A. Discovery of novel reactivity under the Sonogashira reaction conditions B. Synthesis of functionalized BODIPYs and BODIPY-sugar conjugates Ravi Shekar Yalagala, M.Phil Department of Chemistry Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Faculty of Mathematics and Science, Brock University St. Catharines, Ontario © 2016 ABSTRACT A. During our attempts to synthesize substituted enediynes, coupling reactions between terminal alkynes and 1,2-cis-dihaloalkenes under the Sonogashira reaction conditions failed to give the corresponding substituted enediynes. Under these conditions, terminal alkynes underwent self-trimerization or tetramerization. In an alternative approach to access substituted enediynes, treatment of alkynes with trisubstituted (Z)- bromoalkenyl-pinacolboronates under Sonogashira coupling conditions was found to give 1,2,4,6-tetrasubstituted benzenes instead of Sonogashira coupled product. The reaction conditions and substrate scopes for these two new reactions were investigated. B. BODIPY core was functionalized with various functional groups such as nitromethyl, nitro, hydroxymethyl, carboxaldehyde by treating 4,4-difluoro-1,3,5,7,8- pentamethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene with copper (II) nitrate trihydrate under different conditions. Further, BODIPY derivatives with alkyne and azido functional groups were synthesized and conjugated to various glycosides by the Click reaction under the microwave conditions. One of the BODIPY–glycan conjugate was found to form liposome upon rehydration. The photochemical properties of BODIPY in these liposomes were characterized by fluorescent confocal microscopy. ii ACKNOWLEDGEMENTS I am extremely grateful to a number of people. Without their help, this document would have never been completed. First and foremost, I would like to thank my supervisor and mentor Professor Tony Yan for his guidance and supervision to make the thesis possible. I really appreciate his patience, help and support throughout this thesis work. He treated me with great patience so that I have learned a lot in the lab throughout the years. I could not have imagined having a better advisor and mentor for my graduate study. I would like to extend my gratitude to other members of my committee, Professor Costa Metallinos, Professor Art van der East for their support over the year and for their valuable advice and insightful comments. I thank Tim Jones, Liqun Qiu and Razvan Simionescu for their assistance in collecting data for mass spectrometry and NMR spectroscopy. I also thanks Prof. Jeff Stuart and Lucas Anthony Maddalena for their assistance in collecting data from fluorescence microscope. My sincere thanks also go to my past and current fellow labmates: Ningzhang Zhou, Qiang Wang, Dhruval Joshi, Jia Li, and Nazanin Heidari, Viola, Fatma Elijabu, Emily Bordeleau, Sina Mazinani, Christopher Oberc, David Nguyen, Meesook Jun, Khatma Ali for the stimulating discussions and all the fun we have had. I specially thank few people I met in Brock University, Ningzhang Zhou, Mikel, Christopher, Sina, and another friend Thirupathi (University of Michigan) with whom I gained lots of knowledge due to the stimulating discussions we had. Special thanks to Saba Pirnia for being a great friend who brought up engaging conversations on variety of chemistry/biology topics and memorable friendship. iii I would like to thank the Natural Sciences and Engineering Research Council of Canada for funding this work. I would like to thank the Government of Ontario for the Trillium scholarship for pursuing my Ph.D. Words cannot express how grateful I am to my parents, brother and wife. Their love provided me inspiration and driving force, without their support finishing this journey was not possible. iv TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v LIST OF TABLES vii LIST OF FIGURES ix LIST OF SCHEMES xi LIST OF ABBREVIATIONS xiv Chapter 1 1.1 Enediyne-containing natural products 2 1.2 Mechanism of action of enediynes 4 1.3 Synthetically prepared enediynes and their biological activities 7 1.4 Potential of enediyne as site-specific DNA cutters 18 1.5 General perspectives and objectives of this project 20 1.5.1 Synthesis and conjugation of enediyne functionality to oligo- 20 nucleotides 1.5.2 Investigation of the self-oligomerization of terminal alkyne 22 1.5.3 Reaction between (Z)-bromoalkenylpinacol boronates and terminal 22 alkyne Chapter 2 2.1 Introduction to fluorophores 24 2.2 BODIPY as a fluorophore 26 2.3 Methodologies for the synthesis of BODIPY 27 2.4 Functionalization of the BODIPY core 29 2.4.1 Electrophilic substitution reactions 29 v 2.4.2 Nucleophilic substitutions of halogenated BODIPYs 33 2.4.3 Palladium catalyzed functionalization of BODIPY compounds 36 2.4.4 Condensation reactions 38 2.4.5 C-H activation 38 2.5 Functional groups on the BODIPY core for conjugation purposes 41 2.5.1 Succinimidyl esters 42 2.5.2 Isothiocyanate BODIPY dyes 43 2.5.3 Iodoacetamide BODIPY dyes 45 2.5.4 Maleimide BODIPY dyes 46 2.5.5 Thiosulfate and thiosulfonate BODIPY dyes 49 2.6. Bio-orthogonal labeling of biomolecules 51 2.6.1 BODIPY-hydrazine, hydrazide or aminooxy compounds 51 2.6.2 Azide alkyne click reaction 54 2.6.3 Tetrazene-alkene click reaction 56 2.7 General perspectives and objectives of the present work 58 Chapter 3 3.1 Homotrimerization and tetramerization of terminal alkynes 60 3.1.1. Synthesis of the designed substituted enediynes 62 3.1.2 Homocoupling of terminal alkynes 65 3.1.3 Optimization of the reaction conditions for the self-trimerization of 67 terminal alkynes 3.1.4 Exploring the substrate scope for the self-trimerization of terminal 69 alkynes 3.1.5 Characterization of the trimerized alkyne products by NOE and 71 HMBC NMR spectroscopy 3.1.6 Conclusion 73 3.2 Reactions of terminal alkynes with trisubstituted (Z)-bromoalkenyl 74 pinacol boronates towards the synthesis of 1,2,4,6-tetrasubstituted benzenes vi 3.2.1 Synthesis of pinacolboronate and bromoalkene 74 3.2.2 Optimization of the reaction conditions for the aromatization 76 reaction 3.2.3 Exploring the substrate scope for the aromatization reaction 78 3.2.4 Mechanistic investigations of the aromatization reaction between a 80 bromoalkene boronate and an alkyne 3.2.5 Conclusion 82 3.3 Reactions of BODIPY with cupric nitrate 83 3.3.1 Various transformations of BODIPY with Cu(NO3)2•3H2O 83 3.3.2 Optimization of the reaction conditions for the synthesis of 3-nitro 85 BODIPY 174b 3.3.3 Conclusion 88 3.4 Microwave-assisted synthesis of BODIPY-sugar conjugates through 89 Click chemistry and assembly of the conjugate into liposomes 3.4.1 Synthesis of BODIPY derivatives with alkyne and azido functional 90 groups 3.4.2 Synthesis of BODIPY-sugar conjugates through click reaction 91 3.4.3 Formation of liposomes by the BODIPY-sugar conjugate 94 3.4.4 Conclusion 97 Chapter 4 4.1 General Experimental Procedures 99 4.2 Detailed Experimental Procedures 100 5. References 140 vii LIST OF TABLES Table 3-1: Optimization of reaction conditions described in Scheme 3- 67 2B Table 3-2: Homocoupling products of terminal alkynes 70 Table 3-3: Optimization of aromatization conditions 77 Table 3-4: Substrate scope of the aromatization reaction 79 Table 3-5: Optimization of conditions for the synthesis of 3-nitro 86 BODIPY 65b viii LIST OF FIGURES Figure 1-1: Natural enediyne compounds 3 Figure 1-2: Bergman cyclization 4 Figure 1-3: Structure of calicheamicin 5 Figure 1-4: The activation and DNA cleavage mechanism of Calicheamicin 5 Figure 1-5: DNA cleavages by abstracting the 5’-H of sugar backbone 6 Figure 1-6: First generation of synthetic enediynes 8 Figure 1-7: Enediynes with substituted amine and the corresponding 9 cyclization temperature Figure 1-8: Enediynes with aromatic and hetero aromatic substitutions 10 Figure 1-9: Enediynes with a hydroxyl side chain 11 Figure 1-10: Analogues of dynemycin 15 Figure 1-11: Covalent bond formation between DNA and a PBD 16 Figure 1-12: PBDs conjugated enediynes 17 Figure 1-13: Enediynes with similar structure of Combretastatin 18 Figure 1-14: EDTA conjugated oligo nucleotide 19 Figure 1-15: EDTA containing thymidine 20 Figure 2-1: Structures of fluorescent dyes 25 Figure 2-2: Numbering scheme for BODIPY framework derived from 26 indacene Figure 2-3: Commercially available BODIPY probes from Molecular Probes 27 Figure 2-4: Mesomeric structures of BODIPY core 29 Figure 3-1: Cleavage of target DNA by enediyne 60 Figure 3-2: Designed enediyne phosphoramidite 61 Figure 3-3: Possible structures of trimerized product 152 b 71 Figure 3-4: 1D NMR spectra of compound 152 b in DMSO-d6 72 ix Figure 3-5: HMBC spectrum of compound 152b in DMSO-d6 72 Figure 3-6: Bromoboronate alkene designed for sequential Sonogashira and 74 Suzuki coupling reactions Figure 3-7: Products generated in the aromatization reactions with various 80 alkene and alkyne substrates Figure 3-8: Two intermediates isolated in the aromatization reaction 81 Figure 3-9: Glycans with either an alkyne or azide 89 Figure 3-10: NOESY spectra of click reaction product 94 Figure 3-11: A cartoon illustrating the bilayer formed by the BODIPY–sugar 95 conjugates in aqueous solutions Figure 3-12: Bright field microscope image of a cluster of liposomes 96 formed from BODIPY-conjugate of sugar 185 x LIST OF SCHEMES Scheme 1-1: Enediynes with phosphine ligands 12 Scheme 1-2: Enendiynes with bypiridyl ligands 13 Scheme 1-3: DNA cleavages by photo enediyne activated by deprotection of 14 a photolabile amino protecting