Vers De Nouveaux Nanosystèmes Pour La Vectorisation De Peptides

Synthetic Molecular Nanodevices for Selective Peptide-Based Therapy by Anthony Fernandes Degree of Doctor of Philosophy Department of Chemistry University of Edinburgh and Laboratoire de Chimie des Substances Naturelles, UMR 6514 Université de Poitiers - April 2009 - Table of Contents Declaration ii Acknowledgements iii General Remarks on Experimental Data v Layout of this Thesis vi Chapter 1: Strategies for Peptide-Based Chemotherapy 1 Chapter 2: Towards a Model Rotaxane-Based Peptide Prodrug 41 Chapter 3: Rotaxane-Based Delivery Systems: Protection and 108 Enzymatic Release of a Bioactive Pentapeptide Chapter 4: Towards a Synthetic Molecular Peptide Synthesiser 138 Conclusion 160 i Declaration The scientific work described in this Thesis was carried out in the Laboratoire de Chimie des Substances Naturelles (UMR 6514) at the Université de Poitiers and in the Department of Chemistry at the University of Edinburgh between November 2005 and September 2008. Unless otherwise stated, it is the work of the author and has not been submitted in whole or in support of an application for another degree or qualification ii Acknowledgements I would like to thank my supervisors Prof. David Leigh and Prof. Jean-Pierre Gesson for giving me the opportunity to do this PhD. These three years, shared between France and Scotland, have been absolutely rewarding both professionally and personally. I would particularly like to thank my french “supervisor” Dr. Sébastien Papot, who helped me all over this PhD, even when I was in Poitiers or in Edinburgh. The discussions we had guided me all along this work. I also thank him for his efforts in proof reading my thesis. I would also thank Dr. Vincent Aucagne who is the chemist behind such an innovative project. I would like to thank Anne-Céline for her support and advices, even if she is not always right…she is just never wrong… I also acknowledge Aurélien Viterisi who was involved with me in this fabulous project and carried out all the HPLC purifications and monitoring of our molecular devices. I would also like to thank Paul McGonigal, a.k.a. “blondie”, who carefully corrected my English…and for the long but even too short hours playing guitar together. I would also like to thank Dr. Dominik Heckmann, another member of the Saturday Guitar Band, for his advices with the peptide synthesiser project. I would also thank Dr. Stephen Goldulp for his help and advices. I would like to thank Isabelle Opalinski, Brigitte Renoux, Claudine Dumiot, Jean-Marie Coustard, Martine Mondon, André Amblès, Bruno Violeau and all the researchers I worked with in Poitiers. I particularly thank my colleagues and friends from France: Nicolas “KCo”, Cédric Charrier, Sébastien Picard, Mickaël Thomas, Julien Célérier, Dorothée Moine, Emilie Vardelle and Sophie Vuong. iii Thanks to Kevin, Edzard (the “man down under”), Satoshi, Vicky, Amaya, Nathalie, Euan, Brian, Daniel, Michael, Mark, Roy, Tao, Max from the Leigh group. I would also thank all the people that dealt with all the administrative struggles associated with this first Franco-Scottish PhD, particularly Amanda Ewing and Annette Burgess. Finally I gratefully thank the Royal Society of Edinburgh for financing this PhD. iv General Remarks on Experimental Data All the melting points (m.p.) were determined using a Sanyo Gallenkamp apparatus and are uncorrected. 1H and 13C NMR spectra were recorded at 400 MHz on a Bruker AV 400 instrument at 298K unless otherwise stated. Chemical shifts (δ) are reported in parts per million from low to high field and referenced to residual solvent. Coupling constants (J) are reported in hertz (Hz). Standard abbreviations indicating multiplicity are used as follows: b = broad, s = singlet, d = doublet, dd= doublet of doublet, t = triplet, q = quartet, m = multiplet, J = coupling constant). Flash column chromatography was carried out using Kiesegel C60 (Fisher Scientific) as the stationary phase. Analytical TLC was performed on aluminium- backed sheets pre-coated with silica 60 F254 adsorbent (0.25 mm thick, Merck, Germany) and visualized under UV light. Mass spectrometry was carried out by the mass spectrometry services at the University of Edinburgh and at the EPSRC National Centre, Swansea. Size exclusion chromatography was performed using Toyopearl HW-405 (Tosoh, Japan) with methanol/chloroform in a 1:1 v/v ratio as the eluent. Unless otherwise stated, all reactions were run under an atmosphere of N2. Prior to use, isophthaloyl dichloride was purified by recrystallization from hexane; p-xylylenediamine was purified by distillation under reduced pressure. Analytical RP-HPLC was carried out on a Gilson instrument composed of 306 pumps, 811C dynamic mixer (100 L), 806 manometric module and an Applied Biosciences 759A UV detector with a Phenomenex C18 (2) Luna column (2 x 250 mm, 5 m, 100A). Preparative RP-HPLC was carried out on a Gilson instrument composed of 306 pumps, 811C dynamic mixer (1.5 ml), 806 manometric module and a 118 UV detector with a Spherisorb ODS2 column (21.2 x 250 mm, 5 m, 100A). LCMS was carried out on a Finnigan Mat system composed of an LCQ mass spectrometer, P4000 pumps, and a UV2000 UV detector with a Phenomenex C18 (2) Luna column (2 x 250 mm, 5, 100A). Chromatograms were recorded at 220 nm unless stated otherwise. v Layout of this Thesis During this thesis we tried to design, synthesise and analyse some novel devices for the selective delivery of peptides. These systems are based on the enzyme-activated anticancer prodrugs developed by Prof. Gesson in Poitiers and the peptide rotaxanes developed by Prof. Leigh in Edinburgh. The innovative rotaxanes we constructed are devised to protect and selectively release a peptide in response to an enzyme-specific stimulus for the targeted therapy of cancer. In Chapter 1 we tried to expose the main synthetic strategies aimed at improving the stability and permeation features of biologically active peptides. We examined some prodrug approaches and particularly the tumour-activated prodrugs (TAPs), largely investigated for use in anticancer chemotherapy. TAPs are generally three-part molecules composed of trigger, spacer and effector units. We also presented the original methodology developed by Prof. Leigh, namely the hydrogen bond-directed assembly of peptide rotaxanes, to protect a peptide thread from external environment. Finally we presented our project which consists of a combination of the peptide prodrug and rotaxane approaches. Therefore, based on the knowledge of both research groups we tried in Chapter 2 to develop some model systems in order to study the influence of the rotaxane architecture upon prodrug molecules. The first step towards such rotaxane-based peptide prodrugs relied on the efficient design of a spacer which has to be bulky enough to work as a stopper for the macrocycle. Much of the work presented in this chapter is based on the design and synthesis of such self-immolative units. We then explored the response of our model rotaxanes under the action of the activating enzyme. After this detailed study, in Chapter 3 we applied our concept to the biologically active peptide Met-enkephalin. In this chapter we presented a comparison between a rotaxane prodrug of Met-enkephalin and its non-interlocked derivative. Thus both compounds were successfully synthesised and evaluated to release the free peptide after enzymatic activation. The protective effect of encapsulating the peptide within a rotaxane assembly was also studied in human plasma and with different proteases. Finally, in Chapter 4, we introduced the construction of a rotaxane-based molecular machine programmed to synthesise a short peptide unit from the amino acids carried on its thread. We synthesised with success a one-station model rotaxane to study the catalyst effect of the macrocycle. Unfortunately this model machine proved not to work and current research is still ongoing to achieve such a synthetic device. vi A mes parents vii CHAPT. 1 | STRATEGIES FOR PEPTIDE-BASED CHEMOTHERAPY I. Strategies to enhance peptide delivery................................................................................ 7 I.1. Peptidomimetics............................................................................................................... 8 I.1.1. Backbone modification ............................................................................................. 8 I.1.2. Secondary structure mimetics ................................................................................. 10 I.1.3. Functional and topographical peptidomimetics...................................................... 11 I.2. Cyclic peptides............................................................................................................... 13 I.3. The prodrug approach .................................................................................................... 15 I.3.1. Prodrugs to improve bioavailability........................................................................ 16 I.3.2. Bioreversible cyclic peptides .................................................................................. 19 II. Tumour-activated prodrugs............................................................................................. 21 II.1. Prodrug monotherapy (PMT) ....................................................................................... 22 II.1.1. Glucuronylated prodrugs ....................................................................................... 23 II.1.2. HMR 1826 ............................................................................................................

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