Science, Irving K. Barber Faculty of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Tardif, Anthony
Master of Science - MSc
University of British Columbia
Kainate receptors (KARs) are classified as one of three main subfamilies of ionotropic glutamate receptors (iGluRs) located in the brain. They have been shown to participate in neurological homeostasis and normal brain development processes such as synapse elimination; however, excessive synapse elimination is a hallmark of neurodegenerative diseases. Despite a strong association between KARs and neurodegeneration, our understanding of the underlying mechanisms that contribute to excessive synapse elimination is hindered by the lack of chemical tools required to study them. The development of new chemical tools to study KARs will provide a better understanding of their role in neurodegenerative processes and help identify therapeutics to treat related symptoms and diseases. This thesis details the synthesis of a fluorescent kainate probe that selectively labels KARs to address this knowledge gap. The kainate probe consists of three smaller molecules; a kainoid ligand, a linker, and a squaraine fluorophore which were all individually synthesized and then coupled to assemble the probe. Chapter 1 provides a brief introduction to kainate receptors and their role in the brain, as well as an overview of fluorescent protein imaging methods and the concept of turn-on fluorescence. Chapter 2 reviews the structural features that are essential to designing kainoid ligands, precedents in the kainoid syntheses, and the synthesis of a novel kainoid analog, (4R)-4-azidokainic acid, to enable late-stage derivatization at the C4 position of kainic acid. Chapter 3 contextualizes the biological applications of squaraine dyes and how they can be utilized to achieve turn-on fluorescence with improved contrast during fluorescent imaging. Next, it describes the syntheses of squaraine and aminosquaraine intermediates, along with an alkyne linker. Chapter 3 concludes with a detailed account of the synthetic steps performed and the challenges faced towards assembling the kainate probe. Overall, the synthesis of a novel kainoid analog, (4R)-4-azidokainic acid, serves as a versatile kainoid intermediate, with late-stage modularity, for the development of new kainate probes. The synthetic routes established in Chapter 3 constitute significant progress towards a unique kainate probe with selective turn-on fluorescence to study kainate receptors.
DEVELOPMENT OF AN AFFINITY-GUIDED PROBE WITH TURN-ON FLUORESCENCE TO SELECTIVELY LABEL KAINATE RECEPTORS by Anthony Tardif B.Sc., University of British Columbia (Okanagan), 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) February 2024 © Anthony Tardif, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: DEVELOPMENT OF AN AFFINITY-GUIDED PROBE WITH TURN-ON FLUORESCENCE TO SELECTIVELY LABEL KAINATE RECEPTORS submitted by Anthony Tardif in partial fulfillment of the requirements of the degree of Master of Science. Dr. Frederic Menard, Associate Professor, Department of Chemistry, UBC Supervisor Dr. Stephen McNeil, Associate Professor, Department of Chemistry, UBC Supervisory Committee Member Dr. Kevin Smith, Professor, Department of Chemistry, UBC Supervisory Committee Member Dr. Adam Ford, Associate Professor, Department of Biology, UBC University Examiner Dr. John-Tyler Binfet, Associate Professor, Okanagan School of Education, UBC Neutral Chair iii Abstract Kainate receptors (KARs) are classified as one of three main subfamilies of ionotropic glutamate receptors (iGluRs) located in the brain. They have been shown to participate in neurological homeostasis and normal brain development processes such as synapse elimination; however, excessive synapse elimination is a hallmark of neurodegenerative diseases. Despite a strong association between KARs and neurodegeneration, our understanding of the underlying mechanisms that contribute to excessive synapse elimination is hindered by the lack of chemical tools required to study them. The development of new chemical tools to study KARs will provide a better understanding of their role in neurodegenerative processes and help identify therapeutics to treat related symptoms and diseases. This thesis details the synthesis of a fluorescent kainate probe that selectively labels KARs to address this knowledge gap. The kainate probe consists of three smaller molecules; a kainoid ligand, a linker, and a squaraine fluorophore which were all individually synthesized and then coupled to assemble the probe. Chapter 1 provides a brief introduction to kainate receptors and their role in the brain, as well as an overview of fluorescent protein imaging methods and the concept of turn-on fluorescence. Chapter 2 reviews the structural features that are essential to designing kainoid ligands, precedents in the kainoid syntheses, and the synthesis of a novel kainoid analog, (4R)-4-azidokainic acid, to enable late-stage derivatization at the C4 position of kainic acid. Chapter 3 contextualizes the biological applications of squaraine dyes and how they can be utilized to achieve turn-on fluorescence with improved contrast during fluorescent imaging. Next, it describes the syntheses of squaraine and aminosquaraine intermediates, along with an alkyne linker. Chapter 3 concludes with a detailed account of the synthetic steps performed and the challenges faced towards assembling the kainate probe. Overall, the synthesis of a novel kainoid analog, (4R)-4-azidokainic acid, serves as a versatile kainoid intermediate, with late-stage modularity, for the development of new kainate probes. The synthetic routes established in Chapter 3 constitute significant progress towards a unique kainate probe with selective turn-on fluorescence to study kainate receptors. iv Lay summary When most people hear the word “protein” they may think of meats, supplements, or muscles. However, from a scientist's point of view, the term has a different meaning. Proteins are large complex molecules composed of smaller molecules. They are essential to nearly every structure and function in the human body. Kainate receptors (KARs) are a type of protein found in the brain and they play an important role in communication between nerve cells. KARs are also known to be involved in neurodegeneration which is prevalent in diseases such as Alzheimer’s and Parkinson’s. Unfortunately, there is a current lack of chemical tools that can selectively target KARs which limits our understanding of their involvement in neurodegenerative processes. My project is on the development of a molecular probe to study KARs using light. The probe is hypothesized to improve typical imaging contrast through its unique turn-on fluorescence after labeling the receptor. v Table of Contents Abstract.............................................................................................................................................................................. iii Lay summary .................................................................................................................................................................... iv List of tables .................................................................................................................................................................... vii List of figures ................................................................................................................................................................. viii List of schemes.................................................................................................................................................................. x List of symbols and abbreviations ................................................................................................................................ xi Acknowledgments .........................................................................................................................................................xiv CHAPTER 1: INTRODUCTION................................................................................................... 1 1.1 Kainate receptors and their neurophysiological function in the brain .................................................................. 1 1.2 Fluorescent protein labeling............................................................................................................................................... 4 1.2.1 Affinity-guided organic fluorescent probes ....................................................................................................... 5 1.2.2 Turn-on fluorescent probes ................................................................................................................................... 6 1.3 Research objectives and thesis overview ........................................................................................................................ 8 1.3.1 Research hypothesis and objectives .................................................................................................................... 8 1.3.2 Thesis overview ...................................................................................................................................................... 9 CHAPTER 2: THE SYNTHESIS OF (4R)-4-AZIDOKAINIC ACID AND OTHER KAINOIDS ............................................................................................................................... 12 2.1 Structure-activity relationship of kainic acid-based analogs towards glutamate receptors........................... 12 2.1.1 Key structural features in biologically active kainoids .................................................................................. 13 2.1.2 Proposed kainoid structural modifications ....................................................................................................... 15 2.2 Precedents in kainoid syntheses ...................................................................................................................................... 15 2.3 Proposed synthesis of (4R)-4-azidokainic acid ............................................................................................................ 18 2.4 Installation of protecting groups and C3 alkylation.................................................................................................. 19 2.5 Stereoselective reduction of the ketodiester ................................................................................................................. 22 2.6 Installation of the C4-azide and global deprotection ................................................................................................ 24 2.7 Derivatizing azidokainoid intermediates with a CuAAC reaction ........................................................................ 25 2.8 Summary ............................................................................................................................................................................... 28 2.9 Experimental ........................................................................................................................................................................ 28 2.9.1 General experimental procedures ...................................................................................................................... 28 2.9.2 Materials................................................................................................................................................................. 29 vi 2.9.3 Instruments ............................................................................................................................................................ 29 2.9.4 Experimental procedures and characterization................................................................................................ 30 CHAPTER 3: SYNTHESIS OF METHOXYSQUARAINE, AN ALKYNE LINKER, AMINOSQUARAINE DERIVATIVES, AND THE SQ-KA PROBE ........................................ 46 3.1 Squaraine dyes: A unique and promising biological fluorophore ......................................................................... 46 3.2 Synthesis of a methoxysquaraine advanced intermediate ....................................................................................... 49 3.2.1 Optimizing the ethylation of 2-methylbenzothiazole ..................................................................................... 49 3.2.2 Formation of the squaraine dimer and O-methylation ................................................................................... 51 3.3 Synthesis of the alkyne linker .......................................................................................................................................... 55 3.4 Synthesis of aminosquaraine derivatives...................................................................................................................... 56 3.5 Synthesis towards a squaraine-kainate probe ............................................................................................................. 57 3.6 Experimental ........................................................................................................................................................................ 59 3.6.1 General experimental procedures ...................................................................................................................... 59 3.6.2 Materials................................................................................................................................................................. 60 3.6.3 Instruments ............................................................................................................................................................ 60 3.6.4 Experimental procedures and characterization................................................................................................ 61 3.6.4.1 Alkyne linker ....................................................................................................................................... 61 3.6.4.2 Methoxysquaraine ............................................................................................................................... 64 3.6.4.3 Aminosquaraines ................................................................................................................................. 67 3.6.4.4 Squaraine-kainate probe..................................................................................................................... 69 CHAPTER 4: CONCLUSION ..................................................................................................... 71 REFERENCES ............................................................................................................................. 73 APPENDIX A: NMR SPECTRA ................................................................................................. 77 A1 Kainoids ..................................................................................................................................................... 79 A2 Alkyne linker ..........................................................................................................................................106 A3 Methoxysquaraine and aminosquaraines ...........................................................................................111 A4 Squaraine-kainate probe........................................................................................................................124 vii List of tables Table 2.1 Reaction conditions for the stereoselective alkylation of ketoproline 6. .......................21 Table 2.2 Edelmann’s reported crude dr at varying reaction temperatures and times. .................22 Table 2.3 Experimental and literature conditions for the reduction and cyclization .....................23 Table 2.4 The ratio of recovered compounds after the displacement reaction determined via NMR...............................................................................................................................25 Table 3.1 Reaction condition for the formation of thiazole 11. .....................................................50 Table 3.2 Reaction conditions for the formation of squaraine dimers with comparison to literature. ........................................................................................................................54 Table 3.3 Reaction conditions for the synthesis of aminosquaraines. ...........................................57 viii List of figures Figure 1.1 A tetrameric iGluR in complex with glutamate. Different colours represent each of the four identical subunits that make up the protein. (PDBs: 6KZM and 4MH5). ....... 2 Figure 1.2 Schematic representation of a synapse containing Glu- and GABA-releasing terminals in which KARs are localized on both pre- and post-synaptic neurons modulating the release of Glu and GABA neurotransmitters. ...................................... 3 Figure 1.3 Kainic acid and domoic acid are natural products exogenous to the mammalian CNS, however, they resemble the neurotransmitter glutamate as conformationally restricted analogs with superior binding affinity to KARs than glutamate. ................. 3 Figure 1.4 Schematic representations of (A) a generic affinity probe and (B) affinity-guided labeling. (C) Relative size comparison of GFP (green, 27 kDa), fused to an example target protein (cyan), next to the popular Cy5 organic fluorophore (orange, ~0.5 kDa). PDBs: 5MFC (left) and 5NS3 (right).................................................................. 6 Figure 1.5 Schematic depiction of ToF probes exhibiting aqueous-induced aggregation and protein-induced disassembly resulting in protein-selective turn-on fluorescence. ....... 7 Figure 1.6 BODIPY (Mizusawa) and squaraine (He) hydrophobic fluorophores utilized for ToF probes.20–22 .................................................................................................................... 7 Figure 2.1 (A) Relative activity of Glu, KA, and DomA towards GluRs and normalized to KA. (B) A relative quantitative activity scale to compare activity induced by kainoids in GluR assays................................................................................................................. 12 Figure 2.2 Examples of kainoids that demonstrated little to no biological activity, highlighting the limited scope of modification for biologically active analogs. ............................. 13 Figure 2.3 GluK2 receptor in complex with (A) kainic acid and (B) domoic acid (3C33, 2PBW). The dotted lines represent ligand-residue interactions (yellow = hydrogen bonding, magenta = π– π interactions)........................................................................ 14 Figure 2.4 Kainate agonists that induce strong activity towards receptors................................... 14 Figure 2.5 (A) Structure of the proposed kainoid ligand incorporated into the probe. (B) Relative biological activity of thiazole kainoids. ........................................................ 15 Figure 2.6 (A) Previously reported syntheses of kainic acid and (B) kainoid derivatives. .......... 17 Figure 2.7 (A) Tian’s divergent synthetic route from 4-hydroxyproline to synthesize the three highlighted kainoid analogs. (B) Tian’s sulfo-Cy5-KA probe synthesized from 4-alkynylkainic acid. ...................................................................................................... 18 Figure 2.8 Proposed mechanism for the formation of lactone 8c from mesylate 9. ..................... 25 Figure 3.1 Generic scaffolds of cyanines and squaraines. The aryl (Ar) groups of squaraines are conjugated through the squarate core and contain at least one nitrogen each, one of which is positively charged......................................................................................... 46 Figure 3.2 Schematic depiction of turn-on fluorescent probes exhibiting aqueous-induced aggregation and protein-induced disassembly resulting in protein-selective turn-on fluorescence. ............................................................................................................... 47 ix Figure 3.3 (A) General BODIPY-based probe reported by Mizusawa et al.20,21 (B) SQ-biotin probe. (C) Relative fluorescence intensity of SQ-biotin (5 μM) in the absence and presence of avidin (3.85 μM), and SQ-biotin (5 μM) with free-biotin (77 μM) and avidin (3.85 μM). (D) Relative fluorescence intensity of a biotin-less SQ (5 μM) in the presence of avidin (0–5.03 μM). Fluorescence intensity graphs were reported by He et al and provided here with additional labels. 22................................................... 48 Figure 3.4 The proposed squaraine-kainate (SQ-KA) probe (1). ................................................. 49 Figure 3.5 Squaraine 12 after recrystallization in DCM/MeOH................................................... 53 Figure 3.6 Tian’s sulfo-Cy5-KA kainate probe and the SQ-KA probe 1 (this work). ................. 58 x List of schemes Scheme 1.1 Retrosynthetic breakdown of the squaraine-kainate probe (1).................................... 8 Scheme 1.2 Synthesis of (4R)-4-azidokainic acid (4): 1) esterification, 2) N-protection, 3) Swern oxidation, 4) C4-regio- and stereoselective alkylation, 5) C4-stereoselective reduction, 6) diastereomer separation, 7) mesylation, 8) C4-stereoinversion, 9) global deprotection. ..................................................................................................... 9 Scheme 1.3 Synthesis of (A) methoxysquaraine: 1) ethylation, 2) dimerization, 3) O-methylation; (B) alkyne linker 3: 1) Boc mono-protection, 2) amide coupling to hexynoic acid, 3) N-deprotection; and (C) aminosquaraines. ................................................................ 10 Scheme 1.4 Three synthetic routes attempted to assemble the SQ-KA probe.............................. 11 Scheme 2.1 Overall synthetic route for azidokainoid 4: 1) esterification, 2) N-protection, 3) Swern oxidation, 4) C4-regio- and stereoselective alkylation, 5) C4-stereoselective reduction, 6) lactonization, 7) mesylation, 8) C4-stereoinversion, 9) global deprotection. .............................................................................................................. 19 Scheme 2.2 Synthesis of the ketodiester 7a. ................................................................................. 20 Scheme 2.3 a) t-BuOH, H2SO4, MgSO4, DCM, 67%. b) NaI, acetone, quantitative yield. ......... 20 Scheme 2.4 Synthesis of 4(R)-4-azidokainic acid (4)................................................................... 24 Scheme 2.5 The proposed CuAAC mechanism (L=neutral ligand). (A) In situ reduction of copper(II) to form the dinuclear copper(I) dimer complex as the catalytic species. (B) The proposed catalytic cycle for the CuAAC reaction. ...................................... 26 Scheme 2.6 (A) Previous work from Edelmann.31 (B) The deprotection of kainoid 27 to provide the novel kainoid 28. ................................................................................................. 27 Scheme 2.7 The synthesis of kainoid 18 from azidokainoid 10. .................................................. 27 Scheme 2.8 Azidokainoids 10 and 4 were provided over 8–9 steps to be used as advanced intermediates for the synthesis of the squaraine-kainate probe described in Chapter 3. ................................................................................................................................ 28 Scheme 3.1 Synthesis of squaraine 2 with an overall yield of 55%. Microscale (<100 mg) routes gave improved overall yields (up to 67%) due to better yields for the formation of squaraine 12 (77%).................................................................................................... 49 Scheme 3.2 A proposed mechanism for the formation of squaraine 12. ...................................... 51 Scheme 3.3 Methylation of squaraine 12 to form methoxysquaraine (2). .................................... 54 Scheme 3.4 Overall synthesis of alkyne linker 3. ......................................................................... 55 Scheme 3.5 Protection of ethylenediamine resulting in the mono- and di-protected products. ... 55 Scheme 3.6 Equilibrium of ethylenediamine in the presence of equimolar HCl. ......................... 56 Scheme 3.7 A strategic plan devised to assemble probe 1 with optimal usage of the limited azidokainoid 10 advanced intermediate. ................................................................... 59 Scheme 4.1 Summary of the syntheses involved in the development of the SQ-KA probe 1...... 71 xi List of symbols and abbreviations °C degrees Celsius 13C carbon-13 1H proton Å angstrom AGOFPs affinity-guided organic fluorescent probes AMPA -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid Bn benzyl BODIPY boron-dipyrromethene BT benzothiazole CHCl3 chloroform cm-1 wavelength CNS central nervous system CuAAC copper-catalyzed azide-alkyne cycloaddition DCC N,N’-dichlorohexylcarbodiimide DCM dichloromethane DIC N,N’-diisopropylcarbodiimide DIPEA diisopropylethylamine DMAP 4-dimethylaminopyridine DMF dimethylformamide DomA domoic acid dr diastereomeric ratio EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride equiv equivalent ESI electrospray ionization FPs fluorescent proteins g gram GABA -aminobutyric acid xii Glu glutamate GluRs glutamate receptors h hour hi-vac high-vacuum HMPA hexamethylphosphoramide HPLC high-performance liquid chromatography HRMS high-resolution mass spectrometry Hz hertz iGluRs ionotropic glutamate receptors IR infrared KA kainic acid KARs kainic acid receptors L litre LBD ligand binding domain M molar mg milligram MHz megahertz min minute mL millilitre mmol millimolar mol mole Ms mesyl n.d. not determined NIR near-infrared NMDA N-methyl-D-aspartate NMR nuclear magnetic resonance OD oven-dried Pf phenyl fluorene xiii POI protein of interest Qdots quantum dots rotavap rotary evaporator rt room temperature s second SQ squaraine TAL traceless-affinity labeling TBTA tris(1-benzyl-4-triazolyl)methyl)amine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin-layer chromatography TMS tetramethylsilane TMSCl trimethylsilyl chloride TOF time-of-flight ToF turn-on fluorescent UV ultraviolet δ chemical shift xiv Acknowledgments First, I would like to thank my supervisor, Dr. Frederic Menard, for accepting me into your group and giving me the opportunity, patience, and knowledge to grow as a chemist. Additionally, I’d like to thank my committee members, Dr. Stephen McNeil and Dr. Kevin Smith for their time and advice throughout my time in graduate school. I would like to thank Zhenlin Tian and Simon Edelmann for laying the groundwork for this project, as well as my past and present lab members Chelsey Brien, Matthew Heard, Omkar Kulkarni, Vojtech Kapras, Christoph Nopper, Darina Vekhova, Marisa Aviani, Olivia Dixon, Victor Hellgren, Raphael Poirier, Megan Schroeder, and Vicente Stranger. You all made my time in graduate school more enjoyable and often helped with my research in and out of the lab. I would like to thank Dr. Wesley Zandberg for providing me access to your LC-MS instrument and especially Nitin for your time and assistance in collecting my HRMS data. I would like to thank Dr. Jennifer Robinson and Ieva Zigg for offering the opportunity of teaching assistantships, lab development, and access to instruments in undergraduate labs. I would like to gratefully acknowledge Graham Lowe and Joanne McKechnie for their financial assistance via the Lowe McKechnie Bursary. I would like to express my deep gratitude to my parents, Mario Tardif and Sandra Cristino, for raising me into the man (boy) I am today and for their never-ending love and support. Lastly, I would like to thank the love of my life, Amanda Darbyshire, for your love and support throughout our years in graduate school. I am glad we could experience this adventure together and look forward to many more.Chapter 1 1 Chapter 1: Introduction 1.1 Kainate receptors and their neurophysiological function in the brain The first cloning of a glutamate receptor was reported in 1989.1 Since then, extensive research has come about implicating their role in the regulation of neuronal excitation and synaptic plasticity, as well as their involvement in the development of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and multiple sclerosis.2–4 The central nervous system (CNS) is largely regulated through a balance of excitatory and inhibitory systems. Glutamate (Glu, aka glutamic acid) and -aminobutyric acid (GABA) are the principal excitatory and inhibitory neurotransmitters in the brain, respectively.5 Regulation of glutamate and GABA is controlled by glutamate receptors (GluRs), which are divided into two classes: metabotropic and ionotropic (mGluRs and iGluRs). iGluRs are ligand-gated ion channels that allow cation influx upon the binding of glutamate; they are divided into three main subfamilies according to their selective agonist: -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate (KA, aka kainic acid) receptors (KARs). iGluRs are composed of four large subunits that form a central ion channel pore.6 Each of the four subunits consists of four well-conserved domains: an amino-terminal, a ligand-binding (LBD), a transmembrane, and an intracellular carboxy-terminal domain (Figure 1.1). The binding of glutamate at the LBD enforces a conformation change that opens the central ion channel pore to allow cation influx across the membrane. Chapter 1 2 Figure 1.1 A tetrameric iGluR in complex with glutamate. Different colours represent each of the four identical subunits that make up the protein. (PDBs: 6KZM and 4MH5). Among the three iGluRs, the NMDA and AMPA receptors are found in the postsynaptic region where they regulate membrane depolarization and neuronal excitability. Unique to the NMDA and AMPA receptors, KARs are ubiquitous in the pre- and postsynaptic regions, although they are predominantly found in the former (Figure 1.2). Postsynaptic KARs function similarly to NMDA and AMPA receptors; however, presynaptic KARs modulate the release of neurotransmitters, Glu and GABA, involving them in short- and long-term synaptic plasticity. Additionally, KARs have been linked to metabotropic signaling pathways despite their role as ionotropic receptors. The multifaceted nature of KARs makes them an attractive target for the development of therapeutics to treat a wide range of neurological conditions.3,7 Chapter 1 3 Figure 1.2 Schematic representation of a synapse containing Glu- and GABA-releasing terminals in which KARs are localized on both pre- and post-synaptic neurons modulating the release of Glu and GABA neurotransmitters. Kainic acid is a naturally occurring nonproteinogenic amino acid originally isolated in 1953 from Digenea simplex, a Japanese marine algae.8 Six years later, domoic acid (DomA) was discovered in another species of Japanese marine algae, Chondria armata.9 Although, non-native to the mammalian CNS, both KA and DomA have been shown to selectively bind KARs inducing stronger activity than the neurotransmitter glutamate, making them potent neurotoxins.10,11 Figure 1.3 Kainic acid and domoic acid are natural products exogenous to the mammalian CNS, however, they resemble the neurotransmitter glutamate as conformationally restricted analogs with superior binding affinity to KARs than glutamate. Chapter 1 4 In 1987, Canadian government agencies received hundreds of reports over four weeks from ill patients who consumed mussels in Eastern Canada. Short-term memory loss, seizures, and rapid onset confusion were among the symptoms reported. Four individuals died within four months post-consumption of the mussels and DomA was eventually identified as the cause of toxicity. Neuropathological studies performed on the four deceased revealed that neuronal necrosis and astrocytosis, predominantly in the hippocampus and amygdala, were prevalent among the patients. The Canadian poisoning incident was an unfortunate natural experiment that provided evidence of the neurotoxic effects induced by kainate agonists in humans. These observations suggest that excessive activation and/or expression of kainate receptors may play a role in detrimental neurological symptoms and diseases.12,13 1.2 Fluorescent protein labeling Fluorescent protein labeling is a vital component in the chemical toolbox employed by researchers to further our understanding of biological processes. Generally, a fluorophore is attached to a recognition moiety to visualize a target protein and provide information on protein interactions, localization, conformational changes, enzymatic activity, and the ability to see individual proteins move in real-time.14 Fluorophores selected for biological imaging should be stable and avoid perturbing biological functions, along with being sufficiently bright to provide signals that overcome those from autofluorescence (background fluorescence from molecules native to the biological system).15 Genetic modification can be used to introduce recombinant fluorescent proteins (FPs), recognition proteins/peptide tags, or biorthogonal handles to achieve fluorescence. However, protein and large peptides can adversely affect protein folding and function making them less applicable for in vivo and live cell imaging.16 Smaller genetic additions like biorthogonal handles or small peptides are better suited for these applications, but genetic modification can be time-consuming and lacks flexibility. Quantum dots (Qdots) are fluorophores composed of inorganic fluorescent cores that achieve exceptional brightness and photostability (20 times brighter and 100 times more photostable than traditional fluorophores), have narrow emission peaks suitable for multifluorescent imaging, and are optically tunable for visualization at various wavelengths. The advantages of Qdots are Chapter 1 5 dampened by their disadvantages; their inorganic cores (typically 2–10 nm in diameter) and semiconductor shells are cytotoxic and require a layer of coating to mitigate toxicity and improve solubility for biological applications. Functionalized Qdots typically range from 15–50 nm in diameter making them magnitudes larger than FPs and organic fluorophores, even potentially exceeding the size of the protein of interest (POI).14,17 Organic fluorophores are advantageous due to their tunable photophysical properties and relatively small size which lessens the likelihood of protein perturbation, an essential component for in vivo and live cell imaging. They can be selectively introduced to genetically modified proteins via biorthogonal chemistry or protein/peptide tags, but face the same previously mentioned downsides.18,19 However, without a sufficient recognition moiety, organic fluorophores can lead to off-target labeling resulting in a high background signal. Affinity-guided organic fluorescent probes (AGOFPs) can be used to selectively introduce an organic fluorophore to a POI without the need for genetic modification. 1.2.1 Affinity-guided organic fluorescent probes Affinity-guided organic fluorescent probes (AGOFPs) are modularly designed, consisting of three interchangeable components: (1) a protein-specific ligand to provide selectivity, (2) a small molecule organic fluorophore to enable visualization, and (3) a linker to distance the fluorophore from the protein (Figure 1.4). Typically, ligands are modified agonists or antagonists of the targeted protein that selectively interact with an orthosteric or allosteric binding site. The fluorophore is distanced from the ligand to reduce potential perturbation of ligand binding affinity or protein function. Organic fluorophores are magnitudes smaller in size than Qdots and FPs (25-30 kDa, Figure 1.4C) making them better suited for the study of native proteins. Chapter 1 6 Figure 1.4 Schematic representations of (A) a generic affinity probe and (B) affinity -guided labeling. (C) Relative size comparison of GFP (green, 27 kDa ), fused to an example target protein (cyan), next to the popular Cy5 organic fluorophore (orange, ~0.5 kDa). PDBs: 5MFC (left) and 5NS3 (right). Perhaps the greatest challenge regarding AGOFPs is achieving high selectivity and low background. Insufficient binding affinity and selectivity towards the POI can result in the probe labeling multiple proteins or agglomerating in the cell membrane causing high background and misleading signals. Additionally, the persistent fluorescent state of typically organic fluorophores requires thorough washing steps after introducing the probe-containing solution for accurate imaging. The introduction of a mechanism that initiates fluorescence upon recognition of a POI can markedly enhance fluorescent imaging contrast. 1.2.2 Turn-on fluorescent probes Turn-on fluorescent (ToF) probes have grown in interest due to their favourable signal-to-noise properties for biological imaging. ToF probes exist in a non-fluorescent state until a specific condition is met, at which point, fluorescence is initiated. Chemical reactions, conformational changes, and recognition moieties are among some of the methods that can initiate turn-on fluorescence depending on the design of the ToF probe. AGOFPs can be made with turn-on fluorescence through the specific incorporation of a hydrophobic fluorophore and a hydrophilic affinity ligand. The hydrophobic nature of the fluorophore causes aggregation in aqueous solution Chapter 1 7 resulting in an equilibrium of the monomeric and aggregated probe that is dominated by the latter. This aqueous-induced aggregation has been shown to efficiently quench fluorescence until the corresponding target protein is introduced (Figure 1.5). Figure 1.5 Schematic depiction of ToF probes exhibiting aqueous-induced aggregation and protein-induced disassembly resulting in protein-selective turn-on fluorescence. Mizusawa and coworkers have previously developed ToF probes with a BODIPY fluorophore that self-assembled into nearly non-fluorescent aggregates; however, the probe required ultraviolet (UV) excitation which can be detrimental to cell viability.20,21 Following this, He and coworkers developed a ToF probe for the detection of avidin using a squaraine as the aggregating fluorophore (Figure 1.6).22 Their promising work demonstrates the ability of ToF probes to significantly improve selective detection in fluorescent protein imaging. Figure 1.6 BODIPY (Mizusawa) and squaraine (He) hydrophobic fluorophores utilized for ToF probes.20–22 Chapter 1 8 1.3 Research objectives and thesis overview 1.3.1 Research hypothesis and objectives Although there is a high interest in investigating the role glutamate receptors play in neurodegenerative processes, most selective chemical tools recently developed are limited to the NMDA and AMPA receptors. Achieving selective detection or activation between KARs and AMPA receptors is especially difficult due to the high homology of their respective binding sites. The only known agonists that can discern between KARs and AMPA receptors are kainoids, a class of nonproteinogenic amino acids named after the naturally occurring neurotoxin, kainic acid.4 The lack of selective chemical tools to investigate KARs prevents us from better understanding their neurophysiological role within the brain. My research aims to address this gap with the development of the squaraine-kainate (SQ-KA) probe (1) for improved live cell and in vivo imaging of KARs. Combining a modified kainoid ligand and a squaraine fluorophore, I hypothesize that probe 1 will provide an enhancement to fluorescent imaging contrast with selective turn-on fluorescence in the presence of KARs. Scheme 1.1 Retrosynthetic breakdown of the squaraine-kainate probe (1). Squaraines were chosen as the aggregating fluorophore for their strong and narrow absorption bands in the near-infrared (NIR, >700 nm) region. NIR excitation benefits from deep tissue penetration and minimal background fluorescence attributed to shortwave-absorbing biological Chapter 1 9 fluorophores (aka autofluorescence), making it an ideal excitation wavelength for live cell protein imaging. In contrast, shortwave excitation results in surface-level tissue penetration, photodamage from higher energy excitation, and unwanted fluorescence from endogenous fluorophores. Kainoids modified at the C4 position have been shown to maintain sufficient, and in some cases improved, binding affinity towards KARs. Additionally, reported structure-activity relationship studies on over 100 kainoids, provided evidence that the incorporation of a flat sp2-hybridized sidechain at the C4 position may benefit binding affinity through a π−π interaction with a tyrosine residue in the LBD of KARs.23 1.3.2 Thesis overview The synthesis of probe 1 converges from three individual syntheses of each essential component. In Chapter 2, I discuss the structure-activity relationships of kainoid analogs with GluRs and propose structural modifications for the probe design. Afterward, I detail a nine-step synthetic route from the commercially available trans-4-hydroxy-L-proline to synthesize a novel kainoid analog, (4R)-4-azidokainic acid (4), an advanced intermediate for the SQ-KA probe (Scheme 1.2). Scheme 1.2 Synthesis of (4R)-4-azidokainic acid (4): 1) esterification, 2) N-protection, 3) Swern oxidation, 4) C4-regio- and stereoselective alkylation, 5) C4-stereoselective reduction, 6) diastereomer separation, 7) mesylation, 8) C4-stereoinversion, 9) global deprotection. Chapter 1 10 In Chapter 3, I discuss squaraine dyes and their promising use as biological fluorophores. Afterwards, I detail three-step syntheses of methoxysquaraine (2) and a simple alkyne linker 3. These advanced intermediates were subsequently used for the synthesis of aminosquaraine derivatives (Scheme 1.3). Scheme 1.3 Synthesis of (A) methoxysquaraine: 1) ethylation, 2) dimerization, 3) O-methylation; (B) alkyne linker 3: 1) Boc mono-protection, 2) amide coupling to hexynoic acid, 3) N-deprotection; and (C) aminosquaraines. Finally, I close out chapter 3 detailing my efforts towards assembling the SQ-KA probe. The challenging synthesis of azidokainoids 10 and 4 yielded a limiting amount of material; thus, I devised a strategic plan for optimal utilization. Three synthetic pathways were investigated, each of which involved a copper-catalyzed azide-alkyne cycloaddition (CuAAC), a nucleophilic substitution, and a global deprotection. Regrettably, each route fell short at the final step and the depletion of azidokainoids prevented me from completing the synthesis of the SQ-KA probe in a reasonable timeframe. However, the accomplished synthesis of the novel kainoid analog, (4R)-4-azidokainic acid, presents a versatile option for the ongoing development of KAR-specific probes. Furthermore, the successful synthesis of the protected probe 19 constitutes significant progress in the development of the squaraine-kainate probe. With more time and larger-scale preparation of Chapter 1 11 the azidokainoid intermediates, completion of the SQ-KA probe synthesis can be achieved, introducing a valuable alternative to fluorescently image KARs. Scheme 1.4 Three synthetic routes attempted to assemble the SQ-KA probe. Chapter 2 12 Chapter 2: The synthesis of (4R)-4-azidokainic acid and other kainoids 2.1 Structure-activity relationship of kainic acid-based analogs towards glutamate receptors Kainoids (meaning “kainate-like”) are a class of nonproteinogenic amino acids named after kainic acid, a natural product and neurotoxin that elicits stronger biological activity in kainate receptors (KARs) than the natural neurotransmitter glutamate (Glu, aka glutamic acid). This section reviews a structure-activity relationship (SAR) study reported by a former Ph.D. student of the Menard lab, Dr. Zhenlin Tian, who surveyed an exhaustive list of kainoids and their ability to trigger biological activity. His review prioritized activity studies of the GluK2 isoform, the most studied of the five KARs (GluK1-5), but also incorporated other glutamate receptor (GluR) assays. This analysis is the foundation for the design process of protein-selective agonists that can be utilized in biological studies or even as therapeutic drugs. A relative qualitative scale based on Tian’s SAR studies is herein used to compare relative GluR activity induced from various kainoids (Figure 2.1).23 Figure 2.1 (A) Relative activity of Glu, KA, and DomA towards GluRs and normalized to KA. (B) A relative quantitative activity scale to compare activity induced by kainoids in GluR assays. Chapter 2 13 2.1.1 Key structural features in biologically active kainoids Kainic acid (KA) and domoic acid (DomA) are naturally occurring kainoids that are exogenous to the mammalian CNS, yet they exhibit exceptionally strong and selective binding affinity to KARs. Their superior ability to activate KARs, in comparison to the neurotransmitter glutamate, served as a starting point in early SAR studies that focused on derivatizing the KA core structure. Activity inferior to that of glutamate was observed with the following structural changes to KA (Figure 2.2): removal or modification of the amine or carboxylic acids (20a–c), inversion of C2 or C3 stereocenters (-KA), substitution at the C5 position regardless of stereochemistry (21a, b), and removal or stereoinversion of the C4 isopropene (allo-KA, 22). These results suggest that modifications are limited to the C4 position if biological activity is to be conserved and that the superior activity exhibited by KA and DomA is likely attributed to more than being a glutamate analog. Figure 2.2 Examples of kainoids that demonstrated little to no biological activity, highlighting the limited scope of modification for biologically active analogs. Chapter 2 14 Tian’s SAR study then focused on the scope of possible modifications to the C4 sidechain for the incorporation of reactive handles or to develop agonists with superior biological activity. Crystal structures of KA and DomA in complex with the GluK2 receptor indicate the C1’-C2’ alkene interacts with a tyrosine residue via π–π orbital overlap (Figure 2.3). Furthermore, the extended conjugation and the C5’ carboxylic acid on the C4 sidechain of DomA may enable stronger interactions with the tyrosine, explaining its enhanced activity. The highly active acromelates A and B further support the importance of π-unsaturation on the C4-sidechain, with the latter being the most potent kainate agonist to date. (Figure 2.4). Figure 2.3 GluK2 receptor in complex with (A) kainic acid and (B) domoic acid (3C33, 2PBW). The dotted lines represent ligand-residue interactions (yellow = hydrogen bonding, magenta = π– π interactions). Figure 2.4 Kainate agonists that induce strong activity towards receptors. Chapter 2 15 2.1.2 Proposed kainoid structural modifications My proposed KAR-specific probe features a kainoid ligand with an aromatic 1,2,3-triazole ring on the C4 sidechain (Figure 2.5A). To gain insight into its potential binding affinity, the class of thiazole kainoids 23a–d (Figure 2.5B) was reviewed as they also contain an aromatic 5-membered heterocyclic ring. Three of the four thiazole kainoids exhibited activity on par or stronger than DomA suggesting that the thiazole ring has a positive impact on binding affinity. These results support the proposed squaraine-kainate probe as a potentially strong and selective chemical tool to label kainate receptors. Figure 2.5 (A) Structure of the proposed kainoid ligand incorporated into the probe. (B) Relative biological activity of thiazole kainoids. 2.2 Precedents in kainoid syntheses Kainic acid has been a highly pursued synthetic target since its isolation in 1953. More than 60 total syntheses have come about since then, however, most of them do not allow for late-stage derivatization of the C4 sidechain. The cis-C3,C4 stereocenters are often more difficult to incorporate in syntheses compared to the trans-C2,C3 stereocenters. This section will review a selection of syntheses that were influential in the development of synthetic kainic acid and kainoid analogs. In 1982, Oppolzer and Thirring developed the first enantioselective total synthesis of kainic acid starting from 5-ethylglutamic acid due to its inherent (S)-carboxylic acid. Their synthesis utilized an intramolecular ene-cyclization to install the crucial cis-C3,C4 stereocenters but resulted in a poor overall yield of 5%.24 The ene-cyclization was optimized over the years with the emergence of the shortest total synthesis of KA over six steps reported by Ohshima et al. in 2015. Their synthesis enabled gram-scale production of KA with an overall yield of 34% proceeding Chapter 2 16 through a one-pot sequential platinum-catalyzed epoxide ring-opening and subsequent ene-cyclization to form the (2S,3R,4R)-pyrrolidine core.25 This provided a good option for the total synthesis of KA; however, it lacked the ability for late-stage C4 modification (Figure 2.6A). The synthesis of kainoids from the natural amino acid, trans-4-hydroxy-L-proline, was introduced by Baldwin and Rudolph in 1994. Since then, 4-hydroxyproline has been a mainstay in kainoid syntheses due to its inherent pyrrolidine core, (2S)-carboxylic acid, and modifiable C4-alcohol. Baldwin and Rudolph’s initial synthesis introduced the C3-sidechain through the regioselective formation of enamine 24 and subsequent alkylation that was predominantly stereoselective (~95%) but gave moderate yields (53%).26 First reported in 1985, the 9-phenyl-9-flurenyl (Pf) protecting group emerged as a strong option for the protection of cyclic secondary amines to control ensuing regioselective alkylation.27 The Pf group has since been used in many syntheses of kainoids that start from 4-hydroxyproline including a recent report in 2014 by Zanato et al. Their synthesis detailed optimized conditions for the C3 alkylation with an 80% yield and a 16:1 diastereomeric ratio (dr). A subsequent alkynylation afforded the alkynylkainoid 25; however, the consequential C4-alcohol was not removable without losing the stereochemistry (Figure 2.6B).28 Chapter 2 17 Figure 2.6 (A) Previously reported syntheses of kainic acid and (B) kainoid derivatives. Most recently, Tian developed a versatile synthetic route from 4-hydroxyproline that affords 4-phenylkainic acid over eight steps, 4-carboxykainoid over 10 steps, and 4-alkynylkainic acid over 15 steps (Figure 2.7A).29 Tian went on to couple the 4-alkynylkainic acid to a sulfo-Cy5 fluorophore, providing the first known fluorescent probe (sulfo-Cy5-KA) with a kainoid-directing ligand reported in the literature (Figure 2.7B). Preliminary in vitro imaging of HEK 293 cells modified to express the GluK2 receptor successfully demonstrated the probe’s ability to selectively visualize kainate receptors with confocal microscopy.30 Chapter 2 18 Figure 2.7 (A) Tian’s divergent synthetic route from 4-hydroxyproline to synthesize the three highlighted kainoid analogs. (B) Tian’s sulfo-Cy5-KA probe synthesized from 4-alkynylkainic acid. 2.3 Proposed synthesis of (4R)-4-azidokainic acid The synthesis of kainoids must follow a set of structural guidelines, based on SAR studies, for the best chance of being biologically active. A pyrrolidine ring with a cis-C2,C3 and trans-C3,C4 relationship is essential to activity. The C2 and C3 positions each possess carboxylic acid substituents with a one- and two-carbon chain respectively. Finally, substitutions at the C5 position are not tolerated. Kainoids with higher binding affinity generally bear a C4 sidechain with sp2 centers at the 1’ and 2’ positions.23 Following these rules, Tian synthesized 4-alkynylkainic acid which provided a flexible intermediate that could be coupled to a variety of fluorophores through a copper-catalyzed azide-alkyne cycloaddition (CuAAC); however, the synthesis was a lengthy 15 steps. Thus, I propose a synthesis of (4R)-4-azidokainic acid (4) over nine steps, providing an alternative kainoid ligand for CuAAC coupling to a corresponding fluorophore. This synthesis followed Tian’s optimized route to the ketodiester 7a advanced intermediate. Ketodiester 7a was stereoselectivity reduced to alcohol 8a with conditions optimized by Edelmann,31 followed by a Chapter 2 19 mesylation and subsequent stereoinversion of the C4 center to afford the azide handle. Finally, a global deprotection would provide the novel kainoid analog, (4R)-4-azidokainic acid (4). Scheme 2.1 Overall synthetic route for azidokainoid 4: 1) esterification, 2) N-protection, 3) Swern oxidation, 4) C4-regio- and stereoselective alkylation, 5) C4-stereoselective reduction, 6) lactonization, 7) mesylation, 8) C4-stereoinversion, 9) global deprotection. 2.4 Installation of protecting groups and C3 alkylation The synthesis of azidokainoid 4 began from the commercially available trans-4-hydroxy-L-proline. Two consecutive protections provided the bis-protected hydroxyproline 5. Protection of the secondary amine was achieved with a rather unique 9-phenyl-9-fluorenyl (Pf) group. Oxidation of the C4-alcohol was performed with a Swern reaction to afford ketoproline 6 which was then alkylated through a regioselective enolate formation and subsequent stereoselective alkylation, providing ketodiester 7a over four steps. (Scheme 2.2). Chapter 2 20 Scheme 2.2 Synthesis of the ketodiester 7a. The bulky fluorenyl protecting group for the secondary amine was chosen for its ability to control regioselectivity in the upcoming alkylation step. The Pf group was first shown to promote regioselective enolate formation in 1985 by Rapoport and Christie in the syntheses of optically pure pipecolates.27 Sharma and Blanco then utilized the Pf group in the regioselective alkylations of 4-ketoproline analogs.32,33 For ketoproline 6, both the C2 and C5 hydrogens are protected from deprotonation, importantly, conserving the C2 stereochemistry and limiting enolate formation to the C3 position. Scheme 2.3 a) t-BuOH, H2SO4, MgSO4, DCM, 67%. b) NaI, acetone, quantitative yield. tert-Butyl iodoacetate (26b) was prepared over two steps from bromoacetic acid and used for the subsequent stereoselective alkylation (Scheme 2.3). Ketoproline 6 was alkylated via the formation of a transient enolate and subsequent addition of 26b. When lithium-enolates are formed in tetrahydrofuran (THF), the solvent tends to aggregate around the enolate through the coordination of the lithium counterion. Typically, this effect is utilized to promote selective C-alkylation; however, the overall rate of both O- and C-alkylation is decreased due to the steric bulk imparted by the aggregation. Hexamethylphosphoramide (HMPA) can be used to increase overall alkylation rates by reducing aggregate formation around the enolate-oxygen; however, this typically causes O-alkylation to be favoured.34 Fortunately, when utilized as a minor co-solvent or Chapter 2 21 in near equimolar amounts, HMPA has been shown to increase C-alkylation rates in THF while avoiding O-alkylation.35 In practice, HMPA concentrations of ~0.2 M in THF provided the best experimental results, however, yields still suffered (< 50%). It is suspected that the quality of HMPA was sub-optimal as it was an older bottle and likely not anhydrous anymore. Distillation to purify was not practical due to the small volume remaining and Covid-related supply issues prevented new anhydrous HMPA from being obtained. In the future, fresh dry HMPA should be used to minimize potential water contamination. Table 2.1 Reaction conditions for the stereoselective alkylation of ketoproline 6 Entry 26b (eq) HMPA (eq/M)a THF (M) Crude dr (a:b) Yield (%, dr > 20:1) 1b 5 1.3/0.43 0.3 1:1 6 2b 5 6.5/0.57 0.1 5:1 39 3b 6 1.3/0.17 0.1 7:1 49 4c 2.5 1.3/0.13 0.1 2:1 27 a Molar equivalents respective to enolate/ molarity respective to THF volume. b Milligram scale. c Gram scale. Concentration with respect to ketoproline 6 in THF seemed to have a significant effect on the resulting diastereomeric ratio (dr). Diluting the reaction led to an increase in the dr by at least five-fold on a milligram scale. Although purification of the two diastereomers by flash chromatography was possible, their close Rf values made partial co-elution a common occurrence. Fortunately, the purified mixture of diastereomers could be triturated with diethyl ether, precipitating the desired ketodiester 7a (dr > 20:1). The resulting supernatant typically contained near-racemic ratios of the diastereomers. Unreacted iodoacetate was consistently recovered during chromatography in upwards of 3–4 molar excess. Equivalents of iodoacetate were reduced in the subsequent reaction; however, the resulting dr was diminished. Unfortunately, the scale of the reaction was also increased fourfold, so it is uncertain exactly which of the variables, if not both, were responsible for the lower yield (Table 2.1). Further investigation on this matter is needed. Although purified yields did not exceed 50%, higher yields of the single diastereomer are achievable through multiple triturations or flash chromatography of the racemic supernatants. The stereochemistry of 7a and Chapter 2 22 7b were determined with comparison to previously reported literature NMR spectra in which X-ray diffraction crystallography was utilized to confirm the relative stereochemistry of an identical C4 analog.28 2.5 Stereoselective reduction of the ketodiester Reductions of ketodiester 7a and similar substrates have been previously reported in the literature to favour the formation of the trans-3,4-hydroxydiester 8a over the cis-hydroxydiester 8b.28,31,36–38 The most successful conditions were reported by Zanato,28 reducing the ketodiester with sodium borohydride in methanol at ambient temperatures to afford 8a in a 90% yield after chromatographic purification. Given the reported success of these conditions, Edelmann, a former student in the Menard lab, attempted to replicate and improve the reduction with modifications to temperature and reaction time (Table 2.2).31 Table 2.2 Edelmann’s reported crude dr at varying reaction temperatures and times Entry Temperature (°C) Time (h) Crude dr (a:b) 1 0 3 4:1 2 -40 6 20:1 3 -78 10 20:1 4 -100 12 20:1 a Crude diastereomeric mixture was subjected to basic conditions to cyclize 8b to 8c for easier separation by flash chromatography. Purified yields after cyclization and flash chromatography were not reported. Temperature decreases below -40 °C had a negligible effect on stereoselectivity but required more time to reach full conversion. In contrast to Zanato, Edelmann observed a significant decrease in dr at warmer temperatures up to 0 °C. This would suggest cooling the reaction down is important for the stereoselective formation of 8a, contradictory to Zanato’s reported success at ambient temperatures. Chapter 2 23 In my own hands, the reduction showed different results. The overall conversion of the ketone to the alcohol ranged between 86–98%, although stereoselectivity matching those of Zanato or Edelmann was not reproducible. Diastereomeric ratios ranged between 4:1 to 5:1 (a:b) despite replicating conditions in which Edelmann achieved dr > 20:1. Flash chromatography to purify the diastereomers was challenging due to their identical Rf values in varying eluents. I investigated the possibility of a dr enrichment via trituration (in methanol and diethyl ether) just like I had done with the ketodiesters 7a and 7b, however, the improvement was minimal and still insufficient for use in the subsequent reaction. An alternative solution was reported by Edelmann where cyclization of the cis-hydroxydiester 8b to the lactone 8c was induced under basic non-nucleophilic conditions, leaving the trans-hydroxydiester 8a unreacted. After the transformation, 8a and 8c were separatable by flash chromatography, enabling the isolation of 8a as a single diastereomer. While this sequence did provide a method for isolating the single desired diastereomer, it resulted in lower yields compared to Zanato. Further investigation into the optimal reaction conditions and diastereomeric separation is needed given the reported results from the Zanato group. Table 2.3 Experimental and literature conditions for the reduction and cyclization Entry NaBH4 (equiv) dr (a:b) NaH (equiv) Yield (%)a 1 4 5:1 1.5 40 2 4 5:1 1.8 39 3 5 5:1 4.5 34 4b 6 4:1 / / Edelmann 4 > 20:1 1.1 n.d. Zanato 1 n.d. / 90 a Reported yield of 8a as a single diastereomer. b The reduced diastereomers were triturated giving an enriched dr of 6:1 with a 77% yield. Chapter 2 24 2.6 Installation of the C4-azide and global deprotection The last steps focused on installing the azide to the C4 position with the proper cis-C3,C4 stereochemistry. Alcohol 8a was activated through a mesylation followed by a nucleophilic displacement that proceeded through a stereoinversion of the C4 substituent to provide azidokainoid 10. Global deprotection of 10 was performed over two telescoped steps to afford 4(R)-4-azidokainic acid (4). Scheme 2.4 Synthesis of 4(R)-4-azidokainic acid (4). Alcohol 8a was mesylated in consistently good yields. Triethylamine and diisopropylethylamine were used interchangeably as non-nucleophilic bases with no noticeable effect on conversion. Displacement of the mesylate group was difficult due to the steric demand of the ring’s substituents. Amine nucleophiles were also considered to create other reactive handles for linkage, however, attempts to displace the mesylate with ethylenediamine were ineffective in both protic and non-protic solvents. Even with the increased nucleophilicity of the azide anion, the displacement never exceeded 50% yield, while still requiring high temperatures, a 10-fold excess of sodium azide, and extended reaction times. These forcing conditions made the kainoid susceptible to degradation and side reactions, such as the observed formation of lactone 8c (Figure 2.8). Under ambient conditions, the orientation of the tert-butyl ester required for this side reaction to proceed would likely not occur due to steric collision with the C4 proton; however, the prolonged increase in thermal energy enables sufficient orbital overlap for the intramolecular displacement to proceed. In solvents like dimethylsulfoxide, this side reaction was exacerbated, as reported by Edelmann, thus dimethylformamide was used instead.31 Overall, the azide Chapter 2 25 stereoinversion failed to fully convert the mesylate while partially losing material to at least one known side reaction (Table 2.4). Optimization is needed to remedy these issues. Figure 2.8 Proposed mechanism for the formation of lactone 8c from mesylate 9. Table 2.4 The ratio of recovered compounds after the displacement reaction determined via NMR Entry Mesylatea Lactone Azide 1 1.2 1.0 5 2 1.1 1.0 6 3 1.0 1.0 4 4 1.0 1.7 7 5 2.2 1.0 7.1 a Mesylate 9 went unreacted by 10–20% and was recoverable sthrough chromatography. Global deprotection of azide 10 was performed over two telescoped steps followed by ion-exchange flash chromatography to afford 4(R)-4-azidokainic acid (4) with satisfactory yields. Deprotection of the tert-butyl ester and the Pf group was performed under acidic conditions with the addition of triethylsilane as a carbocation scavenger.39 It should be noted that performing the deprotection steps in reverse order is ineffective. The methyl ester is significantly less accessible in the presence of the tert-butyl and Pf groups, preventing the hydrolysis from occurring. 2.7 Derivatizing azidokainoid intermediates with a CuAAC reaction The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a variant of the Huisgen cycloaddition broadly used to couple two chemical species bearing an azide and alkyne to facilitate the regioselective formation of a 1,4-substituted triazole. Optimal conditions for the CuAAC reaction were previously investigated by Edelmann in the synthesis of kainoid 27.31 The click reaction was most productive with copper sulfate as the source of copper (II), sodium ascorbate as Chapter 2 26 the reducing agent to form the catalytic copper (I) species, and tris(benzoyltriazolylmethyl)amine (TBTA) as the copper-coordinating ligand to stabilize the transient copper (I) species from disproportionation and re-oxidation. A proposed mechanism is shown below (Scheme 2.5); however, the exact mechanism and catalytic species have not yet been fully elucidated. Mechanistic studies suggest that acetylide-copper (I) species tend to form aggregates that greatly benefit catalysis due to the involvement of at least two copper (I) species.40,41 Scheme 2.5 The proposed CuAAC mechanism (L=neutral ligand). (A) In situ reduction of copper(II) to form the dinuclear copper(I) dimer complex as the catalytic species. (B) The proposed catalytic cycle for the CuAAC reaction. Edelmann’s previous efforts afforded the 1,4-triazole kainoid 27 with 100 percent regioselectivity, avoiding the formation of the 1,5-triazole isomer (Scheme 2.6A). To conclude Edelmann’s work, 27 was deprotecting to afford kainoid 28, a potentially potent agonist for kainate receptors; however, its biological activity is not explored in this work (Scheme 2.6B). Chapter 2 27 Scheme 2.6 (A) Previous work of Edelmann.31 (B) My deprotection of kainoid 27 to provide the novel kainoid 28. Modifications to Edelmann’s CuAAC conditions were made to enable microscale-friendly quantities of reagents. Azidokainoid 10 was coupled to hexynoic acid to furnish the carboxykainoid 29 for the subsequent amide coupling to diamine 13 to afford kainoid 30. Global deprotection of 30 over two telescoped steps followed by ion-exchange chromatography provided kainoid 18 over three steps with a 58% overall yield. It should be noted that alkyne linker 3 can be directly coupled to either azidokainoid 10 or 4; however, this was not done due to a shortage of the azidokainoid advanced intermediates. Scheme 2.7 The synthesis of kainoid 18 from azidokainoid 10. Chapter 2 28 2.8 Summary In conclusion, I have demonstrated a nine-step synthetic route from commercially available trans-4-hydroxy-L-proline to afford (4R)-4-azidokainic acid (4), a kainoid analog that provides flexible coupling via the CuAAC reaction to a variety of chemical species bearing an alkyne reactive handle. Additionally, I completed the synthesis of the novel kainoid analogs 18 and 28. In Chapter 3, kainoids 4, 10, and 18 are used as advanced intermediates towards the title squaraine-kainate probe. Scheme 2.8 Azidokainoids 10 and 4 were provided over 8–9 steps to be used as advanced intermediates for the synthesis of the squaraine-kainate probe described in Chapter 3. 2.9 Experimental 2.9.1 General experimental procedures Unless otherwise noted, reactions run under inert conditions were first evacuated in vacuo and then backfilled with an inert gas (argon or nitrogen, three times), using a balloon adaptor to maintain the environment throughout the reaction. Reactions performed at -78 °C or -40 °C were cooled using an acetone/dry ice and MeCN/dry ice bath respectively. Reactions performed at 0 °C were cooled using an ice-brine bath. When reactions were heated above rt, the reaction vessel was immersed in a heated mineral oil bath unless stated otherwise. The addition of reagents under 10 μL was performed using a VWR Ergonomic High-Performance 10 ± 0.6 μL micropipettor. Solutions were concentrated under reduced pressure using rotary evaporation (50–200 torr) unless stated otherwise. High-vacuum (hi-vac) conditions (< 0.001 torr) were achieved with a Welch 1400B-01 DuoSeal Vacuum Pump. Thin layer chromatography (TLC) was performed on glass plates precoated with silica gel (F254, 250 μm, 60 Å, Sigma Aldrich). TLC plates were visualized Chapter 2 29 under a 254 or 365 nm UV light source, then stained by immersion in either acidic ethanolic ninhydrin, acidic aqueous ethanolic vanillin solution, or potassium permanganate, followed by heating using a heat gun. Flash column chromatography was performed using silica gel (230–400 mesh, SiliaFlash R12030B, P60, 40–63 μm, 60 Å). Ion-exchange chromatography was performed using Dowex®, 50W-X8, Ion Exchange Resin, hydrogen form, 200–400 mesh, matrix acetic group: sulfonic acid. 2.9.2 Materials Commercial reagents and starting materials were purchased from Sigma-Aldrich, Oakwood Chemicals, Alfa Aesar, Acros Organics, TCI America, or Fisher Scientific and were used as received unless otherwise noted. Anhydrous solvents were dried over activated 4 Å molecular sieves and let stand for at least 24 h before use unless stated otherwise. Solvents that needed to be exceptionally dry were purified on a glass contour solvent purification system under an argon atmosphere. Diisopropylethylamine, pyridine, and triethylamine were distilled over calcium hydride and used immediately, otherwise, they were stored over 4 Å molecular sieves in light-protected amber bottles until discolouration. The molarity of commercially obtained n-butyllithium solutions was determined by titration against diphenylacetic acid as an indicator. Supplemental characterization is provided for some compounds previously synthesized by Menard lab alumni Dr. Zhenlin Tian or Simon Edelmann. This spectral information is indicated as follows: (Tian) or (Edelmann) after the end of the listed values. Title compounds are numbered for the assignment of protons and do not always match numbering for nomenclature. 2.9.3 Instruments 1H and 13C NMR spectra were recorded on a 400 MHz Varian NMR AS400 equipped with an ATB-400 probe at 25 °C. NMR spectra were analyzed with MestReNova version 6.0.2-5475 from Mestrelab Research. Proton chemical shifts are reported in parts per million (ppm, δ scale) and are referenced to either tetramethylsilane (defined at 0.00 ppm) or residual protium in the NMR solvent (CHCl3: δ 7.26 ppm, methanol-d3: δ 3.31, dimethylsulfoxide-d5: δ 2.50 ppm, HOD: δ 4.79 ppm, pyridine-d5: δ 8.74, 7.58, 7.22 ppm). Carbon chemical shifts are reported in parts per million (ppm, δ scale) and are referenced to either tetramethylsilane (defined at 0.00 ppm) or the carbon resonance of the NMR solvent (CDCl3: δ 77.16 ppm, methanol-d4: δ 49.00, dimethylsulfoxide-d6: Chapter 2 30 δ 39.52 ppm. Fluorine chemical shifts were not referenced to a standard; however, chemical shift values were consistent to the hundredth decimal over multiple experiments. Spectral data are listed as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet), coupling constant (Hz), and assignment (if known). Infrared (IR) spectra were obtained on a PerkinElmer FT-IR spectrometer Spectrum two, with a globar light source, a Michelson moving-mirror and a pyroelectric detector working at room temperature at a range of (4000 cm-1 to 700 cm-1). The attenuated total reflectance (ATR) with one reflection used was a PIKE MIRacle which contained material composed of a ZnSe mixture. High-resolution mass spectra were obtained using an Agilent 6530 qTOF mass spectrometer with a 1290 infinity II HPLC (Zandberg lab, ESI-positive mode, 5 μL direct injections of sample in methanol with 0.1% formic acid). 2.9.4 Experimental procedures and characterization tert-Butyl bromoacetate (26a). Dichloromethane (250 mL) was charged in a 1 L round-bottom flask and stirred vigorously. Magnesium sulfate (46 g, 38 mmol) was slowly added to prevent clumping and seizing of the stir bar. Concentrated H2SO4 (4 mL, 0.07 mol) was added dropwise. Bromoacetic acid (10 mL, 0.14 mol) and tert-butanol (37 mL, 0.39 mol) were melted by gently heating with a heat gun and then added to the suspension. The reaction was stirred at rt for 24 h, then a second aliquot of tert-butanol (20 mL, 0.21 mol) was added, and the reaction was stirred at rt for another 72 h. The magnesium sulfate was vacuum-filtered, and the filtrate was washed with brine (1 x 75 mL) and extracted with dichloromethane (3 x 50 mL). The combined organic layer was washed once with water (50 mL), dried over magnesium sulfate, and concentrated in vacuo yielding the crude bromoacetate (22.82 g, 67%) as a yellow liquid which was used without further purification. The spectrum was consistent with that reported in the literature.31 Molecular formula/weight: C2H3O2Br: 138.95 g/mol. 1H NMR (400 MHz, CDCl3): δ 3.75 (s, 2H, H2), 1.48 (s, 9H, H1) ppm. Chapter 2 31 tert-Butyl iodoacetate (26b). Sodium iodide (1.823 g, 12.16 mmol) was charged in a 100 mL oven-dried round-bottom flask under an argon atmosphere. Acetone (50 mL) was added and the mixture was stirred until homogenous. tert-Butyl bromoacetate (1.2 mL, 8.1 mmol) was added, immediately producing sodium bromide (NaBr) as a white precipitate. The flask was wrapped with foil to protect it from light and stirred at rt for 7 h. The mixture was vacuum-filtered to remove the NaBr precipitate. Diethyl ether was added to precipitate more NaBr and a second filtration was performed. A saturated aqueous solution of sodium thiosulphate was added to the filtrate and stirred for 5 min. The biphasic mixture was transferred to a separatory funnel and extracted with diethyl ether (2 x 50 mL). The combined organic layer was washed with brine (1 x 50 mL), dried over magnesium sulfate, and concentrated in vacuo yielding the crude iodoacetate (1.98 g, quantitative yield) as a pale-yellow liquid which was used without further purification. When not used immediately the iodoacetate was stored in a foil-wrapped, oven-dried vial over 4 Å sieves molecular sieves. The spectrum was consistent with that reported in the literature.31 Molecular formula/weight: C2H3O2I: 185.95 g/mol. 1H NMR (400 MHz, CDCl3): δ 3.61 (s, 2H, H2), 1.46 (s, 9H, H1) ppm. trans-4-Hydroxy-(S)-proline methyl ester HCl (5a). trans-4-Hydroxy-L-proline (10.02 g, 76.41 mmol) was charged in a 500 mL round-bottom flask and suspended in methanol (150 mL). The suspension was cooled to 0 °C and placed under an argon atmosphere. TMSCl (34 mL, 0.27 mol) was added at a rate of 0.2 mL/ 20 sec. The reaction was stirred at 0 °C for 15 mins, then warmed to rt and stirred for 24 h. The solvent was removed in vacuo, producing a white clumpy solid that was resuspended in diethyl ether and vacuum-filtered. The product was noticeably hygroscopic and needed extensive drying, so it was suspended in benzene and concentrated (3 x 20 mL) in vacuo, then concentrated to dryness under hi-vac in a warm water bath (40°C). The crude proline Chapter 2 32 salt 5a was recovered as a white powder (12.93 g, 93%), and stored in a vacuum desiccator to be used without further purification. Spectra were consistent with those reported in the literature.30,31 Molecular formula/weight: C6H12NO3Cl: 181.62 g/mol. 1H NMR (400 MHz, D2O): δ 4.74-4.70 (m, 2H, H2), 3.87 (s, 3H, H6), 3.55 (dd, J = 12.4, 3.6 Hz, 1H), 3.45–3.42 (m, 1H), 2.52 (dd, J = 14.2, 7.8 Hz, 1H), 2.36-2.27 (m, 1H) ppm. 13C{1H} NMR (101 MHz, D2O): δ 172.3, 70.0, 58.7, 53.7, 49.3, 37.3 ppm. IR (ATR): 3323, 2933, 2861, 2700, 1741, 1593, 1441, 1242, 1076 cm-1. (Edelmann) HRMS (ESI-TOF) m/z: [M+H]+ calcd for C6H12NO3 146.0817, found 146.0817. (Tian) trans-4-Hydroxy-N-(Pf)-(S)-proline methyl ester (5). The proline salt (1.023 g, 5.633 mmol) and anhydrous dichloromethane were added to a flame-dried 100 mL round-bottom flask under an argon atmosphere. The solution was cooled to 0 °C and freshly distilled triethylamine (3 mL, 0.02 mol) was added. Trimethylsilyl chloride (TMSCl, 1.8 mL, 14 mmol) was diluted in dichloromethane (10 mL) and the solution was slowly added by syringe. The reaction was brought to reflux using a water bath and stirred for 2 h, then cooled to 0 °C. Anhydrous methanol (0.34 mL, 8.4 mmol) was slowly added by syringe to quench the excess TMSCl, and the reaction was warmed to rt and stirred for 2 h. Lead (II) nitrate (2.60 g, 7.85 mmol) and 9-bromo-9-phenyl-fluorenyl (PfBr, 2.03 g, 6.32 mmol) were added followed by freshly distilled triethylamine (1 mL, 7 mmol). The reaction mixture was stirred for 5 days, then vacuum filtered to remove triethylammonium chloride as a white solid. The filtrate was concentrated in vacuo and the residue yellow oil was stirred with citric acid (3.32 g, 17.3 mmol) in methanol (40 mL) as a suspension at rt for 3h. The mixture was concentrated in vacuo, redissolved in dichloromethane, and stirred with an aqueous solution of sodium bicarbonate (0.4 M, 45 mL, 18 mmol) until gas evolution ceased. The resulting biphasic mixture was extracted with dichloromethane (3 x 50 mL) and the combined organic layer was washed with brine, dried over magnesium sulfate, and concentrated in vacuo. The crude protected proline 5b was recovered as a pale-yellow foamy solid (1.909 g, 89%) which Chapter 2 33 was used without further purification. Spectra were consistent with those reported in the literature.30,31 Molecular formula/weight: C25H23NO3: 385.45 g/mol. 1H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 7.6 Hz, 1H, Pf), 7.64 (d, J = 7.6 Hz, 1H, Pf), 7.55 –7.53 (m, 3H, Pf), 7.42 (t, 1H, Pf), 7.33-7.21 (m, 5H, Pf), 7.15 (t, 1H, Pf), 4.50 (p, J = 5.2 Hz, 1H, H4), 3.57 (dd, J = 10.0, 5.2 Hz, 1H, H2), 3.28 (dd, J = 8.8, 5.2 Hz, 1H, H5), 3.24 (s, 3H, H6), 2.91 (dd, J = 10.0, 5.2 Hz, 1H, H5’), 2.01-1.94 (m, 1H, H3’), 1.82-1.75 (m, 1H, H3) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 175.9, 147.2, 146.1, 142.7, 141.4, 139.9, 128.7, 128.4, 128.3, 127.6, 127.3, 127.3, 127.2, 127.1, 126.4, 120.0, 119.8, 70.3, 59.3, 56.8, 51.3, 39.9 ppm. (Edelmann) MP: 85–86 °C. (Tian) FTIR (thin film): 3443, 3057, 2946, 1727 cm-1. (Tian) HRMS (ESI) m/z: [M+H]+ calcd. for C25H24NO3 386.1756, found 386.1750. (Tian) 4-Oxo-N-(Pf)-(S)-proline methyl ester (6). A 25 mL oven-dried round-bottom flask was placed under an argon atmosphere. Anhydrous dichloromethane (2 mL) and oxalyl chloride (0.20 mL, 2.3 mmol) were charged to the flask and cooled to -78 °C. Dimethylsulfoxide (0.26 mL, 3.7 mmol) was diluted in anhydrous dichloromethane (2 mL) and added to the dilute oxalyl chloride solution slowly to avoid warming. After stirring at -78 °C for 30 min, alcohol 5 (283 mg, 0.734 mmol) was charged in an oven-dried vial, dissolved in anhydrous dichloromethane (4 mL), and slowly added to the reaction mixture which was then stirred at -78 °C for 3 h. Freshly distilled triethylamine (0.3 mL, 2 mmol) was added by syringe, and the reaction was allowed to warm to rt over 30 min. The reaction mixture was diluted in dichloromethane, washed with a saturated aqueous solution of sodium bicarbonate (1 x 50 mL), and extracted with dichloromethane (3 x 50 mL). The combined organic layer was washed with brine (1 x 100 mL), dried over magnesium sulfate, and concentrated in vacuo yielding a thick yellow oil with suspended solid. The crude product was diluted in minimal benzene and purified by flash chromatography on silica gel (20 to 25% acetone/hexane Chapter 2 34 gradient). Ketoproline 6 was recovered as a white foamy solid. (230 mg, 82%). Spectra were consistent with those reported in the literature.30,31 Molecular formula/weight: C25H21NO3: 383.44 g/mol. TLC: 25% acetone/hexane. 1H NMR (400 MHz, CDCl3): δ 7.73-7.70 (m, 2H, Pf), 7.47–7.37 (m, 6H, Pf), 7.31–7.27 (m, 2H, Pf), 7.25-7.22 (m, 2H, Pf), 3.78-3.74 (m, 2H, H5, H2), 3.47 (d, J = 18.0 Hz, 1H, H5’), 3.21 (s, 3H, H6) 2.44 (dd, J = 18.0, 8.6 Hz, 1H, H3), 2.28 (dd, J = 18.0, 2.8 Hz, 1H, H3’) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 212.7, 173.1, 146.5, 145.4, 141.9, 140.9, 140.4, 129.0, 128.9, 128.6, 128.1, 127.7, 127.6, 127.0, 126.9, 125.5, 120.3, 120.1, 76.0, 58.2, 55.2, 51.5, 41.7 ppm. (Edelmann) MP: 128–129 °C. (Tian) FTIR (thin film): 3060, 2949, 2838, 1758, 1739 cm-1. (Tian) HRMS (ESI) m/z: [M+H]+ calcd. for C25H22NO3 384.1600, found 384.1602. (Tian) trans-3-(2’-tert-Butoxyl)-4-oxo-N-(Pf)-(S)-proline methyl ester (7a). Ketoproline 6 (354 mg, 0.923 mmol) was charged in a 100 mL oven-dried round-bottom flask, dissolved in benzene, and concentrated in vacuo (2 x 10 mL) to remove trace water due to the hygroscopic nature of the compound. The flask was sealed with a rubber septum and placed under an argon atmosphere. Anhydrous tetrahydrofuran (6 mL) and anhydrous hexamethylphosphoramide (0.21 mL, 1.2 mmol) were added to the flask and the solution was cooled to -78 °C. n-BuLi (2.5 M, 0.4 mL, 0.001 mol) was added dropwise into the reaction and stirred at -78 °C for 40 min. Iodoacetate 26b (1.36 g, 5.55 mmol) was added at -78°C and then warmed to -40 °C after 15 mins. The mixture was stirred at -40 °C for 4 h, at which point thin-layer chromatography indicated complete consumption of starting material. The mixture was quenched with an aqueous solution of phosphoric acid (10% w/v, 10 mL) at -40 °C then extracted with diethyl ether (3 x 20 mL). Brine was added sparingly to help break up emulsions during the first extraction. The combined organic layer was washed with brine (1 x 50 mL), dried over magnesium sulfate, and concentrated in vacuo yielding the crude diastereomers as a bourbon-coloured sweet-smelling oil (1.45 g, 316%). Chapter 2 35 Spectroscopic analysis by proton NMR showed a 7:1 diastereomeric ratio and a large presence of unreacted iodoacetate causing the sweet smell. The crude oil was diluted in minimal benzene (sonication sometimes required) and purified by flash chromatography on silica gel (15 to 20 to 25% EtOAc/hexane gradient). The diastereomers were collected in two fractions with 6:1 (346 mg) and > 20:1 dr (115 mg) respectively. The coeluted diastereomers were dissolved in minimal diethyl ether with the aid of sonication and stored at -20 °C overnight. The supernatant liquid was removed via syringe, and the solid was washed with cooled hexanes (2 x 5 mL), removing the supernatant by syringe after each wash. The remaining solid was dried in vacuo yielding ketodiester 7a (227 mg, 49%) as a white solid with an enriched (dr > 20:1. The concentrated supernatant contained the diastereomers in a 1.6:1 ratio that could be further purified by flash chromatography. Spectra were consistent with those reported in the literature.30,31 On gram-scale, the crude dr ratio decreased to 2:1 with a lowered yield of ketodiester 7a (529 mg, 27%, dr > 20:1). Molecular formula/weight: C31H31NO5: 497.58 g/mol. TLC: 25% EtOAc/hexane. trans-Ketodiester (7a) 1H NMR (400 MHz, CDCl3): δ 7.71–7.69 (m, 2H, Pf), 7.52–7.48 (m, 3H, Pf), 7.43–7.41 (m, 1H, Pf), 7.38 (tt, J = 7.6, 1.2 Hz, 2H, Pf), 7.29–7.26 (m, 3H, Pf), 7.25–7.22 (m, 2H, Pf), 3.78 (d, J = 18.0 Hz, 1H, H2), 3.47–3.42 (m, 2H, H5), 3.11 (s, 3H, H6), 2.84–2.79 (m, 1H, H3), 2.42–2.39 (m, 2H, H7), 1.31 (s, 9H, H8) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 211.8, 172.9, 169.6, 146.0, 144.4, 141.4, 141.1, 140.4, 128.9, 128.5, 127.9, 127.8, 127.7, 127.3, 127.0, 126.0, 120.2, 120.0, 81.4, 75.8, 63.7, 55.7, 51.5, 49.2, 34.2, 27.9 ppm. (Tian) FTIR (thin film): 2974, 1758, 1730 cm-1. (Tian) HRMS (ESI-TOF) m/z: [M+H]+ calcd for C31H32NO5 498.2280, found 498.2278. (Tian) cis-Ketodiester (7b) 1H NMR (400 MHz, CDCl3): δ 7.80–7.23 (m, Pf), 4.02 (d, J = 8.2 Hz, 1H), 3.80 (d, J = 17.7 Hz, 1H), 3.58 (d, J = 17.7 Hz, 1H), 3.14 (s, 3H), 3.10–3.05 (m, 1H), 2.57 (dd, J = 17.4, 5.2 Hz, 1H), 1.83 (dd, J = 17.4, 8.9 Hz, 1H), 1.34 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3): δ 212.2, 171.7, 170.3, 146.9, 145.3, 141.7, 141.3, 139.8, 128.9, 128.8, 128.6, 128.1, 127.7, 127.6, 126.8, 126.7, 125.4, 120.1, 81.1, 75.4, 61.9, 53.7, 51.1, 48.0, 31.1, 28.0. (Tian) FTIR (thin film): 2974, 1758, 1727 cm-1. (Tian) Chapter 2 36 HRMS (ESI-TOF) m/z: [M+H]+ calcd for C31H32NO5 498.2280, found 498.2275. (Tian) (2S,3R,4S)-3-(2’-tert-Butoxyl)-4-hydroxy-N-(Pf)-S-proline methyl ester (8a). Ketodiester 7a (192 mg, 0.386 mmol) was charged in a 20 mL vial, suspended in anhydrous methanol (5 mL), and sonicated until a moderately homogenous mixture was achieved. With vigorous stirring, the mixture was cooled to -40 °C followed by the addition of sodium borohydride (76 mg, 2.0 mmol) as a solid in one portion. The vial was placed under an argon atmosphere and sealed with a rubber septum. The mixture was stirred at -40 °C for 4 h, at which point thin-layer chromatography indicated complete consumption of starting material. After warming to rt, the reaction was quenched with a saturated aqueous solution of ammonium chloride, diluted in ethyl acetate, and solvent volume was reduced in vacuo. The aqueous mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic layer was washed with an aqueous solution of HCl (0.1 M, 1 x 20 mL), washed with brine (1 x 50 mL), dried over magnesium sulfate, and concentrated in vacuo. The crude (165 mg, 86% conversion) was recovered as a clear-colourless oil containing diastereomers 8a and 8b in a 5:1 ratio (by NMR analysis) which was used without further purification. Diastereomers 8a/8b (0.386 mmol) were charged in a 50 ml round-bottom flask and dried in vacuo with benzene (2 x 10 mL) to azeotrope water due to the hygroscopic nature of the compound. The flask was placed under an argon atmosphere and anhydrous tetrahydrofuran (3 mL) was added. The mixture was cooled to 0 °C and 60% w/w sodium hydride in oil (70 mg, 1.8 mmol) was added as a powder in one portion. After 10 min, the reaction was warmed to rt, stirred for 4 h, then diluted in diethyl ether and quenched with a saturated aqueous solution of ammonium chloride (30 mL). The aqueous layer was extracted with diethyl ether (3 x 30 mL) and the combined organic layer was washed with brine (1 x 60 mL), dried over magnesium sulfate, and concentrated in vacuo yielding a light-yellow oil with some suspended solid (126 mg). The crude product was diluted in minimal benzene (sonication sometimes required) and purified by flash chromatography on silica Chapter 2 37 gel (40% EtOAc/hexane). Lactone 8c was recovered as an oily film (8 mg) and hydroxydiester 8a was recovered as a foamy white solid (65 mg, 34% over two steps). Spectra were consistent with those reported in the literature.28,31 Molecular formula/weight: C31H33NO5: 499.60 g/mol. TLC: 100% DCM or 40% EtOAc/hexane, Rf = 0.2 and 0.5 respectively. trans-3,4-Hydroxydiester (8a) 1H NMR (400 MHz, CDCl3): δ 7.77 (d, J = 7.4 Hz, 1H, Pf), 7.63 (d, J = 7.6 Hz, 1H, Pf), 7.56 (d, J = 7.2 Hz, 2H, Pf), 7.46 (t, J = 7.6 Hz, 2H, Pf), 7.38–7.34 (m, 1H, Pf), 7.31–7.22 (m, 5H, Pf), 7.12 (t, J = 7.6 Hz, 1H, Pf), 4.44 (d, J = 10.4 Hz, 1H, OH), 3.83–3.79 (m, 1H, H4), 3.41 (d, J = 10.0 Hz, 1H, H5’), 3.30 (s, 3H, H6), 3.18 (dd, J = 10.0, 4.0 Hz, 1H, H5), 2.68 (d, J = 2.8 Hz, 1H, H2), 2.34–2.29 (m, 1H, H3), 1.88 (dd, J = 15.6, 7.6 Hz, 1H, H7), 1.71 (dd, J = 15.6, 8.4 Hz, 1H, H7’), 1.32 (s, 9H, H8) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 177.6, 170.5, 147.9, 145.2, 141.8, 141.8, 139.4, 128.9, 128.5, 127.8, 127.4, 127.2, 126.9, 126.2, 120.3, 120.0, 81.1, 75.2, 75.4, 64.1, 55.2, 52.0, 48.3, 38.5, 27.9 ppm. (Edelmann) FTIR (thin film): 3400, 2927, 1728, 1154, 741 cm-1. (Edelmann) HRMS (ESI-qTOF) m/z: [M]+ calcd. for C31H33NO5 (499.2359); observed (499.2319). cis-3,4-Hydroxydiester (8b) 1H NMR (400 MHz, CDCl3): δ 7.78–7.10 (m, Pf), 4.42 (m, 1H), 3.66 (dd, J = 11.2, 4.8 Hz, 1H), 3.22 (s, 3H, H6), 3.09 (dd, J = 11.2, 3.6 Hz, 1H), 2.87 (d, J = 7.6 Hz, 1H), 2.54–2.47 (m, 1H), 2.34–2.27 (m, 1H), 1.94 (dd, J = 16.0, 5.2 Hz, 1H), 1.36 (s, 9H, H10) ppm. Lactone (8c) 1H NMR (400 MHz, CDCl3): δ 7.78–7.17 (m, 1H, Pf), 5.21–5.18 (m, 1H), 3.73 (dd, J = 11.2, 4.8 Hz, 1H), 3.54 (d, J = 11.2 Hz, 1H), 3.33 (s, 1H), 2.91 (s, 3H, H6), 2.89–2.84 (m, 2H), 2.60–2.52 (m, 1H) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 176.6, 172.1, 147.6, 144.3, 141.9, 141.8, 139.3, 128.7, 128.6, 128.2, 127.6, 127.22, 127.17, 126.3, 124.9, 120.0, 120.0, 83.2, 73.4, 66.4, 51.4, 51.1, 41.1, 34.9 ppm. (Edelmann) FTIR (thin film): 2926, 2854, 1770, 1731, 1455, 1451, 1176, 740 cm-1. (Edelmann) Chapter 2 38 (2S,3R,4S)-3-(2’-tert-Butoxyl)-4-mesyl-N-(Pf)-S-proline methyl ester (9). Alcohol 8a (57 mg, 0.11 mmol) was charged in a 20 mL vial, and dried in vacuo with benzene (2 x 10 mL) to azeotrope water due to the hygroscopic nature of the compound. The vial was placed under an argon atmosphere, then a solution of 0.5 M methanesulfonyl chloride in dichloromethane (1 mL, 0.5 mmol) and DIPEA (0.1 mL, 0.5 mmol) was added. The reaction mixture was stirred at rt for 3 h, quenched with a saturated aqueous solution of ammonium chloride (10 mL), and extracted with dichloromethane (3 x 10 mL). The combined organic layer was washed with brine (1 x 30 mL), dried over magnesium sulfate, and concentrated in vacuo yielding the crude mesylate (75 mg) as a yellow oil. The crude was diluted in minimal benzene and purified by flash chromatography on silica gel (20% EtOAc/hexane). Mesylate 9 was recovered as a foamy white solid (52 mg, 79%). Spectra were consistent with those reported in the literature.31 Molecular formula/weight: C32H35NO7S: 577.69 g/mol. TLC: 20% EtOAc/hexane. 1H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 7.6 Hz, 1H, Pf), 7.62 (d, J = 7.6 Hz, 1H, Pf), 7.53–7.50 (m, 2H, Pf), 7.47–7.43 (m, 2H, Pf), 7.37–7.33 (m, 1H, Pf), 7.31–7.27 (m, 3H, Pf), 7.25–7.23 (m, 1H, Pf), 7.14 (td, J = 7.6, 1.2 Hz, 1H, Pf), 4.67 (q, J = 6.0 Hz, 1H, H4), 3.56 (d, J = 6.0 Hz, 2H, H2), 3.25 (s, 3H, H6), 3.03 (s, 3H, H9), 2.75–2.68 (m, 2H, H3), 2.17–2.03 (m, 2H, H7), 1.32 (s, 9H, H8) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 174.0, 169.9, 146.7, 145.9, 142.5, 141.8, 139.7, 129.2, 128.71, 128.68, 128.3, 127.7, 127.5, 127.3, 127.1, 125.8, 120.4, 120.0, 81.4, 80.9, 76.4, 64.3, 53.8, 51.7, 45.8, 38.6, 36.7, 28.0 ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C32H35NO7S (577.2134); observed (577.2096). Chapter 2 39 (2S,3R,4R)-3-(2’-tert-Butoxyl)-4-azido-N-(Pf)-S-proline methyl ester (10). Mesylate 9 (172 mg, 0.297 mmol) was charged to a 50 mL oven-dried round-bottom flask and dried in vacuo with benzene (2 x 15 mL) to azeotrope water due to the hygroscopic nature of the compound. The flask was purged and sealed under nitrogen and anhydrous dimethylformamide (DMF, 5 mL) was added to dissolve the mesylate. Sodium azide (192 mg, 2.95 mmol) was quickly added in one portion and the flask was re-purged with nitrogen. The reaction was stirred at 75 °C for 48 h, cooled to rt, and diluted in ethyl acetate. The organic layer was washed with water (1 x 30 mL), then washed with an aqueous solution of HCl (0.5 M, 1 x 30 mL), extracted with ethyl acetate (3 x 30 mL), and dried over magnesium sulfate. The dried solution was concentrated in vacuo yielding a yellow liquid (280 mg). 1H NMR analysis showed a large amount of DMF remained, so the crude was diluted in ethyl acetate, washed with brine (2 x 30 mL), dried over magnesium sulfate, and concentrated in vacuo yielding the crude azidokainoid 10 (159 mg) as a yellow liquid. The crude product was diluted in minimal benzene and purified by flash chromatography on silica gel (10 to 20 to 30% EtOAc/hexane gradient). Azidokainoid 10 was recovered as a pale-yellow oil (76 mg, 49%). Spectra were consistent with those reported in the literature.31 Molecular formula/weight: C31H32N4O4: 524.61 g/mol. TLC: 30% EtOAc/hexane, Rf = 0.75. 1H NMR (400 MHz, CDCl3): δ 7.73 (d, J = 7.4 Hz, 1H, Pf), 7.62 (d, J = 7.5 Hz, 1H, Pf), 7.56–7.51 (m, 3H, Pf), 7.44 (td, J = 7.2 Hz, 1H, Pf), 7.35 (td, J = 7.2 Hz, 1H, Pf), 7.30–7.21 (m, 5H, Pf), 7.13 (td, J = 7.3 Hz, 1H, Pf), 4.39–4.35 (m, 1H, H4), 3.67 (dd, J = 10.7, 5.6 Hz, 1H, H5’), 3.22 (s, 3H, H6), 3.12 (dd, J = 10.7, 4.6 Hz, 1H, H5), 2.85 (d, J = 6.3 Hz, 1H, H2), 2.61 (dq, J = 8.8, 6.2 Hz 1H, H3), 2.24 (dd, J = 16.7, 8.8 Hz, 1H, H7’), 1.83 (dd, J = 16.7, 6.2 Hz, 1H, H7), 1.36 (s, 9H, H8) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 175.0, 170.8, 147.0, 145.7, 142.3, 141.5, 139.9, 129.0, 128.7, 128.6, 127.68, 127.65, 127.5, 127.5, 127.2, 126.6, 120.1, 119.9, 80.9, 76.2, 64.9, 62.1, 53.4, 51.62, 43.9, 33.7, 28.1 ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C31H32N4O4 (524.2424); observed (524.2381). Chapter 2 40 (4R)-4-Azidokainic acid (4). Azidokainoid 10 (10 mg, 0.019 mmol) was charged in a 2 mL polypropylene Eppendorf tube with an oven-dried flea-stir bar and dissolved in anhydrous dichloromethane (0.5 mL, 0.04 M). Trifluoroacetic acid (TFA, 3 μL, 0.04 mmol) and triethylsilane (Et3SiH, 7 μL, 0.04 mmol) were added with a 10 μL micropipette. After 3h, starting material was still observed by thin-layer chromatography (TLC) so a second aliquot of TFA (5 μL, 0.07 mmol) and Et3SiH (7 μL, 0.04 mmol) was added. After 5 h of total reaction time, volatiles were removed in vacuo and the residue was dissolved in methanol (0.5 mL). An aqueous solution of lithium hydroxide (0.6 M, 0.4 mL, 0.2 mmol) was added and the suspension was stirred at rt for 22 h. The reaction mixture was diluted in water and methanol was removed in vacuo. The aqueous mixture was washed with petroleum ether (3 x 10 mL) to remove the 9-phenylfluorene byproduct. The aqueous mixture was concentrated to dryness in vacuo (rotavap then hi-vac) producing a white solid residue that was purified by flash chromatography on Dowex 50WX8 ion-exchange resin (0.5 M aqueous ammonia solution). Fractions were evaluated by spotting on a silica-coated TLC plate, evaporating the bulk of the ammonia with gentle heating, and staining with ninhydrin. Background ammonia stains gave a wide-pink spot while the product spotted a yellow/orange colour. Azidokainoid 4 (3 mg, 75%) was recovered as a white solid. Molecular formula/weight: C7H10N4O4: 214.18 g/mol. TLC: 30% EtOAc/hexane, Rf = 0. 1H NMR (400 MHz, D2O): δ 4.65–4.63 (m, 1H, H4), 3.74 (d, J = 10.8 Hz, 1H, H2), 3.65 (dd, J = 12.8, 4.4 Hz, 1H, H5), 3.56 (d, J = 12.4 Hz, 1H, H5), 2.80-2.70 (m, 2H, H3, H6), 2.57-2.49 (m, 1H, H6) ppm. 13C{1H} NMR (101 MHz, D2O): δ 175.2, 172.4, 113.6, 83.4, 62.9, 50.2, 50.2, 43.0, 32.3, 27.1 ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C7H10N4O4 (214.0702); observed (214.0695). Chapter 2 41 4-[1’,2’,3’-Triazoyl-4’-(4’’-butanoic acid)] kainoid (29). Azidokainoid 10 (12 mg, 0.023 mmol) and hexynoic acid (6 mg, 0.05 mmol) were charged in a 1 mL strawberry-flask and dissolved in a 5:1 solution of tert-butanol:water (0.6 mL). Using a 10 μL micropipette, solutions of CuSO4 in water (1.2 M, 2 μL, 2 μmol), tris(benzyltriazolylmethyl)amine in dimethylsulfoxide (TBTA, 0.7 M, 3 μL, 2 μmol), and sodium ascorbate in water (2.2 M, 3 μL, 7 μmol) were added and the reaction was stirred at rt for 21 h. Solvents were removed in vacuo and the residue was redissolved in water. The aqueous mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic layer was washed with brine (1 x 10 mL), dried over sodium sulfate, and concentrated in vacuo yielding an oily residue that was diluted in minimal benzene and purified by flash chromatography on silica gel (40 to 70% EtOAc/hexane gradient with 1% acetic acid). Kainoid 29 (12 mg, 80 %) was recovered as a white solid. Molecular formula/weight: C37H40N4O6: 636.74 g/mol. TLC: 40% EtOAc/hexane with 1% AcOH. 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = Hz, 1H, Pf), 7.66 (d, J = Hz, 1H, Pf), 7.62–7.59 (m, 3H, Pf), 7.48–7.43 (m, 1H, Pf), 7.41–7.38 (m, 2H, Pf), 7.36 (m, 3H, Pf), 7.25–7.18 (m, 2H, Pf), 7.09 (s, 1H, H9), 5.38 (q, J = 7.2 Hz, 1H, H4), 3.83 (dd, J = 9.2, 6.8 Hz, 1H, H5), 3.69–3.64 (m, 1H, H5’), 3.27 (s, 3H, H6), 3.14 (d, J = 3.6 Hz, 1H, H2), 2.86–2.80 (m, 1H, H3), 2.73 (t, J = 7.6 Hz, 2H, H10/12), 2.39 (t, J = 7.2 Hz, 2H, H10/12), 1.97 (p, J = 7.2 Hz, 2H, H11), 1.71 (dd, J = 16.4, 8.8 Hz, 2H, H7), 1.61 (dd, J = 16.4, 6.4 Hz, 2H, H7’), 1.42 (s, 9H, H8) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 177.0, 174.8, 170.6, 146.8, 146.7, 145.6, 141.4, 141.3, 140.2, 129.1, 128.9, 128.6, 128.2, 127.8, 127.6, 127.4, 127.2, 126.5, 121.8, 120.4, 120.2, 81.3, 77.4, 75.6, 64.6, 59.6, 51.8, 50.3, 43.8, 33.9, 33.3, 27.9, 24.8, 24.4 ppm. Chapter 2 42 4-[1’,2’,3’-Triazoyl-4’-((N-(N’-Boc-ethylenediamine))-4’’-butamide)] kainoid (30). Kainoid 29 (12 mg, 0.019 mmol), hydroxybenzotriazole (4 mg, 0.03 mmol), and EDC hydrochloride (4 mg, 0.02 mmol) were charged in a 2 mL high-recovery vial. Anhydrous dichloromethane (0.3 mL) was added and stirred at rt for 20 min. Diamine 13 (3 μL, 0.02 mmol) was diluted in anhydrous dichloromethane (0.2 mL) and added to the reaction followed by triethylamine (8 μL, 0.06 mmol). The vial was placed under an argon atmosphere and stirred at rt for 42 h. The reaction was diluted in dichloromethane, washed with a saturated aqueous solution of sodium bicarbonate (1 x 10 mL), and extracted with dichloromethane (3 x 10 mL). The combined organic layer was washed with brine (1 x 30 mL), dried over magnesium sulfate, and concentrated in vacuo yielding the crude product as a thin white solid film (13 mg, 87%) which was used without further purification. Molecular formula/weight: C44H54N6O7: 778.93 g/mol. TLC: 40% EtOAc/hexane. 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.2 Hz, 1H, Pf), 7.66 (d, J = 7.6 Hz, 1H, Pf), 7.62–7.59 (m, 3H, Pf), 7.46 (t, J = 7.6 Hz, 1H, Pf), 7.41–7.28 (m, 5H, Pf), 7.25–7.18 (m, 2H, Pf), 7.10 (s, 1H, H9), 6.45 (br s, 1H, amide), 5.38 (q, J = 7.2 Hz, 1H, H4), 5.10 (br s, 1H, amide), 3.83 (dd, J = 9.6, 6.8 Hz, 1H, H5), 5.38 (t, J = 9.2 Hz, 1H, H5’), 3.38–3.33 (m, 2H, H13), 3.27–3.23 (m, 5H, H6,14), 3.12 (d, J = 3.2 Hz, 1H, H2), 2.88–2.82 (m, 1H, H3), 2.68 (t, J = 7.2 Hz, 2H, H10), 2.18 (m, 2H, H12), 2.00–1.90 (m, 3H, H11), 1.72 (dd, J = 16.4, 8.8 Hz, 1H, H7), 1.62 (dd, J = 16.4, 6.4 Hz, 1H, H7’), 1.42 (s, 9H, H15), 1.24 (s, 9H, H8) ppm. Chapter 2 43 4-[1’,2’,3’-Triazoyl-4’-((N-2’’-aminoethyl)-4’-butanamide)] kainoid (18). Kainoid 30 (13 mg, 0.017 mmol) was charged in a 5 mL vial and dissolved in anhydrous dichloromethane (0.5 mL). Using a 10 μL micropipette, TFA (4 μL, 0.05 mmol) and Et3SiH (5 μL, 0.03 mmol) were added in that order. The vial was placed under an argon atmosphere, sealed with a vial cap, and stirred at rt. After 3h, starting material was still observed by thin-layer chromatography (TLC) so a second aliquot of TFA (4 μL, 0.05 mmol) and Et3SiH (5 μL, 0.03 mmol) was added. After 5 h of total reaction time, a third aliquot of TFA (15 μL, 0.20 mmol) was added to help deprotect the remaining N-Boc and the reaction was stirred overnight for 23 h. Volatiles were removed in vacuo yielding a white solid that was redissolved in methanol (0.5 mL) followed by 0.6 M solution of lithium hydroxide (0.32 mL, 0.19 mmol) creating a suspension that was stirred at rt for 25 h. Methanol was removed in vacuo and the resulting white solid was dissolved in water. The undissolved solid believed to be PfH byproduct was filtered through a cotton-pipette. The remaining crude was purified by flash chromatography on Dowex 50W-X8 ion exchange resin (0.5 M aqueous ammonia solution). Fractions were evaluated by spotting on a silica-coated TLC plate, evaporating the bulk of the ammonia with gentle heating, and staining with ninhydrin. Background ammonia stains gave a wide-pink spot while the product spotted a yellow/orange colour. Kainoid 18 was recovered as a white solid (5 mg, 83%). Ammonium trifluoroacetate co-eluted with the product and was present in the carbon and fluorine NMRs but not quantified. The product was used as is for the next step. Molecular formula/weight: C15H24N6O5: 386.39 g/mol. TLC: 40% EtOAc/hexane, Rf = 0. 1H NMR (400 MHz, D2O): δ 7.70 (s, 1H, H7), 5.63–5.59 (m, 1H, H4), 4.10 (m, 2H, H2,5), 3.92 (d, J = 13.6 Hz, 1H, H5’), 3.51 (t, J = 5.6 Hz, 2H, H13), 3.15 (t, J = 6.0 Hz, 2H, H12), 3.08–3.00 (m, 1H, H3), 2.79–2.71 (m, 3H, H6,9), 2.28–2.21 (m, 2H, H11), 2.01–1.94 (m, 1H, H10), 1.38 (dd, J = 17.2, 11.6 Hz, 1H, H6’) ppm. Chapter 2 44 13C{1H} NMR (101 MHz, D2O): δ 178.1, 177.1, 160.2, 146.7, 125.0, 90.3, 64.2, 61.9, 50.0, 45.3, 39.1, 36.8, 34.7, 34.2, 24.3, 23.4 ppm. {13C-19F}: δ 116.3 (q, 1J = 293 Hz, NH4CF3COO); 162.9 (q, 1J = 36 Hz, NH4CF3COO) ppm. 19F-NMR (376 MHz, D2O): δ -75.65 (ammonium trifluoroacetate). (4R)-4-(1’,2’,3’-triazole-4’-(methanol)) kainic acid (28). Kainoid 27 was previously synthesized by Edelmann.31 Kainoid 27 (25 mg, 0.043 mmol) was charged in an oven-dried 25 mL strawberry-flask, dissolved in anhydrous dichloromethane (1 mL), and placed under a nitrogen atmosphere. Trifluoroacetic acid (TFA, 10 μL, 0.13 mmol) and triethylsilane (10 μL, 63 μmol) were added. Aliquots of anhydrous dichloromethane (5 x 0.8 mL) were added every 1–2 h to replace evaporated solvent. After 3h, starting material was still observed by thin-layer chromatography (TLC) so a second aliquot of TFA (15 μL, 0.20 mmol) and triethylsilane (30 μL, 0.19 mmol) were added. After 3 h, a final aliquot of triethylsilane (20 μL, 1.3 mmol) and TFA (20 μL, 0.26 mmol) were added and the reaction was stirred at rt for another 3 h at which point TLC indicated complete consumption of starting material. Volatiles were removed in vacuo and the residue was redissolved in methanol (1 mL). An aqueous solution of lithium hydroxide (2.5 M, 1 mL, 2.5 mmol) was added and the suspension was stirred at rt for 20 h. The reaction mixture was cooled to 0 °C, quenched with an aqueous solution of HCl (1 M), and diluted in water. The aqueous mixture was washed with petroleum ether (2 x 10 mL) to remove the 9-phenylfluorene byproduct. The mixture was concentrated to dryness in vacuo (rotavap then hi-vac) producing a white solid residue that was purified by flash chromatography on Dowex 50W-X8 ion-exchange resin (0.5 M aqueous ammonia solution). Fractions were evaluated by spotting on a silica-coated TLC plate, evaporating the bulk of the ammonia with gentle heating, and staining with ninhydrin. Molecular formula/weight: C10H14N4O5: 270.24 g/mol. TLC: 50% acetone/hexane. Chapter 2 45 1H NMR (400 MHz, D2O): δ 7.97 (s, 1H, H8), 5.71 (t, J = 5.6 Hz, 1H, H4), 4.19 (m, 2H, H2 and H5’), 4.04 (d, J = 13.2 Hz, 1H, H5), 3.21–3.12 (m, 1H, H3), 2.80 (dd, J = 17.2, 4.0 Hz, 1H, H6’), 1.55 (dd, J = 17.2, 11.2 Hz, 1H, H6) ppm. Note: H10 is masked by the solvent residual peak of HOD. 13C{1H} NMR (101 MHz, D2O): δ 174.7, 172.1, 146.8, 125.7, 63.8, 61.7, 54.6, 50.2, 43.7, 31.9 ppm. IR (thin film): 3390, 3287, 2510, 1716, 1634, 1419, 1047, 713 cm-1. Chapter 3 46 Chapter 3: Synthesis of methoxysquaraine, an alkyne linker, aminosquaraine derivatives, and the SQ-KA probe 3.1 Squaraine dyes: A unique and promising biological fluorophore Cyanines are a class of synthetic organic dyes with a conjugated π-system between two nitrogen atoms, one of which is oxidized as an iminium.42,43 Squaraines (SQs) are a subclass of cyanines that contain an electron-deficient squarate core and electron-donating aryl groups on either side which can be symmetric or asymmetric (Figure 3.1). Cyanines and SQs alike are utilized in biological applications due to their strong and narrow absorption bands in the near-infrared (NIR, >700 nm) region. For in vivo applications, NIR excitation is especially beneficial as it results in less photodamage over time and is capable of deep-tissue penetration. In general, NIR excitation also benefits from minimal background fluorescence due to shortwave-absorbing biological fluorophores. In contrast, shortwave excitation is limited to imaging surface-level tissue due to its shallow penetration, results in unwanted fluorescence from endogenous fluorophores, and produces higher energy photons that can be damaging to cells.44–46 Figure 3.1 Generic scaffolds of cyanines and squaraines. The aryl (Ar) groups of squaraines are conjugated through the squarate core and contain at least one nitrogen each, one of which is positively charged. Chapter 3 47 Initially, squaraines found limited use in biological applications due to their planar and hydrophobic nature which caused aggregation in aqueous media and effectively quenched fluorescence. However, this made squaraines an ideal fluorophore to incorporate into affinity-guided turn-on fluorescent (ToF) probes. Affinity-guided ToF probes are composed of a hydrophobic fluorophore and hydrophilic ligand. In the presence of aqueous media, an equilibrium is established that is dominated by non-fluorescent aggregates. When the target protein is introduced, the affinity of the probe towards the protein drives the disassembly of probe aggregates (Figure 3.2). Figure 3.2 Schematic depiction of turn-on fluorescent probes exhibiting aqueous-induced aggregation and protein-induced disassembly resulting in protein-selective turn-on fluorescence. This method was demonstrated in the literature by Mizusawa and coworkers when they reported their BODIPY-based probes (Figure 3.3A) which remained non-fluorescent in aqueous media until the corresponding target protein was introduced.20,21 Subsequently, He and coworkers demonstrated their version of an affinity-guided ToF probe when they developed their squaraine-biotin (SQ-biotin, Figure 3.3B) probe to monitor fluorescence emission in the absence and presence of avidin.22 They reported that the introduction of avidin resulted in a nine-fold increase in fluorescence intensity (Figure 3.3C); however, given the hydrophobic nature of the binding site, they needed to confirm if the change in fluorescence was driven by protein-ligand interactions or hydrophobic interactions between the SQ fluorophore and the binding site. Fluorescence emission of a biotin-less SQ was monitored while being subjected to increasing concentrations of avidin. Although a relatively small increase in fluorescence intensity was observed, it was also accompanied by a bathochromic shift (Figure 3.3D) which was characteristic of increased rigidity Chapter 3 48 of the SQ molecule induced by hydrophobic interactions with the binding site. Their control experiment confirms that the disassembly of non-fluorescent aggregates is mainly driven by protein-ligand interactions as the SQ-biotin produced a more stark increase in fluorescence intensity and lacked the bathochromic shift observed with the biotin-less SQ. Figure 3.3 (A) General BODIPY-based probe reported by Mizusawa et al.20,21 (B) SQ-biotin probe. (C) Relative fluorescence intensity of SQ-biotin (5 μM) in the absence and presence of avidin (3.85 μM), and SQ-biotin (5 μM) with free-biotin (77 μM) and avidin (3.85 μM). (D) Relative fluorescence intensity of a biotin-less SQ (5 μM) in the presence of avidin (0–5.03 μM). Fluorescence intensity graphs were reported by He et al and provided here with additional labels. 22 The work of He and colleagues served as a proof of concept that squaraines could be paired with affinity ligands to selectively label a protein of interest with minimal background fluorescence for enhanced image contrast. Thus, I sent out to synthesize the proposed squaraine-kainate (SQ-KA) probe (1, Figure 3.4), a new chemical tool to study kainate receptors (KARs). Chapter 3 49 Figure 3.4 The proposed squaraine-kainate (SQ-KA) probe (1). 3.2 Synthesis of a methoxysquaraine advanced intermediate Methoxysquaraine (2) was synthesized over three steps and required only one chromatographic purification with an overall yield of 55% (Scheme 3.1). Squaraine 2 served as the advanced intermediate for all the aminosquaraine derivatives in Section 3.4. Scheme 3.1 Synthesis of squaraine 2 with an overall yield of 55%. Microscale (<100 mg) routes gave improved overall yields (up to 67%) due to better yields for the formation of squaraine 12 (77%). 3.2.1 Optimizing the ethylation of 2-methylbenzothiazole The synthesis of methoxysquaraine (2) began with an ethylation of the commercially available 2-methylbenzothiazole (MeBT). Prior work on this reaction was performed in the Menard lab; however, improvement was still needed as benzothiazolium 11 was never produced in yields Chapter 3 50 greater than 47 percent, and even lower yields were observed at gram scale.31 My initial attempts were performed in a pressurized flask where the pressure was a result of solvent evaporation from prolonged elevated temperature. The miscibility of MeBT with iodoethane allowed for reactions to be run neat, with iodoethane in a large excess to favour product formation; however, this resulted in unproductive yields. Next, I attempted a procedure reported by Xu et al,47 where iodoethane and MeBT were refluxed in acetonitrile, although I modified the procedure with the addition of sodium triflate. I theorized that the iodide counterion was acting as a nucleophile towards the ammonium resulting in the reverse reaction. If true, then conversion to the ammonium product would depend on the equilibrium between the forward and reverse reaction. The addition of sodium triflate would facilitate an ion exchange to form a more stable ion pair and potentially decrease the reverse reaction. Unfortunately, this resulted in no product formation and was not explored further. I then decided to incorporate acetonitrile as a solvent in the pressure reaction. Iodoethane (3 equiv) and MeBT were diluted in acetonitrile and heated in a pressure vessel providing benzothiazolium 11 with improved yields that remained consistent regardless of scale. Near-quantitative yields of 11 were later achieved by cooling the suspension to -20 °C after the addition of diethyl ether and filtering under reduced pressure. Table 3.1 Reaction condition for the formation of thiazole 11 Entry Iodoethane (equiv) Temp. (°C) Reaction time (h) Yield (%) 1a 30 30–80 30 7 2a 30 30–80 72 36 3b 3 82 24 0 4c 3 80 71 65 5c 3 80 72 56 6c 3 80 95 52 7c 3 80 72 74 8d 3 80 70 94 a Ran neat, unstable hot plate. b Refluxed in MeCN, no pressure flask, NaOTf added. c Anhydrous MeCN solvent, pressure vessel. d Anhydrous MeCN solvent, pressure vessel, suspension cooled to -20 °C before isolating the product. Chapter 3 51 3.2.2 Formation of the squaraine dimer and O-methylation The consensus among reported literature suggests that the formation of squaraine dimers is accelerated through the activation of squaric acid. Initially, this was facilitated by refluxing squaric acid in alkyl alcohols. Treibs and Jacob, the pioneers of synthetic squaraine dyes, initially reported the reflux of squaric acid in ethanol, presumably forming the activated squarate ester.48 Shortly after, Sprenger and Ziegenbein improved upon this idea with the incorporation of n-butanol/benzene co-solvents for azeotropic distillation of the condensation byproduct.49 Modern adaptations of this synthetic protocol utilize n-butanol/pyridine as co-solvents under reflux resulting in enhanced yields compared to the former methods.50 A proposed mechanism for the formation of the squaraine dimer is depicted below (Scheme 3.2). First, an -deprotonation of the iminium forms a transient nucleophilic enamine. The addition of two enamines to the squarate core proceeds through a condensation reaction, resulting in the elimination of two water molecules. Throughout the reaction pyridine and n-butanol provide a means for proton transfers. Scheme 3.2 A proposed mechanism for the formation of squaraine 12. Chapter 3 52 My initial attempts to synthesize squaraine 12 were based on modern reported procedures in which the reagents were refluxed in n-butanol/pyridine co-solvents (5:1 ratio) over approximately 18–24 hours; however, this resulted in low yields due to the challenges associated with chromatographic purification. The crude reaction mixture was sparsely soluble in typically chromatographic eluents which resulted in the co-elution of product with impurities; necessitating multiple purifications to achieve mediocre pure yields. To overcome this, the crude was instead dry-loaded to a column after adhering to silica gel which provided significant improvements to yields after one purification attempt. Despite improvements with dry-loading, when the reactions were scaled up, yields suffered significantly (Table 3.2). The detriment to yield with upscaled reactions is believed to be a purification issue rather than a reactivity issue. It is hypothesized that the crude contained π-conjugated polymerized byproducts that resulted from the high heat and long reaction times. These byproducts can aggregate with the desired squaraine and prevent it from effectively passing through the silica gel. Additionally, when performing chromatography on larger portions of crude material, column diameter may not have been increased sufficiently to accommodate the upscaled reaction, resulting in thick bands and co-elution. Two procedures have since been reported by Mandim et al. and Lima et al. for the synthesis of squaraine dimers that procure the zwitterionic compound in significantly improved yields (93 and 73%, respectively) from previously reported methods, without necessitating chromatographic purification.50,51 Their procedures utilize the n-butanol/pyridine co-solvents in a 9:1 ratio over a four-hour reaction period. The more sparing use of pyridine as well as lower reaction times, likely reduces the potential for side reactions and demonstrates that lower reaction times are sufficient in achieving optimal conversion. Perhaps the biggest factor in their enhancement of yield was the exclusion of chromatographic purification. Mandim and colleagues reported the isolation and purification of their squaraine product via precipitation from the reaction mixture at 0 °C, washing with water and diethyl ether, and filtering under reduced pressure to afford a chromatographically pure product. Lima and colleagues report a similar method, although they went on to further purify their product by recrystallization in dichloromethane/diethyl ether. Given this new-found information, I decided to adapt their methods for the synthesis of 12 in hopes of achieving enhanced yields while avoiding the challenges associated with chromatographic purification. The first complication was identifying if a precipitate was forming Chapter 3 53 in the cooled reaction mixture due to its dark and opaque nature. Attempts to shine light through the mixture to monitor precipitation were unsuccessful. In hindsight, the reaction mixture could have been transferred to a large crystallization dish for more efficient cooling and better visibility of the precipitate formation. With the reaction mixture at 0 °C, diethyl ether was added to help force the product out of the solution. The suspension was filtrated under reduced pressure and subsequently washed with diethyl ether and water leaving behind a dark green residue. I then attempted to recrystallize the residue in dichloromethane and diethyl ether according to Lima et al.51 However, boiling dichloromethane was not sufficient to fully solubilize the solids, so I began adding methanol while maintaining a boil. Upon full dissolution of the solids, diethyl ether was slowly added until precipitation was observed, at which point the solution was cooled to -40 °C, filtered under reduced pressure, and washed with ether to provide the product as a remarkably lustrous green solid in a 24 percent yield (Figure 3.5). The resulting filtrate was diluted with more ether, cooled to -40 °C for 5–10 minutes, then filtered under reduced pressure. This was done twice increasing the yield to 33 and 42% respectively. Although my yield falls below that reported by Mandim and Lima, it demonstrates the ability to isolate the pure squaraine 12 without resorting to chromatography. Optimization of the crystallization procedure could easily elevate yields within the reported range and should be pursued. Figure 3.5 Squaraine 12 after recrystallization in DCM/MeOH. Chapter 3 54 Table 3.2 Reaction conditions for the formation of squaraine dimers with comparison to literature Entry Squaric acid (mmol) BuOH-pyridine (mL) Time (h) [M]a Yield (%) 1 11.0 6:1 22 0.05 4 2 4.38 5:1 24 0.04 13 3 3.68 5:1 18 0.03 32 4 2.53 5:1 22 0.03 47 5 1.68 5:1 19 0.04 61 6 0.62 5:1 22 0.03 77 7b 2.11 9:1 3.5 0.01 42 Lima c 4.12 9:1 5.5 0.08 73 Mandimc 2.32 9:1 4 0.01 93 a Concentration of squaric acid (mol) in the total solvent volume of n-BuOH + pyridine (L). b Experimental results after replicating reported procedures for the synthesis of squaraine dimers. c Reported literature conditions from Lima and Mandim.50,51 The last step to procure squaraine 2 was performed easily through the methylation of squaraine 12 with methyl triflate. Trituration of the crude residue provided methoxysquaraine in excellent yields without the need for chromatographic purification (Scheme 3.3). Scheme 3.3 Methylation of squaraine 12 to form methoxysquaraine (2). Chapter 3 55 3.3 Synthesis of the alkyne linker To couple the kainoid and fluorophore together, a simple alkyne linker 3 was synthesized from ethylenediamine over three steps. First, one of the amines was protected with a tert-butoxycarbonyl (Boc) group to provide diamine 13 for a subsequent amide coupling to hexynoic acid. A deprotection in the presence of excess trifluoroacetic acid afforded the alkyne linker 3 in a 66% overall yield (Scheme 3.4). Scheme 3.4 Overall synthesis of alkyne linker 3. The Boc protection was performed at 0 °C with gradual additions of a dilute Boc anhydride solution over two hours to achieve mono-protection while minimizing the formation of the diamine 31. Initially, ethylenediamine was added in a 2-fold excess resulting in a 15–25% formation of 31. Optimal conditions were achieved with three equivalents of ethylenediamine and slight adjustments to the reaction conditions (described in Section 3.6.4.1), providing diamine 13 as the sole product with an 81% yield. Scheme 3.5 Protection of ethylenediamine resulting in the mono- and di-protected products. Attempts were made to achieve selective mono-protection without the use of excess ethylenediamine to improve reaction efficiency. A procedure was reported by Lee et al. where unsymmetrical and symmetrical diamines, including ethylenediamine, were selectively mono-protected with Boc anhydride in the presence of one equimolar hydrochloric acid (HCl).52 An equimolar amount of HCl, when allowed to reach equilibrium, would contain the mono-protonated Chapter 3 56 amine as the dominant species (Scheme 3.6). This procedure was adapted to introduce the acid via an aqueous solution of HCl, whereas Lee introduced HCl as a gas instead. In practice, these conditions produced diamine 13 and 31 in 1:1 ratios, and further investigation to improve the conversion was not continued. Scheme 3.6 Equilibrium of ethylenediamine in the presence of equimolar HCl. Two classical coupling reagents were used to test their influence on conversion. N,N’-Dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (EDC) was used to couple 13 to hexynoic acid with 94% and 77% yields respectively. Typically, EDC is the preferred coupling reagent due to the water-soluble urea byproduct being easily removed during an aqueous workup, as opposed to the DCC urea byproduct that is not water-soluble and usually requires chromatography to remove. Fortunately, the majority of the DCC urea byproduct could be precipitated with 10% hexanes in dichloromethane at -20 °C and filtered out, simplifying the subsequent chromatographic purification. 3.4 Synthesis of aminosquaraine derivatives The synthesis of aminosquaraines with unique reactive handles was sought out to enable multiple coupling options to a ligand of choice (Table 3.3). Aminosquaraine 15 was easily attained in good yields by stirring methoxysquaraine (2) in an excess of ethylenediamine. The synthesis of aminosquaraine 16 proceeded at a slower rate, requiring heat and extended reaction times, but still resulted in lower yields. I decided to optimize the nucleophilic displacement when coupling the more precious alkyne linker 3. Ultimately, the use of pyridine as the reaction solvent was found to increase coupling efficiency despite ambient temperatures, reduced reaction times, and near-stoichiometric quantities of the amine. Pyridine is hypothesized to help reactivity for four reasons: (1) the basicity of pyridine increases the nucleophilicity (reactivity) of the amine, (2) pyridine can participate as a nucleophilic catalyst through the displacement of the methoxy group to form a more reactive pyridinium-Chapter 3 57 squarate intermediate, (3) pyridine can stabilize ionic transition states as an electron donor (pyridine) or acceptor (pyridinium conjugate acid), (4) pyridine can disrupt π- π stacking between squaraine-squaraine aggregates, which would increase the effective concentration of monomeric squaraine available to participate in the reaction. Table 3.3 Reaction conditions for the synthesis of aminosquaraines Aminosquaraine R-NH2 (equiv) Solvent Temp/Time Yield (%) 15 5.0 dichloromethane rt, 3 h 73 16 1.2 methanol 40 °C, 44 h 21 17 1.3 pyridine rt, 24 h 84 3.5 Synthesis towards a squaraine-kainate probe As discussed in Section 2.2, Tian developed the first known fluorescent probe for KARs in his synthesis of sulfo-Cy5-KA. Tian’s probe demonstrated that 4-triazolekainoids can be used as effective ligands for selective labeling when incorporated into a fluorescent probe.30 Triazolekainoids can be accessed through the installation of an alkyne (Tian) or azide (this work) at the C4-position of kainic acid enabling a copper-catalyzed azide-alkyne cycloaddition (CuAAC) to a fluorophore with the opposing reactive handle. To the best of my knowledge, no other fluorescent probe has since been developed for KARs. Herein, I describe my efforts towards a novel squaraine-kainate (SQ-KA) turn-on fluorescent probe (1), adding to the limited arsenal of chemical tools to study KARs and their role in neurological health and disease (Figure 3.6). Chapter 3 58 Figure 3.6 Tian’s sulfo-Cy5-KA kainate probe and the SQ-KA probe 1 (this work). I believe it is important to preface that I went through multiple iterations of the azidokainoid synthesis and subsequent depletion of material which required me to go back and procure more of the azidokainoid advanced intermediates over many months. Initial difficulties with the CuAAC reaction led me to exhaust other coupling options, however, those were unsuccessful, motivating me to instead go back and resolve the CuAAC reaction issues. At this stage I possessed a limiting amount of azidokainoid 10 (30 mg); thus, I devised a strategic plan for optimal utilization splitting the material into three equal portions for three unique routes (Scheme 3.7). Luckily, I was able to immediately resolve the CuAAC conditions with the successful synthesis of kainoid 18, described in Section 2.7. Next, I attempted another CuAAC between azidokainoid 10 and aminosquaraine 17, successfully yielding the protected SQ-KA probe 19. A global deprotection of probe 19 was performed which unfortunately led to its suspected complete degradation and no product or starting material was able to be recovered. I then attempted to couple kainoid 18 to methoxysquaraine (2) using the same conditions described in Section 3.4 for the optimized synthesis of aminosquaraine 17. Unfortunately, despite various attempts at characterizing the reaction mixture by NMR, no conclusive information led to the identification or recovery of any product(s). This left me with minimal information on potential reaction optimizations, and I was forced to abandon this route. Lastly, I deprotected the remaining azidokainoid 10 and subsequently attempted a CuAAC reaction between azidokainoid 4 and aminosquaraine 17. Despite the previous success of the CuAAC reaction, at this final stage, the reaction failed to produce the desired SQ-KA probe. Characterization of the reaction mixture with various NMR solvents (methanol-d4, pyridine-d6, Chapter 3 59 and deuterium oxide) was conclusive in identifying only the unreacted aminosquaraine 17. Unfortunately, the depletion of azidokainoid advanced intermediates prevented me from completing the synthesis of the SQ-KA probe 1 in a reasonable timeframe. Scheme 3.7 A strategic plan devised to assemble probe 1 with optimal usage of the limited azidokainoid 10 advanced intermediate. 3.6 Experimental 3.6.1 General experimental procedures Unless otherwise noted, reactions run under inert conditions were first evacuated in vacuo and then backfilled with an inert gas (argon or nitrogen, three times), using a balloon adaptor to maintain the environment throughout the reaction. Reactions performed at -78 °C or -40 °C were cooled using an acetone/dry ice and MeCN/dry ice bath respectively. Reactions performed at 0 °C were cooled using an ice-brine bath. When reactions were heated above rt, the reaction vessel was Chapter 3 60 immersed in a heated mineral oil bath unless stated otherwise. The addition of reagents under 10 μL was performed using a VWR Ergonomic High-Performance 10 ± 0.6 μL micropipettor. Solutions were concentrated under reduced pressure using rotary evaporation (50–200 torr) unless stated otherwise. High-vacuum (hi-vac) conditions (< 0.001 torr) were achieved with a Welch 1400B-01 DuoSeal Vacuum Pump. Thin layer chromatography (TLC) was performed on glass plates precoated with silica gel (F254, 250 μm, 60 Å, Sigma Aldrich). TLC plates were visualized under a 254 or 365 nm UV light source, then stained by immersion in either acidic ethanolic ninhydrin, acidic aqueous ethanolic vanillin solution, or potassium permanganate, followed by heating using a heat gun. Flash column chromatography was performed using silica gel (230–400 mesh, SiliaFlash R12030B, P60, 40–63 μm, 60 Å). Ion-exchange chromatography was performed using Dowex®, 50W-X8, Ion Exchange Resin, hydrogen form, 200–400 mesh, matrix acetic group: sulfonic acid. 3.6.2 Materials Commercial reagents and starting materials were purchased from Sigma-Aldrich, Oakwood Chemicals, Alfa Aesar, Acros Organics, TCI America, or Fisher Scientific and were used as received unless otherwise noted. Anhydrous solvents were dried over activated 4 Å molecular sieves and let stand for at least 24 h before use unless stated otherwise. Solvents that needed to be exceptionally dry were purified on a glass contour solvent purification system under an argon atmosphere. Diisopropylethylamine, pyridine, and triethylamine were distilled over calcium hydride and used immediately, otherwise, they were stored over 4 Å molecular sieves in light-protected amber bottles until discolouration. The molarity of commercially obtained n-butyllithium solutions was determined by titration against diphenylacetic acid as an indicator. Supplemental characterization is provided for some compounds previously synthesized by Menard lab alumni Simon Edelmann. This spectral information is indicated as follows: (Edelmann) after the end of the listed values. Title compounds are numbered for the assignment of protons and do not always match numbering for nomenclature. 3.6.3 Instruments 1H and 13C NMR spectra were recorded on a 400 MHz Varian NMR AS400 equipped with an ATB-400 probe at 25 °C. NMR spectra were analyzed with MestReNova version 6.0.2-5475 from Chapter 3 61 Mestrelab Research. Proton chemical shifts are reported in parts per million (ppm, δ scale) and are referenced to either tetramethylsilane (defined at 0.00 ppm) or residual protium in the NMR solvent (CHCl3: δ 7.26 ppm, methanol-d3: δ 3.31, dimethylsulfoxide-d5: δ 2.50 ppm, HOD: δ 4.79 ppm, pyridine-d5: δ 8.74, 7.58, 7.22 ppm). Carbon chemical shifts are reported in parts per million (ppm, δ scale) and are referenced to either tetramethylsilane (defined at 0.00 ppm) or the carbon resonance of the NMR solvent (CDCl3: δ 77.16 ppm, methanol-d4: δ 49.00, dimethylsulfoxide-d6: δ 39.52 ppm. Fluorine chemical shifts were not referenced to a standard ; however, chemical shift values were consistent to the hundredth decimal over multiple experiments. Spectral data are listed as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet), coupling constant (Hz), and assignment (if known). Infrared (IR) spectra were obtained on a PerkinElmer FT-IR spectrometer Spectrum two, with a globar light source, a Michelson moving-mirror and a pyroelectric detector working at room temperature at a range of (4000 cm-1 to 700 cm-1). The attenuated total reflectance (ATR) with one reflection used was a PIKE MIRacle which contained material composed of a ZnSe mixture. High-resolution mass spectra were obtained using an Agilent 6530 Q-TOF mass spectrometer with a 1290 infinity II HPLC (Zandberg lab, ESI-positive mode, 5 μL direct injections of sample in methanol with 0.1% formic acid). 3.6.4 Experimental procedures and characterization 3.6.4.1 Alkyne linker N-(2-tert-Butylcarbamate)-ethylenediamine (13). Ethylene-1,2-diamine (1.8 mL, 27 mmol) and anhydrous dichloromethane (65 mL) were charged in a 250 mL oven-dried round-bottom flask and cooled to 0 °C. Di-tert-butyl dicarbonate (Boc anhydride, 1.79 g, 8.20 mmol) was charged in a 125 mL addition funnel and dissolved in dichloromethane (50 mL). Approximately half of the dilute Boc-solution was added to the chilled diamine in small aliquots over 1.5 h (note: addit ion of the Boc-solution induced cloudiness to the reaction mixture that would slowly clear up over time). The reaction mixture was warmed to rt and stirred for 30 min without additions of the Boc-Chapter 3 62 solution. The remaining half of the Boc-solution was then added in larger aliquots over 1 hr at rt, after which the reaction was stirred for 30 min. The reaction was quenched with a saturated aqueous solution of sodium bicarbonate (150 mL) then the solvent volume was reduced in vacuo. The biphasic mixture was separated, and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic layer was dried over sodium sulfate and concentrated in vacuo yielding the crude mono-protected diamine 13 (1.06 g, 81%) as a colourless oil which was used without further purification. Spectra were consistent with those reported in the literature.31 Molecular formula/weight: C7H16N2O2: 160.21 g/mol. 1H NMR (400 MHz, CDCl3): δ 3.17 (br. q, J = 6.0 Hz, 2H, H2), 2.80 (t, J = 6.0 Hz, 2H, H3), 1.45 (s, 9H, H1) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 156.2, 79.2, 43.2, 41.7, 28.4 ppm. (Edelmann) FTIR (thin film): 3347, 2977, 1688, 1517, 1367, 1168, 873 cm-1. (Edelmann) N-(N’-(tert-Butylcarbamate)-2’-amidoethyl))-hex-5-ynamide (14). A 100 mL oven-dried round-bottom flask was charged with hexynoic acid (609 mg, 5.43 mmol) and anhydrous dichloromethane (15 mL) under a nitrogen atmosphere. The flask was cooled to 0 °C, and then 4-dimethylaminopyridine (1.715 g, 14.04 mmol) and N-Boc-ethylenediamine (3.51 mmol) were added. In an addition funnel, DCC (1.0 mL, 6.4 mmol) was dissolved in anhydrous dichloromethane (17 mL) and added dropwise. The reaction mixture was warmed to rt, stirred for 22 h, then quenched with a saturated aqueous solution of sodium bicarbonate (1 x 50 mL), and extracted with dichloromethane (3 x 50 mL). The combined organic layer was washed with brine (1 x 50 mL), dried over magnesium sulfate, and concentrated in vacuo yielding a white powder that was redissolved in 10% hexanes/dichloromethane (100 mL) and stored at -20 °C overnight precipitating the urea byproduct. The suspension was cold vacuum filtered and the solid was washed with cooled dichloromethane. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography on silica gel (20% EtOAc/hexane). Alkyne 14 (774 mg, 87%) was recovered as a white powder. Spectra were consistent with those reported in the literature.31 Chapter 3 63 Molecular formula/weight: C13H22N2O3: 254.32 g/mol. TLC 20% EtOAc/hexane, Rf = 0.45. 1H NMR (400 MHz, CDCl3): δ 6.45 (br s, 1H, NH), 5.13 (br s, 1H, NH), 3.38–3.34 (m, 2H, H2/3), 3.31–3.23 (m, 2H, H2/3), 2.32 (t, J = 7.4 Hz, 2H, H4), 2.26 (td, J = 6.8, 2.6 Hz, 2H, H6), 1.98–1.97 (m, 1H, H7), 1.86 (p, J = 7.1 Hz, 2H, H5), 1.44 (s, 9H, H1) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 173.1, 157.0, 83.6, 79.7, 69.3, 40.7, 40.4, 35.2, 28.5, 24.3, 18.0 ppm. FTIR (thin film): 3316, 3290, 2977, 2120, 1702, 1645, 1536, 1288, 1246, 976, 641 cm-1. (Edelmann) N-(2’-Aminoethyl)-hex-5-ynamide trifluoroacetic acid (3). Alkyne 14 (774 mg, 3.04 mmol) was charged in an oven-dried 100 mL round-bottom flask and placed under a nitrogen atmosphere. Anhydrous dichloromethane (20 mL) was added followed by TFA (2.3 mL, 30 mmol) and the reaction was stirred at rt until consumption of starting material was indicated from TLC after 21 h. Volitiles were removed in vacuo yielding a thick yellow oil that was diluted in ether and stored at -20 °C overnight. The resulting suspension was cold vacuum filtered and washed with cold hexanes. Alkyne linker 3 was recovered as a white crystalline solid (764 mg, 94%). Spectra were consistent with those reported in the literature.31 Molecular formula/weight: C10H15F3N2O3: 268.23 g/mol. TLC: (4:1 DCM/MeOH), Rf = 0.6. 1H NMR (400 MHz, MeOD): δ 3.44 (t, J = 6.0 Hz, 2H, H2/3), 3.04 (t, J = 6.0 Hz, 2H, H2/3), 2.39–2.34 (m, 2H, C4), 2.27–2.21 (m, 3H, H6,7), 1.88–1.78 (m, 2H, H5) ppm. 13C{1H} NMR (101 MHz, MeOD): δ 176.7, 84.1, 70.3, 40.8, 38.2, 35.6, 25.5, 18.7 ppm. FTIR (thin film): 3290, 3070, 2941, 2118, 1674, 1550, 1204, 1136, 834, 800, 722 cm-1. (Edelmann) HRMS (ESI-qTOF) m/z: [M]+ calcd. for C8H15N2O (155.1179); observed (155.1182). Chapter 3 64 3.6.4.2 Methoxysquaraine N-Ethyl-2-methylbenzothiazolium iodide (11). Acetonitrile was dried over CaCl2 and purged with argon for 20 min before use. 2-Methylbenzothiazole (3.53 g, 23.7 mmol), iodoethane (7.4 mL, 71 mmol), and MeCN (30 mL) were charged in a 150 mL oven-dried pressure flask and placed under an argon atmosphere. The reaction was maintained between 80-90 °C over 72 h with vigorous stirring. After cooling to rt, 30 mL of diethyl ether was added to precipitate the product as a white solid. The suspension was stored at -20 °C for 3 h, then vacuum filtered and washed with cold hexanes. Benzothiazolium 11 (6.80 g, 94%) was recovered as a white powdery solid which was used without further purification. Spectra were consistent with those reported in the literature.31,47,53 Molecular formula/weight: C10H12INS: 305.18 g/mol. 1H NMR (400 MHz, DMSO-d6): δ 8.44 (m, 1H, benzothiazole), 8.34 (d, J = 8.4 Hz, 1H, benzo.), 7.90 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H, benzo.), 7.81 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H, benzo), 4.76 (q, J = 7.3 Hz, 2H, H2), 3.21 (s, 3H, H3), 1.46 (t, J = 7.3 Hz, 3H, H1) ppm. 1H NMR (400 MHz, MeOD): δ 8.33 (m, 1H, benzothiazole), 8.29 (d, J = 8.4 Hz, 1H, benzo.), 7.93 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H, benzo.), 7.82 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H, benzo.), 4.85 (q, J = 7.2 Hz, 2H, H2), 3.25 (s, 3H, H3), 1.60 (t, J = 7.2 Hz, 3H, H1) ppm. 13C{1H} NMR (101 MHz, MeOD): δ 177.7, 142.3, 131.0, 130.8, 129.7, 125.4, 117.8, 46.5, 17.2, 13.8 ppm. IR (thin film): 2959, 2924, 1513 1420, 733 cm-1. (Edelmann) HRMS (ESI-qTOF) m/z: [M]+ calcd. for C10H12NS (178.0685); observed (178.0701). Chapter 3 65 Squaraine (12). Benzothiazolium 11 (992 mg, 3.25 mmol), anhydrous n-butanol (40 mL), and squaric acid (192 mg, 1.68 mmol) were charged in a 250 mL flame-dried round-bottom flask and placed under an argon atmosphere. An oven-dried reflux condenser was flushed with argon, fitted to the reaction flask, and sealed with a rubber septum. Freshly distilled pyridine (8 mL, 0.1 mol) was added by syringe, and the mixture was brought to reflux (note: as the temperature increases the reaction mixture goes from a cloudy white suspension to a solution with multiple color changes: pink–brown–black–green–dark blue). After 19 h the reaction mixture was cooled to rt, and concentrated in vacuo, using toluene as an azeotrope for the butanol and pyridine. The dark blue, almost black, residue was redissolved in dichloromethane and minimal methanol, then silica gel was added to create a slurry. The dichloromethane and methanol were removed in vacuo and the crude product/silica powder was dry-loaded and purified by flash chromatography on silica gel (2% to 5% methanol/dichloromethane gradient). Squaraine 12 was recovered as a lustrous green/blue solid (426 mg, 61%). Spectra were consistent with those reported in the literature.31,47,53 Note: (1) Thin-layers of squaraine 12 appeared greener in colour (see Section 3.2.2) while thicker layers appeared more blue. (2) Smaller scale reactions resulted in higher yields and reactions under 100 mg of squaric acid did not typically require dry-loading for chromatographic purification. (Ex., 0.622 mmol squaric acid gave 77% yield of squaraine 12). Molecular formula/weight: C24H20N2O2S2: 432.56 g/mol. TLC (5% MeOH/DCM), Rf = 0.45. 1H NMR (400 MHz, CDCl3): δ 7.52 (d, J = 8.0 Hz, 2H, benzothiazole), 7.35 (ddd, J = 8.4, 7.6, 1.2 Hz, 2H, benzo.), 7.18 (t, J = 7.6 Hz, 2H, benzo.), 7.13 (d, J = 8.0 Hz, 2H, benzo.), 5.86 (s, 2H, H3), 4.14 (q, J = 6.8 Hz, 4H, H2), 1.41 (t, J = 6.8 Hz, 6H, H1) ppm. 13C{1H} NMR (101 MHz, CDCl3): δ 175.6, 159.1, 140.7, 128.8, 127.0, 123.89, 122.2, 111.0, 85.0, 40.9, 29.7, 12.4 ppm. (Edelmann) IR (thin film): 3099, 1588, 1459, 1253, 1097, 755 cm-1. (Edelmann) HRMS (ESI-qTOF) m/z: [M]+ calcd. for C24H20N2O2S2 (432.0966); observed (432.0948). Chapter 3 66 Methoxysquaraine (2). Squaraine 12 (287 mg, 0.663 mmol) was charged in a 100 mL oven-dried round-bottom flask, placed under an argon atmosphere, then dissolved in anhydrous dichloromethane (11 mL). Methyl triflate (0.4 mL, 4 mmol) was added and the reaction was stirred at rt for 25 h. The reaction mixture was quenched with a 5% aqueous solution of sodium bicarbonate, extracted with dichloromethane (3 x 30 mL), and dried over sodium sulfate. The dried solution was concentrated in vacuo yielding a dark blue solid that was subsequently purified by triturating with toluene providing methoxysquaraine (376 mg, 95%) as a lustrous purple/blue solid. Spectra were consistent with those reported in the literature.31,47,53 Molecular formula/weight: C26H23F3N2O5S3: 596.66 g/mol. 1H NMR (400 MHz, DMSO–d6): δ 8.06 (d, J = 7.6 Hz, 2H, benzothiazole), 7.79 (d, J = 8.4 Hz, 2H, benzo.), 7.60 (t, J = 7.8 Hz, 2H, benzo.), 7.44 (t, J = 7.7 Hz, 2H, benzo.), 6.09 (s, 2H, H3), 4.55 (s, 3H, H4), 4.51 (q, J = 7.3 Hz, 4H, H2), 1.32 (t, J = 7.1 Hz, 6H, H1) ppm. 13C{1H} NMR (101 MHz, DMSO–d6): δ 177.3, 174.2, 161.2, 157.7, 140.2, 128.1, 127.5, 125.4, 123.1, 113.7, 85.2, 60.9, 41.6, 12.7 ppm. {13C-19F} (not always visible): δ 120.7 (q, 1J = 323 Hz, OTf) ppm. 19F NMR (376 MHz, DMSO–d6): δ -77.77 (s, OTf) ppm. IR (thin film): 1645, 1460, 1264, 1030, 751 cm-1. (Edelmann) HRMS (ESI-qTOF) m/z: [M]+ calcd. for C25H23N2O2S2 (447.1195); observed (447.1161). Chapter 3 67 3.6.4.3 Aminosquaraines N-(2-aminoethyl) aminosquaraine (15). Squaraine 2 (25 mg, 0.042 mmol) was charged in a 50 mL round-bottom flask, dissolved in anhydrous dichloromethane (8 mL), and placed under an argon atmosphere. The flask was wrapped in foil to protect it from light. Ethylenediamine (14 μL, 0.2 mmol) was added by syringe and the reaction was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo yielding a black/purple solid that was purified by triturating with diethyl ether, water, and benzene producing a colourless, red, and black supernatant respectively. The supernatants were filtered through a pipette with cotton and the remaining solids were dissolved in methanol, and then concentrated in vacuo providing the aminosquaraine (19 mg, 73%) as a dark purple solid. Molecular formula/weight: C27H27F3N4O4S3: 624.72 g/mol. TLC: 10% MeOH/DCM. 1H NMR (400 MHz, CDCl3): δ 7.59 (d, J = 8.0 Hz, 1H, benzothiazole), 7.49 (d, J = 8.0 Hz, 1H, benzo.), 7.45–7.36 (m, 3H, benzo.), 7.32–7.25 (m, 1H, benzo.), 7.21–7.17 (m, 1H, benzo.), 6.48 (s, 1H, H3), 5.85 (s, 1H, H3’), 4.44–4.39 (m, 2H, H2), 4.21–4.16 (m, 2H, H2’), 3.74–3.71 (m, 2H, H4/5), 3.14–3.11 (m, 2H, H4/5), 1.49–1.40 (m, 6H, H1) ppm. 19F NMR (376 MHz, CDCl3): δ -78.23 (s, OTf) ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C26H27N4OS2 (475.1621); observed (475.1596). Chapter 3 68 N-(6’-Hexanoic acid) aminosquaraine (16). Squaraine 2 (24 mg, 0.040 mmol), 6-aminohexanoic acid (AHA, 6 mg, 0.05 mmol), and anhydrous methanol (10 mL) were charged in a 20 mL vial under an argon atmosphere and stirred at rt for 24 h. Thin-layer chromatography indicated unreacted 2 and AHA, so the reaction temperature was raised to 40 °C and stirred for an additional 20 h at which point thin-layer chromatography indicated consumption of starting materials. The reaction mixture was concentrated in vacuo yielding a blue solid (17 mg) that was subsequently purified by flash chromatography on silica gel (5% methanol/dichloromethane) to recover aminosquaraine 16 (7 mg, 25%) as a blue solid. Flushing the column with methanol provided an additional fraction containing aminosquaraine 16 and hexanoic acid in a 1:1 ratio. Molecular formula/weight: C31H32F3N3O6S3: 695.79 g/mol. TLC: 10% MeOH/DCM. 1H NMR (400 MHz, MeOD): δ 7.83 (d, J = 8.0 Hz, 1H, benzothiazole), 7.79 (d, J = 8.0 Hz, 1H, benzo.), 7.62–7.49 (m, 4H, benzo.), 7.42–7.34 (m, 2H, benzo.), 6.06 (s, 1H, H3), 5.91 (s, 1H, H3’), 4.40–4.34 (m, 4H, H2), 3.68 (t, J = 7.2 Hz, 2H, H4), 2.36 (t, J = 7.6 Hz, 2H, H4), 1.81 (p, J = 7.2 Hz, 2H), 1.72 (p, J = 7.6 Hz, 2H), 1.58–1.52 (m, 2H), 1.48–1.41 (m, 6H, H1) ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C30H32N3O3S2 (546.1880); observed (546.1843). N-(N’-(5’’-alkynylcarbamate)-2’-aminoethyl) aminosquaraine (17). Squaraine 2 (192 mg, 0.322 mmol) and alkyne linker 3 (115 mg, 0.429 mmol) were charged in a 25 mL oven-dried round-bottom flask and placed under an argon atmosphere. Freshly distilled pyridine (4.5 mL, 58 mmol) and triethylamine (100 μL, 0.717 mmol) were injected and the reaction mixture was stirred at room temperature for 24 h. The mixture was diluted in dichloromethane-methanol (1:1) and silica gel was added to make a slurry. The dichloromethane and methanol were removed in vacuo and the crude/silica powder was dry-loaded and purified by flash chromatography on silica gel Chapter 3 69 (10% to 20% methanol/dichloromethane gradient) providing aminosquaraine 17 (194 mg, 84%) as a lustrous blue solid. Molecular formula/weight: C33H33F3N4O5S3: 718.83 g/mol. TLC (20% MeOH/DCM), Rf = 0.8. 1H NMR (400 MHz, CDCl3): δ 7.63 (d, J = 7.6 Hz, 1H, benzothiazole), 7.56 (d, J = 7.8 Hz, 1H, benzo.), 7.49 (t, J = 7.9 Hz, 1H, benzo.), 7.43 (t, J = 7.8 Hz, 1H, benzo.), 7.35 –7.32 (m, 2H, benzo.), 7.28–7.21 (m, 2H, benzo.), 6.60 (s, 1H, H3), 5.89 (s, 1H, H3’), 4.44 (q, J = 6.8 Hz, 2H, H2), 4.31 (q, J = 7.2 Hz, 2H, H2’), 3.82 (q, J = 5.6 Hz, 2H, H4/5), 3.60 (q, J = 5.6 Hz, 2H, H4/5), 2.37 (t, J = 7.2 Hz, 2H, H6), 2.23 (td, J = 7.2, 2.4 Hz, 2H, H8), 1.89 (t, J = 2.4 Hz, 1H, H9), 1.84 (p, J = 7.2 Hz, 2H, H7), 1.50 (t, J = 6.8 Hz, 3H, H1), 1.45 (t, J = 7.2 Hz, 3H, H1’) ppm. Protons influenced by concentration-based aggregation: δ 8.7–8.2 (m, 1H, amide), 7.40–7.11 (m, 1H, amide), 6.68–6.44 (s, 1H, H3), 5.91–5.88 (s, 1H, H3) ppm. See Appendix A3. 13C{1H} NMR (101 MHz, CDCl3): δ 175.0, 173.8, 164.5, 162.6, 159.9, 159.1, 156.1, 140.6, 128.9, 128.6, 128.1, 127.6, 125.4, 124.6, 122.6, 122.34, 112.8, 111.8, 87.78, 87.75, 85.8, 83.7, 69.1, 44.0, 42.6, 41.5, 39.7, 35.0, 24.5, 18.1, 13.0, 12.7 ppm. {13C-19F} (not always visible): δ 120.7 (q, 1J = 321 Hz, OTf) ppm. 19F NMR (376 MHz, CDCl3): δ -78.26 (s, OTf) ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C32H33N4O2S2 (569.2039); observed (569.2003). 3.6.4.4 Squaraine-kainate probe Protected squaraine-kainate probe (19). Aminosquaraine 17 (14 mg, 0.019 mmol), azidokainoid 10 (9.0 mg, 0.017 mmol), and a solution of tert-butanol/water (5:1, 1 mL) were charged in a 20 mL vial with a cross-stir bar. The mixture was sonicated to promote dissolution, but a partial Chapter 3 70 suspension remained. Solutions of CuSO4 in water (1.2 M, 2 μL, 2 μmol), tris(benzyltriazolylmethyl)amine in dimethylsulfoxide (0.7 M, 3 μL, 2 μmol), and sodium ascorbate in water (2.2 M, 3 μL, 7 μmol) were added with a 10 μL micropipette and the reaction mixture was stirred at rt for 24 h at which point thin-layer chromatography indicated complete consumption of starting material. The mixture was concentrated in vacuo and the aqueous mixture was diluted in water and extracted with dichloromethane (5 x 5 mL). The combined organic layer was washed with brine (1 x 25 mL), dried over sodium sulfate, and concentrated in vacuo yielding a blue solid (23 mg) that was purified by flash chromatography on silica gel (10% methanol/dichloromethane). The protected squaraine-kainate probe was recovered as a blue solid (14 mg, 67%). A separate fraction was isolated (2 mg) and determined to be the product that had undergone an ion exchange, losing the triflate anion, determined by 19F-NMR (no signal). Molecular formula/weight: C64H65F3N8O9S3: 1243.44 g/mol. TLC: 10% MeOH/DCM, Rf = 0.3. 1H NMR (400 MHz, CDCl3): δ 7.74 (d, 1H, J = 7.2 Hz, Pf), 7.66–7.30 (m, 17H, aromatics), 7.25–7.19 (m, 4H, aromatics), 5.34 (q, 1H, J = 7.2 Hz, H10), 4.46 (q, 2H, J = 7.6 Hz, H2), 4.29 (q, 2H, J = 7.2 Hz, H2), 3.83–3.77 (m, 3H, H4/5,11), 3.67–3.59 (m, 3H, H4/5,11’), 3.27 (s, 3H, H16), 3.10 (d, 1H, J = 3.2 Hz, H12), 2.86–2.80 (m, 1H, H13), 2.69–2.65 (m, 2H, H8), 2.30 (t, 2H, J = 7.2 Hz, H6), 1.94 (p, 2H, J = 7.2 Hz, H7), 1.70 (dd, 1H, J = 16.8, 9.2 Hz, H14’), 1.61 (dd, 1H, J = 16.4, 6 Hz, H14), 1.51 (t, 3H, H1), 1.44 (t, 3H, H1), 1.21 (s, 9H, H15) ppm. Protons influenced by concentration-based aggregation: 9.65–8.61 (br s, 1H, amide), 7.96–6.62 (br s, 1H, amide), 7.17–7.13 (s, 1H, H9), 5.91–5.89 (s, 1H, H3) ppm. See Appendix A4. 13C{1H} NMR (101 MHz, CDCl3): δ 175.0, 174.7, 174.0, 170.1, 164.6, 159.8, 159.1, 156.0, 146.7, 145.6, 141.6, 141.3, 140.7, 140.2, 129.1, 128.9, 128.8, 128.6, 128.2, 128.0, 127.8, 127.61, 127.55, 127.3, 127.2, 126.5, 125.4, 124.6, 122.6, 122.4, 120.4, 120.1, 112.8, 111.8, 80.9, 76.6, 75.6, 64.7, 59.4, 51.7, 50.3, 44.1, 43.6, 42.7, 41.5, 39.6, 35.8, 34.0, 29.9, 27.9, 25.4, 25.1, 13.1, 12.7 ppm. 19F-NMR (376 MHz, CDCl3): δ -78.25 (s, OTf) ppm. HRMS (ESI-qTOF) m/z: [M]+ calcd. for C63H64N8O6S2 (1093.4463); observed (1093.4384). Chapter 4 71 Chapter 4: Conclusion The overarching objective of this work was the design and synthesis of the squaraine-kainate (SQ-KA) probe 1 to label kainate receptors (KARs) with enhanced imaging contrast by limiting fluorescent background signals through fluorescence quenching. Although this work focused on KARs, the concept of fluorescence quenching can be applied to most proteins of interest by exchanging the ligand. The SQ-KA probe consists of three components: a squaraine fluorophore, alkyne linker, and kainoid ligand, synthesized individually and then pieced together (Scheme 4.1). Scheme 4.1 Summary of the syntheses involved in the development of the SQ-KA probe 1. Chapter 2 detailed important structural-activity relationships (SAR) for the development of biologically active kainoid analogs and a brief review of kainoid syntheses reported in the literature. The design of the kainoid ligand was inspired by such SAR studies that revealed a pattern among kainoids possessing aromatic C4-sidechains, inducing strong activation of Chapter 4 72 glutamate receptors (GluRs) due to a favourable interaction with a tyrosine residue in the binding site of GluRs. Synthesis of the novel kainoid analog, (4R)-4-azidokainic acid (4), provided the first component for probe 1 in addition to being a flexible ligand option for the general development of KAR-specific chemical probes through the robust copper-catalyzed azide-alkyne cycloaddition (CuAAC). Chapter 3 entails the optimized three-step syntheses of methoxysquaraine (2) and the alkyne linker 3, the second and third components of probe 1. The synthesis of three aminosquaraine analogs (15, 16, 17) from methoxysquaraine demonstrated the ability to easily derivatize the squaraine advanced intermediate, providing aminosquaraines with unique reactive handles for multiple coupling options. The latter portion of the chapter describes the culmination of my efforts toward the assembly of the SQ-KA probe 1. A limited quantity of azidokainoids 4 and 10 motivated me to devise a synthetic strategy for optimal usage. 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(referenced to literature). ...................................................................................... 82 Spectrum 5. 4(R)-Hydroxy-N-(Pf)-(S)-proline methyl ester (5b), 1H NMR (CDCl3, 400 MHz). .............................................................................................................................. 83 Spectrum 6. 4-Oxo-N-(Pf)-(S)-proline methyl ester (6), 1H NMR (CDCl3, 400 MHz).............. 84 Spectrum 7. 3(R)-(2’-tert-Butoxyl)-4-Oxo-N-(Pf)-(S)-proline methyl ester (7a), 1H NMR (CDCl3, 400 MHz)................................................................................................ 85 Spectrum 8. 3(S)-(2’-tert-Butoxyl)-4-Oxo-N-(Pf)-(S)-proline methyl ester (7b), 1H NMR (CDCl3, 400 MHz)................................................................................................ 86 Spectrum 9. 3(R)-(2’-tert-Butoxyl)-4(S)-hydroxy-N-(Pf)-(S)-proline methyl ester (8a), 1H NMR (CDCl3, 400 MHz)................................................................................................ 87 Spectrum 10. 3(S)-(2’-tert-Butoxyl)-4(S)-hydroxy-N-(Pf)-(S)-proline methyl ester (8b), 1H NMR (CDCl3, 400 MHz). .................................................................................... 88 Spectrum 11. Lactone 8c, 1H NMR (CDCl3, 400 MHz)............................................................. 89 Spectrum 12. 3(R)-(2’-tert-Butoxyl)-4(S)-mesyl-N-(Pf)-(S)-proline methyl ester (9), 1H NMR (CDCl3, 400 MHz)................................................................................................ 90 Spectrum 13. 3(R)-(2’-tert-Butoxyl)-4(S)-mesyl-N-(Pf)-(S)-proline methyl ester (9), 13C NMR (CDCl3, 101 MHz)................................................................................................ 91 Spectrum 14. 4(R)-Azido-3(R)-(2’-tert-butoxyl)-N-(Pf)-(S)-proline methyl ester (10), 1H NMR (CDCl3, 400 MHz)................................................................................................ 92 Spectrum 15. 4(R)-Azido-3(R)-(2’-tert-butoxyl)-N-(Pf)-(S)-proline methyl ester (10), 13C NMR (CDCl3, 101 MHz)................................................................................................ 93 Spectrum 16. 4(R)-4-Azidokainic acid (4), 1H NMR (D2O, 400 MHz). .................................... 94 Spectrum 17. 4(R)-4-Azidokainic acid (4), 13C NMR (D2O, 101 MHz). ................................... 95 Spectrum 18. Kainoid 29, 1H NMR (CDCl3, 400 MHz). ........................................................... 96 Spectrum 19. Kainoid 29, 1H gCOSY NMR (CDCl3, 400 MHz). .............................................. 97 Spectrum 20. Kainoid 29, 13C NMR (CDCl3, 101 MHz). .......................................................... 98 Spectrum 21. Kainoid 30, 1H NMR (CDCl3, 400 MHz). ........................................................... 99 Spectrum 22. Kainoid 30, 1H gCOSY NMR (CDCl3, 400 MHz). ............................................ 100 Spectrum 23. Kainoid 18, 1H NMR (D2O, 400 MHz). ............................................................. 101 Spectrum 24. Kainoid 18, 1H NMR (D2O, 400 MHz, PRESAT at 4.79 ppm). ........................ 102 Spectrum 25. Kainoid 18, 13C NMR (D2O, 101 MHz). *Ammonium trifluoroacetate. ........... 103 Spectrum 26. Kainoid 28, 1H NMR (D2O, 400 MHz). ............................................................. 104 Spectrum 27. Kainoid 28, 13C NMR (D2O, 101 MHz). ............................................................ 105 Spectrum 28. N-(2-tert-butylcarbamate)-ethylenediamine (13), 1H NMR (CDCl3, 400 MHz). ............................................................................................................................ 106 Appendix A 78 Spectrum 29. N-(N’-(tert-Butylcarbamate)-2’-amidoethyl))-hex-5-ynamide (14), 1H NMR (CDCl3, 400 MHz).............................................................................................. 107 Spectrum 30. N-(N’-(tert-Butylcarbamate)-2’-amidoethyl))-hex-5-ynamide (14), 13C NMR (CDCl3, 101 MHz).............................................................................................. 108 Spectrum 31. N-(2’-Aminoethyl)-hex-5-ynamide trifluoroacetic acid (3), 1H NMR (MeOD, 400 MHz)................................................................................................................... 109 Spectrum 32. N-(2’-Aminoethyl)-hex-5-ynamide trifluoroacetic acid (3), 13C NMR (MeOD, 101 MHz)............................................................................................................ 110 Spectrum 33. N-Ethyl-2-methylbenzothiazolium iodide (11), 1H NMR (DMSO-d6, 400 MHz). ............................................................................................................................ 111 Spectrum 34. N-Ethyl-2-methylbenzothiazolium iodide (11), 13C NMR (MeOD, 101 MHz). 112 Spectrum 35. Squaraine 12, 1H NMR (CDCl3, 400 MHz). ...................................................... 113 Spectrum 36. Methoxysquaraine (2), 1H NMR (DMSO-d6, 400 MHz). .................................. 114 Spectrum 37. Methoxysquaraine (2), 13C NMR (DMSO-d6, 101 MHz). ................................. 115 Spectrum 38. Aminosquaraine 15, 1H NMR (CDCl3, 400 MHz). ............................................ 116 Spectrum 39. Aminosquaraine 15, 1H gCOSY NMR (CDCl3, 400 MHz). .............................. 117 Spectrum 40. Aminosquaraine 16, 1H NMR (MeOD, 400 MHz). ........................................... 118 Spectrum 41. Aminosquaraine 17, 1H NMR (CDCl3, 400 MHz). ............................................ 119 Spectrum 42. Aminosquaraine 17, stacked 1H NMR (CDCl3, 400 MHz) of four samples. ..... 120 Spectrum 43. Aminosquaraine 17, stacked 1H NMR (CDCl3, 400 MHz) from four samples.. 121 Spectrum 44. Aminosquaraine 17, 1H gCOSY NMR (CDCl3, 400 MHz). .............................. 122 Spectrum 45. Aminosquaraine 17, 13C NMR (CDCl3, 101 MHz). ........................................... 123 Spectrum 46. Protected squaraine-kainate probe 19, 1H NMR (CDCl3, 400 MHz). ................ 124 Spectrum 47. Protected squaraine-kainate probe 19, 1H NMR (CDCl3, 400 MHz) from two samples. .............................................................................................................. 125 Spectrum 48. Protected squaraine-kainate probe 19, 1H gCOSY NMR (CDCl3, 400 MHz). .. 126 Spectrum 49. Protected squaraine-kainate probe 19, 13C NMR (CDCl3, 101 MHz). ............... 127 Appendix A 79 A1 Kainoids Spectrum 1. tert-Butyl bromoacetate (26a), 1H NMR (CDCl3, 400 MHz). Appendix A 80 Spectrum 2. tert-Butyl iodoacetate (26b), 1H NMR (CDCl3, 400 MHz). Appendix A 81 Spectrum 3. 4(R)-Hydroxy-(S)-proline methyl ester HCl (5a), 1H NMR (D2O, 400 MHz). Appendix A 82 Spectrum 4. 4(R)-Hydroxy-(S)-proline methyl ester HCl (5a), 13C NMR (D2O, 101 MHz). (referenced to literature). Appendix A 83 Spectrum 5. 4(R)-Hydroxy-N-(Pf)-(S)-proline methyl ester (5b), 1H NMR (CDCl3, 400 MHz). Appendix A 84 Spectrum 6. 4-Oxo-N-(Pf)-(S)-proline methyl ester (6), 1H NMR (CDCl3, 400 MHz). Appendix A 85 Spectrum 7. 3(R)-(2’-tert-Butoxyl)-4-Oxo-N-(Pf)-(S)-pro line methyl ester (7a), 1H NMR (CDCl3, 400 MHz). Appendix A 86 Spectrum 8. 3(S)-(2’-tert-Butoxyl)-4-Oxo-N-(Pf)-(S)-proline methyl ester (7b), 1H NMR (CDCl3, 400 MHz). Appendix A 87 Spectrum 9. 3(R)-(2’-tert-Butoxyl)-4(S)-hydroxy-N-(Pf)-(S)-pro line methyl ester (8a), 1H NMR (CDCl3, 400 MHz). Appendix A 88 Spectrum 10. 3(S)-(2’-tert-Butoxyl)-4(S)-hydroxy-N-(Pf)-(S)-proline methyl ester (8b), 1H NMR (CDCl3, 400 MHz). Appendix A 89 Spectrum 11. Lactone 8c, 1H NMR (CDCl3, 400 MHz). Appendix A 90 Spectrum 12. 3(R)-(2’-tert-Butoxyl)-4(S)-mesyl-N-(Pf)-(S)-p roline methyl ester (9), 1H NMR (CDCl3, 400 MHz). Appendix A 91 Spectrum 13. 3(R)-(2’-tert-Butoxyl)-4(S)-mesyl-N-(Pf)-(S)-p roline methyl ester (9), 13C NMR (CDCl3, 101 MHz). Appendix A 92 Spectrum 14. 4(R)-Azido-3(R)-(2’-tert-butoxyl)-N-(Pf)-(S)-pro line methyl ester (10), 1H NMR (CDCl3, 400 MHz). Appendix A 93 Spectrum 15. 4(R)-Azido-3(R)-(2’-tert-butoxyl)-N-(Pf)-(S)-pro line methyl ester (10), 13C NMR (CDCl3, 101 MHz). Appendix A 94 Spectrum 16. 4(R)-4-Azidokainic acid (4), 1H NMR (D2O, 400 MHz). Appendix A 95 Spectrum 17. 4(R)-4-Azidokainic acid (4), 13C NMR (D2O, 101 MHz). Appendix A 96 Spectrum 18. Kainoid 29, 1H NMR (CDCl3, 400 MHz). Appendix A 97 Spectrum 19. Kainoid 29, 1H gCOSY NMR (CDCl3, 400 MHz). Appendix A 98 Spectrum 20. Kainoid 29, 13C NMR (CDCl3, 101 MHz). Appendix A 99 Spectrum 21. Kainoid 30, 1H NMR (CDCl3, 400 MHz). Appendix A 100 Spectrum 22. Kainoid 30, 1H gCOSY NMR (CDCl3, 400 MHz). Appendix A 101 Spectrum 23. Kainoid 18, 1H NMR (D2O, 400 MHz). Appendix A 102 Spectrum 24. Kainoid 18, 1H NMR (D2O, 400 MHz, PRESAT at 4.79 ppm). Appendix A 103 Spectrum 25. Kainoid 18, 13C NMR (D2O, 101 MHz). *Ammonium trifluoroacetate. Appendix A 104 Spectrum 26. Kainoid 28, 1H NMR (D2O, 400 MHz). Appendix A 105 Spectrum 27. Kainoid 28, 13C NMR (D2O, 101 MHz). Appendix A 106 A2 Alkyne linker Spectrum 28. N-(2-tert-butylcarbamate)-ethylenediamine (13), 1H NMR (CDCl3, 400 MHz). Appendix A 107 Spectrum 29. N-(N’-(tert-Butylcarbamate)-2’-amidoethyl))-hex-5-ynamide (14), 1H NMR (CDCl3, 400 MHz). Appendix A 108 Spectrum 30. N-(N’-(tert-Butylcarbamate)-2’-amidoethyl))-hex-5-ynamide (14), 13C NMR (CDCl3, 101 MHz). Appendix A 109 Spectrum 31. N-(2’-Aminoethyl)-hex-5-ynamide trifluoroacetic acid (3), 1H NMR (MeOD, 400 MHz). Appendix A 110 Spectrum 32. N-(2’-Aminoethyl)-hex-5-ynamide trifluoroacetic acid (3), 13C NMR (MeOD, 101 MHz). Appendix A 111 A3 Methoxysquaraine and aminosquaraines Spectrum 33. N-Ethyl-2-methylbenzothiazolium iodide (11), 1H NMR (DMSO-d6, 400 MHz). Appendix A 112 Spectrum 34. N-Ethyl-2-methylbenzothiazolium iodide (11), 13C NMR (MeOD, 101 MHz). Appendix A 113 Spectrum 35. Squaraine 12, 1H NMR (CDCl3, 400 MHz). Appendix A 114 Spectrum 36. Methoxysquaraine (2), 1H NMR (DMSO-d6, 400 MHz). Appendix A 115 Spectrum 37. Methoxysquaraine (2), 13C NMR (DMSO-d6, 101 MHz). Appendix A 116 Spectrum 38. Aminosquaraine 15, 1H NMR (CDCl3, 400 MHz). Appendix A 117 Spectrum 39. Aminosquaraine 15, 1H gCOSY NMR (CDCl3, 400 MHz). Appendix A 118 Spectrum 40. Aminosquaraine 16, 1H NMR (MeOD, 400 MHz). Appendix A 119 Spectrum 41. Aminosquaraine 17, 1H NMR (CDCl3, 400 MHz). Appendix A 120 Spectrum 42. Aminosquaraine 17, stacked 1H NMR (CDCl3, 400 MHz) of four samples. Protons influenced by concentration-based aggregation: δ 8.7–8.2 (m, 1H, amide), 7.40–7.11 (m, 1H, amide), 6.68–6.44 (s, 1H, H3), 5.91–5.88 (s, 1H, H3) ppm. Appendix A 121 Spectrum 43. Aminosquaraine 17, stacked 1H NMR (CDCl3, 400 MHz) from four samples. Shifts between 1.2–5.0 ppm remain consistent despite concentration-based aggregation. Appendix A 122 Spectrum 44. Aminosquaraine 17, 1H gCOSY NMR (CDCl3, 400 MHz). Appendix A 123 Spectrum 45. Aminosquaraine 17, 13C NMR (CDCl3, 101 MHz). Appendix A 124 A4 Squaraine-kainate probe Spectrum 46. Protected squaraine-kainate probe 19, 1H NMR (CDCl3, 400 MHz). Appendix A 125 Spectrum 47. Protected squaraine-kainate probe 19, 1H NMR (CDCl3, 400 MHz) from two samples. Protons influenced by concentration-based aggregation: 9.65–8.61 (br s, 1H, amide), 7.96–6.62 (br s, 1H, amide), 7.17–7.13 (s, 1H, H9), 5.91–5.89 (s, 1H, H3) ppm. Appendix A 126 Spectrum 48. Protected squaraine-kainate probe 19, 1H gCOSY NMR (CDCl3, 400 MHz). Appendix A 127 Spectrum 49. Protected squaraine-kainate probe 19, 13C NMR (CDCl3, 101 MHz).
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Menard, Frederic
Development of an affinity-guided probe with turn-on fluorescence to selectively label kainate receptors
Text