Table of contents. Bibliografische Informationen digitalisiert durch

Transcription

1 1. Research scope: The role of structure rigid'tf'ication in nature and chemistry 1 2. Establishing a Dha=Tap backbone scan in order to elucidate structural properties of the N-terminusofNPY Introduction. A seemingly endless conundrum: understanding and controlling the receptor promiscuity of NPY The NPY family NPY in the human organism: perspectives and problems The multi-talented NPY: receptor affinities and physiological roles NPY/receptor complexes: analytical options The app crystal structure and its transferability to NPY The discussion of the NPY structure in solution NPY in membrane-mimetic environment Water-suppression techniques The theory of membrane-assisted receptor selection The underestimation of the role of the N-terminus: a motivation for a novel scan The concept of the Dha=Tap backbone scan Research context: The art to gain maximum information from designed peptide analogs Earlier work accomplished for NPY analogs Motivation: The need for novel scanning strategies The dipeptide backbone scan: A consequent advancement of existing scanning strategies Results and discussion The backbone scan reveals distinct receptor trends Overview: The Yj and Y s subtypes differentiate matched- and mismatched substitutions Further influences: Substitution number, total charge, and aromaticity of the N-terminus Substitution variation patterns reveal additive, over-additive and compensative effects A key puzzle piece: Lining up Dha=Tap units towards the C-terminus results in Y 5 selectivity Probing the NPY receptor selectivity with NMR studies Peptide and protein structure elucidation by NMR: strategy and methodology NMR experiments: General considerations and experimental setup Native pnpy: signal assignment and secondary structure 59 Bibliografische Informationen digitalisiert durch

2 ii Table of Contents 'H DPFGSE-NMR spectrum Signal assignment by 2D NMR experiments: NOESY, TOCSY, and HSQC Single-substituted NPY analogs [2=3], [5=6], and [8=9]: The Dha=Tap unit influences the backbone conformation according to its position Overview The analog [5=6] The analogs [2=3] and [8=9] The triple-substituted analog [2=3,5=6,8=9] The position-selective line-broadening in the 'H NMR: reasons and implications The structural impact of triple Dha=Tap incorporation: Short-, medium- and longe-range effects Molecular modeling: The role of Pro" amide conformation and implications on the term "flexibility" A hypothesis about the determinants of Y ( and Y 5 receptor binding Summary and outlook 93 3 Iminopeptides as key to the thermodynamic analysis of macrocylizations: The ring-chain equilibria of peptide aldehydes Introduction: The role of macrocyclizations in biology and medicine Examples of macrocyclizations in natural product biosynthesis Macrocyclizations in medicinal chemistry and chemical synthesis The lack of experimental setups for the thermodynamic characterization of biological macrocyclizations Previous work: kinetic experiments Rotamer incrementation as theoretical basis for an experimental setup Ill The complexity of molecular systems: from rotamers to conformers to dynamics "Flexibility" and "rigidity": How to use terms from the macroscopic world for microscopic systems From chains to rings: the macrocyclization process Our approach: an experimental setup combined with the simple numerical model The Nostocyclopeptides: rare examples of natural products with imino functions Imines in natural products and in dynamic covalent chemistry Previous work on nostocyclopeptide biosynthesis and in vitro macrocyclization Task of this study Results and discussion: Ring-chain equilibria of the nostocyclopeptides 127 Ill

3 iii Synthesis of the peptide aldehydes NMR assignment and structure elucidation General considerations: nomenclature and experimental setup The linear peptides Macrocyclic mine formation: ph dependence of the equilibria Spontaneous cyclizations: monitoring the selective macrocyclization process by NMR Quantitative investigations: The ph dependence of ncpal and ncpa2 equilibrium Structure elucidation Hydrogen bonding Conformational differences of the C-termini in the ncpal and ncpa2 cydization precursor Side-chain orientations in the cyclopeptides determined by rptamer distributions NOE-based molecular modeling The entropy balance of ncp macrocyclization General considerations and experimental setup Experiments and results Determination of the temperature-dependent equilibrium compositions Calculation of the macrocyclization enthalpy and entropy balances The entropy balances of the peptide chains Discussion of the entropy values: implications for the role of preorganization Interim conclusion: The carbonyl substitution as logical next step Results and discussion: The ring-chain equilibrium of Tyrocidine A aldehyde Tyrocidine Aas test substrate for carbonyl functionalization Experimental setup Cyclization studies Stability of the hemiaminal Stereopurity of the hemiaminal Thermodynamics of TycA macrocyclization Structural determinants of macrocyclization entropy Comparison of the ncp and TycA-CHO rnacrocyclization processes Preliminary studies: The side chain-to-backbone cydization of segetalinal A Summary and outlook 191

4 4 Synthesis of polyhydroxylated thiazoles from sugar/amino acid condensation products Thiazole units in peptidic natural products Ribosomal and non-ribosomal heterocycle formation Biological roles of thiazoles Thiamine (Vitamin Bl): nucleophilic catalysis Tubulysins, hectochlorin and microcin B17: protein interaction Bleomycin A and the patellamides: DNA intercalation and redox catalysis Thiopeptide antibiotics Chemical thiazole synthesis: Strategies and problems Two-component reactions Cyclizations of linear precursors C-C couplings and multicomponent reactions Total syntheses of Xaa<Thz dipeptides present in thiopeptide antibiotics Motivation and aims of this study Results and discussion Synthesis and isolation of the thiazolidine condensation products Hydroxyl protection and oxidation to the thiazole lactones Deprotection and lactone opening Interim summary and outlook: depsidipeptide targets Selective hydroxyl protections and further lactone opening experiments D-gluco configured substrates D-arabino configured substrates Lactone opening of protected substrates Chain tailoring experiments Interim summary and outlook: dipeptide targets Thiazole dipeptide synthesis with hexose sugar chain substrates Experiments with D-gluco derivatives A thiazolidine lactam as synthetic back-door: synthesis of altrosamine<thz dipeptides Thiazole dipeptide synthesis with pentose sugar chain substrates The stereoconfiguration of Dyg<Thz in nocathiacin Activation and azide introduction: Identification of the appropriate protection pattern Synthesis of 25 and 2R configured Dyg<Thz dipeptide motifs Summary and outlook 307

5 Table of Contents 5 Experimental section General: Materials and methods Experimental data: chapter Synthesis of the Dha=Tap building block Peptide synthesis and purification Binding assays NMR measurements and data Molecular modeling Experimental data: chapter Peptide aldehyde syntheses and purification procedures Preparation of ncpal-cho, ncpa2-cho, and TycA-CHO Preparation of SegA'-CHO and SegA'-CO^H Synthesis of W'-Fmoc-Gly W.O-dimethylhydroxamicacid Synthesis of W'-Fmoc-Gly-H SPPS protocol A: Loading of H-Thr-GlyNovaSyn TG resin with C-terminal amino acid aldehyde and capping of oxazolidine nitrogen as Boc carbamate SPPS protocol B: Manual synthesis of peptide aldehydes SPPS protocol C: Manual synthesis of peptides Peptide purification 3nd analysis NMR measurements and data Molecular modeling data ph and temperature dependence of the macrocyclization equilibria ncpal and ncpa2 equilibria TycA ring/chain equilibrium SegA side chain-to-backbone cydization equilibrium Experimental data: chapter 4 - synthetic procedures References 494