, quella dell'alluminio e dei fertilizzanti. L'azienda è il secondo più grande operatore del gas e del petrolio sulla piattaforma continentale norvegese ed è una grande azienda integrata dell'alluminio, quarta nel mondo. Nel 1999 la Norsk Hydro ha acquistato un'altra azienda norvegese del gas e del petrolio, “Petrolio Saga” e nel 2002 la Norsk Hydro ha acquistato il produttore tedesco principale dell'alluminio. Ha anche acquisito dal gruppo Eni tutta la produzione di fertilizzanti in Italia acquisendo un ruolo dominante in Europa. La Norsk Hydro ha impianti in circa 40 paesi intorno al mondo ed è attiva su tutti i continenti. Lo stato norvegese dichiara di possedere la proprietà di 43.8 per cento dell'azienda. Il numero dei dipendenti è 36.000. La Norsk Hydro ha una divisione Biotech che ha dato origine ad una serie di spin-off aziendali su prodotti biotech specifici. Il progetto “Cancer Vaccine” è stato incorporato nella società GEMVAX. 15 Prof. Vincenzo Pavone COLLABORAZIONI INTERNAZIONALI DOCUMENTATE DA LAVORI IN COLLABORAZIONE Svizzera: 1. Altmann Eva Novartis Institutes for BioMedical Research, CH4002 Basel; 2. Lorenzi Gian Paolo Technische-Chemische Laboratorium, ETH - Zentrum 8092, Zurich; 3. Mutter Manfred Institute of Molecular and Biological ChemistryEcole Polytechnique Fédérale de Lausanne, 1015 Lausanne; USA: 4. Mapelli Claudio Oncology and Peptide Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Lawrenceville; 5. DeGrado William F. Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA; 6. Di Costanzo Luigi Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania; 7. Schechter Alan N. Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD; 8. Luskey Kenneth L. Department of Molecular Genetics and Department of Internal Medicine University of Texas Health Science Center at Dallas Southwestern Medical School, Dallas, Texas; 16 Prof. Vincenzo Pavone 9. Dutton Leslie P. Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania; 10. Goodman Murray Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA; 11. Felix Arthur School of Theoretical and Applied Science, Ramapo College of New Jersey, Mahwah, NJ; 12. Lahr Stephen Department of Protein Design, Centocor, Radnor, PA; 13. Mierke Dale F. Department of Chemistry, Dartmouth College, Hanover, NH; 14. Summa Christopher M. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA; 15. Giovanna Ghirlanda Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona; Norvegia: 16. Ericsen Jon Amund GemVax AS, Oslo; 17. Naess Hilde Merete Pronova Biopharma; Giappone: 18. Nagai Ukon Mitsubishi Kasei Institute of Life Sciences, Tokyo; 19. Yamada Takashi Department of Chemistry, Faculty of Science, Konan University, Kobe; 20. Miyazawa Toshifumi Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Okamoto, Higashinada-ku, Kobe; Francia: 21. Chottard Genevieve Laboratoire de Chimie Inorganique et Matériaux Moléculaires, ESA CNRS 7071, Université Pierre 17 Prof. Vincenzo Pavone et Marie Curie, Paris;. 22. Mansuy Daniel Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601, Université Paris Descartes, 45 Rue des Saints Pères, Paris; Inghilterra: 23. Fraternali Franca Randall Division of Cell and Molecular Biophysics, King's College London, London; Polonia: 24. Leplawy Miroslaw T. Institute of Organic Chemistry, Technical University of ód , eromskiego 116, 90-924 ód ; India: 25. Kishore Raghuvansh Institute of Microbial Technology, Sector 39-A, Chandigarh; Germania: 26. Wieland Theodor Max-Planck-Institut ffir Medizinische Forschung, Jahnstrasse 29, D-6900 Heidelberg 18 Prof. Vincenzo Pavone RICERCATORI STRANIERI CHE HANNO LAVORATO A NAPOLI SOTTO LA GUIDA DEL PROF. VINCENZO PAVONE 1. Wonnemann Jörg Organisch-Chemisches Institut Westfälische Wilhelms-Universität, Corrensstrasse 40, 48149 Münster, Germany. 2. Hengelen Mirelle Leiden Institute of Chemistry, Gorlaeus Laboraties, Leiden University, 2300 RA Leiden, The Netherlands. 3. Lataika Rafal Wroclaw University of Technology, Institute of Organic Chemistry, Biochemistry and Biotechnology, Wybrzeze Wyspianskiego, 27 50370 WROCLAW, Poland. 4. Torres Rafael School of Chemistry, The University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland. 5. Tepper Armand Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. 19 Prof. Vincenzo Pavone SOMMARIO DELLA PRODUZIONE SCIENTIFICA • Totale delle review: n. 12. Review più significative: 1. Chemical Reviews (2001), 101(10), 3165-3189. 2. Annual Review of Biochemistry (1999), 68, 779-819. 3. Biological Chemistry (1998), 379(8/9), 987-1006. • Totale dei lavori pubblicati su riviste ad alta diffusione: n. 142. Lavori più significativi: 1. Nature Structural Biology (2001), 8(7), 611-615. 2. PNAS USA (2003), 100(7), 3772-3777. 3. PNAS USA (2000), 97(12), 6298-6305. 4. PNAS USA (2000), 97(22), 11922-11927. 5. PNAS USA (1992), 89(15), 7218-21. 6. PNAS USA (1986), 83(7), 1988-92. 7. PNAS USA (1982), 79(24), 7951-4. 8. Angewandte Chemie, International Edition (2003), 42(4), 417-420. 9. Angewandte Chemie, International Edition (2001), 40(22), 4245-4247. 10. Journal of Molecular Biology (1990), 214(3), 633-5. 11. Journal of the American Chemical Society (2001), 123(51), 12749-12757. 12. Journal of the American Chemical Society (1998), 120(24), 5879-5886. 13. Journal of the American Chemical Society (1992), 114(21), 8277-83. 14. Journal of the American Chemical Society (1992), 114(16), 6273-7. 15. Journal of the American Chemical Society (1990), 112(24), 8909-12. 16. Journal of the American Chemical Society (1989), 111(25), 9089-98. 17. Journal of the American Chemical Society (1984), 106(26), 8146-52. 18. Journal of the American Chemical Society (1984), 106(26), 8152-6. 20 Prof. Vincenzo Pavone 19. Journal of the American Chemical Society (1982), 104(9), 2437-44. 20. Journal of Pharmacology and Experimental Therapeutics (1994), 271(3), 1489-500. 21. Journal of Biological Chemistry (1983), 258(23), 14725-32. 22. Journal of Biological Chemistry (1981), 256(17), 9229-34. 23. Protein Science (1999), 8(1), 91-95. 24. Protein Science (1998), 7(2), 243-253. 25. Journal of Medicinal Chemistry (1996), 39(10), 2008-17. 26. Journal of Medicinal Chemistry (1994), 37(21), 3630-8. • Totale dei brevetti: n. 7 Brevetti internazionali: 1. WO2008017372 2. WO2004065406 3. WO2002012278 4. WO2000027880 5. WO9731941 6. WO9321227 • Totale dei proceedings: n. 33 21 Prof. Vincenzo Pavone SOMMARIO DELLE ATTIVITA’ Il prof. Pavone, laureato con lode in chimica all’università di Napoli, ha iniziato la sua carriera nel 1976 ed ha avuto numerose esperienze formative all’estero (USA e Giappone) e presso Aziende (Montedison e Mitsubishi). La sua produzione scientifica è stata particolarmente intensa e negli ultimi 10 anni il prof. Pavone ha assunto incarichi di responsabilità via via crescenti. In particolare la direzione del Dipartimento di chimica, che per dimensione e tipologia organizzativa e gestionale corrisponde alla direzione di in Istituto CNR di medio-grandi dimensioni, è stata caratterizzata da un particolare impegno che ha consentito il raggiungimento di significativi obiettivi, come la buona produzione scientifica dell’intero dipartimento ed un raddoppio delle entrate finalizzate alla ricerca. Questo impegno ha, tuttavia, prodotto un rallentamento della sua produzione scientifica non potendo contare su un gruppo di ricerca di dimensioni adeguate. Ciononostante, i risultati scientifici accumulati in questi ultimi anni saranno presto oggetto di numerose ed importanti pubblicazioni. Il prof. Pavone è stato ed è particolarmente attento alle potenzialità applicative delle sue ricerche, come testimoniato dai brevetti internazionali depositati e di cui è inventore. Questo aspetto ha consentito al prof. Pavone di intrattenere numerose collaborazioni con gruppi industriali italiani e stranieri. Le ricerche del prof. Pavone sono motivo di attrazione a livello internazionale, come testimoniato anche dai ricercatori stranieri che hanno voluto unirsi al suo gruppo. La dimensione internazionale è anche testimoniata da importanti review su invito (Chemical Reviews e Annual Review of Biochemistry). Il prof. Pavone ha anche una buona capacità attrattiva di risorse economiche. Negli ultimi 10 anni ha potuto contare su finanziamenti pubblici per circa 3.000.000 di euro e circa 500.000 euro da soggetti privati. Le sue ricerche sono state in parte anche finanziate dal CNR. Il prof. Pavone intrattiene anche numerose collaborazioni con altri Istituti del CNR. 22 Prof. Vincenzo Pavone ATTIVITÀ SCIENTIFICA L’attività scientifica del Prof. Vincenzo Pavone è iniziata nel 1976 e si è sviluppata secondo linee di ricerca di grande attualità. Le principali tematiche di ricerca sono di seguito elencate: 1. METALLO-PROTEINE ARTIFICIALI FERRO-PROTEINE NON-EME (2000 a tutt’oggi) EMOPROTEINE (1997 a tutt’oggi) PROTEINE FERRO-ZOLFO (1995 - 2002) 2. PROTEINE UPAR (2006 a tutt’oggi) RANTES (2000 a tutt’oggi) TROMBINA (1996 - 1999) STRUTTURA SECONDARIA DELLE PROTEINE (1985 - 1996) COMPOSTI MODELLO DI STRUTTURE SECONDARIE(1990 – 1996) 3. PEPTIDI BIOATTIVI NEUROKININA A (1994 - 1998) 4. AMMINOACIDI NON NATURALI E PEPTIDI (1983-2003) 5. INTERAZIONE DI AMMINOACIDI E PEPTIDE CON IONI METALLICI (1986-1996) 6. MISCELLANEA Il lavoro scientifico svolto si è concretizzato in 194 pubblicazioni (12 review, 142 lavori, 7 brevetti e 33 proceeding). I lavori a stampa sono tutti su riviste internazionali ad alta diffusione. Le ricerche sono state portate avanti interagendo proficuamente con numerosi ricercatori italiani e stranieri, avvalendosi in tal modo di una collaborazione 1 Prof. Vincenzo Pavone multidisciplinare e sfruttando nel modo migliore le risorse disponibili. I risultati scientifici ottenuti sono stati presentati in occasione di convegni nazionali ed internazionali, come testimoniato dalle numerose comunicazioni inviate. Il prof. Pavone è stato, inoltre, invitato a tenere numerose conferenze e seminari sull’attività di ricerca svolta sia in Italia sia all’estero. L’interesse scientifico del prof. Pavone nel corso degli anni ha riguardato inizialmente lo studio del riconoscimento molecolare di molecole a base peptidica da parte di recettori e/o partner proteici e dagli anni ‘90 si occupa prevalentemente dello studio delle relazioni struttura-funzione delle proteine. Da un punto di vista metodologico, il prof. Pavone ha una lunga esperienza nella caratterizzazione strutturale di peptidi e proteine mediante diffrazione di raggi X, risonanza magnetica nucleare (NMR) e dinamica molecolare. I sistemi studiati da un punto di vista strutturale sono anche stati caratterizzati da un punto di vista funzionale. Le ricerche condotte non solo hanno dato un contributo all’identificazione dei motivi strutturali fondamentali per l’attività biologica di alcune classi di proteine, ma anche allo sviluppo di nuovi e potenti farmaci (oggetto di brevetti internazionali), (antiasmatici), quali antagonisti inibitori immunosoppressori (utili della nella del recettore trombina terapia dei della neurochinina (antitrombotici), trapianti, delle A agenti malattie autoimmunitarie, dei tumori, ecc.), agenti antivirali (anti-HIV). Molto di recente è stata anche sviluppata una nuova classe di anti-tumorali basati sul recettore dell’urochinasi. In questi ultimissimi anni, le ricerche del Prof. Pavone sono rivolte alla progettazione e sintesi di proteine artificiali che mostrano un’attività comparabile o superiore alle proteine naturali. In particolare, una classe di tali molecole rappresenta un nuovo tipo di potenti e specifici biosensori elettrochimici. Nelle pagine seguenti saranno descritti in maniera più dettagliata le principali 2 Prof. Vincenzo Pavone e recenti linee di ricerche (dal punto 1 al punto 3) ed i risultati più rilevanti, senza voler sminuire gli altri lavori, che hanno dato un contributo significativo alla comprensione delle proprietà strutturali e funzionali dei sistemi studiati. Per completezza nell’ultima sezione sono riportati gli abstract di tutte le review, brevetti, pubblicazioni e proceeding. 3 Prof. Vincenzo Pavone FERRO-PROTEINE NON-EME Reviews: 1. Comptes Rendus Chimie (2007), 10(8), 703-720. 2. Biopolymers (2005), 80(2 and 3), 264-278. 3. Annual Review of Biochemistry (1999), 68 779-819. Pubblicazioni: 4. Journal of Biological Inorganic Chemistry (2005), 10(5), 539-549. 5. Proceedings of the National Academy of Sciences of the United States of America (2003), 100(7), 3772-3777. 6. Angewandte Chemie, International Edition (2003), 42(4), 417-420. 7. Journal of the American Chemical Society (2001), 123(51), 12749-12757. 8. Proceedings of the National Academy of Sciences of the United States of America (2000), 97(12), 6298-6305. Oltre che nelle eme-proteine, il ferro è presente nei sistemi naturali in proteine contenenti un centro bimetallico, in cui i due atomi di metallo sono spesso legati tra loro attraverso uno o più atomi di ossigeno. Questi sistemi sono caratterizzati dall'assenza di un legante macrociclico rigido e, pertanto, in essi la coordinazione del metallo avviene attraverso le catene laterali di aminoacidi quali His, Asp e Glu. In queste molecole, quindi, il metallo costituisce un elemento strutturale determinante nell'indurre o nello stabilizzare il folding della proteina. Questi sistemi, unitamente all'uso di modelli di più piccole dimensioni, offrono buone possibilità per esaminare il processo di inserzione dei metalli nelle proteine naturali ed il ruolo che tali metalli hanno nel determinare il folding e la stabilità della proteina stessa. Emeritrina, Ribonucleotide reduttasi e Metano- monossigenasi sono esempi di questa classe di proteine. L'Emeritrina è un trasportatore reversibile di ossigeno, mentre le ultime due catalizzano rispettivamente la riduzione di ribonucleotidi e l'ossidazione del metano. Le 4 Prof. Vincenzo Pavone Ferritine costituiscono un altro esempio attraente di “metal-binding proteins” contenenti ferro; esse contengono un centro binucleare ed uno polinucleare. Le Bacterioferritine contengono anche un ferro-eme. Il motivo strutturale ricorrente in tutte le proteine citate è il cosiddetto four-helix bundle, caratterizzato da α-eliche che aggregano in maniera parallela o antiparallela in strutture tetrameriche. L'impacchettamento di tali aggregati porta alla costituzione di cavità in cui si sistema il metallo. I sistemi four helix-bundle rappresentano, quindi, un motivo strutturale utile ed interessante sia per lo studio dei fattori responsabili del folding delle proteine, sia perchè sono essenziali building block per la costruzione di molte proteine biologicamente attive. Nella Bacterioferritina da Escherichia Coli, nota anche come citocromo b1, di cui è stata determinata la struttura tridimensionale mediante diffrazione di raggi X, il sito di binding binucleare è localizzato tra quattro eliche allineate in maniera antiparallela. Ciascuno dei due atomi di ferro, costituenti il sito bimetallico, è coordinato attraverso un residuo di His e tre residui di Glu. Due residui di Glu coordinano entrambi gli ioni metallici formando due ponti tra di essi. Al fine di ottenere un semplice sistema modello di proteine contenenti il motivo strutturale four-helix bundle e sfruttando le conoscenze relative alle proprietà conformazionali degli amminoacidi ed alla loro propensione a dare strutture 5 Prof. Vincenzo Pavone elicoidali, sono state progettate, usando la metodologia del de novo design, sequenze peptidiche capaci di dare strutture tetrameriche. In particolare, è stato progettato un sistema del tipo α2 che dimerizzando porta alla formazione dell’aggregato tetramerico. Il sistema α2 è costituito da un helix-loop-helix, cioè da da due eliche antiparallele legate da un loop. Nel design di un appropriato sistema α2 è stato necessario stabilizzare una corretta conformazione del linker per ottimizzare specifiche interazioni favorevoli tra le due eliche. L’aggregazione di eliche singole e di sistemi α2 può avvenire dando luogo ad orientazione parallela od antiparallela delle unità monomeriche costituenti. Quindi, affinchè l'assemblaggio avvenisse nella orientazione desiderata, particolare attenzione nel design è stata rivolta alla costruzione di sequenze capaci di dar luogo ad un buon packing idrofobico e ad interazioni elettrostatiche che favorissero una orientazione rispetto all'altra. Infine, è stato costruito in questo scaffold elicoidale il sito di binding dinucleare, costituito da due residui di His e quattro residui di Glu. Il primo modello ottenuto è rappresentato schematicamente nelle figure seguenti, in cui si evidenzia il sito di binding, le interazioni elettrostatiche e il packing idrofobico. Il sistema così progettato, denominato DF1 (Due Ferri 1) è stato sintetizzato mediante sintesi peptidica in fase solida, utilizzando le metodologie classiche della chimica Fmoc (fluorenilmetossicarbolnile). È stato, quindi, intrapreso un studio sistematico della capacità di DF1 a legare differenti ioni metallici, attraverso misure di dicroismo circolare e di spettroscopia uv-vis. 6 Prof. Vincenzo Pavone I risultati ottenuti con il Co2+ sono molto incoraggianti, in quanto, come si può vedere dalla tabella seguente, le proprietà spettroscopiche del modello nella regione del visibile sono molto simili a quelle della proteina naturale ricostituita con il cobalto. λmax ε λmax ε λmax ε (nm) (M-1cm-1) (nm) (M-1cm-1) (nm) (M-1cm-1) Modello 525 120 565 150 605 130 Bacterioferritina 520 126 555 155 600 107 Proteina Inoltre, studi di folding condotti sia in presenza che in assenza del metallo hanno dimostrato che la coordinazione influenza positivamente la stabilità del sistema, per tutta la classe di composti esaminati. Il sistema modello è anche in grado di legare ioni ferro o zinco. La proteina nella forma apo è stata completamente caratterizzata in soluzione mediante NMR. Inoltre, sono stati ottenuti cristalli del complesso con lo zinco, adatti ad uno studio diffrattometrico. È stata effettuata una raccolta dati al Centro Elettra, in Trieste, usando luce di sincrotone (risoluzione 2.5 Å, gruppo spaziale C2221, a = 35.97 Å, b = 89.01 Å, c = 79.66 Å). I risultati ottenuti hanno confermato pienamente il modello, sia per quanto riguarda la struttura complessiva della molecola, sia in particolare il sito di coordinazione. 7 Prof. Vincenzo Pavone Sovrapposizione (in stereo) della struttura cristallina del complesso di-Zn-DF1 (azzurro) con il modello (violetto). Studi successivi di mutazioni effettuate nella seconda sfera di coordinazione hanno dimostrato che essa svolge un ruolo chiave nel modulare l’accesso del metallo al sito attivo. Infatti, Ala13-DF1, che contiene in prossimità del centro metallico un residuo di alanina al posto della più ingombrante leucina, lega il ferro più facilmente, anche in condizioni non denaturanti. Inoltre, Ala13-DF1 è stata cristallizata come complesso di di-Mn(II). Sono stati raccolti i dati di diffrazione mediante luce di sincrotrone, ad una risoluzione di 1.7 Å. La densità elettronica è ben definita in prossimità del centro bimetallico, ed è possibile osservare la presenza di un legante esogeno. Una cavità di dimensioni maggiori è stata osservata nel composto di-Mn(II)-Gly13-DF1, in cui il residuo di leucina in posizione 13 è stato sostituito dall’amminoacido meno ingombrato stericamente. 8 Prof. Vincenzo Pavone EMOPROTEINE Reviews: 1. Chemical Reviews (Washington, D. C.) (2001), 101(10), 3165-3189 2. Biopolymers (1998), 47(1), 5-22. Pubblicazioni: 1. Journal of Biological Inorganic Chemistry (2004), 9(8), 1017-1027. 2. Chemistry -A European Journal (2003), 9(22), 5643-5654. 3. Journal of Biological Inorganic Chemistry (1998), 3(6), 671-681. 4. Inorganica Chimica Acta (1998), 275-276(1,2), 301-313. 5. Chemistry--A European Journal (1997), 3(3), 350-362. 6. Chemistry--A European Journal (1997), 3(3), 340-349. L'attività di ricerca svolta dal prof. Pavone nel campo delle eme-proteine è stata rivolta alla realizzazione di sistemi modello, in cui catene peptidiche di differente struttura e composizione potessero costituire per l’eme un intorno chimico simile a quello presente nei sistemi naturali, e riprodurne le loro caratteristiche essenziali, quali regio- e/o stereo-selettività. Lo studio è stato rivolto ad una migliore comprensione di come la matrice proteica possa modulare le proprietà e la reattività del centro metallico. Composizione e struttura della catena proteica in prossimità dell’eme, sostituenti presenti sull’anello porfirinico, natura dei leganti assiali e geometria di coordinazione rappresentano tutti fattori importanti nel determinare le differenti funzioni dell’eme, il cui ruolo può essere meglio razionalizzato attraverso lo studio di piccole molecole. È stata, pertanto, sviluppata una nuova classe di sistemi modello di emeproteine, denominati Mimochrome, il cui motivo strutturale caratteristico è un “sandwich” del tipo elica-eme-elica. Infatti, tali mini-proteine sono costituite da due segmenti peptidici α-elicoidali legati covalentemente ai gruppi propionici della deuteroporfirina IX attraverso l'ε-ammino gruppo di un residuo di Lys. In una 9 Prof. Vincenzo Pavone o in entrambe le catene peptidiche è presente un residuo di His quale legante assiale del metallo inserito nel nucleo porfirinico. Le strutture schematiche delle molecole Mimochrome I, Mimochrome II, Mimochrome III e Mimochrome IV sono riportate di seguito. R Mimochrome N N Fe N N O O R1 R, R1 I R=R1 Ac-L-A-Q-L-H-A-N-K-L-NH2 II R=R1 Ac-D-L-S-D-L-H-S-K-K-L-K-I-T-L-NH2 IV R=R1 Ac-E-S-Q-L-H-S-N-K-R-NH2 III, V, VI R= R1= Ac-D-E-H-K-L-H-S-K-K-R-K-I-T-L-NH2 Ac-D-E-H-K-L-Y-S-K-K-R-K-I-T-L-NH2 Rappresentazione schematica delle molecole Mimochrome È ben noto che in molte eme-proteine l'α-elica è una delle strutture secondarie ricorrenti in prossimità del sito attivo. Uno studio approfondito e dettagliato delle strutture proteiche in prossimità dell'eme nei sistemi naturali ha consentito: 1. di individuare la minima sequenza aminoacidica in grado di stabilizzare una conformazione elicoidale della catena peptidica intorno all’eme; 2. di posizionare correttamente il legante assiale e di renderlo disponibile alla coordinazione del metallo; 3. di costituire un intorno sufficientemente idrofobico per l'eme. Il prototipo della classe, Mimochrome I [3,7,12,17-tetrametilporfirina-2-18-di- N8ε-(Ac-Leu1-Ala2-Gln3-Leu4-His5-Ala6-Asn7-Lys8-Leu9-NH2)-propionammide] è 10 Prof. Vincenzo Pavone stato progettato usando l’elica F della catena β della deossiemoglibina come struttura di riferimento. Studi di modeling molecolare hanno mostrato che cambiando la conformazione del gruppo propionico dell’eme e della catena laterale della Lys95 da trans a gauche, il gruppo carbossilico del propionile e l’ε-ammino gruppo della lisina si sarebbero portati a distanza di legame. Tale legame covalente ci ha consentito di posizionare lo scaffold elicoidale in prossimità del centro metallico, in modo che il segmento peptidico fosse in grado di coprire l’eme in seguito alla coordinazione assiale dell’istidina. Opportune sostituzioni apportate nella sequenza originaria, ed una operazione di simmetria C2 ha condotto alla costruzione del sandwich elica-eme elica. L’analisi degli spettri UV-visibili delle molecole come base libera e nella forma coordinata a ioni metallici nelle regioni caratteristiche del nucleo porfirinico (regione di Soret e regione β/α) ha evidenziato l’inserzione di ioni metallici (Fe(II), Fe(III), Co(III)) nell'anello porfirinico ed una coordinazione del tipo bis- 11 Prof. Vincenzo Pavone istidina, come previsto dal modello. Le proprietà conformazionali della parte peptidica sono stata analizzate mediante tecniche di dicroismo circolare (CD). Secondo le nostre aspettative, le catene peptidiche assumono una conformazione elicoidale, sia nella base libera che nei sistemi contenenti il metallo, come dimostrato dai loro spettri CD tipici di αeliche. A differenza del complesso con il ferro, il complesso Co(III)-Mimochrome I si è presentato sotto forma di due isomeri stabili, separabili in HPLC; essi hanno però evidenziavano effetti Cotton nella regione di Soret di diversa intensità e di segno opposto. I due isomeri sono stati definitivamente identificati via NMR, come diastereoisomeri di tipo ∆ e Λ del complesso ottaedrico. Le catene peptidiche dell’isomero ∆ adottano una conformazione αelicoidale piuttosto regolare, con una parziale distorsione verso l’elica 310 nella parte C-terminale. Le due eliche sono circa antiparallele e correlate da uno pseudo asse di simmetria C2. Nell’isomero Λ, invece, la conformazione delle due catene peptidiche risulta meno regolare. L’insieme dei risultati ottenuti su Mimochrome I e del suo derivato contenente cobalto hanno costituito una base rilevante per la progettazione e la sintesi di una seconda generazione di molecole. Si è cercato di migliorare il modello iniziale favorendo una singola topologia strutturale e cercando così di evitare la formazione dei due possibili isomeri. 12 Prof. Vincenzo Pavone Mimochrome II e Mimochrome III, sono costituiti da due catene peptidiche di 14 residui. I primi 10 residui sono stati modellati in conformazione elicoidale, mentre la parte C-terminale in conformazione estesa. Le due catene peptidiche sono in grado di avvolgere completamente la porfirina, in modo da renderla inaccessibile al solvente e da sfavorire fenomeni di aggregazione tra i nuclei porfirinici. L’analisi delle proprietà spettroscopiche e dicroiche del composto Co(III)Mimochrome II ha evidenziato l’esistenza di una singola specie. Inoltre, un confronto con l’analogo complesso Mimochrome I ha suggerito che l’organizzazione strutturale del composto Co(III)-Mimochrome II corrisponda all’isomero ∆. Mimochrome III si differenzia dalle due precedenti molecole per la differente composizione amminoacidica delle due catene peptidiche. Essa è stata progettata allo scopo di stabilizzare un complesso del ferro pentacoordinato e, pertanto, un residuo di istidina in una catena è stato sostituito con un residuo di serina, non adatto a coordinare il metallo. Nella cavità che si 13 Prof. Vincenzo Pavone crea in prossimità del centro metallico potrebbe essere ospitato un legante esogeno, quale una molecola di CO, NO, O2, H2O2. Una preliminare caratterizzazione ha messo in evidenza anche in questa nuova molecola l’esistenza di una singola specie. Un differente approccio per la stabilizzazione di una singola specie ha riguardato l'introduzione alle estremità N- e C-terminali di residui atti a stabilizzare la struttura a sandwich mediante la formazione di coppie ioniche intercatena. A tale scopo è stata progettata e sintetizzata una nuova molecola, Mimochrome IV, le cui catene peptidiche sono costituite da 9 ammino acidi, come in Mimochrome I, ma che si differenzia da quest’ultima per la presenza di residui di acido glutammico e di arginina rispettivamente nelle posizioni 1 e 9. Anche in questo caso la caratterizzazione mediante CD nelle regione di Soret ha evidenziato la presenza preponderante di una unica specie; questo risultato è stato poi confermato da una caratterizzazione strutturale in soluzione mediante NMR. Gli effetti della parte peptidica nella modulazione delle proprietà del centro metallico sono stati analizzati mediante studi di reattività nei confronti di diversi leganti (quali, CO, NO ed alchi-idrossil-ammine) e di catalisi di particolari reazioni. Gli studi di attività catalitica hanno dimostrato la capacità del complesso Fe(III)-Mimochrome I di catalizzare reazioni di idrossilazione in mezzo acquoso in presenza di un agente ossidante pulito, quale l’acqua ossigenata; inoltre, la catena peptidica svolge un ruolo fondamentale nel proteggere l'anello porfirinico da reazioni di degradazione. Sebbene questi dati siano solo preliminari, ciò rappresenta un risultato di notevole rilevanza e prova che è possibile costruire catalizzatori basati su semplici porfirine naturali, modulandone le proprietà, quali solubilità e resistenza alla degradazione, progettando in modo opportuno uno specifico intorno peptidico. 14 Prof. Vincenzo Pavone PROTEINE FERRO-ZOLFO Miniaturized Electron Transfer Proteins (METP) Brevetti: 1. Pseudo-metalloproteins, their preparation and use in biosensors. WO2002012278 A2 20020214 Pubblicazioni: 2. Proceedings of the National Academy of Sciences of the United States of America (2000), 97(22), 11922-11927. L’interesse scientifico del prof. Pavone è stato rivolto verso le proteine ferrozolfo. I centri Fe/S in alcuni casi sono presenti come sistemi isolati all'interno delle proteine; più spesso, però, mostrano forti interazioni con altri gruppi prostetici o altri centri metallici (Ni, Mo, V, Fe-eme, flavine). La loro principale caratteristica strutturale è rappresentata dalla coordinazione dello ione ferro da parte dello zolfo delle cisteine della catena proteica (RS-), e, nei centri polinucleari, da solfuro inorganico acido-labile (S2-). I centri Fe/S possono essere classificati, in accordo con il loro grado di aggregazione nel cluster, in [1Fe-0S], [2Fe-2S], [3Fe4S] e [4Fe-4S] in base al numero di atomi di ferro e di zolfo non proteico presenti. Le rubredossine sono le più semplici proteine ferro-zolfo, esse sono piccole proteine presenti in alcuni batteri anaerobici, dotate di un potenziale di riduzione compreso tra -57 e 6 mV. Il centro redox attivo nelle rubredossine, [1Fe-0S], consiste in uno ione ferro ad alto spin, coordinato da quattro residui di cisteine in una geometria tetraedrica distorta. Dall’analisi delle strutture di alcune rubredossine le sequenze amminoacidiche mostrano un pattern dei residui coordinanti il metallo che è costituito da due set di coppie di Cys, ciascuna separata da due amminoacidi in una sequenza del tipo -Cys-x-y-Cys-Gly-z. Il lavoro del prof. Pavone nello studio delle proteine ferro-zolfo ha avuto inizio con un’analisi attenta della struttura della rubredossina da Desulfovibrio Vulgaris, 15 Prof. Vincenzo Pavone osservando che, enucleando dall’intera proteina i residui compresi in una sfera di 9 Å intorno al centro metallico, organizzata la in catena modo peptidica è simmetrico rispetto al centro metallico. Un asse di simmetria C2 del tetraedro di coordinazione relaziona due segmenti peptidici di circa 10 amminoacidi ciascuno. Struttura ai raggi X della rubredossina da Desulfovibrio Vulgaris. (a) (b) (a) Segmenti della sequenza amminoacidica della rubredossina da Desulfovibrio Vulgaris contenenti i residui di Cys coordinanti in cui è evidenziata la simmetria del sito metallico; (b) modelli molecolari derivanti dalla rotazione binaria di ciascun segmento di catena, di dieci amminoacidi, contenenti i residui coordinanti. L’applicazione delle metodologie di design, seguendo un approccio minimalistico, ha consentito la progettazione di una prima molecola, denominata METP (Miniaturized Electron Transfer Protein). Tale molecola è costituita da un omodimero formato da due sequenze peptidiche costituite da 11 residui 16 Prof. Vincenzo Pavone aminoacidici; la dimerizzazione ha luogo a seguito della coordinazione dei residui di cisteina a ioni metallici, riproducendo le proprietà della rubredossina naturale. METP Ac- Cys-Thr-Lys-Cys-Gly-Ala-Asn-Aib-Ser-Glu-Ile-NH2 Rd[5-16] Val-Cys-Thr-Val-Cys-Gly-Tyr-Glu-Tyr-Asp-Pro-Ala- Rd[38-49] Val-Cys-Pro-Val-Cys-Gly-Ala-Pro-Lys-Ser- Glu-Phe- Il complesso omodimerico è stabilizzato da interazioni inter-catena di tipo idrofobico tra i residui di Ile C-terminali e di tipo ionico tra il residuo di Lys di una catena e quello di Glu dell’altra. La molecola dimerizza in presenza di ferro, cobalto e zinco, mostrando proprietà spettroscopiche del tutto analoghe a quelle della rubredossina naturale, e tipiche di un complesso con geometria di coordinazione tertraedrica. Spettri UV/Vis Co(II)-METP (sinistra) e Fe(II)/Fe(III)-METP (destra). La corretta stechiometria del complesso Co(II)-METP e la maggiore affinità di METP per lo Zn(II), rispetto al Co(II), confermano il modello e sono in pieno accordo con una coordinazione tetraedrica. Il complesso Zn(II)-METP è stato anche caratterizzato strutturalmente in soluzione mediante NMR. Lo spettro monodimensionale, riportato di seguito, ha mostrato un’ottima dispersione di segnali, indice di strutturazione della molecola. Inoltre, lo spettro mostra un unico set di segnali, in accordo con la simmetria C2 della molecola. L’analisi degli spettri bidimensionali ha confermato che la molecola adotta in soluzione una struttura molto simile a quella della proteina naturale ed al modello proposto. 17 Prof. Vincenzo Pavone Regione amminica dello spettro protonico del complesso Zn(II)-METP in CD3OH a 298 K Gli incoraggianti risultati ottenuti con il primo modello hanno indotto a proseguire le ricerche, cercando di costruire una collezione di modelli analoghi, ML1L2L3L4, con differenti residui coordinanti (L= Cys, Dap, His, Glu, Asp, ecc), e contenenti ioni metallici con diverse proprietà elettroniche, redox e di coordinazione (M= Co, Fe, Mn, Zn, Cd, Ni, ecc.). A tale scopo è risultata necessaria la progettazione di sistemi eterodimeri in cui il complesso metallico si formi in seguito alla dimerizzazione di due catene peptidiche diverse. Sono state già progettate e sintetizzate sequenze peptidiche opportunamente modificate rispetto al prototipo METP sia nella prima che nella secoda sfera di coordinazione. Modelli molecolari dei complessi METP omo- ed etero-dimeri. 18 Prof. Vincenzo Pavone IL RECETTORE DELL’UROCHINASI (UPAR) Pubblicazioni: 1. FEBS Letters (2008), 582(7), 1141-1146. Brevetti: 2. WO 2008017372 A1 20080214 Il recettore dell’urochinasi (uPAR) gioca un ruolo chiave nei processi fisiologici e patologici sostenuti da una alterata migrazione cellulare. Esso è formato da tre domini ed è ancorato alla membrana cellulare mediante un gruppo GPI. uPAR forma complessi con diverse proteine transmembrana e questa interazione consente la trasmissione del segnale. Pertanto, uPAR può svolgere molteplici funzioni a seconda della proteina transmembrana con cui interagisce. uPAR è attivato dall’urochinasi (uPA) che causa una idrolisi dell’uPAR liberando il domino N-terminale. L’interazione uPAR-uPA causa l’esposizione del segmento peptidico Ser88-Arg-Ser-Arg-Tyr92 (SRSRY) che possiede proprietà chemotattiche. E’ stato individuato un peptide di 5 amminoacidi che antagonizza l’interazione di uPAR o del peptide SRSRY con il recettore transmembrana FPR. Questo antagonismo si riflette in una ridotta motilità cellulare direzionata. E’ stata successivamente identificata una nuova classe di peptidi che può trovare applicazione in numerose patologie come i processi infiammatori cronici e nei tumori. 19 Prof. Vincenzo Pavone RANTES Pubblicazioni: 1. Biochemical and Biophysical Research Communications (2006), 351(3), 664668. 2. Nature Structural Biology (2001), 8(7), 611-615. Brevetti: 3. WO 2004065406 A2 20040805 4. WO 2000027880 A2 20000518 Le terapie attualmente in uso per l’infezione da HIV mostrano severe limitazioni. In particolare esse richiedono l’uso concertato di una miscela di farmaci che agiscono a vari stadi della replicazione del virus, ma non sono capaci di bloccare l’ingresso del virus in nuove cellule linfocitarie. Ai farmaci attualmente in uso sono associate tossicità piuttosto elevate che in alcuni casi ne limtano l’uso. Numerosi sforzi sono stati fatti per lo sviluppo di vaccini, ma attualmente non esistono preparati soddisfacenti. È, infatti, da considerare che i pazienti affetti da infezione da HIV sono immunodepressi e, quindi, la reazione di stimolazione immunitaria da vaccino può essere piuttosto ridotta. Da questo quadro di riferimento è emersa la necessità di sviluppare metodi alternativi per sconfiggere l’AIDS. La strategia intrapresa dal prof. Pavone è stata quella di realizzare un sistema che impedisce all’HIV di entrare nelle cellule linfocitarie. Il virus adopera due recettori per entrare nella cellula linfocitaria: CCR5 e CD4. Per impedire l’ingresso del virus nella cellule linfocitarie basterebbe occupare il recettore CCR5 che, quando pieno, non potrebbe assistere il virus per penetrare nelle cellule. La chemochina specifica per il recettore CCR5 è RANTES, una piccola proteina che stimola il sistema immunitario. Studiando RANTES è stato possibile individuare due regioni idrofobiche della sua superficie responsabili 20 Prof. Vincenzo Pavone dell’interazione specifica con CCR5. Sulla base dell’orientazione relativa nello spazio di questi due domini idrofobici, come ricavato dalla struttura tridimensionale di RANTES, è stato possibile composti sviluppare che una serie beneficamente di non stimolano il sistema immunitario e parallelamente inibiscono la fusione del virus con la cellula linfocitaria. Queste nuove molecole possono trovare numerose terapeutiche. altre Ad applicazioni esempio, possono essere adoperate come antivirali. La classe dei mimetici del CD4 possono in aggiunta trovare applicazione nelle malattie autoimmuni, come l’artrite reumatoide, la sclerosi multipla e nel trattamento post-operatorio di trapianto d’organo. 21 Prof. Vincenzo Pavone TROMBINA Review: 1. Biopolymers (1999), 51(1), 19-39. 2. Biological Chemistry (1998), 379(8/9), 987-1006. Pubblicazioni: 3. Protein Science (1999), 8(1), 91-95. 4. Protein Science (1998), 7(2), 243-253. 5. Journal of Medicinal Chemistry (1996), 39(10), 2008-17. Risultati di rilievo sono stati anche ottenuti nello sviluppo di una nuova classe di potenti inibitori sintetici della trombina, denominati Hirunorms, potenziali candidati in applicazioni terapeutiche. Vista stereo della sovrapposizione degli atomi del backbone tra l’irudina (da Ile1’ ad Asp5’ e da Glu49’ a Gln65’, in blu) ed hirunorm IV (da Chg1” ad Asp5” e da Glu10” a D-Glu26”, in rosso) Il più potente inibitore naturale della trombina è l’irudina, una piccola proteina 22 Prof. Vincenzo Pavone di 65 amminoacidi, che lega specificamente la trombina con un valore di Ki di 2.2 . 10-14 M, inibendo il taglio del fibrinogeno e la formazione di coaguli di fibrina. Sulla base della recente risoluzione delle strutture cristalline del complesso irudina-trombina e di complessi della trombina con inibitori sintetici, abbiamo “razionalmente” progettato e sintetizzato una serie di composti peptidici, di basso peso molecolare, capaci di agire, in maniera simile all’irudina, da inibitori “multisito” della trombina. Le strutture cristalline dei complessi della trombina con gli inibitori hirunorm IV ed hirunorm V sono state recentemente risolte. Esse hanno pienamente confermato il nostro modello iniziale ed il modo di binding previsto, simile a quello dell’irudina. Infatti, le molecole Hirunorms, in maniera analoga all’irudina, interagiscono con la trombina con il tetrapeptide N-terminale, allineando il backbone in maniera parallela a quello dell’enzima. Allo stesso tempo l’inibitore si lega in maniera specifica con la parte C-terminale al sito di riconoscimento del fibrinogeno. I risultati ottenuti hanno dimostrato ancora una volta la stretta correlazione esistente tra struttura e funzioni e la possibilità di poter ottenere molecole sintetiche dotate di proprietà analoghe rispetto ai composti naturali. Struttura del complesso Hirunorm IV – α-trombina, come ottenuta da analisi diffrattometrica 23 Prof. Vincenzo Pavone STRUTTURA SECONDARIA DELLE PROTEINE Review: 1. Biopolymers (1993), 33(7), 1037-49. Pubblicazioni: 2. Biopolymers (1996), 38(6), 705-721. 3. Biopolymers (1996), 38(6), 693-703. 4. Biopolymers (1996), 38(6), 683-691. 5. Biopolymers (1994), 34(11), 1517-26. 6. Biopolymers (1994), 34(11), 1505-15. 7. Biopolymers (1993), 33(4), 621-31. 8. Journal of the American Chemical Society (1992), 114(16), 6273-7. 9. Biopolymers (1992), 32(2), 173-83. 10. Gazzetta Chimica Italiana (1991), 121(1), 21-7. 11. Biopolymers (1991), 31(1), 129-38. 12. Biopolymers (1991), 31(10), 1181-8. 13. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1990), (11), 1829-37. 14. Journal of Biomolecular Structure & Dynamics (1990), 7(6), 1321-31. 15. International Journal of Biological Macromolecules (1988), 10(4), 238-40. 16. Journal of Molecular Biology (1990), 214(3), 633-5. 17. Biopolymers (1990), 30(1-2), 189-96. 18. Journal of Biomolecular Structure & Dynamics (1988), 5(4), 803-17. 19. Biopolymers (1989), 28(1), 215-23. 20. Biopolymers (1989), 28(1), 203-14. 21. Biopolymers (1989), 28(1), 193-201. 22. Proceedings of the National Academy of Sciences of the United States of America (1986), 83(7), 1988-92. 24 Prof. Vincenzo Pavone L’esame di un gran numero di strutture tridimensionali di proteine è stata utilizzato dal prof. Pavone per uno studio dettagliato sulla conformazione di “αturn” isolati. Strutture secondarie irregolari, quali appunto “α-turn” isolati (che non fanno cioè parte di α-eliche), sono importanti domini strutturali coinvolti in processi di riconoscimento molecolare e nel “folding” di proteine. L’analisi condotta sulle strutture di proteine riportate nel “Protein Data Bank” ha consentito una razionale classificazione di tale motivo strutturale in 9 classi distinte, sulla base del segno degli angoli diedri φi+1, φi+2, e φi+3. È stato, inoltre, condotto uno studio preliminare per analizzare la frequenza dei vari amminoacidi in particolari posizioni del “turn”. Nomenclatura proposta per gli α-turn sulla base del segno degli angoli φi+1, φi+2, φi+3 . I-αRS φi+1 - φi+2 - φi+3 - I-αLS + + + II-αRS - + - II-αLS + - + I-αLU - + + I-αRU + - - II-αLU - - + II-αRU + + - names Il prof. Pavone ha anche proposto una nuova nomenclatura e classificazione per i β-hairpin. In particolare, è stato proposto un nuovo criterio per distingure i residui amminoacidici appartenenti al loop da quelli caratteristici del tratto βantiparallelo, basato sui possibili schemi di legame idrogeno osservati nelle strutture proteiche. Questa analisi ha permesso di classificare i β-hairpin in 4 differenti classi ed è stato possibile proporre una semplice relazione esistente tra i 25 Prof. Vincenzo Pavone membri di una stessa classe. Inoltre, durante i suoi studi, il prof. Pavone ha individuato strutture molto particolari e, all’epoca, poco conosciute, come ad esempio il β-bend ribbon spiral, l’elica 310, doppie eliche o strutture tubulari tipo nanotubi. Struttura del β-bend ribbon spiral Elica 310 Una ulteriore strategia adoperata per individuare le particolarità di strutture secondarie è consistito nello studio di peptidi ciclici. Tuttavia, anche se il vincolo topologoco della ciclizzazione riduce considerevolmente la flessibilità conformazionale in confronto al precursore lineare, i peptidi ciclici sono ancora 26 Prof. Vincenzo Pavone molecole flessibili. β-Amminoacidi incorporati in peptidi ciclici possono fornire domini strutturali ben definiti e rappresentano importanti “attrezzi molecolari” per la stabilizzazione di particolari conformazioni. E’ stata intrapresa una analisi sistematica sulle preferenze conformazionali di peptidi ciclici contenenti βalanine. Abbiamo ipotizzato e successivamente verificato sperimentalmente che: • Il segmento dipeptidico β-alanil-β-alanina può assumere le posizioni 1 ed n+1 di (a) un “γ-turn” in un tripeptide ciclico (n=2); (b) un “β-turn” in un tetrapeptide ciclico (n=3); (c) un “α-turn” in un pentapeptide ciclico (n=4). • Il segmento tripeptidico β-Ala-Pro-β-Ala può stabilizzare un “γ-turn” intorno al residuo di prolina e, quando incorporato in un tetrapeptide ciclico, può forzare il quarto amminoacido ad adottare una conformazione “γ-turn”. β L’analisi strutturale condotta allo stato solido sul peptide ciclo-(L-ProL-Phe-β-Ala-β-Ala) ha dimostrato che il dipeptide -β-Ala-β-Ala può essere convenientemente utilizzato per forzare il resto della molecola in una conformazione di tipo “β-turn”. Infatti il composto mostra un legame idrogeno intramolecolare tra il gruppo CO di βAla4 e l’NH di β-Ala3, stabilizzando un “β-turn” di tipo I, in cui i residui di prolina e fenilalanina occupano rispettivamente le posizioni 2 e 3. 27 Prof. Vincenzo Pavone I tetrapeptidi ciclici c-(L-Pro-β-Ala-L-Pro-βAla) e c-(L-Pro-β-Ala-L-Val-β-Ala) sono stati caratterizzati sia in soluzione che allo stato solido. I dati NMR ottenuti in soluzione di acetonitrile hanno pienamente confermato le nostre ipotesi, mostrando che la conformazione, in entrambe le molecole, è caratterizzata da due “γi-turn”, in cui gli αamminoacidi occupano la posizione centrale. Allo stato solido, mentre il primo composto conserva la conformazione trovata in soluzione, il secondo presenta una inaspettata conformazione, stabilizzata da legami idrogeno intermolecolari; nessun legame idrogeno intramolecolare è, invece, presente. In conclusione, gli studi condotti sui peptidi ciclici contenenti β-alanine hanno dimostrato che questi residui sono capaci di indurre peculiari conformazioni, suggerendo che β-amminoacidi possono essere utilizzati come convenienti “tool” molecolari e possono essere incorporati in molecole di dimensioni maggiori, per stabilizzare ben definiti elementi strutturali. 28 Prof. Vincenzo Pavone NEUROKININA A Review: 1. Regulatory Peptides (1996), 65(1), 55-59. Brevetti: 2. WO 9731941 A2 19970904 3. WO 9321227 A1 19931028 Pubblicazioni: 4. Bioorganic & Medicinal Chemistry Letters (1998), 8(13), 1735-1740. 5. Bioorganic & Medicinal Chemistry Letters (1998), 8(10), 1153-1156. 6. Journal of the American Chemical Society (1998), 120(24), 5879-5886. 7. Journal of Peptide Science (1995), 1(4), 236-40. 8. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1995), (5), 987-93. 9. Journal of Pharmacology and Experimental Therapeutics (1994), 271(3), 1489-500. 10. Journal of Medicinal Chemistry (1994), 37(21), 3630-8. I risultati ottenuti nei primi anni dell’attività di ricerca sulle preferenze conformazionali di peptidi ciclici contenenti β-amminoacidi sono stati opportunamente adoperati nella progettazione di un peptide biciclico, denominato Men 10627 [ciclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6)ciclo(2β-5β)], potente e selettivo antagonista della Neurochinina A. La realizzazione di tale composto è stato anche oggetto di brevetti internazionali. La Neurochinina A (NKA) appartiene alla famiglia delle tachichinine, peptidi largamente distribuiti nel sistema nervoso centrale e periferico. Essi rivestono una varietà di ruoli importanti e ciò ha stimolato un grande interesse 29 Prof. Vincenzo Pavone verso la possibilità di modulare o di bloccare la loro azione attraverso lo sviluppo di specifici antagonisti. Le tachichinine riconoscono, anche se con diversa affinità, tre differenti recettori: NK1, NK2 ed NK3; questa osservazione ci ha portato ad ipotizzare che queste molecole possono assumere in soluzione almeno tre differenti conformazioni, ciascuna delle quali si accomoda in un differente sito recettoriale. Tale ipotesi è supportata da una analisi strutturale condotta su diversi agonisti ed antagonisti, che ha mostrato che queste molecole sono estremamente flessibili in soluzione e distribuite in un gran numero di famiglie conformazionali. Si è, pertanto, cercato di “costruire” un antagonista della NKA, costretto in una ben determinata conformazione, che interagisse con maggiore affinità e selettività con il suo recettore primario NK2. Sulla base dei dati disponibili in letteratura, si è ipotizzato che una struttura contenente due “β-turn” fosse responsabile dell’interazione della NKA con il recettore NK2 ed abbiamo, quindi, cercato di riprodurre tale struttura in una “impalcatura” rigida. Utilizzando le nostre conoscenze sulle preferenze confomazionali di tetrapeptidi ciclici contenenti due β-alanine consecutive, abbiamo sviluppato un modello corrispondente a due tetrapeptidi ciclici “fusi” insieme, ciascuno contenente un “β-turn”. Men 10627 assume la conformazione prevista nella fase di progettazione e si è rivelato il più potente e selettivo antagonista della NKA, dimostrando che è una tridimensionale rigida struttura è requisito il essenziale per lo sviluppo di molecole dotate di specifiche funzioni. Successivamente, è stato sviluppato un nuovo analogo denominato 30 Prof. Vincenzo Pavone Neuronorm. Esso è un composto di grande interesse applicativo, in quanto, oltre a possedere elevata potenza e selettività, presenta anche una buona solubilità in acqua. Quest’ultima importante caratteristica è conferita dalla presenza di un’unita glicosilica. Lo sviluppo di questo composto è stato oggetto di un brevetto internazionale. 31 Prof. Vincenzo Pavone REVIEWS 1. Diiron-containing metalloproteins: Developing functional models. Maglio, Ornella, Nastri, Flavia, Martin de Rosales, Rafael Torres, Faiella, Marina, Pavone, Vincenzo, DeGrado, William F., Lombardi, Angela. Comptes Rendus Chimie (2007), 10(8), 703-720. Abstract: A review. A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technol., such as in degrdn. of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochem. functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biol., with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-contg. proteins, and we developed models for heme proteins and diiron-oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins. 2. Artificial diiron proteins: From structure to function. Calhoun, Jennifer R., Nastri, Flavia, Maglio, Ornella, Pavone, Vincenzo, Lombardi, Angela, DeGrado, William F. Biopolymers (2005), 80(2 and 3), 264-278. Abstract: A review. De novo protein design provides an attractive approach for the construction of models to probe the features required for the function of complex metalloproteins. These minimal models contain the essential elements believed necessary for activity of the protein. Here, the authors summarize the design, structure detn., and functional properties of a family of artificial diiron proteins which include DF1, DF2, DF2t, and DFsc. 3. Peptide-Based Heme-Protein Models. Lombardi, Angela, Nastri, Flavia, Pavone, Vincenzo. Chemical Reviews (Washington, D. C.) (2001), 101(10), 3165-3189. Abstract: A review, with refs. Topics discussed include: developing heme- 32 Prof. Vincenzo Pavone protein mimetics; spectral properties of metalloporphyrins and hemeproteins; covalent peptide-porphyrin systems; and noncovalent peptideporphyrin systems. 4. From natural to synthetic multisite thrombin inhibitors. Lombardi Angela, De Simone Giuseppina, Galdiero Stefania, Staiano Norma, Nastri Flavia, Pavone Vincenzo. Biopolymers (1999), 51(1), 19-39. Abstract: A large number of potent and selective therapeutic agents, useful for the treatment of several diseases, have been isolated from natural sources. For example, the most active thrombin inhibitors are those secreted by the salivary glands of leeches. One peculiar feature of these agents is the lack of any significant inhibitory cross-reaction with other serine proteinases. Hence, the knowledge of the exact mechanism of action of these molecules provides the basis for the development of new and efficient synthetic drugs. For this reason, many studies have been undertaken on the structure-activity relationships of natural thrombin inhibitors, and a large amount of detailed information has been obtained by the crystal structures of these inhibitors when complexed with thrombin. In this paper, we review natural and synthetic multisite thrombin inhibitors, whose structural aspects have been determined in detail. We also report here the approach used by us to develop a new class of synthetic, multisite directed thrombin inhibitors, named hirunorms, designed to mimic the distinctive binding mode of hirudin. 5. De novo design and structural characterization of proteins and metalloproteins. DeGrado, William F., Summa, Christopher M., Pavone, Vincenzo, Nastri, Flavia, Lombardi, Angela. Annual Review of Biochemistry (1999), 68 779-819. Abstract: A review with 309 refs. De novo protein design has recently emerged as an attractive approach for studying the structure and function of proteins. This approach critically tests our understanding of the principles of protein folding; only in de novo design must one truly confront the issue of how to specify a protein's fold and function. If proteins can be truly understood, it should be possible to design receptors, enzymes, and ion channels from scratch. Further, as this understanding evolves and is further refined, it should be possible to design proteins and biomimetic polymers with properties unprecedented in nature. 6. Miniaturized hemoproteins. Nastri, Flavia, Lombardi, Angela, D'Andrea, Luca D., Sanseverino, Marina, Maglio, Ornella, Pavone, Vincenzo. Biopolymers (1998), 47(1), 5-22. 33 Prof. Vincenzo Pavone Abstract: A review, with .apprx.114 refs. The present paper highlights and reviews current research in the field of hemoprotein models. Hemoproteins have been extensively studied in order to understand structure-function relationships, and to design new mols. with desired functions. A wide no. of synthetic analogs have been developed, using quite different approaches. They differ in mol. structures, ranging from simple meso-substituted tetraaryl-metalloporphyrins and peptide-porphyrin conjugates. In this paper we summarize the state of the art on peptide based hemoprotein models. We also report here the approach used by us to develop a new class of mols., named mimochromes. They can be regarded as miniaturized hemoproteins, because mimochromes are low mol. wt. compds. with some structural and functional properties common to those of the parent high mol. wt. protein. The basic structure of mimochromes is a deutero-porphyrin ring covalently linked to two helical peptide chains. Two mols. of this series have been fully characterized. All the information derived from their structural anal. has been applied to the design of new analogs with addnl. functions. 7. Multiple binding mode of reversible synthetic thrombin inhibitors. A comparative structural analysis. Pavone, Vincenzo, De Simone, Giuseppina, Nastri, Flavia, Galdiero, Stefania, Staiano, Norma, Lombardi, Angela, Pedone, Carlo. Biological Chemistry (1998), 379(8/9), 987-1006. Abstract: A review is given with many refs. on the state of the art on reversible thrombin inhibitors. The authors discuss some structural aspects of thrombin-inhibitor interaction, which account for the different affinity and potency of these mols. The central role of the Ser protease thrombin in hemostasis and thrombosis brought the development of highly potent and selective thrombin inhibitors. Thrombin-inhibitor complexes have extensively been studied to understand structure-function relationships, and to design new inhibitors that can be used with broader efficacy over existing antithrombotic agents. The authors also report the approach to develop a new class of synthetic, multisite-directed thrombin inhibitors, named hirunorms, designed to mimic the distinctive binding mode of hirudin. The authors emphasize that, despite the high specificity of thrombin action, the interaction of inhibitors in its active site may occur with quite different mechanisms. 8. A review of the design, synthesis and biological activity of the bicyclic hexapeptide tachykinin NK2 antagonist MEN10627. Quartara, Laura, Pavone, Vincenzo, Pedone, Carlo, Lombardi, Angelina, Renzetti, Anna Rita, Maggi, Carlo Alberto. Regulatory Peptides (1996), 65(1), 55-59. 34 Prof. Vincenzo Pavone Abstract: A review with 29 refs. We review the reported data on the design, the conformational features and the pharmacol. properties of the bicyclic peptide tachykinin NK2 receptor antagonist MEN 10,627 or cyclo(Met-Asp -Trp-Phe-Dap-Leu)cyclo(2β-5β). MEN 10,627 possesses a highly constrained structure characterized by two consecutive β-turns, as confirmed by the almost coincident results of NMR and x-ray analyses. The compd. has been efficiently synthesized by solid-phase methodol. using either Boc or Fmoc strategies. It is quite stable to metabolic degrdn. and is endowed with high affinity and selectivity for NK2 receptor expressed in various species. At the hamster NK2 receptor MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist, SR 48,968, while the converse is true for the rabbit NK2 receptor. MEN 10,627 and SR 48,968 show comparable affinities for the human NK2 receptor. MEN 10,627 produces a long lasting inhibition of the response to the selective NK2 receptor agonist [βAla8]NKA(4-10) in the rat urinary bladder in vivo after i.v., intranasal and intraduodenal administration. Therefore different administration routes are possible for this compd. that overcomes the usual drawbacks for the application of peptides as drugs. 9. Noncoded residues as building blocks in the design of specific secondary structures: symmetrically disubstituted glycines and β -alanine. Di Blasio, Benedetto, Pavone, Vincenzo, Lombardi, Angela, Pedone, Carlo, Benedetti, Ettore. Biopolymers (1993), 33(7), 1037-49. Abstract: A review with 98 refs. Structural changes can be induced in a peptide by selective substitution of coded α-amino acid residues by noncoded α-amino acid residues and the consequent prodn. of analogs with modified structure and conformational preferences. This review summarizes the solid state structural results and the conformational preferences of two classes of "building blocks": (a) the linear and cyclic sym. α,α-disubstituted glycines in which either two identical n-alkyl groups replace the hydrogen atoms of the glycine residue or a cyclic aliph. sidechain system is formed by linking the two α-carbon side chains, resp.; and (b) the β-alanine residue. Examples, whenever possible, of the use of these residues for the elucidation of the bioactive conformation in the appropriate biol. systems are given. 10. Structure-activity relationships in bioactive peptides. Benedetti, Ettore, Di Blasio, Benedetto, Pavone, Vincenzo, Pedone, Carlo. Colloque INSERM (1989), 174, 27-40. Abstract: A review and discussion with 66 refs., on bioactive peptide functions (e.g., mol. recognition, membrane interactions) dependence 35 Prof. Vincenzo Pavone on conformation and structure, giving as examples (1) antibiotic ionophores, gramicidin A and alamethicin and (2) toxins or toxin antagonists from Amanita mushrooms. Methods for correlation of physicochem. data with structural and conformational parameters are included. 11. Preferred structures of constrained peptides from achiral α,α-dialkylated glycyl residues with acyclic side chains. Benedetti, Ettore, Di Blasio, Benedetto, Pavone, Vincenzo, Pedone, Carlo, Bavoso, Alfonso, Toniolo, Claudio, Bonora, Gian Maria, Leplawy, Miroslaw T, Hardy, Paul M. Journal of Biosciences (Bangalore, India) (1985), 8(1-2), 253-62. Abstract: A review with 28 refs. on conformational analyses of the title peptides by conformational energy computation, IR and H-NMR spectroscopy, and x-ray diffraction. The relation of conformational preference to side-chain bulkiness is discussed. 12. Preferred conformations of peptides containing α,α-disubstituted α-amino acids. Toniolo, Claudio; Bonora, Gian Maria; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Biopolymers (1983), 22(1), 205-15. Abstract: A review with 63 refs. on the conformations of αaminoisobutyrate- and isovaline-substituted peptides. 36 Prof. Vincenzo Pavone BREVETTI 1. Peptides having pharmacological activity for treating disorders associated with altered cell migration, such as cancer. Carriero, Maria Vincenza; De Rosa, Mario; Pavone, Vincenzo. (Italy). PCT Int. Appl. (2008), 35pp. CODEN: PIXXD2 WO 2008017372 A1 20080214 Designated States W: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, TR, TT, TZ. Designated States RW: AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IS, IT, LU, MC, MT, NL, PT, SE, TR, BF, BJ, CF, CG, CI, CM, GA, ML, MR, NE, SN, TD, TG. Patent written in English. Application: WO 2007-EP6424 20070719. Priority: IT 2006-1607 20060809. CAN 148:230115 AN 2008:192190 Patent Family Information Patent No. WO2008017372 Kind A1 Date 20080214 Application No. WO2007-EP6424 Date 20070719 W: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW RW: AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL, PT, RO, SE, SI, SK, TR, BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG, BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW, AM, AZ, BY, KG, KZ, MD, RU, TJ, TM Priority Application IT2006-MI1607 A 20060809 Abstract: Peptides and their functionally equiv. derivs. are disclosed, in salified or non-salified form, with the general formula L1-X1-X2-X3-X4, wherein: L1 is H, or acyl, or any natural or non-natural amino acid, optionally N-acylated, N-alkylated 37 Prof. Vincenzo Pavone and/or Cα-alkylated; X1 and X3, which are equal or different, are any natural or non-natural basic amino acid, optionally N-alkylated and/or Cα-alkylated. X2 is any natural or non-natural amino acid, optionally N-alkylated and/or Cαalkylated, with the proviso that it is not glycine and amino acids mono-substituted on the α carbon atom with a linear or cyclic alkyl group, from 1 to 10 carbon atoms, and amino acids mono-substituted on the α carbon atom with a linear or cyclic alkyl group contg. 4 to 10 carbon atoms, or amino acids mono-substituted on the α carbon atom with an alkyl group contg. 1 to 8 carbon atoms, optionally substituted with a carbamoyl, hydroxyl or arom. group; X4 is any natural or nonnatural hydrophobic amino acid, optionally Cα-alkylated and/or amidated at the C-terminal end, or any hydrophobic amino alc., or a hydrophobic gem-diamine, optionally N'-alkylated or N'-acylated. 2. RANTES-derived peptides for treatment of diseases involving RANTES receptors. Pavone, Vincenzo; Lusso, Paolo. (Primm S.R.L., Italy). PCT Int. Appl. (2004), 18 pp. CODEN: PIXXD2 WO 2004065406 A2 20040805 Designated States W: AE. Patent written in English. Application: WO 2004IB155 20040122. Priority: IT 2003-107 20030124. CAN 141:167845 AN 2004:633944 Patent Family Information Patent No. WO2004065406 WO2004065406 Kind A2 A3 Date 20040805 20040916 Application No. WO2004-IB155 Date 20040122 W: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NA, NI Patent No. EP1585764 Kind A2 Date 20051019 Application No. EP2004-704297 Date 20040122 R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT, IE, SI, LT, LV, FI, RO, MK, CY, AL, TR, BG, CZ, EE, HU, SK Patent No. US20060165650 Kind A1 Priority Application IT2003-MI107 A WO2004-IB155 W Date 20060727 Application No. US2005-542857 Date 20050721 20030124 20040122 38 Prof. Vincenzo Pavone Abstract: This invention describes the prepn. of RANTES-derived peptides of general formula Ac-C-x1-PYI-x2-x3-Y-NH2 (x1, x3 = Phe, Tyr, Nal, Cha; x2 = spacer of 2 to 12 amino acidic residues) for treatment of diseases in which RANTES receptor is involved. Peptides of the invention are useful for prevention or treatment of AIDS and other viral diseases, inflammatory, allergic, degenerative, and neoplastic diseases and all diseases in which chemokines or their receptors are involved. 3. New cyclic peptides, their preparation and pharmaceutical use as immunosuppressants. Lombardi, Angelina; Pavone, Vincenzo. (Universita' degli Studi di Napoli Federico II, Italy). Ital. Appl. (2002), 51pp. CODEN: ITXXCZ IT 2001RM0285 A1 20021128 Patent written in Italian. Application: IT 2001-285 20010528. Priority: . CAN 146:380315 AN 2007:413297 Patent Family Information Patent No. IT2001RM0285 Kind A1 Priority Application IT2001-RM285 Date 20021128 Application No. IT2001-RM285 Date 20010528 20010528 Abstract: Cyclic peptides cyclo[X1-X2-X3-X4-X5-X6] and cyclo[Y6-Y5-Y4-Y3Y2-Y1] [X1, X2 = independently hydrophobic natural or synthetic L-amino acid; X3, X5 = independently hydrophilic natural or synthetic L-amino acid or Gly; X4, Y4 = independently natural or synthetic D-amino acid or Gly, or αmethylalanine, α-aminocyclobutanecarboxylic acid, etc.; X6, Y6 = independently natural or synthetic L-amino acid or Gly, or α-methylalanine, αaminocyclobutanecarboxylic acid, etc.; Y1, Y2 = independently hydrophobic natural or synthetic D-amino acid; Y3, Y5 = independently hydrophilic natural or synthetic D-amino acid or Gly] were prepd. as immunosuppressants useful for treating pathologies involving activation of T-lymphocytes. Their immunosuppressant activity was detd. in vitro and in vivo in human or murine mixed lymphocyte cultures by measuring lymphocyte proliferation by [3H]-TdR incorporation assay and the prodn. of interferon γ and interleukin-5 by ELISA assay. Thus, cyclo[(β-1-Nal)-(β-1-Nal)-Glu-(D-Pro)-His-Asn] (β-1-Nal = β-1naphthylalanine), prepd. by a solid phase synthesis, significantly decreased lymphocyte proliferation and inhibited prodn. of interferon γ and interleukin-5. 4. Pseudo-metalloproteins, their preparation and use in biosensors. Lombardi, Angelina; Pavone, Vincenzo. (Universita' Degli Studi di Napoli "Federico II", Italy). PCT Int. Appl. (2002), 22 pp. CODEN: PIXXD2 WO 2002012278 A2 20020214 Designated States W: AE, AG, AL, AM, AT, AU, AZ, BA, BB, 39 Prof. Vincenzo Pavone BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW, AM, AZ, BY, KG, KZ, MD, RU, TJ, TM. Designated States RW: AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR, BF, BJ, CF, CG, CI, CM, GA, ML, MR, NE, SN, TD, TG. Patent written in English. Application: WO 2001-IB1427 20010809. Priority: IT 2000-454 20000810. CAN 136:180121 AN 2002:123042 Patent Family Information Patent No. WO2002012278 WO2002012278 Kind A2 A3 Date 20020214 20020613 Application No. WO2001-IB1427 Date 20010809 W: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW RW: GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW, AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR, BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG Patent No. IT2000RM0454 IT1317895 CA2419019 AU2001076606 EP1309612 Kind A1 B1 A1 A A2 Date 20020211 20030715 20020214 20020218 20030514 Application No. IT2000-RM454 Date 20000810 CA2001-2419019 AU2001-76606 EP2001-954265 20010809 20010809 20010809 R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT, IE, SI, LT, LV, FI, RO, MK, CY, AL, TR Patent No. NZ524643 AU2001276606 US20050090649 Kind A B2 A1 Date 20040924 20070118 20050428 Application No. NZ2001-524643 AU2001-276606 US2003-344329 Date 20010809 20010809 20030731 40 Prof. Vincenzo Pavone Priority Application IT2000-RM454 A WO2001-IB1427 W 20000810 20010809 Abstract: Described herein are Pseudo-metalloproteins (M = metal selected among Fe, Mn, Ti, Mo, Co, Ni, Cu, Pd, Pt, Au, Ru, Cr, V, Tb, Yb, Rh, Ir, Os; X1 = antigen, or else a functional group that enables assocn. to a biomol.; X2 = functional group that enables assocn. to an electrode; S1 and S2 = spacer groups made up of a chain of 3-12 atoms of C, N, O, S and corresponding mixts.; all the other substituents have an amino acid nature), their prepn., and electrochem. biosensors contg. them. The biosensors can be used in various assays such as diagnostic assays, immunodiagnostic assays, detn. of pollutants in water, etc. A peptide-metal complex, contg. Fe3+ as M; substance P sequence as X1; Cys as X2 and C1-4; Gly-Gly as S1 and S2, was prepd. The peptides were synthesized on an automatic peptide synthesizer and then complexed with Fe(SO4)2(NH4)2. 5. RANTES-derived peptides with anti-HIV, anti-allergic, and antiinflammatory activity. Lusso, Paolo; Pavone, Vincenzo. (Fondazione Centro San Raffaele del Monte Tabor, Italy; Primm S.R.L.). PCT Int. Appl. (2000), 23 pp. CODEN: PIXXD2 WO 2000027880 A2 20000518 Designated States W: AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW, AM, AZ, BY, KG, KZ, MD, RU, TJ, TM. Designated States RW: AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, BF, BJ, CF, CG, CI, CM, GA, ML, MR, NE, SN, TD, TG. Patent written in English. Application: WO 99-EP8651 19991111. Priority: IT 98-2441 19981111. CAN 133:824 AN 2000:335443 Patent Family Information Patent No. WO2000027880 WO2000027880 Kind A2 A3 Date 20000518 20001005 Application No. WO1999-EP8651 Date 19991111 W: AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW 41 Prof. Vincenzo Pavone RW: GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW, AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG Patent No. IT98MI2441 IT1303736 CA2348308 EP1131352 Kind A1 B1 A1 A2 Date 20000511 20010223 20000518 20010912 Application No. IT1998-MI2441 Date 19981111 CA1999-2348308 EP1999-968785 19991111 19991111 R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT, IE, SI, LT, LV, FI, RO Patent No. JP2003500334 US6709649 Kind T B1 Priority Application IT1998-MI2441 A WO1999-EP8651 W Date 20030107 20040323 Application No. JP2000-581057 US2001-831500 Date 19991111 20010622 19981111 19991111 Abstract: Peptides are provided which have 12-30 amino acids with a sequence homologous or corresponding to the sequence 10-34 of RANTES and have inhibitory activity against the human immunodeficiency virus (HIV) as well as anti-allergic and anti-inflammatory activity. 6. Preparation of soluble peptide tachykinin antagonists. Pavone, Vincenzo; Lombardi, Angelina; Pedone, Carlo; De Rosa, Mario; Rossi, Mose. (Centro Interuniversitario Di Ricerca Sui Peptidi Bioattivi-Universita Degli, Italy). PCT Int. Appl. (1997), 15 pp. CODEN: PIXXD2 WO 9731941 A2 19970904 Designated States W: AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, AM, AZ, BY, KG, KZ, MD, RU, TJ, TM. Designated States RW: AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, BF, BJ, CF, CG, CI, CM, GA, ML, MR, NE, SN, TD, TG. Patent written in English. Application: WO 97-EP917 19970226. Priority: IT 96-1401 19960301. CAN 127:248426 AN 1997:594751 Patent Family Information Patent No. Kind Date Application No. Date 42 Prof. Vincenzo Pavone WO9731941 WO9731941 A2 A3 19970904 19971023 WO1997-EP917 19970226 W: AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU RW: GH, KE, LS, MW, SD, SZ, UG, AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG Patent No. CA2247802 AU9718771 EP894093 EP894093 Kind A1 A A2 B1 Date 19970904 19970916 19990203 20020522 Application No. CA1997-2247802 AU1997-18771 EP1997-905089 Date 19970226 19970226 19970226 R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT, IE, F Patent No. JP2001502656 AT217886 US6075006 Kind T T A Priority Application IT1996-MI401 A WO1997-EP917 W Date 20010227 20020615 20000613 Application No. JP1997-530587 AT1997-905089 US1998-125542 Date 19970226 19970226 19980828 19960301 19970226 Abstract: Glycosated cyclopeptides I [X1 = D- or L-Cys(Y), D- or L-SeCys(Y); Z1 = Asp, Z2 = Dap; Z1 = Dap, Z2 = Asp; X2, X3, X4 = natural or synthetic hydrophobic amino acids, having Z1, Z2, X2, X3, and X4 the same D- or Lconfiguration; Y = glycosidic group selected from the aldo and keto hexoses in the furanose or pyranose form bound to the Cys residue with an α- or β- thioacetal bond or a cyclitol or a polyvinyl alc. or PEG group contg. 5-10 monomers, bound to the Cys with a thioether bond; SeCys = selenocysteine; Dap = 2,3diaminopropanoic acid], are endowed of high soly. and of potent tachykininantagonistic activity. Thus, galactocysteine cyclopeptide II, prepd. by std. solidphase methods, inhibited neurokinin A response to an agonist with pA2 = 8.4 and had a soly. of 1.8 µg/mL. 43 Prof. Vincenzo Pavone X 1 - Z 1 -X 2 -X 3 -Z 2 -X 4 H-C ys-A sp-Trp-Phe-D OH O I ap-Leu-OH OH HO HO 7. II Preparation of cyclic peptide tachykinin antagonists. Pavone, Vincenzo; Lombardi, Angelina; Pedone, Carlo; Maggi, Carlo Alberto; Quartara, Laura. (Menarini A., Industrie Farmaceutiche Riunite S.r.L., Italy; Laboratori Guidotti S.p.A.; Malesci - Istituto Farmacobiologico S.p.A.). PCT Int. Appl. (1993), 29 pp. CODEN: PIXXD2 WO 9321227 A1 19931028 Designated States W: AU, BB, BG, BR, CA, CZ, FI, HU, JP, KP, KR, KZ, LK, MG, MN, MW, NO, NZ, PL, RO, RU, SD, SK, UA, US, VN. Designated States RW: AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, BF, BJ, CF, CG, CI, CM, GA, ML, MR, NE, SN, TD, TG. Patent written in English. Application: WO 93-EP893 19930413. Priority: IT 92-89 19920415. CAN 121:158200 AN 1994:558200 Patent Family Information Patent No. WO9321227 Kind A1 Date 19931028 Application No. WO1993-EP893 Date 19930413 W: AU, BB, BG, BR, CA, CZ, FI, HU, JP, KP, KR, KZ, LK, MG, MN, MW, NO, NZ, PL, RO, RU, SD, SK, UA, US, VN RW: AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG Patent No. AU9340395 AU671118 EP636146 EP636146 Kind A B2 A1 B1 Date 19931118 19960815 19950201 19970730 Application No. AU1993-40395 Date 19930413 EP1993-911459 19930413 R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LI, LU, MC, NL, PT, SE Patent No. HU70189 JP08500331 IN177038 Kind A2 T A1 Date 19950928 19960116 19961026 Application No. HU1994-2977 JP1993-517974 IN1993-CA212 Date 19930413 19930413 19930413 44 Prof. Vincenzo Pavone AT156135 ES2105265 RO112872 BR9306248 CN1080916 CN1039126 IL105381 ZA9302644 NO9403861 FI9404838 IN1995CA00332 US5731285 T T3 B1 A A B A A A A A A 19970815 19971016 19980130 19980623 19940119 19980715 19980222 19931022 19941013 19941014 20050304 19980324 Priority Application IT1992-FI89 A WO1993-EP893 A US1994-318669 B1 AT1993-911459 ES1993-911459 RO1994-1666 BR1993-6248 CN1993-104453 19930413 19930413 19930413 19930413 19930414 IL1993-105381 ZA1993-2644 NO1994-3861 FI1994-4838 IN1995-CA332 US1996-731709 19930414 19930415 19941013 19941014 19950327 19961017 19920415 19930413 19941006 Abstract: Title compds. (I; X1-X6 = NR'CO, CONR'; R, R' = H, alkyl; Y = CONR, NRCO, OCO, COO, CH2R, NRCH2, SS, CH2CH2, CH:CH; R1-R4 = hydrophobic group; n, m = 1-4), were prepd. Thus, title compd. II (Dpr = 2,3diaminopropionyl residue), prepd. via cyclization of intermediate III using PyBOP/DIEA in DMF, at 10 µM gave 100% inhibition of response to substance P Me ester by isolated guinea pig ileum. R 1 CH X2 CH X3 CHR 2 CH 2 ) n X1 X4 Y CH 2 ) m R 4 CH X6 CH X5 CHR 3 H-Met-A sp-Trp-Phe-D I Met-A sp-Trp-Phe-D pr-Leu-OH pr-L eu II III 45 Prof. Vincenzo Pavone PUBBLICAZIONI 1. An urokinase receptor antagonist that inhibits cell migration by blocking the formyl peptide receptor. Bifulco, Katia; Longanesi-Cattani, Immacolata; Gargiulo, Lucia; Maglio, Ornella; Cataldi, Mauro; De Rosa, Mario; Stoppelli, Maria Patrizia; Pavone, Vincenzo; Carriero, Maria Vincenza. FEBS Letters (2008), 582(7), 1141-1146. Abstract: Urokinase receptor (uPAR) plays a key role in physiol. and pathol. processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser88-Arg-Ser-ArgTyr92 (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH2) shares the same binding site with SRSRY and competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the G-protein-coupled N-formyl-peptide receptor (FPR). pERERY-NH2 is a dose-dependent inhibitor of both SRSRY- and fMLF-directed cell migration, and prevents agonist-induced FPR internalization and fMLF-dependent ERK1/2 phosphorylation. pERERYNH2 is a new and potent uPAR inhibitor which may suggest the generation of new pharmacol. treatments for pathol. conditions involving increased cell migration. 2. Critical role of the N-loop and β1-strand hydrophobic clusters of RANTESderived peptides in anti-HIV activity. Vangelista, Luca; Longhi, Renato; Sironi, Francesca; Pavone, Vincenzo; Lusso, Paolo. Biochemical and Biophysical Research Communications (2006), 351(3), 664-668. Abstract: HIV initiates its infectious cycle by docking to CD4 and a chemokine receptor, most commonly CCR5. RANTES, a natural CCR5 ligand, is a potent inhibitor of HIV-1. Despite the lack of structural information on the RANTES-CCR5 complex, determinants of HIV blockade were previously identified within the RANTES N-loop and β1strand regions. A prototype N-loop/β1-strand peptide, named R11-29, contains two terminal hydrophobic stretches sepd. by a central hydrophilic region. Here, the role of the terminal hydrophobic clusters was investigated by means of amino acid substitutions or deletions. Most hydrophobic residues in these clusters were shown to be fundamental for the anti-HIV activity. However, increasing the hydrophobicity of the two clusters using non-natural amino acids did not significantly improve the potency of the peptides. These results may provide instrumental knowledge for the rational design of RANTES-deriv. mols. with increased anti-HIV activity. 3. Artificial di-iron proteins: solution characterization of four helix bundles 46 Prof. Vincenzo Pavone containing two distinct types of inter-helical loops. Maglio, Ornella; Nastri, Flavia; Calhoun, Jennifer R.; Lahr, Stephen; Wade, Herschel; Pavone, Vincenzo; DeGrado, William F.; Lombardi, Angela. JBIC, Journal of Biological Inorganic Chemistry (2005), 10(5), 539-549. Abstract: Peptide-based models have an enormous impact for the development of metalloprotein models, as they seem appropriate candidates to mimic both the structural characteristics and reactivity of the natural systems. Through the de novo design of four-helix bundles, we developed the DF (Due Ferri) family of artificial proteins, as models of di-iron and dimanganese metalloproteins. The goal of our research is to elucidate how the electrostatic environment, polarity and solvent accessibility of the metalbinding site, influence the functional properties of di-iron proteins. The first two subsets of the DF protein family, DF1 and DF2, consist of two non-covalently assocd. helix-loop-helix motifs, which bind the di-metal cofactor near the center of the structure. The DF2 subset was designed to improve the properties of DF1: DF2 and DF2t have several changes in their sequences to improve soly. and metal ion access, as well as a change in the loop connecting the two helixes. In order to evaluate how these changes affect the overall structure of the model proteins, we solved the NMR structures of the di-Zn(II) complexes of DF2 and DF2t, and compared these structures with those recently obtained from X-ray crystallog. Further, we examd. the thermodn. consequences assocd. with the mutations, by measuring the stability of DF2t in the presence of different metal ions, and comparing the results with the data already obtained for DF2. Taken together, anal. of all the data showed the importance of the turn conformation in the design and stability of four-helix bundle. 4. Miniaturized heme proteins: crystal structure of Co(III)-mimochrome IV. Costanzo, Luigi; Geremia, Silvano; Randaccio, Lucio; Nastri, Flavia; Maglio, Ornella; Lombardi, Angela; Pavone, Vincenzo. JBIC, Journal of Biological Inorganic Chemistry (2004), 9(8), 1017-1027. Abstract: Protein design provides an attractive approach to test the essential features required for folding and function. Previously, we described the design and structural characterization in soln. of mimochromes, a series of miniaturized metalloproteins, patterned after the F-helix of the Hb β-chain. Mimochromes consist of two medium-sized helical peptides, covalently linked to the deuteroporphyrin. CD and NMR characterization of the prototype, mimochrome I, revealed that the overall structure conforms well to the design. However, formation of ∆ and Λ diastereomers was obsd. To overcome the problem of diastereomer formation, we re-designed mimochrome I, by engineering intramol., interchain interactions. The 47 Prof. Vincenzo Pavone resulting model was mimochrome IV: the soln. structural characterization showed the presence of the Λ isomer as a unique form. To examine the extent to which the stereochem. stability and uniqueness of mimochrome IV was retained in the solid state, the crystal structure of Co(III)mimochrome IV was solved by x-ray diffraction, and compared to the soln. structure of the same deriv. Co(III)-mimochrome IV structures, both in soln. and in the solid state, are characterized by the following common features: a bis-His axial coordination, a Λ configuration around the metal ion, and a predominant helical conformation of the peptide chains. However, in the crystal structure, intrachain Glu1-Arg9 ion pairs are preferred over the designed, and exptl. found in soln., interchain interactions. This ion pairing switch may be related to strong packing interactions. 5. Design of a new mimochrome with unique topology. Lombardi, Angela; Nastri, Flavia; Marasco, Daniela; Maglio, Ornella; De Sanctis, Giampiero; Sinibaldi, Federica; Santucci, Roberto; Coletta, Massimo; Pavone, Vincenzo. Chemistry -A European Journal (2003), 9(22), 5643-5654. Abstract: Peptide-based metalloprotein models represent useful systems to help understand how metalloproteins can support different functions, using similar metal ion cofactors. To shed light on the role of the protein matrix in modulating the heme properties, the authors developed new models: mimochromes. They are pseudo-C2 sym. systems, composed of two helical peptides covalently linked to the deuteroporphyrin. The use of C2 symmetry is particularly advantageous, because it simplifies the design, synthesis and characterization. However, it leaves the problem of possible diastereomeric forms. In the cobalt complex of the first deriv., mimochrome I, Λ and ∆ isomers were indeed exptl. obsd. All the insights derived from the CoIII-mimochrome I structure were used to obtain a re-designed mol., mimochrome IV. The spectroscopic characterization of the iron and cobalt derivs. suggested the Λ isomer as unique species. The NMR soln. structure of the diamagnetic CoIII-mimochrome IV confirmed the ability of the mol. to adopt a unique topol., and revealed the peptide chains to be in helical conformation, as designed. The insertion of intramol., inter-chain interactions was successful in favoring the formation of one of the two possible diastereomers. The stereochem. stable structure of mimochrome IV provides an attractive model for modulating the redox potential of the heme, by simple changing the peptide chain compn. around the heme. 6. Preorganization of molecular binding sites in designed diiron proteins. Maglio, Ornella; Nastri, Flavia; Pavone, Vincenzo; Lombardi, Angela; DeGrado, William F. Proceedings of the National Academy of Sciences of the 48 Prof. Vincenzo Pavone United States of America (2003), 100(7), 3772-3777. Abstract: De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallog. characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences assocd. with the introduction of these binding sites. To det. the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in soln. was solved by NMR and compared with the crystal structure of the di-Zn(II) deriv. The overall fold of the apo protein was highly similar to that of the di-Zn(II) deriv., although there was a rotation of one of the helixes. We also examd. the thermodn. consequences assocd. with building a small mol.-binding site within the protein. The protein exists in an equil. between folded dimers and unfolded monomers. DF1 is a highly stable protein (Kdiss = 0.001 fM), but the dissocn. const. increases to 0.6 nM (∆∆G = 5.4 kcal/mol monomer) as the active-site cavity is increased to accommodate small mols. 7. Conformational and coordination properties of a peptide containing the novel α,α-bis(2-pyridyl)glycine amino acid. Di Costanzo, Luigi; Geremia, Silvano; Randaccio, Lucio; Ichino, Tomoyuki; Yanagihara, Ryoji; Yamada, Takashi; Marasco, Daniela; Lombardi, Angela; Pavone, Vincenzo. Dalton Transactions (2003), (5), 787-792. Abstract: A fully protected tripeptide contg. a novel Cα,α-disubstituted glycine-α,α-di(2-pyridyl)glycine (2Dpy)-in the central position and Cα,αdimethylglycine (Aib) in positions 1 and 3 was synthesized and structurally characterized by single crystal x-ray anal. The 2Dpy residue bears two 2pyridyl moieties, which may coordinate metal ions, and promote selfassembly of peptide units. The tripeptide Z-Aib1-2Dpy2-Aib3-OCH3 (Z: benzyloxycarbonyl) adopts in the solid state a folded type III (III') β-turn conformation (with Aib1-2Dpy2 as corner residues), stabilized by an i ← i + 3 H bond between the Aib3 NH and the urethane CO group. The peptide was able to self-assemble in the presence of Cu(II) ions, giving rise to an octahedral complex with a 2 : 1 peptide/Cu(II) stoichiometry, which was structurally characterized. The metal ion, arranged on a crystallog. symmetry center, is coordinated by two symmetry related 2Dpy residues, 49 Prof. Vincenzo Pavone which act as tridentate ligands: each 2Dpy residue donates to the Cu(II) the N atoms of both pyridyl moieties in the equatorial positions, and the carbonyl O atom in the axial positions. A very similar folded conformation characterizes the peptide in both the free and metal-bound states. The overall peptide structure is ready to coordinate the metal ion without substantial conformational changes. Metal binding of the tri-peptide toward Cu ions was analyzed by UV-visible spectroscopy performed in MeOH soln. The exptl. data agree with an equil. between two species in soln., one in the ratio Cu/peptide 1:1, the other 1:2. The formation consts. are 9.7 × 103 M-1 and 3.5 × 102 M-1, resp. The molar absorptivities are 66 ± 3 M-1 cm-1 (590 nm) for the 2:1 complex and 17 ± 2 M-1 cm-1 (690 nm) for the other. All the data obtained in the present work suggest that the 2Dpy residue is well suited to be accommodated into folded conformations. The spontaneous formation of an ordered structure by the 2Dpy contg. tri-peptide and Cu ions may guide toward the design of more elaborate self-assembling systems. 8. Sliding helix and change of coordination geometry in a model Di-MnII protein. DeGrado, William F.; Di Costanzo, Luigi; Geremia, Silvano; Lombardi, Angela; Pavone, Vincenzo; Randaccio, Lucio. Angewandte Chemie, International Edition (2003), 42(4), 417-420. Abstract: A sliding helix mechanism for stabilizing a change in the ligand environment of DF1, a model di-MnII protein is described. DF1 is a dimer of helix loop motifs with a dimetal site near the center of the four-helixbundle structure. The DF1-L13G-DF1 has an active site cavity that has been expanded by further reducing the bulk of residue 13 to Gly. The structure of di-MnII-L13G-DF1 displays four crystallog. independent dimers in the asym. unit. Anal. of the differences in crystal-lattice packing among the four helix bundles revealed no significant relationship between the packing environment and the different structural behavior of these mols. 9. Toward the de novo design of a catalytically active helix bundle: A substrate-accessible carboxylate-bridged dinuclear metal center. Di Costanzo, Luigi; Wade, Herschel; Geremia, Silvano; Randaccio, Lucio; Pavone, Vincenzo; DeGrado, William F.; Lombardi, Angela. Journal of the American Chemical Society (2001), 123(51), 12749-12757. Abstract: De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, the authors described the design and crystal structure detn. of a di-Zn(II) complex of "due-ferri-1" (DF1), a protein patterned after the diirondimanganese class of redox-active proteins. The overall structure of DF1, 50 Prof. Vincenzo Pavone which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13'), which prevent facile entry of metal ions and small mols. The authors have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water mols. and a ligating DMSO mol., which forms a monat. bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a deriv. of L13A-DF1, DF2, is shown to bind azide, acetate, and small arom. mols. 10. Calixarene-porphyrin supramolecular complexes: pH-tuning of the complex stoichiometry. Di Costanzo, Luigi; Geremia, Silvano; Randaccio, Lucio; Purrello, Roberto; Lauceri, Rosaria; Sciotto, Domenico; Gulino, Fabio Giuseppe; Pavone, Vincenzo. Angewandte Chemie, International Edition (2001), 40(22), 4245-4247. Abstract: The formation of stable supramol. complexes, both in solid state and in aq. soln., from the cationic porphyrin and the anionic sulfonated calixarene (C4TsTc) blocked in a cone conformation and bearing carboxylic functions is described. The supramol. complexes were prepd. and crystd. by vapor-diffusion with hanging drops at 18 °C by using Linbro multiwell tissue plates as containers for the reservoir soln. Single crystals of porphyrin-calixarene complexes were obtained at pH 2 and pH 6. The central H2T4 unit of a trimeric H2T4 ensemble was arranged on a crystallog. center of symmetry and each of its methylpyridinium moieties was inserted into a tetra-anionic C4TsTc mol. from the sulfonated side of the calixarene. The crystals' asym. unit at pH 6 contained two heptaanionic calixarene mols., three tetracationic porphyrin rings, and two sodium ions, with a H2T4:(C4TsTc+H2T4) molar ratio of 0.6. The organization of cationic porphyrin was obtained due to the presence of sulfonated and carboxylate groups on the upper and lower rim, resp. 11. Structural determinants of CCR5 recognition and HIV-1 blockade in RANTES. Nardese, Vanessa; Longhi, Renato; Polo, Simona; Sironi, Francesca; Arcelloni, Cinzia; Paroni, Rita; DeSantis, Claudio; Sarmientos, Paolo; Rizzi, Menico; Bolognesi, Martino; Pavone, Vincenzo; Lusso, Paolo. Nature Structural Biology (2001), 8(7), 611-615. Abstract: Certain chemokines act as natural antagonists of human immunodeficiency virus (HIV) by blocking key viral coreceptors, such as 51 Prof. Vincenzo Pavone CCR5 and CXCR4, on the surface of susceptible cells. Elucidating the structural determinants of the receptor-binding and HIV-inhibitory functions of these chemokines is essential for the rational design of deriv. mols. of therapeutic value. Here the authors identify the structural determinants of CCR5 recognition and antiviral activity of the CC chemokine RANTES, showing that crit. residues form a solvent-exposed hydrophobic patch on the surface of the mol. Moreover, the authors demonstrate that the biol. function is critically dependent on dimerization, resulting in the exposure of a large (.apprx.180 .ANG.2), continuous hydrophobic surface. Relevant to the development of novel therapeutic approaches, the authors designed a retro inverted RANTES peptide mimetic that maintained both HIV- and chemotaxis-antagonistic functions. 12. The crystal structure of Afc-containing peptides. Lombardi, Angela; De Simone, Giuseppina; Galdiero, Stefania; Nastri, Flavia; Di Costanzo, Luigi; Makihira, Kazunari; Yamada, Takashi; Pavone, Vincenzo. Biopolymers (2000), 53(2), 150-160. Abstract: A systematic structural anal. of Afc (9-amino-fluorene-9carboxylic acid) contg. peptides is reported. The crystal structures of four fully protected tripeptides Cbz-X-Afc-Y-OMe, (peptide a: X = Y = Gly; peptide b: X = Aib, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib; Aib = Cα,α-dimethylglycine; Cbz = benzyloxycarbonyl) have been solved by x-ray crystallog. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in peptides a-c. In contrast, Afc is found in a folded conformation in the 310-helical region only for peptide d, in which it is both preceded and followed by the strong helix promoting Aib. 13. Conformational behavior of Cα,α-diphenyl glycine: extended conformation in tripeptides containing consecutive Dϕg residues. Pavone, Vincenzo; Lombardi, Angela; Saviano, Michele; De Simone, Giuseppina; Nastri, Flavia; Maglio, Ornella; Omote, Yuichiro; Yamanaka, Yoshinori; Yamada, Takashi. Biopolymers (2000), 53(2), 161-168. Abstract: Recent studies on the conformational preferences of the Dϕg (Cα,α-diphenylglycine) residue showed that this Cα,α-disubstituted glycine has a structural versatility. Dϕg can assume either folded or extended conformations depending on the nature of the following or preceding residue. The conformational preferences of the Dϕg residue in tripeptides contg. consecutive Dϕg residues are analyzed. The crystal structures of Z-Dϕg-Dϕg-Dϕg-OMe (Z = benzyloxycarbonyl), Z-Aib- 52 Prof. Vincenzo Pavone Dϕg-Dϕg-OMe (Aib = α-aminoisobutyric acid), and Z-Ac3c-Dϕg-DϕgOMe (Ac3c = α-aminocyclopropanecarboxylic acid), are reported. The Dϕg residues adopt the fully extended conformation in the three tripeptides examd. Both previous and current data indicate that the presence of adjacent Dϕg residue in the peptide further stabilizes the extended conformation. 14. The crystal structure of a Dcp-containing peptide. De Simone, Giuseppina; Lombardi, Angela; Galdiero, Stefania; Nastri, Flavia; Di Costanzo, Luigi; Gohda, Shin; Sano, Atsuiro; Yamada, Takashi; Pavone, Vincenzo. Biopolymers (2000), 53(2), 182-188. Abstract: The conformational preferences of a newly synthesized Cα,α sym. disubstituted glycine, α,α-dicyclopropylglycine (Dcp), have been investigated. The crystal structure of a fully protected dipeptide contg. Dcp, namely Z-Dcp1-Dcp2-OCH3 (Z = benzyloxycarbonyl) is reported. Both Dcp residues are in a folded conformation. The overall peptide structural organization corresponds to an α-pleated sheet conformation, similar to that obsd. in linear peptides made up of alternating D- and L-residues and in ZAib-Aib-OCH3 (Aib = α,α-dimethylglycine). These preliminary data suggest that the Dcp could represent an alternative as mol. tool to stabilize folded conformations. 15. Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins. Lombardi, Angela; Summa, Christopher M.; Geremia, Silvano; Randaccio, Lucio; Pavone, Vincenzo; DeGrado, William F. Proceedings of the National Academy of Sciences of the United States of America (2000), 97(12), 6298-6305. Abstract: De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approx. 1 .ANG. rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallog., by using data that extend to 2.5 .ANG.. This four-helix bundle protein is comprised of two noncovalently assocd. helix-loop-helix motifs. The dinuclear center 53 Prof. Vincenzo Pavone is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-contg. R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidn. 16. Miniaturized metalloproteins: application to iron-sulfur proteins. Lombardi, Angela; Marasco, Daniela; Maglio, Ornella; Di Costanzo, Luigi; Nastri, Flavia; Pavone, Vincenzo. Proceedings of the National Academy of Sciences of the United States of America (2000), 97(22), 11922-11927. Abstract: The miniaturization process applied to rubredoxins generated a class of peptide-based metalloprotein models, named METP (miniaturized electron transfer protein). The crystal structure of Desulfovibrio vulgaris rubredoxin was selected as a template for the construction of a tetrahedral (Sγ-Cys)4 iron-binding site. Anal. of the structure showed that a sphere of 17 .ANG. in diam., centered on the metal, circumscribes two unconnected approx. C2 symmetry related β-hairpins, each contg. the -Cys-(Aaa)2-Cyssequence. These observations provided a starting point for the design of an undecapeptide, which self assembles in the presence of tetrahedrally coordinating metal ions. The METP peptide was synthesized in good yield by std. methodologies. Successful assembly of the METP peptide with Co(II), Zn(II), Fe(II/III), in the expected 2:1 stoichiometry, was proven by UV-visible and CD spectroscopies. UV-visible anal. of the metal complexes indicated the four Cys ligands tetrahedrally arrange around the metal ion, as designed. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. NMR characterization of the Zn(II)-METP complex fully supported the structure of the designed model. These results prove that METP reproduces the main features of rubredoxin. 17. Crystallization and preliminary x-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. De Simone, Giuseppina; Manco, Giuseppe; Galdiero, Stefania; Lombardi, Angela; Rossi, Mose; Pavone, Vincenzo. Acta Crystallographica, Section D: Biological Crystallography (1999), D55(7), 1348-1349. Abstract: EST2, a thermophilic carboxylesterase from A. acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, was crystd. 54 Prof. Vincenzo Pavone for the 1st time. (NH4)2SO4 was used as a precipitant and the crystn. proceeded at pH 7.8. The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106.4 .ANG.. A complete data set was collected at the synchrotron source Elettra in Trieste to 2.4 .ANG. resoln., using a single frozen crystal. 18. Maroteaux-Lamy syndrome: five novel mutations and their structural localization. Villani, G. R. D.; Balzano, N.; Vitale, D.; Saviano, M.; Pavone, V.; Di Natale, P. Biochimica et Biophysica Acta, Molecular Basis of Disease (1999), 1453(2), 185-192. Abstract: Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation anal. in Maroteaux-Lamy syndrome resulted in the identification of approx. 40 mol. defects underlying a great genetic heterogeneity. Here the authors report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) anal. The authors also performed a three-dimensional (3-D) structure anal. of the alterations identified by the authors', and of an addnl. 22 point mutations reported by other groups, to draw helpful information about their possible effects on protein conformation. 19. The crystal structure of α-thrombin-hirunorm IV complex reveals a novel specificity site recognition mode. Lombardi, Angela; De Simone, Giuseppina; Nastri, Flavia; Galdiero, Stefania; Della Morte, Rossella; Staiano, Norma; Pedone, Carlo; Bolognesi, Martino; Pavone, Vincenzo. Protein Science (1999), 8(1), 91-95. Abstract: The x-ray crystal structure of the human α-thrombin (I)hirunorm IV (II) complex was detd. at 2.5 .ANG. resoln., and refined to an R-factor of 0.173. The structure revealed an inhibitor binding mode distinctive of a true hirudin mimetic, which justified the high inhibitory potency and the selectivity of II. This novel inhibitor, II, composed of 26 amino acids, interacted through the N-terminal end with the I active site in a nonsubstrate mode, and bound specifically to the fibrinogen recognition exosite through the C-terminal end. The backbone of the N-terminal tripeptide, Chg-1"-Arg-2"-2Nal-3" [Chg = cyclohexylglycine; 2Nal = β-(2naphthyl)alanine] formed a parallel β-strand to the thrombin main-chain segment, Ser-214-Gly-216. The Chg-1" side-chain occupied the S2 site, Arg-2" penetrated into the S1 specificity site, whereas the 2Nal-3" side-chain occupied the aryl binding site. The Arg-2" side-chain entered the S1 55 Prof. Vincenzo Pavone specificity pocket from a position quite apart from the canonical P1 site. This notwithstanding, the Arg2" side-chain established the typical ion pair with the carboxylate group of Asp-189. 20. Hemoprotein models based on a covalent helix-heme-helix sandwich. 3. Coordination properties, reactivity and catalytic application of Fe(III)- and Fe(II)-mimochrome I. Nastri, Flavia; Lombardi, Angela; Morelli, Giancarlo; Pedone, Carlo; Pavone, Vincenzo; Chottard, Genevieve; Battioni, Pierrette; Mansuy, Daniel. JBIC, Journal of Biological Inorganic Chemistry (1998), 3(6), 671-681. Abstract: The coordination state of Fe(III)- and Fe(II)-mimochrome I, a covalent peptide-deuteroheme sandwich involving two nonapeptides bearing a histidine residue in a central position, was studied by UV-visible, EPR, and resonance Raman spectroscopy. The ferric and ferrous states of this new iron species mainly exist, at pH7, in a low-spin hexacoordinate form with two axial histidine ligands coming from the peptide chains. A minor amt. of high-spin form for the ferric state is also present at pH 7. However, it is mainly high-spin at pH 2 or in DMSO. Fe(II)-mimochrome I binds CO with an affinity comparable to that of myoglobin and Hb. Fe(III)-mimochrome I reacts with alkylhydroxylamine and arylhydrazines, leading to the corresponding Fe(II)-nitrosoalkyl and Fe(III)-σ-aryl complexes, resp. These reactions were greatly dependent on the solvent used and on the pH, and were much slower than the corresponding reactions performed by deuterohemin in the presence of excess imidazole. All these results indicate that the reactivity of iron-mimochrome I is controlled by the binding of the peptide chains to the iron. The reactivity shown by this complex at neutral pH is intermediate between that obsd. for iron porphyrins in the presence of excess imidazole and that of hemoproteins characterized by a strong bis-histidine axial coordination, such as cytochrome b5. Fe(III)-mimochrome I is able to catalyze styrene epoxidn. by using a [Fe(III)-mimochrome I]/[H2O2]/[styrene] ratio of 1:10:2000 in phosphate buffer soln. (pH 7.2) contg. 2% CTAB both under strictly anaerobic conditions and in the presence of oxygen, at 0°C. 21. Neuronorm is a potent and water soluble neurokinin A receptor antagonist. Lombardi, Angela; D'agostino, Bruno; Filippelli, Amelia; Pedone, Carlo; Matera, Maria Gabriella; Falciani, Maddalena; De Rosa, Mario; Rossi, Francesco; Pavone, Vincenzo. Bioorganic & Medicinal Chemistry Letters (1998), 8(13), 1735-1740. Abstract: The authors report the synthesis and preliminary pharmacol. characterization of a novel water sol. Neurokinin A (NKA) receptor 56 Prof. Vincenzo Pavone antagonist named Neuronorm, cyclo[Cys1(β-D-Gal)-Asp2-Trp3-Phe4Dap5-Leu6]cyclo(2β-5β). The synthesis was achieved in high yield by a combination of classical peptide synthesis methodologies, in both soln. and solid phase. The pharmacol. properties as neurokinin A receptor antagonist were assessed in in vitro expts. on rat vas deferens and guinea pig trachea, and were compared to those of MEN10627. 22. A Novel Rigid β -Turn Molecular Scaffold. Lombardi, Angela; D'Auria, Gabriella; Maglio, Ornella; Nastri, Flavia; Quartara, Laura; Pedone, Carlo; Pavone, Vincenzo. Journal of the American Chemical Society (1998), 120(24), 5879-5886. Abstract: We describe here the soln. 1H NMR anal., restrained and unrestrained mol. dynamic simulations of the bicyclic peptide cyclo(Met1asp2-Trp3-Phe4-dap5-Leu6)cyclo(2β-5β) (MEN10701) [dap = (2R)-2,3diaminopropionic acid]. This compd. is an analog of cyclo(Met1-Asp2-Trp3Phe4-Dap5-Leu6)cyclo(2β-5β) (MEN10627) [Dap = (2S)-2,3diaminopropionic acid], which is the most potent and selective, peptidebased NK2 receptor antagonist known to date. MEN10701 differs from MEN10627 in the D chirality of the Asp2 and Dap5 residues; it was designed to better understand the role of the lactam bridge in detg. the shape of the mol. and to elucidate whether its position, above or below the plane contg. the pharmacophores (Met1, Trp3, Phe4, and Leu6 side chains), could modulate the biol. response. Despite our expectations, the uncoercible bicyclic structure of MEN10627 is dramatically coerced into a novel conformation, by the replacement of the lactam bridge forming units (Asp2 and Dap5) with residues of opposite chirality. The overall shape of MEN10701 is also quite unique because of its compactness. It is ellipsoidal instead of being rectangle-like, and the structure is stabilized by two intramol. hydrogen bonds encompassing two type I' β-turns. This structure can be added to the repertoire of rigid β-turn scaffolds for the design of bioactive mols., which require turned motifs to elicit potency and specificity. 23. Miniaturized hemoproteins: design, synthesis and characterization of mimochrome II. Lombardi, Angela; Nastri, Flavia; Sanseverino, Marina; Maglio, Ornella; Pedone, Carlo; Pavone, Vincenzo. Inorganica Chimica Acta (1998), 275-276(1,2), 301-313. Abstract: The present paper describes the design, synthesis and spectroscopic characterization of mimochrome II, namely 3,7,12,17tetramethylporphyrin-2,18-di-N9.vepsiln.-(Ac-Asp1-Leu2-Ser3-Asp4-Leu5His6-Ser7-Lys8-Lys9-Leu10-Lys11-Ile12-Thr13-Leu14-NH2) 57 Prof. Vincenzo Pavone propionamide, and its cobalt deriv. Mimochrome II belongs to a new class of hemoprotein models, called mimochromes. We have developed these mols. in order to investigate the effect of protein structure and compn. on modulation of the activity of the heme center. These mols. are composed of two helical peptide chains linked covalently to the deuteroporphyrin IX. The synthetic procedure we developed for synthesizing the prototype mol. mimochrome I, together with the information derived from the structural characterization of the Fe(III)- and Co(III)-mimochrome I complexes, enabled us to design mols. having more precise properties. By comparing the behavior of Co(III)-mimochrome II with that of the prototype mol. mimochrome I, we show here that the compn. and folding of the peptide chains affect critically the overall structural organization of the models. 24. A novel super-potent neurokinin A receptor antagonist containing dehydroalanine. Lombardi, Angela; D'agostino, Bruno; Nastri, Flavia; D'andrea, Luca D.; Filippelli, Amelia; Falciani, Maddalena; Rossi, Francesco; Pavone, Vincenzo. Bioorganic & Medicinal Chemistry Letters (1998), 8(10), 1153-1156. Abstract: The authors report the synthesis and preliminary pharmacol. characterization of a novel Neurokinin A receptor antagonist. This peptide, ∆-Ala1-Neuronorm, contains a dehydroalanine residue; it displays a high conformational rigidity and possesses very high activity. Its pharmacol. properties as a neurokinin A receptor antagonist were assessed in in-vitro expts. on rat vas deferens and were compared to those of Neuronorm and MEN10627. 25. Conformational behavior of Cα,α-diphenylglycine: folded vs. extended structures in DΦg-containing tripeptides. Pavone, Vincenzo; Lombardi, Angela; Saviano, Michele; Nastri, Flavia; Zaccaro, Laura; Maglio, Ornella; Pedone, Carlo; Omote, Yuichiro; Yamanaka, Yoshinori; Yamada, Takashi. Journal of Peptide Science (1998), 4(1), 21-32. Abstract: The crystal structures of three fully protected tripeptides contg. the DΦg residue (Cα,α-diphenylglycine) in the central position are reported, namely Z-Gly-DΦg-Gly-OMe (a), Z-Gly-DΦg-Aib-OMe (b) and Z-Aib-DΦg-Aib-OMe (c). The mol. conformations are quite unusual because the DΦg residue adopts a folded conformation in the 310-helical region when the following residue adopts a folded conformation of opposite handedness (peptides b and c). In contrast, the DΦg residue adopts the more frequently obsd. fully extended conformation when the following residue adopts a semi-extended conformation (peptide a). These findings are in agreement with the theor. calcns. on Ac-DΦg-Aib-NHCH3 and Ac- 58 Prof. Vincenzo Pavone Aib-DΦg-NHCH3 also reported in this work. 26. Hirunorms are true hirudin mimetics. The crystal structure of human αthrombin-hirunorm V complex. De Simone, Giuseppina; Lombardi, Angela; Galdiero, Stefania; Nastri, Flavia; Morte, Rossella Della; Staiano, Norma; Pedone, Carlo; Bolognesi, Martino; Pavone, Vincenzo. Protein Science (1998), 7(2), 243-253. Abstract: A novel class of synthetic, multisite-directed thrombin inhibitors, known as hirunorms, has been described recently. These compds. were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here the authors report the crystal structure of the complex formed by human α-thrombin and hirunorm V, a 26-residue polypeptide contg. non-natural amino acids, detd. at 2.1 .ANG. resoln. and refined to an R-factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the mol. basis of the high inhibitory potency (Ki is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C-terminal segment to the fibrinogen recognition exosite. The backbone of the N-terminal tetrapeptide Chg1"Val2"-2-Nal3"-Thr4" (Chg, cyclohexyl-glycine; 2-Nal, β-(2-naphthyl)alanine) forms a short β-strand parallel to thrombin main-chain residues Ser214-Gly219. The Chg1" side chain fills the S2 subsite, Val2" is located at the entrance of S1, whereas 2-Nal3" side chain occupies the aryl-binding site. Such backbone orientation is very close to that obsd. for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C-terminal segment binds to the fibrinogen recognition exosite, similarly to what is obsd. for hirudin C-terminal tail and related compds. The linker polypeptide segment connecting hirunorm V N-and C-terminal regions is not observable in the electron d. maps. The crystallog. anal. proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisitedirected synthetic thrombin inhibitors. 27. Hemoprotein models based on a covalent helix-heme-helix sandwich: 2. Structural characterization of CoIIImimochrome I ∆ and Λ isomers. D'Auria, Gabriella; Maglio, Ornella; Nastri, Flavia; Lombardi, Angela; Mazzeo, Marco; Morelli, Giancarlo; Paolillo, Livio; Pedone, Carlo; Pavone, Vincenzo. Chemistry--A European Journal (1997), 3(3), 350-362. 59 Prof. Vincenzo Pavone Abstract: FeIII mimochrome I is the prototype of a new class of hemoprotein models characterized by a covalent helix-heme-helix sandwich. It contains deuterohemin bound through two propionyl groups to two identical N- and C-terminal protected α-helical nonapeptides, each of which bears a His residue (a potential axial ligand of the iron ion) in the central position. To understand better the three-dimensional structure of FeIII mimochrome I and its correlation with spectral properties, we have characterized the fully diamagnetic parent compd. CoIII mimochrome I by UV/visible, CD, and NMR spectroscopy, coupled with conformational energy calcns. CoIII mimochrome I is a highly water-sol. compd. present in soln. as two isomers, which slowly interconvert only at very low pH values. These isomers were isolated and sep. characterized. Their UV/visible spectral properties are very similar, while their CD spectral properties differ markedly in both the far UV and Soret regions. The isomers were identified by 1H NMR spectroscopy as diastereomers of the ∆ and Λ type. This is the first example of an accurate three-dimensional structure detn. in soln. of a hemoprotein mimetic that allows a straightforward correlation between structure and spectral properties. 28. Hemoprotein models based on a covalent helix-heme-helix sandwich: 1. Design, synthesis, and characterization. Nastri, Flavia; Lombardi, Angela; Morelli, Giancarlo; Maglio, Ornella; D'Auria, Gabriella; Pedone, Carlo; Pavone, Vincenzo. Chemistry--A European Journal (1997), 3(3), 340-349. Abstract: In this paper we describe design, synthesis, and spectroscopic characterization of a covalent helix-heme-helix sandwich named FeIII mimochrome I. It contains deuterohemin bound through both propionyl groups to two identical N- and C-terminal protected nonapeptides as αhelical scaffolds. Each peptide moiety bears a His residue in the central position, which acts as axial ligand to the metal ion. The newly developed synthetic strategy is based on a combination of soln. and solid-phase methodologies. It represents a powerful method for obtaining a large variety of analogs contg. two sym. or unsym. peptide chains covalently bound to the deuteroporphyrin ring. UV/Visible spectroscopic characterization in buffered 2,2,2-trifluoroethanol/water soln. proves low-spin bis(histidine) iron(III) coordination; CD (CD) measurements show an α-helical conformation for the peptide moieties. Thus, all the data are in agreement with the designed hypothetical model regarding both the iron(III) coordination and the peptide chain structural organization. 29. A racemic bicyclic acylamidine from a tripeptide derivative. Saviano, Michele; Lombardi, Angelina; Pavone, Vincenzo; Yamada, Takashi; 60 Prof. Vincenzo Pavone Yanagi, Takashi; Iwamoto, Akira; Kuwata, Shigeru. Acta Crystallographica, Section C: Crystal Structure Communications (1996), C52(7), 1705-1708. Abstract: 2,2,8-Triisopropyl-4,5,7,8-tetrahydroimidazo[1,2-a]pyrazine-3,6dione is monoclinic, space group P2/n, with a 12.533(2), b 7.6536(9), c 16.804(2) .ANG., and β 101.03(1)°; Z = 4, dc = 1.173; R = 0.045, Rw = 0.042 for 2700 reflections. At. coordinates are given. The mol. has a double bond and two partial double bonds in the bicyclic skeleton, with some π-electron delocalization along C'1-N3-C'2. The conformation parameters of the diisopropyl (Dip) residue reveal that it is in an unusually high-energy conformation. The peptide bond between the glycine and valine residues is cis [Cα3-C'3-N1-Cα1 = -7.0(3)°]. In the crystal, the mols. are held together in the ac plane by intermol. H bonds formed around a 2-fold axis by mols. related by symmetry centers. 30. Crystal and Molecular Structure of the [6-Deoxy-6-[(2-(4imidazolyl)ethyl)amino]cyclomaltoheptaose]copper(II) Ternary Complex with L-Tryptophanate. Role of Weak Forces in the Chiral Recognition Process Assisted by a Metallocyclodextrin. Bonomo, Raffaele P.; Di Blasio, Benedetto; Maccarrone, Giuseppe; Pavone, Vincenzo; Pedone, Carlo; Rizzarelli, Enrico; Saviano, Michele; Vecchio, Graziella. Inorganic Chemistry (1996), 35(15), 4497-4504. Abstract: The ternary Cu(II) complex of 6-deoxy-6-[(2-(4imidazolyl)ethyl)amino]cyclomaltoheptaose (CDhm) and L-tryptophanate (L-TrpO-) was prepd. characterized by ESR and x-ray diffraction. The solid state structure of [Cu(CDhm)(L-TrpO)]+ shows that the arom. side chain of TrpO- is outside the cavity and that the two amino N atoms, one from the histamine mol. and one from the amino acidate, are in a cis disposition. The two amino nitrogens, the imidazole N, and the carboxylate O atoms form the base of a square pyramid, which surrounds the Cu(II) ion, a H2O mol. occupying an apical position. At. distances suggest for this complex that π-π and d-π interactions could occur in the solid state. The [Cu(CDhm)(L-TrpO)]+ has a self-assembled structure in which a CDhm mol. behaves as host and as guest. The imidazole and the indole ring are directed into the cavity of an adjacent CDhm mol. from the wider cyclodextrin rim, thus forming a polymeric column structure. ESR spectra were run on the Cu(II) ternary complexes with L- or D-tryptophanate and L- or D-alaninate in frozen aq. soln. and on the former pair of enantiomers in the solid state, as well. While in the case of the ternary complex with Lor D-alaninate no differences are obsd. in their frozen soln. spectra, in the case of complexes with TrpO- subtle differences are found. These differences, which disappear when excess MeOH was used, are ascribed to 61 Prof. Vincenzo Pavone the presence of weak forces, such as hydrophobic or d-π interactions. 31. Discovering protein secondary structures: classification and description of isolated α-turns. Pavone, Vincenzo; Gaeta, Girolamo; Lombardi, Angela; Nastri, Flavia; Maglio, Ornella; Isernia, Carla; Saviano, Michele. Biopolymers (1996), 38(6), 705-721. Abstract: Irregular protein secondary structures are believed to be important structural domains involved in mol. recognition processes between proteins, in interactions between peptide substrates and receptors, and in protein folding. In these respects, tight turns are being studied in detail. They also represent template structures for the design of new mols. such as drugs, pesticides, or antigens. Isolated α-turns, not participating in α-helical structures, have received little attention due to the overwhelming presence of other types of tight turns in peptide and protein structures. The growing no. of protein x-ray structures allowed the authors to undertake a systematic search into the Protein Data Bank of this uncharacterized protein secondary structure. A classification of isolated α-turns into different types, based on conformational similarity, is reported here. A preliminary anal. on the occurrence of some particular amino acids in certain positions of the turned structure is also presented. 32. Solvent-mediated conformational transition in β -alanine-containing cyclic peptides. VIII. Lombardi, Angela; Saviano, Michele; Nastri, Flavia; Maglio, Ornella; Mazzeo, Marco; Isernia, Carla; Paolillo, Livio; Pavone, Vincenzo. Biopolymers (1996), 38(6), 693-703. Abstract: We describe the soln. NMR structural anal. and restrained mol. dynamics simulation of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-β-Alaβ-Ala). The conformational anal., carried out in CD3CN and DMSO solns. by NMR spectroscopy, was based on interproton distances derived from rotating frame nuclear Overhauser effect spectroscopy and homonuclear coupling consts. A restrained mol. dynamics simulation in vacuo was also performed to build refined mol. models. The mol. is present in both solvent systems as two slowly interconverting conformers, characterized by a cistrans isomerism around the β-Ala5-Pro1 peptide bond. In CD3CN soln., the conformer with a cis peptide bond is quite similar to that obsd. in the solid state, while the conformer contg. all trans peptide bonds is characterized by an intramol. hydrogen bond stabilizing a C10- and a C13-ring structure. In DMSO soln., the trans isomer is partly similar to that obsd. in CD3CN soln. while the cis isomer is different from that obsd. in the solid state. The effect of the solvent in stabilizing different conformations was also investigated in DMSO-CD3CN solvent mixts. 62 Prof. Vincenzo Pavone 33. Unusual conformational preferences of β-alanine containing cyclic peptides. VII. Lombardi, Angela; Saviano, Michele; Nastri, Flavia; Maglio, Ornella; Mazzeo, Marco; Pedone, Carlo; Isernia, Carla; Pavone, Vincenzo. Biopolymers (1996), 38(6), 683-691. Abstract: The synthesis, purifn., and single crystal X-ray anal. of cyclic pentapeptide cyclo(Pro-Phe-Phe-β-Ala-β-Ala) were carried out. In the solid state this compd. adopts an unusual conformation characterized by a cis βAla5-pro1 peptide bond and by an intramol. hydrogen bond stabilizing a C11and a C12-ring structure. The C11 structure contains the Phe3 and the β-Ala4 at the corner position of the turn; it is the first observation of a type II βturn enlargement due to the insertion of an extra methylene group of the βalanine residue. The rest of the mol. participates in a newly characterized C12-ring structure, which incorporates a β-Ala residue at position i of the turn. 34. A modified cyclodextrin with a fully encapsulated dansyl group: selfinclusion in the solid state and in solution. Corradini, Roberto; Dossena, Arnaldo; Marchelli, Rosangela; Panagia, Anna; Sartor, Giorgio; Saviano, Michele; Lombardi, Angela; Pavone, Vincenzo. Chemistry--A European Journal (1996), 2(4), 373-81. Abstract: A monofunctionalized β-cyclodextrin contg. a dansyl moiety, 6deoxy-6-N-[N'-(5-dimethylamino-1-naphthalenesulfonyl)diaminoethane]β-cyclodextrin (I), was synthesized and its crystal structure was detd. It was shown that the dansyl group is fully encapsulated within the cyclodextrin cavity, with the dimethylamino and sulfonyl groups emerging from opposite sides. The conformation of the diaminoethane linker was found to be detd. by the inclusion of the dansyl group and by a hydrogen bond between the sulfonamide NH and one of the O(6)-H groups on the cyclodextrin rim. Fluorescence spectra showed that the inclusion of the dansyl group in the cyclodextrin cavity considerably increases the quantum yield; time-resolved fluorescence expts. showed the presence of a longlifetime component (16.1 ns), which was attributed to the included fluorophore. The ability of I to act as a fluorescence sensor was evaluated by the addn. of several guests of different shape: fluorescence intensity was lowered, esp. upon addn. of adamantane-carboxylic acid. Copper(II) was shown to enhance the difference in the fluorescence of I in the presence of guests by addnl. static quenching. 35. Rational Design of True Hirudin Mimetics: Synthesis and Characterization 63 Prof. Vincenzo Pavone of Multisite-Directed α -Thrombin Inhibitors. Lombardi, Angela; Nastri, Flavia; Della Morte, Rossella; Rossi, Armando; De Rosa, Alfredo; Staiano, Norma; Pedone, Carlo; Pavone, Vincenzo. Journal of Medicinal Chemistry (1996), 39(10), 2008-17. Abstract: We describe here the design, synthesis, and activity of a novel class of α-thrombin inhibitors named hirunorms. They were rationally designed to interact through their N-terminal end with the α-thrombin active site in a nonsubstrate mode and to specifically bind the fibrinogen recognition exosite. An appropriate spacer that is able to properly orient the N-terminal end in the active site was also selected. This spacer allowed the size of the inhibitors to be reduced to about one-third of the amino acid residues in the hirudin sequence. Hirunorms specifically inhibit the amidolytic action of human α-thrombin toward a small chromogenic substrate. The most active compds. of the series, hirunorms IV and V, inhibit α-thrombin catalyzed hydrolysis of Tos-Gly-Pro-Arg-p-nitroanilide with Ki = 0.09 and Ki = 0.21 nM, resp. Comparison of the anticoagulant properties of hirunorms, natural hirudin from the European leech Hirudo medicinalis, and the synthetic analog hirulog-1 revealed that hirunorm IV is about 10-fold and 3-fold more active, on a molar base, than hirudin and hirulog-1 in increasing the aPTT, PT, and TT of normal human plasma. The peculiar structure of hirunorms makes them stable to the amidolytic action of thrombin without the introduction of any peptide bond modification. These mols. display long-lasting activity in human plasma, due to the presence of several unnatural amino acids in susceptible positions. Hirunorms are potential candidates for injectable anticoagulants, due to their potency, specificity of action, long-lasting activity, and safety profiles. 36. Bicyclic peptides as type I/type II beta-turn scaffolds. Lombardi A; D'Auria G; Saviano M; Maglio O; Nastri F; Quartara L; Pedone C; Pavone V Biopolymers (1996), 40(5), 505-18. Abstract: We recently reported the rational design, synthetics, and structural characterization of the most potent and selective peptide-based neurokinin A antagonist thus far described: cyclo(Met1-Asp2-Trp3-Phe4-Dap5Leu6)cyclo(2 beta-5 beta). Its bicyclic structure is characterized by a type I and a type II two beta-turn around Trp3-Phe4 and Leu6-Met1, respectively. In order to understand whether the two different beta-turned structures are determined by the bicyclic structure or by the amino acid type at the corner positions, we have synthesized the pseudo-symmetrical analogue cyclo(Phe1-Asp2-Trp3-Phe4-Dap5-Trp6)cyclo(2β-5β). The structural characterization in the crystal state and in solution, here reported, gives an 64 Prof. Vincenzo Pavone experimental evidence that the backbone of the bicyclic structure is a rigid scaffold that can be used to build both a type I and type II beta-turn independently from the amino acid composition. 37. Conformational behavior of a cyclolinopeptide A analog: two-dimensional NMR study of cyclo(Pro1-Pro-Phe-Phe-Ac6c-Ile-ala-Val8). Mazzeo, Marco; Isernia, Carla; Fossi, Filomena; Saviano, Michele; Pedone, Carlo; Paolillo, Livo; Benedetti, Ettore; Pavone, Vincenzo. Journal of Peptide Science (1995), 1(5), 330-40. Abstract: The title cyclic octapeptide cyclo(Pro1-Pro-Phe-Phe-Ac6c-Ile-DAla-Val8) (I; Ac6c = 1-aminocyclohexane-1-carboxylic acid), contg. the Pro1-Pro-Phe-Phe sequence, followed by a bulky helicogenic Cα,αdialkylated glycine residue Ac6c and a D-Ala residue at position 7 has been synthesized. Peptide Iis a deletion analog of the naturally occurring cyclic nonapeptide cyclolinopeptide A(CLA). I has been designed with the aim of studying the role that the Ac6c and D-Ala residues play on the conformational behavior of the whole mol. and their influence on the conformation of the Pro1-Pro-Phe-Phe sequence when compared with cyclolinopeptide A. I has been investigated in chloroform and acetonitrile solns. by 2-dimensional NMR techniques. Only one set of sharp signals is obsd. in both solvents. The evidence strongly supports the hypothesis that only one conformational state exists in the chosen solvents. The interpretation of the exptl. data points to the existence of a very rigid structure stabilized by intramol. hydrogen bonds. The measured NOE effects allow the calcn. of internuclear distances, which have been used as restraints in mol. dynamic calcns. The proposed conformation of the mol. shows that the Pro-Pro-Phe segment retains the conformation obsd. in natural CLA both in soln. and in the solid state and that the Ac6c residue indeed reinforces the ring rigidity not permitting the formation of any appropriate cavity in which inorg. cations could be complexes. 38. Design of metal ion binding peptides. Fattorusso, R.; Morelli, G.; Lombardi, A.; Nastri, F.; Maglio, O.; D'Auria, G.; Pedone, C.; Pavone, V.. Biopolymers (1995), 37(6), 401-10. Abstract: Two cyclic and branched peptides (PLA and AZU) were synthesized with the aim of reproducing the active site of the blue copper proteins plastocyanin and azurin. Both peptides, designed on the basis of the X-ray structures of Poplar plastocyanin and Alcaligenes denitrificans azurin, contain the same coordinating residues of the parent native proteins. The visible spectra of PLA in the presence of equimolar amt. of Cu(II) strongly support the interaction between the peptide and copper(II) ion. 65 Prof. Vincenzo Pavone The CD titrn. of AZU with the Hg(II) ion indicates the formation of two species, [AZUHg]+ and [AZUHg2]3+ having binding consts. (Keq) of 3.106 and 2.104 M-1, resp. 39. Conformational rigidity versus flexibility in a novel peptidic neurokinin A receptor antagonist. Pavone, Vincenzo; Lombardi, Angelina; Maggi, Carlo Alberto; Quartara, Laura; Pedone, Carlo. Journal of Peptide Science (1995), 1(4), 236-40. Abstract: Neurokinin A receptor antagonists have been proposed as a new class of drugs for several applications in humans (asthma, intestinal motility, etc.). The rational design, synthesis, structural characterization and biol. activity evaluation of a new potent, highly selective, long-lasting, peptide based receptor antagonist are reported. The structure-activity relation indicates that the conformational rigidity dets. potency, specificity and esp. the long life of the mol. in the living body. MEN 10627 is the prototype of a new class of cyclic, peptide-based, neurokinin A receptor antagonists and it is a suitable candidate for clin. testing in humans. 40. Design and structure of a novel Neurokinin A receptor antagonist cyclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6-)cyclo(2β-5β). Pavone, Vincenzo; Lombardi, Angelina; Nastri, Flavia; Saviano, Michele; Maglio, Ornella; D'Auria, Gabriella; Quartara, Laura; Maggi, Carlo Alberto; Pedone, Carlo. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1995), (5), 987-93. Abstract: The rational design, synthesis and solid state and soln. structural characterization of title compd. I (Dap = L-2,3-diaminopropanoic acid) (MEN 10627), the most potent and selective peptide-based neurokinin A (NKA) antagonist thus far described is reported. The bioactive conformation of the known NKA antagonist cyclo(Met1-Gln2-Trp3-Phe4Gly5-Leu6) was predicted by comparison with the known structures of other cyclohexapeptides. On this basis the authors designed the highly constrained I. I was synthesized efficiently, using a combined soln. and solid phase strategy. I was fully characterized in the solid state by x-ray crystallog. and I is shown to adopt an almost identical conformation in acetonitrile soln. by NMR spectroscopy. This structure fully confirms the author's hypothetical model. The structure and conformational rigidity of I in soln. explain the high potency and selectivity and the resistance to proteolytic degrdn. Therefore the structural requirements for NKA antagonistic activity are clarified. 66 Prof. Vincenzo Pavone Me t-A sp-Trp-Phe-D a p-L eu I 41. MEN 10,627, a novel polycyclic peptide antagonist of tachykinin NK2 receptors. Maggi, Carlo Alberto; Astolfi, Mara; Giuliani, Sandro; Goso, Cristina; Manzini, Stefano; Meini, Stefania; Patacchini, Riccardo; Pavone, Vincenzo; Pedone, Carlo; et al. Journal of Pharmacology and Experimental Therapeutics (1994), 271(3), 1489-500. Abstract: We describe the in vitro and in vivo pharmacol. properties of MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β-5β), the first example of a polycyclic peptide tachykinin NK2 receptor antagonist. MEN 10,627 is endowed with high affinity for NK2 receptor expressed in various species with pKB values ranging between 10.1 (hamster trachea) and 8.1 (rabbit pulmonary artery). The antagonism is of competitive type in both functional and radioligand binding assays. A 100- to 10,000-fold selectivity was found vs. NK1 or NK3 receptors expressed in various species. As an NK2 receptor antagonist, MEN 10,627 is 10- to 100-fold more potent than the monocyclic peptide antagonist L 659,877 or cyclo(Met-Gln-Trp-Phe-GlyLeu). At the hamster NK2 receptor, MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist SR 48,968 [(S)-Nmethyl-N(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4dichlorophenyl)butyl)benzamide], whereas the converse is true for the rabbit NK2 receptor. Furthermore, MEN 10,627 is, up to micromolar concns., devoid of antagonist properties toward a wide range of transmitters of both peptide and nonpeptide nature. In urethane-anesthetized rats in vivo, MEN 10,627 (10-100 nmol/kg i.v.) produced long-lasting inhibition of contraction of the urinary bladder and duodenum produced by i.v. administration of the NK2 receptor agonist [βAla8]NKA(4-10), without affecting the responses produced by i.v. administration of the NK1 receptor agonist [Sar9]SP sulfone or acetylcholine. In anesthetized rats, both MEN 10,627 and SR 48,968 blocked urinary bladder contraction induced by the NK2 receptor agonist after i.v., intranasal or intraduodenal administration. Equieffective doses of MEN 10,627 producing about 50% inhibition of the response to [βAla8]NKA(4-10) in the rat urinary bladder in vivo, were 0.01, 0.03 and 3 µmol/kg after i.v., intranasal and intraduodenal administration, resp. The corresponding doses of SR 48,968 were 0.03, 0.1 and 1 µmol/kg, after i.v., intranasal and intraduodenal administration, resp. In conclusion, MEN 10,627 is a potent and selective NK2 receptor antagonist, endowed with high potency and long duration of action in vivo, which is not restricted to parenteral administration. Therefore, MEN 10,627 overcomes 67 Prof. Vincenzo Pavone some of the most common drawbacks for the use of peptides as drugs, and appears as a serious candidate for testing the potential usefulness of NK2 receptor antagonists in humans. 42. Mixed conformation in Cα,α-disubstituted tripeptides: x-ray crystal structures of Z-Aib-Dph-Gly-OMe and Bz-Dph-Dph-Gly-OMe. Pavone, V.; Lombardi, A.; Saviano, M.; DiBlasio, B.; Nastri, F.; Fattorusso, R.; Zaccaro, L.; Maglio, O.; Yamada, T.; et al. Biopolymers (1994), 34(12), 1595604. Abstract: The synthesis and solid-state mol. structure of fully protected tripeptides contg. Cα,α-diphenylglycine (Dph), namely Z-Aib-Dph-GlyOMe (Z = PhCH2O2C, Aib = Cα,α-dimethylglycine) and Bz-Dph-DphGly-OMe are reported. The mol. conformation around the Dph residue, contg. two bulky substituents, is fully extended, while the Aib residue, contg. two smaller groups on the Cα atom, adopts the typical 310/α-helical conformation. Gly residues, without substituents on the Cα atom, show different conformational preferences. Each residue seems to behave, from a conformational point of view, independently from the presence of the other residues, and thus mixed local conformations (folded and extended) are present in the crystals. A nonconventional peptide synthesis, using the Ugi reaction, is also reported. 43. β-Alanine containing cyclic peptides with turned structure: the "pseudo type II β -turn." VI. Pavone, Vincenzo; Lombardi, Angelina; Saviano, Michele; Nastri, Flavia; Fattorusso, Roberto; Maglio, Ornella; Isernia, Carla; Paolillo, Livio; Pedone, Carlo. Biopolymers (1994), 34(11), 1517-26. Abstract: We describe the synthesis, purifn., single crystal x-ray anal., and NMR soln. characterization, combined with restrained mol. dynamic simulations, of the cyclic hexapeptide cyclo(L-Pro-L-Phe-β-Ala)2. The peptide was synthesized by classical soln. methods and the cyclization of the free hexapeptide was accomplished in good yields in dild. methylene chloride soln. using DCC. The compd. crystallizes in the monoclinic space group P21 from methanol-dichloromethane soln. The two identical halves of the mol. adopt in the solid state two different conformations. One β-Ala-LPro peptide bond is trans, while the second is cis. The mol. is present in dimethylsulfoxide d6 solns. as a mixt. of conformational families. One of these corresponds to a C2 sym. mol. with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that obsd. in the solid state. All Pro-Phe segments, both in the solid state and the sym. and unsym. soln. conformations, display φ,ψ angles close to that of position i + 1 68 Prof. Vincenzo Pavone and i + 2 of type II β-turns. In addn., the segments preceded by a trans βAla-pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer contg. a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the "pseudo type II β-turn.". 44. β-Alanine containing cyclic peptides with predetermined turned structure. V. Pavone, Vincenzo; Lombardi, Angelina; Saviano, Michele; Di Blasio, Benedetto; Nastri, Flavia; Fattorusso, Roberto; Maglio, Ornella; Isernia, Carla. Biopolymers (1994), 34(11), 1505-15. Abstract: We describe the synthesis, purifn., single crystal x-ray anal., and soln. structural characterization by NMR spectroscopy, combined with restrained mol. dynamic simulations, of the cyclic hexapeptide cyclo(ProPhe-β-Ala-Phe-Phe-β-Ala). The peptide was synthesized by classical soln. methods and the cyclization of the free hexapeptide was accomplished in good yields in dild. methylenechloride soln. using DCC. The compd. crystallizes in the monoclinic space group P21 from methanol/ethyl acetate. The mol. adopts in the solid state a conformation characterized by cis βAla6-Pro1 peptide bond. The α-amino acid residues are at the corner positions of turned structures. The Pro1-Phe2 segment is incorporated in a pseudo type I β-turn, while Phe4-Phe5 is in a typical type I β-turn. Assignment of all 1H and 13C resonances was achieved by homo- and heteronuclear two-dimensional techniques in DMSO solns. The conformational anal. was based on interproton distances derived from rotating frame nuclear Overhauser effect spectroscopy spectra and homonuclear coupling consts. Restrained mol. dynamic simulation in vacuo was also performed to built refined mol. models. The mol. is present in DMSO soln. as two slowly interconverting conformers, characterized by a cis-trans isomerism around the β-Ala6-Pro1 peptide bond. This work confirms our expectations on the low propensity of β-alanyl residues to be positioned at the corners of turned structure. 45. Influence of Lipophilicity on the Biological Activity of Cyclic Pseudopeptide NK-2 Receptor Antagonists. Quartara, Laura; Fabbri, Gaetano; Ricci, Renzo; Patacchini, Riccardo; Pestellini, Vittorio; Maggi, Carlo Alberto; Pavone, Vincenzo; Giachetti, Antonio; Arcamone, Federico. Journal of Medicinal Chemistry (1994), 37(21), 3630-8. Abstract: A series of cyclic pseudopeptides cyclo(Leuψ[CH2NH]Xaa-GlnTrp-Phe-β-Ala) (I; Xaa = α-amino acid residue), was prepd. to establish the role of the Xaa side chain for tachykinin NK-2 receptor antagonist activity. 69 Prof. Vincenzo Pavone Syntheses have been carried out in solid phase with either 9fluorenylmethoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc) strategy. The antagonist potency on NK-2 receptors in the hamster isolated trachea (HT) and the rabbit isolated pulmonary artery (RPA) bioassays increases with Xaa lipophilicity; I [Xaa = β-cyclohexylalanine, Asp(NHCH2Ph)] were the two most active antagonists (pA2 = 9.06 and 9.26 on HT, resp.). A significant linear correlation was found between pA2 values detd. in HT and RPA bioassays and capacity factors measured in reversed phase HPLC. The comparison between the biol. activities of cyclic hexapeptides contg. or not contg. the aminomethylene moiety proved the crucial role of the pseudopeptide bond for detg. high antagonist potency at the NK-2 receptor. 46. Conformational studies on peptides as enzyme inhibitors: chymotrypsin inhibitors using Bowman-Birk type as models. Pavone, Vincenzo; isernia, Carla; Saviano, Michele; Falcigno, Lucia; Lombardi, Angelina; Paolillo, Livio; Pedone, Carlo; Buoen, Solfrid; Naess, Hilde Merete; et al. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (19721999) (1994), (5), 1047-53. Abstract: A complete structural characterization in soln., by NMR spectroscopy, and in vacuo, by mol. dynamic simulations, of two synthetic peptide fragments from SBB (soybean Bowman-Birk inhibitor) is reported. Peptide 197 I, corresponding to the SBBI(41-49) chymotrypsin recognition site, has free N- and C-terminal groups, while peptide 212 II, corresponding to the Leu-16-SBBI(14-22) has uncharged and fully protected terminal ends. Peptide 212 shows significant antichymotryptic activity while peptide 197 is inactive. Neither of the two peptides shows anti-tryptic activity. The structural information obtained in the present paper suggests a quant. structure-activity relationship which may help both in understanding the mechanism of action of protease inhibitors, and in providing new directions for the rational design of more specific and potent inhibitors. H-C ys-Ala-Leu-Ser-Tyr-Pro-Ala-Gln-C Ac-C ys-Thr-Leu-Ser-Asn-Pro-Pro-Gln-C 47. ys-OH ys-NH 2 I II Conformational versatility of the Nα-acylated tripeptide amide tail of oxytocin. Synthesis and crystallographic characterization of three C2α backbone modified, conformationally restricted analogs. Fabiano, N.; Valle, G.; Crisma, M.; Toniolo, C.; Saviano, M.; Lombardi, A.; Isernia, C.; Pavone, V.; Di Blasio, B.; et al. International Journal of Peptide & Protein Research (1993), 42(5), 459-65. 70 Prof. Vincenzo Pavone Abstract: The synthesis, phys. and anal. characterization, and crystal-state structural anal. by x-ray diffraction of R-Pro-X-Gly-NH2 [I; R = Me3CO2C (Boc), X = 1-aminocyclopropane-1carboxylic acid (Ac3c); R = PhCH2O2C (Z), X = 1-aminocyclopentane-1-carboxylic acid (Ac5c), 1aminocyclohexane-1-carboxylic acid (Ac6c)], analogs of the Nα-acylated tripeptide amide tail of oxytocin, each contg. a cyclic Cα,α-disubstituted glycine at position 2, have been performed. While I (R = Boc, X = Ac3c) is folded in a type-II β-turn conformation at the Pro-Ac3c sequence, I (R = Z, X = Ac5c, Ac6c) form two consecutive (type-II, type-I') β-turns. I (R = Z, X = Ac5c, Ac6c) are the first examples of such a highly folded structural combination in a position-2 analog of the Nα-acylated Pro-Leu-Gly-NH2 sequence. 48. β-Alanine containing peptides: γ -turns in cyclotetrapeptides. Di Blasio, B.; Lombardi, A.; D'Auria, G.; Saviano, M.; Isernia, C.; Maglio, O.; Paolillo, L.; Pedone, C.; Pavone, V.. Biopolymers (1993), 33(4), 621-31. Abstract: The synthesis, purifn., single-crystal x-ray anal., soln. conformational characterization, and conformational energy calcns. of the cyclic tetrapeptide cyclo(β-Ala-L-Pro-β-Ala-L-Val) are described.. The peptide was synthesized by classical soln. methods and the cyclization of the free tetrapeptide was accomplished in good yields in dild. methylene chloride soln. using DCC. The compd. crystallizes in the monoclinic space group P21 from ethanol with two independent mols. in the unit cell. All peptide bonds are trans. The NMR mol. conformation in the acetonitrile soln. as well as that derived from the mol. dynamic simulation in vacuo is quite different from those obsd. in the solid state and is very similar to that previously obsd. for the parent compd. cyclo(β-Ala-L-Pro-β-Ala-L-Pro). 49. Platinum(II) complexes of amino acids and peptides. III. X-ray diffraction study of [Cl(Ph3P)Pt(H-Aib-O)]. Lombardi, Angela; Maglio, Ornella; Pavone, Vincenzo; Di Blasio, Benedetto; Saviano, Michele; Nastri, Flavia; Pedone, Carlo; Benedetti, Ettore. Inorganica Chimica Acta (1993), 204(1), 8792. Abstract: [Cl(Ph3P)Pt(L)] (I; HL = α-aminoisobutyric acid) was synthesized and characterized in the solid state by x-ray diffraction anal. I was characterized by x-ray crystallog. Crystal data: I.H2O; triclinic, space group P.hivin.1, a 9.020(1), b 10.011(2), c 13.748(1) .ANG., α 91.74(1), β 90.29(1), γ 111.26(1)°, Z = 2, R = 0.038, Rw = 0.042. The Pt(II) displays the square planar coordination with L- acting as a bidentate ligand. The N atom 71 Prof. Vincenzo Pavone of L- is trans to the P atom of the PPh3, and the O atom of the carboxylate is trans to the Cl atom. The obsd. distortions in the bond angles around the Pt(II) atom and the conformation assumed by PPh3 and L- are explained from the need to release the intramol. nonbonded interactions between atoms of the various ligands. Three intermol. H bonds, involving as donors the co-crystd. H2O mol. and the 2 H atoms of the N-H groups and as acceptors the Cl, the O of the H2O mol., and 1 of the O atoms of the carboxylic acid group, together with van der Waals interactions between hydrophobic groups held the mols. in the crystal state. 50. Non coded Cα,α-disubstituted amino acids. X-ray diffraction analysis of a dipeptide containing (S)-α -methylserine. Pavone, V.; Di Blasio, B.; Lombardi, A.; Maglio, O.; Isernia, C.; Pedone, C.; Benedetti, E.; Altmann, E.; Mutter, M. International Journal of Peptide & Protein Research (1993), 41(1), 15-20. Abstract: The crystal and mol. structure of Boc-Val-(S)-α-MeSer-OMe (Boc = Me3CO2C) has been detd. by x-ray diffraction techniques. Both chiral centers have the S configuration. The dipeptide assumes in the solid state an S shape. The urethane moiety is in the cis conformation, while the amide bond is in the common trans conformation. The conformational angles φ1, ψ1 of the Val and φ2, and ψ2 of the (S)-αMeSer fall in the F region of the φψ map. The iso-Pr side chain of the Val residue has the (t, g-) conformation, while the Ser side chain has a g+ conformation. The hydrogen bond donor groups are all involved in intermol. H-bond interactions. Along the quaternary axis the dipeptide mols. are linked to each other with the formation of infinite rows. 51. Platinum(II) complexes of amino acids and peptides. II. Structural analysis of trans-[Cl2Pt(H-Aib-OH)2] and trans-[Pt(H-Aib-O-)2]. Lombardi, Angela; Maglio, Ornella; Benedetti, Ettore; Di Blasio, Benedetto; Saviano, Michele; Nastri, Flavia; Pedone, Carlo; Pavone, Vincenzo. Inorganica Chimica Acta (1992), 196(2), 241-6. Abstract: trans-[PtCl2(H-Aib-OH)2] and trans-[Pt(H-Aib-O-)2] were prepd. and characterized in soln. and in the solid state. Their crystal and mol. structures were detd. by x-ray diffraction. The complexes show a distorted planar coordination around the heavy atom. The org. moiety in the 2 complexes shows different conformations: in trans-[Pt(H-Aib-O-)2] the amino acid residue, acting as a bidentate, is forced to assume on unusual conformation. But in trans-[PtCl2(H-Aib-OH)2], in which the amino acid acts as a monodentate ligand, the α-aminoisobutyric acid assumes an extended conformation, which in general for α-monosubstituted amino 72 Prof. Vincenzo Pavone acid residues coordinated to the Pt should be the preferred conformation. In both crystal structures H bonds are formed between the donor and acceptor groups with further stabilization deriving from van der Waals interactions between hydrophobic moieties. 52. Conformation of diastereomeric peptide sequences: structural analysis of ZD-Val-Ac6c-Gly-L-Phe-OMe. Di Blasio, B.; Lombardi, A.; Nastri, F.; Saviano, M.; Pedone, C.; Yamada, T.; Nakao, M.; Kuwata, S.; Pavone, V.. Biopolymers (1992), 32(9), 1155-61. Abstract: A systematic structural anal. of fully protected tetrapeptides contg. at the N- and C-terminus either homo- or heterochiral amino acids, spaced by an achiral dipeptide segment was undertaken. The interest for this class of peptides derives from the observation that, on reverse-phase HPLC, the homo- and heterochiral sequences have a markedly different retention times. Therefore, following preliminary studies in soln., the detailed x-ray anal. of the tetrapeptide Z-D-Val-Ac6c-Gly-L-Phe-OMe (Z = PhCH2O2C, Ac6c = 1-aminocyclohexane-1-carboxylic acid) are reported to understand the structural features governing the overall hydrophobicity of linear fully protected tetrapeptides. 53. Conformation for a β-cyclodextrin monosubstituted with a cyclic dipeptide. Di Blasio, Benedetto; Pavone, Vincenzo; Nastri, Flavia; Isernia, Carla; Saviano, Michele; Pedone, Carlo; Cucinotta, Vincenzo; Impellizzeri, Giuseppe; Rizzarelli, Enrico; Vecchio, Graziella. Proceedings of the National Academy of Sciences of the United States of America (1992), 89(15), 7218-21. Abstract: The structural characterization of 6-deoxy-6-cyclo(L-histidyl-Lleucyl)-β-cyclodextrin is reported. This work provides an x-ray example of a covalently bound group that folds in such a way that the terminal apolar side chain is retained in the hydrophobic interior of the cone-shaped cyclodextrin cavity. The mol. structure consists of two independent mols. with the formula C54H86N4O36⋅7.25H2O. Each mol. assumes a "sleeping swan"-like overall shape with the hydrophobic leucine side chain inserted inside the cavity of the macrocycle. The two independent units give rise to a head-to-tail dimer linked by hydrogen bonds occurring between primary and secondary hydroxyl groups of the two monomers. The packing of the dimers produces cavities contg. water mols. There are infinite hydrophilic channels running in the crystal, which is similar to what is found in the structures of cyclic peptides. 54. Linear oligopeptides. CCLXV. Synthesis and solution conformational analysis of β -bend ribbon forming peptides. Crisma, Marco; Anzolin, 73 Prof. Vincenzo Pavone Michele; Bonora, Gian Maria; Toniolo, Claudio; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Saviano, Michele; Lombardi, Angelina; et al. Gazzetta Chimica Italiana (1992), 122(7), 239-44. Abstract: Peptides Z-(Pro-Aib)n-OMe (Z = PhCH2O2C, Aib = αaminoisobutyric acid, n = 1-4), p-BrZ-(Pro-Aib)n-OMe (n = 1-5) and pBrBz-Aib-(Pro-Aib)n-OMe (n = 1-4) were prepd. as potential model compds. for the β-bend ribbon spiral. A conformational anal. in chloroform soln., performed by using IR absorption and 1H NMR, strongly supports the view that this ordered secondary structure, first obsd. at the -Aib-L-ProAib- tripeptide level, is extensively adopted by the longest oligomers. 55. β-Alanine and β -bends. X-ray diffraction structures of three linear oligopeptides. Pavone, Vincenzo; Di Blasio, Benedetto; Lombardi, Angela; Isernia, Carla; Pedone, Carlo; Benedetti, Ettore; Valle, Giovanni; Crisma, Marco; Toniolo, Claudio; Kishore, Raghuvansh. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1992), (8), 1233-7. Abstract: A crystal-state structural anal. of Boc-L-Ala-β-Ala-NHMe (Boc = Me3CO2C), Boc-Aib-β-Ala-NHMe (Aib =, α-aminoisobutyric acid), and Boc-Aib-Aib-β-Ala-NHMe has been performed by x-ray diffraction. While the conformation adopted by Boc-L-Ala-β-Ala-NHMe and Boc-Aib-β-AlaNHMe is essentially extended, Boc-Aib-Aib-β-Ala-NHMe is folded into two consecutive intramolecularly hydrogen-bonded structures of the i + 3 → i type (β-bends), with Aib(1)-Aib(2) and Aib(2)-β-Ala(3), resp., as corner residues. Owning to the presence of the β-amino acid, the latter β-bend is characterized by an unusual C11 hydrogen-bonded ring. These results indicate that: (i) a β-amino acid may be incorporated into a β-bend without a major perturbation of the overall geometry of this folded conformation, and (ii) the propensity of the β-Ala residue for β-bend formation is rather low, unless other conformational constraints (e.g. a preceding β-bend) are present in the linear peptide mol. 56. Bioactive peptides: x-ray and NMR conformational study of [Aib5,6-DAla8]cyclolinopeptide A. Di Blasio, Benedetto; Rossi, Filomena; Benedetti, Ettore; Pavone, Vincenzo; Saviano, Michele; Pedone, Carlo; Zanotti, Giancarlo; Tancredi, Teodorico. Journal of the American Chemical Society (1992), 114(21), 8277-83. Abstract: The conformational anal. of [Aib5,6-D-Ala8]cyclolinopeptide A, cyclo(Pro-Pro-Phe-Phe-Aib-Aib-Ile-D-Ala-Val), (I; Aib = α- 74 Prof. Vincenzo Pavone aminoisobutyric acid) in the solid state and soln., has been carried out by xray diffraction and NMR spectroscopy. The structure of the orthorhombic form of I, obtained from aq. MeOH, shows the presence of 5 intramol. NHOC hydrogen bonds, with formation of one C17 ring structure, one α-turn (C13), one γ-turn (C7), and two β-turns (C10, one of type III and one of type I). The Pro1-Pro2 peptide unit is cis (ω = 9°); all other units are trans. The conformational study in soln. by NMR indicates that, even at room temp., the peptide is conformationally homogeneous; the structure detd. is almost identical to that obsd. in the solid state. The soln. study reveals, also, that the constraints imposed by the two Aib and D-Ala residues are particularly strong, because the NMR conformational parameters are only slightly affected by wide temp. variations and salt addn. 57. Molecular dynamics simulation in vacuo and in solution of [Aib5,6-D-Ala8] cyclolinopeptide A: a conformational and comparative study. Saviano, Michele; Rossi, Filomena; Pavone, Vincenzo; Di Blasio, Benedetto; Pedone, Carlo. Journal of Biomolecular Structure & Dynamics (1992), 9(6), 1045-60. Abstract: The conformation of cyclolinopeptide A analog cyclo(Pro-ProPhe-Phe-Aib-Aib-Ile-D-Ala-Val) (Aib = α-aminoisobutyric acid) was investigated by mol. dynamics simulations in various mol. environments. The mol. dynamics results are compared with that obtained for cyclolinopeptide A and a detailed anal. of the different behavior for the two compds. is reported. A complete anal. of hydrogen bonds is presented. 58. First observation of a helical peptide containing chiral α-monosubstituted residues without a preferred screw sense. Pavone, Vincenzo; Lombardi, Angelina; Nastri, Flavia; Saviano, Michele; Di Blasio, Benedetto; Fraternali, Franca; Pedone, Carlo; Yamada, Takashi. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1992), (6), 971-7. Abstract: The detailed x-ray structure of the fully blocked tetrapeptide PhCH2O2C-D-Val-(Aib)2-L-Phe-OMe (Aib = α-aminoisobutyric acid) is reported. There is a regular alternation of right- and left-handed 310-helices hydrogen bonded head-to-tail along this axis. Pairs of mols. with the same handedness differ in the conformation of the side chains and of the N- and C-terminal blocking groups. This is the first observation of a helical peptide contg. chiral α-monosubstituted α-amino acids without a preferred screw sense. Conformational energy computations confirmed that those helices with different handedness have comparable stabilities. This work furthers the understanding of structural features responsible for the diastereoselective sepn. by reversed-phase HPLC. 75 Prof. Vincenzo Pavone 59. Structural characterization of the β -bend ribbon spiral: crystallographic analysis of two long (L-Pro-Aib)n sequential peptides. Di Blasio, B.; Pavone, V.; Saviano, M.; Lombardi, A.; Nastri, F.; Pedone, C.; Benedetti, E.; Crisma, M.; Anzolin, M.; Toniolo, C. Journal of the American Chemical Society (1992), 114(16), 6273-7. Abstract: The mol. and crystal structures of title peptides p-BrBz-Aib-(LPro-Aib)n-OMe (Aib = α-aminoisobutyric acid; n = 3, 4) were detd. by xray diffraction. In both crystals two mols. in the asym. unit are found. Either mol. in the asym. unit of each structure shows a right-handed β-bend ribbon spiral, stabilized by the max. possible no. of intramol. N-H⋅⋅⋅O:C Hbonds. Thus, for the first time it was possible to characterize at at. resoln. this polypeptide conformation. 60. A helical Dpg homopeptide. Di Blasio, Benedetto; Pavone, Vincenzo; Isernia, Carla; Pedone, Carlo; Benedetti, Ettore; Toniolo, Claudio; Hardy, Paul M.; Lingham, Ian N. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1992), (4), 523-6. Abstract: The first x-ray diffraction structure anal. of a helical homopeptide of Cα,α-dipropylglycine (Dpg) has been performed for Tfa-(Dpg)3-DBH monohydrate (Tfa = trifluoroacetyl, DBH = N',N'-dibenzylhydrazido). 61. Characterization at atomic resolution of peptide helical structures. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Toniolo, C.; Crisma, M. Biopolymers (1992), 32(4), 453-6. Abstract: A survey of literature for the various types of helices exptl. obsd. in high-resoln. single crystal x-ray diffraction analyses of peptides allowed the detn. of accurate conformational and helical parameters for the various secondary structures such as the α-helix, the 310-helix, the fully extended conformation (25-helix), and the β-bend ribbon spiral. For each of these structures the characteristic ϕ,ψ conformational parameters, n, the no. of residues per turn, h, the height per residues, and p, the pitch of the helix are described. 62. β-Alanine containing peptides: a novel molecular tool for the design of γ turns. Pavone, V.; Lombardi, A.; D'Auria, G.; Saviano, M.; Nastri, F.; Paolillo, L.; Di Blasio, B.; Pedone, C. Biopolymers (1992), 32(2), 173-83. Abstract: The synthesis, purifn., single crystal x-ray anal., and soln. conformational characterization of the cyclic tetrapeptide cyclo(Pro-β-Ala- 76 Prof. Vincenzo Pavone Pro-β-Ala) is described. This peptide was synthesized by classical soln. methods and the cyclization of the free tetrapeptide was accomplished in good yields using DCC. The mol. conformation is stabilized by two intramol. hydrogen bonds between the CO and NH groups of the two βalanine residues. These hydrogen bonds take part in a C7 structure in which both proline residues occupy the 2 position of an inverse γ-turn. The two βalanine residues have a typical folded conformation (around the Cα-Cβ bond) obsd. in other cyclic peptides contg. this residue. A detailed 1H-NMR anal. in CD3CN soln. has been carried out. The mol. assumes a twofold symmetry in soln. with a mol. conformation consistent with that obsd. in the solid state. 63. β-Alanyl-β-alanine in cyclic β-turned peptides. Di Blasio, B.; Lombardi, A.; Yang, X.; Pedone, C.; Pavone, V.. Biopolymers (1991), 31(10), 1181-8. Abstract: The synthesis, purifn., and single crystal x-ray anal. of the cyclic pentapeptide cyclo(L-Pro-L-Pro-L-Phe-β-Ala-β-Ala) is described). The peptide was synthesized by classical soln. methods and the cyclization of the free pentapeptide was accomplished in good yields in dil. CH2Cl2 using DCC. The Pro1-Pro2 peptide bond is cis and the mol. conformation is stabilized by an intramol. hydrogen bond between the CO group of β-Ala5 and the NH of the Phe3. The Pro1-Pro2 segment occupies the relative positions 2 and 3 of a type VIa β-turn, while the L-phenylalanyl-β-alanyl-βalanine segment is incorporated in a C13-like ring structure. The crystal packing is characterized by a network of 11 intermol. hydrogen bonds involving all the remaining CO, NH, and water mols. 64. Crystal-state conformation of homo-oligomers of α -aminoisobutyric acid: molecular and crystal structure of pBrBz-(Aib)6-OMe. Di Blasio, Benedetto; Santini, Antonello; Pavone, Vincenzo; Pedone, Carlo; Benedetti, Ettore; Moretto, Vittorio; Crisma, Marco; Toniolo, Claudio. Structural Chemistry (1991), 2(5), 523-7. Abstract: The title terminally blocked homohexapeptide 4BrC6H4CO(NHCMe2CO)6OMe was examd. by x-ray crystallog. The compd. is folded into almost two turns of a 310-helix, stabilized by four consecutive intramol. NH-OC hydrogen bonds. An asym. covalent geometry for the α-aminoisobutyric acid residues, known to be responsible for the onset of the 310-helix on the basis of a theor. conformational anal., has been exptl. verified in this structure. 65. Linear oligopeptides. CCXXXI. Preferred conformation of homo-oligomers 77 Prof. Vincenzo Pavone of α-aminoisobutyric acid: molecular and crystal structure of Z-(Aib)7OMe. Pavone, Vincenzo; Di Blasio, Benedetto; Pedone, Carlo; Santini, Antonello; Benedetti, Ettore; Formaggio, Fernando; Crisma, Marco; Toniolo, Claudio. Gazzetta Chimica Italiana (1991), 121(1), 21-7. Abstract: The structure of the title compd., 2-(Aib)7-OMe (Z = PhCH2O2C, Aib = α-aminoisobutyric acid) was detd. by x-ray crystallog. The compd. is folded into two complete turns of a regular 310-helix, stabilized by five consecutive intramol. NH-OCH bonds of the β bend III (III') type. This structure completes the series of the oligomers of α-aminoisobutyric acid to the octapeptide level analyzed at at. resoln. by x-ray diffraction. 66. Linear oligopeptides. 229. Preferred conformation of the terminally blocked (Aib)10 homo-oligopeptide: a long, regular 310-helix. Toniolo, Claudio; Crisma, Marco; Bonora, Gian Maria; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Santini, Antonello. Biopolymers (1991), 31(1), 129-38. Abstract: The decapeptide 4-BrC6H4CO-(Aib)10-OCMe3 (I; Aib = αaminoisobutyric acid) was prepd. by the 5(4H)-oxazolone method, and its crystal structure detd. I adopts a regular 310-helical structure stabilized by 8 NH-OC intramol. 1←4 (or C10) H bonds. This study has allowed the characterization this important peptide secondary structure in great detail. The crystal-state conformation agrees well with proposals made on the basis of an IR absorption and 1H-NMR study in soln. 67. Bicyclic peptides: solid state conformation of cyclo(Glu-Leu-Pro-Gly-LysLeu-Pro-Gly)cyclo(1γ-5ε)Gly. Di Blasio, Benedetto; Benedetti, Ettore; Pavone, Vincenzo; Pedone, Carlo; Saviano, Michele; Zanotti, Giancarlo; Blout, Elkan R. Biopolymers (1990), 30(5-6), 509-16. Abstract: The solid state conformational anal. of the title ionophoric homodetic bicyclic peptide I (BCP3) has been carried out by x-ray diffraction. The structure characterized by all trans peptide bonds is stabilized by three intramol. H bonds: a type II β-turn, a mixed type I-type III β-turn, and a pseudo γ-turn, which involves the side chain CO and the main-chain NH groups of the glutamic acid residue. The resulting globular mol. presents a rather hydrophilic surface with most of the CO groups available to hydration by the solvent mols., which are linked through H bonds of the NH-O or OH-O types in a complicated H-bonding scheme. The conformation obsd. in the solid state is rather different from the conformation proposed in soln. for both the free and the Ca2+-complexed mol. 78 Prof. Vincenzo Pavone 68. Linear oligopeptides. Part 227. X-ray crystal and molecular structures of two α -helix-forming (Aib-L-Ala) sequential oligopeptides, pBrBz-(Aib-LAla)5-OMe and pBrBz-(Aib-L-Ala)6-OMe. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Santini, Antonello; Bavoso, Alfonso; Toniolo, Claudio; Crisma, Marco; Sartore, Luciana. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1990), (11), 1829-37. Abstract: A crystal-state structural anal. of the title compds. 4-BrC6H4CO(NHCMe2CO-Ala)n-OMe (n = 5,6) has been performed by x-ray diffraction. The decapeptide and dodecapeptide mols. are both basically αhelical with five and seven 1 ← 5 intramol. H-bonds, resp. A similarity between the two structures is also seen near the C-terminus, where regularity of the α-helix is disrupted in favor of formation of intramol. Hbonds of the 1 ← 4 and 1 ← 6 types. A brief comparison with parameters and interactions characteristic of the helices present in globular proteins has been made. 69. Stereochemical behavior of acyclic peptide-cation complexes. Grimaldi, Maria; Rossi, Filomena; Saviano, Michele; Benedetti, Ettore; Pavone, Vincenzo; Pedone, Carlo. Biopolymers (1990), 30(1-2), 197-204. Abstract: The role of the β-turns in the peptide interaction with cations was investigated for PhCH2O2C-Aib-Aib-Val-OMe (Aib = NHCMe2CO), Boc-D-aIle-Ile-Ile-OMe (Boc = Me3CO2C), Boc-Leu-Leu-Leu-Leu-OMe, and Boc-Phe-D-Phe-Phe-D-Phe-OMe. CD and 1H-NMR spectra reveal the presence of multiple ion-bonding equil. The stoichiometry and binding const. of the four peptides in the presence of Ca2+ ions in acetonitrile soln. has been detd. Variable-temp. NMR spectra in the absence and in the presence of a large excess of cation have shown that the complexation process is not critically dependent on the conformation of the free peptide. 70. Cyclic β -alanyl-β -alanine containing peptides: a new molecular tool for β turned peptides. Pavone, V.; Lombardi, A.; Yang, X.; Pedone, C.; Di Blasio, B. Biopolymers (1990), 30(1-2), 189-96. Abstract: The synthesis, purifn., and single-crystal x-ray anal. of the cyclic tetrapeptide cyclo-(L-Pro-L-Phe-β-Ala-β-Ala) (I) is described. I contains the β-alanyl-β-alanine dipeptide as putative cyclization arm to force the remaining dipeptide-L-Pro-L-Phe- in a β-turned conformation. I was synthesized by classical methods and cyclization of the free linear 79 Prof. Vincenzo Pavone tetrapeptide was accomplished in reasonable yields in dil. CH2Cl2. In the solid state I shows an intramol. hydrogen bond between the CO group of the β-Ala4 and the NH group of the β-Ala3 residue, stabilizing a type I βturn conformation in which Pro1 and Phe2 occupy the relative position 2 and 3 of the turn, resp. 71. Crystal structure of two retro-inverso sweeteners. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Fuller, William D.; Mierke, Dale F.; Goodman, Murray. Journal of the American Chemical Society (1990), 112(24), 8909-12. Abstract: The structure of a crystal composed of 2 diastereomers, N-(Laspartyl)-N'-[(2,2,5,5-tetramethylcyclopentanyl)carbonyl]-(R)-1,1diaminoethane(L,R isomer) and N-(L-aspartyl)-N'-[(2,2,5,5tetramethylcyclopentanyl)carbonyl]-(S)-1,1-diaminoethane (L,S isomer), was solved. Both diastereomers are intensely sweet and are retro-inverso stereoisomers of dipeptides. The crystal structure of these mols. was related to the model to explain the sweetness of peptide-based ligands. The L shape postulated on the basis of mol. mechanics and NMR spectroscopy is in full agreement with the crystal structures. 72. The longest, regular polypeptide 310 helix at atomic resolution. Pavone, Vincenzo; Di Blasio, Benedetto; Santini, Antonello; Benedetti, Ettore; Pedone, Carlo; Toniolo, Claudio; Crisma, Marco. Journal of Molecular Biology (1990), 214(3), 633-5. Abstract: A synthetic, terminally blocked homodecapeptide from the Cα,αdimethylated glycyl residue α-aminoisobutyric acid was analyzed by singlecrystal x-ray diffraction and the structure refined to R = 0.073. The compd. crystallizes as a perfect 310 helix, stabilized by 8 consecutive intramol. NH...O:C hydrogen bonds. This is the first observation at at. resoln. of a regular polypeptide 310 helix as long as 3 complete turns. 73. Linear oligopeptides. CCXXV. Critical main-chain length for conformational conversion from 310-helix to α-helix in polypeptides. Pavone, Vincenzo; Benedetti, Ettore; Di Blasio, Benedetto; Pedone, Carlo; Santini, Antonello; Bavoso, Alfonso; Toniolo, Claudio; Crisma, Marco; Sartore, Luciana. Journal of Biomolecular Structure & Dynamics (1990), 7(6), 1321-31. Abstract: To assess the minimal peptide length required for the stabilization of the α-helix relative to the 310-helix in α-aminoisobutyric acid (Aib)-rich peptides, the x-ray diffraction structures of terminally blocked sequential 80 Prof. Vincenzo Pavone hexa- and octapeptides with the general formula, -(Aib-L-Ala)n- (where n = 3 and 4, resp.), were solved. The hexapeptides were completely 310-helical with 4 1←4 intramol. N-H…O:C H-bonds. On the other hand, the octapeptides were essentially α-helical with 4 1←5 H-bonds; however, the helix was elongated at the N-terminus, with 2 1←4 H-bonds, giving these mols. a mixed α/310-helical character. In both compds., the right-handed screw sense of the helix was dictated by the presence of the L-alanine residues. This study represents the 1st exptl. proof for a 310 → α-helix conversion in the crystal state induced by peptide backbone lengthening only. 74. Structure of clathridine zinc complex, a metabolite of the marine sponge Clathrina clathrus. Ciminiello, Patrizia; Fattorusso, Ernesto; Mangoni, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo. Tetrahedron (1990), 46(12), 4387-92. Abstract: The solid state structure of the Zn complex of clathridine, a genuine metabolite of the sponge Clathrina clathrus, was established by xray diffraction anal. The conformation of this complex in CDCl3 soln. was investigated by NOE difference NMR expts. 75. Linear oligopeptides. Part 202. Structural versatility of peptides containing Cα,α-dialkylated glycines. An x-ray diffraction study of six 1aminocyclopropane-1-carboxylic acid rich peptides. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, A.; Barone, V.; Fraternali, F.; Lelj, F.; Bavoso, A.; et al. International Journal of Biological Macromolecules (1989), 11(6), 353-60. Abstract: The crystal structures of 6 fully blocked 1-aminocyclopropane-1carboxylic acid (Acc)-rich peptides were detd. by x-ray diffraction. The peptides are Fmoc-(Acc)2-OMe, Ac-(Acc)2-OMe, Boc-Acc-L-Phe-OMe, 4BrC6H4CO-(Acc)3-OMe, Z-Gly-Acc-Gly-OTmb, and Boc-(Acc)4-OMe [fmoc = 9-fluorenylmethoxycarbonyl; Boc = Me3CO2C; Z = PhCH2O2C; Tmb = CH2C6H2(OMe)3-2,4,6]. Type-I (I') β-bends and distorted 310helices were found to be typical of the tri- and tetrapeptides, resp. In the dipeptides too short to form β-bend conformations, other less common structural features may be obsd. The av. geometry of the cyclopropyl moiety of the Acc residue is asym. and the N-Cα-C' bond angle is significantly expanded form the regular tetrahedral value. A comparison with the structural preferences of other extensively investigated Cα,αdialkylated α-amino acids is made and the implications for the use of the Acc residue in conformational design are examd. 81 Prof. Vincenzo Pavone 76. Linear oligopeptides. Part 201. Structural versatility of peptides containing Cα,α-dialkylated glycines: conformational energy computations, IR absorption and proton NMR analysis of 1-aminocyclopropane-1-carboxylic acid homopeptides. Crisma, M.; Bonora, G. M.; Toniolo, C.; Barone, V.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, A.; et al. International Journal of Biological Macromolecules (1989), 11(6), 345-52. Abstract: Conformational energy computations on the 1-aminocyclopropane1-carboxylic acid mono-, di-, and tripeptide amides, Ac(Ac3C)nNHMe (n = 1-3), indicate that this Cα,α-dialkylated, cyclic acid residue is conformationally restricted and that type-(I') β-bends and distorted 310helices are particularly stable conformations for the di- and tripeptide amides, resp. The results of the theor. anal. are in agreement with those obtained in a IR and 1H NMR investigation in CHCl3 to form soln. of Ac3C-rich tri- and tetrapeptide esters. A comparison is also made with the conclusions extd. from previous work on peptides rich in α-aminoisobutyric acid, 1-aminocyclopentane-1-carboxylic acid, and 1-aminocyclohexane-1carboxylic acid. 77. Preparation of all four diastereomers of β-phenylcysteine methyl ester through chromatographic optical resolution of the 2,2-dimethylthiazolidine derivatives. Nagai, Ukon; Pavone, Vincenzo. Heterocycles (1989), 28(2), 58992. Abstract: The cis and trans isomers of thiazolidine deriv. I, prepd. from the corresponding erythro- and threo-β-phenylcysteines, were resolved into the enantiomers by a chiral HPLC column, from which all four chiral βphenylcysteine Me esters were obtained. Ph S CO 2 Me NH Me Me 78. I Structure-toxicity relationships in the amatoxin series. Synthesis of Sdeoxy[γ (R)-hydroxy-Ile3]-amaninamide, its crystal and molecular structure and inhibitory efficiency. Zanotti, Giancarlo; Wieland, Theodor; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. International Journal of Peptide & Protein Research (1989), 34(3), 222-8. Abstract: The title compd. (I), bearing a γ-hydroxyl group in the isoleucine side chain, was synthesized. The compd. had about the same inhibitory 82 Prof. Vincenzo Pavone effect on RNA polymerase II from Drosophila embryo as amanullin and the Ile3-analog. Structure anal. by x-ray diffraction revealed that the hydroxyl group at the carbon atom of side chain-3 has the [R]-configuration, the new analog thus being -deoxo[δ(R)-hydroxy-[Ile3]-amaninamide. It follows that the [S]-configuration of this chiral center is a prerequisite to maximal toxicity. Crystallog. data demonstrating great similarity between the peptide backbones of the new analog and those of natural amatoxins are given. HO H C H C Me Me NH CHCONH CH OC CO NH CH 2 CO NH CH 2 CHCHMeEt HO N OC S CH 2 CHNH CO CH CH 2 CO 2 NH 79. N H NH CO CO CH 2 NH I Bioactive peptides: solid-state and solution conformation of cyclolinopeptide A. Di Blasio, Benedetto; Rossi, Filomena; Benedetti, Ettore; Pavone, Vincenzo; Pedone, Carlo; Temussi, Piero Andrea; Zanotti, Giancarlo; Tancredi, Teodorico. Journal of the American Chemical Society (1989), 111(25), 9089-98. Abstract: The solid-state and soln. conformational anal. of cyclolinopeptide A, a cyclic nonapeptide isolated from linseed, is reported. The x-ray crystal structure shows the presence of five strong transannular intramol. hydrogen bonds with the formation of one C7, two C10 (one type I and one type III), one C13, and one C17 ring structures. One peptide unit (linking the two proline residues) is cis (ω = 10°); all others are trans. The conformational study in soln. by NMR spectroscopy indicates that provided one selects the right environment, solid-state and soln. conformations are essentially identical, even if this cyclic system tends to give rise to a complex mixt. of quasi-isoenergetic conformations, favored by the flexibility of the ring enhancement by the isomerism of the proline-proline bond and by polar solvents. 80. Structural versatility of peptides from Cα,α dialkylated glycines: linear Ac3c homo-oligopeptides. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, A.; Crisma, M.; Valle, G.; Toniolo, C. Biopolymers (1989), 28(1), 175-84. Abstract: The crystal structures of five linear Ac3c homooligopeptides R- 83 Prof. Vincenzo Pavone (Ac3c)n-OMe (n = 2, R = H, 9-fluorenylmethoxycarbonyl, Ac; n = 3, R = 4BrC6H4CO; n = 4, R = Me3CO2C; Ac3c = 1-amino-1-cyclopropane carboxylate). The results indicate the propensity of the tri- and tetrapeptides to fold into type I β-bends and distorted 310-helices, resp., in partial contrast to aminoisobutyrate, 1-amino-1-cyclopentane-, and cyclohexanecarboxylate homopeptides of comparable main-chain length, were regular type III β-bends and 310-helical structures were found. When the influence of the constraints produced by the intramol. H bonds of the C10-type is absent, other less common structural features may be obsd. The av. geometry of the cyclopropyl group of the Ac3c residue is asym. and the N-Cα-C' bond angle is significantly expanded from the regular tetrahedral value. 81. Regularly alternating L,D-peptides. III. Hexacyclic peptides from valine or phenylalanine. Pavone, Vincenzo; Benedetti, Ettore; Di Blasio, Benedetto; Lombardi, Angela; Pedone, Carlo; Tomasich, Lera; Lorenzi, Gian Paolo. Biopolymers (1989), 28(1), 215-23. Abstract: The single-crystal x-ray analyses of 2 cyclic hexapeptides contg. an equal no. of alternating L,D-residues as putative analogs of the metal binding compds. enniatin and beauvericine are described. Both cyclo(L-ValD-Val)3 and cyclo(L-Phe-D-Phe)3 retain in the solid state the center of symmetry and crystallize with 6 and 8 CF3CO2H mols., resp. The peptides are strongly hydrogen bonded to the solvent mols. On the basis of the mol. geometry and spatial arrangement of the peptide carbonyl groups and in comparison with other metal binding cyclic peptides, the ability of these mols. to interact with metal ions as 1:1 complexes was estd. 82. Regularly alternating L,D-peptides. II. The double-stranded right-handed antiparallel β -helix in the structure of Boc-(L-Phe-D-Phe)4-OMe. Di Blasio, Benedetto; Benedetti, Ettore; Pavone, Vincenzo; Pedone, Carlo; Gerber, Cristoph; Lorenzi, Gian Paolo. Biopolymers (1989), 28(1), 203-14. Abstract: The crystal structure of Boc-(L-Phe-D-Phe)4-OMe (Boc = Me3CO2C) has been detd. by x-ray diffraction anal. The 2 independent octapeptide mol. form a dimer in the solid state: the 2 chains are assocd. by interstrand hydrogen bonds (12 of the type NH-OC) with the formation of a double-stranded antiparallel right-handed ↑↓ β5,6-helix. These double helices can be represented as a cylinder with a hydrophilic inner core represented by the peptide units and a hydrophobic exterior constituted by the arom. moieties. The dimensions of the cylinder are equal to those obsd. for Boc-(L-Val-D-Val)4-OMe. In the solid state, the dimers pack with each other in an hexagonal fashion with the formation of layers; between the 84 Prof. Vincenzo Pavone layers, solvent mols. fill empty spaces. 83. Regularly alternating L,D-peptides. I. The double-stranded left-handed antiparallel β-helix in the structure of Boc-(L-Val-D-Val)4-OMe. Di Blasio, Benedetto; Benedetti, Ettore; Pavone, Vincenzo; Pedone, Carlo; Spiniello, Ottavia; Lorenzi, Gian Paolo. Biopolymers (1989), 28(1), 193-201. Abstract: The structure of Boc-(L-Val-D-Val)4-OMe (Boc = Me3CO2C) has been detd. by x-ray single-crystal diffraction anal. Two octapeptide chains, related by a crystallog. binary axis, wind up around each other giving rise to a double-stranded left-handed antiparallel ↑↓ β5,6-helix. The dimer, stabilized by 14 interstrand NH-OC hydrogen bonds, can be regarded as a cylinder with an hydrophilic inner core represented by the peptide units as a hydrophobic exterior of iso-Pr groups. 84. Linear oligopeptides. 197. Structure of the linear oligopeptide tert-butyl 1-[1(benzyloxycarbonyl)amino-1-cyclohexanecarboxamido]-1cyclohexanecarboxylate. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, A.; Crisma, M.; Toniolo, C. Acta Crystallographica, Section C: Crystal Structure Communications (1989), C45(4), 634-8. Abstract: The title compd. is triclinic, space group P.hivin.1, with a 5.971(5), b 14.033(5), c 16.011(11) .ANG., α 103.30(39), β 92.97(65), and γ 93.25(44)°; dm = 1.16 and dc = 1.171 for Z = 2. The final R = 0.068 for 2298 reflections. At. coordinates are given. The conformations of the urethane and peptide CONH groups are trans. The 2 Ac6c residues show ϕ,ψ sets of torsion angles both falling in the region of the conformational energy map where αand 310-helixes are found, but their handedness is opposite. The 2 cyclohexyl rings adopt a slightly distorted chair conformation with the NH group in the axial position. 85. Structural versatility of peptides from Cα,α-dialkylated glycines: an infrared absorption and proton NMR study of homopeptides from 1aminocyclopentane-1-carboxylic acid. Crisma, M.; Bonora, G. M.; Toniolo, C.; Benedetti, E.; Bavoso, A.; Di Blasio, B.; Pavone, V.; Pedone, C. International Journal of Biological Macromolecules (1988), 10(5), 300-4. Abstract: The conformational preferences of terminally-blocked homopeptides R-(Acc5)n-OCMe3 (R = PhCH2O2C, n = 1-6; R = 4BrC6H4CO, n = 4, 5; Acc5 = 1-aminocyclopentane-1-carboxylic acid residue) in CDCl3 were assessed by IR and 1H NMR as a function of concn., temp., and addn. of perturbing agents. The results show that an incipient 310-helix is first formed at the tripeptide level. A comparison is made with the 85 Prof. Vincenzo Pavone preferred conformation of homopeptides from the higher homolog 1aminocyclohexane-1-carboxylic acid and the open chain analog Cα,αdiethylglycine. 86. Structural versatility of peptides from Cα,α-dialkylated glycines: a conformational energy calculation and x-ray diffraction study of homopeptides from 1-aminocyclopentane-1-carboxylic acid. Santini, A.; Barone, V.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Fraternali, F.; Lelj, F.; Pavone, V.; Pedone, C.; et al. International Journal of Biological Macromolecules (1988), 10(5), 292-9. Abstract: Conformational energy calcns. on the 1-aminocyclopentane-1carboxylic acid monopeptide Ac-Acc5-NHMe indicate that this Cα,αdialkylated, cyclic α-amino acid residue is conformationally restricted and that its min. energy conformation falls in the α/310-helical region. The results of the theor. anal. are in agreement with the crystal-state structural tendency of R-(Acc5)n-OCMe3 (R = 4-BrC6H4CO, n = 4, 5; R = PhCH2O2C, n = 6), which were detd. by x-ray diffraction and which also form 310-helices. The implications for the use of Acc5 residues in designing conformationally constrained analogs of bioactive peptides are briefly discussed. 87. Platinum(II) complexes of amino acids and peptides. I. Structural analysis of trans-bis(L-alanine)dichloroplatinum. Pavone, Vincenzo; Lombardi, Angela; Di Blasio, Benedetto; Benedetti, Ettore; Pedone, Carlo. Inorganica Chimica Acta (1988), 153(3), 171-4. Abstract: trans-PtCl2(L-HAlaOH)2 (HAlaOH = alanine) was prepd. by a described literature procedure and was characterized by IR and 1H NMR and by x-ray crystallog. Crystals are orthorhombic, space group P21221, with a 7.460(1), b 8.544(1), c 9.754(1) .ANG., Z = 2, R = 0.038, and Rw = 0.050 for 681 reflections with I > 3.0σ(I). There is an extensive network of H bonds. The structure is mainly characterized by double layers, held together by H bonds, whereas the double layers interact with each other by van der Waals interactions. 88. Linear oligopeptides. Part CLXXII. Incorporation of a guest residue into a host (Aib)n homooligopeptide chain: conformational analysis in chloroform. Toniolo, Claudio; Bonora, Gian Maria; Formaggio, Fernando; Crisma, Marco; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Gazzetta Chimica Italiana (1988), 118(1), 4753. 86 Prof. Vincenzo Pavone Abstract: IR absorption spectra of R-(Aib)n-L-Leu-(Aib)2-OMe [I; R = PhCH2O2C (Z), Aib = α-aminoisobutyric acid, n = 0-5] in CDCl3 have been compared with those of the ref. homopeptide series Z-(Aaib)m-OMe (m = 3-8) in the amide-A and amide-I regions. In the former series, total development of the 310-helical structure seems to require, at least, the full length of the highest oligomer (8 residues), but IR spectra of a similar band shape with significant magnitude appear as early as n = 1, which is equiv. to two consecutive type-III β-bends. The detn. of this type of ordered conformation has been confirmed by 1H NMR. The incorporation of a leucine residue, albeit helicogenic, into a host (Aib)m chain tends to decrease the stability of the 310-helices. A comparison of I (n = 5, R = Z, Ac, 4-BrC6H4CO) indicates closely similar conformational preferences. 89. On β -hairpin classification. Pavone, Vincenzo. International Journal of Biological Macromolecules (1988), 10(4), 238-40. Abstract: In the present report, some ambiguities in naming and grouping the various β-hairpin conformational types of peptides and globular proteins are discussed. A new criterion to distinguish the residues in the loop from those in the β-ladder, based on different H-bond schemes, is presented. A more rational way of naming the residues in a β-hairpin, in terms of loop residues, allows an unambiguous assignment of different β-hairpins into 4 distinct classes, and a simple relationship existing between the members of the same class is reported. An alternative approach to identify the various βhairpin types, in terms of ring structure Cm (where m is the no. of atoms in the ring structure) of the loop part, also fulfills the requirement of a complete description, an unambiguous assignment, and grouping into 4 distinct classes. 90. Structural versatility of peptides from Cα,α-dialkylated glycines. II. An IR absorption and proton NMR study of homooligopeptides from Cα,αdiethylglycine. Toniolo, C.; Bonora, G. M.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Barone, V.; Lelj, F.; et al. Biopolymers (1988), 27(3), 373-9. Abstract: The conformational preferences of the N-trifluoroacetylated diethylglycine homopeptides F3CCO(NHCEt2CO)nOR (n = 1, R = H; n = 2-5, R = CMe3) in chloroform were detd. by IR and 1H NMR. Intramol. H bonding was found to be the dominant factor for all NH groups. The likely absence of a conformational transition upon increasing main-chain length, and the remarkable stability to diln., heating, and addn. of perturbing agents, are addnl. relevant findings of this study. These results are in 87 Prof. Vincenzo Pavone agreement with those of the fully extended, C5-conformation-forming homopeptides from the higher homolog Cα,α-dipropylglycine, but contrast dramatically to those of the homopeptides from the lower homol. Cα,αdimethylglycine, which have been shown to adopt the 310-helical structure. Ethylglycine homopeptide conformation IR NMR. 91. Structural versatility of peptides from Cα,α-dialkylated glycines. I. A conformational energy computation and x-ray diffraction study of homopeptides from Cα,α-diethylgylcine. Benedetti, E.; Barone, V.; Bavoso, A.; Di Blasio, B.; Lelj, F.; Pavone, V.; Pedone, C.; Bonora, G. M.; Toniolo, C.; et al. Biopolymers (1988), 27(3), 357-71. Abstract: Conformational energy computations on diethylglycine derivs. Ac(NHCEt2CO)nNHEt (n = 1, 2) were performed. In both cases, the Cα,αdiethylglycine residues are conformationally restricted and the min. energy conformation corresponds to the fully extended C5 structure when the NCα-C' bond angle is smaller than 108°, as is exptl. obsd. The results of the theor. anal. are in agreement with the crystal-state structural propensity of the complete series of N-trifluoroacetylated homopeptides CF3CO(NHCEt2CO)mOR (I; m = 1, R = H; m = 2-5, R = CMe3), as detd. by x-ray diffraction. A crystallog. planar peptide backbone was obsd. for I (m = 3, R = CMe3). A comparison with peptides Cα,α-dimethylglycine, CαMe,Cα-ethylglycine, and Cα,α-dipropylglycine indicates that the fully extended conformation becomes more stable than the helical structures when both amino acid side-chain Cβ atoms are substituted. 92. Structural studies of cyclopeptides. Solid state and solution conformation of cyclo(L-histidyl-D-histidyl). Benedetti, E.; Bavoso, A.; Di Blasio, B.; Pavone, V.; Pedone, C.; Paolillo, L.; D'Alagni, M. International Journal of Peptide & Protein Research (1988), 31(2), 220-4. Abstract: The solid-state and soln. conformations of cycle(L-His-D-His) were detd. by x-ray diffraction anal. and NMR spectroscopy, resp. The cyclic dipeptide retains in the solid-state a crystallog. center of symmetry: each mol. sitting on it presents a very flat chair conformation with alternating pos. and neg. conformational angles, whose values are in the range 5-6°. The imidazole groups of the side chains are planar, making an angle of 78° with the av. plane of the 2,5-piperazinedione ring. These arom. groups are folded back over the 2,5-piperazinedione ring and they sandwich it from opposite sides. Packing is obtained through an H-bonding scheme which involves the N-H donors of the peptide groups as well as the N-H groups of the imidazole moieties. In water soln., at pHs equal to 2.55 and 8, 88 Prof. Vincenzo Pavone the mol. retains on the av. an element of symmetry since only one set of signals is obsd. for the His protons in the NMR spectrum. From coupling consts., the predominant conformation was calcd. to be all gauche in excellent agreement with the solid-state results. 93. Long, chiral polypeptide 310-helixes at atomic resolution. Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria; Formaggio, Fernando; Crisma, Marco. Journal of Biomolecular Structure & Dynamics (1988), 5(4), 803-17. Abstract: The crystal-state preferred conformation of the terminally blocked hepta- and octapeptides with the general formula -(Aib)n-L-Leu-(Aib)2(where Aib = α-aminoisobutyric acid and Leu = leucine) (n = 4 and 5, resp.), detd. by x-ray diffraction was found to be right-handed 310-helix stabilized by 5 and 6 consecutive intramol. NH⋅⋅⋅O:C H-bonds of the C10-III type, resp. The octapeptide structure represents the first observation at at. resoln. of a regular, chiral 310-helix larger than 2 complete turns. In both cases the right-handed screw sense of the helix is dictated by the presence of the single, internal L-residue. This study confirms the propensity of short peptides rich in Aib, the prototype of the amino acid residues dialkylated at the α C, to adopt a 310-helical structure and is expected to help the understanding of the conformational preferences of the membrane-active, channel-forming, ion-transporting peptaibol antibiotics. 94. Structural versatility of peptides from Cα,α-dialkylated glycines. An infrared absorption and 1H nuclear magnetic resonance study of homopeptides from 1-aminocyclohexane-1-carboxylic acid1. Crisma, M.; Bonora, G. M.; Toniolo, C.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C. Macromolecules (1988), 21(7), 2071-4. Abstract: The preferred conformation of an N- and C-protected 1aminocyclohexane-1-carboxylic acid homopeptide series, from monomer to pentamer, in chloroform soln. was detd. by IR and 1H NMR as a function of concn., temp., and addn. of perturbing agents. The results are in favor of the onset of an incipent 310 helix at the tripeptide level, as found in the crystal state. A comparison is also made with the conformational propensities of homopeptides of 1-aminocyclopentane-1-carboxylic acid and the Cα,αdialkylated glycyl residues with linear side chains. 95. Structural versatility of peptides from Cα,α-dialkylated glycines. A conformational energy computation and x-ray diffraction study of homopeptides from 1-aminocyclohexane-1-carboxylic acid1. Pavone, V.; Benedetti, E.; Barone, V.; Di Blasio, B.; Lelj, F.; Pedone, C.; Santini, A.; 89 Prof. Vincenzo Pavone Crisma, M.; Bonora, G. M.; Toniolo, C. Macromolecules (1988), 21(7), 206470. Abstract: Conformational energy calcns. on Ac-Acc6-NHMe (Acc6 = 1aminocyclohexane-1-carboxylic acid residue) indicated that the Acc6 residue is conformationally restricted and that its min. energy conformation falls in the α/310 helical region, irresp. of the position (either axial or equatorial) or the α-amino group. The results of the theor. anal. are in agreement with the crystal-state structural tendency of PhCH2O2C-(Acc6)4-OCMe3 and pBrC6H4CO-(Acc6)4-OCMe3. The implications for the use of Acc6 residues in designing conformationally constrained analogs of bioactive peptides are briefly discussed. 96. Long, chiral polypeptide 310-helixes. Crisma, M.; Formaggio, F.; Bonora, G. M.; Toniolo, C.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C. Protides of the Biological Fluids (1987), 35 465-8. Abstract: The IR absorption and 1H NMR analyses of well-characterized, monodispersed, terminally blocked -(Aib)n-L-Leu-(Aib)2- (n = 0-5) peptides are strongly in favor of the full development of a stable 310-helix for the highest oligomer in CDCl3 soln. The x-ray structures of the hepta- and octapeptides (n = 4 and 5, resp.) indicate the formation of right-handed 310helixes in the crystal state. The octapeptide structure represents the 1st observation at at. resoln. of a regular, chiral 310-helix longer than 2 complete turns. 97. Conformations of bioactive peptides: cycloinopeptide a. Di Blasio, B.; Benedetti, E.; Pavone, V.; Pedone, C.; Goodman, M. Biopolymers (1987), 26(12), 2099-101. Abstract: The results of the structure detn. of cyclolinopeptide A in the solid state by x-ray diffraction are briefly reported. 98. Stereostructure and formation mechanism of a new substituted benzofuran from phomenone. Capasso, Renato; Palumbo, Giovanni; Randazzo, Giacomino; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo. Tetrahedron (1986), 42(16), 4493-8. Abstract: Treating phomenone (I), a known phytotoxic and mycotoxic sesquiterpene, with H2SO4-MeOH 15 min at room temp. gave 65% of a new substituted benzofuran II, whose structure was confirmed by spectral and xray anal. 90 Prof. Vincenzo Pavone OH Me O O CH 2 OH HO Me 99. Me O CH 2 Me I CH 2 OMe II Linear oligopeptides. Part 157. A novel peptide conformation: first unequivocal observation of the oxy-analog of a β -bend. Toniolo, Claudio; Valle, Giovanni; Bonora, Gian Maria; Crisma, Marco; Formaggio, Fernando; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Biopolymers (1986), 25(12), 2237-53. Abstract: The x-ray diffraction anal. of Z-(Aib)3-OH (I; Z = PhCH2O2C, Aib = NHCMe2CO) showed the occurrence of an incipient 310-helix characterized by one type-III (or type-III') β-bend followed by one oxyanalog of the same type of β-bend. This represents the first unequivocal observation of the latter conformation, where the OH group of the CO2H moiety present at the C-terminus of the peptide main chain plays the role of the hydrogen-bonding donor. These results have been compared with those of I⋅H2O and Z-(Aib)3-OMe. 100. Isolation and structure determination of norsphaerol, a bis-nor-diterpene from the red alga Sphaerococcus coronopifolius. Bavoso, Alfonso; Cafieri, Francesco; De Napoli, Lorenzo; Di Blasio, Benedetto; Fattorusso, Ernesto; Pavone, Vincenzo; Santacroce, Ciro. Gazzetta Chimica Italiana (1987), 117(2), 87-9. Abstract: On the basis of spectroscopic evidence structure (I) is proposed for norsphaerol, a bromo-bis-nor-diterpene isolated from the marine alga S. coronopifolius. Final proof of the correctness of this formula and the detn. of the relative stereochem. has been provided by x-ray crystallog. anal. performed on a single crystal of I. H H Me 2 C H 101. Me H Br I Linear oligopeptides. Part 147. Chemical and crystallographic study of the reaction between benzyloxycarbonyl chloride and α-aminoisobutyric acid. Valle, Giovanni; Formaggio, Fernando; Crisma, Marco; Bonora, Gian Maria; Toniolo, Claudio; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Journal of the Chemical 91 Prof. Vincenzo Pavone Society, Perkin Transactions 2: Physical Organic Chemistry (1972-1999) (1986), (9), 1371-6. Abstract: The reaction of PhCH2O2CCl (ZCl) with α-aminoisobutyric acid (Aib) gave 3 cryst. products, which were characterized by x-ray diffraction. Two of the products were the α- and β-forms of Z-Aib-OH (67 and 27% yield, resp.), which differ in the orientation of the Ph ring relative to the urethane moiety and in the packing modes. of the mols., including the intramol. H-bonding arrangements. The third product was Z-Aib-Aib-OH (1.5% yield). The crystal structure of (Z-Aib)2O was also detd. 102. Cyclic peptide metal salt adducts. II. Crystal structure of the silver nitrate cyclosarcosylsarcosine 2:1 adduct. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Rossi, Filomena. Inorganica Chimica Acta (1986), 116(1), 31-5. Abstract: The title adduct is triclinic, space group P1, with a 5.410(4), b 7.562(4), c 8.020(6) .ANG., α 92.06(4), β 105.07(4), and γ 104.60(4)°; dm = 2.62(1) and dc = 2.625 for Z = 2. The final R = 0.058. The Ag ion directly interacts with the carbonyl O atoms of the peptide moiety. The independent unit is composed of a half cyclosarcosylsarcosine mol., which sits on a crystallog. center of symmetry, per each AgNO3 unit. The crystal is held together by strong coulombic interactions between the Ag and the NO3 ions and by ion-dipole interactions between the Ag ion and the org. mol. The coordination at the Ag ion cannot be described in terms of a regular geometry; each Ag ion experiences different types of contacts with the surrounding O atoms. Six Ag-O interactions are 2.35-2.68 .ANG.; a 7th Ag-O interaction presents a distance of 2.90 .ANG.. This latter contact is perhaps the cause of the severe distortion from the ideal octahedral geometry obsd. exptl. The NO3 ion and the cyclic peptide mol. are both nearly planar. At. coordinates are given. 103. Cyclic peptide-metal salt adducts. I. Crystal structure of the hexaquocopper(II) perchlorate cyclosarcosylsarcosine 1:2 adducts. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Inorganica Chimica Acta (1986), 123(3), 155-9. Abstract: The title adduct is monoclinic, space group P21/c, with a 13.879(7), b 14.504(7), c 13.083(8) .ANG., and β 90.98(10)°; dm = 1.65 and dc = 1.652 for Z = 4. The final R = 0.083. At. coordinates are given. The independent unit is composed of 6 H2O mols. octahedrally coordinated to the Cu(II) ion, 2 tetrahedral ClO4- ions and 4 independent halves of cyclosarcosylsarcosine mols. lying on crystallog. centers of symmetry. All available H atoms of 92 Prof. Vincenzo Pavone H2O mols. are involved in H bonding as donors and all carbonyl O atoms of the cyclic peptide mols. function as acceptors. Two other O atoms for each ClO4- participate in the H bonding scheme, which leads to the absence of the orientational disorder usually obsd. in these ions. Layers of inorg. material and layers of org. material, roughly parallel to the ab plane, pack alternatively with each other. Electrostatic and ion-dipole interactions together with H bonds are responsible for the building up of the crystals. 104. Linear oligopeptides. CXLII. Molecular structure of peptaibol antibiotics: solution conformation and crystal structure of the octapeptide corresponding to the 2-9 sequence of emerimicins III and IV. Toniolo, Claudio; Bonora, Gian Maria; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Journal of Biomolecular Structure & Dynamics (1985), 3(3), 585-98. Abstract: The IR absorption and 1H NMR of title peptide sequence pBrC6H4CO-(Aib)3-Val-Gly-Leu-(Aib)2-OMe (I, Aib = NHCMe2CO) in CHCl3 indicated a 310-helical structure of high thermal stability. The crystal structure of I indicated the formation of a right-handed 310-helix stabilized by 6 consecutive intramol. NH...OC H bonds, slightly distorted at the level of the Leu residue. 105. Long polypeptide 310-helixes at atomic resolution. Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Proceedings of the National Academy of Sciences of the United States of America (1986), 83(7), 1988-92. Abstract: The crystal-state preferred conformation of the terminally blocked homooctapeptide of Cα,α-dimethylated α-aminoisobutyric acid (Aib), pBrBz(Aib)8-OBut (where pBrBz is p-bromobenzoyl and OBut is tertbutoxy), detd. by x-ray diffraction anal. using direct methods, was a 310helix stabilized by 6 consecutive intramol. N-H⋅⋅⋅O=C H bonds of the C10III (or III') type. This is the 1st observation at at. resoln. of a regular 310helix longer than 2 complete turns. The solid-state structural anal. was extended to the terminally blocked, Aib-rich octapeptide corresponding to the 2-9 sequence of the peptaibol antibiotics emerimicins III and IV, pBrBzAib3-L-Val-Gly-L-Leu-Aib2-OMe. Again, this peptide adopts a (righthanded) 310-helical structure, although slightly distorted at the level of the L-leucine residue. The role of specific amino acid sequence and peptide main-chain length in stabilizing either the 310- or the α-helical conformation and their possible implications on the nature of the channel formed by peptaibol antibiotics in the membrane are also briefly discussed. 93 Prof. Vincenzo Pavone 106. Linear oligopeptides: peptaibol antibiotics - preferred conformation of the 29 segment of emerimicins III and IV and all related short sequences. Toniolo, Claudio; Bonora, Gian Maria; Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. International Journal of Biological Macromolecules (1985), 7(6), 357-62. Abstract: With the aim of obtaining information on the effect induced by main-chain length and amino acid sequence on the type of helical structure adopted by naturally occurring peptides rich in Cα,α-dialkylated residues, an IR absorption and 1H NMR anal. of CHCl3 solns. of the protected 2-9 segment of the peptaibol antibiotics emerimicins III and IV (-(Aib)3-L-ValGly-L-Leu-(Aib)2-) (Aib = aminoisobutyrate) and all related short sequences starting from both the N- and C-termini was performed. The results are consistent with the presence of folded structures of the β-bend type (in the shorter peptides) or 310-helixes (in the longer peptides). Extent of formation and stability of the inter- and intramol. H bonds were assessed as a function of concn., temp., and addn. of DMSO and the free radical Tempo. At high peptide concn. both folded and helical structures tend to self-assoc. extensively. In the self-assocn. process, the N(1)H and N(2)H groups are those acting as H-bonding donors. These results agree well with those obtained in the solid state by x-ray diffraction on the octapeptide itself and selected short sequences. 107. A long, regular polypeptide 310-helix. Toniolo, C.; Bonora, G. M.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C. Macromolecules (1986), 19(2), 472-9. Abstract: The IR absorption and 1H NMR spectral analyses of CHCl3 solns. of the terminally blocked homooctapeptide from the Cα,α-dimethylated αaminoisobutyric acid residue were consistent with the presence of a 310helical structure of high thermal stability. The crystal structure of the octapeptide, obtained by x-ray diffraction, indicated the formation of a 310helix, stabilized by 6 consecutive intramol. N-H⋅⋅⋅O:C H-bonds of the C10III (or -III') type. This represents the 1st observation at at. resoln. of a regular 310-helix larger than 2 complete turns. The packing of the octapeptide mols. gives rise to a channel in which the solvent (MeOH and H2O) mols. are accommodated. 108. Conformational behavior of α,α-dialkylated peptides. Barone, Vincenzo; Lelj, Francesco; Bavoso, Alfonso; Di Blasio, Benedetto; Grimaldi, Patrizio; Pavone, Vincenzo; Pedone, Carlo. Biopolymers (1985), 24(9), 1759-67. Abstract: The preferred conformations of N-acetyl-N'-Me amides of some 94 Prof. Vincenzo Pavone dialkylglycines were detd. by empirical conformational-energy calcns.; min.-energy conformations were located by minimizing the energy with respect to all the dihedral angles of the mols. The conformational space of these compds. is sterically restricted, and low-energy conformations are found only in the regions of fully extended and helical structures. On increasing the bulkiness of the substituents on the Cα atom, the fully extended conformation becomes gradually more stable than the helical structure preferred in the cases of dimethylglycine. This trend is, however, strongly dependent on the bond angles between the substituents on the Cα atom: In particular, helical structures are favored by std. values (111°) of the N-Cα-C' angle, whereas fully extended conformations are favored by smaller values of the same angle, as exptl. obsd., for instance, in the case of α,α-di-n-propylglycine. 109. Linear oligopeptides - effect of lengthening of the main chain by one tetrahedral carbon atom in the Aib-L-Ala- sequence: a solid-state conformational analysis of segments of polypeptide antibiotics. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Grimaldi, Patrizio; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. International Journal of Biological Macromolecules (1985), 7(2), 81-8. Abstract: The IR and x-ray diffraction conformational anal. of Boc-Aib-LAla-Aib-OMe (Boc = Me3CO2C, Aib = NHCMe2CO) showed the presence of the type-I 4 → 1 intramolecularly H-bonded peptide conformation (βbend) in the solid state. Lengthening the chain by one tetrahedral carbon atom, as in Boc-Aib-β-Ala-Aib-OMe, has a disruptive effect on this folded structure. Two water mols. co-crystallize with each mol. of the L-Ala contg. tripeptide. These results are discussed in comparison with those, previously reported, obtained in chloroform soln. 110. Dipeptides as inhibitors of the gelation of sickle hemoglobin. Noguchi, Constance Tom; Luskey, Kenneth L.; Pavone, Vincenzo. Molecular Pharmacology (1985), 28(1), 40-4. Abstract: To examine in detail a class of peptides that inhibit the polymn. of deoxygenated Hb S [9035-22-7], L-amino acids and 22 dipeptides were assayed for their effect on deoxyHb S soly. Of the amino acids, the aroms. (phenylalanine [63-91-2], tyrosine [60-18-4], and tryptophan [73-22-3]) increased deoxyHb S soly., as did high concns. of arginine [74-79-3]. Combinations of the hydrophobic (specifically the arom.) amino acids with a hydrophilic amino acid, such as arginine or lysine [56-87-1], resulted in dipeptides which were much more sol. than the hydrophobic or arom. amino acid alone, and also inhibited polymn. Furthermore, samples of 95 Prof. Vincenzo Pavone deoxyHb S at 26 to 27 g/dL contg. some of these dipeptides such as Arg-Trp [25615-38-7], Arg-Phe [2047-13-4], and Lys-Trp [50674-18-5] in excess of 50 to 100 mM did not polymerize, indicating a 1.4- to 1.6-fold increase in deoxyHb S soly. The enhancement of polymn., i.e., decrease in deoxyHb S soly., obsd. by the addn. of aspartic acid [56-84-8], glycine [56-40-6], or lysine was obsd. or was reduced in the dipeptides contg. these hydrophilic amino acids combined with hydrophobic amino acids. The effects of these dipeptides on deoxyHb S soly. were mostly linear with concn. However, the changes in deoxyHb S soly. by addn. of a dipeptide was not simply the sum of the effects obsd. with the individual amino acids as exemplified by the differential effect of reversing the dipeptide sequence. These data provide further evidence as to the stereospecific nature of this class of noncovalent inhibitors of deoxyHb S polymn. 111. Conformation of pleionomers of α-aminoisobutyric acid. Toniolo, Claudio; Bonora, Gian Maria; Barone, Vincenzo; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Grimaldi, Patrizio; Lelj, Francesco; Pavone, Vincenzo; Pedone, Carlo. Macromolecules (1985), 18(5), 895-902. Abstract: The IR spectra and 1H NMR of the peptides PhCH2OCO[NHC(Me)2CO]nOBu-tert (n = 1-12) indicates the formation of fully developed, stable 310 helices for those with n = 8-12 (pleionomers). Spectra were studied in the solid state, and in CDCl3 as a function of concn., temp., and addn. of DMSO and the radical 2,2,6,6tetramethylpiperidinyl-1-oxyl. The mode of self-assocn. of the helical structures was detd. A theor. study of AcNMC(Me)2COHNMe [42037-263] by conformational energy calcn. indicates that the fully extended structure is less stable than the helical structures, regardless of the size of the N-Cα-C' valence angle. 112. Structure of dichloro[1,2-ethanedione bis(dimethylhydrazone)](ηethylene)platinum(II), [PtCl2(C2H4)(C6H14N4)]. Bavoso, A.; Funicello, M.; Morelli, G.; Pavone, V. Acta Crystallographica, Section C: Crystal Structure Communications (1984), C40(12), 2035-7. Abstract: The title compd., is monoclinic, space group P21, with a 8.998(3), b 8.133(4), c 9.872(2) .ANG., and β 106.72(3)°; dm = 2.09 and dc = 2.094 for R = 0.050 and Rw = 0.057 for 1404 reflections. At. coordinates are given. The compd. has a trigonal-bipyramidal structure with the Cl atoms in apical positions and the bidentate ligand (which coordinates through its α-diimine units) and ethylene in the equatorial plane. The bidentate ligand is in a cis conformation with some distortion from planarity. The Pt-Cl and Pt-olefin bond distances are usual for 5-coordinate complexes of PtII. 96 Prof. Vincenzo Pavone 113. Folded and extended structures of homooligopeptides from α,α-dialkylated glycines. A conformational energy computation and x-ray diffraction study. Benedetti, Ettore; Toniolo, Claudio; Hardy, Paul; Barone, Vincenzo; Bavoso, Alfonso; Di Blasio, Benedetto; Grimaldi, Patrizio; Lelj, Franceso; Pavone, Vincenzo; et al. Journal of the American Chemical Society (1984), 106(26), 8146-52. Abstract: Conformational energies were calcd. for Ac-(NHCPr2CO)nNHMe (I, n = 1, 2) and Ac-D-Iva-NHMe (II, Iva = isovaline residue) and the results were compared to literature data for AcNHCMe2CONHMe (III). The min. energy conformations of I correspond to fully extended conformations, and a comparison with II and III indicated that the preference from a folded to a fully extended conformation increases with increasing bulkiness of the Cα-substituents. Theor. results agreed with conformational properties in the solid state as detd. by x-ray diffraction. 114. Folded and extended structures of homooligopeptides from α,α-dialkylated α-amino acids. An infrared absorption and proton nuclear magnetic resonance study. Bonora, Gian Maria; Toniolo, Claudio; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Benedetti, Ettore; Lingham, Ian; Hardy, Paul. Journal of the American Chemical Society (1984), 106(26), 8152-6. Abstract: The conformational preferences of Tfa-(NHCPr2CO)n-OCMe3 (Tfa = CF3CO, n = 2-5) in the solid state and in CHCl3 soln. were assessed by IR and 1H NMR. A comparison is made with the conformations adopted by the corresponding series from α-aminoisobutyric acid, also dialkylated at the α-carbon, and from L-norvaline, in which the single alkyl side chain is the same as those in α,α-di-n-propylglycine. The highest L-norvaline homopeptides exhibit a significant tendency for adopting an intermolecularly H-bonded β-structure, in contrast to the α,α-di-npropylglycine and α-aminoisobutyric acid peptides where intramol. Hbonding is the dominating factor. The likely absence of a conformational transition with increasing chain length and the exceptional structural stability upon heating of all the α,α-di-n-propylglycine homopeptides represent an addnl. relevant finding. 115. Modified calmodulin calcium binding domain III. Solid phase synthesis, purification and proton NMR characterization. Pavone, Vincenzo; Di Nola, Angela; Andini, Salvatore; Ferrara, Luciano; Di Blasio, Benedetto; Benedetti, Ettore; Pucci, Piero. International Journal of Peptide & Protein 97 Prof. Vincenzo Pavone Research (1984), 23(5), 454-61. Abstract: Title dodecapeptide Ac-Asp-Lys-Asp-Gly-Asn-Gly-Tyr-Ile-SerAla-Ala-Gaba-OH [I, Gaba = NH(CH2)3CO] was prepd. by the solid phase method with a total protection scheme using PAM-resin. Purified I was characterized by 1H NMR spectroscopy, both in the presence and in the absence of calcium ions at various pH values. No strong specific interaction occurred between I and Ca2+ in aq. solns. 116. Studies on gliadin related peptides. I. Synthesis, purification and proton NMR characterization of the pentapeptide H-Tyr-(Gln)3-Pro-OH. Pavone, V.; Rossi, F.; Pucci, P.; Andini, S.; Ferrara, L.; Di Blasio, B.; Pedone, C. International Journal of Peptide & Protein Research (1983), 22(4), 482-8. Abstract: The title pentapeptide (I) was prepd. by the solid-phase method on the PAM-resin. I was characterized by its NMR. I has been postulated as the basic repetitive unit of a sequential polypeptide contained in wheat bread α-gliadins, which could be the toxic factors in coeliac disease. 117. Protected 1-3 segment of the peptaibol antibiotics alamethicin and hypelcin. Solid-state and solution study of a stereochemically constrained linear peptide. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria; Crisma, Marco. International Journal of Peptide & Protein Research (1983), 22(4), 385-97. Abstract: Title peptide PhCH2O2C-Aib-Pro-OMe (Aib = NHCMe2CO) was prepd. and its modes of folding and self-assocn. in the solid state were detd. using IR absorption and x-ray diffraction. Stereochem.-constrained tripeptide mols. adopt a 4 → 1 intramol. H-bonded form (β-turn), where the single intramol. H bond is found between the peptide NH group of the Aib3 residue and the urethane CO group of the N-blocking benzyloxycarbonyl moiety. This folded structure is stabilized by an intermol. H bond between the urethane NH group of the Aib1 residue and the peptide CO group of the Pro2 residue of a symmetry related mol. In CH2Cl2 and TMP solns. the same intramol. H bonded form occurs as that found in solid state. Compared to the solid state, CH2Cl2 and TMP solvation of the urethane NH group replaces self-assocn. (through the same NH group). The results are discussed in relation to those obtained for other protected -Aib-X-Aib(X = Aib, Ala, Val) tripeptide segments of peptaibol antibiotics. 118. Bioorganic stereochemistry. A study of the peptide oxazolones from Z(Aib)n-OH (n = 2-4) in the solid state. Toniolo, Claudio; Bonora, Gian Maria; Crisma, Marco; Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, 98 Prof. Vincenzo Pavone Benedetto; Pavone, Vincenzo; Pedone, Carlo. International Journal of Peptide & Protein Research (1983), 22(5), 603-10. Abstract: The conformations and modes of self-assocn. of title oxazolones (Z = PhCH2O2C, Aib = NHCMe2CO) in the solid state were detd. by IR spectroscopy. The structure of oxazolone from Z-(-Aib-)3-OH was obtained by x-ray diffraction. In this compd. the conformations of the first two Aib residues differ substantially, only the N-terminal one was in the usual 310 (or α) helical region of the Ramachandran map. The C:N bond of the oxazolone group is not conjugated with the lactone moiety. A very weak intermol. interaction occurs between the urethane NH and the carbonyl group of the oxazolone ring. 119. Structure of N-tert-butyloxycarbonyl-D-leucyl-Lphenylalanylethanolamide. An Nα-protected analog of the carboxy-terminal dipeptide of linear gramicidins. Bonora, Gian Maria; Toniolo, Claudio; Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Journal of Biological Chemistry (1983), 258(23), 14725-32. Abstract: Solid state conformational anal. of N-tert-butyloxycarbonyl-Dleucyl-L-phenylalanylethanolamide (I) an Nα-protected analog of the Cterminal dipeptide of linear gramicidins, carried out by x-ray diffraction, has indicated that the mols. are characterized by an N-H...O:C intramolecularly H-bonded chain reversal of the β-turn II' type. One of the 2 independent mols. in the asym. unit shows an addnl. intramol. H bond of the O-H...O:C type, linking the hydroxyl function of the C-terminal ethanolamide moiety to the carbonyl O of the urethane N-protecting group. This is the 1st exptl. evidence for a β-turn conformation fused with the oxy analog of an α-turn. The results of an investigation in a solvent of low polarity (deuteriochloroform), using IR absorption and 1H NMR strongly support the view that an intramolecularly H-bonded β-turn conformation is the most populated conformation of I mols. at high diln. In the self-assocn. process, taking place at high concn., the urethane and peptide NH groups are involved as H-bonding pairs. 120. Linear oligopeptides. 90. Peptaibol antibiotics: conformational preferences of synthetic emerimicin fragments. Toniolo, Claudio; Bonora, Gian Maria; Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Biopolymers (1983), 22(5), 1335-56. Abstract: The IR absorption and CD conformational analyses of solns. of the protected 2-9 fragment of the peptaibol antibiotics emerimicins III and IV, -(Aib)3-Val-Gly-(Aib)2- (Aib = NHCMe2CO), and related short 99 Prof. Vincenzo Pavone sequences are consistent with the presence of a right-handed α-helix for the octapeptide, while the tri-, tetra-, and pentapeptides adopt a 310-helix, either right- or left-handed, depending on the amino acid sequences. The structural preferences of solid-state Z(Aib)-Val-OMe (Z = PhCH2O2C) and Z(Aib)-Val-Gly-OMe were detd. by x-ray diffraction. In accord with the soln. data. incipient 310-helices, formed by 2 and 3 β-turns, were found for the tetra- and pentapeptides, resp. The tetrapeptide helix has the lefthanded screw sense, while that of the pentapeptide is right-handed, thus confirming the conclusions of the CD anal. in soln. 121. First observation of a β -turn conformation fused with the oxy-analog of an α -turn: the molecular structure of a model peptide of the C-terminal part of gramicidin A. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Biochemical and Biophysical Research Communications (1983), 112(3), 1056-60. Abstract: The mol. structure of Me3CO2C-D-Leu-Phe-NHCH2CH2OH, a protected analog of the C-terminal dipeptide of gramicidin A, was detd. by x-ray diffraction. One of the two independent mols. in the asym. unit is characterized by a chain reversal stabilized by intramol., three-center, double hydrogen bonding. This is the first exptl. evidence for a β-turn conformation fused with the oxy-analog of an α-turn. 122. Structure and conformation of regularly alternating LD linear peptides. Pedone, Carlo; Benedetti, Ettore; Di Blasio, Benedetto; Mattia, Carlo Andrea; Morelli, Giancarlo; Pavone, Vincenzo; Lorenzi, Gian Paolo; Gerher, Christofer. Biopolymers (1983), 22(1), 323-5. Abstract: Boc(L-Val-D-Val)4OMe (Boc = Me3CO2C) was shown by x-ray anal. to have a dimeric left-handed β-structure stabilized by 14 interstrand H bonds. Boc(D-Phe-L-Phe)4OMe has a similar structure. Boc(D-allo-IleL-Ile)OMe has a chain-reversed structure, stabilized by multiple intramol. H bonds, in which a β-turn is fused to an α-turn. 123. Solid-state geometry and conformation of linear, diastereoisomeric oligoprolines. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Biopolymers (1983), 22(1), 305-17. Abstract: The solid-state conformation preferences of a no. of linear homooligoprolines (to the tetramer) were detd. by anal. of IR and x-ray data. The peptides present different chiral sequences (tacticities), various types (urethane and amide) of N-protecting groups, and free and 100 Prof. Vincenzo Pavone blocked C-termini (which imply different capabilities of forming H-bonds). The following conclusions can be drawn: (1) values for the geometry of the prolyl residue and the peptide bond in the cis and in the trans conformations are proposed; (2) in general the conformational angles φ and ψ in the linear homooligoprolines have values appropriate for the polyproline II structure; (3) the pyrrolidine ring shows various types of puckering with no apparent relation to the backbone conformation; (4) ProPro peptide bonds generally take the trans conformation, the few cases of cis conformation being formed by Pro residues of different chirality; (5) the single H-bond donor OH, when present, is always bonded to H-acceptors, which can be either the urethane or the amide or the peptide carbonyl but never the carbonyl group of the CO2H moiety. 124. Linear oligopeptide. Part 87. Conformation of linear homooligoprolines. VII. N-Pivaloyl-L-prolyl-D-proline methyl ester in the solid state. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. International Journal of Peptide & Protein Research (1982), 20(4), 312-19. Abstract: A conformational anal. of Me3CCO-L-Pro-D-Pro-OMe was performed in the solid state using IR absorption and x-ray diffraction. The tertiary amide bond is in the trans conformation, whereas the tertiary peptide bond is in the cis conformation. The preferred conformations of the pivaloylamino group, the pyrrolidine rings, and the ester moiety are also discussed. 125. Peptaibol antibiotics: a study on the helical structure of the 2-9 sequence of emerimicins III and IV. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Proceedings of the National Academy of Sciences of the United States of America (1982), 79(24), 7951-4. Abstract: Soln. conformations of the protected 2-9 segment of the peptaibol antibiotics emerimicins III and IV [α-aminoisobutyric acid (Aib)]3-L-ValGly-L-Leu-(Aib)2 and the related short sequences benzyloxycarbonyl(Aib)3-L-Val-OMe and benzyloxycarbonyl-(Aib)3-L-Val-Gly-OMe have been investigated by CD studies. For the latter 2 compds. the structural preferences in the solid state have been assayed by x-ray diffraction analyses. These data and those previously reported support the view that the shortest Aib-contg. segments (from tri- through pentapeptides) adopt the 310-helical structure both in soln. and in the solid state. In contrast, the octapeptide appears to adopt the α-helical structure in soln. The role of peptide chain length and specific amino acid sequences in stabilizing either 101 Prof. Vincenzo Pavone of the 2 helical structures and, hence, their possible implications on the nature of the channel formed by peptaibol antibiotics in the membrane are also briefly outlined. 126. A hairpin-shaped peptide conformation stabilized by multiple intramolecular hydrogen bonds for a linear alternating D,L hexapeptide. Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Lorenzi, Gian Paolo; Muri-Valle, Valentina. Biochemical and Biophysical Research Communications (1982), 107(3), 910-13. Abstract: A linear hexapeptide with alternating D,L residues, butoxycarbonyl-(D-alloIle--L-Ile)3-OMe (alloIle = alloisoleucine), which in the solid state give rise to a chain reversal stabilized by multiple intramol. H bonds, was conformationally characterized by x-ray anal. This is the 1st description of a β-turn (C10) and an α-turn (C13) fused together and included in a larger 17-membered ring occurring in a linear peptide. 127. Linear oligopeptides. 81. Solid-state and solution conformation of homooligo(α-aminoisobutyric acids) from tripeptide to pentapeptide: evidence for a 310 helix. Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Crisma, Marco; Bonora, Gian Maria; Toniolo, Claudio. Journal of the American Chemical Society (1982), 104(9), 2437-44. Abstract: Anal. of the x-ray diffraction and IR absorption data for PhCH2O2C(NHCMe2CO)nOR (I; R = CMe3, n = 3, 5; R = H, n = 4) showed the occurrence of incipient 310 helices formed by type III (or type III') β-turns. The intramol. NH to CO H-bonding schemes of I (n = 4, 5) are not compatible with α-helical structures. The IR of I in solvents of low polarity indicated the occurrence of the same intramol. H-bonded forms as found in the solid state. These structures undergo aggregation at high concns. The 310-helical structure must be considered when developing a model of folding for alamethicin. 128. Linear oligopeptides. Part 85. Preferred conformations of linear homooligoprolines. N-tert-butyloxycarbonyl-D-prolyl-D-prolyl-L-proline. Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Macromolecules (1982), 15(1), 54-9. Abstract: The title peptide (I) was prepd. and its solid-state and soln. conformations were detd. by x-ray diffraction and IR absorption. In the solid state, the mols. of I do not form an intramol. H-bonded oxy-C10 102 Prof. Vincenzo Pavone peptide conformation, but they are held together by intermol. H bonds between the peptide carbonyl of D-Pro1-D-Pro3 and the carboxyl OH. For concn. solns. of I in solvents of low polarity, the type of assocn. is different than that in the solid state, whereas in dil. solns. there is no onset of intramol. H bonding. In solvents with strong H-bonding acceptor and donor properties, only solvated species of I exist. 129. Structure of N-acetyl-D-alanyl-D-alanine hydrate. An analog of the carboxy-terminal segment of peptidoglycan of bacterial cell walls. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Journal of Biological Chemistry (1981), 256(17), 9229-34. Abstract: The solid state conformational anal. of Ac-D-Ala-D-AlaOH.H2O, carried out by IR absorption and x-ray diffraction, has indicated that the mols. are not extended in a regular β conformation, but rather that they are partially folded, the ϕ,ψ* torsional angles of the C-terminal residue in particular being in the region of the left-handed α helix of the Ramachandran map. The acetylamino and peptide groups are found in the usual trans conformation, the latter, however, exhibiting a deviation from rigid planarity. Only intermol. H bonds occur in the crystal state. The soln. conformational anal., performed by IR absorption and CD, has revealed that the amt. of intramol. N-H...O:C hydrogen-bonded folded forms, if any, should be extremely small, even in C2HCl3 at high diln. In water, solvated, unordered species largely predominate. 130. Self-association of N-protected α-amino acids. Optically active and racemic N-tert-butyloxycarbonyl-alanine. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Biopolymers (1981), 20(8), 1635-49. Abstract: The IR absorption and x-ray diffraction anal. of N-tertbutyloxycarbonyl-DL-alanine (t-Boc-DL-Ala-OH) in the solid state has revealed a new mode of self-assocn. for a N-urethanyl-α-amino acid, i.e., ribbons of H-bonded cyclic dimers formed through the COOH groups. In contrast to the racemate, a water mol., incorporated into the crystal of the chiral t-Boc-D-Ala-OH, alters in part that H-bonding scheme. In the 2 independent mols. of the unit cell of the optically active alanine deriv., as in that of the racemic deriv.: (1) the conformation of the CONH group is trans, and (2) the overall conformation is quasi-extended. These findings exclude the occurrence of an oxy-C7 peptide conformation. In solvents of high polarity, strongly solvated species predominate, as shown by IR absorption spectroscopy. In CDCl3 nonassocd. and assocd. species occur 103 Prof. Vincenzo Pavone simultaneously. No differences were obsd. between the optically active and racemic derivs. The type of self-assocn. near satn. seems to differ, at least in part, from that found in the anhyd. racemic compd. in the crystal state. 131. Conformational studies of peptides: crystal and molecular structures of L3,4-dehydroproline and its tert-butoxycarbonyl and acetyl amide derivatives. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Felix, Arthur; Goodman, Murray. Biopolymers (1981), 20(2), 283-302. Abstract: The crystal structure of the title compds. were detd. by x-ray data; 3,4-dehydroproline exists as the zwitterion in the cryst. state. The conformations of the 2 amide derivs. have 2 planes: one for the primary amide and the other for the remaining atoms of the mol. Acetyl-3,4dehydroproline amide contains a tertiary amide bond in the cis conformation. Both amide derivs. have φ and ψ values corresponding to a collagen-like conformation. 132. Linear oligopeptides. 65. Conformational analysis of the N-protected aromatic α -amino acid N-tert-butyloxycarbonyl-L-phenylalanine by x-ray diffraction and infrared absorption. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. International Journal of Biological Macromolecules (1980), 2(4), 217-24. Abstract: The x-ray diffraction and IR absorption conformational anal. of N-tert-butyloxycarbonyl-L-phenylalanine showed the absence of intramolecularly H-bonded peptide conformations in the solid state. The mols. are held together in rows of cyclic dimer motifs through intermol. NH...O:C(acid) and O-H...O:C (urethane) H bonds, the secondary amidelike group of the urethane moiety being in the unusual cis conformation, whereas the COOH group in the common syn conformation. The 2 mols. in the unit cell present a centrosym. set of ϕ, ψ1, and ψ2 values. In polar solvents, solvated species largely predominate. In satd. hydrocarbon soln., nonassocd. and assocd. (mostly involving the carboxylic acid C:O as the proton acceptor) species simultaneously occur. The extent of assocn. decreases with diln. The amt. of intramolecularly H-bonded oxy-C7 and C5 forms if any, should be extremely small. The type of assocn. at satn. apparently differs from that found in the cryst. compd. obtained by pptn. with satd. aliph. hydrocarbons (from a Et2O soln.). 133. Conformations of diastereoisomeric peptides. N-(tert-butyloxycarbonyl)-Lprolyl-D-proline and its methyl ester in the solid state and in solution. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; 104 Prof. Vincenzo Pavone Toniolo, Claudio; Bonora, Gian Maria. Macromolecules (1980), 13(6), 1454-62. Abstract: X-ray diffraction and IR data showed that the solid-state conformations of Me3CO2C-Pro-D-Pro-OR (I; R = H, Me) have a cis tertiary urethane bond and a trans tertiary amide bond. The mols. of I (R = H) in the solid state are linked by an intermol. OH...OC (urethane) H bond. The conformations of I in soln. were also detd. In solvents of high polarity, solvated species predominate. For I (R = H), even in a solvent of low polarity at high diln., the extent of intramol. H bonding was small. 134. Conformational analysis of N-(tert-amyloxycarbonyl-L-proline in the solid state and in solution. Benedetti, Ettore; Ciajolo, Anna; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. International Journal of Peptide & Protein Research (1979), 14(2), 130-42. Abstract: X-ray and IR data showed that the mols. of the title compd. (I) in the solid state are not folded into an oxy-C7 peptide conformation, but are held together through intermol. urethane H bonds, and the tertiary amide bond has the cis configuration. CO and IR data showed that strongly solvated species predominate for I in highly polar solvents. In cyclohexane nonassociated and assocd. species of I are simultaneously present and the amt. of oxy-C7 form is extremely small. CD measurements alone can lead to an incorrect conformation of amino acid derivs. and small peptides in soln. 135. Structure and conformation of peptides: N-benzyloxycarbonyl-(γ -ethyl)-Lglutamyl-(γ -ethyl)-L-glutamic acid ethyl ester. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Germain, Gabriel; Goodman, Murray. Biopolymers (1979), 18(3), 517-22. Abstract: The crystal structure of the title compd. was detd. The urethane and amide bonds and all ester groups are in the trans configuration, and the Φ and ψ angles of the glutamyl residue fall in the β-structure region of the Ramachandran plot. The mol. is flat with the amide plane almost parallel to the c axis along which 2 H bonds hold the mols. together in a parallel pleated sheet. 136. Linear oligopeptides. 59. Stereochemical analysis of N-tertbutyloxycarbonyl-L-prolylsarcosine and N-tertbutyloxycarbonylsarcosylsarcosine in the solid state and in solution. Benedetti, Ettore; Ciajolo, Anna; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria. Macromolecules (1979), 12(3), 438-45. 105 Prof. Vincenzo Pavone Abstract: The conformation of the title peptides in the solid state and in soln. were analyzed by IR and x-ray diffraction data. In the solid state, a cistert-urethane bond and an intermol. H bond between the OH of the CO2H and an amide O characterized the mol. and crystal structure. In soln. the formation of intramol. H bonds was negligible, even in a solvent of low polarity and high diln. 137. L-Methionine hydrochloride. De Blasio, B.; Pavone, V.; Pedone, C. Crystal Structure Communications (1977), 6(4), 845-8. Abstract: Mols. of the title compd. are disposed in crystals according to the general mode of packing for this type of mol. The conformation of the side chain moiety is different from that of the same group in α and β forms of free DL-methionine. The mol. is more compact as the HCl salt due to the presence of a gauche bound around the C2-C3 atoms, whereas this rotation angle is trans in the α and β forms. 138. Glycine hydrochloride C2H5NO2.HCl [and] L-alanine hydrochloride C3H7NO2.HCl. Di Blasio, B.; Pavone, V.; Pedone, C. Crystal Structure Communications (1977), 6(4), 745-8. Abstract: The crystal structures of the title compds. consist of layers contg. Cl- and the appropriate amino acid ammonium ion in which the Cl- forms 2 H bonds with N atoms and 1 H bond with an O-H group. This is the usual packing of mols. for crystals of anhyd. amino acid hydrochlorides with a non-polar side chain. 139. Solid-state and solution conformation of N-tert-butyloxycarbonyl-Lprolylglycine. Benedetti, E.; Pavone, V.; Toniolo, C.; Bonora, G. M.; Palumbo, M. Macromolecules (1977), 10(6), 1350-6. Abstract: The proposed folding of the title compd. (I) in the solid state to form an intramol. H-bonded 10-membered oxy analog of the trans II β turn, which was based on IR data, was rejected on x-ray detn. The low-frequency value of the urethane CO stretching vibration in the IR spectrum of I in the solid state can be explained by intermol. H bonds. This type of intramol. folding is also absent in polar solvents; a strong neg. Cotton effect at 225 nm in the CO of I in solvents of low polarity could be due to this type of folding. 140. N,N'-bis(β -chloroethyl)pimelamide. Ciajolo, M. R.; Pavone, V.; Benedetti, E. Acta Crystallographica, Section B: Structural Crystallography and Crystal 106 Prof. Vincenzo Pavone Chemistry (1977), B33(4), 1295-7. Abstract: The title compd. is monoclinic, space group P21, with a 4.941(5), b 32.425(30), c 4.817(5) .ANG., and β 113°37(5)'; d.(calcd.) = 1.331 for Z = 2. The mol. conformation is not fully extended. Each mol. forms H bonds along two directions (almost the a and c directions). A comparison with N,N'bis(β-chloroethyl)glutaramide is made. 141. Bis(n-dodecylammonium) tetrachlorozincate. Ciajolo, M. R.; Corradini, P.; Pavone, V.. Acta Crystallographica, Section B: Structural Crystallography and Crystal Chemistry (1977), B33(2), 553-5. Abstract: The title compd. is monoclinic, space group P21/c, with a 7.409(3), b 10.379(5), c 44.399(10) .ANG., and β 105.56(5)°; d.(calcd.) = 1.159 for Z = 4. The structure is characterized by ionic layers sandwiched between layers of paraffinic chains. 142. Comparative studies of layer structures: the crystal structure of bis(monodecylammonium)tetrachloromanganate(II). Ciajolo, Maria R.; Corradini, Paolo; Pavone, Vincenzo. Gazzetta Chimica Italiana (1976), 106(910), 807-16. Abstract: The structure of the title compd. was detd. by x-ray diffraction, solved by the Patterson method, and refined by least-squares calcns. to a R of 0.086. The crystals are monoclinic, space group P21/a, with a 7.213(8), b 7.337(2), c 26.747(21) .ANG., and b 94.64(5)°; Z = 2. Bond lenghts and angles are given and the mol. packing is discussed. The MnCl42- anion is polymeric and has D4h symmetry. Individual anions are bridged by Cl atoms. The NH3 groups are placed at the center above the squares formed by 4 axial Cl atoms giving the Mn ions a square pyramidal conformation. 107 Prof. Vincenzo Pavone PROCEEDINGS 1. Developing synthetic hemoprotein mimetics: design, synthesis and characterization of heme-peptide conjugates. Lombardi, Angela; Nastri, Flavia; D'Andrea, Luca D.; Maglio, Ornella; D'Auria, Gabriella; Pedone, Carlo; Pavone, Vincenzo. Editor(s): Tam, James P.; Kaumaya, Pravin T. P. Peptides: Frontiers of Peptide Science, Proceedings of the American Peptide Symposium, 15th, Nashville, June 14-19, 1997 (1999), Meeting Date 1997, 9193. Publisher: Kluwer, Dordrecht, Neth., Abstract: A symposium with five refs. The properties of mimochrome II, a hemoprotein model compd., were evaluated in relation to the peptide chain compn. and length and its importance in controlling heme properties. 2. A novel class of calmodulin mimetics: De novo designed proteins in molecular recognition. Lombardi, Angela; Ghirlanda, Giovanna; Zaccaro, Laura; Pavone, Vincenzo; DeGrado, William F. Editor(s): Tam, James P.; Kaumaya, Pravin T. P. Peptides: Frontiers of Peptide Science, Proceedings of the American Peptide Symposium, 15th, Nashville, June 14-19, 1997 (1999), Meeting Date 1997, 94-96. Publisher: Kluwer, Dordrecht, Neth., Abstract: This symposium report describes the designing of an α-helical dimer, covalently linked in a parallel or antiparallel orientation, that specifically interacts with only one of the target enzymes recognized by calmodulin (CaM). The chosen protein sequence was from the CaM binding domain of calcineurin. 3. Induced helical structure in highly charged peptides. Lombardi, A.; Zaccaro, L.; Ansanelli, G.; Pedone, C.; Pavone, V.. Editor(s): Shimonishi, Yasutsugu. Peptide Science: Present and Future, Proceedings of the International Peptide Symposium, 1st, Kyoto, Nov. 30-Dec. 5, 1997 (1999), Meeting Date 1997, 385-387. Publisher: Kluwer, Dordrecht, Neth., Abstract: A symposium on the properties and capacity of a synthetic highlycharged peptide (no data) to bind with a polynucleotide sequence contg. the GCG recognition triplet. 4. Miniaturized hemoproteins: a covalent asymmetric peptide-porphyrin system. D'Andrea, Luca D.; Nastri, Flavia; Lombardi, Angela; Maglio, Ornella; Pavone, Vincenzo. Editor(s): Bajusz, Sandor; Hudecz, Ferenc. Peptides 1998, Proceedings of the European Peptide Symposium, 25th, Budapest, Aug. 30-Sept. 4, 1998 (1999), Meeting Date 1998, 304-305. 108 Prof. Vincenzo Pavone Publisher: Akademiai Kiado, Budapest, Hung., Abstract: A symposium report. The solid-phase synthesis of a hemoprotein mimetic, mimochrome III, is reported. 5. Topology of porphyrins linked to de novo four-helix bundle proteins: towards the design and synthesis of multicofactor redox enzymes. Rabanal, Francesc; Lombardi, Angela; Pavone, Vincenzo; DeGrado, William F.; Dutton, P. Leslie. Editor(s): Ramage, Robert; Epton, Roger. Peptides 1996, Proceedings of the European Peptide Symposium, 24th, Edinburgh, Sept. 813, 1996 (1998), Meeting Date 1996, 741-742. Publisher: Mayflower Scientific, Kingswinford, UK, Abstract: A symposium report on the prepn. of a designed four-α-helix bundle protein with histidine residues at selected locations for complexation of a deuteroporphyrin residue to be attached to a defined lysine side chain. 6. Rational design of enzymically resistant, peptide based, multi-site directed, α -thrombin inhibitors. Lombardi, A.; Nastri, F.; Galdiero, S.; Della Morte, R.; Staiano, N.; Pedone, C.; Pavone, V.. Editor(s): Kaumaya, Pravin T. P.; Hodges, Robert S. Peptides: Chemistry, Structure and Biology, Proceedings of the American Peptide Symposium, 14th, Columbus, Ohio, June 18-23, 1995 (1996), Meeting Date 1995, 374-375. Publisher: Mayflower Scientific, Kingswinford, UK, Abstract: A symposium report on the synthesis of hirudin analogs for use as α-thrombin inhibitors. 7. A novel strategy for the synthesis of heme-peptide conjugates. Nastri, Flavia; Lombardi, Angela; Maglio, Ornella; Morelli, Giancarlo; D'auria, Gabriella; Pedone, Carlo; Pavone, Vincenzo. Editor(s): Epton, Roger. Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries: Peptides, Proteins and Nucleic Acids--Small Molecule Organic Chemical Diversity, Collected Papers, International Symposium, 4th, Edinburgh, Sept. 12-16, 1995 (1996), Meeting Date 1995, 491-494. Publisher: Mayflower Scientific, Birmingham, UK, Abstract: A symposium report. We report here a novel strategy for the synthesis of heme-peptide conjugates. This synthetic procedure allows the synthesis of a wide variety of analogs made up of either two equal or different peptide chains linked to the porphyrin ring. 8. Conformation features of functionalized cyclodextrins: A ternary complex 109 Prof. Vincenzo Pavone of histamino β -cyclodextrin, copper(II) and L-tryptophan. Saviano, M.; Di Blasio, B.; Pavone, V.; Pedone, C. Editor(s): Maia, Hernani L. S. Peptides 1994, Proceedings of the European Peptide Symposium, 23rd, Braga, Port., Sept. 4-10, 1994 (1995), Meeting Date 1994, 568-569. Publisher: ESCOM, Leiden, Neth., Abstract: The structure of the ternary complex of 6-deoxy-6-N-histamino-βcyclodextrin, copper(II), and L-tryptophan (no prepn. given) has been solved by direct methods in order to gain conformational information about interactions responsible for chiral recognition of arom. amino acids by the copper histamino-β-cyclodextrin receptor. Structure data: monoclinic space group P21, a 15.145, b 17.296, c 16.420 .ANG., β 105.2°, Z = 2. One nitrate and 15 co-crystd. H2O mols. were detected in the unit cell. Both tryptophan and the histamino β-cyclodextrin act as bidentate ligands. The Cu(II) ion assumes a distorted pentacoordination in which two N atoms of the histamino moiety and of the amide, and an O atom of the carboxyl group of the L-amino acid, occupy the four square-planar positions. The remaining apical position is occupied by a water mol. 9. Structural requirements for antagonist activity at tachykinin NK2 receptor in a series of bicyclic hexapeptides. Quartara, L.; Fabbri, G.; Patacchini, R.; Maggi, C. A.; Astolfi, M.; D'Auria, G.; Maglio, O.; Lombardi, A.; Pedone, C.; Pavone, V.. Editor(s): Maia, Hernani L. S. Peptides 1994, Proceedings of the European Peptide Symposium, 23rd, Braga, Port., Sept. 4-10, 1994 (1995), Meeting Date 1994, 591-592. Publisher: ESCOM, Leiden, Neth., Abstract: The authors describe here the conformational and configurational requirements for the high affinity interaction of MEN 10627 with tachykinin NK2 receptor. The remarkable differences in the antagonist potencies shown by the peptides described herein are strictly related to their different structures. Less active compds. are characterized by different orientations of the amino acid side chains. In the homochiral sequences, the bridge residues Asp2 and Dap5 are located in the i and i+3 positions of two β-turns, while in the heterochiral sequences the bridge residues occupy the i+2 positions of two β-turns. This shift of β-turn corner positions dets. a completely different mol. shape and a dramatic drop in biol. activity. 10. Specific interaction between cyclophilin and cyclic peptides. Gallo, P.; Saviano, M.; Rossi, F.; Pavone, V.; Pedone, C.; Ragone, R.; Stiuso, P.; Colonna, G. Editor(s): Maia, Hernani L. S. Peptides 1994, Proceedings of the European Peptide Symposium, 23rd, Braga, Port., Sept. 4-10, 1994 (1995), Meeting Date 1994, 654-655. Publisher: ESCOM, Leiden, Neth., 110 Prof. Vincenzo Pavone Abstract: Cyclolinopeptide A, a cyclic nonapeptide from linseed and its synthetic analog CLAIB bind to bovine cyclophilin A and inhibit peptidylprolyl cis-trans isomerase activity. The dissocn. consts. and enzymic 50% inhibition values of cyclolinopeptide A, CLAIB, and the immunosuppressant cyclosporin A (which also binds with cyclophilin A). 11. A new potent and highly selective, long lasting, peptide-based neurokinin A antagonist: rational design of MEN 10627. Pavone, V.; Lombardi, A.; Pedone, C.; Quartara, L.; Maggi. C. A. Editor(s): Hodges, Robert S.; Smith, John A. Pept.: Chem., Struct. Biol., Proc. Am. Pept. Symp., 13th (1994), Meeting Date 1993, 487-9. Publisher: ESCOM, Leiden, Neth., Abstract: MEN 10627 is the prototype of a new class of cyclic peptide-based NK-2 receptor antagonists. Owing to its high potency and long lasting activity in vivo, MEN 10627 and its analogs are suitable candidates for clin. testing in humans. 12. β-cyclodextrins as potent bioactive peptide delivery systems. Rossi, Filomena; Zaccaro, Laura; Di Blasio, Benedetto; Pavone, Vincenzo; Maglio, Ornella; Saviano, Michele; Pedone, Carlo; Cucinotta, Vincenzo; Impellizzeri, Giuseppe; et al. Editor(s): Schneider, Conrad H.; Eberle, Alex N. Pept. 1992, Proc. Eur. Pept. Symp., 22nd (1993), Meeting Date 1992, 577-8. Publisher: ESCOM, Leiden, Neth., Abstract: Solid-state studies on 6-deoxy-6-cyclo(L-histidyl-L-leucyl)-βcyclodextrin and 6-deoxy-6-N-(2-methylhexahydropyrimidine)-βcyclodextrin complexes are reported. 13. Molecular tools for the design of γ -turn in peptides. Pavone, V.; Lombardi, A.; D'Auria, G.; Saviano, M.; Di Blasio, B.; Paolillo, L.; Pedone, C. Editor(s): Smith, John A.; Rivier, Jean E. Pept.: Chem. Biol., Proc. Am. Pept. Symp., 12th (1992), Meeting Date 1991, 366-7. Publisher: ESCOM, Leiden, Neth., Abstract: A symposium report on the synthesis and structural characterization both by NMR in CD3CN soln. and by x-ray diffraction of the cyclic tetrapeptides cyclo(β-Ala-L-Pro-β-Ala-Aaa) (Aaa = L-Pro, L-Val) in order to verify the usefulness of the sequence β-Ala-Pro-β-Ala as mol. tool to force the peptide in a γ-turn conformation. 14. The fully extended polypeptide conformation. Toniolo, C.; Valle, G.; Crisma, M.; Benedetti, E.; Pedone, C.; Di Blasio, B.; Pavone, V.. Editor(s): Smith, John A.; Rivier, Jean E. Pept.: Chem. Biol., Proc. Am. Pept. 111 Prof. Vincenzo Pavone Symp., 12th (1992), Meeting Date 1991, 276-7. Publisher: ESCOM, Leiden, Neth., Abstract: A report from a symposium on conformational energy calcns. on disubstituted glycine derivs. AcNHCR2CONHMe (R = Et, Pr, Ph, CH2Ph) and solid-state conformations of their corresponding peptide derivs. by xray crystallog. 15. Structural characterization of the β -bend ribbon spiral: Crystallographic analysis of two long (L-Pro-Aib)n sequential peptides. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Crisma, M.; Anzolin, L.; Toniolo, C. Editor(s): Smith, John A.; Rivier, Jean E. Pept.: Chem. Biol., Proc. Am. Pept. Symp., 12th (1992), Meeting Date 1991, 290-1. Publisher: ESCOM, Leiden, Neth., Abstract: A report from a symposium on the prepn. of the title compds. 4BrC6H4CO-Aib-(Pro-Aib)n-OMe (I; n = 2-5; Aib = α-aminoisobutyric acid) and the crystal structure of I (n = 2, 3). 16. The polypeptide 310-helix. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Santini, Antonello; Toniolo, Claudio; Crisma, Marco; Formaggio, Fernando; Sartore, Luciana. Editor(s): Renugoplakrishnan, Venkatesan. Proteins (1991), 302-4. Publisher: ESCOM, Leiden, Neth., Abstract: A report from a symposium on the relative stabilities of 310- and αhelicies of α-aminoisobutyric acid (Aib) residues in the solid state by anal. of crystal structures of 3 (Aib)n (n = 6, 8, 10) homooligopeptides and 3 (AibL-Ala)m (m = 3-5) sequential oligopeptides. 17. Helical structures in peptides. Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Editor(s): Giralt, Ernest; Andreu, David. Pept. 1990, Proc. Eur. Pept. Symp., 21st (1991), Meeting Date 1990, 454-5. Publisher: ESCOM Sci. Publ., Leiden, Neth., Abstract: A report from a symposium on a statistical anal. of 54 independent helical mols. using highly refined crystal structure detns. A total of 407 residues were taken into account, of which 258 were considered in helical conformation, being part of at least 4 successive amino acid residues with ϕ,ψ values pertinent to either the α or the 310 helix. The min. peptide length required for onset of α-helixes is 7 residues. 18. β-Ala residues for molecular design of cyclic peptides containing 112 Prof. Vincenzo Pavone hydrogen-bonding turns. Di Blasio, B.; Pavone, V.; Yang, X.; Lombardi, A.; Benedetti, E.; Pedone, C. Editor(s): Rivier, Jean E.; Marshall, Garland R. Pept.: Chem., Struct. Biol., Proc. Am. Pept. Symp., 11th (1990), Meeting Date 1989, 667-8. Publisher: ESCOM Sci. Pub., Leiden, Neth., Abstract: A report from a symposium on the prepn. and structures of βalanine-contg. cyclic peptides cyclo-[(Pro)n-Phe-βAla-βAla] (n = 1, 2). 19. Symmetry in synthetic and natural peptides. Benedetti, E.; Di Blasio, B.; Lombardi, A.; Pavone, V.; Pedone, C. Editor(s): Gruber, Bruno; Yopp, John H. Symmetries Sci. 4 [Proc. Symp.] (1990), Meeting Date 1989, 1-14. Publisher: Plenum, New York, N. Y., Abstract: A review with 38 refs. on the symmetry and conformations of cyclic peptides, linear peptides, and helical structures 20. Reactivity and structure of platinum(II) complexes in peptide synthesis. Lombardi, A.; Pavone, V.; Pedone, C.; Di Blasio, B.; Benedetti, E. Colloque INSERM (1989), 174(Forum Pept., 2nd, 1988), 207-10. Abstract: A report from a forum on peptides. Pt amino acid complexes, e.g. trans-[Cl2PtL2] (L = alanine, valine, α-aminoisobutyric acid), were prepd. The reactivity of the Pt complexes in peptide synthesis was studied. 21. Cyclic regularly alternating L,D peptides. Pavone, V.; Benedetti, E.; Di Blasio, B.; Pedone, C.; Lombardi, A.; Lorenzi, G. P. Editor(s): Jung, Guenther; Bayer, Ernst. Pept., Proc. Eur. Pept. Symp., 20th (1989), Meeting Date 1988, 447-9. Publisher: de Gruyter, Berlin, Fed. Rep. Ger., Abstract: A symposium report on the crystal structure and conformation of title peptides cyclo(L-Val-D-Val)3 and cyclo(L-Phe-D-Phe)3. 22. Metal ion binding peptides: solid-state conformation of cyclo(Glu-Leu-ProGly-Lys-Leu-Pro-Gly)cyclo(1γ-5ε)Gly. Di Blasio, B.; Pavone, V.; Pedone, C.; Benedetti, E.; Spiniello, O.; Zanotti, G.; Blout, E. R. Editor(s): Jung, Guenther; Bayer, Ernst. Pept., Proc. Eur. Pept. Symp., 20th (1989), Meeting Date 1988, 453-5. Publisher: de Gruyter, Berlin, Fed. Rep. Ger., Abstract: A symposium report on the crystal structure and solid-state conformation of the title homodetic bicyclic nonapeptide. 23. Crystal-state 310-α -helix conformational transition induced by peptide chain lengthening. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, 113 Prof. Vincenzo Pavone A.; Bavoso, A.; Toniolo, C.; Crisma, M.; Sartore, L. Editor(s): Jung, Guenther; Bayer, Ernst. Pept., Proc. Eur. Pept. Symp., 20th (1989), Meeting Date 1988, 465-7. Publisher: de Gruyter, Berlin, Fed. Rep. Ger., Abstract: A symposium report on the detn. of the solid-state conformation of p-BrC6H4CO-(Aib-Ala)n-OMe (Aib - NHCMe2CO; n = 4, 5, 6) by xray diffraction anal. All peptides in the solid state have a helical structure stabilized by NH...OC H bonds. In all peptides the central core of the helix is always of the α-helical type with the formation of consecutive intramol. H-bonded C13 ring structures. At both terminal ends in all peptides, C10 ring structures are obsd. 24. Crystal and molecular structure of S-deoxo [γ (R) hydroxy-Ile3]amaninamide: a synthetic analog of Amanita toxins. Zanotti, Giancarlo; Wieland, Theodor; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Editor(s): Marshall, Garland R. Pept.: Chem. Biol., Proc. Am. Pept. Symp. 10th (1988), Meeting Date 1987, 93-4. Publisher: ESCOM Sci. Pub., Leiden, Neth., Abstract: A symposium on the crystal structure of the title compd. 25. Structural versatility of peptides from Cα,α-dialkylated glycines: ACC3rich peptides. Toniolo, Claudio; Crisma, Marco; Valle, Giovanni; Bonora, Gian Maria; Barone, Vincenzo; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; et al. Peptide Chemistry (1988), Volume Date 1987, 45-8. Abstract: A report on the structure of 1-aminocyclopropane-1-carboxylic acid (Acc3)-rich peptides. Crystal and mol. structures and conformational energy calcns. are discussed. 26. Structural versatility of peptides from Cα,α-dialkylated glycines: Acc5- and Acc6-containing peptides. Benedetti, E.; Barone, V.; Bavoso, A.; Di Blasio, B.; Lelj, F.; Pavone, V.; Pedone, C.; Toniolo, C.; Crisma, M.; Bonora, G. M. Editor(s): Theodoropoulos, Dimitrios. Pept., Proc. Eur. Pept. Symp., 19th (1987), Meeting Date 1986, 315-18. Publisher: de Gruyter, Berlin, Fed. Rep. Ger., Abstract: A symposium on conformational energy calcns., x-ray diffraction anal., and IR and 1H NMR spectra of the title peptides (Acc5 = αaminopentanecarboxylic acid, Acc6 = α-aminocyclohexanecarboxylic acid). 27. Thiolation and epimerization of α-hydroxy α-amino acid derivatives by 114 Prof. Vincenzo Pavone the Mitsunobu reaction. Nagai, Ukon; Kato, Rika; Pavone, Vincenzo. Peptide Chemistry (1988), Volume Date 1987, 187-90. Abstract: A report on the thiolation and epimerization of α-hydroxy αamino acid derivs. by the Mitsunobu reaction. 28. Folded, helical and extended structures of peptides from Cα ,α -dialkylated α -amino acids. Solid-state and solution conformation of homopeptides from Cα ,α -diethylglycine. Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria; Leplawy, Miroslaw T.; Redlinski, Adam; Kaczmarek, Krzysztof. D. Pept.: Struct. Funct., Proc. Am. Pept. Symp., 9th (1985), 193-6. Abstract: Structure of diethylglycine (Deg) homopeptides were detd. by (1) conformational energy calcns. for Ac-Deg-NHMe, (2) diffraction anal. of CF3CO-(Deg)n-OCMe3 (I; n = 3-5), and (3) IR and 1H NMR of I (n = 2-5). The intramol. H bonded C5 structures found were remarkably stable to diln., heating, and addn. of polar solvent. 29. Folded and extended structures of homo-peptides from α ,α -dialkylated α amino acids. Benedetti, Ettore; Barone, V.; Bavoso, A.; Di Blasio, B.; Grimaldi, P.; Lely, F.; Pavone, V.; Pedone, C.; Bonora, G. M.; et al. Editor(s): Ragnarsson, Ulf. Pept., Proc. Eur. Pept. Symp., 18th (1984), 603-6. Publisher: Almqvist & Wiksell, Stockholm, Swed., Abstract: Conformational preferences of α,α-dipropylglycine (Dpg) and αmethyl-α-ethylglycine derivs. and homopeptides were compared with those of the α,α-dimethylglycine and α-aminoisobutyric acid (Aib) series. The IR and 1H NMR results in CDCl3 soln. indicate that the Aib peptides assume a 310-helix conformation and the Dpg peptides assume a multiple-C5 conformation. 30. Membrane-active peptaibol antibiotics: conformational preferences of the 29 segment of emerimicins III and IV and all related short sequences. Toniolo, Claudio; Bonora, Gian Maria; Mapelli, Claudio; Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Editor(s): Hruby, Victor J.; Rich, Daniel H. Pept.: Struct. Funct., Proc. Am. Pept. Symp., 8th (1983), 495-8. Publisher: Pierce Chem. Co., Rockford, Ill, Abstract: IR and CD spectra of Z-(Aib)3-Val-Gly-Leu-(Aib)2-OMe (Aib = α-aminoisobutyric acid residue)(I) and of several fragments in soln. indicated that I assumes a right-handed α-helical conformation. 115 Prof. Vincenzo Pavone 31. Channel-forming molecules: conformation of peptides with alternating L and D residues as models of gramicidin A. Pedone, Carlo; Barone, Vincenzo; Benedetti, Ettore; Di Blasio, Benedetto; Esposito, Gennaro; Garolla, Francesco Lelj; Pavone, Vincenzo; Lorenzi, Gian Paolo. Editor(s): Hruby, Victor J.; Rich, Daniel H. Pept.: Struct. Funct., Proc. Am. Pept. Symp., 8th (1983), 473-6. Publisher: Pierce Chem. Co., Rockford, Ill, Abstract: Semiempirical calcns. of the conformations of peptides contg. alternating D and L amino acids were performed, and the results compared with the results of x-ray crystallog. data. The contributions of electrostatic and nonbonded interactions to the conformational stability of these peptides are discussed. 32. Conformational features of alternating L,D peptides. Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Barone, Vincenzo; Esposito, Gennaro; Lelj, Francesco; Lorenzi, Gian Paolo. Editor(s): Blaha, Karel; Malon, Petr. Pept., Proc. Eur. Pept. Symp., 17th (1983), Meeting Date 1982, 699-704. Publisher: de Gruyter, Berlin, Fed. Rep. Ger., Abstract: The conformations of Boc-D-Leu-L-Leu-OMe (I, Boc = Me3CO2C), Boc-L-Ile-D-aIle-OMe (II), Boc-(D-aIle-L-Ile)3-OMe (III), Boc-(L-Val-D-Val)4-OMe (IV), and Boc-(D-Phe-L-Phe)4-OMe were studied by crystallog. data and theor. calcns. I and II present an V-shaped structure with ϕ and ψ torsion angles falling in the calcd. min. energy regions. III adopts a folded shape due to the formation of a type II β-turn fused with an α-turn in which both are included in a C17-membered intramol. H-bonded ring structure. The crystal structure of IV indicated double-stranded β-helical dimers with antiparallel chains. 33. Peptaibol antibiotics: a study on the helical structure of emerimicins. Toniolo, Claudio; Bonora, Gian Maria; Benedetti, Ettore; Bavoso, Alfonso; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo. Editor(s): Blaha, Karel; Malon, Petr. Pept., Proc. Eur. Pept. Symp., 17th (1983), Meeting Date 1982, 741-4. Publisher: de Gruyter, Berlin, Fed. Rep. Ger., Abstract: The conformational preferences in soln. of the protected 2-9 segment of emerimicins III and IV, and several related short sequences, were studied by IR and CD spectroscopies. The structural preferences of Z(Aib)3-OCMe3 (Z = PhCH2O2C, Aib = α-aminoisobutyric acid residue) (I) and Z-(Aib)3-L-Val-X-OMe [X = bond (II) or Gly (III)] were confirmed by x-ray diffraction anal. The structures of I, II, and III are characterized by 116 Prof. Vincenzo Pavone C10-conformations [one type-III (or type III'), a double type-III', and triple, resp.]. 117