PhD THESIS OF THE UNIVERSITY OF CERGY-PONTOISE CO-TUTORED WITH THE UNIVERSITY OF FLORENCE Non conventional synthetic strategies of stapled peptides: modulation of secondary structures to optimise biological recognition presented by : Chiara TESTA PhD discussed on March 26th 2012, Composition of the evaluation committee: Pr. Solange LAVIELLE Rapporteur Pr. Luis MORODER Rapporteur Pr. Anna Maria PAPINI Directrice de thèse Pr. Nadège GERMAIN Directrice de thèse Pr. Paolo ROVERO Directeur de thèse Pr. Michael CHOREV Examinateur Pr. Jacques AUGE Examinateur Pr Andrea GOTI Examinateur Chiara Testa, PhD Thesis Table of contents INTRODUCTION .................................................................................................................. 1 1 Difficult peptide synthesis optimized by microwave-assisted approach: a case study of PTHrP(1–34)NH2 ......................................................................................... 18 1.1 The Parathyroid hormone (PTH) and the Parathyroid hormone-related protein (PTHrP) .................................................................................... 18 1.2 Microwave assisted peptide synthesis .................................................. 20 1.2.1 Thermal effects ..................................................................................... 21 1.2.2 Specific non-thermal effects ................................................................. 22 1.2.3 Modes .................................................................................................... 22 1.3 Synthetic strategies for difficult peptide sequences .............................. 23 1.4 Synthesis of PTHrP(1-34) ..................................................................... 25 1.5 MW-assisted mini-cleavage of PTHrP(1-34)NH2 fragments ............... 26 1.5.1 MW-assisted mini-cleavage protocol of 19-34 fragment of ................. 27 PTHrP(1-34)NH2: a case study of multi-arginine containing peptides. 27 1.5.2 Charachterization of PTHrP(1-34)NH2 fragments by UPLC-ESI-MS analysis .................................................................................................. 29 1.5.3 Characterization of the 12-34 fragment of PTHrP(1-34)NH2 by UPLS- ESI-MS/MS analysis ............................................................................. 32 1.6 RP-HPLC Semi-preparative of PTHrP(1–34)NH2 ............................... 33 1.7 Results ................................................................................................... 33 1.8 Advantages of MW irradiation in SPPS ............................................... 34 2 Intramolecular i-to-(i+5) Side-Chain-to-Side Chain Cyclization to stabilize β-turn Secondary Structures: the melanocortin peptides hormones ................................. 38 2.1 The Endocrine System .......................................................................... 38 2.1.1 Peptide hormones .................................................................................. 41 2.1.2 The Nervous and Endocrine Systems ................................................... 43 2.2 Melanocortin peptide hormones ........................................................... 44 2.2.1 G Protein-coupled Melanocortin receptors: MCRs .............................. 47 2.2.2 Development of Agonist for MCRs ...................................................... 51 2.3 Stabilization of β-turn conformation in melanocortin like peptide by Click Chemistry .................................................................................... 54 i Chiara Testa, PhD Thesis 2.4 Click Chemistry .................................................................................... 56 2.5 Synthetic Strategy ................................................................................. 60 2.6 Synthesis of non-coded amino acids ..................................................... 61 2.6.1 Synthesis of Nα-Fmoc-ω-azido-α-amino acids ..................................... 61 2.6.2 Synthesis of Nα-Fmoc-ω-alkynyl-α-amino acids .................................. 63 2.6.3 Collection of linear peptide precursor .................................................. 65 2.6.4 Collection of triazole containing cyclo-peptides .................................. 67 2.6.5 Solid Phase Synthesis of lactam bridge-containing cyclopeptide ........ 69 2.7 Biological study .................................................................................... 70 2.8 Conformational study. .......................................................................... 72 2.9 NMR analysis ....................................................................................... 73 2.10 NMR Structure Calculations ................................................................. 75 2.11 Conclusion ............................................................................................ 78 3 PART B: Impact of Endocrine disruptor Chemicals on human health ................ 84 3.1 Phthalates .............................................................................................. 85 3.1.1 Metabolism of DEHP in humans .......................................................... 87 3.2 Health effect .......................................................................................... 90 3.2.1 Endocrine disruptor (EDs) .................................................................... 90 3.2.2 PPAR-mediated activity of phthalates .................................................. 91 3.2.3 Alteration of Thyroid Hormone Levels ................................................ 93 3.2.4 Exposure to DEHP in fetuses and infants ............................................. 94 3.3 Detection of DEHP metabolites in urine .............................................. 95 3.4 Synthesis of DEHP metabolites ............................................................ 96 3.5 Quantitative analysis ............................................................................. 97 3.6 Relationships between urinary concentrations of DEHP metabolites, obesity and insulin sensitivity in obese children ................................ 101 3.6.1 Obesity ................................................................................................ 101 3.6.2 Causes ................................................................................................. 102 3.6.3 Mechanism of action ........................................................................... 103 3.7 Detection of DEHP metabolites in obese children ............................. 104 3.8 Results ................................................................................................. 106 3.9 Association between urinary excretion of di(2-ethylhexyl)phthalate secondary metabolites and Autism Spectrum Disorders in children .. 109 ii Chiara Testa, PhD Thesis 3.9.1 Autism Spectrum Disorders ................................................................ 109 3.9.2 Etiology of ASDs ................................................................................ 110 3.9.3 Subjects’ population ........................................................................... 111 3.9.4 Statistical analysis ............................................................................... 112 3.9.5 Results ................................................................................................. 112 4 EXPERIMENTAL PART ....................................................................................... 122 4.1 Materials and methods ........................................................................ 122 5 Solid Phase Peptide Synthesis (SPPS). ................................................................... 124 5.1 General procedure for in batch SPPS on a manual synthesizer .......... 124 5.1.1 Conventional and MW-assisted synthesis of PTHrP(1–34)NH2 ........ 125 5.2 Amino Terminal Acetylation. General Procedure .............................. 126 5.3 Deprotection, Cleavage and Purification of Free Peptide. General Procedure ............................................................................................ 126 5.4 General procedure for solid-phase extraction SPE ............................. 127 5.5 MW-assisted Mini-cleavage. General Procedure ............................... 127 5.6 HPLC Analysis of PTHrP(1–34)NH2 ................................................ 128 5.7 UPLC-ESI-MS Analysis of intermediate fragments of PTHrP(1– 34)NH2 ................................................................................................ 128 6 Synthesis of Nα-Fmoc-ω-azido-α-amino acids and Nα-Fmoc-ω-alkynyl-α-amino acids ........................................................................................................................... 129 6.1 Deprotection of Boc-protecting group. General procedure ............... 129 6.2 Fmoc Protection of the Free Amino Acid. General Procedure ........... 129 6.3 Diazotransfert on ω-aminic group of Nα-Boc or Nα-Fmoc-amin acids with triflic anhydride. General procedure ........................................... 129 6.4 Synthesis of imidazole-1-sulfonyl azide hydrochloride as diazotransfer reagent ................................................................................................. 130 6.5 Diazotransfert on ω-aminic group of Nα-Fmoc- amino acids with Imidazole-1-sulfonyl azide hydrochloride. General Procedure .......... 130 6.5.1 6–Azido–2S-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino]hexanoic acid (6-Azido-Fmoc-L-norleucine) (m=4) .......................................... 131 6.5.2 5-Azido-2S-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]