UNIVERSITAT POLITÈCNICA DE CATALUNYA DEPARTAMENT D’ENGINYERIA QUÍMICA “CONFORMATIONAL PROPERTIES OF CONSTRAINED PROLINE ANALOGUES AND THEIR APPLICATION IN NANOBIOLOGY” Alejandra Flores Ortega Supervisors: Dr. Carlos Alemán Llansó and Dr. Jordi Casanovas Salas. th Barcelona, 27 January 2009 “Chance is a word void of sense; nothing can exist without a cause”. François-Marie Arouet, Voltaire “Imagination will often carry us to worlds that never were. But without it, we go nowhere”. Carl Sagan iii ACKNOWLEDGEMENTS I would like to acknowledge to Dr. Carlos Aleman and Dr. Jordi Cassanovas Salas for an interesting research theme, and scientific support. I gratefully acknowledge to Dr. David Zanuy for interesting suggestions and strong discussions, without their support this would be an unfulfilled task. Also I, would like to address my thanks to all my colleagues in my group and department, specially Elaine Armelin for assiting me in many different ways. I thank not only my friends, but also colleagues for helping me to overcome the stressful time, without whom it would have been difficult to cope up. I wish to express my gratefulness to my parents, specially to my mother, María Esther, for all his care, and support. Also I will like to thanks to my friends and specially Jesus, Merches, Laura y Arturo. My PhD thesis have been finished for all this support. I am greatly indepted to Dr. Ruth Nussinov at NCI, Dr. Carlos Cativiela at the University of Zaragoza and Ana I. Jiménez at the “Instituto de Ciencias de Materiales de Aragon” for a collaborative effort. I wish to thank all my colleague in the “Chimie et Biochimie Théoriques, Faculté des Sciences et Techniques” in Nancy France, I will be grateful to have worked with : Pr. Xavier.Assfeld and PhD Adele Laurent. I gratefully acknowledge the financial support provided by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. v OBJECTIVES (1) Examine the conformational preferences of proline analogs having one or more double bonds in the pyrrolidine ring. Analyze the influence of the insaturations on: (i) the stability of the cis arrangement of the peptide bond involving the pyrrolidine nitrogen; and (ii) the conformational flexibility of the backbone. (2) Analyze the intrinsic conformational preferences of two representative α- tetrasubstituted proline analogs (α-methylproline and α-phenylproline) and compare them with those of conventional proline. Understand the effects of the substituent incorporated at the α position on the preferred backbone conformation, the puckering of the pyrrolidine ring and the cis/trans disposition of the amide bonds. (3) Compare the conformational properties of different aminated and dimethylaminated derivatives of proline. Examine how the formation of side chain···backbone hydrogen bonds affects not only the conformational flexibility but also the trans/cis disposition of the peptide bond involving the pyrrolidine nitrogen. (4) Determine the conformational preferences of the aminoproline analogs protonated at the amino side group. Analyze the influence of the pH on the relative stability of the different possible isomers, the backbone flexibility and the disposition of the peptide bond. (5) Characterize the conformational profile of the CREKA sequence, which defines a very efficient tumor-homing pentapentide, and identify the corresponding bioactive conformation. A satisfactory achievement of this objective is essential for designing of synthetic analogs able to provide protection from proteases, which is an important step before the development of potential applications of tumor-homing peptides. (6) Improve the biological performance and pharmacological profile of CREKA by engineering an analogue that incorporates a non-proteinogenic amino acid. This residue should be conceived to retain the most relevant characteristics of the conformational profile of the natural peptide and simultaneously impart stability against proteolytic cleavage. vii GLOSSARY. A Puckering Amplitude αMePro Methylproline Amp Aminoproline Dipeptides αPhPro Phenylproline Aze L-azetidine-2-carboxylic acid azPro azaproline B3 Becke´s three-parameter hybrid functional Seven Memberedintramolecular Hydrogen C7 Bond ∆Egp Relative Energy DFT Density Functional Theory ∆Ggp Gibbs free energies in the gas phase Dmp Dimethylaminoproline E Energy of the System HF Hartree-Fock Hyp 4R-Hydroxyproline ϕ Fi LYP Lee, Yang and Parr MP Moller-Plesset NHMe N-Methyl Amide Group Oxa (S)-oxazolidine-4-carboxylicacid P State of Puckering Pip (S)-piperidine-2-carboxylic acid Pro Proline Simulated Annealing - Molecular SA Dynamics SCF Self Consist Field SPIO Dextran-Coated Iron Oxide Thz ((R)-thiazolidine-4-carboxylicacid UHF Unrestricted Hartree-Fock ZPVE Zero-Point Vibrational Energies ρ electronic density Ψ Wave Function ψ Psi ix TABLE OF CONTEN T S 1 Introduction ........................................................................................................ 1 1.1 Prolin estructure and properties .................................................................... 1 1.2Survey of modified Proline residues. Conformational features ..................... 3 1.3 Peptide design: improving nature for bionanotechnological applications .... 5 1.4 References ..................................................................................................... 8 2 Methods ............................................................................................................. 11 2.1 Introduction ................................................................................................. 11 2.2 Quantum Mechanical Methods ................................................................... 11 2.2.1 Ab Initio Methods .................................................................................... 12 2.2.2 Hartree-Fock Method ............................................................................... 13 2.2.3 Other Ab Initio Methods .......................................................................... 14 2.2.4 DFT Methods ........................................................................................... 15 2.2.4.1 Correlation Term ................................................................................... 16 2.2.4.2 Hybrid Functionals ................................................................................ 16 2.2.5 Solvent Effects ......................................................................................... 16 2.3 Molecular Dynamics Simulation................................................................. 18 2.3.1 Force Field ............................................................................................... 18 2.3.2 Classical Dynamics .................................................................................. 19 2.3.3 Periodic boundary conditions ................................................................... 20 2.3.4 Temperature and pressure ........................................................................ 20 2.4 Conformational Search Methods................................................................. 21 2.5 References ................................................................................................... 24 3 Intrinsic Conformational Properties of synthetic Proline Analogues ......... 27 3.1 Conformation of Proline Analogs Having Double Bonds in the Ring ...... 29 3.1.1 Introduction .............................................................................................. 29 3.1.2 Methods .................................................................................................... 32 3.1.2.1 Computational Details ........................................................................... 32 3.1.2.2 Nomenclature and Pseudorotational Parameters ................................... 33 3.1.3 Results and Discussion ............................................................................. 34 3.1.3.1 Ac-L-Pro-NHMe ................................................................................... 34 3.1.3.2 Ac-∆α,βPro-NHMe ................................................................................ 36 3.1.3.3 Ac-L-∆β,γPro-NHMe ............................................................................. 41 3.1.3.4 Ac-L-∆γ,δPro-NHMe ............................................................................. 42 3.1.3.5 Ac-Py-NHMe ........................................................................................ 43 3.1.3.6 Relative Stability of the Cis Conformers .............................................. 44 3.1.4 Conclusions .............................................................................................. 47 3.1.5 References ................................................................................................ 48 3.2 Conformational Preferences of α-Substituted Proline Analogues ............ 53 3.2.1 Introduction .............................................................................................. 53 3.2.2 Methods .................................................................................................... 56 3.2.2.1 Computational Details. .......................................................................... 56 xi 3.2.2.2 Nomenclature and Pseudorotational Parameters. .................................. 59 3.2.3 Results and Discussion ............................................................................. 60 3.2.3.1 Ac-L-Pro-NHMe. .................................................................................