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Joana Salta Phd Thesis to Submitt ADVANCES IN THE SYNTHESIS OF AMINOPYRAN -BASED CARBOHYDRATE MIMETICS AND THEIR EVALUATION AS SELECTIN INHIBITORS Inaugural-Dissertation Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt dem Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin Eingereicht von M. Sc. JOANA SALTA aus Lissabon, Portugal September 2014 1. Gutachter: Prof. Dr. Hans-Ulrich Reißig 2. Gutachter: Prof. Dr. Christian P. R. Hackenberger Promotionsdatum: 30.10.2014 To my parents The submitted doctoral thesis has been carried out under the supervision of Professor Dr. Hans-Ulrich Reißig in the period from August 2010 to September 2014 at the Institut für Chemie und Biochemie des Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin. Content Abbreviations 1 Abstract 3 German abstract 4 Summary 5 1. Introduction 12 1.1. Carbohydrate chemistry 12 1.1.1. Sulfated carbohydrates 12 1.1.2. Carbohydrate mimetics 13 1.1.3. Low molecular weight carbohydrate mimetic inhibitors 14 1.2. Multivalent interactions 16 1.2.1. Multivalent binding effects 16 1.3. Selectin-carbohydrate interactions 17 1.3.1. Selectin role during the inflammation process 17 1.3.2. Selectin structure and ligands 18 2. Scientific background 21 3. Aim of the present work 26 4. Results and Discussion 28 4.1. Synthesis of starting materials 28 4.1.1. syn-1,2-Oxazine synthesis 28 4.1.2. Lewis acid-induced rearrangement 28 4.1.3. Stereoselective reduction with sodium borohydride 29 4.1.4. Hydrogenolysis 33 4.1.5. Copper(II)-catalyzed diazotransfer 37 4.1.6. Introduction of protecting groups 39 4.1.7. Conversion of azidopyrans into aminopyrans 41 4.2. Multivalent compounds by amide bond formation 45 4.2.1. Amide bond formation by Schotten-Baumann reaction 45 4.2.1.1. Synthesis of monovalent carbohydrate mimetics 46 4.2.1.2. Synthesis of divalent carbohydrate mimetics 46 4.2.1.3. Synthesis of divalent 2-amino-1,3-diol derivatives 50 4.2.2. Amide bond formation by HATU coupling reagent 52 4.2.2.1. Synthesis of monovalent carbohydrate mimetics 54 4.2.2.2. Synthesis of divalent carbohydrate mimetics 54 4.2.2.3. Synthesis of trivalent carbohydrate mimetics 55 4.2.2.4. Synthesis of trivalent 2-amino-1,3-diol derivatives 56 4.3. Multivalent compounds by reductive amination 57 4.3.1. Synthesis of monovalent carbohydrate mimetics 58 4.3.2. Synthesis of divalent carbohydrate mimetics 59 4.3.3. Synthesis of divalent 2-amino-1,3-diol derivatives 63 4.4. Multivalent compounds by “click” chemistry 64 4.4.1. 1,3-Dipolar cycloaddition 64 4.4.2. Acid/Base-catalyzed azide-alkyne cycloadditions 68 4.4.3. Copper on charcoal-catalyzed azide-alkyne cycloadditions 68 4.4.4. Synthesis of monovalent carbohydrate mimetics 69 4.4.4.1. Evaluation of trace metal content by ICP-MS 73 4.4.5. Synthesis of divalent carbohydrate mimetics 75 4.4.6. Synthesis of trivalent carbohydrate mimetics 79 4.4.7. Synthesis of tetravalent carbohydrate mimetics 81 4.4.8. Synthesis of bigger architectures 82 4.4.9. Metal-free 1,2,3-triazole formation 91 4.5. Polysulfation of carbohydrate mimetics 97 4.5.1. 2-Amino-1,3-diol derivatives 101 4.5.2. Monovalent carbohydrate mimetics 104 4.5.3. Divalent carbohydrate mimetics 105 4.5.4. Trivalent carbohydrate mimetics 107 4.5.5. Tetravalent carbohydrate mimetics 109 4.5.6. Other oligovalent compounds 110 4.6. Selectin inhibition 113 4.6.1. Surface plasmon resonance 113 4.6.2. L-Selectin 115 4.6.2.1. Screening of compounds 115 4.6.2.2. Monovalent carbohydrate mimetics 118 4.6.2.3. 2-Amino-1,3-diol derivatives 119 4.6.2.4. Divalent carbohydrate mimetics 119 4.6.2.5. Trivalent carbohydrate mimetics 122 4.6.3. P-Selectin 124 4.6.4. E-Selectin 125 4.6.5. Comparison with previously reported selectin inhibitors 127 5. Summarizing discussion and Outlook 129 6. Experimental Part 133 6.1. General information 133 6.1.1. Analytical methods 133 6.1.2. Chromatography 133 6.1.3. Preparative methods, solvents and reagents 134 6.1.4. General procedures 135 6.2. Experimental procedures 138 6.2.1. Synthesis of starting materials 138 6.2.1.1. Hydrogenolysis 139 6.2.1.2. Copper(II)-catalyzed diazotransfer 141 6.2.1.3. Introduction of protecting groups 143 6.2.1.4. Conversion of azidopyran into aminopyran 147 6.2.2. Amide bond formation 149 6.2.2.1. Schotten-Baumann reaction 149 6.2.2.2. HATU coupling reagent 167 6.2.3. Reductive amination 174 6.2.4. Copper(I)-catalyzed azide alkyne cycloaddition 187 6.2.5. Metal-free 1,2,3-triazole formation 209 6.2.6. Miscellaneous subsequent reactions 213 6.2.7. Polysulfation 215 6.3. Surface plasmon resonance (SPR) assay 226 6.3.1. Assay protocol 226 6.3.2. Data evaluation 227 6.3.3. Experimental data 228 Literature 235 Acknowledgment 239 Curriculum Vitae 241 Abbreviations Abbreviations In the text, numbers of the compounds are given in bold font. In the experimental part, experiments are indicated as JNS#, as appears in the laboratory notebooks. The following abbreviations are used throughout the entire text. Ac Acetyl Ar Aryl Bn Benzyl bp Boiling point brs Broad singlet Bu n-Butyl t-Bu tert -Butyl COSY 1H-NMR correlated spectroscopy Cu/C Copper on charcoal CuAAC Copper(I)-catalyzed azide alkyne cycloaddition δ Chemical shift d Day(s) or Doublet DIPEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF N,N-Dimethylformamide DMSO Dimethylsulfoxide equiv. Stoichiometric equivalent(s) ESI-TOF Electrospray ionization time of flight mass spectrometry Et Ethyl EtOAc Ethyl Acetate g Gram GP General Procedure h Hour(s) HMQC Heteronuclear multiple quantum correlation HOAc Acetic Acid HRMS High resolution mass spectrometry Hz Hertz 1 Abbreviations IR Infrared J Coupling constant lit. Literature value m Multiplet M Molarity MHz Megahertz Me Methyl min Minute(s) Nf Nonaflyl MS Mass spectrometry NMR Nuclear magnetic resonance spectroscopy Pd/C Palladium on charcoal Ph Phenyl ppm Parts per million q Quartet quant. Quantitative, yield > 99.5% R Organic substituent r.t. Room temperature s Singlet sLe x Sialyl-Lewis-X SPR Surface plasmon resonance t Triplet TBTA Tris-[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl]amine TBS tert -Butyldimethylsilyl THF Tetrahydrofuran TLC Thin layer chromatography TMSE Trimethylsilylethyl ν Wave number 2 Abstract Abstract The aim of this dissertation was the development of new enantiopure multivalent carbohydrate mimetics and their study as potential selectin inhibitors. The main building blocks have been synthetized following a previously established synthetic route, starting from lithiated 2-(trimethylsilyl)ethoxyallene and a chiral D-glyceraldehyde-derived aldonitrone. The obtained 1,2-oxazine undergoes a stereoselective Lewis acid-promoted rearrangement leading to a bicyclic ketone which can be transformed into the corresponding carbohydrate mimetic with two straightforward steps: reduction of the carbonyl group followed by the cleavage of the N-benzyl and N-O bonds leads to an enantiopure aminopyran derivative. In the present work several transformations were made from this key building block including the conversion to the corresponding stable azidopyran prepared by a copper-catalyzed diazotransfer. Using the amino group and choosing adequate linkers, a library of multivalent compounds was prepared via Schotten-Baumann reaction or reductive amination. Additionally, different conditions for the Huisgen-Sharpless-Meldal cycloaddition were used to synthesize carbohydrate mimetics with different degrees of multivalency. In this approach, aspects that can influence multivalency binding were considered as linker length, symmetry and number of pyran ligands. As final and most challenging step, some of the obtained carbohydrate mimetics could successfully be polysulfated and tested with and without sulfate groups in a surface plasmon resonance (SPR) inhibition assay as potential selectin inhibitors. The tested compounds showed IC 50 values from micro- to nanomolar range (for L- and P- selectin), which are promising results for such conjugates. Overall, a straightforward synthetic route towards new multivalent carbohydrate mimetics was developed and the first results were obtained from the SPR inhibition assays providing some leads to further development of selectin inhibitors. 3 German abstract German abstract Ziel dieser Arbeit war die Darstellung neuer enantiomerenreiner multivalenter Kohlenhydratmimetika und die Evaluierung ihre Aktivität als potentielle Selektin-Inhibitoren. Der zentrale Baustein wurde ausgehend von einer zuvor etablierten Methode aus lithiiertem 2- (Trimethylsilyl)ethoxyallen und einem chiralen von D-Glyceraldehyd abgeleiteten Aldonitron synthetisiert. Das erhaltene 1,2-Oxazin wurde durch eine stereoselektive Lewis-Säure- induzierte Umlagerung in ein bicyclisches Keton umgewandelt, welches dann in zwei direkten Schritten in das Kohlenhydratmimetika übergeführt wurde: die Reduktion der Carbonyl-Gruppe und die anschließende Spaltung der N-Benzyl- und der N-O-Bindungen liefert ein Aminopyran. In der vorliegenden Arbeit wurden verschiedene Transformationen mit diesem Schlüsselbaustein durchgeführt, zum Beispiel die Darstellung des entsprechenden stabilen Azidopyrans durch Kupfer-katalysierten Diazotransfer. Durch die freie Aminogruppe und adequate Linker konnte eine Vielzahl von multivalenten Verbindungen durch Schotten- Baumann-Reaktion oder durch reduktive Aminierung dargestellt werden. Zusätzlich wurden unter verschiedenen Reaktionsbedingungen der Huisgen-Meldal-Sharpless-Reaktion Verbindungen mit unterschiedlichem Grad an Multivalenz synthetisiert. Dabei wurden verschiedene
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