Arts and Sciences, Irving K. Barber School of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Tian, Zhenlin
Doctor of Philosophy - PhD
University of British Columbia
Kainate receptors play important roles in the central nervous system. However, investigating their role in physiology is hampered by the current lack of tools to study them in dynamic cellular contexts. This thesis describes the synthesis of C4-kainoids and their derivatives, which are designed to serve as chemical probes to study kainate receptors in neurobiology. Chapter 1 provides a brief introduction of kainate receptors’ roles in central nervous systems and an overview of existing probes for glutamate receptors. Chapter 2 presented a comprehensive structure/activity analysis for efficient KARs ligands. This chapter reviews all reported kainic acid analogs that have been biologically tested and identifies systematic trends in biological activity of these analogs. These trends combined with protein crystallographic studies led us to identify structural guidelines that must be respected to design the next generation of efficient ligands for kainate receptors. These guidelines informed our synthetic design of the kainoid chemical probes presented in subsequent chapters. Chapter 3 focuses on the development of a synthetic strategy of kainic acid analogs that: (1) is short, (2) is high-yielding and scalable, and (3) allows easy variation of substituents at the C4 position of kainic acid. It resulted in a general stereoselective synthesis of 4-substituted kainoids: kainic acid and its natural isomers were synthesized in 8 to 11 steps from the commercially available 4hydroxyproline. The sequence also enables a late-stage modification of C4 substituents with sp 2 nucleophiles. Stereoselective steps include a cerium-promoted nucleophilic addition and a palladium-catalyzed reduction. A 10-step route to a carboxylic acid derivative was also established to enable ready functionalization of the C4 position. This work represents the first unified synthetic route to C4 derivatives of kainic acid. Chapter 4 describes the synthesis of kainoid derivatives that bear a fluorescent moiety that is used to visualize kainate receptors in living cells. A key intermediate from our general synthesis was exploited to create a novel alkynyl kainoid at the C4 position. This alkynyl kainoid was then coupled with a sulfo-Cy5-azide linker chain using a copper-catalyzed reaction to construct a fluorescent probe. In vitro fluorescence imaging performed by confocal microscopy with this far-red fluorescent probe confirmed the capability of labeling kainate receptors. This work reports the synthesis and use of the first fluorescent probe for kainate receptors. Chapter 5 describes the synthesis of two photo-controllable agonists for kainate receptors: a photocaged and a photoswitchable kainoids. First, our synthesis of one of the most potent unnatural kainoids (phenylkainic acid) was modified to install a photo-cleavable group on the nitrogen atom. The coumarin-type photo-cleavable group can be removed rapidly upon blue light irradiation, which results in the irreversible release of the agonist to trigger kainate receptor activity. Second, a kainoid derivative was synthesized where the C4 side chain integrates an azobenzene that can be isomerized between its cis or trans configuration using light. From the structural analyses presented in Chapter 2, only the trans-azobenzene form should be able to bind to kainate receptors. Since this photoisomerization is reversible using visible light (i.e., photoswitch), this kainoid probe can serve as an on/off switch to control the ion conductance of kainate receptors in cells. Kinetic studies by UV-Vis and 1 H NMR spectroscopy confirmed that the photoswitch does isomerize upon irradiation. Overall, a set of new chemical tools for kainate receptors is reported that expands the range of means to perform neurobiological experiments. Along the way, a practical synthetic route to new analogs of kainic acid was established. The compounds presented in this thesis are now being used by colleagues and collaborators to answer questions about the role of kainate receptors in neurons and glial cells that cannot be addressed using current neurobiology methods.
SYNTHESIS OF KAINIC ACID DERIVATIVES AND THEIR APPLICATION AS KAINATE RECEPTORS PROBES by Zhenlin Tian B.Sc., Hubei University of Technology, 2010 M.Sc., East China University of Science and Technology, 2013 A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) July 2019 © Zhenlin Tian, 2019 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Synthesis of kainic acid derivatives and their application as kainate receptors probes submitted by Zhenlin Tian in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Chemistry . Dr. Fred Menard, Department of Chemistry, UBC Okanagan Supervisor Dr. Paul Shipley, Department of Chemistry, UBC Okanagan Supervisory Committee Member Dr. Isaac T.S. Li, Department of Chemistry, UBC Okanagan Supervisory Committee Member Dr. Gregory Dake, Department of Chemistry, UBC Vancouver University Examiner Dr. Jeffrey Keillor, Department of Chemistry and Biomolecular Sciences, University of Ottawa External Examiner ii Abstract Kainate receptors play important roles in the central nervous system. However, investigating their role in physiology is hampered by the current lack of tools to study them in dynamic cellular contexts. This thesis describes the synthesis of C4-kainoids and their derivatives, which are designed to serve as chemical probes to study kainate receptors in neurobiology. Chapter 1 provides a brief introduction of kainate receptors’ roles in central nervous systems and an overview of existing probes for glutamate receptors. Chapter 2 presented a comprehensive structure/activity analysis for efficient KARs ligands. This chapter reviews all reported kainic acid analogs that have been biologically tested and identifies systematic trends in biological activity of these analogs. These trends combined with protein crystallographic studies led us to identify structural guidelines that must be respected to design the next generation of efficient ligands for kainate receptors. These guidelines informed our synthetic design of the kainoid chemical probes presented in subsequent chapters. Chapter 3 focuses on the development of a synthetic strategy of kainic acid analogs that: (1) is short, (2) is high-yielding and scalable, and (3) allows easy variation of substituents at the C4 position of kainic acid. It resulted in a general stereoselective synthesis of 4-substituted kainoids: kainic acid and its natural isomers were synthesized in 8 to 11 steps from the commercially available 4-hydroxyproline. The sequence also enables a late-stage modification of C4 substituents with sp2 nucleophiles. Stereoselective steps include a cerium-promoted nucleophilic addition and a palladium-catalyzed reduction. A 10-step route to a carboxylic acid derivative was also established to enable ready functionalization of the C4 position. This work represents the first unified synthetic route to C4 derivatives of kainic acid. Chapter 4 describes the synthesis of kainoid derivatives that bear a fluorescent moiety that is used to visualize kainate receptors in living cells. A key intermediate from our general synthesis was exploited to create a novel alkynyl kainoid at the C4 position. This alkynyl kainoid was then coupled with a sulfo-Cy5-azide linker chain using a copper-catalyzed reaction to construct a fluorescent probe. In vitro fluorescence imaging performed by confocal microscopy with this far-red fluorescent probe confirmed the capability of labeling kainate receptors. This work reports the synthesis and use of the first fluorescent probe for kainate receptors. Chapter 5 describes the synthesis of two photo-controllable agonists for kainate receptors: a photocaged and a photoswitchable kainoids. First, our synthesis of one of the most potent unnatural iii kainoids (phenylkainic acid) was modified to install a photo-cleavable group on the nitrogen atom. The coumarin-type photo-cleavable group can be removed rapidly upon blue light irradiation, which results in the irreversible release of the agonist to trigger kainate receptor activity. Second, a kainoid derivative was synthesized where the C4 side chain integrates an azobenzene that can be isomerized between its cis or trans configuration using light. From the structural analyses presented in Chapter 2, only the trans-azobenzene form should be able to bind to kainate receptors. Since this photo-isomerization is reversible using visible light (i.e., photoswitch), this kainoid probe can serve as an on/off switch to control the ion conductance of kainate receptors in cells. Kinetic studies by UV-Vis and 1H NMR spectroscopy confirmed that the photoswitch does isomerize upon irradiation. Overall, a set of new chemical tools for kainate receptors is reported that expands the range of means to perform neurobiological experiments. Along the way, a practical synthetic route to new analogs of kainic acid was established. The compounds presented in this thesis are now being used by colleagues and collaborators to answer questions about the role of kainate receptors in neurons and glial cells that cannot be addressed using current neurobiology methods. iv Lay Summary Kainate receptors (KARs) are crucial for maintaining the function of neurons in the brain. However, the lack of tools that can target KARs has prevented the progress of research about the physiological role of these receptors in neurons. Accordingly, my project aimed at developing chemical biology tools that can solve the limitations of KARs research. First, I developed a set of guidelines to design better chemical tools. Next, I developed a unified strategy to build the tools using organic synthesis. With further modifications, I developed a method to create three types of chemical tools that can be used to probe the activity of KARs in cells: a fluorescent kainoid, a photocaged kainoid, and a photoswitchable kainoid. These probes all have in common that they use light either track, activate, or control KARs in living cells. My compounds will accelerate the research on kainate receptors in neurobiology, which will help find treatments for neurodegenerative diseases. ii Preface This research project was conceived equally by Zhenlin Tian and Dr. Menard, with contributions also provided by Briana Clark and Simon Edelmann. As outlined below, the results of this thesis have led to, or are in the process of being written for, five publications in peer-reviewed journals. Zhenlin Tian wrote all of the manuscripts, including this dissertation. Chapter 2 Zhenlin Tian, Brianna Clark, Frederic Menard. Kainic acid-based agonists of glutamate receptors – Structural design guidelines emerge from a SAR analysis. ACS Chemical Neuroscience 2019 (Under review). Zhenlin Tian and Dr. Menard defined the scope of the manuscript. Zhenlin Tian: (1) conducted the literature search and structure analysis of all the reported kainoids; (2) compared the bioactivities of kainoids systematically; (3) wrote the manuscript and prepared the figures with the help of Dr. Menard; (4) assembled and compiled the tables listing all literature assays in the supplementary information files. Brianna Clark and Dr. Menard researched, analyzed and created the gene sequence alignments. Chapter 3 Zhenlin Tian, Frederic Menard. Synthesis of kainoids and C4 derivatives. Journal of Organic Chemistry 2018, 83, 6162. Zhenlin Tian and Dr. Menard defined the scope of this manuscript. Zhenlin Tian: (1) conducted the synthetic experiments for all the compounds included in this paper; (2) conducted all characterization experiments, including NMR, IR and MP; (3) processed and analyzed all data the experimental data; (4) wrote the manuscript and prepared the submission with the help of Dr. Menard. Chapter 4 Zhenlin Tian, Frederic Menard. An affinity probe for kainate receptors, Journal of the American Chemical Society (manuscript in preparation). Zhenlin Tian and Dr. Menard defined the scope of this manuscript. Zhenlin Tian: (1) conducted the synthetic experiments for all the compounds included in this paper; (2) performed all iii characterization experiments, including NMR, IR and MP; (3) conducted molecular biology experiments, including plasmid propagation and transfection; (4) carried out the confocal imaging experiments in living cells with the help of Dr. Menard; (5) processed and analyzed all data the experimental data; (6) wrote the manuscript and prepared the submission with the help of Dr. Menard. Chapter 5 Zhenlin Tian, Simon Edelmann, Frederic Menard. A coumarin-caged kainoid as a blue-light releasable agonist for kainate receptors. (manuscript in preparation). Zhenlin Tian and Dr. Menard defined the scope of this manuscript. Zhenlin Tian: (1) conducted the synthetic experiments with the help of Simon Edelmann for several precursor synthesis; (2) performed all characterization and photochemistry experiments; (3) processed and analyzed all data the experimental data; (4) wrote the manuscript and prepared the submission with the help of Dr. Menard and Simon Edelmann for reviewing. Zhenlin Tian, Frederic Menard. A visible light switchable agonist for kainate receptors. (manuscript in preparation). Zhenlin Tian and Dr. Menard defined the scope of this manuscript. Zhenlin Tian: (1) conducted the synthetic experiments for all the compounds involved; (2) performed all characterization and photochemistry experiments; (3) processed and analyzed all data the experimental data; (4) wrote the manuscript and prepared the submission with the help of Dr. Menard. iv Table of Contents Abstract ...................................................................................................................................................................................... ii Lay Summary ............................................................................................................................................................................. iv Preface ....................................................................................................................................................................................... ii Abbreviations ............................................................................................................................................................................. vii List of Schemes ........................................................................................................................................................................ viii List of Figures ............................................................................................................................................................................ ix List of Tables .............................................................................................................................................................................. x Acknowledgments ..................................................................................................................................................................... xi Dedication ................................................................................................................................................................................. xii CHAPTER 1. INTRODUCTION…………………………………………………. 1 1.1 Distinct role of kainic acid receptors in CNS………………………………………………………….. 2 1.2 Therapeutic potential of kainic acid receptors……………………………………………………….. 4 1.3 Limitations in research on kainic acid receptors…………………………………………………….. 6 1.4 Precedents on biosensors and switchable probes for GluRs……………………………………… 6 1.5 Thesis overview…………………………………………………………………………………………….. 9 CHAPTER 2. A STRUCTURE-ACTIVITY RELATIONSHIP ANALYSIS OF KAINIC ACID-BASED AGONISTS……………………………………………. 11 2.1 Background………………………………………………………………………………………………….11 2.2 Structural considerations………………………………………………………………………………... 12 2.2.1 The activity grading system ............................................................................................................................................. 12 2.2.2 Early SAR studies on kainic acid ..................................................................................................................................... 13 2.2.3 Structural variation on the C4 position ............................................................................................................................. 14 2.2.4 C4 π-π stacking ............................................................................................................................................................... 16 2.2.5 Domoic acid derivatives .................................................................................................................................................. 18 2.3 Protein–ligand interaction: the binding site of kainic acid………………………………………… 20 2.3.1 Crystallographic structures of glutamate receptors ........................................................................................................ 20 2.3.2 Structural analysis of GluK2-bound kainoids .................................................................................................................. 21 2.3.3 Notes on selectivity across KAR subtypes ...................................................................................................................... 23 2.4 Molecular design rules for potent kainic acid analogs……………………………………………... 23 2.5 Summary…………………………………………………………………………………………………….. 24 2.6 Data analysis……………………………………………………………………………………………….. 25 2.6.1 Calculation method .......................................................................................................................................................... 25 2.6.2 Calculated ratios and grades .......................................................................................................................................... 25 CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES………... 27 3.1 Background……………………………………………………………………………………………….. 27 v 3.2 Restrosynthetic analysis of 4-substituted kainoids………………………………………………… 28 3.3 Optimization of C3 intermediate synthesis…………………………………………………………… 29 3.4 Installation of C4 sidechain……………………………………………………………………………… 30 3.4.1 Stereoselective addition of sp2 nucleophiles ............................................................................................................ 30 3.4.2 Stereoselective deoxygenation of the C4 alcohol ..................................................................................................... 31 3.4.3 Protecting group swap: Pf for Boc ............................................................................................................................ 32 3.4.3 Deoxygenation to kainoids with modified selectivity ................................................................................................ 33 3.5 Synthesis of phenylkainic acid & more C4 functionalization……………………………………… 34 3.6 Summary……………………………………………………………………………………………………. 36 3.7 Experimental Section……………………………………………………………………………………... 37 3.7.1 General Experimental Procedures ............................................................................................................................ 37 3.7.2 Materials .................................................................................................................................................................... 37 3.7.3 Instruments ............................................................................................................................................................... 37 3.7.4 Experimental Procedures and Characterization ....................................................................................................... 38 CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS………………………………………………………… 56 4.1 Background………………………………………………………………………………………………… 56 4.2 Probe design……………………………………………………………………………………………….. 57 4.3 Probe synthesis…………………………………………………………………………………………… 58 4.4 Preliminary imaging experiments of KAR with the Sulfo-Cy5-KA probe……………………….. 62 4.5 Summary……………………………………………………………………………………………………. 66 4.6 Experimental Section……………………………………………………………………………………... 67 4.6.1 General Experimental Procedures ............................................................................................................................ 67 4.6.2 Materials .................................................................................................................................................................... 67 4.6.3 Instruments ............................................................................................................................................................... 67 4.6.4 Statistical analysis and data processing .................................................................................................................. 68 4.6.5 Chemical synthesis procedures & characterization ................................................................................................. 68 4.6.5 Biological experiments .............................................................................................................................................. 71 CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS………………. 73 5.1 Background………………………………………………………………………………………………… 73 5.2 Research gap………………………………………………………………………………………………. 75 5.3 Design of photo-controllable agonists for KARs……………………………………………………. 75 5.4 Synthesis of photocaged agonists…………………………………………………………………….. 77 5.4.1 Synthesis of MNI-Glu ................................................................................................................................................ 77 5.4.2 Synthesis of DECM-Glu and DECM-PhKA ................................................................................................................ 77 5.5 Synthesis of photoswitchable kainoids 79 vi 5.5.1 Synthesis of a new heteroaromatic kainoid .............................................................................................................. 79 5.5.2 Synthesis of an azobenzene kainoid photoswitch .................................................................................................... 80 5.6 Photochemical characterization of photo-controllable kainoids 82 5.6.1 Photochemical characterization of photocaged glutamates and kainoids ............................................................... 83 5.6.2 Photochemical characterization of kainoid photoswitch 136 .................................................................................... 88 5.7 Summary 93 5.8 Experimental section 95 5.8.1 General Experimental Procedures ............................................................................................................................ 95 5.8.2 Materials .................................................................................................................................................................... 95 5.8.3 Statistical analysis and data processing .................................................................................................................. 95 5.8.4 Instruments ............................................................................................................................................................... 95 5.8.5 Synthetic procedures & characterization .................................................................................................................. 96 CHAPTER 6. GENERAL CONCLUSION …………………………………… 110 APPENDIX A…………………………………………………………………... 112 Biological data for kainoids collected from reported studies…………………………………………...112 APPENDIX B…………………………………………………………………... 125 NMR data of kainoids and comparison……………………………………………………………………... 125 1D NOE Experiments and Spectra…………………………………………………………………………… 127 NMR spectra………………………………………………………………………………………………………130 APPENDIX C………………………………………………………………… 162 1D NOE spectra of compounds in Chapter 4………………………………………………………………. 162 NMR spectra of compounds in Chapter 4………………………………………………………………….. 165 APPENDIX D………………………………………………………………… 174 NMR spectra of photolysis……………………………………………………………………………………. 174 1D NOE spectra of compounds in Chapter 5………………………………………………………………. 177 NMR spectra of compounds in Chapter 5………………………………………………………………….. 178 REFERENCESÉ É É É É É É É É É É É É É É É É É É É É É É É É .203 vii AbbreviationsGeneral Abbreviations in Schemes: aq aqueous atm atmosphere c concentration cat. catalytic/catalyst conv. conversion d chemical shift D heat d day(s) (e.g., 2 d) dr diastereomeric ratio ee enantiomeric excess eq equation equiv. molar equivalent(s) EDG electron donating group EWG electron withdrawing group h hour(s) (e.g., 3 h) h light irradiation min minute(s) MS molecular sieves NMR nuclear magnetic resonance quant. quantitative yield Rf retardation factor rt (rt) room temperature (22 oC) sat. saturated UV ultraviolet Vis visible NOE nuclear Overhauser effect LED light-emitting diode CNS central nervous system PLC phospholipase C PKC protein kinase C LBD ligand binding domain SAR structure–activity relationship KAR kainate receptor GluR glutamate receptor Chemical Abbreviations Ac acetyl AcOH acetic acid Ac2O acetic anhydride Ar aryl Boc tert-butoxycarbonyl Bu butyl t-Bu tert-butyl Bz benzoyl Cbz benzyloxycarbonyl Cy cyclohexyl Pf. 9-Phenyl-9-fluorenyl dba dibenzylideneacetone DCM dichloromethane DCE 1,2-dichloroethane DIPEA N,N’-diisopropylethylamine Diox. 1,4-dioxane DMSO dimethylsulfoxide TMSCl trimethylsilyl chloride Et ethyl Bu butyl Et3N triethylamine Et2O diethyl ether EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide DMAP 4-dimethylaminopyridine HOBT hydroxybenzotriazole Pf 9-phenyl-9-fluorenyl DMP Dess–Martin periodinane TBTA tris(benzyltriazolylmethyl)amine DECM 7-(diethylamino)coumarinyl MNI 4-Methoxy-7-nitroindolinyl EtOAc ethyl acetate Hex hexanes MCPBA m-chloroperoxybenzoic acid Me methyl MeCN acetonitrile MeOH methanol Ph phenyl i-Pr isopropyl py pyridine p-Ns 4-nitrobenzenesulfonyl R undefined substituent TFA trifluoroacetate THF tetrahydrofuran TLC thin layer chromatography TMS trimethylsilyl Tf trifluoromethansulfonyl Ts tosyl AMPA α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid NMDA N-Methyl-D-aspartic acid KA kainic acid DA domoic acid PhKA phenylkainic acid GABA γ-aminobutyric acid Glu Glutamic acid DMA-DMF N,N-dimethylformamide dimethyl acetal CNQX 6-cyano-7-nitroquinoxaline-2,3-dione PFQX 6-pyrrolyl-7-trifluoromethyl- quinoxaline- 2,3-dione Cy5 cyanine 5 viii List of Schemes Scheme 3–1. Retrosynthesis analysis of 4-substituted kainoids ........................................................................................ 29 Scheme 3–2. Synthesis of pyrrolidin-4-one 62a. ................................................................................................................ 29 Scheme 3–3. Synthesis of C4 tertiary alcohols and NOE analyses ..................................................................................... 30 Scheme 3–4. Palladium-catalyzed allylic deoxygenation ..................................................................................................... 31 Scheme 3–5. Proposed mechanism for allylic deoxygenation ............................................................................................. 32 Scheme 3–6. Synthesis of allo-kainic acid and kainic acid precursors ................................................................................ 33 Scheme 3–7. Deprotection to kainic acid and isomers ....................................................................................................... 34 Scheme 3–8. First attempt at phenylkainic acid synthesis ................................................................................................. 34 Scheme 3–9. Synthesis and Functionalization of Phenylkainic Acid. (p-Ns: p-nitrobenzenesulfonyl) ................................. 35 Scheme 3–10. A unified synthesis strategy of C4-kainoid derivatives ............................................................................... 36 Scheme 4–1 Synthesis of kainoid alcohol 85 ...................................................................................................................... 59 Scheme 4–2. Synthesis and stereochemistry assignment for aldehyde 87 ....................................................................... 60 Scheme 4–3. Synthesis of novel alkynylkainic acid 89 and probe 90 ................................................................................ 61 Scheme 4–4. Synthesis of a clickable kainoid and conjugated fluorescent probes ............................................................ 66 Scheme 5–1. Synthesis of MNI-Glu (101) .......................................................................................................................... 77 Scheme 5–2. Synthesis of DECM cage reagents ................................................................................................................ 78 Scheme 5–3. Synthesis of DECM-Glu (102) ...................................................................................................................... 78 Scheme 5–4. Synthesis of DECM-PhKA (100) ................................................................................................................... 79 Scheme 5–5. Synthesis C4 kainoid derivative 124 ............................................................................................................ 80 Scheme 5–6. Synthesis of a photoisomerizable tetracholoroazobenzoic acid. ................................................................. 81 Scheme 5–7. First attempt at the synthesis of kainoid photoswitch 134 ........................................................................ 81 Scheme 5–8. Synthesis of a photoisomerizable tetracholoroazobenzene semicarbazide. ................................................ 82 Scheme 5–9. Synthesis of kainoid photoswitch 136 ......................................................................................................... 82 Scheme 5–10. Schematic diagram of photoisomerization mechanism of azobenzenes ..................................................... 89 Scheme 5–12. Summary of synthesized new kainoids ....................................................................................................... 93 ix List of Figures Figure 1–1. (A) In neurons, iGluRs are present at both the pre- and post-synapse. (B) Naturally occurring agonists of iGluRs: AMPA, NMDA, and kainic acid. (C) A functional iGluR protein is composed of four monomers that assemble to create a central channel pore to allow passage of cationic ions through the cell membrane. ................................ 1 Figure 1–2. Common antagonists used in GluRs neurobiology research. .............................................................................. 3 Figure 1–3. Two proposed kainate receptor-mediated signalling pathways. ........................................................................ 4 Figure 1–4. Examples of reported fluorescent probes for iGluRs. ......................................................................................... 7 Figure 1–5. (A) Examples of photocaged glutamate molecules. (B) Upon light irradiation, the photosensitive MNI or DECM moieties (photocages) are cleaved and yield the parent carboxylates as formal hydrolysis products equivalents. .................................................................................................................................................................. 7 Figure 1–6. Photoswitches for GluRs modulation. ................................................................................................................ 8 Figure 1–7. General structure of the proposed probes to study kainic acid receptors. ...................................................... 9 Figure 2–1. Natural kainoids and L-glutamic acid .............................................................................................................. 12 Figure 2–2. Kainiods for binding moiety study. .................................................................................................................. 14 Figure 2–3. Early kainoids derivatized from KA .................................................................................................................. 15 Figure 2-4. Early kainoids with C4 sp2 substituents ........................................................................................................... 15 Figure 2–5. Aromatic kainoids obtained from total synthesis ............................................................................................ 16 Figure 2–6. Plot of 36 kainoids as a function of Ki versus Hammett coefficients and the aromatic substitutents. .......... 17 Figure 2–7. Domoic acid and iso-domoic acid derivatives. ................................................................................................. 18 Figure 2-8. 3D structures of domoic acid and its analogues. ............................................................................................ 19 Figure 2–9. Structures of GluRs. ......................................................................................................................................... 21 Figure 2–10. Kainic and domoic acids make a π–π interac-tion with Tyr488 in GluK receptors.. ...................................... 22 Figure 2–11. Sequence homology of kainic acid’s binding pocket for GluKs. ..................................................................... 23 Figure 3–1. Naturally occurring (1, 6, 55, 26) and synthetic kainoids (32a, 33c). ........................................................... 28 Figure 3–2. Preparation CeCl3×2LiCl THF solution. ............................................................................................................... 39 Figure 4–1. Structures of GluK receptors, domoic acid, and the proposed fluorescent probe.. ......................................... 58 Figure 4–2. pcDNA3-GRIK2-EGFP plasmid gene map. ......................................................................................................... 62 Figure 4–3. Fluorescence labelling experiments design with probe 90. .............................................................................. 63 Figure 4–4. Representative images of HEK-293 cells after application of probe 90. ......................................................... 64 Figure 5–1. Cartoon examples of photopharmacology tools for the control of ion channels function. .............................. 74 Figure 5–2. Examples of photocontrollable KAR agonists. ................................................................................................ 75 Figure 5–3. Overlay of the two photoreactors irradiadiation ranges (blue-shaded areas) of the absorption spectra of caged compounds 100-102.. .................................................................................................................................... 83 Figure 5–4. Time-dependent photolysis of caged compounds under blue LED light irradiation (445 nm): the absorbance of the compounds decrease as the photocage is released. ....................................................................................... 84 Figure 5–6. Overlaid 1H NMR spectra for progressive photolysis of DECM-Glu. ................................................................. 85 Figure 5–7. 1H NMR (400 MHz, D2O) spectra for progressive photolysis of DECM-PhKA. .................................................. 86 Figure 5–8. Photolysis rates of DECM-Glu (102) and DECM-PhKA (100).. ......................................................................... 87 Figure 5–9. Trans / cis- isomerization of 136 and its UV-vis spectra. .............................................................................. 89 Figure 5–10. 1H NMR spectra (CD3OD) of 136 before and after light irradiation. .............................................................. 91 Figure 5–11. Sequential photoswitching of 136 using green (535 nm) and blue (405 nm) lasers, followed by an extended period of relaxation in the dark.. ................................................................................................................ 91 Figure 6–1. Overview of the SAR analysis, probes synthesis, and their application. ....................................................... 110 Figure S5–1. 1H NMR spectra for progressive photolysis of DECM-Glu by 465 nm photoreactor ................................... 174 Figure S5–2. Photolysis rate of DECM-Glu under 465 nm photoreactore ........................................................................ 174 Figure S5–3. 1H NMR spectra for progressive photolysis of DECM-Glu by 445 nm photoreactor ................................... 175 Figure S5–4. 1H NMR spectra for progressive photolysis of DECM-PhKA by 445 nm photoreactor ............................... 176 x List of Tables Table 2–1. Qualitative scale used in this analysis ............................................................................................................... 13 Table 2–2. Qualitative scale ................................................................................................................................................ 25 Table 2–3. Potency scales for kainoid analogues ............................................................................................................... 26 Table 3–1. Stereoselective alkylation of ketone 61 ........................................................................................................... 30 Table 3–2. Nucleophilic addition of ketone 11 ................................................................................................................... 31 Table 3–3. Deprotection of N-phenylfluorenyl (N-Pf) ......................................................................................................... 32 Table 4–1 Conditions for reduction of acid 72a to alcohol 85 ........................................................................................... 59 Table 5–1. Photochemical properties of caged Glu and PhKA compounds ........................................................................ 88 Table S2–1. Original literature data for the activity of kainic acid analogs in functional assays ..................................... 112 Table S2–2. Original literature data for the activity of kainic acid analogs in functional assays. (Continued) ............... 113 Table S2-3. Correlation of the binding affinity of analogs of compounds 34, 35, and 36 with the Hammett constants of their aromatic substituents. ................................................................................................................................... 115 Table S2–4. Kainate receptors reference sequences for the most commonly used organisms. .................................... 116 Table S2–5. Homology analysis of GluK1 between mouse, rat, and human isoforms. ................................................... 117 Table S2–6. Homology analysis of GluK2, GluK3, GluK4, and GluK5 between mouse, rat, and human isoforms. .......... 118 Table S2–7. Reported crystallized protein structures of ionotropic glutamate receptors: kainate receptors (GluK1-5), AMPA receptors (GluA1-4), and NMDA receptors (GluN1-2B). .............................................................................. 120 Table S3–1. NMR comparison between obtained kainoids and reported data ................................................................. 125 Table S3–2 Rules for assigning C3,4 configuration of Kainoids ....................................................................................... 126 xi Acknowledgments I would like to thank my supervisor, Dr. Frederic Menard, for patient guidance in chemistry learning and assistance on the troubleshooting for the research projects. I appreciate your continuing support during my Ph.D. learning. And a huge thanks to your great efforts on bio-experiments and manuscript publication. I am grateful to Dr. Vojtech Kapras for insightful discussions. Thanks to Dr. Zandberg and Matt Noestedem for HPLC and mass spectrometry analyses. Thanks to Simon Edelmann for the synthesis of precursors. Thanks to Mitra Tabatabaee for the help with biological experiments, also to Brianna Clark for the gene analysis of kainate receptors project. A huge thanks to my committee members, Dr. Paul Shipley and Dr. Issac Li. Thanks, Dr. Shipley for NMR support and guidance. Thank you to Dr. Li for excellent suggestions on the biological experiments. To my collaborators, Zongjie Wang, Dr. Xian Jian and Morgan A. Alford, thank you for your efficient work and productivity on the projects. And thanks for allowing me to be involved in the novel materials projects. I gratefully acknowledge UBC for the financial support of University Graduate Fellowships and Graduate Dean’s Thesis Fellowship. Also, thanks to UBC for the travel grants that enabled me to attend chemistry conferences and have chances to access the Canadian Chemistry community. Great thanks to Dr. Sandra Mecklenburg for offering the opportunity of teaching assistantships and the access to instruments in undergraduate labs. xii Dedication To Beibei Chen, my wife, thank you for your patience, resilience and selfless support during these four years. I appreciate your continuous learning to adapt to the local work and life. It is one of the most important driving forces for me cracking all the difficulties in my projects. Please forgive my absence during your holidays due to experiments. Without you, I would not be able to finish my Ph.D. 献给我的爱人–陈贝贝，以及我的父亲母亲。 Zhenlin Tian Kelowna, March 2019 1 Chapter 1 Introduction Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate the principal excitatory neurotransmission in the brain (Figure 1–1).1 They are categorized into three subgroups based on their sensitivity to agonists: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate or kainic acid (KA).1 Each iGluR subtype has modular domains that assemble into tetramers to form ligand-gated ion channels; they include an amino-terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxy-terminal domain (CTD, Figure 1–1c).2 Figure 1–1. (A) In neurons, iGluRs are present at both the pre- and post-synapse.3 (B) Naturally occurring agonists of iGluRs: AMPA, NMDA, and kainic acid. (C) A functional iGluR protein is composed of four monomers that assemble to create a central channel pore to allow passage of cationic ions through the cell membrane.4 a cNH2OHOOOHGlutamic acidNHCO2HCO2HHNOOHOHONMDANOHONH2OOHAMPA Kainic acidNative ligand:bCHAPTER 1. INTRODUCTION 2 2 In mammals, cationic ion channels of iGluRs open upon glutamate binding to generate synaptic currents for neuron communication.2 These events induce long-term potentiation (LTP) and long-term depression (LTD), which are fundamental to learning and memory formation.5 The dysfunction of GluRs leads to neurological disorders and neurodegenerative diseases. Over the past three decades, scientists have done extensive studies on the molecular mechanism of iGluRs. A variety of X-ray crystallography and cryogenic electron microscopy (cryo-EM) studies of GluRs have been reported.2,6,7 They show that conformational changes underlying activation and desensitization occur mainly in the LBD and ion channel regions. Upon binding of glutamate, the clamshell closure of the LBD imparts a rotation of the pore helices, which opens GluRs for Ca2+ inflow. In other words, the conformational change of LBD leads to the open state of iGluRs.8 This model can also predict the conformation changes of iGluR domains that lead to different functional states.8,9 However, the exact regulation pathways of different iGluRs members remain uncertain. The expression level and trafficking of iGluRs in CNS is another factor that impacts neurotransmission.5 There is now extensive evidence confirming that expression of GluRs can be activity-induced in either positive or negative feedback. For instance, immature synapse models have been shown to display NMDARs-mediated currents only,10 but an AMPA-type response was observed after being subjected to LTP.11 This rapid unsilencing of synapses was caused by AMPARs “appearing” at initially AMPARs-void postsynapses.12 This finding has sparked interest in differential GluRs expression and trafficking events at synaptic termini.5 The basic mechanism of this trafficking is thought to involve phosphorylation and dephosphorylation of GluRs with their subsequent insertion at synapse sites. However, due to the lack of tracking tools, the molecular mechanisms governing the movement of GluRs in and out of synapse boutons are still elusive.13 1.1 Distinct role of kainic acid receptors in CNS Among the ionotropic family of glutamate receptors (iGluR), the AMPA and NMDA receptors have been studied the most. In contrast, the role of kainic acid-sensitive receptors (KAR) in CNS is still not well understood.14 There are five subtypes of KAR proteins (Table 1–1).15 For consistency, only the more recent nomenclature will be used in this thesis, i.e., GluK1-5. Unlike AMPA and NMDA receptors that are located on the postsynaptic bouton, KARs are present at both the pre- and post-synapse—although higher densities are found at the pre-synapse.16 This ubiquitous feature allows KARs to contribute to several functions: presynaptic modulation of excitatory and inhibitory currents, postsynaptic depolarization of excitatory CHAPTER 1. INTRODUCTION 3 3 synapses, refinement of synaptic strength (during development and synaptic plasticity), and enhancement of neuronal excitability. The bidirectional modulation role of KARs at synapses has also been confirmed in mossy fibers and CA3 region of the hippocampus.16 This dual role affects the balance between excitation and inhibition in the neuronal network and has important implications for cognition.16 Table 1–1. NC-IUPHAR* nomenclature of kainic acid receptors. The receptor used in this thesis is shown in bold. Receptor Family NC-IUPHAR subunit nomenclature Previous + unofficial names Human gene Kainate receptors GluK1 GluR5 GRIK1 GluK2 GluR6 GRIK2 GluK3 GluR7 GRIK3 GluK4 GluR KA1 GRIK4 GluK5 GluR KA2 GRIK5 * NC-IUPHAR: Nomenclature Committee of the International Union of Pharmacology on receptor and drug classification. The predominant presynaptic localization of KARs suggests that they can facilitate excitatory synaptic plasticity. In early research on GluK2 and GluK3 knockouts mice models, both rapid activation and long-term potentiation (LTP) were impaired in mossy fibers.17 The same effect was observed by blocking KARs in mossy fibers: LTP was significantly blocked by the selective antagonist LY382884 (Figure 1–2). Surprisingly, this LTP could be still induced by cyanquixaline (CNQX), a non-selective antagonist of AMPARs and KARs.3,18 These results show that wide-spectrum antagonists are unable to prevent LTP phenomena.19 As LY382884 is selective for the GluK1 subtype, it suggests that LTP is induced primarily by GluK1. Similarly, Contractor et al. have demonstrated that LTP is not abolished in GluK2 knockout mice, while GluK1-deficient mice showed normal LTP at synapses.5 While much evidence supports a crucial role for KARs in synaptic plasticity, there is no clear explanation for this difference observed in subtype function.3,19 Figure 1–2. Common antagonists used in GluRs neurobiology research. Besides the regulation roles in ionotropic pathways, KARs are also involved in a metabotropic modulation that is distinct from AMPARs and NMDARs.19 Studies suggest that KARs can trigger downstream neuron cell signals through an unconventional metabotropic mechanism involving G NHOOHOHOHHLY382884NNOONCCNQXO2N NHOOHONHNN NHHLY377770CHAPTER 1. INTRODUCTION 4 4 protein and secondary messengers (Figure 1–3a). This signaling inhibits gamma-aminobutyric acid (GABA) at pre-synapses, correlates with G protein activation, phospholipase C (PLC) and protein kinase C (PKC) activity, and is followed by signaling modes in dorsal root ganglion neurons. This activity is independent of Ca2+ ion flux,19 but the molecular mechanisms underlying this signaling are unknown. This proposed pathway raises important questions, such as how do KARs activate G proteins and trigger downstream effects. Regardless of the exact mechanisms, an increasing number of physiological functions seem to be regulated by KARs through this unconventional signal. So far, this putative dual signaling appears to be a feature unique to KARs. Figure 1–3. Two proposed kainate receptor-mediated signalling pathways. (A) Kainate receptors (KARs) can act via ionotropic and metabotropic pathways.20 Neto1 and Neto2 auxiliary subunits co-assemble with KARs to modulate their function. In the hippocampus, Neto1 enhances the amplitude and prolongs the kinetics of KAR-mediated currents at synapses between mossy fibers–CA3 pyramidal cells.21 (B) Kainate receptors-mediated modulation of Glu and GABA release in hippocampus.16 ESPC: excitatory postsynaptic current. Neto: Neuropilin and tolloid-like protein. KARs also play important roles in the mediation of GABA transmission in the CNS (Figure 1–3b). Early research has shown that activation of KARs by glutamate in the hippocampus leads to the release of GABA.16 For example, KAR agonists enhanced the action potential-independent GABA release in both CA1 interneurons and pyramidal cells. The diversity of the select KAR functions presented above offer a glimpse at to why they appear involved in so many neurological diseases. Finding compounds that can modulate KAR activity is an attractive medicinal goal. 1.2 Therapeutic potential of kainic acid receptors Neuropathology studies have shown that KARs are involved in acute and chronic neurodegenerative diseases, including epilepsy, pain and psychiatric disorders.20,22 Among these a bCHAPTER 1. INTRODUCTION 5 5 diseases, KARs have been most prominently associated with epilepsy. This is likely due to the excitatory/inhibitory imbalances caused by abnormal KARs activity. KAR & epilepsy. It is well known that kainic acid (KA) injection in animal models produces epilepsy symptoms. In fact, KA is routinely used to model epileptic diseases22 and to evaluate neuroprotective drugs.23 Moreover, the elimination of GluK2 in knockout mice reduces their seizures after KA injection. These reports indicate that KARs contribute to overexcitability in neurons, which can cause epilepsy. Antagonists of GluK1, e.g., LY377770 and LY382884, demonstrated the ability to prevent the development of epileptic activities in hippocampal slices (Figure 1–2). These agonists reduced seizures both in vitro and vivo. KARs & pain. KARs are also expressed in dorsal root ganglion (DRG) and dorsal horn neurons, where they play roles in sensory transmission and pain. For instance, GluK5 is the only ionotropic GluR expressed in DRG and may be involved in nociception in DRG neurons (pain transmission). 19 While pain perception is not a disease per say, chronic pain (persistent recurrent pain) is now a prevalent debilitating neurological disorder. Biological assays with KAR antagonists support their involvement in pain sensation. For example, in a paw-licking experiment induced by formalin, the KAR antagonist LY382884 decreased the pain response in rodents. Similarly, GRIK1 knockout animal models showed mitigation of pain-associated behaviours.19 Additional experimental data that are beyond the scope of this review also support a role for KARs in neurophysiological pathologies. KARs & other disorders. Genetic epidemiology has identified several correlations between KARs and neuropsychiatric disorders. For instance, aberrant GluK4 function has been found to be linked to schizophrenia in some populations. GluK2 and GluK3 are associated with the obsessive-compulsive disorder. GluK3 was also found to be associated with depression, and GluK2 to be associated with autism.22 However, these epidemiological studies are limited to suggestive correlations, further experimental research is required to identify a causative link to human disease. A recent genetic study also found that loss-of-function mutations in GluK2 correlated with non-syndromic autosomal recessive mental retardation in a consanguineous family, thereby suggesting that GluK2 receptors are essential to brain function in humans.22 The above genetic associations with neurodevelopmental disorders are consistent with KARs playing an important role in the development/stability of neuronal circuitry. 19 These intriguing associations still await confirmation from molecular and cellular studies, yet they offer clear research opportunities with applications to human disease. CHAPTER 1. INTRODUCTION 6 6 1.3 Limitations in research on kainic acid receptors Current KAR research relies on agonist and antagonist-induced cell models, and on gene knock-out animal models.24 The potent agonist neurotoxin kainic acid (KA) is widely used to mimic the effect of glutamate in neurodegenerative models, as well as to distinguish KARs activity from AMPARs and NMDARs.25 Kainic acid is typically used in cells or tissues by bath perfusions or by local micro-injections. In animals, the injection of KA leads to seizures, followed by deficiencies in short-term memory and long-term learning, and by depression-like behaviours.24 In neurons, KA causes an excessive Ca2+ influx that eventually induces acute neuron death. The Ca2+ influx also increases oxidative stress in neuron cells (via positive feedback loops of reactive oxygen species).26 In microglia, KA triggers an inflammatory-like response that causes their deleterious activation, leading to outcomes that resemble Parkinson and Alzheimer’s diseases. While KARs’ phenotypic role in neurodegenerative diseases has been explored extensively with GRIK gene knockout models, our understanding of their biochemical role in these diseases is still superficial.27 Most current biological methods rely on recombinant gene manipulations and prevent the precise spatio-temporal control of KARs. Moreover, since these techniques can only be used in animal models, their translation to clinical research is also very limited.24 More importantly, distinguishing the individual contributions of the three classes of GluRs is difficult; a challenge made even greater for KA and AMPA receptors due to the high homology of their binding site for glutamate. Indeed, besides the kainoid neurotoxins, there is still no agonist or antagonist that is selective for KARs over AMPARs.22 1.4 Precedents on biosensors and switchable probes for GluRs A few biosensors have been reported to study iGluRs in neurons. For example, Hamachi et al. reported fluorescent probes based on PFQX as selective ligand to label AMPARs in cells (Ax488-5s, Figure 1–4).28,29 Similarly, Stromgaard et al. employed ariotoxin-636 to visualize glutamate channels in hippocampal neurons.30 These molecules solve two limitations of biological alternatives: they enable the real-time tracking of native glutamate receptors in neurons by simple bath application, that is, without genetic modification. CHAPTER 1. INTRODUCTION 7 7 Figure 1–4. Examples of reported fluorescent probes for iGluRs. (A) Structures of Ax488-5s and iGluR probe-1. (B) Representative fluorescence imaging of AMPA-sensitive GluRs in living cells using Ax488-5s (green dye); mCherry was used as transfection marker for AMPARs.28 With the advent of modern photochemistry, new photoactive pharmacology tools have been emerging in ion channels research.31 The more established photoactive chemical tools for iGluRs study are the photo-caged glutamates pioneered by Ellis-Davies (Figure 1–5).32-34 For example, the MNI-Glu and DECM-Glu photo-caged compounds were invented by assembling glutamate and a photolabile protecting group. These molecules enable the photo-inducible activation of iGluRs. Upon local light irradiation, the caged glutamate can be released with spatial precision. These photo-caged glutamates have been widely used in neurobiology as tools to activate select neurons or tissues. An important limitation of these compounds is that they release glutamate which by definition activates all GluRs. While useful for physiological studies, these compounds do not allow one to study the contributions of individual classes of ion channels. Figure 1–5. (A) Examples of photocaged glutamate molecules. (B) Upon light irradiation, the photosensitive MNI or DECM moieties (photocages) are cleaved and yield the parent carboxylates as formal hydrolysis products equivalents. OH2N-O3S-O3SNH2+CO2HHN O O NO ONHNOHNNNHNF3COOOHONBNFF NNN NHNHONH2NHNH2NHPFQX (AMPAR ligand)Alexa 488 dyeBODIPY dye Argiotoxin-636 analogue(ion channel blocker)a bLinkerMeONO2MNI-GluNH2ONOOHNH2OOOOHOOEt2NDECM-GluPhoto-cleavable moiety GlutamateaNa+Ca2+LigCageNeuronal activation(by blue or purple laser)bCHAPTER 1. INTRODUCTION 8 8 Photocaged glutamates are “turn-on” chemical probes: the formation of the active agonist is irreversible once the protecting group is cleaved. A complementary type of chemical probes are photo-switches: they rely on an agonist that can be turned on/off reversibly with light. Examples of “photo-switches” for GluRs are depicted in Figure 1–6a. The photoswitch component of the molecules is an azobenzene sidechain attached to known GluRs ligands. Light irradiation triggers a reversible cis/trans isomerization of the azo double bond. In GluRs, the binding site of the agonists possessed a narrow channel that can only accommodate the trans-azobenzene sidechain; consequently, the reversible photoswitching of the cis/trans sidechain controls the activation « deactivation mode of the receptor. Due to their well characterized binding site, several photoswitches have been developed for iGluRs.34,35 For instance, ligands that exert selectivity towards AMPARs, such as AMPA or ShuBQX, were modified with azobenzene components and have been demonstrated to reversibly control AMPAR activation in living systems.35-37 Furthermore, in vivo experiments with photoswitchable compounds in zebrafish suggest that they can even trigger photo-dependent behaviours.38 Figure 1–6. Photoswitches for GluRs modulation. (A) Examples of reported GluR photoswitches. (B) The cis configuration of the azobenzene in the photoswitches prevents proper binding of the ligand. (C) The trans configuration of the azobenzene sidechain matches the LBD’s channel and allows the ligand to reach its high affinity binding pocket. Thermodynamically, the azobenzene group prefers to exist in the trans configuration under dark condition (thermal), and cis confirmation under purple light irradiation (photochemical). NNaNH2OHOOOHNNONOOHNH2NNNNNNRNHN OOPHO OHOF3CNNShuBQX-3ATA-3NHONOO L-MAG0Photo-switchable moiety LigandNN Switchable moietyLigNa+Ca2+Switchable moietyLigNeuronal activation(by purple laser)Relaxation under dark conditionNeuron deactivationb cPhoto-switchable moiety Ligand Photo-switchable moiety LigandNa+Ca2+CHAPTER 1. INTRODUCTION 9 9 1.5 Thesis overview While chemical probes have been reported to study GluRs generally, or AMPARs selectively, no tool exists yet that enables the selective visualization or control of KARs. The objective of my research is thus to develop chemical probes to study KARs in living cells without needing the recourse to genetic modifications. Ideally, a set of probes will allow the fluorescence imaging of KARS, and a different set will enable the selective activation or inactivation of KARs. As common structural features, they consist of a ligand, a linker, and a functional moiety (Figure 1–7a). The ligand that will impart selectivity for the KARs—over the NMDA or AMP receptors—is the high affinity neurotoxin kainic acid. This thesis will focus on two types of probes: affinity probes for the visualization of KARs and photo-pharmacological tools for KAR control. Figure 1–7. General structure of the proposed probes to study kainic acid receptors. (A). Generic structure of proposed probes. (B) The structure of fluorescent probe for KARs. (C). The structure of proposed photocage for KARs. (D). The structure of proposed photoswitch for KARs. The first challenge in designing probes based on kainic acid required identification the most appropriate site for chemical modification that would still maintain a high binding affinity for KARs. Accordingly, I surveyed all the reported kainoid derivatives that have been tested for their biological activity. Then, I analyzed the relationship between their structure and their activity, and identified a set of guidelines for the best ligand design (Chapter 2). Structural features required for high affinity were extracted from over 100 KARs agonists, they revealed that modifications at C4 of the pyrrolidine ring is the best option. A complementary crystallographic analysis of iGluR proteins complexed with kainoid agonists supported the conclusions. Phtotocage:Fluorescent probe:Photoswitch:NNCyanine dyeO ONR2CO2HCO2HO ONN ONHR1R1 n1 n2NHCO2HCO2HXOHNn3NHCO2HCO2HYGeneric structure:R3R4 R5abcdKainoid ligandLinkerCoumarin cage(photocleavable)Kainoid ligandLinkerKainoid ligandLinkerAzobenzene switch(photoisomerable)LigandFluo tag orSwitchable moietyLinkerCHAPTER 1. INTRODUCTION 10 10 Next, a practical, scalable synthesis of kainic acid derivatives that can be easily modified at C4 had to be developed. While more than 50 syntheses of kainoids have been reported, none allow for late-stage modification of C4 substituents or are realistically scalable. Thus, I developed a unified synthetic strategy for C4 kainoids (Chapter 3). The synthesis begins with commercially available materials and can diverge into several C4 derivatives. Its effectiveness was demonstrated by preparing natural and unnatural kainoids. The goal of creating novel chemical probes for KAR studies would be all the more powerful if any probe type could be assembled from a flexible kainoid precursor and the desired reporter tag or switch (Figure 1–7b). Since click chemistry provides a convenient option, I synthesized an alkynyl kainoid. This clickable kainoid was then utilized to construct fluorescent probes for KARs visualization in living cells (Chapter 4). A proof of concept was demonstrated using a red fluorescent probe with GluK2 expressed in model cells. Finally, my synthetic efforts culminated with the creation of photo-active chemical probes to manipulate KARs function with visible light. Thus, I designed and synthesized photo-controllable KARs agonists (Chapter 5). A coumarin-caged kainoid was synthesized to control KARs with blue light (Figure 1–7c). Also, a photo-switchable kainoid was synthesized to reversibly control KARs (Figure 1–7d). The photo-induced kinetics of cleavage or switching of the probes were evaluated by extensive spectroscopic studies. At the moment of writing, these photoactive probes were being applied in living cells. 11 Chapter 2 A structure-activity relationship analysis of kainic acid-based agonists A comprehensive survey is presented of all kainic acid analogs that have been tested for their biological activity. Specifically, this chapter: (1) gathers in a single location all structural variations of kainic acids reported thus far, (2) exposes design rules that must be met to optimize affinity for kainate receptors, and (3) suggests structural insights toward the design of next-generation KA analogs. The available structure-activity relationship (SAR) data were systematically analyzed and combined with the most recent crystallographic studies. From the exercise emerged a set of structural guidelines to help design high-affinity analogs. Given the renewed interest in neuroactive molecules, this chapter aims to guide the synthetic efforts of organic synthesis and facilitate the design of KAR probes. 2.1 Background Due to KAR selective excitation by kainic acid, KARs were identified as a distinct family.17 Besides KA, its natural analog-domoic acid (DA, 2, Figure 2–1) also shows strong affinity with KARs.39 Those kainoids were first isolated in the 1950s from the red algae.43,44 Kainoids were initially used as anthelmintic for children in Japan, Southeastern Asia and Taiwan for decades.40 Owing to the induced epilepsy and seizures by kainic acid and domoic acid,41 they were abandoned for medicine use in the 1980s. However, their potent action on KARs made them widespread tools in neurobiology study.3 In neuro research, KA is utilized in tissue culture and animal to stimulate neurodegenerative models mediated by KARs.42-44 It is also employed as an activator to validate their identity to distinguish other inotropic glutamate receptors (like AMPA and NMDA receptors). When binding to KARs, KA induces the influx of cellular Ca2+, production of reactive oxygen species and CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 12 mitochondrial dysfunction, which leads to neuronal apoptosis and necrosis.45 Although extensive studies have been conducted on KA induced models, their physiological functions in CNS is still obscure due to the lack of selective tools.22 Figure 2–1. Natural kainoids and L-glutamic acid As part of our research program on calcium signalling in glia, we needed to create a KARs–selective chemical probe. Opting to design a kainic acid analog, we looked to the literature to inform our structural design. However, we were surprised to find that, despite the wealth of SAR information that has been accumulated on kainic acid analogs over the past three decades, a systemic analysis of KA analogs' (kainoids) biological effects had never been reported. In this analysis, we attempt to meet this need: we have gathered in one article, all the KA derivatives that have been biologically tested from natural or synthetic sources. We focus on the SAR of KA derivatives that have been tested for their biological activity. By juxtaposing this dense information, trends emerged in terms of SAR. These trends are supported by crystallographic data from recent reports. 2.2 Structural considerations The unique activity of kainic acid (1) has led to several medicinal chemistry efforts aiming to create kainate analogs with improved potency and increased selectivity toward the KAR subset of glutamate receptors. This section summarizes how structural changes made to KA derivatives influence their biological activity. Results from over 50 studies were gathered and analyzed to identify correlations in structure-activity relationship. 2.2.1 The activity grading system Direct comparisons between analogs can be difficult to make, as no standardized assay is used in the field. The biological data are reported from assays that use different techniques, different cells/tissue/organism, different iGluR proteins, etc. Consequently, we have opted for a qualitative kainic acid (1)NHCO2HCO2Hdomoic acid (2)NHCO2HCO2HCO2HL-glutamic acid (3)H2NCO2HCO2H23451'+ + + + + +CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 13 system indicating the activity of the compounds (Table 2–1). To enable a systematic comparison of analogs, a relative activity scale was calculated instead of using absolute values (see the original data in Table S2–1 and S2–2, Appendix A). The scale was normalized to positive controls used in each assay, i.e., glutamate, KA or DA. Calculations were always based on the most recent data for each compound. When available, priority was given to Ki or EC50 values with the most studied isoform GluK2 (also known as GluR6). Otherwise, [3H] displacement assays in cells, or activity in neurons were used. If more than one value was found for the same isoform, the average was used. All quantitative data and assay details are listed in a comprehensive table in the accompanying data analysis section (Tables 2–3). Table 2–1. Qualitative scale used in this analysis 2.2.2 Early SAR studies on kainic acid Before the age of elucidation of KARs’ crystal structures, early SAR studies focused on direct derivatization of glutamic acid and kainic acid (Figure 2–2) to get insight into the features responsible for its binding activity. The acid and amine blocked kainoids (bisester 4 and amide 5) showed no biological activity in crayfish opener muscle.51,52 The natural occurring allo-kainic acid (6) differs from KA only by its epimeric C4 stereocenter and is essentially inactive when compared to the neurotoxin.51,53 Similarly, inverting the configuration at C2 resulted in the loss of activity (kainoid 7).46 Together, these studies revealed that the amine, both carboxylic acids, and the stereocenters are all indispensable for receptor binding. Interestingly, any steric bulk at C5 is not tolerated. For example, even the addition of a simple methyl group abolished the compounds’ binding affinity, irrespective of the substituent’s stereochemistry (kainoid 8 and 9).47 This suggested a closely nested interaction between KA’s basic nitrogen and the protein’s binding site. However, 10 and 11 lacking the C4 iso-propene are active for GluRs, but lost most of its activity on KARs. 55,56 It suggested that there is more to KA than simply being a rigid form of glutamic acid. domoic acid– ––+ + ++ ++kainic acidEqual or stronger than DABetween L-Glu and KABetween KA and DALess active than L-GluInactiveL-Glu0 < RA < 0.065Relative Activity (RA)0.065 ≤ RA < 11 ≤ RA < 2.892.89 ≤ RAInactive10.0652.89CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 14 Figure 2–2. Kainiods for binding moiety study. Moieties different than KA are highlight as red. 2.2.3 Structural variation on the C4 position Due to the necessity of C2 and C3 moieties, and the C5 being sterically intolerant, synthetic modification of kainoids focused on the C4 variations. Early medicinal chemistry research modified the C4 side-chain of kainic acid to provide more complex derivatives (Figure 2–3) for biological studies. These early kainoids share a common feature – they have same backbone with different C4 functional groups derived from functionalization on the iso-propenyl group of kainic acid. A C4 double bond retained kainoid 12a exhibits comparable activity as KA, 14 which also shows the C4 tolerable feature for the binding. Surprisingly, the simple hydrogenation of the alkene led to an inactive DHKA (13).57-59 Also, kainoids 14, 15 and 17 lost almost all activity for the glutamate receptors.48,49 And the more sterically demanding lactone 16 saw activity reduced by order of magnitude.48 The fully oxidized carboxyl kainic acid 14a and its C4 inverted acid 14b were inactive because of the hydrophilic moiety on C4.49 While ketone derivative 12a is potent towards iGluRs, the C4 inverted derivative 12b is inactive.49 These observations point to the importance of maintaining an sp2 hybridized centre on the C4 side-chain and C3,4-cis relative configuration. NHCO2CH3CO2CH3Kainic aciddimethyl ester (4)- -NRCO2HCO2H5a R = acetyl5b R = Boc- -NHCO2HCO2Hallo-Kainic acid (allo-KA, 6)- -NHCO2HCO2Hβ-Kainic acid (β-KA, 7)- -NHCO2HCO2HR58a R = Me8b R = Ph- -NHCO2HCO2HR59a R = Me9b R = Ph- -NHCO2HCO2H2-carboxy-3-pyrrolidine-3-acetic acid (CPAA, 11)NHO CO2HPyroglutamic acid (10)- -CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 15 Figure 2–3. Early kainoids derivatized from KA Perhaps the most intriguing analog is the homologated terminal alkene 18 (Figure 2-4). Similar to DHK, its poor potency 16 initially suggested that the steric constraints around the C4 side chain are very stringent since its hydrophobicity is essentially the same as KA. With extended sidechain kainoids,50-52 higher potency was observed for the isomer bearing the C1’-C2’ double bond in the Z configuration (kainoid 20) than the E configuration isomer 21.49 Bulky substituents on or close to the C1’ alkene (analog 22, 23, 24 and 25b) decrease the activity greatly.49,53-55 However, this argument does not hold up to the fact that 25a possesses a bulky sidechain, and yet is more potent than KA,55 which point to the potential contribution of the planar benzene-alkene conjugation system for the binding affinity. Figure 2-4. Early kainoids with C4 sp2 substituents NHCO2HCO2HDihydrokainic acid(DHK, 13)-NHCO2HCO2HHO2C14a : 3,4-cis - -14b : 3,4-trans - -NHCO2HOORNHCO2HCO2HO12a : 3,4-cis ++12b : 3,4-trans - -NHCO2HCO2HR HO15a : R = H -15b : R = OH -15c : R = PhS - -16a : R = H - -16b : R = OH - -16c : R = I - -16d : R = PhS - -NHCO2HCH3HO2C CO2H17 R = H - -NHCO2HCH3CO2HNHCO2HCH3CO2HHO2CNHCO2HCO2HNHCO2HCH3CO2HSeO2NNHCO2HCO2HOOHOz* S, Domoilactone A (24a)* R, Domoilactone B (24b)NHCO2HCO2HONHCO2HCO2HCH2OHHOH2CCH318 - -22+23- -- -- -H3C*19 20 - -21- -NHCO2HCO2HO2N25b -NHCO2HCO2H25a +O2NCHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 16 2.2.4 C4 π-π stacking With the advances of total synthesis, the more recently synthetic chemists were able to create more complex kainoids bearing various C4 sp2 hybridized side chains (Figure 2–5). The activity of a series of congeners (26-32) were systemically measured by Shinozaki group.56 While a general trend was that electron-poor analogs were more potent, a surprising number of variations were reported to be highly active: from the highly polar acromelate A and B (26 and 27) to the simple phenyl kainic acid (32a). All these kainoid bearing aromatic and heteroaromatic ring (26-33) displayed comparable or enhanced activities as KA.56 Figure 2–5. Aromatic kainoids obtained from total synthesis A more comprehensive series of aryl kainoids and their activities were reported by Bleakman group (kainoids 34-36).57 Consistently with the rules we concluded from early kainoids, the C1’ saturated (34) and C4 inverted analogs (35) were virtually inactive in the binding assays. Intriguingly, most of the C1’ conjugation extended (alkene-benzene conjugation) kainoids 36 are potent agonist for KARs.57 This may be due to the extended conjugation allowing for a better π– π overlap with the tyrosine residue in binding pockets (see Section 2.3, the crystal structure analysis). To some extent, the C4 side chain is bulkiness tolerable, eg. kainoids 36l, 36m, 36o and 36q (disubstituents on meta and para positions) with bulky substituents on benzene ring but still show equal affinity as KA. While disubstituents on ortho and meta position (kainoid 36i) and oversized substituents (kainoid 36x – 36z) decrease the affinity significantly. Although the bulkiness effect for NHCO2HCO2HHNOHO2CNHCO2HCO2HHNOCO2HAcromelic acid A (26) Acromelic acid B (27)++ +++NHCO2HCH2CO2HNHCO2HCH2CO2HR1R2R1R2 R1 R235a -H -H35b 4-C(CH3)=CH2 -H35c 3-Ph -H35d 3-O(CH2)2CH(Ph)2 -H R1 R236a -H -H36b -CH3 -H36c -H -CH336d -OCH3 -HNHCO2HCO2HNHO2CCPPA (30)NHCO2HCO2HNHO2CCNOPA (31)ONHCO2HCO2H33a: R = Me 33b: R = Ph33c: R = NH2 33d: R = NHMeNHCO2HCH3CO2HR1R2 R1 R234a -H -H34b -Ph -HNSRNHCO2HCO2HNMPPA (28)NHCO2HCO2HNHMPPA (29)HONHCO2HAr CO2H32a: Ar = Ph32b: Ar = 2-MeOPh32c: Ar = 3-MeOPh32d: Ar = 4-MeOPh32e: Ar = 2-HOPh32+ ++++-+++++++++++--+ +----++++++++++++++-++++++++++-++++++++---++ R1 R236e -H -OCH336f -H -Cl36g -H -F36h -CH3 -CH336i -CH3 -CH3(meta)*36j -CH3 -F36k -H -CH2CH336l -H -CH2CH2CH336m -H -CH2(CH2)2CH336n -H -C(CH3)336o -H -CH(CH3)=CH236p -H -C(CH2CH3)=CH2 R1 R236q -Ph -H36r -H -Ph36s -O(CH2)3CH3 -H36t -H -O(CH2)3CH336u -H -OPh36v -O(CH2)2Ph -H36w -H -O(CH2)2Ph36x -H -O(CH2)5Ph36y -O(CH2)2CH(Ph)2 -H36z -OCH3 -O(CH2)2Ph36aa -CH3 -O(CH2)2Ph36ab -CH3 -O(CH2)4CH333CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 17 approaching the binding sites of iGluRs, the substituents may also affect the electronic density of the conjugation system and further affect the non-covalent overlapping tyrosine residue in the binding site. To better understand the electronic factors governing the binding affinity of kainoid 36 with iGluRs, we plotted the potency of derivatives 36 against their Hammett constants when they were available (Figure 2–6a).58-60 While no clear electronic trend emerged from the plots, it can be noted that all compounds with lower Ki’s than KA only have substituents in the para position; plotted compounds with meta-substituents were less potent than KA. Slightly lower activity for electron-rich aromatic compounds can arguably be observed (suggesting a potential electronic destabilization due to the electron-rich tyrosine)—however, this is admittedly a weak correlation. This weak correlation is also observed when we use the electrophilic constant parameters (Fig. 2–6b). Overall, this analysis does not invalidate a π-π stacking hypothesis, but it rules out a cation-π interaction. Figure 2–6. Plot of 36 kainoids as a function of Ki versus Hammett coefficients and the aromatic substitutents. Taken together, the above observations converge toward a few structural features that are essential to the activity of KA derivatives. Both C1- and C2-carboxylic acids and the secondary amine must remain intact, and substitution at C5 is not tolerated. KA can be modified at C4 position, as long as: (1) the stereochemistry is kept syn relative to the C3 acetic acid side chain; (2) the C1’ atom is sp2 hybridized; and (3) if a substituted C1’–C2’ alkene is present, that the cis geometry will facilitate the efficient binding. These “permissible” structural modifications are deduced from synthetic kainic acid analogs. They are further supported by the potent activity of the natural kainoid, domoic acid. The next section examines the effect of structural variation of DA on the GluR’s activity. -0.2 0.0 0.201234σ Hammett substituent constantsLog 10KiMeOMeMeMeOMeEtMeFMeMeMeClFKainic acidDomoic acid-0.8 -0.6 -0.4 -0.2 0.0 0.201234σ+ Electrophic substituent constantsLog 10Ki OMe Me MeMeEtMeFMeMeMeFOMeCl Domoic acidKainic acidEDG EWGa bCHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 18 2.2.5 Domoic acid derivatives Domoic acid (2, DA) was first isolated in 1959 from the marine red algae, Chondria armata.61 It is a natural toxin produced by marine diatoms (Pseudo-nitzschia). Pseudo-nitzschia was reported poisonous and the cause of the death of sea birds and sea lions.62 The diatoms produced DAs can also bioaccumulate in marine organisms which feed on phytoplankton, such as shellfish, anchovies, and sardines.63 Consumption of DAs contaminated seafood may eventually lead the neurotoxin accumulation in human body. In 1987, three deaths and over 100 illnesses outbroke in Princess Edward Island, the Canadian East coast as a result of the ingestion of DA contaminated blue mussels.64 Those people suffered short-term memory loss (in addition to the normal clinical signs of food poisoning), which was later attributed to domoic acid.64 In the brain, domoic acid destroys neuron cells by over-activating KARs, which leads to uncontrolled calcium influxes and causes neuron cells to degenerate.39 Figure 2–7. Domoic acid and iso-domoic acid derivatives. Compared to domioic acid, different moieties of iso-domoic acids were labeled as red. Domoic acid is a KA analog, where the iso-propene sidechain is elongated by five carbons and bears a terminal carboxylic acid (Figure 2–7). DA’s higher potency over KA shows not only that substituents on the alkene are tolerable, but that it is possible to increase the potency. While the structure of DA has been known since the 1950s, the first total synthesis was achieved by the Tomita group in 1982.65 More recently, a comprehensive synthesis strategy of domoic acid and its isomers +++domoic acid (2)NHCO2HCO2HCO2H++iso-domoic acid A (37)NHCO2HCO2HCO2H-iso-domoic acid B (38)NHCO2HCO2HCO2HZEZEEE-iso-domoic acid C (39)NHCO2HCO2HCO2HE-iso-domoic acid D (40)NHCO2HCO2HZZCO2H-iso-domoic acid E (41)NHCO2HCO2HEECO2H+iso-domoic acid F (42)NHCO2HCO2HCO2HEZiso-domoic acid G (43)NHCO2HCO2HCO2HN/AEEisodomoic acid H (44)NHCO2HCO2HCO2HEENHCO2HCO2HOH45N/A N/ACHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 19 B, C, E, F (38-42) were established by the Clayden and Baldwin groups. It remains the sole effort to prepare domoids to investigate the effect of DA’s sidechain. Figure 2-8. 3D structures of domoic acid and its analogues. (3D structures were produced by CORINA. The generated figures were processed by Pymol and Chemdraw.) More recent biological studies revealed that the configuration of the C1’-alkene must be (Z) to maintain high affinity. Indeed, iso-domoic acid E (41) is 240 times less potent than DA—this recapitulates the observations made with KA derivatives 21. Similarly, iso-domoic acids A and B (37 and 38) show that isomerizing the C1’-alkenes from Z- to E- resulted in a more than a 1000-fold decrease in Ki. This difference in activity is likely caused by an unfavourable interaction with the protein, as the exo C1’-alkene in the more flexible iso-domoic acid C (39) results in a Ki of 171 nM (close to Glu according to the grading system). This less dramatic loss than for 41 is most likely due to the ability of the sidechain to rotate around C1’-C2’ position (Figure 2–8). For this reason, iso-domoic acid F (42) is active in binding with KARs. Overall, the most important sidechain parameter to maintain high potency appears to be the ability of the side chain to adopt a flexible (linear) conformation. NHCOOHCOOHDomoic acid (DA)Isodomoic acid-AIsodomoic acid-BIsodomoic acid-CIsodomoic acid-DIsodomoic acid-EIsodomoic acid-FIsodomoic acid-G Isodomoic acid-HNHCOOHCOOHCOOHNHCOOHCOOHCOOHNHCOOHCOOHCOOHNHCOOHCOOHCOOHNHCOOHCOOHCOOHNHCOOHCOOHNHCOOHCOOHCOOHZEEEEZZEEZENHCOOHCOOHCOOHEECOOHEZCOOHZECHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 20 The position of the distal alkene at C4’ or C5’ does not appear to be critical. The regioisomer 37 shows virtually the same Ki as domoic acid. However, its configuration affects binding: the (Z)-C4’-alkene analog 40 is 250-fold less potent than domoic acid. In this case, the favoured trans configuration of the diene significantly increases the steric demand around the side chain compared that of domoic acid. This is supported by the (E, Z)-isomer 41 which is only ~30 times less potent than DA. Interestingly, the configuration of the C6’ centre does not appear to be important. Ablation of the stereocentre in iso-domoic acid A (38) still maintained activity. Two more diene isomers were synthesized by the Montgomery group (43 and 44), but their activity has not yet been reported. Given previous trials with KA analogs showing that stereogenicity at C4 is critical to its binding, we predict that their activity would be poor. Consistent with our prediction, another domoid 45 was reported with moderate activity for KARs (selective for KARs and non-active for NMDARs) by the Baldwin group.66 2.3 Protein–ligand interaction: the binding site of kainic acid The KAR family consists of five proteins members: GluK1–GluK5. Like all glutamate receptors, individual GluK proteins assemble as tetramers to create a functional channel. The crystal structure of a full-length ionotropic glutamate receptor has remained elusive for over four decades due to their large size. However, progress in crystallization techniques has recently yielded structures for AMPA, NMDA, and KA receptors in rapid succession.7,9 The structural features are then correlated with the SAR trends observed from the synthetic derivatives of KA and DA. 2.3.1 Crystallographic structures of glutamate receptors Most knowledge about the binding site of glutamate and kainoid agonists has been gleaned from truncated proteins and chimera constructs. Indeed, close to 400 fragmentary structures of KA, AMPA, and NMDA receptors have been entered in the PDB since Gouaux’s seminal report of the AMPA receptor GluA2’s ligand binding domain (1GR2).1 Subsequently, many fragments of kainate receptors have been reported, mostly about the ligand-binding domain (LBD). In 2014, full-length structures of (truncated) ionotropic glutamate receptors were simultaneously reported for the first time: kainate receptor GluK2 by the Mayer lab (4UQQ, Fig. 2–9a, b),67 and the AMPA receptor GluA2 by the Gouaux lab (4U1W, Fig. 2–9c, d). 7 Moreover, these structures provided long-awaited evidence confirming that structure-activity relationship deductions based on truncated LBD’s closely matched the conformations of the complete receptors. CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 21 While it provided insight into how Glu (or KA) acts on the channel, the mechanism about how exactly it activates the channel to allow Ca2+ entry remains to be revealed. Figure 2–9. Structures of GluRs. (a) Kainate receptor GluK2 (iGluR6; 4UQQ). (b) Ligand-binding domain of kainate receptor iGluR6 for size comparison (4UQQ). (c) AMPA receptor GluA2 (iGluR2, 4U1W). (d) Zoomed-in portion of the full-length GluK2’s LBD. 2.3.2 Structural analysis of GluK2-bound kainoids Several truncated glutamate receptors have been cocrystallized with kainic acid. The LBDs have been reported for GluK1, GluK2, GluK3, and GluK5 as monomers, dimers, and trimers with typical resolutions ranging from 1.5 to 2.5 Å (~257, ~514, and ~790 aa, respectively).68-72 From these structures, we know that KA’s pyrrolidine basic nitrogen is involved in hydrogen bonding with residue Tyr764. Similarly, both C2 and C3 carboxylic acids make strong stabilizing electrostatic interactions with residues Arg523, Thr690, and Ser689. 73 This structural evidence explains and supports the empirical SAR data that arose from the early medicinal chemistry studies discussed in Section 2.2. The LBD of all glutamate receptors has a flexible clam-like shape, with glutamate’s binding pocket at the cleft's bottom. Binding of the ligand stabilizes a “closed” conformation of the clam-like domain, which induces the opening of the ion channel’s pore, and allows the flow of Ca2+ ions through the cell’s membrane. Similarly, the structures also reveal why the alkene configuration of domoic acid’s sidechain is crucial. For instance, it explains the difference in activity between iso-domoic acids A and B (38 and 39): the Z geometry of the C1’-alkene in 38 is required for the chain to extend in the cleft toward the solvent-exposed region. In contrast, the E alkene of 39 would force the aromatic system of tyrosine 488 within 3.9 Å distance. The residues making direct contact with the ligand are highlighted in green. Inset: full-length GluK2 tetramer displaying expanded LBD area (PDB 4uqq). The C4 sidechain of domoic acid extends into a space occupied by a semi-helical rigid portion of the protein (Figure 2–10). Interestingly, the geometry of the sidechain’s past the C3’ position seems a) c)b) d)CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 22 more forgiving as it lies in an outer region of the domain—a region mostly constituted of flexible loops. This is reflected in the affinity of domoic acids analogs 38, 40, and 43: as long as the sidechain can adopt a somewhat extended linear conformation, the Ki remains in the low nanomolar range. And high affinity derivatives iso-domoic acid A (37) and F (42) can all adopt a conformation similar to domoic acid's C-4 side chain. Such orientation of the side chain is prevented for iso-domic acid B (38), C (39), D (40) and E (41) (Figure 2–8). Figure 2–10. Kainic and domoic acids make a π–π interac-tion with Tyr488 in GluK receptors. (a) Side-view of GluK receptor cocrystallized with domoic acid (GluK1•DA; PDB 2pbw). (b,c) Top views of bound kainic acid and domoic acid; the alkene sidechain orientation is identical for both compounds (PDB 3c33, 2pbw, respectively). (d) Side-view of bound domoic acid shows perfect parallel alignment of the C4-alkene with the Try488. It has been proposed that the carboxylic acid on domoic acid’s sidechain participates in hydrogen bonding with an NH group from the receptor's main chain.73 While this is a plausible interaction, structural analyses indicate it is unlikely to contribute much to the overall affinity of the compound. However, the hydrophilicity of the carboxylate may ensure proper orientation of the sidechain toward the protein surface. No terminal ester of DA (or an analog lacking the terminal carboxylic acid) has been reported to date. From observing all crystal structure of iGluRs complexed to kainic acid or domoic acid, it is clear that the C4-isopropene engages in π–π stacking with tyrosine 488 (Fig. 2–10). Intriguingly, CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 23 Tyr488 is conserved in all three classes of GluR’s despite it apparently not being involved in the binding of glutamate. 2.3.3 Notes on selectivity across KAR subtypes The high level of homology between full sequences of kainate receptors GluK1–5 would seem to pose a tremendous challenge to design subtype-specific inhibitors (typically from 94% to 99.9% identity, Tables S2–4 – S2–7). However, close inspection of the agonist binding sites of KARs56-60 shows that variations exist within the binding pocket’s residues (Figure 2–11). These differences may offer the possibility to design subtype-selective agonists. * # # # * ## *# GluK1 501 DGKYGAQNDK 528 AVAPLTITYVREK 700 VRDGSTMTFF 749 ALLMESTSIEY GluK2 485 DGKYGAQDDV 513 AVAPLAITYVREK 685 VEDGATMTFF 734 AFLMESTTIEY GluK3 488 DGKYGAQDDK 515 AVAPLTITHVREK 687 VKDGATMTFF 735 ALLMESTTIEY GluK4 470 DGVYGVPEAN 497 AVAGLTITAEREK 670 IHGGSSMTFF 719 AFLLESTMNEY GluK5 469 DGLYGAPEPN 496 AVAAFTITAEREK 669 IHAGSTMTFF 718 AFLLESTMNEY Figure 2–11. Sequence homology of kainic acid’s binding pocket for GluKs. Domains 1 and 2 provide two contact surfaces each with KA; residues involved in hydrogen bonding with kainic acid are indicated with a pound sign (#), and those contributing to van der Waals interactions with a star sign (*). Conserved residues are highlighted in green, similar residues in yellow (reference numbering: GRIK2/rat, P42260). 2.4 Molecular design rules for potent kainic acid analogs In summary, we proposed the key factor for kainoids’ affinity towards KARs are: (1) alkene group is necessary for activity due to the potential stacking with Try488 in the binding site; (2) bulky unsaturated or aromatic groups neighbouring the alkene would not affect the activity greatly; (3) configuration of iso-propene is very important for activity; (4) bulky group attached to distant group from KA core greatly decreases activity; (5) C4 side chains of high affinity domoids share a similar conformation as domoic acid, for instance analogue 45 compound (its flexible C4 side chain enables the rotation to a similar conformation as DA). D1 D2CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 24 2.5 Summary Kainic acid remains the most widespread tool in neuro research. With the systematic SAR analysis presented above, we hope it stimulates the creation of novel next-generation KAR agonists by synthetic scientists. We anticipate that those novel KA analogs will also offer the neurobiology community a convenient testing bed for KARs research. Finally, as part of our research program in the mechanisms of synapse elimination, we have used these design rules to design and synthesize new agonists and molecular imaging probes to study iGluRs in living cells. CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 25 2.6 Data analysis 2.6.1 Calculation method The literature values of binding, activity or affinity of KAR agonists are reported using a wide range of biochemical and biological assays. In order to compare the activity of kainic acid derivatives presented in different studies, a qualitative scale was established. Glutamate, kainic acid and domoic acid were set as benchmarks for this scale system. Table 2–2. Qualitative scale The activity of each compound was first normalized to the positive control used in a given study (typically, glutamic acid or kainic acid). If needed, the resulting activity ratio was then further standardized to kainic acid. When a compound was tested in more than one assay, priority was given to data from GluRs binding assay (Ki or IC50). Otherwise, data from [3H]-KA displacement assays was used for calculations. If those data were not available, calculations were performed with the reported activity in neurons or in animal experiments. When multiple data were available for a single agonist, the most recent data is used. Calculation methods: 1. Relative activity = Ki (or IC50) of KA / Ki (or IC50) of compounds 2. Relative activity = Effect of compounds / effect of KA 2. Relative activity (iso-DAs) = (Ki of DA / Ki of iso-DAs) ´ Ratio potency of DA 2.6.2 Calculated ratios and grades The potency ratios of agonists are calculated according to the originally reported data (see Table S2-1 and S2–2, Appendix A). The calculated results are tabulated as following. Relative Activity (RA) Grade Notes Inactive -- Inactive 0 < RA < 0.065 - Weaker than L-Glu, but active 0.065 £ RA < 1 + Between L-Glu and KA 1 £ RA < 2.89 ++ Between KA and DA 2.89 £ RA +++ Being equal as or stronger than DA CHAPTER 2. STRUCTURE-ACTIVITY RELATIONSHIPS ANALYSIS OF KAINIC ACID-BASED AGONISTS 26 Table 2–3. Activity scales for kainoid analogues Compound Potency Ratio (Relative to KA) RA Level Ref. Compound Potency Ratio (Relative to KA) RA Level Ref. Kainic acid (1) –Reference 1 ++ 51, 52, 62, 63, 65, 81-88 32d 0.03 - 74 Domoic acid (2) 2.89 +++ 75 32e 1.6 ++ 74 L-Glu (3) 0.065 + 76 33a 5.67 +++ 74 4 Inactive -- 77 33b 0.12 + 74 5a Inactive -- 77 33c 6.8 +++ 74 5b Inactive -- 77 33d 2.13 ++ 74 6 Inactive -- 78 34a Inactive -- 57 7 Inactive -- 46 34b Inactive -- 57 8a Inactive -- 79 35a 0.016 - 57 8b Inactive -- 79 35b 0.023 - 57 9a Inactive -- 79 35c 0.017 - 57 9b Inactive -- 79 35d 0.001 - 57 10 0.049 - 80 36a 2.3 ++ 57 11 0.0063 - 81 36b 1.7 ++ 57 12a 1 ++ 78 36c 1.4 ++ 57 12b 0.299 + 78 36d 1.1 ++ 57 13 0.00063 - 82 36e 0.95 + 57 14a Inactive -- 48 36f 0.95 + 57 14b Inactive -- 48 36g 1.4 ++ 57 15a 0.013 - 52 36h 1.6 ++ 57 15b 0.00065 - 52 36i 0.40 - 57 15c Inactive -- 48 36j 1.94 ++ 57 16a Inactive -- 48 36k 0.48 + 57 16b Inactive -- 48 36l 1.02 ++ 57 16c Inactive -- 52 36m 1.94 ++ 57 16d Inactive -- 83 36n 0.20 + 57 17 Inactive -- 78 36o 1.02 ++ 57 18 Inactive -- 78 36p 0.41 - 57 19 Inactive -- 78 36q 1.76 ++ 57 20 0.68 + 78 36r 0.41 + 57 21 Inactive -- 78 36s 0.16 + 57 22 Inactive -- 84 36t 1.32 + 57 23 Inactive -- 78 36u 0.31 + 57 24a Inactive -- 54 36v 0.21 + 57 24b Inactive -- 54 36w 0.29 + 57 25a 0.22 + 55 36x 0.029 - 57 25b 0.043 - 55 36y 0.018 - 57 Acro A (26) 2.2 ++ 76 36z 0.041 - 57 Acro B (27) 7.2 +++ 76 36aa 0.41 + 57 MPPA (28) 0.31 + 76 36ab 0.08 + 57 HMPPA (29) 0.68 + 76 iso DA A (37) 1.62 ++ 81 CPPA (30) 0.87 + 76 iso DA B (38) 0.001 - 81 CNOPA (31) 0.27 + 76 iso DA C (39) 0.040 - 81 32a 0.34 + 74 iso DA D (40) 0.004 - 81 32b 4.0 + 74 iso DA E (41) 0.004 - 81 32c 0.85 + 74 iso DA F (42) 0.10 + 81 27 27 Chapter 3 Synthesis of Kainoids and C4 Derivatives Developing a general synthetic route to C4 kainoids is required as they are the most potent agonists of kainic acid receptors (KAR). While several syntheses of kainic acid and analogs have been reported since the discovery of this neurotoxin, none offers a practical route to kainic acid and its C4 derivatives simultaneously. This chapter describes a general synthesis of kainoids that allows access to C4 derivatives via late-stage divergence. 3.1 Background Kainoids are a group of non-proteinogenic amino acids displaying strong neurological activity.85 The title compound of the family, kainic acid (1) and its epimer allo-kainic acid (6) were isolated from the marine algae Digenea simplex in 1953 (Figure 3–1).86 The unique selectivity of kainic acid for a subset of ionotropic glutamate receptors led to naming the class as kainate receptors (GluK1-5).87 Kainic acid is now commonly used in neurobiology research to study conditions such as epilepsy, Huntington’s, Parkinson’s and Alzheimer’s diseases.45,88,89 As discussed in Chapter 2, biological testing of KA analogs revealed that the neuroactivity of KA is highly dependent on its C4 substituent. For instance, synthetic derivatives such as phenylkainic acid (32a),46 acromelic acid A (26),46 and thiazole analog 33c74 all display equal or higher potency than kainic acid. CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 28 Figure 3–1. Naturally occurring (1, 6, 55, 26) and synthetic kainoids (32a, 33c). The affinity is indicated as “+” or “-” according to the grading system in Chapter 2. Kainoids are a popular synthetic target: close to 70 syntheses of kainic acid, allo-kainic acid, iso-kainic acid, and phenylkainic acid have been reported.85,86,90-95 The shortest synthesis of kainic acid was accomplished by Ohshima in six steps, however, it does not allow for ready variation of the C4 position.96 Structure-activity relationship studies have confirmed that the C4 substituent of kainoids is the only position that can be exploited to enhance the activity of kainic acid (Chapter 2).56,97 Moreover, these studies revealed that only sp2-hybridized C4 substituents display a high affinity for GluK receptors. As part of our research program in neuron-glia communication, we required a GluK chemical probe bearing a reporter tag at C4. We reasoned that an ideal approach would enable late-stage introduction of sp2-hybridized nucleophiles at the C4 position. A route to phenylkainic acid reported by Baldwin in 1995 provided a potential framework that could be modified for our purpose.98 Accordingly, we developed a pragmatic route that can deliver large amounts of C4 kainoid derivatives for cell and animal assays.99 3.2 Restrosynthetic analysis of 4-substituted kainoids The proposed approach prioritizes the late-stage introduction of C4 substituents as sp2-hybridized nucleophiles (Scheme 3–1). Installing C4 side chains was achieved via a sequence of stereoselective addition/reduction onto pyrrolidinone 56, which is the pivotal intermediate of our strategy. Access to pyrrolidinone 57 from 4-hydroxyproline 58 was optimized from select precedents. This path would provide one of the shortest access to kainoid analogs. NHCO2HCO2Hα-iso-kainic acid (55)NHCO2HCO2HNHCO2HCO2H(+)-allo-kainic acid (6)234(–)-kainic acid (1)NHCO2HCO2Hacromelic acid A (26)NHNHCO2HCO2H33cOHO2CNSNH2NHCO2HCO2Hphenylkainic acid (32a)+ + ++ + ++ + - - n/a6CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 29 Scheme 3–1. Retrosynthesis analysis of 4-substituted kainoids 3.3 Optimization of C3 intermediate synthesis The synthesis began with a two-step protection of trans-4-hydroxy-L-proline, followed by a Swern oxidation to obtain pyrrolidinone 61 (Scheme 3–2).100-102 The choice of protecting group for the amine is crucial for the subsequent C3 alkylation leading to 62.101 Indeed, Lubell and Rapoport’s phenylfluorenyl (Pf) protecting group103,104 was unique in achieving enolate alkylation regioselectively at the more hindered alpha position of ketone 61. Initial trials with Boc or Cbz were unproductive, leading to poor regioselectivity or stereoselectivity. While Pf’s large steric bulk was essential to control selectivity, it would later offer its share of challenges. Scheme 3–2. Synthesis of pyrrolidin-4-one 62a. Alkylation of the C3 ester side chain to obtain the desired trans diester 62a is highly sensitive to reactions conditions (Table 3–1). We selected a tert-butyl ester because it allowed us to unambiguously assign the stereochemistry of subsequent compounds 63–65 (reduced conformational flexibility greatly enhances NOE). Adapting conditions for a related alkylation using a catalytic amount of NaI101,102 yielded a 5:1 ratio when the reaction was performed on >5 grams scale using BrCH2CO2tBu (entry 1). Instead, using tert-butyl iodoacetate directly, the ratio was improved to 14:1, although the conversion was sluggish (entry 4). Increasing equivalents and N CO2R1PGCO2R2RN CO2R1PGCO2R2ONHHOCO2HC4 kainoids5657 trans-4-hydroxy-L-proline (58)Stereoselectiveaddition &reductioncommonintermediate4NHHOCO2H N CO2MePf60NOCO2MePfCO2tBu62b (8%)ii. Conditionsi. n-BuLi, THF/HMPA, –78 oCTMSCl, MeOH58i. DMSO, (COCl)2, DCM, -78 oCNOCO2MePfCO2tBu2362a (86%)i. TMSCl, Et3N, DCM; ii. MeOH, Pb(NO3)2, PfBr, Et3N;NHHOCO2CH3· HCliii. 10% citric acid, MeOHHO59ii. Et3N, -78 oC to rt N CO2MePf61OOOOOCbz =Boc =Pf =95% 89%91%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 30 temperature afforded an acceptable compromise in terms of conversion and selectivity (entries 2 and 3). The stereochemistry of 62a and 62b was determined by 1H NMR analysis: the empirical JH2-3 value for the 2,3-trans product 62a is 6.1 Hz, and that of 62b is 8.2 Hz (these C2,3 configurations were further confirmed by crystal structures of their derivatives in the reported studies). 100,102,105 Table 3–1. Stereoselective alkylation of ketone 61 Entry Halide Conditions Conversion (%)a dra 62a:62b 1b BrCH2CO2tBu (3.5 eq) –41 ºC, 3h 100 5:1 2c ICH2CO2tBu (5.0 eq) –41 ºC, 5.5 h 100 9:1d 3c ICH2CO2tBu (6.0 eq) –78 ºC, 5 h 60 10:1 4c ICH2CO2tBu (3.5 eq) –78 ºC, 5 h 50 14:1 a Determined by 1H NMR analysis of crude products. b Reaction conditions: 1:5 HMPA/THF; 0.5 equiv. NaI, halide (Method B in supp. info.). c 1:10 HMPA/THF; halide (Method A in supp. info.). d After purification, the ratio of isolated products was 10.8:1. 3.4 Installation of C4 sidechain 3.4.1 Stereoselective addition of sp2 nucleophiles Next, we turned to the key installation of side chains at the C4 position of 62a (Scheme 3–3). When ketone 62a was treated with alkenyl or aromatic Grignard reagents, only starting material 62a epimerized at C3 was recovered. We explored less basic organocerium reagents to promote the addition. We found that using CeCl3×2LiCl (1.3 equiv. for 62a, 3.0 equiv. for 63b) afforded alcohols 63 as single diastereomers with an almost quantitative yield.106 The presence of LiCl is essential to full obtain conversion with 62a, and the reaction worked equally well with i-Pr- and Ph- organometallic reagents (Table 3–2). Baldwin et al. reported a similar strategy using N-Bz proline ketone with PhMgBr and CeCl3 that afforded a 52% yield.98 However, the more sterically demanding N-Pf protected 62a led to poor conversion with CeCl3. For instance, using i-PrMgBr/CeCl3 gave only 5% yield, and PhMgBr/CeCl3 gave 63b in 16% yield with C3 epimerized. NOE experiments confirmed the configuration of the C4 centers (Scheme 3–3). Scheme 3–3. Synthesis of C4 tertiary alcohols and NOE analyses N CO2MePfCO2tBuHOR63a: R = 2-propenyl63b : R = PhNOCO2MePfCO2tBu62aConditionsNCO2MePfCO2tBuOHH3CHHHbHaNOENCO2MePfCO2tBuOHPh HHHbHaNOENOE63a 63bCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 31 Table 3–2. Nucleophilic addition of ketone 11 Entry Conditions Conversion (%) 1 RMgBr, THF, rt NAa 2 RMgBr (1.0 eq), CeCl3 (1.0 eq), THF, 0 oC 63a: 5%; 63b: 16% 3 RMgBr (3 eq), CeCl3×2LiCl (1.5 eq), THF, -78 oC 63a: 98%; 63b: 95% a No desired conversion was observed. 3.4.2 Stereoselective deoxygenation of the C4 alcohol Deoxygenation of tertiary alcohols 63a and 63b posed a significant challenge of chemoselectivity. Traditional radical-based deoxygenation methods failed, so we examined the reactivity of the parent carbonates (Scheme 3–4). Allylic carbonate 64a was reduced via a catalytic transfer hydrogenation using ammonium formate and palladium acetate 64a to afford a 3:1 regioisomeric mixture of allo–kainic acid precursor 65a and iso–kainic acid precursor 65b.107 No conversion was observed on the 64b with the same condition, which confirmed the limited application scope of this type reaction on allylic substrates. Scheme 3–4. Palladium-catalyzed allylic deoxygenation The C3,4–cis diastereomer of 65 (precursor of kainic acid) was not observed under these reduction conditions. The steric bulk of the N-Pf protecting group likely prevents the catalyst from approaching the top face of the allylic carbonate (viz. path B, Scheme 3–5). We surmise that competing catalytic pathways take place. For instance, the sterically congested substrate 64a may slow the antiperiplanar addition of Pd(0) to the allylic system, and a Pd(II) complex may act as Lewis acid toward the carbonate group. The resulting transient allylic carbocation then gets reduced from the same face as the leaving group. N CO2MePfCO2tBuHORN CO2MePfCO2tBuORLiHMDS, THF, - 78 oC;N CO2MePfCO2tBu65a, 51%N CO2MePfCO2tBu65b, 16%THF, 60 oC64a, Pd(OAc)2Ph3P, NH4HCO2OMeO64a: R = 2-propenyl, 96%64b: R = Ph, 95%ClCO2Me63a: R = 2-propenyl63b: R = PhCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 32 Scheme 3–5. Proposed mechanism for allylic deoxygenation 3.4.3 Protecting group swap: Pf for Boc To change this stereoselectivity, the amine’s Pf protecting group was replaced by a less bulky Boc. Extensive optimization was needed to selectively cleave the Pf group without losing the tert-butyl ester or epimerizing C3. We found buffered reductive conditions that are compatible with: tertiary alcohols, allylic carbonates, tert-butyl carbonates, and esters (summarized in Table 3–3). We settled on using Et3SiH with TFA in dichloromethane, which led to quantitative removal of N-Pf (entries 2 and 3). Alternatively, Et3SiH with I2 in acetonitrile was also efficient, but led to the loss of the tert-butyl group (entry 4). Using Rapoport’s original conditions (excess TFA)108 removed the Pf group, however the tert-butyl ester was quite prone to elimination and high yields were difficult to reproduce (entry 1). Table 3–3. Deprotection of N-phenylfluorenyl (N-Pf) Entry Cmpd Conditions Yield Byproduct 1 63a/b TFA (15 eq.), DCM, 1 h 90%a PfOH 2 63a/b Et3SiH (3 eq.), TFA (5 eq.), DCM, 1 h Quant. PfH 3 64a/b Et3SiH (3 eq.), TFA (5 eq.), DCM, 1 h Quant. PfH 4 64a/b Et3SiH (2 eq.), I2 (1 eq.), MeCN, 10 min Quant.b PfH (a) ~10% tert-Butyl ester was converted to the parent acid. (b) tert-Butyl ester was also deprotected under these conditions. NOOO OOOMeOPdIIHNOOO OOCO2MePd0LnNOOO ONOOO OPdIIHMeO2COPdIIH3,4-cisnot observedinternal terminal internalA BN CO2MePfCO2tBuOOMeOH[PdII]65a 65bNOENOEN CO2MePfH2C CO2tBuH3C HHHbHaH65a64aCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 33 3.4.3 Deoxygenation to kainoids with modified selectivity The pyrrolidine resulting from Pf deprotection of 64 was directly subjected to standard Boc protection conditions to afford carbamates 66a and 66b in excellent yields (Scheme 3–6). The catalytic hydrogenolysis of allylic carbonate 66a with Pd(OAc)2 and formate afforded 67a and 67b as a 1.1:1 mixture of C4 epimers. Improving the ratio for kainic acid precursor 67b remained elusive, despite several attempts. Scheme 3–6. Synthesis of allo-kainic acid and kainic acid precursors Kainic acid (1), allo-kainic acid (6), and iso-kainic acid (55) were obtained via global deprotection of 67a, 67b, 65a and 65b, respectively (Scheme 3–7). A telescoped sequence was used for 67a and 67b: the reductive Pf deprotection was followed by a mildly alkaline hydrolysis. Natural products 6 and 55 were obtained in 78% and 79% yields respectively. Intriguingly, no epimer was observed in this ester hydrolysis reaction. 65 and 67 have epimerizable of C2 stereocentre, but the C3 and C4 stereocenters are not epimerizable under aqeous basic conditions. Due to thermodynamically favoured C2,3-trans configuration, it posts low probability for 65 and 67 to undergo C2 epimerization during the global deprotection. For this reason, aqeous basic conditions were employed to invert the C2 configuration when people obtained undesired C2,3-cis epimers.109-111 Moreover, NMR spectra of 6 and 55 are consistent with reported data (Table S–1, Appendix C). The ~1:1 mixture of N-Boc protected diesters 67a and 67b was also deprotected with a telescoped sequence: alkaline hydrolysis followed by anhydrous TFA treatment led to kainic acid (1, 35%) and allo-kainic acid (6, 46%). Separation of 1 and 6 by crystallization and HPLC proved challenging. While it was possible to obtain analytically pure samples for characterization, the bulk of the products remained a mixture. Regardless, this mixture was useful to us—and we expect to others—as it enables the gram-scale synthesis of thiazole 33c in four steps, currently the most potent GluK receptor agonist.74 N CO2MePfCO2tBuORi. Et3SiH, TFA, DCMii. Boc2O, DMAP66a, Pd(OAc)2Ph3PNH4HCO2 THF, 60 oCN CO2MeBocCO2tBuN CO2MeBocCO2tBu67a, 50%67b, 46%OMeON CO2MeBocCO2tBuOROMeO64a: R = 2-propenyl64b: R = Ph66a: R = 2-propenyl, 96%66b: R = Ph, 95%NOOO OOCO2MeOOPdIIHNOOO OOOPd0Lnterminal via Ainternalnot observedCCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 34 Scheme 3–7. Deprotection to kainic acid and isomers 3.5 Synthesis of phenylkainic acid & more C4 functionalization Inspired by the lactone hydrogenolysis reaction developed by Baldwin et al.,98 we attempted to invert the C4 stereocentre with a hydrogenation reaction (Scheme 3–8). Upon full deprotection, carbonate 64b spontaneously cyclized to lactone 68 (sparingly soluble in DMSO). This lactone was then ring-opened with inversion of configuration under standard hydrogenation conditions using Pd/C. Phenylkainic acid (32a) was obtained as a single diastereomer in 90% yield from 68. Scheme 3–8. First attempt at phenylkainic acid synthesis Alternatively, we shortened the path above by directly cyclizing alcohol 63b to lactone 69a under excess of TFA. Lactone 69 was hydrogenolyzed to yield phenylkainic ester 70 as a single diastereomer (Scheme 3–9). Compared to Baldwin’s synthesis of phenylkainic acid (9 steps, 11% overall yield),98 our route fares well: 32a is obtained in 8 steps with an overall yield of 54%. The configuration of C4 in 32a was confirmed by 1H NMR spectroscopic analyses and is consistent both with literature data (Table S3–1, Appendix B)112 and with configuration assignment rules (Table N CO2MePfCO2tBu65a, 51%N CO2MePfCO2tBu65b, 16%N CO2MeBocCO2tBuN CO2MeBocCO2tBu67a, 50%67b, 46%1. LiOH aq, CH3OH2. TFA, DCMNHCO2HCO2HNHCO2HCO2H81%dr = 1 : 1.31 61. Et3SiH, TFA, DCM2. LiOH aq, CH3OHNHCO2HCO2HNHCO2HCO2H6 5578% 79%64b1. LiOH aq, CH3OH2. TFA, DCMN CO2MePfOPhOPd/C, H2CH3OH/AcOH90% 90%NHCO2HCO2H32aPh68N CO2MePfCO2tBuOPhOMeOCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 35 S3–2, Appendix B).113 The C3,4–cis configuration was further confirmed by NOE analysis of the subsequent products 71a and 71b (Scheme 3–8). Scheme 3–9. Synthesis and Functionalization of Phenylkainic Acid. (p-Ns: p-nitrobenzenesulfonyl) Our need for kainic acid-based chemical probes motivated us to exploit our most efficient route: that leading to intermediate 70a in 7 steps (Scheme 3–9). Chemoselective oxidation of the C4-phenyl group to a carboxylic acid would provide a versatile synthetic handle to append any side chain at a late stage. Accordingly, diester amine 70a was converted to the N- Boc diester 71b in two steps. However, when 71b was presented to RuCl3 and NaIO4, the phenyl group remained untouched and only the C5 position of the pyrrolidine was oxidized to pyrrolidinone 72b. Instead of Boc, a p-nitrobenzenesulfonyl (Ns) protecting group was selected for its ability to deactivate the N-alpha position and its mild deprotection condition (ArSH, K2CO3, DMF).114 Thus 70a was converted to the N-Ns amide 71a in two steps and almost quantitative yield. The phenyl group of 71a was oxidized selectively and afforded the desired acid 72a in 78% yield without epimerization (stereochemistry confirmed by NOE analysis). The C4-acid kainoid 72a provides easy access to a variety of kainoid derivatives. It enables the synthesis of numbers of heteroaromatic kainoids.115 In addition, our work toward kainoid-based GluK imaging probes— presented in the following Chapters—confirms that 72a can be readily coupled to fluorescent dyes. N CO2MeCO2MePhRNaIO4, RuCl372a, 78%NHCO2MeR2R1HHCO2MeXNOENHCO2HCO2HPh32aNOOPhCO2MeN CO2MeCO2HPh63bquant.69a: R = H70a: R = H19a, LiOH aq, CH3OH86%1. 70a, TMSCl, CH3OH2. DMAP, Et3N, DCM, p-NsCl for 71a or Boc2O for 71b71a: R = Ns, 96%71b: R = Boc, 92%CH3CN/CCl4/H2O71a: R1 = Ns, R2 = Ph71b: R1 = Boc, R2 = Ph72a: R1 = Ns, R2 = CO2H69b: R = BocRDMAP, Et3N, Boc2O, DCMRONR1R2OOO70b: R = BocTFA, DCM Pd/C, H2quant.CH3OH/AcOHPdHorAN CO2MeCO2MePhBocO72b, 85%N CO2MeCO2MeNsOHON COOCH3OCOOCH3O OHNONH73 NsEt2NOEt2N O HNONH2Et3N,HBTU,DCM92%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 36 3.6 Summary In conclusion, a unified diastereoselective route to 4-substituted kainoids was demonstrated from commercially available trans-4-hydroxy-L-proline (Scheme 3–10). The sequence affords: kainic acid (11 steps, 19%), allo-kainic acid (9 steps, 24%), iso-kainic acid (9 steps, 8%), and phenylkainic acid (8 steps, 54%). In addition, a mild oxidation step has been developed for C4 derivatization from phenyl kainic acid. The novel acid 72a (10 steps, 47%) is a readily modifiable intermediate that can give rapid access to non-natural kainoids via amide couplings, including KARs chemical probes. This report provides a general access to a range of biologically active 4-substituted kainoids. Scheme 3–10. A unified synthesis strategy of C4-kainoid derivatives NHHOCOOH4-hydroxyprolineNHCO2HCO2HR = Alkenyl, ArylR8–11 steps47–54 % yield472a3 steps N CO2MeCO2MeOHONsCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 37 3.7 Experimental Section 3.7.1 General Experimental Procedures Unless otherwise noted, reactions were carried out under an argon atmosphere, in flame-dried single-neck, round bottom flasks fitted with a rubber septum and with magnetic stirring. Air or water sensitive liquids and solutions were transferred via syringe or stainless-steel cannula. Organic solutions were concentrated by rotary evaporation at 25–45 °C at 50–200 torr. Thin layer chromatography (TLC) was performed on glass plates precoated with Silica gel F254, 250 µm, 60 Å, from EMD Chemicals Inc (EMD 5715-1). TLC plates were visualized under a 254 or 365 nm UV light source, then stained by immersion in either acidic aqueous-ethanolic vanillin solution, potassium permanganate, or acidic ethanolic ninhydrin, followed by heating using a heat gun. Purification was performed with 230-400 mesh silica gel from Silicycle, Quebec (SiliaFlash R12030B, P60, 40-63 µm, 60 Å). 3.7.2 Materials 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. Tetrahydrofuran, dichloromethane, hexanes, toluene, and diethyl ether were purified on a glass contour solvent purification system under an argon atmosphere. Methanol was dried by allowing it to stand over freshly activated 4 Å molecular sieves for 48 h prior to use. Solvents used for chromatographic purifications were obtained from Fischer Scientific or VWR and used without further purification. All new compounds (63-68) and final products (1, 6, 32a, 26 and 73) were characterized with 1D (1H NMR and 13C NMR) and 2D NMR (COSY, HSQC, HMBC) spectroscopic analyses. 3.7.3 Instruments 1 H and 13 C 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 10.0.2-15465 from Mestrelab Research. Chemical shifts are reported in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to residual proton signals in the NMR solvents (CHCl3: δ 7.26, C 2 HD 5 SO: δ 2.50), for carbons (CDCl3: δ 77.0, C 2HD5SO: δ 39.5). Spectral data are listed as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s= broad singlet), and coupling constant (J, Hz). Infrared spectra (IR) were obtained using a Nicolet 6700 FT-IR spectrometer as a neat film on a NaCl plate, or a Perkin-Elmer FT-IR Spectrum Two IR spectrometer. High resolution mass spectra were obtained from the UBC Mass Spectrometry Facility using a HCTultra PTM discovery system spectrometer (ESI), or from a Waters Micromass LCT Premier TOF Mass Spectrometer (ESI, Zandberg lab, UBCO). Melting points of solid samples were measured with a a IA9200 melting point apparatus (Electrothermal). The final products were purified HPLC on a C18 column (Eclipse Plus C18, 4.6 ´ 250mm, 5 µm, Agilent) with a Varian Prostar HPLC system (e.g., kainic acid and allo-kainic acid). HPLC condition for the purification was described in the corresponding procedure. CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 38 3.7.4 Experimental Procedures and Characterization Synthesis of C2 precursors tert-Butyl bromoacetate (50). To a rigorously stirred solution of magnesium sulfate (52.0 g, 432 mmol) in dichloromethane (300 mL) was slowly added concentrated sulfuric acid (3.8 mL, 72 mmol). After 10 min, bromoacetic acid (20.0 g, 144 mmol) was added at rt, followed by the addition of tert-butyl alcohol (41.2 mL, 432 mmol). After 24 h, a second portion of tert-butyl alcohol (20.5 mL, 216 mmol) was added to take the reaction to completion in an additional 24 h. The insoluble matter in the resulting reaction solution was removed by vacuum filtration. The filtrate was poured into water and extracted with dichloromethane (3 × 50 mL). The combined organic layer was washed with sodium bicarbonate solution, brine and dried over MgSO4. The solvent was evaporated under reduced pressure to afford the crude tert-butyl bromoacetate as a light-yellow liquid (26.9 g, 96%), which was used for the following reaction without further purification. 1H NMR (400 MHz, CDCl3): δ 3.75 (s, 2H), 1.48 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 166.2, 82.9, 27.8, 27.7 ppm. tert-Butyl iodoacetate (51). tert-Butyl bromoacetate (25.2 g, 128 mmol) was added at rt to a suspension of NaI (23.1 g, 154 mmol) in acetone (200 mL) under a nitrogen atmosphere; the resulting suspension was stirred for 5 h. The insoluble salt was removed by vacuum filtration, and the filtrate was concentrated under reduced pressure. The residue was redissolved in diethyl ether (150 mL) and washed with 100 mL of a basic Na2S2O3 aqueous solution (5% wt in aqueous saturated sodium bicarbonate) and brine (100 mL). The organic layer was dried over MgSO4 and filtered under a vacuum. The resulting solution was concentrated under reduced pressure to afford tert-butyl iodoacetate as a pale-yellow liquid (29.4 g, 95%). The crude tert-butyl iodoacetate, was dried over 4 Å molecular sieves to remove the trace water residue before use. 1H NMR (400 MHz, CDCl3): δ 3.61 (s, 2H), 1.46 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 167.8, 82.3, 27.6, –2.6 ppm. Preparation of a CeCl3×2LiCl solution The CeCl3×2LiCl complex THF solution was prepared according to a literature procedure from Knochel et al.106 A 50 mL Schlenk flask was charged cerium chloride heptahydrate (1.512 g, 4.1 mmol), anhydrous lithium chloride (0.345 g, 8.1 mmol—LiCl was pre-dried in oven at 100 oC for 5 hours) and a stir bar. The top joint was sealed, the side arm of the flask was connected to a vacuum line, and the flask was immersed in an oil bath (Figure 3–2a). The solids were stirred under high vacuum, and the temperature was increased slowly (+10 oC increases every 2 h). Finally, it was maintained at a temperature of 140 oC for 3 hours. The oil bath was removed and the mixture was allowed to cool to rt under vacuum. The vacuum was disconnected and replaced with argon inflow. Anhydrous THF (42 mL) was then added to the solids (Figure 3–2b) and stirred BrOHO t-BuOH, H2SO4MgSO4, DCM96%BrOO49 50NaI, acetone95%IOO51CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 39 for 72 hours, at which point the solution was colorless and transparent (~0.1 M, Figure 3–2c). Note: this solution is very hygroscopic and should be used up within 2 weeks. Figure 3–2. Preparation CeCl3×2LiCl THF solution. (a) Drying of CeCl3×2LiCl under hi-vac. (b) CeCl3×2LiCl mixed with THF under argon. (c) CeCl3×2LiCl is a colourless solution after 72 hours stirring at rt. Synthesis of 9-bromo-9-phenylfluorene 9-Hydroxyl-9-phenyluorene (PfOH, 53). To a suspension of magnesium turnings (9.2 g, 382 mmol) in anhydrous THF (5 mL) under an argon atmosphere was added 0.5 mL of bromobenzene neat at rt in one portion to initiate the reaction. The reaction vessel was cooled to 0 °C on an ice bath, followed by the dropwise addition of a THF solution of bromobenzene (1.2 M, 270 mL) at 0 °C. The resulting reaction solution was allowed to reach rt and stirred vigorously for an additional 2 h. The exact concentration of the resulting phenyl magnesium bromide solution was determined by titration with salicylaldehyde phenylhydrazone. 25 This phenyl magnesium bromide solution (180 mL, 216 mmol) was transferred into a flame-dried single-neck flask by a syringe. To this solution were added CeCl3×2LiCl (15 mL, 0.1 M) THF solution in one portion and 9-fluorenone (30.1 g, 186 mmol) by portions. After 15 min, TLC analysis showed full conversion. The reaction was quenched with diluted hydrochloric acid (1.0 M, 100 mL) at 0 °C. The resulting solution was extracted with Et2O (3 × 150 mL). The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to yield PfOH as pale-yellow crystals (40.8 g, 95%). Mp 108–109 °C. FTIR (thin film): 3544, 3423, 3059, 1603, 1448, 774, 732, 701 cm –1. 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.5 Hz, 2H), 7.53–7.41 (m, 4H), 7.34 (dq, J = 15.1, 7.5 Hz, 7H), 2.79 (s, 1H) ppm. Br1. Mg, THF2. 9-fluorenone,CeCl3·2LiClHO525348% HBr aqToluene95% 90%Br54CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 40 13C NMR (101 MHz, CDCl3): δ 150.4, 143.2, 139.5, 129.0, 128.4, 128.2, 127.2, 125.4, 124.8, 120.1, 83.6 ppm. 9-Bromo-9-phenyluorene (PfBr, 54). PfBr was prepared according to the reported procedure. 26 The above PfOH (36.1 g, 140 mmol) was dissolved in toluene (150 mL), and aqueous HBr (48% w/w, 50 mL) was added at rt. This heterogeneous mixture was stirred vigorously at rt for 48 h, away from light. The mixture was then extracted with toluene (3 × 100 mL). The combined organic layer was washed with brine (6 × 150 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to afford the crude product as a light-yellow solid. Recrystallization with hexane afforded pale-yellow crystals (40.2 g, 90%). Mp 100–101 °C. FTIR (thin film): 3056, 1603, 837, 738, 694 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.67 (dt, J = 7.6, 0.9 Hz, 2H), 7.60–7.49 (m, 4H), 7.36 (td, J = 7.5, 1.2 Hz, 2H), 7.35–7.18 (m, 5H) ppm. 13C NMR (101 MHz, CDCl3): δ 149.6, 141.1, 138.1, 129.0, 128.5, 128.3, 128.0, 127.4, 126.1, 120.3, 67.5 ppm. (2S,4R)-4-Hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride (59). trans-4-Hydroxyl-L-proline (58, 10.0 g, 76 mmol) was suspended in methanol (150 mL) under argon. The suspension was cooled to 0 °C in an ice bath, followed by the dropwise addition of chlorotrimethylsilane (33.9 mL, 267 mmol). After 30 min, it was allowed to warm up to rt and stirred overnight. The solvent was removed under reduced pressure, the residue was triturated with Et2O (100 mL), and ester 59 was obtained as white crystals (13.1 g, 95%). 1H NMR (400 MHz, D2O): δ 4.78–4.68 (m, 2H), 3.88 (s, 3H), 3.56 (dd, J = 12.6, 3.8 Hz, 1H), 3.44 (dt, J = 12.5, 1.6 Hz, 1H), 2.52 (ddt, J = 14.3, 7.8, 1.8 Hz, 1H), 2.32 (ddd, J = 14.5, 10.5, 4.3 Hz, 1H) ppm. 13C NMR (101 MHz, D2O): δ 170.2, 69.4, 58.1, 53.8, 53.4, 36.6 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C6H12NO3, 146.0817; found, 146.0817. Methyl (2S)-4-oxo-1-(9-phenyl-9H-fluoren-9-yl) pyrrolidine-2-carboxylate (60). 60 was prepared according to the reported procedures.100 The amine salt 59 (8.0 g, 44 mmol) was suspended in chloroform (300 mL) under argon. The reaction vessel was cooled to 0 oC in an ice bath, and trimethylamine (22.7 mL, 154 mmol) was added, followed by dropwise addition of chlorotrimethylsilane (14.7 mL, 110 mmol). The reaction was NHHOCO2HNOCO2MePfNOCO2MePfCO2tBu(62b, 8%)ii. MeOH; Pb(NO3)2, PfBr, Et3N; iii. 10% citric acid, MeOHNOCO2MePfCO2tBu(62a, 86%)TMSCl, MeOHNHOCO2HPfi. DMSO, (COCl)2, DCMii. Et3Ni. n-BuLi, THF/HMPA, THF, -78 oCii. ICH2CO2tBuNHHOCO2CH3独HCli. TMSCl, Et3N, DCM;95% 81%91%+58 59 6061CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 41 stirred for 10 min at 0 ºC, then allowed to warm up to rt, followed by heating to reflux for 2 h with vigorous stirring. The reaction mixture was cooled to 0 o C and methanol (2.7 mL, 66 mmol) was added to quench the excess TMSCl. The reaction was allowed to warm up to rt and stirred for 2 h. Anhydrous triethylamine (6.2 mL, 44 mmol) was added, followed 5 min. later by Pb(NO3)2 (11.9 g, 35 mmol) and PfBr (18.8 g, 57 mmol). The heterogeneous mixture was stirred vigorously at rt for 5 days. After full conversion, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The residue was dissolved in a 10 % w/v solution of citric acid in methanol (250 mL), and allowed to react for an additional 1.5 h. The solvents were removed under reduced pressure. The residue was purified by column chromatography (25–50% acetone/hexane as elution gradient). The N-Pf-pyrrolidine 60 was recovered as a white foam-like solid (15.1 g, 89%). Mp 85–86 oC. FTIR (thin film): 3443, 3057, 2946, 1727 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.73 (dt, J = 7.5, 0.9 Hz, 1H), 7.64 (dt, J = 7.5, 1.0 Hz, 1H), 7.59–7.50 (m, 3H), 7.42 (td, J = 7.5, 1.1 Hz, 1H), 7.37–7.18 (m, 6H), 7.14 (td, J = 7.5, 1.2 Hz, 1H), 4.48 (q, J = 5.5 Hz, 1H), 3.56 (dd, J = 10.0, 5.3 Hz, 1H), 3.27 (dd, J = 8.9, 5.2 Hz, 1H), 3.23 (s, 3H), 2.90 (dd, J = 10.0, 5.0 Hz, 1H), 1.96 (dt, J = 12.8, 5.7 Hz, 1H), 1.77 (ddd, J = 13.0, 8.9, 6.0 Hz, 1H) ppm. 13 C 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, 120.1, 119.8, 70.3, 59.3, 56.8, 51.3, 39.9 ppm. HRMS (ESI) m/z (M+H) + calcd. for [C 25H23NO3+H]+ 386.1756, found 386.1750. Methyl (2S)-4-oxo-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate (61). Preparation of ketone 61 was modified from a literature procedure.102 Oxalyl chloride (5.7 mL, 66 mmol) was added to DCM (anhydrous, 150 mL) at - 78 ºC under argon atmosphere, followed by dropwise addition of anhydrous DMSO (9.3 mL, 131 mmol). After 20 mins, a solution of 60 (10.1 g, 26 mmol) in DCM (50 mL) was added dropwise. The mixture was stirred for an additional 2 h. The reaction mixture was then treated with triethylamine (anhydrous, 60 mL) and allowed warm up to rt A saturated aqueous solution of NaHCO3 (200 mL) was added, and the biphasic mixture was extracted with Et2O (3 ´ 150 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (25% acetone/hexane). Ketone 61 was recovered as a white crystalline solid (9.1 g, 91%). Mp 128–129 oC. FTIR (thin film): 3060, 2949, 2838, 1758, 1739 cm–1. 1 H NMR (400 MHz, CDCl3): δ 7.83–7.62 (m, 2H), 7.49–7.40 (m, 3H), 7.44–7.35 (m, 3H), 7.35 –7.16 (m, 5H), 3.76 (d, J = 17.8 Hz, 1H), 3.76 (dd, J = 8.6, 2.9 Hz, 1H), 3.48 (dt, J = 17.8, 1.2 Hz, 1H), 3.20 (s, 3H), 2.44 (dd, J = 18.1, 8.6 Hz, 1H), 2.29 (dd, J = 18.1, 2.9 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 212.9, 173.1, 146.5, 145.3, 141.8, 140.9, 140.3, 128.9, 128.9, 128.6, 128.0, 127.7, 127.6, 127.0, 126.9, 125.5, 120.3, 120.1, 76.0, 58.2, 55.2, 51.5, 51.5, 41.6 ppm. CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 42 HRMS (ESI) m/z (M+H) + calcd. for [C25H21NO3+H]+ 384.1600, found 384.1602. Methyl (2S,3R)-3-[2-tert-butoxy]-2-oxoethyl]-4-oxo-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate (62a) and Methyl (2S,3S)-3-[2-(tert-butoxy)-2-oxoethyl]-4-oxo-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate (62b). Method A. Ketone 10 (6.0 g, 16 mmol) was dissolved in THF (41.0 mL) and anhydrous HMPA (4.1 mL) in a single-neck ask and under an Ar atmosphere. The solution was cooled to – 78 °C on a dry ice/acetone bath, followed by a dropwise addition of n-butyllithium (2.1 M in cyclohexane, 7.8 mL, 16 mmol). After 30 min, tert-butyl iodoacetate (18.9 g, 79 mmol) was added dropwise to the reaction. The solution was allowed to warm up to - 41 °C (dry ice/acetonitrile bath) and stirred for an additional 5.5 h. The reaction was quenched at - 41 °C by a quick addition of phosphoric acid (10% wt, 15 mL) and allowed to warm up to rt. Water (50 mL) was added, and the mixture was extracted with Et2O (3 × 100 mL). The combined organic layers were washed with brine, dried over MgSO4, and ltered. The solvents were evaporated under reduced pressure to yield the alkylated products 11a and 11b. The diastereomeric ratio was determined with 1H NMR of the crude product; dr = 9:1 with ketones 11a as the major isomer. The crude products from Method A were retaken in MeOH (5 mL) and stored at - 20 °C overnight in a freezer. The formed white solid was filtered and washed with cold methanol (5 mL) to yield 11a as a white solid (6.2 g). The mother liquor’s solvents were removed, and the residue was purified by column chromatography on silica gel (15–25% EtOAc/ hexane as elution gradient) to yield 11a (525 mg) and 11b (621 mg, 8%) as a white foam-like solid. Method B. This method was adapted from a reported procedure 14 using BrCH2COOtBu (3.5 equiv)/NaI (0.5 equiv). The alkylated ketones 11a and 11b were recovered with a dr = 5:1 (1H NMR analysis of the crude). trans-Ketodiester 62a (Major, 6.725 g, 86%). FTIR (thin film): 2974, 1758, 1730 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.70 (dd, J = 7.6, 1.0 Hz, 2H), 7.50 (dt, J = 7.6, 1.6 Hz, 3H), 7.43 (dt, J = 7.5, 0.8 Hz, 1H), 7.43–7.33 (m, 2H), 7.32–7.21 (m, 5H), 3.78 (d, J = 17.6 Hz, 1H), 3.45 (d, J = 17.6 Hz, 1H), 3.44 (d, J = 6.1 Hz, 1H), 3.10 (s, 3H), 2.82 (dd, J = 12.4, 6.2 Hz, 1H), 2.45–2.38 (m, 2H), 1.31 (s, 9H) ppm. 13C 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. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C31H32NO5, 498.2280; found, 498.2278. cis-Ketodiester 62b (Minor, 621 mg, 8%). FTIR (thin film): 2974, 1758, 1727 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.76–7.71 (m, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.41 (ddt, J = 9.0, 5.1, 1.6 Hz, 5H), 7.33 (dtd, J = 17.9, 7.4, 1.1 Hz, 3H), 7.24 (dt, J = 7.0, 1.1 Hz, 3H), 4.02 (d, J = 8.2 Hz, 1H), 3.80 (d, J = 17.6 Hz, 1H), 3.58 (dd, J = 17.6, 1.1 Hz, 1H), 3.15 (s, 3H), 3.11–3.01 (m, 1H), 2.57 (dd, J = 17.4, 5.2 Hz, 1H), 1.84 (dd, J = 17.4, 8.9 Hz, 1H), 1.34 (s, 9H) ppm. CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 43 13C 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 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C31H32NO5 , 498.2280; found, 498.2275. Methyl (2S,3R,4S)-3-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxy-1-(9-phenyl-9H-fluoren-9-yl)-4-(prop-1-en-2-yl)pyrrolidine-2-carboxylate (63a). A single-neck flask was charged with magnesium turnings (322 mg, 13.1 mmol) and dry THF (20 mL) under an argon atmosphere, followed by the dropwise addition of 2-bromopropene (1.10 mL, 12.4 mmol) at rt. The suspension was stirred for 2 h and gradually turned to a pale-yellow solution. The resulting isopropenyl magnesium bromide solution was titrated using salicylaldehyde phenylhydrazone. To a single-neck flask was added ketone 11a (4.105 g, 8.25 mmol), purged with argon, and a CeCl3×2LiCl solution in THF (107 mL, 0.1 M) was added via syringe at rt. The clear solution was cooled to –78 °C and stirred for 15 min. The i-PrMgBr solution (19.5 mL, 0.55 M) was added dropwise at –78 °C. After 2 h, the reaction was quenched at – 78 °C with a diluted 0.1 M HCl aqueous solution (10 mL). The biphasic mixture was extracted with Et2O (2 × 100 mL), washed with brine, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure to afford tertiary alcohol 63a as a white foamlike solid (4.305 g, 98%), which was used without further purification. FTIR (thin film): 3510, 3060, 2978, 2949, 2885, 1715, 1706 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 7.6 Hz, 1H), 7.67–7.50 (m, 4H), 7.44 (t, J = 7.5 Hz, 1H), 7.39–7.28 (m, 5H), 7.23–7.09 (m, 2H), 5.13 (s, 1H), 4.91 (s, 1H), 3.72 (d, J = 12.1 Hz, 1H), 3.18 (s, 3H), 3.07 (d, J = 3.9 Hz, 1H), 3.06 (d, J = 17.7 Hz, 1H), 2.85 (dt, J = 9.7, 6.2 Hz, 1H), 2.22 (dd, J = 16.3, 5.4 Hz, 1H), 1.99 (dd, J = 16.3, 6.7 Hz, 1H), 1.82 (s, 3H), 1.78 (s, 1H), 1.30 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 174.9, 171.5, 146.8, 146.4, 144.6, 142.8, 141.7, 139.7, 129.0, 128.4, 128.3, 127.9, 127.7, 127.4, 127.3, 127.0, 126.2, 120.1, 119.7, 113.0, 81.9, 80.6, 66.3, 61.2, 51.3, 47.0, 32.3, 29.7, 27.9, 19.6 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C34H38NO5, 540.2744; found, 540.2730. Methyl (2S,3R,4S)-3-(2-(tert-Butoxy)-2-oxoethyl)-4-hydroxy-4phenyl-1-(9-phenyl-9H-fluoren-9-yl) pyrrolidine-2-carboxylate (63b). This compound proceeded as described for 12a with the following modifications: 62a (2.530 g, 5.08 mmol), phenyl magnesium bromide (12 mL, 1.1 M), and CeCl3×2LiCl (127 mL, 0.1 M). The crude product was purified by silica gel chromatography with ethyl acetate/hexanes as eluent (15–25% gradient). Alcohol 63b was recovered as a white foam-like solid (2.780 g, 95%). FTIR (thin film): 3506, 3059, 2977, 2946, 2879, 1729, 742, 704 cm–1. CeCl3独2LiCl, RMgBrN CO2MePfCO2tBuHOR63a : R = 2-propenyl, 98%63b : R = Ph, 95%N CO2MePfCO2tBuORi. LiHMDS, THF, -78 oC;OMeONOCO2MePfCO2tBu64a: R = 2-propenyl, 96%64b: R = Ph, 95%ii. ClCO2MeTHF, –78 oC62aCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 44 1H NMR (400 MHz, CDCl3): δ 7.75 (ddd, J = 7.5, 1.2, 0.6 Hz, 1H), 7.67–7.62 (m, 2H), 7.59–7.53 (m, 4H), 7.45 (dt, J = 7.5, 1.1 Hz, 1H), 7.40–7.36 (m, 2H), 7.35–7.28 (m, 4H), 7.25–7.24 (m, 1H), 7.24–7.22 (m, 1H), 7.22–7.19 (m, 1H), 7.19–7.16 (m, 1H), 3.86 (d, J = 12.1 Hz, 1H), 3.35 (d, J = 12.1 Hz, 1H), 3.22 (d, J = 12.0 Hz), 3.20 (s, 3H), 3.09–3.01 (m, 1H), 2.20 (dd, J = 16.3, 5.6 Hz, 1H), 2.08 (dd, J = 16.3, 6.7 Hz, 1H), 1.18 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 174.9, 171.4, 146.7, 146.5, 142.8, 141.8, 141.6, 139.8, 129.0, 128.5, 128.4, 128.2, 127.9, 127.8, 127.4, 127.3, 127.1, 127.1, 126.1, 125.6, 120.2, 119.8, 81.1, 80.7, 66.4, 65.3, 51.4, 51.1, 32.5, 27.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C37H38NO5, 576.2744; found, 576.2738. Methyl (2S,3R,4S)-3-(2-(tert-butoxy)-2-oxoethyl)-4-((methoxycarbonyl)oxy)-1-(9-phenyl-9H-fluoren-9-yl)-4-(prop-1-en-2-yl)pyrrolidine-2-carboxylate (64a). Allylic alcohol 63a (3.8 g, 7.0 mmol) was dissolved in THF (40 mL) under an argon atmosphere, cooled to –78 °C. Lithium bis(trimethylsilyl)-amide solution (8.5 mL, 1.0 M) was added dropwise, and the reaction was stirred for 20 min. Methyl chloroformate (1.6 mL, 21.1 mmol) was added slowly, and the solution was allowed to react for 2 h. The reaction was quenched at – 78 °C with a saturated ammonium chloride aqueous solution (20 mL) and extracted with Et2O (2 × 100 mL). The combined organic layer was washed with brine, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure to obtain carbonate 13a as a white foam-like solid. (4.1 g, 95%). Purity of the crude product was excellent, and 64a was used as is for the next step. FTIR (thin film): 2978, 2950, 1753, 1731 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.70 (d, J = 7.5 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.57–7.49 (m, 2H), 7.44 (d, J = 7.6 Hz, 1H), 7.41–7.34 (m, 2H), 7.33–7.26 (m, 1H), 7.24–7.09 (m, 5H), 4.96 (s, 1H), 4.87 (s, 1H), 3.98 (d, J = 12.9 Hz, 1H), 3.68 (s, 3H), 3.65–3.68 (m, 1H), 3.18 (d, J = 8.5 Hz, 1H), 3.15 (s, 3H), 2.80 (dt, J = 8.1, 4.7 Hz, 1H), 2.42 (dd, J = 16.8, 4.8 Hz, 1H), 2.07 (dd, J = 16.9, 7.8 Hz, 1H), 1.90 (s, 3H), 1.31 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 174.0, 170.8, 153.6, 146.2, 146.1, 142.5, 141.9, 141.1, 140.1, 128.6, 128.4, 128.3, 128.1, 127.4, 127.4, 127.3, 126.9, 126.0, 120.0, 119.7, 90.1, 80.5, 66.0, 55.6, 54.4, 51.3, 48.2, 32.5, 27.9, 20.4 ppm. HRMS (ESI-TOF) m/z: [M + H] + calcd for C36H40NO7, 598.2805; found, 598.2797. Methyl (2S,3R,4S)-3-(2-(tert-butoxy)-2-oxoethyl)-4((methoxycarbonyl)oxy)-4-phenyl-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate (64b). This compound proceeded as described for 64a. Allylic alcohol (450 mg, 0.782 mmol) afforded carbonate 64b (472 mg, 95%) as a foam-like solid. FTIR (thin film): 3060, 2977, 2952, 1749, 1733 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.72 (ddd, J = 7.6, 1.1, 0.6 Hz, 1H), 7.65–7.58 (m, 3H), 7.52–7.48 (m, 1H), 7.45–7.30 (m, 7H), 7.30–7.24 (m, 4H), 7.24–7.15 (m, 2H), 4.34 (d, J = 13.0 Hz, 1H), 3.92 (d, J = 13.0 Hz, 1H), 3.67 (s, 3H), 3.28 (d, J = 8.2 Hz, 1H), 3.12 (s, 3H), 2.87 (dt, J = 8.0, 5.0 Hz, 1H), 2.38 (dd, J = 16.8, 5.0 Hz, 1H), 2.13 (dd, J = 16.8, 7.9 Hz, 1H), 1.22 (s, 9H) ppm. CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 45 13C NMR (101 MHz, CDCl3): δ 173.8, 170.3, 153.7, 146.2, 146.0, 142.4, 141.3, 140.1, 139.2, 128.7, 128.5, 128.4, 128.4, 128.1, 127.6, 127.5, 127.4, 127.3, 126.9, 126.1, 125.0, 120.1, 119.7, 89.5, 80.4, 66.4, 57.8, 54.5, 52.7, 51.3, 32.5, 27.9 ppm. HRMS (ESI) m/z: [M + H]+ calcd for C39H40 NO7 , 634.2799; found, 634.2802. Methyl (2S,3S,4R)-3-(2-(tert-butoxy)-2-oxoethyl)-1-(9-phenyl-9H-fluoren-9-yl)-4-(prop-1-en-2-yl)pyrrolidine-2-carboxyl-ate (65a) and Methyl (2S,3S)-3-(2-(tert-butoxy)-2-oxoethyl)-1-(9-phenyl-9H-fluoren-9-yl)-4-(propan-2-ylidene)pyrrolidine-2-carboxylate (65b). Allylic carbonate 13a (621 mg, 1.36 mmol), palladium acetate (15 mg, 0.066 mmol), triphenyl phosphine (71 mg, 0.27 mmol), ammonium formate (427 mg, 6.78 mmol), and THF (10 mL) were charged into a single-neck flask equipped with a condenser. The flask was purged with three vacuum/argon cycles. The reaction vessel was heated at 60 °C with stirring until full conversion was observed by TLC (∼5 h). The reaction mixture was filtered on a Celite pad and washed with Et2O (20 mL). The filtrate was concentrated under reduced pressure to a brown oil. The crude oil was purified by silica gel chromatography with methyl tert-butyl ether/hexane as an eluent (15–25% gradient). The reduced diesters were recovered as white foam-like solids: 14a (263 mg, 51%) and 14b (82 mg, 16%). Diester 65a: FTIR (thin film): 3059, 2977, 2930, 2855, 1728 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.74 (dd, J = 7.6, 1.1 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.587.48 (m, 3H), 7.44 (dt, J = 7.5, 1.1 Hz, 1H), 7.37–7.27 (m, 2H), 7.25–7.17 (m, 2H), 7.11 (dt, J = 7.5, 1.1 Hz, 1H), 4.75 (s, 2H), 3.43 (t, J = 11.1 Hz, 1H), 3.38–3.29 (m, 1H), 3.22 (s, 3H), 2.73–2.67 (m, 1H), 2.60 (dddd, J = 14.3, 8.9, 7.0, 5.2 Hz, 1H), 2.27 (dt, J = 11.0, 7.9 Hz, 1H), 2.07 (dd, J = 16.1, 5.2 Hz, 1H), 1.96 (dd, J = 16.1, 7.0 Hz, 1H), 1.69 (s, 3H), 1.29 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 175.4, 170.7, 147.6, 146.8, 143.9, 142.7, 142.1, 139.3, 128.7, 128.3, 128.2, 128.0, 127.3, 127.2, 127.2, 127.1, 125.7, 120.0, 119.7, 113.5, 80.3, 67.5, 55.1, 52.2, 51.3, 43.9, 37.3, 27.9, 19.0 ppm. HRMS (ESI) m/z: [M + H]+ calcd for C34H38NO4 , 524.2801; found, 524.2805. Diester 65b: FTIR (thin film): 3061, 2978, 2934, 2867, 1732 cm–1. N CO2MePfCO2tBuOOMeO Pd(OAc)2, Ph3P, NH4HCO2THF, 60 oC N CO2MePfCO2tBuN CO2MePfCO2tBu+64a 65a: 51% 65b: 16%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 46 1H NMR (400 MHz, CDCl3): δ 7.66 (dd, J = 12.5, 7.5 Hz, 2H), 7.53–7.42 (m, 3H), 7.42–7.27 (m, 4H), 7.25–7.16 (m, 5H), 4.01 (d, J = 12.5 Hz, 1H), 3.76 (d, J = 12.5 Hz, 1H), 3.38–3.25 (m, 1H), 3.02 (s, 3H), 2.93–2.81 (m, 1H), 2.44–2.23 (m, 2H), 1.62 (s, 3H), 1.57 (s, 3H), 1.33 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 173.9, 171.2, 147.8, 146.1, 142.7, 140.9, 139.8, 133.2, 128.4, 128.3, 128.2, 127.7, 127.4, 127.3, 127.1, 126.9, 125.6, 123.4, 119.9, 119.7, 80.6, 65.0, 50.7, 49.2, 43.0, 40.1, 27.9, 21.3, 20.4 ppm. HRMS (ESI) m/z: [M + H]+ calcd for C34H38NO4 , 524.2801; found, 524.2809. allo-Kainic Acid (6). N-Pf protected diester 65a (103 mg, 0.197 mmol) was dissolved in dichloromethane (1 mL) at rt under argon. Triethyl silane (0.30 mL, 2.0 mmol) was added, followed by trifluoroacetic acid (0.19 mL, 3.0 mmol). The reaction was stirred at rt for 3 h, at which point a second portion of triethyl silane (0.63 mL, 3.9 mmol) and trifluoroacetic acid (0.39 mL, 5.9 mmol) was added. TLC showed full deprotection of the N-Pf group after an additional 4 h. The reaction was evaporated to dryness under reduced pressure. The residue was retaken in methanol (3 mL), a LiOH aqueous solution (1 mL, 2.5 M) was added, and the reaction was stirred at rt overnight. The reaction was then neutralized at 0 °C with a 1.0 M HCl aqueous solution, and the mixture was diluted with water (20 mL). The Pf-H byproduct was removed by extraction with petroleum ether (2 × 50 mL). The aqueous layer was concentrated to dryness under a high vacuum. The residue was retaken in water (5 mL) and purified by ion-exchange chromatography: ion-exchange resin Dowex 50WX4 100200, eluting with 0.5 N aqueous ammonia. The eluting fractions were collected and analyzed by TLC for the presence of the desired product. (TLC plates were dried gently with a heat gun before being stained with ninhydrin; further heating revealed the presence of the amino acid 2 as yellow spots.) The fractions containing the product were combined and flash frozen, and the solvents were removed by lyophilization to yield a pale-yellow solid. This product was recrystallized with aqueous ethanol. The product was dissolved with minimal water, followed by the dropwise addition of ethanol until the crystal formed. It was then left standing at 0 °C, and allo-kainic acid was recovered after trituration as a white crystalline solid (33 mg, 78%). Mp 239–242 °C. FTIR (thin film): 3451, 2971, 1578 cm–1. 1H NMR (400 MHz, D2O): δ 4.95 (bs, 1H), 4.94 (m, 1H), 3.92 (d, J = 8.1 Hz, 1H), 3.51 (dd, J = 11.8, 7.8 Hz, 1H), 3.33 (dd, J = 11.8, 10.6 Hz, 1H), 2.86 (dt, J = 10.5, 8.5 Hz, 1H), 2.72–2.59 (m, 2H), 2.41–2.32 (m, 1H), 1.73 (bs, 3H) ppm. 13C NMR (101 MHz, D2O): δ 179.3, 173.7, 140.6, 114.6, 64.9, 51.5, 48.2, 42.6, 39.7, 17.7 ppm. HRMS (ESI-TOF): m/z: [M + H]+ calcd for C10H16NO4 , 214.1074; found, 214.1074. 1. EtSiH, TFA, DCM2. LiOH aq, CH3OH NHCO2HCO2HN CO2MePfCO2tBu78%65a 6CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 47 iso–Kainic Acid (55). The compound proceeded as described for 2. Diester 65b (50 mg, 0.95 mmol) was fully deprotected to afford iso–kainic 55 acid as a white crystalline solid (16 mg, 79%). Mp 241–244 °C. FTIR (thin film): 3421, 3182, 2920, 1732, 1554 cm–1. 1H NMR (400 MHz, D2O): δ 4.16 (d, J = 1.6 Hz, 1H), 4.07 (d, J = 14.6, 1H), 3.96 (dd, J = 14.6, 1.9 Hz, 1H), 3.55 (t, J = 6.9 Hz, 1H), 2.51 (dd, J = 14.7, 6.9 Hz, 1H), 2.44 (dd, J = 14.6, 6.9 Hz, 1H), 1.74 (bs, 3H), 1.65 (bs, 3H) ppm. 13C NMR (101 MHz, D2O): δ 179.7, 173.7, 129.8, 125.7, 66.4, 46.7, 42.1, 41.2, 20.4, 20.3 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C10H16NO4, 214.1074; found, 214.1075. 1-(tert-butyl)-2-methyl-(2S,3R,4S)-3-(2-(tert-butoxy)-2-oxo-ethyl)4-((methoxycarbonyl)oxy)-4-(prop-1-en-2-yl)pyrrolidine-1,2-dicarboxylate (66a). N-Pf protected allylic carbonate 64a (3.010 g, 5.04 mmol) was dissolved in dichloromethane (50 mL) at rt under argon. Triethyl silane (2.4 mL, 15 mmol) was added, followed by the dropwise addition of trifluoroacetic acid (1.9 mL, 25 mmol). The reaction was stirred at rt for 1 h. Solvents were evaporated in vacuo; the TFA ammonium salt was recovered as a light-yellow waxy solid. The residue was retaken in dichloromethane (50 mL). Di-tert-butyl dicarbonate (1.8 g, 8.1 mmol) and 4-dimethylaminopyridine (1.5 g, 10 mmol) were added sequentially, and the reaction was allowed to stir at rt overnight. The reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by column chromatography using ethyl acetate/hexanes as an eluent (15–25% gradient). N-Boc carbonate 66a was recovered as a colorless oil (2.226 g, 97%). FTIR (thin film): 2979, 1758, 1730, 1707 cm–1. 1H NMR (400 MHz, CDCl3, two rotamers): δ 5.03 (s, 1H), 4.88 (s, 1H), 4.44 (d, J = 13.2 Hz, 1H), 4.00 (dd, J = 26.5, 9.6 Hz, 1H), 3.87–3.79 (m, 2H), 3.71 (s, 3H), 3.68 (s, 3H), 2.84–2.73 (m, 1H), 2.62 (dd, J = 17.5, 4.3 Hz, 1H), 2.39 (ddd, J = 17.6, 14.9, 7.6 Hz, 1H), 1.81 (s, 3H), 1.46–1.29 (m, 19H) ppm. 13C NMR (101 MHz, CDCl3, two rotamers): δ 172.5, 172.4, 170.7, 170.6, 154.2, 153.5, 153.4, 153.3, 139.5, 139.4, 114.1, 90.5, 89.7, 81.0, 80.7, 63.5, 63.0, 54.8, 54.7, 52.5, 52.3, 52.2, 52.1, 47.2, 46.6, 31.9, 31.6, 28.3, 28.1, 20.5, 20.4 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C22H35NO9Na, 480.2204; found, 480.2221 NHCO2HCO2HN CO2MePfCO2tBu 1. EtSiH, TFA, DCM2. LiOH aq, CH3OH79%65b 55N CO2MePfCO2tBuOOMeO i. EtSiH, TFA, DCMii. Boc2O, DMAP, DCM N CO2MeBocCO2tBuOOMeO64a 66a96%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 48 (2S,3R,4S)-1-(tert-Butyl)-2-methyl-3-(2-(tert-butoxy)-2-oxoethyl)4-((methoxycarbonyl)oxy)-4-phenyl-pyrrolidine-1,2-dicarboxylate (66b). This compound proceeded as described for 66a. Allylic carbonate 64b (451 mg, 0.712 mmol) afforded N-Boc carbonate 66b as a colorless liquid (331 mg, 95%). FTIR (thin film): 2974, 1755, 1727, 1705 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.41–7.35 (m, 2H), 7.34–7.28 (m, 3H), 4.66 (d, J = 13.3 Hz, 1H), 4.27 (t, J = 13.0 Hz, 1H), 4.16 (dd, J = 23.4, 9.5 Hz, 1H), 3.75 (s, 3H), 3.70 (s, 3H), 2.89 (ddd, J = 9.6, 7.8, 4.3 Hz, 1H), 2.61–2.43 (m, 2H), 1.45 (s, 9H), 1.31 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 172.3, 172.2, 170.1, 170.0, 154.1, 153.5, 153.4, 153.3, 136.2, 128.8, 128.8, 128.2, 128.2, 124.7, 124.7, 90.0, 89.3, 80.9, 80.9, 80.7, 80.6, 64.0, 63.5, 54.8, 54.7, 53.9, 53.6, 52.4, 52.2, 52.0, 51.4, 31.7, 31.4, 30.9, 28.3, 28.2, 28.0, 27.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C25H36NO9, 494.2385; found, 494.2408. 1-(tert-Butyl)-2-methyl-(2S,3S,4R)-3-(2-(tert-butoxy)-2-oxoethyl)4-(prop-1-en-2-yl)pyrrolidine-1,2-dicarboxylate (67a) and 1-(tert-Butyl) 2-methyl (2S,3S,4S)-3-(2-(tert-butoxy)-2-oxoethyl)-4-(prop1-en-2-yl)pyrrolidine-1,2-dicarboxy-late (67b). These compounds proceeded as described for 14a and 14b. Allylic carbonate 66a (2.010 g, 4.84 mmol) afforded a crude oil, which was purified by silica gel chromatography with ethyl acetate/hexane as an eluent (15–25% gradient). Diesters 67a and 67b were recovered as an inseparable mixture: colorless oil (1.592 g, 96%). The combined diastereomers were used as is for the next step. HRMS (ESI) m/z: [M + Na]+ calcd for C20H33NO6Na, 406.2200; found, 406.2232. Kainic Acid (1) and allo-kainic Acid (6). The diastereomeric mixture of 67a and 67b (605 mg, 1.46 mmol) was dissolved in methanol (20 mL), and a LiOH aqueous solution (2.5 N, 9.4 mL) was added. The mixture N CO2MePfCO2tBuOPhOMeO i. EtSiH, TFA, DCMii. Boc2O, DMAP, DCM N CO2MeBocCO2tBuOPhOMeO64b 66b95%N CO2MeBocCO2tBuOOMeO Pd(OAc)2, Ph3P, NH4HCO2THF, 60 oC N CO2MeBocCO2tBuN CO2MeBocCO2tBu+66a 67a, 50% 67b, 46%1. LiOH aq, CH3OH2. TFA, DCM NHCO2HCO2HNHCO2HCO2H+N CO2MeBocCO2tBuN CO2MeBocCO2tBu+81% 1 6dr = 1 : 1.367a 67bCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 49 was stirred at rt for 5 h. The reaction was neutralized at 0 °C with a diluted 1.0 N HCl aqueous solution and extracted with ethyl acetate (3 × 50 mL). The combined organic layer was dried over MgSO4 and filtered. The solvents were evaporated under reduced pressure to obtain a colorless oil. The crude oil was retaken in dichloromethane (5.0 mL) under an argon atmosphere, and triuoroacetic acid (3.0 mL) was added at rt. The solution was stirred for 3 h and then was concentrated to dryness under reduced pressure to afford a light-yellow soild. This crude product was purified and recrystallized in methanol and water. The product was recovered as a white powder (273 mg, 81%) and consisted of kainic acid (1, 43%) and allo-kainic acid (6, 57%), as determined by 1H NMR. The mixture of diastereomers proved to be inseparable with HPLC. The following data are for kainic acid (1). 1H NMR (400 MHz, D2O): δ 5.08 (bs, 1H), 4.78 (bs, 1H), 4.18 (d, J = 3.5 Hz, 1H), 3.67 (dd, J = 11.9, 7.3 Hz, 1H), 3.46 (t, J = 11.4 Hz, 1H), 3.18–3.00 (m, 2H), 2.53 (dd, J = 16.9, 6.3 Hz, 1H), 2.44 (dd, J = 16.8, 8.2 Hz, 1H), 1.78 (s, 3H) ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C10H16NO4, 214.1074; found, 214.1076. MS analysis was conducted with the mixture of 1 and 6. (3R,4S,6S)-4-Carboxy-2-oxo-6a-phenyl-5-(2,2,2-trifluoro-acetyl)hexahydro-2H-furo[2,3-c]pyrrol-5-ium (68). Benzylic carbonate 64b (205 mg, 0.356 mmol) was dissolved in methanol (5 mL); a LiOH aqueous solution (1 mL, 2.5 N) was added at rt, and the mixture was stirred for 6 h. The reaction was neutralized at 0 °C with a diluted 1.0 N HCl aqueous solution and extracted with ethyl acetate (3 × 10 mL). The combined organic layer was dried over MgSO4 and filtered. The solvents were evaporated under reduced pressure. This residue was retaken in dichloromethane (5 mL) under an argon atmosphere, trifluoroacetic acid (1 mL) was added, and the solution was stirred at rt for 3 h. The reaction mixture was concentrated to dryness under reduced pressure to obtain a light-yellow solid. This crude product was triturated with acetone (5 mL) and afforded lactone 68 as a white powder (90 mg, 90%). The solubility of lactone 68 is extremely poor in either H2O, acetone, chloroform, or DMSO. FTIR (thin film): 3521, 3017, 2983, 1783, 1605 cm–1. 1H NMR (400 MHz, D2O): δ 7.59–7.46 (m, 5H), 4.36 (d, J = 4.8 Hz, 1H), 4.14 (d, J = 13.7 Hz, 1H), 3.92 (d, J = 13.7 Hz, 1H), 3.76–3.64 (m, 1H), 3.21 (dd, J = 19.1, 8.7 Hz, 1H), 2.98 (d, J = 19.2 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 175.5, 171.6, 139.5, 128.7, 128.2, 124.7, 95.0, 67.6, 58.1, 49.5, 34.7 ppm. 19F NMR (376 MHz, DMSO-d6): δ –73.45 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C13H14NO4, 248.0917; found, 248.0912. NHCO2HOOPh1. LiOH aq, CH3OH2. TFA, DCM独TFAN CO2MePfCO2tBuOPhOMeO64b 6890%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 50 (3R,4S,6S)-4-(Methoxycarbonyl)-2-oxo-6a-phenyl-5-(2,2,2-trifluoroacetyl)hexahydro-2H-furo [2,3-c]pyrrol-5-ium (69a). To a stirred solution of allylic alcohol 63b (2.268 g, 3.94 mmol) in dichloromethane (25 mL) was added trifluoroacetic acid (9.2 mL, 0.12 mol). After 3 h, TLC showed the full conversion. The reaction mixture was concentrated to dryness under reduced pressure. The residue was triturated in methanol (20 mL), and the insoluble byproduct (PfOH) was removed by filtration. The filtrate was concentrated to afford the trifluoroacetate salt of 69a as a pale-yellow oil (1.465 g, 99%), which was used as is for the next step. FTIR (thin film): 2955, 2914, 2847, 1777, 1733 cm–1. 1H NMR (400 MHz, CD3OD): δ 7.27–7.21 (m, 4H), 7.21–7.15 (m, 1H), 4.50 (d, J = 6.6 Hz, 1H), 3.86 (d, J = 13.5 Hz, 1H), 3.69 (d, J = 13.5 Hz, 1H), 3.69 (s, 3H), 3.48 (ddd, J = 8.3, 7.1, 1.9 Hz 1H), 2.74 (dd, J = 18.7, 7.5 Hz, 1H), 2.67 (dd, J = 18.7, 2.0 Hz, 1H) ppm. 13C NMR (101 MHz, CD3OD): δ 175.0, 169.0, 137.3, 130.5, 130.3, 126.0, 94.4, 65.5, 57.6, 54.4, 50.4, 49.6, 49.4, 49.2, 49.0, 49.0, 48.6, 48.4, 34.2 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C14H16NO4, 262.1079; found, 262.1070. 5-(tert-Butyl)-4-methyl-(3R,4S,6S)-2-oxo-6a-phenylhexa-hydro-5H-furo[2,3-c]pyrrole-4,5-dicarboxylate (69b). To a solution of 69a (TFA salt, 215 mg, 0.573 mmol) in dichloromethane (2 mL) was added DMAP (7 mg, 0.06 mmol), triethylamine (145 μL, 1.43 mmol), and Boc2O (188 mg, 0.859 mmol). After 6 h, the reaction mixture was concentrated to dryness under reduced pressure. The crude residue was purified by column chromatography using dichloromethane/ methanol as an eluent (10–20% gradient). N-Boc-lactone 69b was recovered as a colorless oil (192 mg, 93%). FTIR (thin film): 2974, 2917, 1793, 1749, 1698 cm–1. 1H NMR (400 MHz, CDCl3, two rotamers): δ 7.41 (m, 7.42–7.40, 4H), 7.39–7.33 (m, 4H), 4.41 (d, J = 4.6 Hz, 0.5H), 4.33–4.25 (m, 4H), 4.25–4.21 (m, 0.5H), 3.89 (dd, J = 13.0, 9.0 Hz, 1H), 3.83 (s, 3H), 3.23–3.17 (m, 1H), 2.90–2.62 (m, 2H), 1.44 and 1.46 (2s, 9H) ppm. 13C NMR (101 MHz, CDCl3, two rotamers): δ 174.2, 172.1, 172.0, 154.0, 153.0, 137.5, 137.2, 129.0, 128.9, 128.8, 124.8, 94.4, 93.4, 81.3, 81.2, 77.4, 77.2, 77.0, 76.7, 65.9, 65.8, 59.2, 58.6, 52.8, 52.6, 51.4, 50.5, 35.1, 34.9, 28.3, 28.2 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C19H23NO6Na, 384.1423; found, 384.1455. NHCO2CH3OOPhN CO2MePfHOPh CO2tBuTFA, DCM独TFA63b 69aBoc2O, Et3NDMAP, DCMN CO2CH3OOPh69bBoc99%93%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 51 (2S,3S,4S)-1-Acetyl-3-(carboxymethyl)-2-(methoxycarbonyl)-4-phenylpyrrolidin-1-ium (70a). To a solution of 69a (TFA salt, 1.126 g, 2.97 mmol) in methanol (30 mL) were added acetic acid (0.71 mL) and Pd/C (56 mg). The reaction mixture was stirred under a hydrogen atmosphere (balloon) for 24 h. The resulting reaction mixture was filtered over a Celite pad. The filtrate was evaporated to dryness under reduced pressure and then a high vacuum to afford the acetate salt of 70a as a pale-yellow powder (956 mg, 99%). FTIR (thin film): 3390, 2521, 1739, 1673, 1635 cm–1. 1H NMR (400 MHz, CD3OD): δ 7.42–7.31 (m, 4H), 7.29–7.22 (m, 2H), 4.59 (d, J = 6.2 Hz, 1H), 4.01–3.93 (m, 1H), 3.91 (s, 3H), 3.88–3.82 (m, 1H), 3.84–3.75 (m, 1H), 3.30–3.19 (m, 1H), 2.33 (ddd, J = 17.0, 7.8, 1.2 Hz, 1H), 2.22 (dd, J = 16.9, 6.1 Hz, 1H), 1.98 (s, 3H) ppm. 13C NMR (101 MHz, CD3OD): δ 173.8, 172.9, 168.9, 134.4, 128.6, 128.2, 127.7, 68.2, 58.1, 53.4, 53.1, 48.2, 48.0, 47.8, 47.6, 47.4, 47.2, 46.9, 43.9, 42.5, 33.0 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C14H18NO4, 264.1236; found, 264.1257. 2-((2S,3S,4S)-1-(tert-Butoxycarbonyl)-2-(methoxycarbonyl)-4-phenylpyrrolidin-3-yl) acetic acid (70b). To a solution of lactone 69b (175 mg, 0.484 mmol) in methanol (20 mL) was added Pd/C (25 mg). The reaction mixture was stirred under a hydrogen atmosphere (balloon) for 24 h. The resulting reaction solution was filtered over a celite pad. The filtrate was concentrated to dryness to under reduced pressure and afford 70b as a colorless oil (174 mg, 99%). FTIR (thin film): 2977, 2917, 1742, 1698 cm–1. 1H NMR (400 MHz, CDCl3, two rotamers): δ 7.33–7.27 (m, 3H), 7.09–7.05 (m, 2H), 4.14 (d, J = 6.6 Hz, 0.4H), 4.02 (d, J = 6.7 Hz, 0.6H), 3.94 (ddd, J = 15.7, 10.9, 6.9 Hz, 1H), 3.84 (dd, J = 11.0, 4.7 Hz, 0.6H), 3.77 (d, J = 3.1 Hz, 3H), 3.74–3.73 (m, 0.4H), 3.71–3.65 (m, 1H), 2.99 (dt, J = 10.7, 6.9 Hz, 1H), 2.30 (ddd, J = 17.4, 14.7, 7.1 Hz, 1H), 2.14–2.02 (m, 1H), 1.45 and 1.49 (2s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 176.8, 172.8, 172.5, 154.3, 153.5, 138.5, 138.4, 128.9, 127.7, 127.7, 127.4, 80.7, 80.6, 77.3, 77.0, 76.7, 63.3, 63.0, 52.5, 52.3, 50.8, 50.4, 45.2, 44.3, 44.1, 43.3, 33.1, 28.4, 28.3 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C19H25NO6Na, 386.1580; found, 386.1556. NHCO2CH3OOPhPd/C, H2CH3OH/AcOH NHCO2CH3Ph CO2H·AcOH99%·TFA69a 70aN CO2CH3OOPhBocPd/C, H2CH3OH/AcOH N CO2CH3Ph CO2HBoc69b 70b99%CHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 52 Phenyl kainic acid (4). Synthetic Route from 68. To a solution of lactone 68 (50 mg, 0.21 mmol), acetic acid (1 mL), and methanol (5 mL) was added Pd/C (5 mg). This reaction mixture was stirred under a hydrogen atmosphere (balloon) for 24 h. The resulting reaction mixture was filtered over a Celite pad, and the ltrate was concentrated to dryness under reduced pressure. The residual acetic acid was removed under a high vacuum. The crude product was purified and recrystallized as described above for allo-kainic acid 6. Phenylkainic acid (32a) was recovered as a white crystalline solid (45 mg, 90%). Synthetic Route from 70a. To a solution of methyl ester 70a (386 mg, 1.21 mmol) in methanol (15 mL) was added a LiOH aqueous solution (8 mL, 2.5 N). Full conversion was observed after 4 h. The reaction was neutralized at 0 °C with 1.0 N aqueous HCl. The solvents were evaporated under a high vacuum. The resulting residue was purified and recrystallized as described above for 1. Phenylkainic acid (32a) was recovered as a white crystalline solid (256 mg, 86%). Mp 255–257 °C. FTIR (thin film): 3429, 3185, 3042, 2920, 1736 cm–1. 1H NMR (400 MHz, D2O): δ 7.49–7.34 (m, 3H), 7.27–7.18 (m, 2H), 4.05 (d, J = 7.3 Hz, 1H), 3.94 (dd, J = 11.4, 7.8 Hz, 1H), 3.87 (q, J = 7.8 Hz, 1H), 3.72 (dd, J = 11.4, 8.1 Hz, 1H), 3.15 (q, J = 7.2 Hz, 1H), 2.38 (dd, J = 16.3, 6.5 Hz, 1H), 2.02 (dd, J = 16.3, 8.7 Hz, 1H) ppm. 13C NMR (101 MHz, D2O): δ 178.5, 173.3, 136.2, 128.8, 128.2, 127.6, 65.1, 48.0, 44.8, 44.0, 35.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C13H16NO4 , 250.1074; found, 250.1086. Methyl (2S,3S,4S)-3-(2-methoxy-2-oxoethyl)-1-((4-nitro-phenyl)sulfonyl)-4-phenylpyrrolidine-2-carboxylate (70a). To a solution of acid 70a (acetate salt, 696 mg, 2.15 mmol) in methanol (5.0 mL) was added TMSCl (690 μL, 5.39 mmol). The solution was stirred at rt for 6 h. The reaction mixture was concentrated to dryness to afford the methyl ester as a white solid. This residue was retaken in dichloromethane (10 mL), and trimethylamine (746 μL, 5.39 mmol) was added, followed by DMAP (26 mg, NHCO2HOOPh独TFA68NHCO2HPh CO2H32aPd/C, H2CH3OH/AcOH90%LiOH aq, CH3OHNHCO2CH3Ph CO2H·AcOH70a86%1. TMSCl, CH3OH2. p-NsCl, DMAP, Et3N, DCMN CO2CH3Ph CO2CH3NsNHCO2CH3Ph CO2H·AcOH70a 96% 71aCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 53 0.22 mmol) and p- NsCl (716 mg, 3.23 mmol). The reaction was stirred at rt overnight. The reaction concentrated to dryness. The resulting residue was purified by column chromatography using ethyl acetate/hexane as an eluent (10–30% gradient). N-Ns-protected diester 71a was recovered as a white foam-like solid (953 mg, 96%). FTIR (thin film): 3100, 2952, 1736, 1527 cm–1. 1H NMR (400 MHz, CDCl3): δ 8.40 (d, J = 9.0 Hz, 1H), 8.13 (d, J = 9.1 Hz, 1H), 7.35–7.27 (m, 3H), 7.09–6.99 (m, 2H), 4.43 (d, J = 4.5 Hz, 1H), 3.81–3.78 (m, 3H), 3.78 (s, 3H), 3.60 (s, 3H), 3.10–2.99 (m, 1H), 2.02 (d, J = 1.8 Hz, 1H), 2.00 (d, J = 3.3 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 171.6, 171.4, 150.2, 144.5, 136.4, 129.0, 128.8, 127.7, 127.7, 124.2, 77.4, 77.0, 76.7, 65.2, 52.9, 51.9, 50.7, 45.2, 44.4, 32.5 ppm. HRMS (ESI) m/z: [M + H]+ calcd for C21H23N2O8S, 463.1175; found, 463.1174. (2S,3S,4S)-1-(tert-Butyloxycarbonyl)-2-methoxycarbonyl-3-(methoxy-2’-oxoethyl)-4-phenylpyrrolidine (71b). To a solution of acid 70a (acetate salt, 136 mg, 0.421 mmol) in methanol (2.5 mL) was added TMSCl (0.1 mL, 1 mmol) at rt. After 6 h, the reaction was concentrated to dryness to afford the methyl ester a white solid residue. This residue was retaken in dichloromethane (3 mL), and trimethylamine (150 μL, 1.08 mmol) was added, followed by DMAP (5 mg, 0.04 mmol) and Boc2O (138 mg, 0.631 mmol). The white suspension was stirred overnight at rt. The solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography using ethyl acetate/hexane as an eluent (10–20% gradient) to afford Boc-protected diester 71b as a colorless oil (146 mg, 92%). FTIR (thin film): 2917, 2854, 1742, 1691 cm–1. 1H NMR (400 MHz, CDCl3, two rotamers): δ 7.37–7.20 (m, 3H), 7.06 (dd, J = 7.9, 3.6 Hz, 2H), 4.07 (dd, J = 39.4, 6.4 Hz, 1H), 3.99–3.89 (m, 1H), 3.87–3.80 (m, 1H), 3.80–3.75 (m, 3H), 3.69 (q, J = 5.8 Hz, 1H), 3.65–3.58 (m, 3H), 3.01 (h, J = 7.2 Hz, 1H), 2.27 (dt, J = 17.7, 7.2 Hz, 1H), 2.04 (ddd, J = 17.2, 7.8, 4.9 Hz, 1H), 1.43 and 1.45 (2s, 9H) ppm. 13C NMR (101 MHz, CDCl3, two rotamers): δ 172.8, 172.5, 172.1, 172.1, 154.2, 153.5, 138.6, 138.4, 128.8, 127.7, 127.7, 127.3, 80.5, 77.3, 77.0, 76.7, 63.4, 63.1, 52.4, 52.2, 51.8, 51.7, 50.7, 50.2, 45.2, 44.3, 44.3, 43.5, 33.3, 33.2, 28.4, 28.3 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C20H27NO6Na, 400.1736; found, 400.1717. 1. TMSCl, CH3OH2. Boc2O, DMAP, Et3N, DCMN CO2CH3Ph CO2CH3BocNHCO2CH3Ph CO2H·AcOH70a92%71bCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 54 (2S,3S,4S)-1-(tert-Butyloxycarbonyl)-2-methoxycarbonyl-3-(methoxy-2’-oxoethyl)-5-oxo-4-phenylpyrrolidine (72b). N-Boc-protected diester 71b (52 mg, 0.14 mmol) was dissolved in a solvent mixture composed of acetonitrile (0.5 mL), carbon tetrachloride (0.5 mL), and water (1 mL). Sodium metaperiodate (471 mg, 2.20 mmol) was added, followed by ruthenium trichloride monohydrate (2 mg, 0.009 mmol). After 8 h, TLC analysis showed only partial conversion. A second portion of metaperiodate (118 mg, 0.552 mmol) and ruthenium trichloride (1 mg, 0.04 mmol) was added. After an additional 24 h of reaction, the mixture was diluted with ethyl acetate (30 mL). The organic layer was washed with a 0.5 N HCl aqueous solution (5 mL) and brine (20 mL). It was then dried over MgSO4 and filtered. The solvent was removed under reduced pressure. The resulting residue was purified by column chromatography using ethyl acetate/hexane as an eluent (10–30% gradient). N-Boc pyrrolidinone 72b was recovered as a colorless oil (45 mg, 85%). FTIR (thin film): 2950, 2912, 2851, 1796, 1743 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.36–7.29 (m, 3H), 7.17–7.10 (m, 2H), 4.43 (d, J = 3.3 Hz, 1H), 4.22 (d, J = 8.7 Hz, 1H), 3.86 (s, 3H), 3.57 (s, 3H), 3.08 (m, 1H), 2.30 (dd, J = 16.9, 8.8 Hz, 1H), 2.09 (dd, J = 16.9, 6.4 Hz, 1H), 1.53 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3): δ 171.9, 171.3, 170.9, 149.5, 133.2, 129.5, 128.8, 127.9, 84.2, 62.0, 52.9, 51.9, 51.4, 37.0, 34.4, 27.9 ppm. HRMS (ESI) m/z: [M + Na]+ calcd for C20H25NO7Na, 414.1529; found, 414.1492. (2S,3S,4S)-1-((4’-Nitrophenyl)sulfonyl)-2-methoxycarbonyl-3-(methoxy-2’-oxoethyl)-4-phenylpyrrolidine-3-carboxylic acid (72a). This synthesis was proceeded according to the procedure described for 72b and started with 71a (901 mg, 1.95 mmol). The resulting reaction mixture was acidified with a 0.5 N aqueous HCl solution at 0 °C until the pH ∼ 2. The resulting mixture was extracted with ethyl acetate (2 × 20 mL), followed by washing with brine (50 mL). The combined organic layers were then extracted with a saturated aqueous sodium bicarbonate solution (3 × 20 mL), and the desired product was thus extracted to the aqueous layers. The aqueous layers were combined and extracted with diethyl ether (3 × 20 mL) to remove impurities. The resulting aqueous layer was then acidified with a 0.5 N aqueous HCl solution at 0 °C until the pH ∼ 2. It was then reextracted with ethyl acetate (3 × 30 mL) and washed with brine. The organic layer was dried over MgSO4 and concentrated to dryness under reduced pressure. The residue was purified by column chromatography using methanol/dichloromethane as an eluent (5–10% gradient) to afford 72a (652 mg, 78%) as a white foamy solid. N CO2CH3Ph CO2CH3BocN CO2CH3Ph CO2CH3BocONaIO4, RuCl3CH3CN/CCl4/H2O85%71b 72bN CO2CH3Ph CO2CH3NsN CO2CH3HO2C CO2CH3NsNaIO4, RuCl3CH3CN/CCl4/H2O78%71a 72aCHAPTER 3. SYNTHESIS OF KAINOIDS AND C4 DERIVATIVES 55 FTIR (thin film): 2914, 2851, 1723, 1591, 1524 cm-1. 1H NMR (400 MHz, CDCl3): δ 8.37 (d, J = 9.1 Hz, 1H), 8.05 (d, J = 9.1 Hz, 1H), 4.14 (d, J = 7.0 Hz, 1H), 3.81 (s, 3H), 3.79–3.74 (m, 1H), 3.69 (s, 3H), 3.67–3.64 (m, 1H), 3.37–3.32 (m, 1H), 3.06–2.98 (m, 1H), 2.47 (dd, J = 17.0, 7.4 Hz, 1H), 2.36 (dd, J = 17.0, 7.5 Hz, 1H) ppm. 13 C NMR (101 MHz, CDCl3): δ 175.8, 170.9, 170.8, 150.3, 143.3, 128.8, 124.2, 64.6, 53.1, 52.2, 49.2, 44.9, 42.0, 32.3 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C16H19N2O10S, 431.0676; found, 431.0648. Methyl (2S,3S,4R)-4-((6-(7-(diethylamino)-2-oxo-2H-chromene3-carboxamido)hexyl) carbamoyl)-3-(2-methoxy-2-oxoethyl)-1-((4nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (73). To a stirred solution of 72a (26 mg, 0.060 mmol) in dichloromethane (1 mL) were added triethylamine (13 μL, 0.090 mmol), HBTU (25 mg, 0.071 mmol), and the coumarin amine (33 mg, 0.091 mmol). After 1 h, 72a was fully converted to 73. The reaction mixture was poured into water and extracted with diethyl ether (3 × 15 mL). The combined ether layer was then washed with an ammonium chloride aqueous solution, sodium bicarbonate aqueous solution, and brine. The organic layer was then dried over MgSO4, filtered, and concentrated to dryness under reduced pressure. The resulting residue was purified by column chromatography on silica gel (10-35% EtOAc/hexane as an elution gradient) to yield 73 (43 mg, 92%) as a yellow solid. FTIR (thin film): 3452, 3338, 2923, 2851, 1672, 1616, 1581, 1533, 1515 cm–1. 1H NMR (400 MHz, CDCl3): δ 8.83 (t, J = 6.0 Hz, 1H), 8.68 (s, 1H), 8.37 (d, J = 8.8 Hz, 2H), 8.06 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.9 Hz, 1H), 6.66 (dd, J = 9.0, 2.5 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.24 (t, J = 5.6 Hz, 1H), 4.22 (d, J = 6.4 Hz, 1H), 3.74–3.78 (m, 4H), 3.68 (t, J = 4.9 Hz, 1H), 3.65 (s, 3H), 3.50–3.39 (m, 6H), 3.28 (td, J = 7.1, 5.2 Hz, 1H), 3.08 (dt, J = 15.0, 6.7 Hz, 2H), 3.03–2.95 (m, 1H), 2.42 (dd, J = 17.2, 8.2 Hz, 1H), 2.26 (dd, J = 17.2, 6.8 Hz, 1H), 1.60 (t, J = 6.7 Hz, 2H), 1.40 (d, J = 6.3 Hz, 2H), 1.37–1.30 (m, 4H), 1.25 (t, J = 7.1 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3): δ 172.0, 171.3, 169.3, 163.3, 162.9, 152.6, 150.2, 148.1, 143.4, 131.2, 129.0, 124.2, 110.0, 108.4, 96.5, 65.3, 52.9, 52.0, 50.0, 45.8, 45.1, 42.8, 38.7, 38.7, 32.3, 29.3, 28.8, 25.7, 25.6, 12.4 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C36H46N5O12S, 772.2864; found, 772.2876. N CO2CH3HO2C CO2CH3NsO OEt2NOHNO OEt2NOHN NH2Et3N, HBTU, DCMNCO2CH3CO2CH3NsNHO72a 7392% 56 56 Chapter 4 An Affinity-based Fluorescence Probe to Study Kainate Receptors The expression of kainate receptors at the surface of neurons is crucial to healthy brain function.16 Being able to visualize kainate receptors in living cells is currently a limitation in elucidating the mechanisms of neuron communication and their associated pathologies. In this chapter, a molecular fluorescence probe for kainate receptors is synthesized to enable the tracking of kainate receptors in model cells. 4.1 Background In the brain, kainate receptors (KAR) play crucial regulatory roles in excitatory neurotransmission. KARs are concentrated at synapses—interface between two neurons—where they translate a chemical stimulus into ion fluxes, thereby creating electrical impulses. Their density at synaptic sites controls the efficiency of neuronal networks, and yet their surface expression is regulated by neuronal activity.116 The mechanisms that govern this feedback-regulated localization are unclear.117 A major challenge to studying these mechanisms is the technological limitations to localize functional KARs in vivo. Current methods for tracking KARs in living cells rely on recombinant protein constructs like KAR fused to a fluorescent protein (e.g., green fluorescent protein, GFP).116 This technique requires several genetic manipulations: cloning the KAR gene (if not available from other labs), fusing it to a GFP marker, inserting the construct in a transfection vector, then knocking down the native KAR in the cells/animal so that only the construct is the active one. An additional limitation is the size of GFP (25 kDa) compared to KARs (~100 kDa)118 which can disturb: the protein’s folding, its membrane trafficking, or its ion channel activity.119 Alternative approaches with smaller tags, such as SNAP-, HA-, or Halo-tagged KARs have been used to minimize perturbations—but they all CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 57 require genetic modification of the protein, so their application is therefore limited to fully sequenced and cloned receptors.120 An ideal strategy to label KARs would be direct visualization without genetic manipulation. Immunochemistry (labelled antibodies) is the most common method to locate proteins, but it requires fixed cells—a process that kills the cell. Additional limitations include that: an antibody must exist for the targeted protein/animal; antibodies may not be selective;121,122 cell permeability may be an issue.123 Chemical labelling of proteins offers an alternative solution to biological methods.124 Such chemical probes are generally based on a strong protein-ligand affinity to locate the POIs.125 A POI can be labelled by its specific ligand and visualized by a fluorescence tag. The protein labelling event can involve a prolonged residence time of its ligand,126 or a reaction between the POI and its ligand.127,128 I proposed to create an affinity-based fluorescence probe to study KARs in living cells. This method should alleviate drawbacks of conventional biological techniques. This probe is composed of a high affinity ligand, a linker of suitable length, and a fluorophore (Figure 4–1). In this chapter, I hypothesized that a KAR protein can bind a kainoid ligand modified with a cyanin dye and that KARs can be visualized by fluorescence microscopy. I expect that the high selectivity of kainoids for their native protein, KAR, will allow us to label selectively KARs in living cells. In addition, since the chemical probe is added after protein expression and it is small (< 1 kDa), it should allow the study of unperturbed receptors in living cells. This KAR-specific chemical probe should enable a broad scope of cellular applications without genetic manipulation. 4.2 Probe design To ensure that the probe can bind to its receptor, I analyzed the crystal structure of the KAR protein complexed with domoic acid (Figure 4–1a, b). The binding site of domoic acid (DA) in GluK1 and GluK2 is conserved: (1) the protein residues interacting with the amine and carboxylic acid groups of DA are the same, (2) DA sits in the same orientation in both binding pockets, and (3) the C4 side chain extends out of the binding pocket. Therefore, the kainoid probe’s C4 side chain should exhibit binding and activity similar to DA if the modification is appropriate. CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 58 Figure 4–1. Structures of GluK receptors, domoic acid, and the proposed fluorescent probe. (A) Ribbon representation of the crystal structure of GluK2 protein complexed with domoic acid (PDB: 1YAE). (B) Ribbon representation of the crystal structure of GluK1 complexed with domoic acid (PDB: 2PBW). (C) 3D structure of domoic acid generated by CORINA. (D) 3D structure of proposed fluorescent probe 90; generated by CORINA. The elements of domoic acid and 90 are shown in CHNOS colour mode. The protein main chains are shown in wheat tint; residues within 5 Å of DA are shown in pale cyan; images created with Pymol. The proposed fluorescent probe, Sulfo-Cy5-KA (90), is expected to mimic the binding of domoic acid with KARs and will be visualized by microscopy via its bis-sulfonylcyanine fluorescent moiety (Figure 4–1d). The length of the linker between the ligand and sulfo-Cy5 was selected to minimize interference of the fluorophore with the binding pocket of KARs. The fluorescent group sulfo-Cy5 was chosen because it was shown to minimize the amount of false positives due to self-embedding within the lipid bilayer of cells—a common issue with lipophilic fluorophores.129 Moreover, sulfo-Cy5 is excited with wavelengths in the red visible range, and therefore does not damage cells, and allows for deep tissue penetration. 4.3 Probe synthesis The synthesis began with the advanced intermediate 72a, reported in the previous chapter. Acid 72a was reduced to alcohol 85 via the one-pot formation of the activated hydroxybenzotriazole ester NHCO2HCO2HHO2CDomoic acidSulfo-Cy5 KA (90)abC4 side chainC4 side chain4 32cdNN ONHHO3S-O3SNHCO2HCO2HNNNCHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 59 72ab, followed by NaBH4 reduction (Scheme 4–1). While extensive optimization was required for this reduction, only the productive conditions are summarized in Table 4–1. Conditions involving the (efficient) conversion of 72a to carbonate 72aa, only led to low yields when presented to the reducing agent (path a, Entry 1). Alternatively, a method using EDCI and HOBT developed more recently converted 72a to the activated ester 72ab smoothly, followed by a slightly more efficient reduction (path b, Entry 2).130 The NaBH4 reduction step was then modified by pre-activating with water before NaBH4 adding to the activated ester, and it gave almost quantitative conversion (path b, Entry 3). Importantly, no epimerization was observed in this process, and NOE experiments confirmed the C3,4-cis configuration of alcohol 85. Scheme 4–1 Synthesis of kainoid alcohol 85 Table 4–1 Conditions for reduction of acid 72a to alcohol 85 Entry Conditions Yield 1 i. ClCO2Et/Et3N/THF, –10 oC; ii. NaBH4/ MeOH 23% 2 i. EDCI/HOBT/DCM, rt; ii. NaBH4, THF/H2O , 0 oC 52% 3 i. EDCI/HOBT/DCM, rt; ii. NaBH4 aq, THF/H2O, 0 oC 95% Next, alcohol 85 was oxidized to aldehyde 86a with Dess-Martin periodinane (Scheme 4–2). It was necessary to use only 1.0 equivalent of DMP to obtain the desired C3,4-cis single diastereomer 86a without C4 epimerization during the reaction. An excess of DMP (3–6 equiv.) gave the undesired 86b C3,4-trans isomer of 86a, which were inseparable by column chromatography. Epimerization of 86a was also observed in presence of basic aqueous solutions, such as LiOH aq and NaOH aq. NHCO2HHO10 steps47% overall yieldchapter 3N CO2CH3HO2C CO2CH3Ns58 72aN CO2CH3CO2CH3NsHO85N CO2CH3CO2CH3NsH HHNOEHO6%2%85ConditionsClCO2Et, Et 3NEDCI, HOBTN CO2CH3CO2CH3NsOOOON CO2CH3CO2CH3NsOPath aPath b[H][H]72aa72abONNNCHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 60 Scheme 4–2. Synthesis and stereochemistry assignment for aldehyde 87 Aldehyde 86a subsequently underwent a one-carbon homologation to alkyne 87a (Scheme 4–2). Bestmann-Ohira’s diazophosphonate reagent converted the aldehyde into the alkyne under optimized mild conditions. Classical reaction conditions, e.g., (MeO)2P(O)C(N2)COMe; K2CO3; MeOH, only epimerized 86a at C4 instead achieving the desired homologation. I optimized the reaction for milder conditions using NaOCH3 at low temperature, which allowed the key ylide intermediate to form without epimerization.131 Conveniently, the (undesired) diastereomeric mixture of 86a and 86b became separable at the alkyne stage. Alkynes 87a and 87b were essential to provide a characterization reference to assign the C3/C4 configuration unambiguously. NOE evidence supports the C3/C4-cis assignment for 86a and 87a. While NOE experiments aiming to confirm the C3/C4-trans configuration for 87b were inconclusive due to chemical shifts overlap, the 1H NMR spectra followed the reported “assignment rules”: (dHb - dHa)–cis > (dHb - dHa)–trans.113 P OCH3ON2OOCH3NaOCH3, CH3OH, -78 oC to r.t.N CO2CH3CO2CH3Ns77% 87aDMP (1.0 eq), DCMN CO2CH3OHC CO2CH3Ns83%86aN CO2CH3CO2CH3NsH HHNOEHO5%2%86aN CO2CH3OHC CO2CH3Ns86aN CO2CH3CO2CH3NsHO85DMP (3.0 eq), DCM86b79%Inseparable mixtureP OCH3ON2OOCH3NaOCH3, CH3OH, -78 oC to r.t.N CO2CH3CO2CH3Ns87a, 53%N CO2CH3CO2CH3Ns87b, 12%2.3 ғ187bN CO2CH3CO2CH3NsH HHNOE3%1%87aN CO2CH3CO2CH3NsHa HbN CO2CH3CO2CH3NsHa Hb87aδHb - δHa = 0.25 0.10N CO2CH3OHC CO2CH3NsCHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 61 Scheme 4–3. Synthesis of novel alkynylkainic acid 89 and probe 90 Finally, alkyne 87a was subjected to a sequence of global deprotection to yield a novel unnatural kainoid: alkynylkainic acid 89 (Scheme 4–3). The nosyl protecting group was removed with thiophenol and sodium bicarbonate, followed by a mild hydrolysis with LiOH. The modest yield of the bis-ester hydrolysis is partially due to the challenge of visualizing 88 and 89 during the purification process (silica-gel, followed by ion-exchange chromatography). NMR analysis of the crude product showed an otherwise clean conversion. With the obtained alkyne 89 in hand, the targeted fluorescent probe 90 was synthesized through a “click” reaction132 coupling with the dye sulfo-cyanine-5-azide. NHCO2CH3CO2CH3CH3CN, 60 oC NHCO2HCO2HLiOH, CH3OH/H2O59%48%88 89Sulfo-Cyanine5 azideNN ONHHO3S-O3SNHCO2HCO2HNNNCu(OAc)2, TBTASodium ascorbate, t-BuOH/H2O72%PhSH, K2CO390N CO2CH3CO2CH3Ns87aCHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 62 4.4 Preliminary imaging experiments of KAR with the Sulfo-Cy5-KA probe The ultimate purpose of developing a KAR imaging probe is to label endogenous kainate receptors present at the surface of neurons (functional KARs). For our proof-of-concept experiments, I opted to test the imaging probe Sulfo-Cy5-KA in model cells that do not express kainate receptors natively, as a means to control for background signal. Thus, experiments were conducted with the human cell line HEK-293; these cells are robust to culture and do not natively express GluK proteins. Ironically, we had to resort to using genetic manipulations to test our fluorescent probe: we opted for a recombinant form of GluK2 accompanied by enhanced GFP (EGFP) as expression marker. Transfecting HEK-293 cells with recombinant pcDNA3-GRIK2-EGFP plasmids (Figure 4–2) enforces the exogenous expression of GluK2-EGFP. GluK2-positive cells can be identified by the fluorescence of EGFP. Transfected HEK cells were then subjected to probe 90, Sulfo-Cy5-KA, for the fluorescence imaging. Figure 4–2. pcDNA3-GRIK2-EGFP plasmid gene map. The fluorescent protein gene for EGFP is downstream of GRIK2 and employed as a marker for GRIK2 gene expression. A spacer sequence separates GRIK2 and EGFP in this plasmid. The successful expression of GRIK2 in mammalian cells can be identified by the presence of EGFP, but they are not fused. CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 63 Assessing the labelling selectivity of the probe relied on the co-localization of green (EGFP) and red (90) fluorescence signals in the same cells. The colocalization protocol used to evaluate the probe 90 for GluK2 labelling is outlined in Figure 4–3. Briefly: HEK-293 cells were transfected with a plasmid encoding for both GluK2 and EGFP proteins (using calcium phosphate precipitate method); and the cells were allowed to incubate for 24-36 h. Typically, 40-60% of cells displayed positive green fluorescence. On a confocal microscope, a solution of the Sulfo-Cy5-KA probe was added to the cells medium and allowed to incubate for a given amount of time; then, the medium was removed and replaced with phosphate-buffered saline (PBS) solution. Cells were then imaged simultaneously in three channels: bright field, EGFP (green) field (488 nm excitation/ 505 nm emission) and Cy5 (far-red) field (635 nm excitation / 675 nm emission). The selectivity level of Gluk2 labelling was assessed by the colocalization of EGFP and probe 90 (Figure 4–3d). Figure 4–3. Fluorescence labelling experiments design with probe 90. (A). HEK293 cells cultivation. (B). GRIK2 plasmid transfection and expression. (C) Fluorescence labelling with probe 90. (D). Confocal imaging experiments. The multiplexed fluorescence imaging experiments using confocal microscopy and HEK-293 cells confirmed that the labelling of GluK2 proteins by probe 90 is selective (Figure 4-4). No red CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 64 fluorescence signal in the Cy5 field was observed above background when probe 90 was applied to non-transfected HEK-293 cells (Figure 4–4a-c). When HEK293 cells transfected with the GRIK2-EGFP plasmid were subjected to probe 90, the cells expressing the green EGFP marker (considered GluK2-positive) were found to exhibit significant red fluorescence (Figure 4–2e). Moreover, cells in the same dish that did not show GFP fluorescence—presumed to be non-transfected—did not display significant red fluorescence (Figure 4–4d-f). This significant difference of signal in Cy5-associated red fluorescence between transfected and non-transfected cells strongly suggests that the probe 90 binds selectively to GluK2 proteins (Figure 4–2g). The fluorescence intensity measured at 675 nm (Cy5 field) in transfected cells was 30 times stronger than in GluK2-negative cells. Finally, the statistical significance difference was verified in repeated experiments of GluK2 transferred cells and control groups (Figure 4–2g) for the labelling probe 90. Figure 4–4. Representative images of HEK-293 cells after application of probe 90. Cells were incubated with fluorescent probe 90 (Sulfo-Cy5 KA) (2.5 µM) for 10 mins at rt, then washed off with PBS buffer. Images were acquired by laser-scanning confocal fluorescence microscopy in green (exc.488 nm, em.505 nm), red (exc. 635 nm, em. 675 nm), and bright fields. Images of merged field were generated by merging images of GFP, Cy5 and bright fields (A–C) Non-transfected HEK-293 cells (control): no Cy5-related signal is discernable above background. (D–F) GRIK2-transfected HEK-293 cells: Cy5-fluorescence signal is observed only in cells that express the exogenous GluK2 protein as identified by presence of the green fluorescence EGFP expression marker. The probe’s Cy5 fluorescence is highly localized to the cell membrane. Scale bar = 10 µM. (G) The fluorescence observed upon Sulfo-Cy5 KA treatment is significantly higher in cells that are GluK2-positive. The probe-labelled cells boundaries were defined manually in the bright field and chose as the region-of-interest (ROI). ROIs were measured in Fiji, and the obtained results were used to calculate the fluorescence intensities. The average fluorescence values were normalized to signal observed in the control cells. Values represent mean ± SD (n1 = 10, n2 = 7, *P < 0.0001). d e fa b cGFP channel Cy5 channel Merged (GFP + Cy5 + Bright channels)gGluk2Probe 90-+++0510152025303540Relative fluorescence intensityGluK2 -Probe 90 +GluK2 +Probe 90 +*CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 65 These preliminary imaging results in living cells demonstrate the potential of Sulfo-Cy5-KA as a KAR fluorescence probe. Two sets of experimental applications in fluorescence labelling are ongoing in the Menard lab; they include (1) unambiguously confirming the selectivity of 90 for KARs proteins by accurate fluorescence colocalization using a fused GRIK2-GFP recombinant chimera, and (2) exploring the use of Sulfo-Cy5 KA as an affinity quantification tool for KARs agonists screening. The results will be submitted for publication upon completion. Despite the labelling efficiency of the Sulfo-Cy5 KA, its binding to the GluK receptors will trigger the opening of their ion channels. In other words, each protein that is visualized and activated by default, which perturbs the cell being observed. An ideal fluorescence probe would allow labelling GluK without perturbing their normal activity. With the general synthesis developed above, the next step will be to construct a traceless labelling probe—one that will label GluKs while leaving the binding site free for its native ligands. CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 66 4.5 Summary An affinity-based fluorescent probe to label kainate receptors, Sulfo-Cy5-KA (90) was synthesized and applied in model cells. The synthesis of Sulfo-Cy5-KA was accomplished in 6 steps from the advanced intermediate 72a in 12% yield. The overall synthesis yield from 4-hydroxyproline was 6% over 16 steps. Preliminary in vitro imaging of GluK2 expressed in HEK 293 cells demonstrated that Sulfo-Cy5-KA can be used to visualize membrane kainate receptors with confocal fluorescence microscopy. Sulfo-Cy5-KA’s far-red emission fluorescence is well suited for applications in neuronal tissue studies. Importantly, the versatility of novel alkynylkainic acid 89 opens the door to the rapid synthesis of any KAR probe for neurobiology studies. It also expands the scope of accessible KAR agonists, e.g., new kainoid analogs 91, via a one-step click reaction for medicinal chemistry research. Scheme 4–4. Synthesis of a clickable kainoid and conjugated fluorescent probes NHCO2HCO2H8990N CO2CH3HO2C CO2CH3Ns72a5 steps17% yield1 step“click” reactionR-N3 “click” reactionNHCO2HCO2HNNNR91R = any functional moietyNN ONHHO3S-O3SNHCO2HCO2HNNNCHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 67 4.6 Experimental Section 4.6.1 General Experimental Procedures Unless otherwise noted, reactions were carried out under an argon atmosphere, in flame-dried single-neck, round bottom flasks fitted with a rubber septum and with magnetic stirring. Air or water sensitive liquids and solutions were transferred via syringe or stainless-steel cannula. Organic solutions were concentrated by rotary evaporation at 25–45 °C at 50–200 torr. Thin layer chromatography (TLC) was performed on glass plates precoated with Silica gel F254, 250 µm, 60 Å, from EMD Chemicals Inc (EMD 5715-1). TLC plates were visualized under a 254 or 365 nm UV light source, then stained by immersion in either acidic aqueous-ethanolic vanillin solution, potassium permanganate, or acidic ethanolic ninhydrin, followed by heating using a heat gun. Purification was performed with 230-400 mesh silica gel from Silicycle, Quebec (SilicaFlash R12030B, P60, 40-63 µm, 60 Å). 4.6.2 Materials 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. Sulfo-Cyanine 5 azide was purchased from Lumiprobe, US. Tetrahydrofuran, dichloromethane, hexanes, toluene, and diethyl ether were purified on a glass contour solvent purification system under an argon atmosphere. Methanol was dried by allowing it to stand over freshly activated 4 Å molecular sieves for 48 h prior to use. Solvents used for chromatographic purifications were obtained from Fischer Scientific or VWR and used without further purification. All new compounds were characterized with 1D (1 H NMR and 13C NMR) and the protons and carbons are assigned by 2D NMR (COSY, HSQC, HMBC) spectroscopy. 4.6.3 Instruments 1 H and 13 C 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 10.0.2-15465 from Mestrelab Research. Chemical shifts are reported in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to residual proton signals in the NMR solvents (CHCl3: δ 7.26, C2HD5SO: δ 2.50), for carbons (CDCl3: δ 77.0, C 2HD5SO: δ 39.5). Spectral data are listed as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s= broad singlet), and coupling constant (J, Hz). Infrared spectra (IR) were obtained using a Perkin-Elmer FT-IR spectrometer. High resolution mass spectra were obtained from the McMaster Regional Centre for Mass spectrometry using a HCTultra PTM discovery system spectrometer (ESI), or from a Waters Micromass LCT Premier TOF Mass. The final product, Sulfo-Cy5 KA, was purified HPLC on a C18 column (Onyx™ Monolithic Semi-PREP C18, LC Column 100 x 10 mm) with a Varian Prostar HPLC system. HPLC condition for the purification was described in the corresponding procedure. CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 68 4.6.4 Statistical analysis and data processing Crystal structures of KARs were downloaded from Protein Data Bank (PDB); the corresponding ID was shown in each figure. These PDB files were processed with PyMol (Education version, Schrödinger, Inc., New York, NY, USA). 3D models of DA and probe 90 were produced by Chemdraw 16.1 (PerkinElmer, Inc., Waltham, Massachusetts, US) and processed by PyMol. GraphPad Prism software (version 8.0, GraphPad Software Inc., La Jolla, CA, USA) was used to conduct statistical analyses. 4.6.5 Chemical synthesis procedures & characterization Methyl (2S,3S,4R)-4-(hydroxymethyl)-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (85). To a solution of acid 72a (290 mg, 0.674 mmol) in dichloromethane (2 mL) were added EDCI (155 mg, 0.809 mmol) and HOBT (124 mg, 0.809 mmol) at rt. After 1.5 h, the solvent was removed under reduced pressure. The resulting residue was redissolved in THF (5 mL) and resulting a suspension. NaBH4 (38 mg, 1.0 mmol) aqueous solution (1.0 mL) was added dropwise. After 30 mins, the reaction mixture was quenched with an ammonium chloride aqueous solution and extracted with ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure and afforded alcohol 85 as a colourless liquid without further purification. FTIR (thin film): 3536, 3103, 2955, 1737, 1531 cm-1. 1H NMR (400 MHz, CDCl3): δ 8.39 – 8.35 (m, 2H), 8.11 – 8.07 (m, 2H), 4.31 (d, J = 5.8 Hz, 1H), 3.74 (s, 3H), 3.70 (s, 3H), 3.59 (dd, J = 11.1, 6.3 Hz, 1H), 3.56 – 3.48 (m, 2H), 3.44 (dd, J = 10.0, 5.6 Hz, 1H), 2.91 (dq, J = 8.3, 6.3 Hz, 1H), 2.65 (dd, J = 12.5, 6.2 Hz, 1H), 2.58 (dd, J = 16.9, 8.4 Hz, 1H), 2.42 (dd, J = 16.9, 6.7 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 172.1, 171.4, 150.2, 144.5, 128.8, 124.2, 65.6, 60.4, 52.8, 52.2, 49.5, 42.1, 41.7, 32.4 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C16H20N2O9SNa, 439.0782; found, 439.0784. Methyl (2S,3S,4R)-4-formyl-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (86). To a solution of alcohol 85 (81 mg, 0.19 mmol) in DCM (2 mL) was added Dess-Martin periodinane (83 mg, 0.19 mmol) at rt. After 1 h, TLC show full conversion. The resulting solution was poured into sodium bicarbonate aqueous solution (5% wt, 20 mL) and extracted with ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with sodium thiosulfate aqueous solution (5% wt, 10 mL) and brine. The resulting organic layer was then dried over MgSO4. The filtrate was concentrated to dryness under reduced N CO2CH3HO2C CO2CH3Nsi. EDCI, HOBT, DCMii. NaBH4/H2O, THF95%72aN CO2CH3CO2CH3NsHO85N CO2CH3CO2CH3NsHODMP, DCMN CO2CH3OHC CO2CH3Ns83%85 86CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 69 pressure. The residue was purified by column chromatography using ethyl acetate / hexane as an eluent (20–40% gradient). Aldehyde product 86 was recovered as a white solid (202 mg, 83%). FTIR (thin film): 3101, 2953, 1742, 1533 cm-1. 1H NMR (400 MHz, CDCl3): δ 9.63 (s, 1H), 8.39 (d, J = 8.9 Hz, 2H), 8.06 (d, J = 8.9 Hz, 2H), 4.24 (d, J = 5.6 Hz, 1H), 3.84 (dd, J = 10.4, 5.5 Hz, 1H), 3.77 (s, 3H), 3.68 (s, 3H), 3.68 – 3.57 (m, 1H), 3.48 (tdd, J = 6.9, 5.3, 1.1 Hz, 1H), 3.12 (p, J = 6.8 Hz, 1H), 2.51 (dd, J = 17.1, 8.0 Hz, 1H), 2.39 (dd, J = 17.1, 7.1 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 198.3, 171.2, 170.7, 150.3, 143.8, 128.7, 124.3, 65.2, 53.0, 52.2, 51.4, 46.5, 41.5, 32.4 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C16H18N2O9SNa, 437.0625; found, 437.0620. Methyl (2S,3S,4S)-4-ethynyl-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (87). To a single-neck flask charged with argon were added a solution of NaOCH3 in methanol (1N, 0.78 mL, freshly prepared) and dry THF (10 mL). The resulting mixture was cooled to -78 oC using a dry ice/acetone bath. To this mixture was added the Bestmann-Ohira reagent (dimethyl-(1-azoacetonyl)phosphonate) in acetonitrile solution (10% wt, 2.1 mL) dropwise under vigorously stirring (slow stirring may lead a frozen solution due to the -45 oC melting point of acetonitrile). 10 mins later, the aldehyde 86 (202 mg, 0.487 mmol) in THF (2 mL) was added dropwise. After 30 mins, the solution was allowed to rt and maintained at rt for 15 mins. The reaction was quenched with ammonium chloride aqueous solution and extracted with ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with sodium bicarbonate, brine, dried over MgSO4 and filtered. The solvent was removed under reduced pressure and afforded a pale-yellow liquid residue. The crude was purified by column chromatography using ethyl acetate / hexane as an eluent (10–50% gradient). The alkyne product 87 was recovered as a white foam solid (153 mg, 77%). FTIR (thin film): 3280, 3103, 3001, 1737, 1538 cm-1. 1H NMR (400 MHz, CDCl3): δ 8.37 (d, J = 8.9 Hz, 2H), 8.07 (d, J = 8.9 Hz, 2H), 4.08 (d, J = 7.6 Hz, 1H), 3.79 (s, 3H), 3.73 (dd, J = 6.4, 3.2 Hz, 1H), 3.69 (s, 3H), 3.63 (dd, J = 10.3, 3.4 Hz, 1H), 3.31 (dh, J = 5.9, 2.6 Hz, 1H), 2.88 – 2.78 (m, 1H), 2.73 (dd, J = 17.0, 8.3 Hz, 1H), 2.46 (dd, J = 17.0, 6.0 Hz, 1H), 1.93 (d, J = 2.4 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 171.3, 171.0, 150.3, 143.8, 128.9, 124.2, 79.0, 64.1, 53.2, 53.0, 52.1, 43.3, 33.6, 32.9 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C17H18N2O8SNa, 433.0682; found, 433.0676. P OCH3ON2OOCH3NaOCH3, CH3OH, -78 oC to r.t. NCO2CH3CO2CH3Ns77% 87N CO2CH3OHC CO2CH3Ns86N CO2CH3CO2CH3Ns87NHCO2CH3CO2CH318-Crown-6, THF, 60 oC59%88PhSHCHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 70 Methyl (2S,3S,4S)-4-ethynyl-3-(2-methoxy-2-oxoethyl)pyrrolidine-2-carboxylate (88). A single-neck flask was charged with alkyne 87 (153 mg, 0.372 mmol) in THF (5 mL), K2CO3 (77 mg, 0.56 mmol) and PhSH (115 µL, 1.12 mmol) at rt. The reaction was heated to 60 oC and maintained for 3 hrs. Then the reaction mixture was poured into water and extracted with ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with sodium bicarbonate and brine. The resulting organic layer was dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure and afforded a pale-yellow liquid. This crude was purified by column chromatography using ethyl acetate / hexane as an eluent (50-100% gradient). The diester product 88 was recovered as a colourless liquid (49 mg, 58%). FTIR (thin film): 3270, 2996, 1738 cm-1. 1H NMR (400 MHz, CDCl3): δ 3.75 (s, 4H), 3.70 (s, 3H), 3.57 (d, J = 7.6 Hz, 1H), 3.33 (dd, J = 10.6, 6.2 Hz, 1H), 3.20 (tdd, J = 6.3, 3.9, 2.5 Hz, 1H), 3.09 (dd, J = 10.6, 4.0 Hz, 1H), 2.89 – 2.79 (m, 1H), 2.72 (dd, J = 8.6, 5.6 Hz, 1H), 2.70 – 2.62 (m, 2H), 2.19 (d, J = 2.5 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 174.4, 172.5, 82.3, 63.8, 52.8, 52.4, 51.8, 43.2, 35.2, 34.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C11H16NO4, 226.1074; found, 226.1076. (2S,3S,4S)-3-(carboxymethyl)-4-ethynylpyrrolidine-2-carboxylic acid (89). To a single-neck flask were added diester (88) (43 mg, 0.07 mmol) in methanol (2 mL) and LiOH aqeous solution (2.5 N, 2 mL) at rt. The reaction was stirred at rt for 5 h. It was then neutralized with hydrochloric acid (1 N) at 0 oC. The resulting solution was concentrated under reduced pressure. The resulting residue was purified by ion-exchange chromotagraphy: ion-exchange resin Dowex 50WX4 100-200 mesh, eluting with 0.5 N aqeous ammonia. The elution fractions were collected and analyzed by TCL for the presence the desired product. (TLC plates were dried gently with a heat gun before being stained with ninhydrin; further heating revealed the presence of the amino acid 89 as yellow spots.) The fractions containing the product were combined and flash frozen, and the solvents were removed by lyophilization to yield 89 as a white solid (18 mg, 48%). FTIR (thin film): 3272, 2996, 1730 cm-1. 1H NMR (400 MHz, D2O): δ 3.86 (d, J = 9.2 Hz, 1H), 3.62 (dd, J = 11.8, 6.4 Hz, 1H), 3.59 – 3.52 (m, 1H), 3.56 – 3.48 (m, 1H), 2.82 – 2.76 (m, 1H), 2.76 – 2.68 (m, 2H), 2.68 – 2.63 (m, 1H) ppm. 13C NMR (101 MHZ, D2O): δ179.4, 173.0, 79.1, 75.5, 63.5, 50.3, 43.3, 37.3, 33.7 ppm. HRMS (ESI-TOF) m/z: [M - H]- calcd for C9H10NO4, 196.0615; found, 196.0612. NHCO2CH3CO2CH3NHCO2HCO2HLiOH, CH3OH/H2O48%88 89NHCO2HCO2H89Cu(OAc)2, TBTA, Sodium ascorbate72%t-BuOH/H2O, Sulfo-Cyanine5 azideNN ONHHO3S-O3SNHCO2HCO2HNNN90CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 71 Sulfo-Cy5-trazole-KA (90). To a single-neck flask charged with argon was added KA alkyne 89 (2 mg, 0.006 mmol), Sulfo-Cy5-azide (7 mg, 0.009 mmol), sodium ascorbate aqueous solution (3.8 mM, 1.1 mL), TBTA aqueous solution (2 mM, 0.4 mL) and CuSO4 aqueous solution (3.4 mM, 0.25 mL) and t-BuOH (1 mL). The temperature was increased to 60 oC and maintained for 24 h. The resulting mixture was concentrated under reduced pressure and purified by HPLC. Sample (injection volume: 0.5 mL) was purified using a mobile phase composed of water/formic acid (0.02%) (solvent A) and acetonitrile/formic acid (0.02%) (solvent B), according to the following gradient steps (flow rate: 2.5 mL / min, detected by UV 235 nm), retention time = 3.6 min. 0 min 65% A- 35% B 10 min 65% A- 35% B FTIR (thin film): 3392, 1702, 1640 cm-1. 1H NMR (400 MHz, D2O): δ 8.07 (t, J = 13.1 Hz, 2H), 7.93 – 7.78 (m, 5H), 7.35 (d, J = 8.4 Hz, 2H), 6.58 (t, J = 12.5 Hz, 1H), 6.28 (dd, J = 13.6, 6.9 Hz, 2H), 4.33 (t, J = 6.7 Hz, 2H), 4.13 – 4.11 (m, 3H), 4.02 (q, J = 6.9 Hz, 1H), 3.80 (dd, J = 12.1, 7.4 Hz, 1H), 3.63 (d, J = 6.3 Hz, 1H), 3.60 (s, 3H), 3.20 – 3.09 (m, 1H), 3.01 (t, J = 6.8 Hz, 2H), 2.68 (dd, J = 17.2, 5.0 Hz, 1H), 2.27 – 2.08 (m, 3H), 2.00 – 1.79 (m, 4H), 1.75 – 1.56 (m, 12H), 1.51 – 1.20 (m, 4H) ppm. HRMS (ESI-TOF) m/z: [M - 2H]2- calcd for C44H53N7O11 S2, 459.6628; found, 459.6633. 4.6.5 Biological experiments Plasmid propagation The GRIK2-GFP cDNA was prepared by Mellissa Hinderle (BSc Biochemistry 2016, UBC Okanagan, see cDNA preparation procedures in her thesis). a. Transformation procedures: The frozen DH10B competent cells were removed from the – 80°C freezer and thawed on ice. The competent cells were gently mixed by lightly flicking tube. 50 µl of cells were pipetted into a chilled falcon tube (Thermo Fisher, plolypylene, 14 mL). Unused cells were refrozen in a dry ice/ ethanol bath and stored in the – 80°C freezer. cDNA solution (3 µl, 298 µg/µL) was added to cell suspension. The resulting mixture was gently swirled for a few seconds to mix, followed by incubation on ice for 30 minutes. The tube(s) was placed in 42°C water bath for 40 seconds without shaking (heat shock). Then the tube(s) was placed on ice for ~2 minutes. The transformation reaction(s) was diluted to 1 mL by addition of super optimal broth (SOC) media. (SOC. medium: 2% Tryptone, 0.5% Yeast Extract, 0.4% glucose, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 & 10 mM MgSO4.) The tube(s) was/were incubated in a shaking incubator at ~200 rpm for 1 hour at 37 °C. The obtained bacteria medium was plated by spreading 30 µL of cell transformation mixture on LB agar plates containing ampicillin and incubated overnight at 37 °C. Single colonies were observed and picked for the subsequent propagation. b. Propagation and cDNA purification procedures: Single colonies were picked and transferred into individual falcon tubes containing 5 mL LB media and 0.5% ampicillin. Falcon tube(s) was/were incubated in a shaking incubator at ~200 rpm for 24 hours at 37°C. The CHAPTER 4. AN AFFINITY-BASED FLUORESCENCE PROBE TO STUDY KAINATE RECEPTORS 72 obtained medium was purified with Plasmid Mini Kit (Omega) by following the enclosed manual. The concentration of cDNA in each tube was determined by NanoPhotometer (Implen, NP80). Cell culture and expression of GluR6. HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 units/mL), streptomycin (100 µg/mL), and incubated in a 5% CO2 humidified chamber at 37 °C. DMEM media with phenol red indicator was used for the culture. Non-phenol red DMEM media was used for the transfection in order to avoid background fluorescence in confocal imaging experiment. HEK293 cells were transfected with the obtained cDNA plasmid. GFP was employed as transfection marker but not fused to the C- or N- terminal of GluK2. Before the induction, the cells were passaged to a 2 mL petri dish with glass bottom (confluence: 20% – 40%). The petri dish was then put in the tissue incubator for 2 h to allow cells to attach to the glass bottom. 5 µL CaCl2 (2.5 M) was pipetted into a 1.5 mL micro centrifuge tube. To this tube was added the obtained plasmid (5 µL, 200–300 µg/µL). This mixture was then diluted with sterile water (50 µL) and mixed with cold HEPES buffer (50 µL). This obtained solution was added dropwise to the culture media of HEK-293 cells. After 24 h, transfection was confirmed by microscopy with the existence of GFP marker. Chemical labelling of HEK-293 cells. The stock solution of probe 90 was prepared by dissolving probe in ultrapure water (prepared by a MilliporeSigma water systems) to the concentration of 100 µM. The fluorescent probe 90 (2.5 µM) was added to medium of HEK293 cells and incubated at rt for 5 mins. After this labeling, the cells were washed with PBS buffer (pH = 7.5) once with a micropipette. Confocal imaging of labelled KAR (GluR6) in HEK-293 cells. Cell imaging was performed with a confocal microscope (FV1000, IX81, Olympus) equipped with a 60× numerical aperture (NA) = 1.35 oil objective. Fluorescence images were acquired using an argon laser for excitation of GFP and a red laser for excitation of Cy5. GluK2 transfected HEK293T cells were identified by the existence of GFP maker. The ROI measurement of fluorescence-labeled cells was performed with Fiji (opensource software). Relative fluorescence intensity = F / F0 F: fluorescence obtained from ROI measurement of GluR 6 transfected HEK293 cells F0: fluorescence obtained from ROI measurement of non-transfected HEK293 cells The data were analyzed by GraphPad Prism. 73 73 Chapter 5 Synthesis and Photochemistry of Light-Controllable Agonists for Kainate Receptors Ion channel agonists and antagonists have been widely used in neurophysiological research over the past three decades. These studies rely on classical methods that lack temporal or spatial precision. In contrast, neurotransmitters that are photocaged or photoswitchable offer attractive alternatives as optically controllable tools for the precise manipulation of neurons. This chapter describes the synthesis and characterization of light-controllable agonists specifically designed to control kainate receptor activity. 5.1 Background Optical control of biological processes is an emerging field, which permits bioactivity manipulations with spatiotemporal resolution.31,133,134 The fundamental concept involves using light to activate or suppress a protein’s activity. Biological approaches include optogenetics, pioneered by Deisseroth and Boyden; this technique controls neuronal activity with genetically encoded light-sensitive ion channels, i.e., channelrhodopsins.135,136 With optogenetics, the gene of a light-gated ion channel is introduced locally into a rodent brain by viral infection. The infected mice subsequently displayed light-responsive behaviours upon blue light irradiation delivered to a brain region by fiber optic.137 These powerful optogenetics tools have been applied to animals to elucidate nervous system functions, such as memory, neurological disorders, and neurodegenerative diseases.138,139 Optogenetic tools are spectacular, but they are restricted to genetically defined proteins and necessitate genetic manipulation for each organism investigated—a complication when studying the human brain. Photopharmacology was thus developed in parallel to optogenetics and offers a complementary set of tools (Figure 5–1).31 CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 74 Photopharmacological tools are based on small molecules; they employ photochromic ligands combined with light irradiation to control an ion channel’s conductance.140 In the context of neurobiology, these photochromic ligands can be classified as photocaged and photoswitchable neurotransmitters (see Chapter 1.4).34,140,141 Figure 5–1. Cartoon examples of photopharmacology tools for the control of ion channels function. (A) Photocaged neurotransimitters: an agonist is made inactive with a light-cleavable protecting group (cage), upon light irradiation the "cage” releases the ligand that can now bind to its associated protein. (B) Photoswitchable neurotransmitters: an agonist is modified with a photo-isomerizable group that either prevents or allows binding to its associated protein, depending on the configuration of the molecule. Photocaged neurotransmitters have two components: a neurotransmitter and the “cage”, which is a photo-cleavable moiety (Figure 5–1a). The cage group must be suitably placed to prevent the neurotransmitter from binding to its receptor. When presented to the appropriate wavelength irradiation, the cage moiety is removed, thereby allowing the neurotransmitter to activate its associated protein receptors. Thus, neuroactivity, e.g. ion channel opening, can be triggered by simple light exposure.142-144 However, this type of activation is irreversible due to the intrinsic nature of the decaging process – an irreversible chemical reaction. Photoswitchable neurotransmitters were proposed as reversible regulators for ion channels (Figure 5–1b).39,127,139-142 The labs of Woolley and Trauner utilized azobenzene groups combined with neurotransmitters to create such probes.143,144 The probes undergo a configurational cis-to-trans CageNNSwitchable moietyLigbNa+Ca2+Ca2+Na+LigCa2+Na+ion channel openingion channel closingZ-configurationNNSwitchable moietyLigion channel openingE-configurationhv.hv.Cage LigNa+Ca2+ion channel closinghv.aCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 75 isomerization upon light irradiation, which gives access to two isomers of a neurotransmitter, with only one being a potent ligand for the receptor. Since the configurational change is reversible, the activation and deactivation of a POI can be reversibly regulated by light. 5.2 Research gap The renewed interest in the role of glutamate receptors in neurodegenerative diseases has led to the recent creation of a number of photo-active tools for AMPA and NMDA receptors.38,145-147 However, no photochromic ligand has been reported specifically for kainate receptors. This is likely due to the relative ease of chemical modification of ligands for AMPA and NMDA receptors. In contrast, the only selective ligands reported for KA receptors are the complex family of kainoid natural products. Given our success in developing a synthesis that allows for easy C4-modification of kainoids, we are uniquely positioned to design new photoactive pharmacological tools to study the function of kainate receptors in neurons. 5.3 Design of photo-controllable agonists for KARs The ideal photochromic ligand will selectively activate—or inhibit—a POI upon visible light irradiation, preferably with long wavelengths like far-red or near-IR (650–900 nm).145,146 High selectivity of the ligand is crucial to minimize off-target effects, and visible light minimizes radical-based damages in living cells. With these considerations in mind, I hypothesized that a photocaged kainoid (DECM-PhKA, 100) and a photoswitchable kainoid (TCAZKA, 103) can be synthesized and will enable the photo-control of kainate receptors (Figure 5–2). Figure 5–2. Examples of photocontrollable KAR agonists. (A) The proposed photocaged agonist for KAR: DECM-PhKA, 100. (B) Simple photocaged glutamates have been reported with UV-cleavable amides or esters. (C) Proposed photoswitchable kainoid: TCAZKA, 103. NHCO2HCO2HNNONNHNClCl ClClCO2HPh CO2HOOEt2NO ONKAR ligandDECM cageByproductCO2HPh CO2HNHPhKADECM-PhKA (100)OOEt2NOHa)NHCO2HCO2HNNONN HNClClClClGreen lightBlue lightc)TCAZKA (103)ON OFFNH2OOOOHOOEt2NDECM-Glu (102)b)OFFONNOMeO CO2HNH2NO2MNI-Glu (101)CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 76 Photocage Design. The proposed DECM-PhKA 100 is composed of phenylkainic acid and a coumarin “cage”. Under blue light radiation, the coumarin cage should be cleaved, and the released phenylkainic acid (PhKA) will be available to bind and activate KARs (Figure 5–2a). The caged carbamate 100 was envisaged inert to KARs as we know from Chapter 2 that (1) the pyrrolidine’s amine is essential to binding, and (2) there is no room in the binding pocket to accommodate the large coumarin group. Since DECM-PhKA is inert when the coumarin cage is in place, the light irradiation will free phenylkainic acid which will exert a “turn-on” effect on the KARs channels. The specific 7-(N,N-diethylamino)coumaryl-3-methlyoxy (DECM) group was selected for its high quantum yield and maximum absorption in the visible blue region (390-410 nm). In order to have a comprehensive understanding on the decaging properties, the reported MNI-Glu (101) and DECM-Glu (102) will also be synthesized for comparison (Figure 5–2b). Since PhKA is at least 100-fold more active than glutamate for KARs, it is expected that DECM-PhKA will be amuch more rapid “turn-on” activator than MNI-Glu and DECM-Glu. This is important for neurobiology applications: intercellular communication in neurons occurs on the millisecond scale, therefore slow activation kinetics combined to diffusion can lead to undiscernable physiological responses. This proposed set of photocaged tools will allow the irreversible activation of GluRs. Photoswitch Design. In contrast, the proposed photoswitch TCAZKA (103) can be reversibly isomerized between configurations that will fit, or not, in the binding site of KARs (Figure 5–2c). Consequently, a KAR probe like TCAZKA is expected to enable more elaborate experiments than photocages. Photoswitch 103 is comprised of a kainoid tethered to a tetracholoroazobenzene. Given that our synthesis makes the C4 position of kainoids easily modifiable (see chapter 4.1), we planned to introduce a tetrachloroazobenze moiety as part of the C4-side chain. Woolley’s tetrachloroazobenze was chosen because it can be isomerized with visible light, at wavelengths that are not damaging to cells.37,147 Indeed, the azo double bond of tetrachloroazobenze can isomerize between the Z (green light) and E (blue light) configuration. The bent Z configuration of the C4 side chain is expected to prevent the kainoid from fitting into the GluK channel’s ligand-bining domain. In contrast, the linear E configuration of the group should allow binding (and possibly increase potency by decreasing Koff). Thus, we hypothesize that the photoswitch TCAZKA (103) can be isomerized reversibly using visible light. We also hypothesize that irradiating 103 with green (Z) light will have an OFF and blue light (E) will have an ON effect on the modulation of KARs channels in living cells. CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 77 5.4 Synthesis of photocaged agonists 5.4.1 Synthesis of MNI-Glu MNI-Glu (101) was synthesized from 4-methoxyindole and glutamic acid in six steps according to literature precedents (Scheme 5–1).143,148 4-Methoxyindole (104) was converted to 4-methoxy-7-nitroindoline (108) via a sequence of reduction, N-Boc protection, nitration, and N-Boc deprotection. The resulting amine 108 was then coupled with N-Boc-Glu-OtBu to give the fully protected amide 109. Acidic conditions using TFA removed both the carbamate and the tert-butyl ester of 109 to afford MNI-Glu as the TFA salt (101). Scheme 5–1. Synthesis of MNI-Glu (101) 5.4.2 Synthesis of DECM-Glu and DECM-PhKA The synthesis began with the preparation of a coumaryl cage precursor since both targets DECM-Glu (102) and DECM-PhKA (100) share this moiety (Scheme 5–2). The direct oxidation of 110149 to obtain aldehyde 112 with selenium dioxide never reached completion, even after overnight reflux in toluene. While a longer reflux time (3 to 5 days) did show consumption of coumarin 110, it also created several impurities. A two-step procedure was thus adopted: coumarin 110 was first converted to enamine 111, followed by a periodate oxidative cleavage to aldehyde 112.150 The aldehyde 112 was then smoothly reduced by NaBH4 to alcohol 113. Coumaryl alcohol 113 is the common fragment that will be used as cage component. The unsymmetrical carbonate 114 was prepared as activated reagent for later amide couplings. This was accomplished by reacting alcohol 113 with 4-nitrophenyl chloroformate. 4-Susbtituted coumarins are highly sensitive to degradation when they bear a potential leaving group at the allylic HNOMeNaBH3CNAcOHHNOMeCO2tBuHN BocHO2CEDCI, Et3N, DCMNOMeO CO2tBuNHBocNO2TFA, DCM NOMeO CO2HNH2NO2109NOMeBoc2O, DMAPDCMBocAgNO2CH3COClNOMeBocNO2TFADCMHNOMeNO2·TFA104 105 106 107108 10192% 93% 58%89% 96%82%CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 78 position. After several attempts, we found that the most stable acylating agent was the para-nitrophenol carbonate. Scheme 5–2. Synthesis of DECM cage reagents With alcohol 113 in hand, Boc-Glu-OtBu was caged by esterification (Scheme 5–3). The N-Boc- and t-butyl ester groups of 116 were removed in a single deprotection step using TFA to afford DECM-Glu (102). We found that ester 102 was surprinsigly stable to daylight exposure (in the lab); enough to purify by column chromatography without requiring special precautions. Scheme 5–3. Synthesis of DECM-Glu (102) The synthesis of DECM-PhKA (100) took advantage of the advanced intermediate pyrrolidine 70a described in Chapter 3 (Scheme 5–4). The carboxylic acid of pyrrolidine 70a was esterified, then its amine was reacted with the unsymmetrical DECM carbonate 114 to give the fully protected 117. However, hydrolysis of dimethyl ester 117 under conventional conditions (LiOH aq/CH3OH) the DECM cage of 117 was cleaved as well. Turning to milder conditions— trimethyltin hydroxide—smoothly provided the desired caged phenylkainic 100. However, the caged product 100 contained residual tin, even after column chromatography purification. Since tin is a concern in biological experiments due to its high cytotoxicity, I looked for an alternative sequence. The successful sequence involved reacting the photoreactive carbonate 114 with PhKA directly. Thus, pyrrolidine 70a was hydrolyzed first to obtain 32a, followed by a direct carbamate O OEt2N O OEt2NDMA-DMFNMe2NaIO4CH3CN/H2O, r.t.O OEt2NONaBH4MeOH, 0 oCO OEt2NOH111 1121134-Nitrophenyl chloroformateDMAP, DCM, r.t., 3dOEt2NOOONO2114reflux94% 83%82% 76%110OOOEt2N OOtBuONHBocOTFA, DCMOOEt2N OOHONH2O113, HBTU, DMAP, DCMHOOtBuONHBocO116 10292% 82%115CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 79 formation with 114. This alternative route circumvented using heavy metal reagents and afforded the desired caged compound DECM-PhKA (100). Scheme 5–4. Synthesis of DECM-PhKA (100) 5.5 Synthesis of photoswitchable kainoids Inspired by the high affinity of C4 aromatic kainoids presented in Chapter 2, I planned to establish a route to new types of aromatic kainoids. Should it be successful, this strategy could be adapted to build kainoid photoswitches. The photoisomerization of simple azobenzes can be realized UV light irradiation.151 For this advantage, azobenzene derivatives have extensively utilized to construct molecular switches.151 Moreover, the switching wavelengths can be extended to visible light region with the introduction substitutents onto the benzene ring, such as methoxyl, fluoro and chloro groups.147,152,153 The tetrachloroazobenze used in this thesis was chosen due this visible-light switchable feature.154 5.5.1 Synthesis of a new heteroaromatic kainoid In modern medicinal chemistry, the strategy of coupling a carboxylic acid to a thiosemi-carbazide155,156 to prepare oxadiazoles in a single step has been widely used to create libraries of compounds.157 This quick chemical assembly was adopted in my synthesis to construct C4 heteroaromatic kainoids and their parent photoswitch. Aminooxazolyl kainoid 124 was selected as a class representative to develop the chemistry for two reasons: its strong electron-deficient quadrupole characteristics would enhance π-π stacking interactions with Tyr488 with the KAR binding site (see Chapter 2), and the 2-amino substituent CO2CH3Ph CO2CH3OONEt2OON Me3SnOH, DCE, 60 oC2. 114, DMAP, DCM, r.t.73%63%117CO2HPh CO2HNH114, NaHCO3, CH3CN/H2O, r.t.CO2HPh CO2HOONEt2OON32a10076%87%i. LiOH, MeOH/H2O, r.t.ii. HCl, 0 oCNHCO2CH3Ph CO2H1. TMSCl, CH3OH70aCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 80 would allow easy attachment of different molecular cargo (e.g., fluorescent tags, biotin, photoswitch, etc.). The synthesis of novel aminooxadiazolyl kainoid 124 began with the preparation of an N-protected thiosemicarbazide (Scheme 5–5). Benzylamine 118 was used to explore the synthesis route since it is removable under acidic conditions. This benzylamine 118 was converted to an isocyanate and then coupled with hydrazine to afford 120. Thiosemicarbazide 120 was condensed with the EDCI-activated acid 72a (Chapter 3) to obtain benzylamine 121, which was surprisingly stable. N-Benzyl-protected kainoid 121 had to be refluxed in TFA to achieve the deprotection. The aminooxadiazolyl product 122 was fully deprotected with additional two steps to afford the desired oxadiazoyl kainic acid (OKA, 124). The C4 configuration of 124 was confirmed by 2D 1H NMR spectrocopy analysis using NOE experiments. Scheme 5–5. Synthesis C4 kainoid derivative 124 5.5.2 Synthesis of an azobenzene kainoid photoswitch The route to substituted oxadiazolyl kainic acid derivative 121 was exploited to create an azobenzene kainic photoswitch. This objective required us to synthesize the appropriate tetrachloroazobenzene precursor (Scheme 5–6). Accordingly, 4-aminobenzoic acid (125) was converted to 4-(phenylazo)benzoic acid 126 through a nitroso/amine condensation. Then a multi- chlorination selective for the ortho positions of 126 was realized via a palladium catalyzed C–H activation to afford tetrachlorinated acid 127 in 82% yield.154 H3CO OCH3NH2 i. CS2, Et3N, DCMii. DMAP (cat.), Boc2O H3CO OCH3NCSiPrOH H3CO OCH3NHNHSNH281% quant.NNsCO2CH3HO2C CO2CH3EDCI, DCMNNsCO2CH3CO2CH3NNOHNH3COOCH372%N2H4独H2OPhSH, K2CO3CH3CN, 60 oCNHCO2CH3CO2CH3NNOH2NLiOHCH3OH/H2O67% 83% NHCO2HCO2HNNOH2NTFA, DCM, reflux90%NNsCO2CH3CO2CH3NNOH2NNH CO2HCO2HH HHNOE4%1%124NNOH2N118 119 12072a121 122123 124CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 81 Scheme 5–6. Synthesis of a photoisomerizable tetracholoroazobenzoic acid. The initial plan involved joining azobenzoic acid 127 with OKA diester 122 (Scheme 5–7). The acid 127 was transiently converted to the acyl chloride 127a, and the poor nucleophilicity of the heteraromatic amine of oxadiazolyl 122 necessitated relative harsh conditions (reflux in pyridine) to provide the amide 133. However, the deprotection of the N-nosyl group in presence of the unusually electrophilic amide 133 proved challenging. Indeed, the tetrachloroazobenzene moiety of 133 was cleaved when presented to PhSH (3.0 eq)/K2CO3 (2.0 eq)/CH3CN at 60 oC. Although extensive optimizations were attempted—e.g., decreasing reagent amounts (1.0 eq PhSH, 1.0 eq K2CO3), decreasing reaction temperature (30 oC), or using additives (1.0 eq 18-crown-6)—the cleavage of tetratcholorobenzene was inevitable. Scheme 5–7. First attempt at the synthesis of kainoid photoswitch 134 We then explored a direct coupling between kainoid acid 72a and an azobenzene thiosemicarbazide to construct the oxadiazole as the coupling key reaction. While this strategy would add several synthetic steps, it was shown to be effective in the synthesis of 121 (Scheme 5–5). H2NN1. Oxone, DCM/H2O2. Benzylamine, AcOHNOHONCS, AcOHSealed tube, 120 oCNNOHOClClClCl78% 82%i. HOBT, EDCI, DCMii. THF, NaBH4/H2ONNOHClClClCli. I2, imidazole, DCMii. NaN3, DMSO NNN3ClClClClPh3P, CH3OHreflux NNNH2ClClClCli. CS2, Et3N, EtOHii. DMAP (cat.), Boc2O NNNCSClClClClN2H4独H2OiPrOH NNNHClClClClNHSNH2125OHO126 127128 129 130131 13283%70% 63%54% 86%NNsCO2CH3CO2CH3NNOHNONNClClClClPhSH, K2CO3, 18-crown-6, CH3CNNHCO2CH3CO2CH3NNOHNONNClClClCl13413361%NNOHOClClClCl127Oxalyl chlorideDMF (cat.), DCM NNClOClClClCl127a122, pyridineDCM, refluxquant.CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 82 Scheme 5–8. Synthesis of a photoisomerizable tetracholoroazobenzene semicarbazide. The required tetracholoroazobenzene semicarbazide presursor 132 was prepared from the azobenzoic acid 127 in seven transformations (Scheme 5–8). Acid 127 was reduced to alcohol 128 through a pre-activation with EDCI. Alcohol 128 was converted to the iodide, then to azide 129 in a one-pot sequence. A Staudinger reaction was used to reduce azide 129 to amine 130 via, which was then converted to thiosemicarbazide 132 by nucleophilic addition of hydrazine. Scheme 5–9. Synthesis of kainoid photoswitch 136 The direct oxadiazole formation between acid 72a and thiosemicarbazide 132 provided the alkylated aminooxadiazolyl kainoid 135 in an acceptable yield (Scheme 5–9). N-Nosyl deprotection of 135 using PhSH/K2CO3/CH3CN at 60 oC gave only a low yield of 136 (~23%) along with impurities. Using a lower temperature and an additive (PhSH/K2CO3/18-crown-6/CH3CN at 30 oC) gave a cleaner reaction profile, and the crude product was telescoped for a methyl ester hydrolysis under basic aqueous condition to obtain the desired kainoid photoswitch 136. 5.6 Photochemical characterization of photo-controllable kainoids In order to use the photoactive chemical probes synthesized above in their physiologogical context adequately requires a comprehensive understanding of their behavior in solution. Measuring their spectral and kinetic properties was accomplished with a number of spectrophysical techniques. NNOHOClClClCli. HOBT, EDCI, DCMii. THF, NaBH4/H2ONNOHClClClCli. I2, imidazole, DCMii. NaN3, DMSO NNN3ClClClClPh3P, CH3OHrefluxNNNH2ClClClCli. CS2, Et3N, EtOHii. DMAP (cat.), Boc2O NNNCSClClClClN2H4独H2OiPrOH NNNHClClClClNHSNH2127 128 129130 131 13283% 70% 63%54% 86%NNsCO2CH3HO2C CO2CH3 1. PhSH, K2CO3, 18-crown-6, CH3CN2. LiOH, H2O/CH3OHNHCO2HCO2HNNONNHNClClClCl68% NNsCO2CH3CO2CH3NNONNHNClClClCl13613572a53%132EDCI, DCMCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 83 5.6.1 Photochemical characterization of photocaged glutamates and kainoids The photochemical properties of caged KAR agonists MNI-Glu (101), DECM-Glu (102) and DECM-PhKA (100) were measured first, followed by their photolysis rates. The photolysis experiments were carried out with two types of blue LED photochemical reactors (custom-made in our lab). One reactor had its maximum irradiation wavelength centered at 465 nm, and the other at 445 nm. Both lamps’ irradiation range overlapped partially with the absorption ranges of DECM-Glu and DECM-PhKA, but there was almost no overlap with MNI-Glu (Figure 5–3). Figure 5–3. Overlay of the two photoreactors irradiadiation ranges (blue-shaded areas) of the absorption spectra of caged compounds 100-102. The absorption of both molecules caged with a DECM coumaryl group overlap with the blue LED light sources. The smaller MNI cage group absorbs mostly in the UV range, and shows very little overlap with the light sources. Photolysis reactions with the caged compounds 100-102 were performed in aqueous solutions to mimick physiological conditions (0.1% DMSO/H2O). Indeed, photochemical reactions in water are notoriously inefficent from a quantum yield standpoint, therefore it was important to measure the efficiency of our photoreactive probes in an unbiased milieu, as close as possible to their intended applications. As expected from the data shown in Figure 5-3, MNI-Glu showed virtually no photolysis upon irradiation with the 445 nm blue LED light source (Figure 5–4a). In contrast, both DECM-Glu and DECM-PhKA showed effective cleavage under same conditions after only five minutes (Figure 5-4b and 4c). 00.20.40.60.81.0300 350 400 450 500 550 600DECM-PhKADECM-GluMNI-GluLED Lampsmax @ 445 nm max @ 465 nmOverlap regionWavelength (nm)NormalizedabsorbanceCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 84 Figure 5–4. Time-dependent photolysis of caged compounds under blue LED light irradiation (445 nm): the absorbance of the compounds decrease as the photocage is released. (A) MNI-Glu in water containing 0.1% DMSO: no change in maximum absorbance is observed after 5 minutes irradiation. (B) DECM-Glu and (C) DEMC-PhKA in water containing 0.1% DMSO: both molecules display a ~10% change in absorbance after 5 minutes due to cleavage of the caged group. (D) DECM-PhKA in DMSO only: no photo-release takes place as water is required in the mechanism of photocehmical cleavage of the DECM coumarin. As a control experiment, 445 nm irradiation of DECM-PhKA did not lead to uncaging in anhydrous DMSO (Figure 5-4d). This lack of reaction is explained by the necessity for a stoichiometric amount of water in the photolysis process (Figure 5–5). A successful lysis process involves the disassociation of [CM-A] to singlet ion pair [CM+A-] and subsequent combination with H2O to yield CM-OH and A-.158 Without H2O, the singlet ion pair [CM+A-] can undergo relaxation and recombine to the ground state of CMA. Figure 5–5. Proposed mechanism for the photolysis of the diethylcoumarin caged compounds under light radiation. [CM-A]*[CM+ A-]AbsCM-ACM+ A-H2OCM-OH + H+ A-+k1kfl + knrkrec kesckhydk2 O OOEt2NACM cageCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 85 Figure 5–6. Overlaid 1H NMR spectra for progressive photolysis of DECM-Glu (400 MHz, D2O/DMSO-d6: 5/1). The top spectrum is the glutamate standard in D2O/DMSO. The cleaved coumarin byproduct is poorly soluble in aqueous solution; it precipitated out of solution and is therefore only visible in trace amount in the spectra. The photolysis process can also be monitored by 1H NMR spectroscopy (Figure 5–6). Initial trials for the uncaging of DECM-Glu (102) were conducted in a photoreactor equiped with a 445 nm or a 465 nm blue LED light source at room temperature. 1H NMR spectra of the photolysis solution were measured at specific intervals. The overlaid 1H NMR spectra presented in Figure 5–6 show that free glutamic acid was gradually released upon 445 nm irradation (peaks a and b). However, DECM-Glu underwent photolysis much more slowly at 465 nm than at 445 nm (Figure S5–1, Appendix D). Under 445 nm irradiation, full cleavage was observed after 140 s (2:20 min). In 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0020406080100120140Chemical shift (ppm)Time(s)D2O DMSOOOEt2NByproductprecipitationDECM-Glu (102)OOEt2NOHNH2OOOOHGlutamic acidH HHHabNH2OHOOOHH HHHabCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 86 contrast, the 465 nm photoreactor required 16 mins irradiation to achieve only 80% conversion (Figure S5–2, Appendix D). Figure 5–7. 1H NMR (400 MHz, D2O) spectra for progressive photolysis of DECM-PhKA. The top row is phenyl kainic acid standard in D2O/DMSO. Since the low solubility of coumarin byproduct in aqueous solution, it gradually precipitated and exhibited low intensity in the spectra. The photolysis of DECM-PhKA (100) was also studied by 1H NMR spectroscopy in D2O (Figure 5–7). The gradual release of free PhKA was found to be completed within 300 s (5:00 min). The CH2 alpha to the carboxylic acid of PhKA (peak b) slightly shifted from 2.29 ppm to 2.20 ppm. Similarly, the ortho hydrogens on the phenyl group of DECM-PhKA shifted from 6.84 ppm to 7.03 ppm of the free PhKA and are key to assessing photolysis completion. The above kinetic CO2HCO2HOOEt2NO ONPhKADECM-PhKA (100)OOEt2NOHbHHHHCO2HCO2HNHHHabByproductprecipitation0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.50204080125175225310PhKAChemical shift (ppm)Time(s)a bD2OCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 87 experiments confirmed the capabilities of releasing Glu and PhKA with visible light irradiation, in aqueous solution at neutral pH. The 1H NMR data presented above were used to determine photolysis rates (Figure 5–8). As expected, the photolysis of DECM cage exhibit an exponential kinetics159(Figure 5–8a), which is also a feature of first-order reactions. DECM-Glu showed slightly faster lysis rate than DECM-PhKA (Figure 5–8a). DECM-Glu exhibits a half-life time of 33.4 s, while that of DECM-PhKA is 60.8 s. The slower lysis rate of DECM-PhKA may due to a the less favoured pathway upon radical cleavage of a carbamate vs. an ester. A similar trend was reported for the lysis of MNI caged glutamate: esters exhibited faster lysis rate than carbamate analogs.160 Figure 5–8. Photolysis rates of DECM-Glu (102) and DECM-PhKA (100). (A, B) DECM-Glu and DECM-PhKA photolysis rate upon 445 nm irradiation calculated from 1H NMR integration data. The conversions versus time were fitted to exponential plateau curves with equations: Y = 100 ´ (1 – e-0.0202x), r2 = 0.994 (DECM-Glu) and Y = 100 ´ (1 – e-0.0107x), r2 = 0.994 (DECM-PhKA). The Ln[C] versus time were fitted to linear functions with equations: Y = -0.0207x + 2.18, r2 = 0.996 (DECM-Glu) and Y = -0.0114x + 2.85, r2 = 0.997 (DECM-PhKA). (C) DECM-Glu and DECM-PhKA photolysis rate under daylight in the lab: less than 10% uncaging occurs after 1 week. (D) Comparison of DECM-PhKA's photolysis rates by a 445 LED light and a 405 nm laser, the process was monitored by UV-Vis absorbance. Absorbances versus time were fitted to exponential plateau curves with equations: Y = (1 – 0.616) ´ e-0.0111x + 0.616 (DECM-Glu), r2 = 0.988 and Y = (1 – 0.719) ´ e-0.0165x + 0.719, r2 = 0.999 (DECM-PhKA). Uncaging rates are similar and cleavage is virtually complete after 5 minutes for both wavelengths. 0 50 100 150 200 250 300 350020406080100120Time (s)Conversion (%)DECM-GluDECM-PhKA50 100 150 200 250 300-0.50.00.51.01.52.02.53.0Time (s)Ln[Caged Glu or PhKA]DECM-GluDECM-PhKA0 2 4 6 8 10020406080100Time (d)Conversion (%)DECM-PhKADECM-Gluacbd0 50 100 150 200 250 300 3500.60.70.80.91.01.1Time (s)Normalized absorbance @ 393 nmIrradiation @ 405 nm laserIrradiation @ 445 nm ledCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 88 The thermal stability of DECM-Glu and DECM-PhKA under normal lab light is vital to their practical use as biological tools: it is inevitable that they will get exposed to light during experiments. Gratifyingly, long-term tracking experiments suggest that they are stable to natural light exposure – only less than 10% cleavage occurred after 8 days of leaving a solution contained in an NMR tube standing in the lab (Figure 5–8c). Finally, the intended release of DECM-PhKA by focal photolysis under 405 nm laser irradiation was evaluated (Figure 5–8d). Using a focused 405 nm laser beam for irradiation caused faster photolysis than a LED photoreactor (global sample irradiation). The photolysis rate was observed with a 405 nm laser was 1.5 times faster than with the LED reactor (Table 5–1). We note that this more efficient photocleavage could also partially be caused by a better spectral overlap with the absorption of DECM-PhKA; however for practical reasons, this aspect was not tested further. Table 5–1. Photochemical properties of caged Glu and PhKA compounds Compounds Abs. lmax (nm) Light source K (s-1) T1/2 (s) MNI-Glu (101) 339 405, 445, or 465 nm – – DECM-Glu (102) 390 465 nm LED lamp 1.66 ´ 10-3 417 390 445 nm LED lamp 2.07 ´ 10-2 33.4 DECM-PhKA (100) 393 445 nm LED lamp 1.14 ´ 10-2 60.8 393 405 nm laser 1.65 ´ 10-2 41.7 5.6.2 Photochemical characterization of kainoid photoswitch 136 The cis /trans isomerization of tetracholorazobenzene groups has been reported to be controllable via blue and green light irradiation.145,155 In our case, it is important to prove that the isomerization of kainoid photoswitch 136 is possible, as well as to understand both the kinetics and thremodynamics of its cis /trans states under conditions that will be used in biological applications. The cis / trans isomerization of photoswitch 136 was measured by UV-Vis spectrophotometirc analysis (Figure 5–9). Under a blue laser irradiation (405 nm), the photoswitch 136 prefers to exist in the trans configuration and showed a local absorbance maximum at ~400 nm. When presented to a green laser irradiation (532 nm), the absorbance of 136 at 400 nm decreased as anticipated. Although it demonstrates a spectral change (modified conjugation) due to configuration switching, a more precise technique than UV-Vis spectroscopy was used to quantify the level of cis / trans transformation. CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 89 Figure 5–9. Trans / cis- isomerization of 136 and its UV-vis spectra. (A) Trans / cis- switching of 136. (B) UV-vis spectra of 136 under irradiation. Blue line: irradiation with 405 nm laser for 3 mins; black line: sample kept in the dark for 24 h; green line: irradiation with 535 nm laser for 3 mins. The mechanism of azobenzene cis / trans photoisomerization has been studied extensively, however its exact details remain controversial.161 The two main proposals involve a bond breaking/rotation of the azobenzene group, or a direct inversion (Scheme 5–10).162 The rotation mechanism involves a rupture of the N=N p bond via a light-generated diradical intermediate (p®p*), followed by a rotation around the N–N bond, terminating with the reformation of the N=N bond in the other configuration. In contrast, the direct inversion mechanism invokes a light-induced rehybridization of one nitrogen atom (n®p*) to form a N=N–C dihedral angle of 180o, followed by the inverted collapse of this dihedral angle to rehybridize the nitrogen in the opposite 120o configuration.161 Scheme 5–10. Schematic diagram of photoisomerization mechanism of azobenzenes NHCO2HCO2HNNOHNNNCl ClClClKAR ligandPhotoswitchable moietyNHCO2HCO2HNNOHNNNClClClCl λ = 405 nmλ = 532 nmKAR Photoswitch300 350 400 450 5000.51.01.52.0Wavelength (nm)AbsorbanceBlue irradiation0 sGreen irradiation360 380 400 420 4400.60.70.80.91.0Wavelength (nm)Absorbancea) b)136N NN NN NR1R2R1 R2R2R1RotationN NInversion (in plane)(out of plane)R1R2CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 90 In order to quantify the ratio of cis and trans isomers of switch 136 upon light irradiation, I turned to analysis of the compounds using 1H NMR spectroscopy experiments (Figure 5–10). Interestingly, the benzylic hydrogens (H13) of the azobenzene 136 presented chemical shifts in the 4.57 to 4.27 ppm range when presented to blue or green light (blue box in Figure 5–10). Upon green light irradiation (Fig. 5-10a), the peak at 4.32 ppm decreased (red box b), while the overlapping 4.28–4.20 multiplets decreased (band c). At the same time, peaks at 4.52 and 4.45 ppm changed only slightly (band a). These shifts caused upon irradiation show trans-to-cis isomerization. The opposite changes were observed upon blue light irradiation (Figure 5–10b): the peaks in band b increased and the peaks in band c decreased. However, it was still challenging to conclusively determine the exact cis / trans ratio by this means, since the bands cannot be unambiguously assigned to the cis / trans isomers. Moreover, the relatively low solubility of 136 in water made it difficult to obtain the kinetic NMR data in D2O; which is more appropriate to the aqueous solution of biological experiments. The reversibility of the photoswitching was measured by UV-Vis spectroscopy analysis. When 136 was presented to alternating pulses of green and blue lasers, fast reversible switching was observed (Figure 5–11). Maximum isomerization from trans to cis was observed after 100 seconds, and quick relaxation from cis to trans was observed after 50 seconds. These isomers exhibited slow thermal relaxation in a dark place (>2 hours). Figure 5–11 also shows that photobleaching of tetrachloroazobenzene 136 is not significant, even after multiple cycles of excitation. All the spectral characterization data presented above confirm that the configuration of photoswitch 136 can be controlled by visible light irradiation. However, none of the spectroscopic techniques could quantify whether a clean 100% conversion occurs between cis and trans isomer. CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 91 Figure 5–10. 1H NMR spectra (CD3OD) of 136 before and after light irradiation. (A). 1H NMR spectrum of 136 after irradiation with a green laser for 10 mins; (B). 1H NMR spectrum of 136 after irradiation with a laser blue for 10 mins; (C). 1H NMR spectrum of 136 after dark-adaption overnight. All the hydrogens were assigned according to the gCOSY spectrum. Figure 5–11. Sequential photoswitching of 136 using green (535 nm) and blue (405 nm) lasers, followed by an extended period of relaxation in the dark. The active kainoid (ON) is the thermodynamically favoured trans-136 isomer and the inactive kainoid (OFF) is cis-136 isomer. 2.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1(ppm)123a. Irradiated with green lightb. Irradiated with blue lightc. Dark-adapted134,5'32,5'' 6' 6''NH1234567N8N910O11NH1213141516171819N20N21222324252627Cl28Cl29Cl30Cl3132O33OH3435O36OH37a b c0 100 200 300 400 500 600 700 800 900 1000 4000 80000.230.240.250.26Time (s)Abs.@400nm max in trans max in cisCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 92 Importantly, it should be noted that the biological experiments where the kainoid photoswitch will be used do not require 100% conversion to the active isomer. As long as the concentration of active compound reaches the activation threshold of the kainate receptors, the calcium influx will be triggered in the cells, which will initiate the downstream propagation of the action potential in neurons. The application concentration and activation irradiation are thus expected to be optimized in biological experiments. Moreover, this incomplete—yet sufficient—photoisomerization is typically observed in other photoswitch activation of ion channels by other research groups.152,163-165 Consequently, we expect that kainoid photoswitch 136 will control its phenotype control in biological experiments (currently being tested, but results were not complete at the time of writing). CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 93 5.7 Summary In summary, a new heteroaromatic kainoid and two new types of pharmacological tools for kainate receptors were synthesized and characterized (Scheme 5–12). The photocaged kainoid DECM-PhKA (100) was obtained in one step with 87% yield from the phenyl kainic acid 32a, which is an overall yield of 47% from 4-hydroxyproline over nine steps. Photoswitchable kainoid 136 was synthesized from crucial intermediate 72a with 36% yield over three steps, which is an overall yield of 17% from 4-hydroxyproline over thirteen steps. The new heteroaromatic kainoid 124 was obtained from 72a with 37%, which is an overall yield of 17% from hydroxyproline over fourteen steps. Scheme 5–12. Summary of synthesized new kainoids The photochemical release of the highly active KAR agonist phenylkainic acid (PhKA, 32a) from DECM-PhKA (100) was demonstrated upon blue light irradiation. The photocage DECM-PhKA displayed acceptable thermal stability under natural light. 100 exerted rapid release of PhKA with a half-life time of 41.7 seconds upon blue laser irradiation. This short time scale is essential for its application in living cells and neurobiology research. 472aN CO2MeCO2MeOHONs NHCO2HCO2HNNOH2NNHCO2HCO2HNNOHNNNClClClCl3 steps4 steps136 NHCO2HCO2HNNOHNNNClCl ClClNHHOCOOHtans-4-hydroxy-L-prolineCO2HCO2HOOEt2NO ON CO2HPh CO2HNHDECM-PhKA (100)PhKACage byproduct10 stepsChapter 3124EZCO2HPh CO2HNHChapter 3 8 steps1 step32aCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 94 The photoswitchable agonist 136 was demonstrated to undergo reversible trans/cis isomerization upon green and blue light irradiation. The photochemical characterization of tetrachloroazobenzene kainoid 136 confirmed that the trans/cis isomerization can manipulated repeatably without photobleaching, and it proved to be resistant to thermal relaxation on practical time scales. From a medicinal chemistry standpoint, our new synthetic route to oxadiazolyl kainoid 124 provides an unprecedented access to a new class of kainoids. Synthesis and testing of this novel class of analogs are being explored, but they are beyond the scope of this thesis. CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 95 5.8 Experimental section 5.8.1 General Experimental Procedures Unless otherwise noted, reactions were carried out under an argon atmosphere, in flame-dried single-neck, round bottom flasks fitted with a rubber septum and with magnetic stirring. Air or water sensitive liquids and solutions were transferred via syringe or stainless-steel cannula. Organic solutions were concentrated by rotary evaporation at 25–45 °C at 50–200 torr. Thin layer chromatography (TLC) was performed on glass plates precoated with Silica gel F254, 250 µm, 60 Å, from EMD Chemicals Inc (EMD 5715-1). TLC plates were visualized under a 254 or 365 nm UV light source, then stained by immersion in either acidic aqueous-ethanolic vanillin solution, potassium permanganate, or acidic ethanolic ninhydrin, followed by heating using a heat gun. Purification was performed with 230-400 mesh silica gel from Silicycle, Quebec (SiliaFlash R12030B, P60, 40-63 µm, 60 Å). 5.8.2 Materials 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. Tetrahydrofuran, dichloromethane, hexanes, toluene, and diethyl ether were purified on a glass contour solvent purification system under an argon atmosphere. Methanol was dried by allowing it to stand over freshly activated 4 Å molecular sieves for 48 h prior to use. Solvents used for chromatographic purifications were obtained from Fisher Scientific or VWR and used without further purification. 5.8.3 Statistical analysis and data processing GraphPad Prism software (version 8.0, GraphPad Software Inc., La Jolla, CA, USA) was used to conduct statistical analyses. Crystal structures of KARs were downloaded from Protein Data Bank (PDB); the corresponding ID was shown in each figure.3D models of compounds were produced by ChemDraw and CORINA and processed by PyMol (Education version, Schrödinger, Inc., New York, NY, USA). These PDB files were processed with PyMol. 5.8.4 Instruments 1H and 13C NMR spectra were recorded on a 400 MHz Varian NMR AS400 equipped with an ATB-400 probe at 25 oC. NMR spectra were analyzed with MestReNova version 10.0.2-15465 from Mestrelab Research. Chemical shifts are reported in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to residual proton signals in the NMR solvents (CHCl3: δ 7.26, C 2 HD 5 SO: δ 2.50), for carbons (CDCl3: δ 77.0, C 2HD5SO: δ 39.5). Spectral data are listed as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s= broad singlet), and coupling constant (J, Hz). Infrared spectra (IR) were obtained using a Nicolet 6700 FT-IR spectrometer as a neat film on a NaCl plate, or a Perkin-Elmer FT-IR Spectrum Two IR spectrometer. High resolution mass spectra were obtained CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 96 from the UBC Mass Spectrometry Facility using a HCTultra PTM discovery system spectrometer (ESI), or from a Waters Micromass LCT Premier TOF Mass Spectrometer (ESI, Zandberg lab, UBCO). UV-VIS spectroscopy UV-VIS spectra were recorded on a Varian 50 Scan UV-Visible Spectropho- tometer. Illumination was provided by a blue laser (405 nm) and a green laser (535 nm) purchased from Armlaser, Quebec, Canada. The spectra were recorded after irradiation of the sample. The absorption is plotted as arbitrary units (A.U.) as a function of wavelength λ (nm). 5.8.5 Synthetic procedures & characterization (S)-2-amino-5-(4-methoxy-7-nitroindolin-1-yl)-5-oxopentanoic acid, MNI-Glu (101) was prepared according to a modified literature procedure. Characterization data matched literature precedents.148 1H NMR (400 MHz, D2O): d = 7.67 (d, J = 9.1 Hz, 1H), 6.78 (d, J = 9.2 Hz, 1H), 4.17 (t, J = 7.9 Hz, 2H), 3.81 (s, 3H), 3.65 (t, J = 6.3 Hz, 1H), 2.96 (t, J = 7.9 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.04 (q, J = 7.1 Hz, 2H) ppm. (E)-7-(diethylamino)-4-(2-(dimethylamino)vinyl)-2H-chromen-2-one (111). To a solution of coumarin 110 (10.00 g, 43.23 mmol) in dry DMF (50 mL) was added N,N-dimethylformamide dimethyl acetal (4.60 mL, 34.6 mmol). The reaction mixture was then refluxed overnight. The resulting mixture was poured to water. It resulted a large amount of brown precipitation, the solid product was collected by vaccum filtration. This solid product was further washed with ether (30 mL) to afford enamine 111 as a dark-yellow solid (11.64, 94%). The crude product was used as is directly for the next step. FTIR (thin film): 2968, 2930, 2876, 2817, 1678, 1605, 1566 cm-1. HNOMeNaBH3CNAcOHHNOMeCO2tBuHN BocHO2CEDCI, Et3N, DCMNOMeO CO2tBuNHBocNO2TFA, DCM NOMeO CO2HNH2NO2109NOMeBoc2O, DMAPDCMBocAgNO2CH3COClNOMeBocNO2TFADCMHNOMeNO2·TFA104 105 106 107108 10192% 93% 58%89% 96%82%O OEt2N O OEt2NDMA-DMFNMe2111reflux94%110CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 97 1H NMR (400 MHz, CDCl3): d 7.53 (d, J = 9.0 Hz, 1H), 7.22 (d, J = 13.0 Hz, 1H), 6.55 (dd, J = 9.0, 2.7 Hz, 1H), 6.49 (d, J = 2.6 Hz, 1H), 5.85 (s, 1H), 5.22 (d, J = 13.0 Hz, 1H), 3.40 (q, J = 7.1 Hz, 4H), 2.99 (s, 6H), 1.20 (t, J = 7.1 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3): d 163.6, 156.4, 152.4, 150.1, 146.6, 124.8, 108.1, 107.9, 98.1, 93.4, 87.5, 44.7, 12.5 ppm. 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (112). Enamine 111 (2.0 g, 6.9 mmol) was dissolved in acetonitrile (5 mL). To this solution was added H2O (5 mL) and NaIO4 (1.6 g, 7.7 mmol). The resulting mixture was stirred at rt for 30 mins, TLC showed the full conversion. The reaction mixture was poured into water and extracted with ethyl acetate (3 ´ 50 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The solvent was removed under reduced pressure. The residue was purified by column chromatography using ethyl acetate / hexane as an eluent (20-60% gradient). Aldehyde 112 was recovered as an orange solid (1.4 g, 83%). 1H NMR (400 MHz, CDCl3): δ 10.03 (s, 1H), 8.31 (d, J = 9.2 Hz, 1H), 6.63 (dd, J = 9.2, 2.6 Hz, 1H), 6.53 (d, J = 2.7 Hz, 1H), 6.45 (s, 1H), 3.43 (q, J = 7.1 Hz, 4H), 1.22 (t, J = 7.1 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3): δ 192.5, 161.9, 157.4, 151.0, 143.9, 127.0, 117.4, 109.5, 103.7, 97.6, 44.8, 12.5 ppm. 7-(diethylamino)-4-(hydroxymethyl)-2H-chromen-2-one (113). To a solution of aldehyde 112 (1.201 g, 4.89 mmol) in methanol (5 mL) was added sodium borohydride (93 mg, 2.5 mmol) at 0 OC. After 30 mins, TLC showed the full conversion. The mixture was quenched with ammonium chloride aqueous solution and extracted with ether (3 ´ 30 mL). The combined organic layer was washed with sodium bicarbonate aqueous solution and brine. The ether layer was dried over MgSO4 and filtered. The filtrate was concentrated to dryness under reduced pressure to afford the alcohol 113 as a yellow solid (993 mg, 82%) without further purification. FTIR (thin film): 3446, 2971, 1739, 1686 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.30 (d, J = 9.0 Hz, 1H), 6.55 (d, J = 9.0 Hz, 1H), 6.45 (s, 1H), 6.28 (s, 1H), 4.82 (s, 2H), 3.38 (q, J = 6.9 Hz, 4H), 1.18 (t, J = 7.3 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3): δ 163.1, 156.1, 155.5, 150.5, 124.4, 108.7, 106.4, 105.2, 97.6, 60.8, 44.7, 12.5 ppm. HRMS (ESI) m/z: [M + Na]+ calcd for C9H10NO4Na, 270.1101; found, 270.1094. O OEt2NNMe2NaIO4CH3CN/H2O, r.t.O OEt2NO111 11283%O OEt2NO112NaBH4MeOH, 0 oC O OEt2NOH11382%CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 98 (7-(diethylamino)-2H-chromen-4-yl)methyl (4-nitrophenyl) carbonate (114). To a single-neck flask were added 4-nitrophenyl chloroformate (1.61 g, 7.99 mmol), alcohol 4 (659 mg, 2.66 mmol), DMAP (326 mg, 2.66 mmol) and dichloromethane (5 mL) at rt. The flask was then charged with argon and covered with tin foil to avoid light irradiation. After 2 d, the reaction mixture was poured into water and extracted with ethyl acetate (3 ´ 50 mL). The combined organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The resulting residue was then washed with diethyl ether (50 mL) to remove excess alcohol and afforded the carbonate 114 (833 mg, 76%) as a yellow solid without further purification. The product was stored in a tin foil covered vial. FTIR (thin film): 2972, 1774, 1711 cm-1. 1H NMR (400 MHz, CDCl3): δ 8.31 (d, J = 9.1 Hz, 2H), 7.42 (d, J = 9.1 Hz, 2H), 7.32 (d, J = 9.0 Hz, 1H), 6.61 (dd, J = 9.0, 2.6 Hz, 1H), 6.54 (d, J = 2.6 Hz, 1H), 6.23 (s, 1H), 5.40 (d, J = 1.2 Hz, 2H), 3.43 (q, J = 7.1 Hz, 4H), 1.22 (t, J = 7.1 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3): δ 161.6, 156.4, 152.2, 150.9, 147.6, 125.4, 124.3, 121.7, 108.8, 107.0, 105.6, 98.0, 65.8, 44.8, 12.4 ppm. 1-(tert-butyl) 5-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl) (tert-butoxycarbonyl)-L-glutamate (116). To a solution of Boc-Glu-OtBu (245 mg, 0.808 mmol) in dichloromethane (5 mL) were added alcohol 113 (241 mg, 0.972 mmol), HBTU (306 mg, 0.807 mmol) and triethylamine (115 µL, 0.808 mmol). The reaction was stirred under argon atmosphere at rt (the flask was cover with tin foil to avoid light irradiation) for 3 d. Then the reaction mixture was poured into water and extracted with ethyl acetate (3 ´ 20 mL). The combined organic layer was washed with ammonium chloride aqueous solution, sodium bicarbonate aqueous solution and brine. The resulting organic layer was dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure (a tin foil was used to cover the flask when removing solvent on the rotavap). The resulting residue was purified by column chromatography using ethyl acetate / hexane as an eluent (10-60% gradient). Ester 116 was recovered as a yellow solid (395 mg, 92%). 1H NMR (400 MHz, CDCl3): δ 7.29 (d, J = 9.4 Hz, 1H), 6.58 (dd, J = 9.0, 2.5 Hz, 1H), 6.52 (d, J = 2.6 Hz, 1H), 6.12 (s, 1H), 5.23 (s, 2H), 5.10 (d, J = 8.3 Hz, 1H), 4.28 – 4.23 (m, 1H), 3.42 (q, J = 7.1 Hz, 4H), 2.64 – 2.44 (m, 2H), 2.27-2.16 (m, 1H), 1.99 -1.90 (m, 1H), 1.44 – 1.49 (m, 18H), 1.21 (t, J = 7.1 Hz, 6H) ppm. O OEt2NOH1134-Nitrophenyl chloroformateDMAP, DCM, r.t., 3dOEt2NOOONO211476%OOEt2N OOtBuONHBocO113, HBTU, DMAP, DCMHOOtBuONHBocO11692%115CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 99 13C NMR (101 MHz, CDCl3): δ 172.1, 161.8, 156.3, 150.7, 149.2, 124.4, 108.7, 106.6, 106.0, 97.8, 82.4, 79.9, 61.6, 53.2, 44.7, 30.1, 28.3, 28.1, 28.0, 12.4 ppm. (S)-2-amino-5-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)-5-oxopentanoic acid (102). Ester 116 (395 mg, 0.742 mmol) was dissolved in dichloromethane (2.0 mL). Trifluoroacetic acid (1.7 mL, 22 mmol) was added dropwise. The resulting mixture was stirred at rt under argon. The flask was covered with tin foil and maintained for 4 h. The solvent was removed under reduced pressure (tin foil was used to cover the flask when removing solvent on the rotavap). The resulting residue was triturated in ether and yielded a yellow precipitation. It was filtered and solid precipitate was collected as the TFA salt of 102 (298 mg, 82%) without further purification. FTIR (thin film): 3461, 3016, 2971, 1738 cm-1. 1H NMR (400 MHz, CD3OD): δ 7.47 (d, J = 9.1 Hz, 1H), 6.74 (dd, J = 9.1, 2.6 Hz, 1H), 6.55 (d, J = 2.7 Hz, 1H), 6.06 (s, 1H), 5.40 – 5.28 (m, 2H), 3.80 (t, J = 6.5 Hz, 1H), 3.48 (q, J = 7.1 Hz, H), 2.84 – 2.67 (m, 2H), 2.21 (dtd, J = 18.3, 14.2, 12.8, 7.3 Hz, 2H), 1.21 (t, J = 7.1 Hz, 6H) ppm. 13C NMR (101 MHz, CD3OD): δ 156.1, 151.2, 151.1, 124.8, 109.0, 105.7, 104.7, 96.9, 61.4, 44.2, 29.2, 26.7, 25.6, 11.3 ppm. 19F NMR (376 MHz, D2O) δ -75.65. 1-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl) 2-methyl (2S,3S,4S)-3-(2-methoxy-2-oxoethyl)-4-phenylpyrrolidine-1,2-dicarboxylate (117). To the solution of 70a (25 mg, 95 µmol) in methanol (0.3 mL) was added TMSCl (30 µL, 0.24 mmol). The mixture was stirred at room temperature for 5 h. Then the solvent was removed under reduced pressure. To the resulting residue were added DMAP (17 mg, 0.14 mmol) and carbonate 114 (38 mg, 0.095 mmol). The mixture was stirred at rt under argon atmosphere for 24 h (tin foil was used to cover the whole flask during the reaction). The resulting mixture was concentrated to dryness. The residue was purified by column chromatography using ethyl acetate / hexane as an eluent (20-60% gradient). The carbamate 117 was recovered as a brown solid (47 mg, 92%). 1H NMR (400 MHz, CDCl3, two rotamers): δ 7.37 – 7.28 (m, 4H), 7.08 (d, J = 7.4 Hz, 2H), 6.59 (q, J = 8.5, 8.1 Hz, 1H), 6.51 (s, 1H), 6.12 (d, J = 37.2 Hz, 1H), 5.44 – 5.15 (m, 2H), 4.25 (t, J = 5.9 Hz, 1H), 4.07 (q, J OOEt2N OOtBuONHBocOTFA, DCMOOEt2N OOHONH2O116 10282%CO2CH3Ph CO2CH3OONEt2OON2. 114, DMAP, DCM, r.t.73%117NHCO2CH3Ph CO2H1. TMSCl, CH3OH70aCHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 100 = 9.1 Hz, 1H), 3.98 – 3.87 (m, 1H), 3.82 (s, 1H), 3.77 (s, 2H), 3.63 (d, J = 3.9 Hz, 3H), 3.41 (d, J = 7.6 Hz, 1H), 3.14 – 3.06 (m, 1H), 2.35 – 2.25 (m, 1H), 2.14 – 2.04 (m, 1H), 1.21 (t, J = 6.7 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3, two rotamers): δ 172.1, 171.9, 171.9, 171.8, 162.1, 162.0, 161.8, 156.3, 156.2, 154.3, 153.9, 153.3, 150.8, 150.7, 150.7, 150.0, 149.9, 148.3, 129.0, 127.6, 127.5, 127.5, 126.1, 124.5, 124.3, 124.3, 115.7, 108.8, 108.7, 108.7, 106.9, 106.0, 105.9, 105.9, 105.8, 105.7, 97.8, 97.8, 65.3, 63.7, 63.2, 62.7, 52.7, 52.7, 51.9, 51.8, 50.7, 50.5, 45.1, 44.8, 44.7, 44.6, 44.2, 43.5, 33.2, 33.1, 12.4 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C30H34N2O8Na, 573.2207; found, 573.2204. (2S,3S,4S)-3-(carboxymethyl)-1-(((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)carbonyl)-4-phenylpyrrolidine-2-carboxylic acid (100). To a solution of carbamate 117 (30 mg, 55 µmol) in dichloroethane (2 mL) was added trimethyltin hydroxide (49 mg, 0.27 mmol). The mixture was refluxed overnight (the flask and condenser were covered with tin foil). The reaction mixture was poured into a hydrochloric acid (0.1 M, 10 mL) and extracted with ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography using methanol / dichloromethane as an eluent (5-20% gradient). Acid 100 was recovered as a yellow solid (18 mg, 88%) with trace tin residues. This method was thus abandoned due to the toxicity of tin for living cells. (2S,3S,4S)-3-(carboxymethyl)-1-(((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)carbonyl)-4-phenylpyrrolidine-2-carboxylic acid (100). To a solution of PhKA (76 mg, 0.30 mmol) in H2O (1.0 mL) and acetonitrile (2.0 mL) were added sodium bicarbonate (77 mg, 0.91 mmol) and carbonate 114 (125 mg, 0.30 mmol). The mixture was stirred at rt for 24 h (the whole flask was covered with tin foils). The resulting solution was then poured into a hydrochloric acid (0.1 M, 10 mL) solution and extracted with ethyl acetate (3 ´ 20 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The filtrate was CO2CH3Ph CO2CH3OONEt2OON Me3SnOH, DCE, 60 oCCO2HPh CO2HOONEt2OON10063%117CO2HPh CO2HNH114, NaHCO3, CH3CN/H2O, r.t. CO2HPh CO2HOONEt2OON32a10087%CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 101 concentrated under reduced pressure (tin foil was used to cover the flask when on the rotavap). The resulting residue was triturated in ether (30 mL) and filtered to afford 100 as a yellow solid. FTIR (thin film): 3414, 2977, 1709, 1601 cm-1. 1H NMR (400 MHz, CD3OD, two rotamers): δ 7.49 (t, J = 9.3 Hz, 1H), 7.32 (dt, J = 7.9, 4.1 Hz, 2H), 7.26 (t, J = 7.2 Hz, 1H), 7.13 (d, J = 7.2 Hz, 2H), 6.74 (ddd, J = 14.2, 9.1, 2.6 Hz, 1H), 6.55 (d, J = 2.5 Hz, 1H), 6.14 (d, J = 15.8 Hz, 1H), 5.47 – 5.28 (m, 2H), 4.31 – 4.09 (m, 1H), 4.07 – 3.84 (m, 1H), 3.77 (bs, 1H), 3.48 (qd, J = 7.0, 4.8 Hz, 4H), 3.07 (bs, 1H), 2.37 (bs, 1H), 2.06 – 1.90 (m, 1H), 1.21 (td, J = 7.1, 4.7 Hz, 6H) ppm. 13C NMR (101 MHz, CD3OD, two rotamers): δ 163.0, 156.1, 156.1, 154.0, 151.8, 151.1, 151.0, 138.5, 138.4, 128.5, 128.4, 127.5, 126.9, 124.8, 124.7, 109.1, 109.1, 105.7, 105.6, 104.8, 104.4, 96.8, 62.5, 45.0, 44.2, 31.3, 11.3 ppm. HRMS (ESI-TOF) m/z: [M - H]- calcd for C28H29N2O8, 521.1929; found, 521.1906. 1-(isothiocyanatomethyl)-2,4-dimethoxybenzene (119). To a solution of benzylamine 118 (1.20 g, 7.18 mmol) and triethylamine (1.09 mL, 7.89 mmol) in dichloromethane (50 mL) was added carbon disulfide (4.34 mL, 71.7 mmol). The mixture was stirred at rt for 1 h. It resulted in a turbid solution. To this suspension were added Boc2O (1.88 g, 8.61 mmol) and DMAP (87 mg, 0.72 mmol). The solution turned clear and was stirred at rt for another 30 mins. The mixture was poured into water and extracted with ether (3 ´ 50 mL). The combined organic layer was washed with 5% citric acid aqueous solution, brine, dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using ethyl acetate / hexane as an eluent (5-10% gradient). The isocyanate product 119 was recovered as a colourless oil (1.221 g, 81%). 1H NMR (400 MHz, CDCl3): δ 7.19 (d, J = 8.1 Hz, 1H), 6.52 – 6.44 (m, 2H), 4.60 (s, 2H), 3.84 (s, 5H), 3.82 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3): δ 161.3, 157.9, 129.4, 115.1, 104.1, 98.6, 55.5, 55.5, 44.1 ppm. N-(2,4-dimethoxybenzyl)hydrazinecarbothioamide (120). To a solution of isocyanate 119 (987 mg, 4.72 mmol) in isopropanol (10 mL) was added hydrazine hydrate (432 µL, 60%, 5.19 mmol) dropwise. It resulted in a white precipitation right after the addition. The mixture was stirred for 20 min at rt. The solid product was recovered by vaccum filtration. It was further washed by ethanol and ether to afford the thiosemicarbozide 120 (1.132 g, 99%) as a white powder without further purification. FTIR (thin film): 3342, 3280, 3188, 3152, 3004, 2938, 1557 cm-1. H3CO OCH3NH2 i. CS2, Et3N, DCMii. DMAP (cat.), Boc2O H3CO OCH3NCS81%118 119H3CO OCH3NCSiPrOH H3CO OCH3NHNHSNH2quant.N2H4独H2O119 120CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 102 1H NMR (400 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.94 (s, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.46 (dd, J = 8.3, 2.3 Hz, 1H), 4.57 (d, J = 5.8 Hz, 2H), 4.49 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 181.3, 159.8, 157.8, 129.0, 119.0, 104.2, 98.3, 55.5, 55.2, 41.6 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C10H15N3O2SNa, 264.0777; found, 264.0786. Methyl (2S,3S,4R)-4-(5-((2,4-dimethoxybenzyl)amino)-1,3,4-oxadiazol-2-yl)-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (122). To a solution of acid 72a (369 mg, 0.857 mmol) in dichloromethane (5 mL) was added thiosemicarbozide 120 (310 mg, 1.29 mmol) and EDCI (575 mg, 3.00 mmol). The mixture was stirred at rt for 24 h. The solvent was removed under reduced pressure. The product was purified by column chromatography using ethyl acetate / hexane as an eluent (10-50% gradient). The oxodiazole product 122 was recovered as a white solid (385 mg, 72%). FTIR (thin film): 3013, 2951, 1742 cm-1. 1H NMR (400 MHz, CDCl3): δ 8.26 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.2 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 6.45 (dd, J = 8.2, 2.4 Hz, 1H), 5.27 (s, 1H), 4.42 – 4.21 (m, 3H), 3.91– 3.83 (m, 1H), 3.86 (s, 3H), 3.80 (s, 6H), 3.79 – 3.70 (m, 2H), 3.60 (s, 3H), 3.07 (p, J = 6.9 Hz, 1H), 2.62 (dd, J = 17.4, 8.1 Hz, 1H), 2.35 (dd, J = 17.5, 6.8 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 171.3, 171.0, 163.5, 161.1, 158.6, 156.5, 150.3, 143.3, 130.7, 128.4, 124.3, 117.9, 103.8, 98.8, 64.5, 55.5, 55.4, 53.0, 52.0, 50.7, 43.5, 42.6, 37.8, 32.0 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C26H29N5O11SNa, 642.1476; found, 642.1483. Methyl (2S,3S,4R)-4-(5-amino-1,3,4-oxadiazol-2-yl)-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfo-nyl)pyrrolidine-2-carboxylate (123). To the solution of oxodiazole (342 mg, 0.426 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (0.163 mL, 2.13 mmol). The mixture was stirred at 50 oC. After 3 h, TLC showed full conversion. The solvent was removed under reduced pressure. The product was purified by column chromatography using ethyl acetate / hexane as an eluent (10-50% gradient). Oxodiazole 123 was recovered as a white solid (181 mg, 90%). FTIR (thin film): 3423, 3363, 3102, 2953, 1733, 1656 cm-1. NNsCO2CH3HO2C CO2CH3H3CO OCH3NHNHSNH2EDCI, DCMNNsCO2CH3CO2CH3NNOHNH3COOCH372%72a122NNsCO2CH3CO2CH3NNOHNH3COOCH3TFA, DCM90%NNsCO2CH3CO2CH3NNOH2N122 123CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 103 1H NMR (400 MHz, CDCl3): δ 8.36 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H), 5.27 (s, 2H), 4.34 (d, J = 6.9 Hz, 1H), 3.94 – 3.86 (m, 1H), 3.84 – 3.78 (m, 1H), 3.81 (s, 3H), 3.64 (s, 3H), 3.35 (s, 1H), 3.09 (p, J = 6.7 Hz, 1H), 2.59 (dd, J = 17.4, 8.3 Hz, 1H), 2.40 (dd, J = 17.4, 6.7 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 171.2, 171.0, 150.4, 143.5, 128.6, 124.3, 64.5, 56.0, 53.1, 52.1, 50.5, 42.7, 37.8, 32.1, 29.7 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C17H19N5O9Na, 492.0796; found, 492.0792. Methyl (2S,3S,4R)-4-(5-amino-1,3,4-oxadiazol-2-yl)-3-(2-methoxy-2-oxoethyl)pyrrolidine-2-carboxylate (124). To a solution of oxodiazole 124 (165 mg, 0.351 mmol) in acetonitrile (3 mL) were added thiophenol (54 µL) and potassium carbonate (73 mg). The mixture was stirred at 60 oC for 3 h. The reaction mixture poured into water and extracted ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was by column chromatography using methanol / dichloromethane as an eluent (5-20% gradient). Oxodiazole amine 124 was recovered as a white solid (67 mg, 67%). FTIR (thin film): 3401, 2931, 2856, 1738, 1659 cm-1. 1H NMR (400 MHz, CDCl3): δ 5.51 (s, 2H), 3.76 (s, 3H), 3.72 (d, J = 7.9 Hz, 1H), 3.71 – 3.66 (m, 1H), 3.65 (s, 3H), 3.43 (dd, J = 11.3, 6.5 Hz, 1H), 3.31 (dd, J = 11.3, 4.1 Hz, 1H), 2.97 (dtd, J = 9.0, 7.7, 6.0 Hz, 1H), 2.73 (s, 1H), 2.64 (dd, J = 17.0, 6.0 Hz, 1H), 2.49 (dd, J = 17.0, 9.0 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 174.1, 172.1, 163.2, 159.9, 64.0, 52.5, 51.9, 50.1, 43.5, 39.8, 34.0 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C11H17N4O5, 285.1193; found, 285.1194. (2S,3S,4R)-4-(5-amino-1,3,4-oxadiazol-2-yl)-3-(carboxymethyl)pyrrolidine-2-carboxylic acid (125). To a solution of oxadiazole amine 124 (63 mg, 0.22 mmol) in methanol (1 mL) was added LiOH aqueous solution (2.5 M, 2.7 mL). The resulting mixture was stirred at rt for 5 h. The solution was neutralized by addition of hydrochloric acid (0.5 M) at 0 oC. The mixture was concentrated under reduced pressure. The residue was dissolved in water (2 mL) and purified by ion-exchange chromatography: ion-exchange resin Dowex 50WX4 100-200 mesh, eluting with 0.5 N aqueous ammonia. The eluting fractions were collected and analyzed by TLC for the presence of the desired product. (TLC plates were dried gently with a heat gun before being stained with ninhydrin; further heating revealed the presence of the amino acid 125 as yellow spots.) The PhSH, K2CO3CH3CN, 60 oC NHCO2CH3CO2CH3NNOH2N67% 124NNsCO2CH3CO2CH3NNOH2N123NHCO2CH3CO2CH3NNOH2NLiOHCH3OH/H2O83%NHCO2HCO2HNNOH2N124 125CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 104 fractions containing the product were combined and flash frozen, and the solvents were removed by lyophilization to yield a pale-yellow solid. This product was recrystallized with aqueous ethanol to afford the final product 125 as a white solid (47 mg, 83%). FTIR (thin film): 3395, 2951, 1742, 1727 cm-1. 1H NMR (400 MHz, D2O): δ 4.03 (q, J = 6.9 Hz, 1H), 3.93 (d, J = 8.9 Hz, 1H), 3.88 (dd, J = 12.4, 7.7 Hz, 1H), 3.77 (dd, J = 12.5, 5.2 Hz, 1H), 3.08 (td, J = 11.3, 10.7, 5.3 Hz, 1H), 2.69 (dd, J = 16.8, 4.6 Hz, 1H), 2.09 (dd, J = 16.8, 10.8 Hz, 1H) ppm. 13C NMR (101 MHz, D2O): δ 178.0, 172.4, 164.7, 158.2, 64.4, 46.5, 43.1, 37.7, 36.3 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C11H17N4O5, 257.0868; found, 257.0868. (E)-4-(phenyldiazenyl)benzoic acid (126). To a solution of 4-aminobenzoic acid (8.1 g, 59.1 mmol) in dichloromethane (30 mL) and water (30 mL) was added oxone (21.8 g, 70.9 mmol). The mixture was stirred vigorously at rt for 2 h. It resulted a suspension mixture. The mixture was filtered, the solid was further washed with water and afforded a dark yellow solid (8.3 g, 93%) product without further purification. The obtained 4-nitrosobenzoic acid (8.3 g, 54.9 mmol) was added to a single-neck flask. To this flask were added benzylamine (6.1 mL, 65.9 mmol) and acetic acid 50 mL. The mixture was stirred at 50 oC overnight. The resulting mixture was poured into water and resulted in a large amount of brown precipitation. The solid precipitation was recovered by vaccum filtration. This obtained solid was then recrystallized in ethanol and gave the 4-(phenylazo)benzoic acid 126 as a red solid (10.5 g, 84%). FTIR (thin film): 3464, 3010, 1739, 1680 cm-1. 1H NMR (400 MHz, DMSO-d6): δ 13.23 (s, 1H), 8.19 – 8.08 (m, 2H), 8.00 – 7.86 (m, 4H), 7.68 – 7.53 (m, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 166.7, 154.3, 151.9, 132.9, 132.2, 130.7, 129.6, 122.8, 122.6 ppm. HRMS (ESI-TOF) m/z: [M - H]+ calcd for C13H9N2O2, 225.0670; found, 225.0666. (E)-3,5-dichloro-4-((2,6-dichlorophenyl)diazenyl)benzoic acid (127). 4-(phenylazo)benzoic acid (5.1 g, 22.5 mmol), N-chlorosuccinimide (15.1 g, 112.7 mmol), palladium acetate (101 mg, 0.451 mmol) and acetic acid (50 mL) was added to a pressure tube. The mixture was degassed by argon and sealed, followed by heating to 120 oC. After 2 days, the tube was cooled to rt and opened. The mixture was poured into water and yielded a H2NN1. Oxone, DCM/H2O2. Benzylamine, AcOHNOHO78%125OHO126NNOHONCS, AcOHSealed tube, 120 oCNNOHOClClClCl82%126 127CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 105 large amount of precipitation. The suspension was filtered. The solid product was recrystallized in methanol and yield 127 as a red solid (6.7 g, 82%) without further purification. FTIR (thin film): 3467, 3016, 2945, 1739 cm-1. 1H NMR (400 MHz, acetone-d6): δ 12.04 (bs, 1H), 8.17 (s, 2H), 7.69 (dd, J = 8.1, 0.6 Hz, 2H), 7.55 (dd, J = 8.7, 7.5 Hz, 1H) ppm. 13C NMR (101 MHz, acetone-d6): δ 164.7, 151.3, 147.8, 133.3, 132.2, 131.24, 130.9, 127.9, 127.4 ppm. HRMS (ESI-TOF) m/z: [M - H]- calcd for C13H5Cl4N2O2, 360.9111; found, 360.9111. (E)-(3,5-dichloro-4-((2,6-dichlorophenyl)diazenyl)phenyl)methanol (128). To a solution of acid 127 (3.5 g, 9.6 mmol) in dichloromethane (10 mL) were added HOBT (1.6 g, 11 mmol) and EDCI (2.0 g, 11 mmol). The mixture was stirred at rt for 1 h. The solvent was then removed under reduced pressure. The resulting mixture was resuspended in THF (10 mL). A solution of sodium borohydride (437 mg, 11.5 mmol) in water (2 mL) was added dropwise into the reaction mixture. After the addition, the mixture was stirred at rt for 30 mins. It resulted in a clear mixture. This mixture was poured into water and extracted with ethyl acetate (3 ´ 20 mL). The combined organic layer was washed with ammonium chloride aqueous solution, brine and dried over MgSO4. It was filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography using ethyl acetate / hexane as an eluent (10-30% gradient). The alcohol product 128 was recovered as a red oil (2.8 g, 83%). FTIR (thin film): 3370, 3070, 1724, 1562 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.44 (bs, 3H), 7.31 – 7.20 (m, 2H), 4.71 (s, 2H) ppm. 13C NMR (101 MHz, CDCl3): δ 147.7, 146.3, 143.5, 129.6, 129.4, 127.6, 127.2, 127.2, 63.4 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C13H9Cl4N2O, 348.9464; found, 348.9459. (E)-1-(4-(azidomethyl)-2,6-dichlorophenyl)-2-(2,6-dichlorophenyl)diazene (129). To a solution of alcohol 128 (1.6 g, 4.6 mmol) in dichloromethane (30 mL) triphenylphosphine (1.4 g, 5.5 mmol), iodine (1.4 g, 5.5 mmol), imidazole (373 mg, 5.5 mmol) were added. The reaction mixture was stirred at rt for 2 h. Then TLC showed full conversion of the alcohol to iodide. To the resulting mixture were added DMSO (20 mL) and sodium azide (357 mg, 5.5 mmol). After 15 min, TLC showed full conversion of iodide to azide. The resulting mixture was poured into water and extracted with ethyl acetate (3 ´ 20 mL). The combined organic layer was NNOHOClClClCli. HOBT, EDCI, DCMii. THF, NaBH4/H2O127 83%NNOHClClClCl128NNOHClClClCli. I2, imidazole, DCMii. NaN3, DMSO NNN3ClClClCl128 12970%CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 106 purified by column chromatography using ethyl acetate / hexane as an eluent (10-20% gradient). The azide 129 was recovered as a red oil (1.2 g, 70%). FTIR (thin film): 3072, 2105, 1562 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.48 – 7.41 (m, 4H), 7.29 – 7.24 (m, 1H), 4.40 (s, 2H) ppm. 13C NMR (101 MHz, cdcl3): δ 147.6, 147.2, 138.0, 129.7, 129.4, 128.6, 127.8, 127.3, 53.2 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C13H8Cl4N5, 373.9528; found, 373.9526. (E)-(3,5-dichloro-4-((2,6-dichlorophenyl)diazenyl)phenyl)methanamine (130).To a solution of azide 129 (1.1 g, 2.9 mmol) in methanol (10 mL) was added triphenylphosphine (1.5 g, 5.9 mmol). The mixture was refluxed for 4 h. The solvent was removed under reduced pressure. The resulting mixture was purified by column chromatography using ether / chloroform (25%) as first eluent to remove triphenylphosphine and triphenylphosphine oxide byproduct, and using chloroform / methanol (10%) to afford the amine 130 as a red liquid (645 mg, 63%). 1H NMR (400 MHz, CDCl3, trans and cis mixture): δ 7.49 – 7.42 (m, 3H), 7.29 – 7.21 (m, 2H), 3.93 (s, 2H), 1.66 (s, 2H) ppm. 13C NMR (101 MHz, CDCl3, trans and cis mixture): δ 148.2, 147.9, 146.6, 146.0, 145.8, 129.5, 129.4, 129.4, 129.3, 129.3, 129.1, 128.0, 128.0, 127.7, 127.6, 127.2, 126.0, 126.0, 45.2, 44.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C13H10Cl4N3, 347.9623; found, 347.9622. (E)-1-(2,6-dichloro-4-(isothiocyanatomethyl)phenyl)-2-(2,6-dichlorophenyl)diazene (131). To a solution of amine 130 (580 mg, 1.66 mmol) in methanol (20 mL) were added triethylamine (0.276 mL, 1.99 mmol) and carbon disulfide (1.0 mL, 16.6 mmol). The mixture was stirred at rt for 1 h. It resulted in a turbid solution. To this suspension were added Boc2O (435 mg, 8.61 mmol) and DMAP (10 mg, 0.083 mmol). The solution then turned to clear solution and was stirred at rt for another 30 mins. The mixture was poured into water and extracted with ether (3 ´ 20 mL). The combined organic layer was washed with 5% citric acid aqueous solution, brine, dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using ethyl acetate / hexane as an eluent (10-20% gradient). The isocyanate 131 was recovered as a red liquid (353 mg, 54%). 1H NMR (400 MHz, CDCl3): δ 7.49 – 7.42 (m, 4H), 7.31 – 7.25 (m, 1H), 4.78 (t, J = 0.7 Hz, 1H) ppm. NNN3ClClClClPh3P, CH3OHreflux NNNH2ClClClCl129 13063%NNNH2ClClClCl130i. CS2, Et3N, EtOHii. DMAP (cat.), Boc2O NNNCSClClClCl13154%CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 107 13C NMR (101 MHz, CDCl3): δ 147.6, 147.4, 136.4, 129.8, 129.5, 128.0, 127.6, 127.4, 47.4 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C14H8Cl4N3S, 389.9188; found, 389.9189. (E)-N-(3,5-dichloro-4-((2,6-dichlorophenyl)diazenyl)benzyl)hydrazinecarbothioamide (132). To a solution of isocyanate 131 (238 mg, 0.608 mmol) in isopropanol (3 mL) was added hydrazine hydrate (101 µL, 60%, 1.22 mmol) dropwise. It resulted yellow in precipitation right after the addition. The mixture was stirred for 20 mins at rt. The solid product was recovered by vaccum filtration. It was further washed by ether and ethanol to afford the thiosemicarbozide 132 (221 mg, 86%) as a yellow powder without further purification. FTIR (thin film): 3402, 2940, 2254, 1660 cm-1. 1H NMR (400 MHz, DMSO-d6): δ 8.90 (bs, 1H), 8.59 (bs, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.58 (s, 2H), 7.50 (t, J = 8.2 Hz, 1H), 4.76 (d, J = 5.3 Hz, 2H), 4.59 (bs, 2H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 146.8, 144.7, 144.6, 131.1, 129.9, 128.4, 126.1, 125.9, 45.0 ppm. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C14H12Cl4N5S, 421.9562; found, 421.9572. Methyl (2S,3S,4R)-4-(5-(3,5-dichloro-4-((E)-(2,6-dichlorophenyl)diazenyl)benzamido)-1,3,4-oxadiazol-2-yl)-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (133). To a suspension of acid 127 (50 mg, 0.14 mmol) in dichloromethane (1 mL) was added oxalyl cholide (35 µL, 0. 42 mmol) at rt. Then a drop of dry DMF was added to the mixture to accelerate the reaction. The reaction mixture turned to a clear dark red solution. After 1 h, the solvent was removed under reduced pressure and yielded a brown solid. The resulting solid was redissolved in dichloromethane (2 mL). To this mixture were added amine 123 (65 mg, 0.14 mmol) and pyridine (11 µL, 0.14 mmol). The resulting mixture was heated to reflux overnight. The resulting mixture was concentrated under reduced pressure. The crude product was purified by column chromatography using ethyl acetate / hexane as an eluent (10-50% gradient). The oxadiazole product 133 was recovered as a red solid (68 mg, 61%). 1H NMR (400 MHz, CD3CN): δ 8.31 (d, J = 8.9 Hz, 2H), 8.25 (s, 2H), 7.99 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.43 (dd, J = 8.6, 7.6 Hz, 1H), 4.31 (d, J = 6.7 Hz, 1H), 3.96 – 3.83 (m, 3H), 3.79 – 3.73 (m, 1H), 3.72 (s, 3H), 3.54 (s, 3H), 3.13 (p, J = 6.9 Hz, 1H), 2.46 (dd, J = 17.2, 6.7 Hz, 1H), 2.31 (dd, J = 17.3, 7.8 Hz, 1H) ppm. NNNCSClClClClN2H4独H2OiPrOH NNNHClClClClNHSNH2131 13286%NNsCO2CH3CO2CH3NNOHNONNClClClCl13361%NNOHOClClClCl127Oxalyl chlorideDMF (cat.), DCM NNClOClClClCl127a123, pyridineDCM, refluxquant.CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 108 13C NMR (101 MHz, CD3CN): δ 172.1, 171.9, 151.3, 149.8, 148.0, 144.4, 131.8, 130.9, 130.8, 129.5, 127.8, 127.3, 125.5, 65.8, 53.5, 52.6, 50.9, 43.4, 39.1, 33.1 ppm. Methyl (2S,3S,4R)-4-(5-((3,5-dichloro-4-((E)-(2,6-dichlorophenyl)diazenyl)benzyl)amino)-1,3,4-oxadiazol-2-yl)-3-(2-methoxy-2-oxoethyl)-1-((4-nitrophenyl)sulfonyl)pyrrolidine-2-carboxylate (135). To a solution of acid 72a (24 mg, 56 µmol) in dichloromethane (2 mL) were added thiosemicarbazide (28 mg, 67 µmol) and EDCI (37 mg, 0.19 mmol). The resulting mixture was stirred at rt for 48 h. The mixture was concentrated under reduced pressure. The resulting mixture was purified by column chromatography using ethyl acetate / hexane as an eluent (10-50% gradient). The oxodiazole amine product was recovered as an orange solid (30 mg, 68%). FTIR (thin film): 3333, 2970, 1739, 1637, 1532 cm-1. 1H NMR (400 MHz, CDCl3): δ 8.36 (d, J = 9.1 Hz, 2H), 8.03 (d, J = 9.1 Hz, 2H), 7.50 – 7.43 (m, 4H), 7.30 – 7.26 (m, 1H), 5.41 (t, J = 6.2 Hz, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.36 (d, J = 6.5 Hz, 1H), 3.87 – 3.80 (m, 3H), 3.78 (s, 3H), 3.61 (s, 3H), 3.08 (dt, J = 12.4, 4.9 Hz, 1H), 2.53 (dd, J = 17.4, 8.2 Hz, 1H), 2.38 (dd, J = 17.4, 6.7 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3): δ 171.1, 171.0, 163.1, 157.3, 150.4, 147.6, 147.1, 143.8, 139.6, 129.8, 129.5, 129.2, 128.6, 128.6, 128.4, 127.8, 127.3, 124.3, 64.6, 53.0, 52.1, 50.2, 46.2, 42.7, 37.9, 32.4 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C30H25Cl4N7O9SNa, 822.0081; found, 822.0074. (2S,3S,4R)-3-(carboxymethyl)-4-(5-((3,5-dichloro-4-((E)-(2,6-dichlorophenyl)diazenyl)benzyl)amino)-1,3,4-oxadiazol-2-yl)pyrrolidine-2-carboxylic acid (136). To a solution of oxodiazole 135 (28 mg, 35 µmol) in acetonitrile (2 mL) were added thiophenol (10 µL, 0.10 mmol), 18-crown-6 (14 mg) and potassium carbonate (7 mg). The mixture was heated to 40 oC. After 3 h, the reaction mixture was poured into water and extracted with ethyl acetate (3 ´ 10 mL). The combined organic layer was washed with brine, dried over MgSO4 and filtered. The filtrate was concentrated under reduced pressure. The resulting mixture was purified by column chromatography using ethyl acetate / hexane as an eluent (10-100% gradient). The oxodiazole amine product was recovered as an orange solid (16 mg, 74%). The obtained solid was redissolved in methanol (1 mL). To this solution was added LiOH aqueous solution (2.5 M, 0.32 mL). The resulting mixture was NNsCO2CH3HO2C CO2CH368% NNsCO2CH3CO2CH3NNONNHNClClClCl13572a132EDCI, DCM1. PhSH, K2CO3, 18-crown-6, CH3CN2. LiOH, H2O/CH3OHNHCO2HCO2HNNONNHNClClClClNNsCO2CH3CO2CH3NNONNHNClClClCl13613553%CHAPTER 5. SYNTHESIS AND PHOTOCHEMISTRY OF LIGHT-CONTROLLABLE AGONISTS FOR KAINATE RECEPTORS 109 stirred at rt for 6 h. The solution was neutralized by addition of hydrochloric acid (0.5 M) at 0 oC, precipitations formed after the neutralization. The solution was centrifuged. The aqueous solution was removed by pipette, the obtained solid was re-suspended in water (3 mL) and re-centrifuged. The aqueous solution was removed and the solid product was dried under high vaccum. It afforded the orange solid (11 mg, 56%) as the azo product 136 without further purification. FTIR (thin film): 3352, 2948, 1643 cm-1. 1H NMR (400 MHz, CD3OD): δ 7.50 – 7.42 (m, 1H), 7.42 – 7.29 (m, 2H), 7.31 – 7.19 (m, 2H), 4.45 – 4.15 (m, 2H), 4.08 – 3.86 (m, 2H), 3.73 – 3.62 (m, 2H), 3.06 – 2.97 (m, 1H), 2.92 – 2.72 (m, 1H), 2.36 – 2.10 (m, 1H) ppm. 13C NMR (101 MHz, CD3OD): δ 172.8, 172.5, 168.7, 168.5, 135.5, 129.6, 129.3, 129.1, 129.0, 128.2, 128.0, 127.0, 61.6, 45.4, 40.7, 32.4 ppm. HRMS (ESI-TOF) m/z: [M - H]- calcd for C22H17Cl4N6O5, 585.0026; found, 585.0020. CHAPTER 6. GENERAL CONCLUSION 110 110 Chapter 6 General Conclusion From a chemical biology perspective, an overarching objective of this thesis is the design of chemical probes to study kainate receptors (KAR) using light. The underlying foundation of this work, however, is the creation and exploitation of synthetic strategies to obtain novel C4 kainoids. The robust synthetic routes presented herein have enabled us to obtain these C4 kainoids on a practical scale. Some C4 kainoid intermediates have been further derivatized to create a number of photoactive KAR probes. Figure 6-1 provides an overview of the synthetic strategies and target molecules developed. Figure 6–1. Overview of the SAR analysis, probes synthesis, and their application. NHHOCOOHtans-4-hydroxy-L-prolineNHCO2HCO2H4 472aN CO2MeCO2MeOHONsNHCO2HCO2H8–11 stepsPhenyl kainic acidKainic acid10 steps1 stepCO2HCO2HOOEt2NO ONNHCO2HCO2H6 steps1 step click reactionNN ONHHO3S-O3SNHCO2HCO2HNNNNHCO2HCO2HNNOH2NNHCO2HCO2HNNOHNNNClClClClCO2HPh CO2HNHDECM-PhKA (100)PhKAIon channels opening ONCage byproductIon channels opening OFF0 100 200 300 400 500 600 700 800 900 1000 4000 80000.230.240.250.26Time (s)Abs.@400nm max in trans max in cis0 50 100 150 200 250 300 350020406080100Time (s)Conversion (%)4 steps4 stepsIon channels opening OFFInactiveNHCO2HCO2HNH2HO2CNHCO2HCO2HHO2CCO2HNHCO2HNHCO2HNHCO2HCO2HNHCO2HCO2HL-Glutamic acid Phenyl Kainic acid (FPA)Phenylacrylic KADomoic acidKainic acidNHCO2HCO2HNHOCO2HAcromelic acid BDHKA Methylpropenyl KACO2H CO2HIncreasingaffinityActiveKainic acid in the binding pocket of GluK2136 NHCO2HCO2HNNOHNNNClCl ClClIon channels opening ONChapter 2: Structures design guidelineChapter 3: C4 kainoids synthesisChapter 4: KARs fluorescent probeChapter 5: photopharmacology tools – photocageChapter 5: photopharmacology tools – photoswitchCHAPTER 6. GENERAL CONCLUSION 111 Chapter 2 described a comprehensive survey of kainic acid analogs that have been tested for their biological activity. The available activity data of kainoids were systematically analyzed and combined with the most recent crystallographic studies. From the exercise emerged a set of structural guidelines to help design high-affinity analogs. We have used this analysis to design the suite of molecules presented in subsequent chapters. We expect it will also help guide the synthetic efforts of organic synthesis and medicinal chemistry research in view of the renewed interest in neuroactive molecules. In our hands In Chapter 3, a unified stereoselective synthesis of 4-substituted kainoids is presented. Four kainic acid analogues were obtained in 8–11 steps with up to 54% overall yields. Starting from trans-4-hydroxy-L-proline, the sequence enables a late-stage modification of C4 substituents with sp2 nucleophiles. Stereoselective steps include a cerium-promoted nucleophilic addition and a palladium-catalyzed reduction. A 10-step route to acid 21a was also established to enable ready functionalization of the C4 position. A versatile alkynyl kainoid was synthesized in Chapter 4 that capitalized on our C4 kainoid synthesis. The easily modifiable nature of the alkyne functional group via “click” chemistry enabled the synthesis of several chemical probes via one-step coupling reaction with azides. A red fluorescent probe was synthesized (sulfoCy5-KA, 90), and confocal microscopy imaging experiments in living cells demonstrated its use for KARs visualization. Biological experiments with this probe are still ongoing in our lab, including binding affinity measurements with biophysical experiments. This probe offers neurobiologists a tool to visualize KARs and study their trafficking that complements genetic methods. Finally, Chapter 5 described two sets of photopharmacology tools for KARs – photocaged and photoswitchable kainoids. The photocaged kainoid, DECM-PhKA is derived from phenyl kainic acid – a selective agonist for KARs. Photochemical studies on DECM-PhKA confirmed the release of the active PhKA upon blue light irradiation, DECM-PhKA is relatively stable under natural light. It will provide a new tool to enable the temporal and spatial activation of KARs by blue light. Moreover, a reversible agonist – photoswitchable kainoid 136 was developed. Photochemical studies of 136 demonstrated the reversible trans/cis isomerization upon blue and green light irradiation. It offers one more reversible modulation tool for KARs with temporal and spatial precision using visible light. The further electrophysiology experiments on these novel tools are still ongoing in our lab. Kelowna July 2, 2019 APPENDIX A 112 112 Appendix A Biological data for kainoids collected from reported studies Table S2–1. Original literature data for the activity of kainic acid analogs in functional assays Compound Potency Potency relative to L-Glu80 Mimimum effective concentration (µM)46 Potency relative to KA neurotoxicity (%)166 Potency relative to L-Glu52 （inducing Na+ efflux from preloaded rat striatum slices） Effect in rat cerebellar slices48,83 Depolarization Potentiation Retina Striatum Electrophysiology EC 50 (mM) KA Antagonist IC50 (mM) (KA 1 mM) L-Glu (3) 1 0.03 Inactivea 0.3 < 0.1 1.0 1.0 Pyroglutamic acid (10) 0.7 – 0.8 CPAA (11) KA (1) 8 - 80 8.0 1.0 allo – KA (6) Inactivea Inactivea DHK (13) 0.06 - 0.6 Inactivea Inactivea < 1.0 < 2.0 Inactivea b-KA (7) Inactivea Inactivea N-acetyl kainic acid (5a) Inactivea Inactivea < 1.0 < 2.0 Inactivea Kainic acid dimethyl ester (4) Inactivea Inactivea < 1.0 < 2.0 Inactivea Carboxyl kainic acid (14a) 2.5 Inactive Inactive Carboxyl allo – kainic acid (14b) 2.3 Inactive Inactive Kainic acid methyl ketone (12a) 0.01 0.003 5.2 allo – kainic acid methyl ketone (12b) 4.6 Hydroxyl kainic acid (15a) 0.2 Dihydroyl kainic acid (15b) 0.1 Phenylthio-hydroxyl kainic acid (15c) 0.1 Inactive Isomer A 0.3 Isomer B 0.5 Kainic acid lactone (16a) Inactivea Inactivea -- 3 (KA 0.1 mM)6 Kainic acid hydroxy-lactone (mixed isomers) (16b) Inactiveb 2.0b Kainic acid iodolactone (16c) Isomer A Very weak Isomer B Inactive Isomer A Inactive Isomer B 0.3 Domoic acid (2) 0.006 0.003 APPENDIX A 113 Table S2–2. Original literature data for the activity of kainic acid analogs in functional assays. (Continued) Compound Potency IC50 (Displacement by kainate analogue of bound [3H] KA (20 nm))51 (µM) Effect on neurons 52-53 Insecticidal Activity54 IC50 for KA receptors56 (µM) Mean molar potency ratios, relative to KA76 Binding to GluR 679 [3H] kainate binding Ki57 (nM) [3H] kainate binding to KA Receptors Ki (µM) 167 Binding to GluR6 Ki 168 (nM) In vivo potencies169 Synaptosomal KA BRA Ki 81(nM) Minimum effective or used concentration ( µM) Effects Dorsal root fibers Motoneurones HumGluR 6 receptors Rat forebrain receptors Spike amplitude EC50 (nM) Spike area EC50 (nM) GluR6 binding assay Ki (nM) L-Glu (3) 0.68 No effect 0.11 ±0.01 0.02 ±0.003 KA (1) 17.8 0.3 - 0.03 Effective 1 1 53.0 ± 23.6 3.7 ± 0.20 0.01 allo-KA (6) 0.47 No effect KA methylketone (12a) 0.3 - 0.03 Effective allo-KA methylketone (12b) 0.47 No effect DA (2) 0.3 – 0.03 Effective 34 ± 2.4 2.2 ± 0.08 18.3 ±10.2 4.9 ± 0.29 228 237 3.35 2.4 DHK (13) 645.6 0.47 No effect < 0.01 < 0.01 16 CPAA (11) 1.6 25a 79.4 25b 416.8 17 0.28 No effect 18 0.26 No effect 19 0.2 No effect 21 0.24 No effect 20 0.24 Effective 23 0.19 No effect Domoilactone A (24a) Inactive Domoilactone B (24b) Inactive 22 > 100 Acro A (26) 1.7 ± 0.08 2.7 ± 0.1 Acro B (27) 13 ± 0.7 1.4 ± 0.04 MPPA (28) 0.045 ± 0.006 0.57 ± 0.05 HMPPA (29) 0.45 ± 0.04 0.91 ± 0.04 CPPA (30) 0.91 ± 0.12 0.82 ± 0.04 CNOPA (31) 0.29 ± 0.06 0.22 ± 0.02 32a 0.22 ± 0.03 0.45 ± 0.33 32b 4.2 ± 0.29 3.8 ± 0.12 32c 1.3 ± 0.13 1.9 ± 0.12 9a No effect 9b No effect 34a > 100,000 NA 34b > 100,000 NA 36a 29.8 ± 6.9 1.6 ± 0.25 36b 122.5 ± 40.9 2.2 ± 0.25 36c 41.1 ±1.8 2.6 ±0.87 36d 93.5 ± 21.5 3.5 ± 0.45 36e 57.4 ± 15.4 3.9 ±1.61 APPENDIX A 114 36f 31.7 ± 5.4 3.9 ± 0.41 36g 38.4 ± 21.7 2.7 ± 0.49 36h 107.8 ± 20.4 2.3 ± 0.90 36i 169.3 ± 23.6 9.5 ± 1.43 36j 54.3 ± 16.2 1.9 ± 0.63 36k 82.2 ± 24.3 7.7 ± 3.02 36l 146.8 ±46.4 3.6 ±2.80 36m 145.6 ± 26.2 1.9 ±0.63 36n 262.7 ± 59.7 18.5 ± 12.75 36o 131.5 ± 46.2 3.6 ± 0.59 36p 288.0 ± 105.0 9.0 ± 0.83 36q 90.6 ± 10.9 2.1 ± 0.57 36r 130.6 ± 29.8 9.1 ± 3.18 36s 275.3 ± 498.5 23.3 ± 0.47 36t 146.6 ± 52.0 2.8 ± 0.64 36u 209.6 ± 60.9 11.9 ± 2.29 36v 667.0 ± 259.8 16.0 ± 6.52 36w 126.2 ± 40.3 6.2 ± 1.10 36x 1295.7 ± 272.2 126.0 ± 34.9 36y 2423.3 ± 300.2 210.3 ± 59.3 36z 1284 ± 131.5 NA 36aa 129.3 ± 13.6 NA 36ab 663.3 ± 300.1 NA 35a 6969.0 ± 1330.8 230.0 ± 38.3 35b 2303.3 ± 802.5 5400 and 2400 35c 3109.2 ± 934.3 NA 35d > 100, 000 3000 ±100 Iso-DA A (37) Effective 887 939 130 4.4 Iso-DA B (38) Effective 5000 Iso-DA C (39) Effective 1176 ± 70 3712 4626 1176 171 Iso-DA D (40) 600 Iso-DA E (41) 600 Iso-DA F (42) 67 APPENDIX A 115 Table S2-3. Correlation of the binding affinity of analogs of compounds 34, 35, and 36 with the Hammett constants of their aromatic substituents. Compound ∑ σ (Hammett substituent constants) ∑ σ+ (Electrophilic substituent constants) [3H] Kainate binding Ki (nM) Log10Ki 1 - - 53 ± 23.6 1.7 ± 1.4 2 - - 18.3 ± 10.2 1.3 ± 1.0 34a - - >100,000 > 5 34b - - >100,000 > 5 35a - - 6,969 ± 1331 3.8 ± 3.1 35b - - 2,303 ± 802 3.4 ± 2.9 35c - - 3,109 ± 934 3.5 ± 3.0 35d - - >100,000 36a 0 0 29.8 ± 6.9 1.5 ± 0.8 36b -0.069 -0.066 122.5 ± 40.9 2.1 ± 1.6 36c -0.170 -0.311 41.1 ± 1.8 1.6 ± 0.3 36d 0.115 0.047 93.5 ± 21.5 2.0 ± 1.3 36e -0.268 -0.778 57.4 ± 15.4 1.8 ± 1.2 36f 0.277 0.114 31.7 ± 5.4 1.5 ± 0.7 36g 0.062 -0.073 38.4 ± 21.7 1.6 ± 1.3 36h -0.239 -0.377 107.8 ± 20.4 2.0 ± 1.3 36i -0.138 -0.132 169.3 ± 23.6 2.2 ± 1.4 36j -0.007 -0.139 54.3 ± 16.2 1.7 ± 1.2 36k -0.151 -0.295 82.2 ± 24.3 1.9 ± 1.4 36l - - 146.8 ± 46.4 2.2 ± 1.7 36m - - 145.6 ± 26.2 2.2 ± 1.4 36n - - 262.7 ± 59.7 2.4 ± 1.8 36o - - 131.5 ± 46.2 2.1 ± 1.7 36p - - 288.0 ± 105.0 2.5 ± 2.0 36q -0.010 0.109 90.6 ± 10.9 2.0 ± 1.0 36r 0.060 -0.179 130.6 ± 29.8 2.1 ± 1.5 36s 0.100 - 275.3 ± 598.5 2.4 ± 2.8 36t -0.320 - 146.6 ± 52.0 2.2 ± 1.7 36u -0.320 -0.500 209.6 ± 60.9 2.3 ± 1.8 36v - - 667 ± 259 2.8 ± 2.4 36w - - 126.2 ± 40.3 2.1 ± 1.6 36x - - 1,296 ± 272 3.1 ± 2.4 36y - - 2,423 ± 300 3.4 ± 2.5 36z - - 1,284 ± 131 3.1 ± 2.1 36aa - - 129.3 ± 13.6 2.1 ± 1.1 36ab - 663 ± 300 2.8 ± 2.5 APPENDIX A 116 Table S2–4. Kainate receptors reference sequences for the most commonly used organisms (mouse, rat, and humans). Ionotropic glutamate receptor organism Catalogued name Accession number Protein size (# a.a.) GluK1 mus musculus m.m. Q60934.2 836 (GluR5) m.m. isoform a NP_666184.2 934 m.m. isoform b NP_034478.1 905 m.m. isoform c NP_001333893.1 903 rattus norvegicus r.n. P22756.3 949 r.n. isoform 1 NP_001104587.1 949 r.n. isoform 2 NP_058937.1 920 r.n. isoform 3 NP_001104584.1 905 homo sapiens h.s. P39086.1 918 h.s. isoform 1 NP_000821.1 918 h.s. isoform 2 NP_783300.1 905 h.s. isoform 3 NP_001307545.1 920 h.s. isoform 4 NP_001307547.1 781 h.s. isoform 5 NP_001307550.1 763 h.s. isoform 7 NP_001317922.1 934 h.s. isoform 8 NP_001317923.1 949 GluK2 mus musculus m.m. isoform 1 NP_001104738.1 908 (GluR6) m.m. isoform 2 NP_034479.2 869 ratus norvegicus r.n. P42260.2 908 r.n. NP_062182.1 908 homo sapiens h.s. isoform 1 NP_068775.1 908 h.s. isoform 2 NP_786944.1 869 h.s. isoform 3 NP_001159719.1 892 GluK3 mus musculus m.m. NP_001074566.1 919 (GluR7) rattus norvegicus r.n. P42264.1 919 r.n. isoform 1 NP_001106187.1 919 r.n. isoform 2 NP_852038.2 910 homo sapiens h.s. NP_000822.2 919 GluK4 mus musculus m.m NP_780690.2 956 (KA1) rattus norvegicus r.n. Q01812.1 956 r.n. NP_036704.1 956 homo sapiens h.s. isoform 1 NP_001269399.1 956 h.s. isofrom 2 NP_001269402.1 956 GluK5 mus musculus m.m. NP_032194.2 979 (KA2) rattus norvegicus r.n. NP_113696.1 979 homo sapiens h.s. isoform 1 NP_001287959.1 981 h.s. isoform 2 NP_002079.3 980 APPENDIX A 117 Table S2–5. Homology analysis of GluK1 between mouse, rat, and human isoforms. APPENDIX A 118 Table S2–6. Homology analysis of GluK2, GluK3, GluK4, and GluK5 between mouse, rat, and human isoforms. Note: The amino acid numbering reference used in this review is highlighted in yellow (GluK2_rat, P42260). APPENDIX A 119 APPENDIX A 120 Table S2–7. Reported crystallized protein structures of ionotropic glutamate receptors: kainate receptors (GluK1-5), AMPA receptors (GluA1-4), and NMDA receptors (GluN1-2B). APPENDIX A 121 APPENDIX A 122 APPENDIX A 123 APPENDIX A 124 APPENDIX B 125 125 Appendix B NMR data of kainoids and comparison Table S3–1. NMR comparison between obtained kainoids and reported data Compound NMR This work Ref 1H NMR (D2O) (400 MHz) δ 5.08 (bs, 1H), 4.78 (bs, 1H), 4.18 (d, J = 3.5 Hz, 1H), 3.67 (dd, J = 11.9, 7.3 Hz, 1H), 3.46 (t, J = 11.4 Hz, 1H), 3.18 – 3.00 (m, 2H), 2.53 (dd, J = 16.9, 6.3 Hz, 1H), 2.44 (dd, J = 16.8, 8.2 Hz, 1H), 1.78 (s, 3H). (300 MHz) δ 5.11 (s, 1H), 4.82 (s, 1H), 4.15 (d, J = 3.1 Hz, 1H), 3.70 (dd, J = 11.7, 7.4 Hz, 1H) 3.50 (dd, J = 11.7, 10.5 Hz, 1H), 3.18−3.03 (m, 2H), 2.45 (dd, J = 15.6, 6.4 Hz, 1H), 2.34 (dd, J = 15.6, 8.2 Hz, 1H), 1.83 (s, 3H). 1H NMR (D2O) (400 MHz) δ 4.95 (bs, 1H), 4.94 (m, 1H), 3.92 (d, J = 8.1 Hz, 1H), 3.51 (dd, J = 11.8, 7.8 Hz, 1H), 3.33 (dd, J = 11.8, 10.6 Hz, 1H), 2.86 (dt, J = 10.5, 8.5 Hz, 1H), 2.72 – 2.59 (m, 2H), 2.41 – 2.32 (m, 1H), 1.73 (bs, 3H). (500 MHz) δ 4.99 (bs, 1H), 4.98 (bs, 1H), 3.93 (d, J = 8.7 Hz, 1H), 3.57 (dd, J = 11.8, 8.1 Hz, 1H), 3.36 (t, J = 11.2 Hz, 1H), 2.89 (dt, J = 10.0, 8.1 Hz, 1H), 2.70 (dd, J = 15.0, 4.4 Hz, 1H), 2.68-2.65 (m, 1H), 2.45 (dd, J = 14.8, 7.2 Hz, 1H), 1.75 (s, 3H). 13C NMR (D2O) (101 MHz) δ 179.3, 173.7, 140.6, 114.6, 64.9, 51.5, 48.2, 42.6, 39.7, 17.7. (125 MHz) δ 178.0, 173.6, 140.5, 115.1, 65.0, 51.5, 48.3, 42.4, 38.9, 18.0. 1H NMR (D2O) (400 MHz) δ 4.16 (d, J = 1.6 Hz, 1H), 4.07 (d, J = 14.6, 1H), 3.96 (dd, J = 14.6, 1.9 Hz, 1H), 3.55 (t, J = 6.9 Hz, 1H), 2.51 (dd, J = 14.7, 6.9 Hz, 1H), 2.44 (dd, J = 14.6, 6.9 Hz, 1H), 1.74 (bs, 3H), 1.65 (bs, 3H). (250 MHz) 4.15 (d, J = 1.2 Hz, 1H), 4.06 (d, J = 14.9 Hz, 1H), 3.94 (d, J = 14.9 Hz, 1H), 3.55 (td, J = 6.8, 1.2 Hz, 1H), 2.54 (dd, J = 14.8, 6.8 Hz, 1H), 2.46 (dd, J = 14.8, 6.8 Hz, 1H), 1.71 (s, 3H), 1.62 (s, 3H). 13C NMR (D2O) (101 MHz) δ 179.7, 173.7, 129.8, 125.7, 66.4, 46.7, 42.1, 41.2, 20.4, 20.3. (126 MHz) δ 178.1, 174.1, 131.0, 126.0, 66.7, 47.4, 42.2, 39.8, 21.2, 20.9. 1H NMR (D2O) (400 MHz) δ 7.49 – 7.34 (m, 3H), 7.27 – 7.18 (m, 2H), 4.05 (d, J = 7.3 Hz, 1H), 3.94 (dd, J = 11.4, 7.8 Hz, 1H), 3.87 (q, J = 7.8 Hz，1H), 3.72 (dd, J = 11.4, 8.1 Hz, 1H), 3.15 (q, J = 7.2 Hz, 1H), 2.38 (dd, J = 16.3, 6.5 Hz, 1H), 2.02 (dd, J = 16.3, 8.7 Hz, 1H). (500 MHz) δ 7.43-7.33 (m, 3H), 7.23 (d, J = 7.5 Hz, 2H), 4.00 (d, J = 6.9 Hz, 1H), 3.91 (dd, J = 12.0, 8.0 Hz, 1H), 3.83 (q, J = 8.0 Hz, 1H), 3.69 (dd, J = 12.0, 8.0 Hz, 1H), 3.09 (ddt, J = 8.6, 8.0, 6.9 Hz, 1H), 2.29 (dd, J = 16.0, 6.9 Hz, 1H), 1.91 (dd, J = 16.0, 8.6 Hz, 1H). 13C NMR (D2O) (101 MHz) δ 178.5, 173.3, 136.2, 128.8, 128.2, 127.6, 65.1, 48.0, 44.8, 44.0, 35.7. (126 MHz) δ 181.9, 176.2, 139.1, 131.2, 131.0, 130.4, 67.9, 50.9, 47.6, 47.0, 38.9. NHCOOHCOOH1, kainic acidNHCOOHCOOH2, allo-kainic acidNHCOOHCOOH3, iso-kainic acidNHCOOHCOOH(+)-Phenylkainic acid, 4PhAPPENDIX B 126 Table S3–2 Rules for assigning C3,4 configuration of Kainoids113 C3,4–cis kainoid C3,4-trans kainoid Rules δHa–δHb (3,4-cis) > δHa–δHb (3,4-trans) Reported data (300 MHz, D2O) δHa-b (3,4-cis) = 0.37 ppm δHa-b (3,4-cis) = 0.18 ppm Experimental data in this work (400 MHz, D2O) δHa-b = 0.36 ppm – NHCO2HCO2HPhHa HbC3,4–cisNHCO2HCO2HPhHa HbC3,4–transAPPENDIX B 127 1D NOE Experiments and Spectra NCO2MePfCO2tBuOHH3CHHHbHaNOE63aCDCl3NCO2MePfCO2tBuOHPh HHHbHaNOENOE63bCDCl3APPENDIX B 128 NOENOEN CO2MePfH2C CO2tBuH3C HHHbHaH65aCDCl3NHCO2MePhNsHHCO2MeXNOE71aCDCl3APPENDIX B 129 NHCO2MePhBocHHCO2MeXNOE71bCDCl3NHCO2MeHO2CNsHHCO2MeXNOE72aCDCl3APPENDIX B 130 NMR spectra 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)05001000150020002500300035004000ZT02-123-19.212.000.000.001.483.757.26-100102030405060708090100110120130140150160170180190f1 (ppm)0100002000030000400005000060000700008000090000ZT02-123-1-CNMR0.0027.8276.6977.0177.3382.87166.24BrOO50CDCl3APPENDIX B 131 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)-10001002003004005006007008009001000110012001300140015001600170018001900ZT02-123-29.322.000.001.463.617.26-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)-20000200040006000800010000120001400016000180002000022000240002600028000300003200034000ZT02-123-2-CNMR-2.580.0027.6576.6977.0177.3382.31167.84IOO51CDCl3APPENDIX B 132 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)-505101520253035404550556065707580ZT01-60-1-HNMR1.007.154.242.062.797.307.317.337.357.377.397.437.457.467.487.487.497.507.747.76OH7.707.757.807.85f1 (ppm)-1001020304050602.067.747.767.257.307.357.407.457.507.55f1 (ppm)-1001020304050607.154.247.307.317.337.357.377.397.437.457.467.487.487.497.500102030405060708090100110120130140150160170180190f1 (ppm)0500010000150002000025000300003500040000ZT01-60-1-CNMR76.8477.1677.4883.56120.05124.84125.40127.15128.19128.41129.01139.54143.22150.43OHHO53CDCl3APPENDIX B 133 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)-20-100102030405060708090100110120130140150160170180190200210220ZT01-61-1-HNMR5.292.083.992.00-0.007.237.237.247.247.247.257.257.257.257.267.267.267.277.277.277.287.287.287.287.297.297.297.317.317.347.357.367.377.387.387.527.527.527.527.547.547.547.557.557.557.567.567.577.577.667.677.677.687.687.687.69Br7.507.557.607.657.70f1 (ppm)0501001502003.992.007.527.527.527.527.547.547.547.557.557.577.577.667.677.677.687.687.687.697.207.257.307.357.40f1 (ppm)0501001502005.292.087.237.257.257.257.257.267.277.277.277.287.297.297.317.317.347.357.367.370102030405060708090100110120130140150160170180190f1 (ppm)-5000050001000015000200002500030000350004000045000ZT01-61-1-CNMR0.0067.4676.6876.9977.31120.34126.09127.43128.01128.31128.51128.99138.09141.12149.61BrBr54CDCl3APPENDIX B 134 0.00.51.01.52.02.53.03.54.04.55.0f1 (ppm)01002003004005006007008009001000ZT01-42-1-HNMR1.001.011.010.982.932.122.292.302.312.322.332.352.362.492.492.502.512.512.522.522.532.532.542.552.553.423.423.433.453.463.463.543.543.573.583.884.794,25''5' 3''3'45N+123H1aH1bOH86O7O9 CH310Cl-11-100102030405060708090100110120130140150160170180190200f1 (ppm)-10000100020003000400050006000700080009000ZT01-42-1-CNMR36.6353.3953.7353.8058.0658.1169.3369.39170.1645N+123H1aH1bOH86O7O9 CH310Cl-11NHHOCO2CH3独HCl59D2OAPPENDIX B 135 -1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)-200020040060080010001200140016001800200022002400ZT01-64-1-HNMR1.000.981.073.040.941.001.010.971.664.181.153.201.111.093.243.263.283.293.307.157.167.177.177.227.237.237.247.247.247.257.267.277.277.287.287.287.297.307.317.327.327.327.327.337.347.347.347.417.417.437.437.537.537.547.547.547.557.557.557.567.567.567.637.647.647.647.657.657.667.667.737.737.737.747.757.757.757.75 1025' 3'5''4 3''45N123OH86O7O9 CH310Pf3.223.243.263.283.303.32f1 (ppm)-10001002003004003.040.943.243.263.283.293.301.751.801.851.901.952.002.05f1 (ppm)01002003001.000.981.761.771.781.791.791.801.811.831.951.961.971.981.982.002.01-100102030405060708090100110120130140150160170180190f1 (ppm)-4000-200002000400060008000100001200014000160001800020000220002400026000280003000032000340003600038000ZT01-64-1-CNMR39.9151.3456.8059.2770.3376.1076.7077.0277.2177.33119.82120.05126.42127.11127.18127.25127.34127.61128.31128.41128.73139.85141.44142.66146.06147.19175.94NHOCO2HPf60CDCl3APPENDIX B 136 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)-50050100150200250300350400450500550600650700750800ZT02-65-1-HNMR1.001.002.870.991.961.543.402.973.392.200.002.312.312.322.322.322.412.412.432.433.213.453.463.463.503.503.503.743.753.753.773.783.797.247.247.257.257.267.377.387.387.397.397.397.407.417.417.417.427.437.437.447.457.457.457.467.467.467.477.477.477.487.707.707.717.717.717.717.727.727.727.737.737.7310523''3'45N123O86O7O9 CH310Pf-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)-2000-10000100020003000400050006000700080009000100001100012000130001400015000160001700018000190002000021000ZT02-65-1-CNMR-0.0041.6451.5051.5255.2258.1875.9876.6877.0077.32120.09120.25125.51126.87126.95127.55127.69128.03128.55128.85128.94140.34140.89141.80145.33146.49173.07212.86NOCO2MePf61CDCl3APPENDIX B 137 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)01002003004005006007008009001000ZT02-67-2-HNMR9.442.031.053.222.061.002.193.223.453.362.19-0.001.311.551.552.392.402.412.422.792.812.812.822.832.843.113.423.443.453.473.753.807.247.247.257.267.267.267.277.277.277.287.297.297.357.367.367.377.387.387.397.397.407.417.437.497.497.507.517.517.697.697.697.697.711385'',25'345N1236O1189O14O1015CH316CH317CH318O12O7CH313Pf3.403.423.443.463.483.50f1 (ppm)-500501001502.063.423.443.453.473.703.743.783.82f1 (ppm)0501001501.003.753.80-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)-500005000100001500020000250003000035000400004500050000550006000065000ZT01-67-2-CNMR27.8734.1949.2051.5155.6863.6775.7576.7177.0277.2277.3481.41119.96120.18125.98126.97127.27127.70127.84127.94128.48128.87140.42141.05141.44144.35145.97169.59172.85211.82NOCO2MePfCO2tBu62aCDCl3APPENDIX B 138 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)0100200300400500600700800900ZT02-67-1-1-HNMR8.821.031.021.173.001.041.011.002.812.925.361.131.110.001.341.801.821.851.872.542.552.582.603.053.073.073.093.103.113.113.153.563.563.603.603.783.824.014.037.237.237.237.247.247.257.257.267.287.297.297.317.317.337.337.347.347.367.367.377.387.397.397.407.407.417.417.427.427.427.437.437.677.697.727.727.727.74-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)-2000-100001000200030004000500060007000800090001000011000120001300014000150001600017000180001900020000ZT02-67-1-1-CNMR-0.0027.8931.0548.0451.1153.7061.8875.3976.6977.0177.2177.3281.07120.14125.35126.70126.77127.59127.72128.12128.61128.83128.85139.78141.25141.73145.26146.90170.26171.65212.16NOOOCH3CH3CH3OO CH3PfNOCO2MePfCO2tBu62bCDCl3APPENDIX B 139 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-100010020030040050060070080090010001100120013001400150016001700180019002000ZT02-73-1-HNMRZT02-73-1-29.211.122.911.101.011.112.133.080.971.041.002.613.801.294.351.130.001.301.781.821.961.982.002.022.192.212.232.252.822.842.852.862.873.043.063.073.093.183.713.744.915.137.137.157.177.217.237.257.257.267.277.287.297.317.337.347.357.367.427.447.457.557.577.587.607.617.637.737.7418,20,2115191113' 2a,5b13''5a 8' 8''3a5N1234OH11128H3aH2a6H5aH5bCH319CH213O7O14CH3159O10O1617CH318CH320CH321Pf0102030405060708090100110120130140150160170180190200f1 (ppm)-50000500010000150002000025000300003500040000450005000055000ZT02-73-1-CNMRZT02-73-1-219.6227.8732.2746.9851.3261.2166.3176.6877.0077.2077.3280.6081.93112.95119.71120.11126.21126.97127.32127.37127.67127.90128.33128.44128.95139.69141.68142.83144.58146.37146.78171.47174.9018,20,212 19315481712 59 1365N1234OH11128H3aH2a6H5aH5bCH319CH213O7O14CH3159O10O1617CH318CH320CH321PfN CO2MePfCO2tBuHO63aCDCl3APPENDIX B 140 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)05000100001500020000250003000035000ZT03-63-1-HNMRZT03-63-19.211.071.041.031.194.171.001.001.151.481.000.953.872.171.204.452.291.130.001.181.542.092.112.172.182.403.203.233.343.373.853.887.187.187.197.207.207.217.217.217.227.237.237.237.237.247.247.247.257.257.267.297.297.307.317.317.317.337.367.367.377.377.397.457.467.547.547.557.557.567.567.567.577.577.587.637.647.647.647.657.667.75 11,12,132,26171415 6'6''3N123456 7O8O910 CH311CH312CH313H14H1516OH17181920212223O24O25 CH326Pf-100102030405060708090100110120130140150160170180190f1 (ppm)-100000100002000030000400005000060000700008000090000100000110000120000130000140000ZT03-63-1-CNMRZT03-63-10.0027.7432.4851.1251.3965.3466.3676.4976.5576.6976.8177.0177.2177.3380.6981.07119.80120.19125.58126.09127.08127.12127.32127.36127.82127.89128.15128.36128.52129.03139.82141.62141.75142.79146.50146.70171.41174.8611,12,13263241065723N123456 7O8O910 CH311CH312CH313H14H1516OH17181920212223O24O25 CH326PfN CO2MePfCO2tBuHO63bCDCl3APPENDIX B 141 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)-200-10001002003004005006007008009001000110012001300140015001600170018001900200021002200ZT02-101-1-HNMRZT02-101-19.023.110.990.991.063.251.061.193.030.900.990.974.871.432.341.262.281.241.201.311.902.042.062.082.102.402.412.442.452.802.813.153.153.173.193.653.673.683.683.693.963.994.874.967.167.167.187.187.197.207.217.217.237.237.247.247.257.267.267.267.287.287.307.307.367.367.377.387.387.397.407.407.437.457.527.527.537.547.557.617.637.697.70 20,24,2517222315''5a15'2a5b8' 8''3a5N1234O11148H3aH2a6H5aH5bO7O16CH3179O18 O1019CH320CH324CH325CH323CH21512O13O21CH322Pf0102030405060708090100110120130140150160170180190200f1 (ppm)050000100000150000200000250000300000350000400000450000ZT02-101-1-CNMRZT02-101-120.3627.9132.5148.1851.2754.3955.6365.9776.1876.7577.0777.2777.3980.4590.08119.66120.00126.04126.86127.27127.35127.41128.05128.28128.43128.61140.14141.14141.89142.46146.12146.15153.64170.77173.9920,24,2523217194 32296 851514125N1234O11148H3aH2a6H5aH5bO7O16CH3179O18 O1019CH320CH324CH325CH323CH21512O13O21CH322PfAPPENDIX B 142 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)050010001500200025003000350040004500ZT03-65-1-HNMRZT03-65-19.021.051.041.063.091.033.081.001.002.194.386.851.113.161.08-0.001.222.363.123.273.293.673.903.934.324.357.177.177.197.217.237.247.247.247.247.257.257.257.257.257.267.267.277.277.277.297.297.297.337.347.357.357.377.377.377.387.397.397.397.417.417.427.427.437.447.447.497.517.607.607.627.627.627.627.627.647.647.717.71 12,13,14282525''5'7' 7''3N12345 67 8O9O1011 CH312CH313CH31415O16171819202122O23O24CH325O26O27CH328Pf-100102030405060708090100110120130140150160170180190f1 (ppm)-50000050000100000150000200000250000300000350000400000450000500000550000600000ZT03-65-1-CNMRZT03-65-1-0.0027.8332.5251.2552.7054.5157.7866.3676.2676.7177.0377.2377.3580.4289.51119.67120.07125.02126.11126.88127.32127.39127.46127.57128.07128.33128.40128.46128.68139.20140.09141.27142.34145.95146.19153.69170.29173.8112,13,142842 3251186 7522N12345 67 8O9O1011 CH312CH313CH31415O16171819202122O23O24CH325O26O27CH328PfN CO2MePfCO2tBuOOMeO64bCDCl3APPENDIX B 143 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)050010001500200025003000350040004500ZT02-102-2-HNMRZT02-102-29.132.941.010.961.021.030.963.001.360.932.051.053.272.951.353.211.451.18-0.001.291.691.941.951.981.992.042.052.082.102.262.282.602.692.713.223.313.333.343.363.403.433.464.754.754.754.767.097.097.117.117.137.137.207.217.227.237.237.237.267.287.307.317.327.337.347.357.357.427.437.447.457.467.467.517.517.527.547.607.617.737.747.7517,19,201418122a5b5a8'8''4a3a5N1234H4a118H3aH2a6H5aH5bCH318CH212O7O13CH3149O10O1516CH317CH319CH320Pf0102030405060708090100110120130140150160170180190f1 (ppm)-100000100002000030000400005000060000700008000090000100000110000120000130000140000150000ZT02-102-2-CNMRZT02-102-20.0019.0027.9137.3043.8751.2552.1555.0567.5276.7077.0177.3380.30113.48119.69120.01125.73127.13127.16127.19127.31127.98128.22128.32128.69139.29142.12142.74143.94146.77147.60170.73175.4317,19,203 18241481211 59 1665N1234H4a118H3aH2a6H5aH5bCH318CH212O7O13CH3149O10O1516CH317CH319CH320PfN CO2MePfCO2tBu65aCDCl3APPENDIX B 144 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)-20002004006008001000120014001600180020002200240026002800ZT02-102-1-HNMRZT02-102-18.703.333.062.111.153.001.350.960.943.044.803.432.13-0.001.331.571.622.282.292.322.332.342.362.382.402.842.862.862.882.893.023.303.303.333.743.774.004.037.287.287.297.297.317.317.337.347.357.357.377.397.447.467.467.477.487.487.647.657.677.69 17,1 ,20141218285' 5'' 35N1234118CH312CH3186O7O13CH3149O10O1516CH317CH319CH320Pf-100102030405060708090100110120130140150160170180190f1 (ppm)-20000020000400006000080000100000120000140000160000180000200000220000240000ZT02-102-1-CNMRZT02-102-10.0020.4321.2927.9440.1442.9849.1950.6764.9674.5276.6977.0177.3380.55119.69119.86123.39125.56126.91127.06127.28127.35127.74128.23128.30128.35133.16139.79140.93142.70146.05147.82171.23173.9017,19,2014 3 12218854 11 166 95N1234118CH312CH3186O7O13CH3149O10O1516CH317CH319CH320PfN CO2MePfCO2tBu65bCDCl3APPENDIX B 145 0.00.51.01.52.02.53.03.54.04.55.05.56.0f1 (ppm)-2000020004000600080001000012000140001600018000200002200024000ZT03-48-1-HNMRZT03-48-13.121.002.150.991.051.010.891.021.091.732.332.352.372.392.602.612.632.632.642.652.662.662.672.672.682.692.702.832.852.862.872.882.903.303.333.333.363.493.513.523.543.913.934.794.934.934.944.944.944.9589''9'3,6'25''5' 6''4NH1234567CH38CH2910O11OH1213O14OH15-100102030405060708090100110120130140150160170180190f1 (ppm)-50000500010000150002000025000300003500040000ZT03-48-1-CNMRZT03-48-117.6939.7142.6248.2151.4564.85114.57140.64173.69179.2634 82 5 69710 13NH1234567CH38CH2910O11OH1213O14OH15NHCO2HCO2H6D2OAPPENDIX B 146 -0.20.20.61.01.41.82.22.63.03.43.84.24.65.05.4f1 (ppm)-50050100150200250300350400450500550ZT03-53-1-HNMRZT03-53-13.003.101.111.001.121.101.130.991.651.742.422.432.452.472.492.512.522.543.543.553.563.943.953.983.984.054.084.164.164.798926''6'5' 35''NH1234567CH38CH3910O11OH1213O14OH15-100102030405060708090100110120130140150160170180190f1 (ppm)-10000-500005000100001500020000250003000035000400004500050000550006000065000700007500080000ZT03-53-1-CNMRZT03-53-120.3020.4441.1742.1546.6566.37125.67129.75173.71179.699832 56471013NH1234567CH38CH3910O11OH1213O14OH15NHCO2HCO2H55D2OAPPENDIX B 147 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)-20002004006008001000120014001600180020002200240026002800ZT03-21-1-HNMRZT02-21-118.403.031.041.031.023.143.051.170.941.021.001.001.371.381.381.401.812.342.362.382.402.402.402.602.612.642.652.762.772.782.792.792.802.812.823.683.713.793.823.833.833.863.953.984.024.044.424.464.885.037.26 20,29,30,25,31,3227222817'17''5' 5''2 10'3 10''45N12 8310166O18O719CH320 CH329CH330CH328CH217O1314O15O911O23O1224CH325CH331CH332O26CH327O21 CH3220102030405060708090100110120130140150160170180190200f1 (ppm)-100000100002000030000400005000060000700008000090000100000110000120000130000140000ZT03-21-1-CNMRZT02-21-10.0620.3520.5228.0728.2931.6431.9346.6347.2252.0752.2452.3252.4554.6754.7763.0163.5476.8477.1677.3677.4880.7081.0389.7090.47114.09139.35139.45153.26153.40153.50154.22170.62170.65172.35172.54 20,29,30,25,31,321728192432 22 105274161186 21445N12 8310166O18O719CH320 CH329CH330CH328CH217O1314O15O911O23O1224CH325CH331CH332O26CH327O21 CH322N CO2MeBocCO2tBuOOMeO66aCDCl3APPENDIX B 148 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)-200-10001002003004005006007008009001000110012001300140015001600170018001900200021002200ZT03-68-1-HNMRZT03-68-18.608.962.041.003.002.891.020.960.983.072.061.311.431.462.172.472.492.522.542.552.562.562.572.582.862.872.882.882.892.892.902.912.923.703.754.114.124.134.144.184.204.244.274.314.654.687.277.287.287.287.287.297.297.297.307.307.317.317.327.327.337.367.367.367.377.387.387.397.397.407.40 29,30,3110,11,1235162'172''4512N3456O7O89CH310CH311CH31213O14O15CH31617O1819202122232425O26O2728CH329CH330CH33132O33O34CH3350102030405060708090100110120130140150160170180190200f1 (ppm)-20000-100000100002000030000400005000060000700008000090000100000110000120000130000140000150000160000170000180000190000200000210000220000230000240000ZT03-68-1-CNMRZT03-68-1-0.0327.9127.9728.2328.3230.8931.3631.7251.3651.9852.1552.3753.6153.8954.7354.8463.4563.9976.7176.9777.0277.1977.2377.3480.6380.7280.8880.9289.2589.95124.69124.73128.17128.20128.83128.84136.15153.25153.38153.51154.13170.03170.07172.19172.34 29,30,3110 11,1220,2421,2322 1649283517225 19133212N3456O7O89CH310CH311CH31213O14O15CH31617O1819202122232425O26O2728CH329CH330CH33132O33O34CH335N CO2MeBocCO2tBuOPhOMeO66bCDCl3APPENDIX B 149 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)-20020406080100120140160180200220240260280300320340ZT03-28-25.822.112.151.951.021.017.371.051.151.001.891.060.001.701.722.993.013.033.313.323.343.353.353.363.373.393.413.423.443.453.473.473.493.733.754.704.884.894.894.914.914.927.291.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.0f2 (ppm)1.01.52.02.53.03.54.04.55.0f1 (ppm)ZT03-28-1-gCOSYN CO2MeBocCO2tBuN CO2MeBocCO2tBu+67a + 67EPL[WXUH&'&OAPPENDIX B 150 1.51.71.92.12.32.52.72.93.13.33.53.73.94.14.34.54.74.95.15.3f1 (ppm)-50050100150200250300350400450500550600650700750ZT03-40-1-HNMRZT03-40-13.000.970.940.971.171.001.031.051.011.782.412.432.452.472.502.512.542.553.033.053.073.083.093.103.113.123.133.143.153.163.443.463.493.643.663.673.694.184.185.075.075.085.085.081.52.02.53.03.54.04.55.0f2 (ppm)1.52.02.53.03.54.04.55.0f1 (ppm)ZT03-40-1-gCOSYZT03-40-1NHCO2HCO2HNHCO2HCO2H+allo-kianic acid (1) + kainic acid (6) mixtureD2OAPPENDIX B 151 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-200002000400060008000100001200014000160001800020000220002400026000ZT03-73-1-HNMRZT03-73-10.960.930.971.021.051.004.802.963.013.183.203.233.253.703.713.723.733.733.743.743.903.934.134.164.364.374.797.477.487.497.497.507.507.517.517.517.527.527.547.557.5611,12,13,14,152''2'4 6''6'512NH34567O8O91011121314 1516O17OH18-100102030405060708090100110120130140150160170180190f1 (ppm)-50000500010000150002000025000300003500040000450005000055000ZT03-73-1-CNMRZT03-73-134.6638.8939.1039.3139.5239.7339.9440.1549.4958.1167.5895.01124.72128.15128.71139.52171.60175.4612,1411,15513616 10 4 27 112NH34567O8O91011121314 1516O17OH18NHCO2HOOPh独TFA68D2ODMSO-d6 APPENDIX B 152 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)-200-100010020030040050060070080090010001100120013001400150016001700180019002000210022002300ZT04-5-1-HNMRZT04-5-11.051.081.064.121.061.001.373.802.642.652.692.692.712.732.752.773.063.063.063.063.073.073.073.073.073.073.353.363.373.373.463.463.473.473.473.483.483.493.493.503.683.683.713.843.874.494.504.877.217.227.227.227.237.237.247.247.247.247.247.247.257.257.267.267.267.26 2'',1911,12,14,152' 6'46''13512NH34567O8O91011121314 1516O17O18 CH3192030405060708090100110120130140150160170180f1 (ppm)01000002000003000004000005000006000007000008000009000001000000ZT04-5-1-CNMRZT04-5-134.2448.3648.5748.7949.0049.2149.4349.6450.4254.4057.5565.4794.36125.98130.31130.46137.32169.01175.001311,1512,1454 62 1917 161012NH34567O8O91011121314 1516O17O18 CH319NHCO2CH3OOPh独TFA69aCD3ODAPPENDIX B 153 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-200020040060080010001200140016001800200022002400260028003000320034003600ZT04-38-1-HNMRZT04-38-19.442.121.003.040.910.361.050.481.103.940.001.441.462.632.662.692.712.732.742.742.762.792.802.812.822.842.863.173.183.193.213.233.833.863.883.893.914.214.244.264.284.314.404.417.277.347.357.367.377.377.387.397.407.417.421321,22,24,2517,18,192'23 72''44 512N345678O910O11O12CH313O14O1516CH317CH318CH3192021222324 25O26-100102030405060708090100110120130140150160170180190f1 (ppm)-100000100002000030000400005000060000700008000090000100000110000120000130000140000150000160000170000180000ZT04-38-1-CNMRZT04-38-10.0028.2028.2634.9235.0650.4851.4452.6052.7558.6459.1565.7565.9176.7277.0377.2377.3581.2081.2793.4194.36124.75124.78128.78128.84128.96128.99137.24137.50152.98153.95171.96172.10174.1917,18,192322,2421,255410 725161318 201612N345678O910O11O12CH313O14O1516CH317CH318CH3192021222324 25O26N CO2CH3OOPh69bBocCDCl3APPENDIX B 154 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-200-1000100200300400500600700800900100011001200130014001500160017001800190020002100220023002400ZT04-8-1-HNMRZT04-8-13.521.271.340.911.141.863.751.041.012.625.221.982.192.212.242.252.302.302.322.322.342.352.362.373.223.243.253.263.273.303.303.303.313.313.783.793.803.813.813.913.953.973.974.584.604.884.887.327.337.337.347.347.347.357.367.367.367.367.377.387.387.397.407.407.40162''4 6''6'1 52'12NH345678910111213O14O15 CH31617O18OH190102030405060708090100110120130140150160170180190f1 (ppm)-20000-100000100002000030000400005000060000700008000090000100000110000120000130000140000150000160000170000180000190000200000210000220000ZT04-8-1-CNMRZT04-8-132.9742.4543.8846.9447.1647.3747.5947.8048.0148.2253.0853.3858.1468.19127.74128.23128.60134.43168.87172.86173.76514 1626131712NH345678910111213O14O15 CH31617O18OH19NHCO2CH3Ph CO2H·AcOH70aCD3ODAPPENDIX B 155 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)-50050100150200250300350400450500550600650700750800850ZT04-39-1-HNMRZT04-39-19.431.161.001.041.190.433.400.641.070.590.442.043.140.001.451.492.052.052.062.072.092.102.112.112.252.272.292.302.312.322.342.352.942.962.972.982.993.003.023.043.063.083.733.743.743.823.833.833.843.863.893.913.923.933.943.953.963.984.014.024.134.157.057.067.077.087.087.097.267.277.307.317.327.33 10,11,122123,24,2522,264 15''12'4 52''2'' 15'12N3456O7O89CH310 CH311CH312131415 16O17OH18O19O20CH3212223242526-100102030405060708090100110120130140150160170180190f1 (ppm)-500005000100001500020000250003000035000400004500050000ZT04-39-1-CNMRZT04-39-10.0028.3028.4333.1143.2944.1244.3045.2150.3850.8352.2652.4563.0363.3276.7077.0277.3480.6380.72127.36127.67127.70128.92138.38138.53153.54154.27172.53172.75176.7510,11,1222,2623,2524921 542141316 611512N3456O7O89CH310 CH311CH312131415 16O17OH18O19O20CH3212223242526N CO2CH3Ph CO2HBoc70bCDCl3APPENDIX B 156 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-200-10001002003004005006007008009001000110012001300140015001600170018001900200021002200ZT04-42-1-HNMRZT04-42-19.451.001.001.082.981.040.273.270.510.980.941.980.722.220.001.431.451.451.491.502.002.012.022.032.042.052.062.072.222.242.262.272.282.312.332.993.003.023.043.613.613.633.633.683.693.713.723.773.783.783.813.813.823.843.853.903.923.933.943.963.963.973.973.994.014.034.114.127.267.297.297.307.317.317.317.337.337.33 10,11,1227264 12' 5 14''2''14'12N3456O7O89CH310 CH311CH312131415161718192021O22O23O24O25CH326CH3270102030405060708090100110120130140150160170180190f1 (ppm)-100000100002000030000400005000060000700008000090000100000110000120000130000140000ZT04-42-1-CNMRZT04-42-1-0.0028.3128.4433.2133.2543.4944.2944.3245.2050.2350.7451.7251.7552.2352.4263.1363.4276.7077.0277.3380.52127.26127.70127.74128.81138.44138.62153.50154.22172.08172.11172.51172.7617,2110,11,1218,201915144 226279275115 212N3456O7O89CH310 CH311CH312131415161718192021O22O23O24O25CH326CH327N CO2CH3Ph CO2CH3Boc71bCDCl3APPENDIX B 157 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)-50050100150200250300350400450500550600650ZT05-40-1-HNMRZT05-40-11.001.031.031.021.051.001.002.041.062.041.881.911.921.952.262.282.302.323.083.083.103.103.113.123.673.693.703.723.813.833.853.873.903.923.933.954.014.024.797.237.237.237.247.257.257.267.357.357.367.377.377.387.387.397.407.417.417.417.427.437.437.447.457.457.4512,1411,152135'5'' 6''6'43NH1234567O8OH9101112131415 16O17OH183.73.83.94.0f1 (ppm)01002003001.021.051.001.003.673.693.703.723.813.833.853.873.903.923.933.954.014.027.207.257.307.357.407.45f1 (ppm)-10001002003004005006002.041.062.047.237.237.237.247.257.257.267.377.387.387.397.417.417.417.427.437.437.450102030405060708090100110120130140150160170180190200210f1 (ppm)-4000-200002000400060008000100001200014000160001800020000220002400026000280003000032000340003600038000ZT05-40-1-CNMRZT05-40-136.2144.3144.8548.0665.13127.50128.21128.74136.37173.43179.25NHCO2HPh CO2H32aD2OAPPENDIX B 158 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)05001000150020002500ZT04-56-1-HNMRZT04-56-11.941.153.043.152.661.002.053.292.142.150.001.992.002.012.023.003.013.023.043.043.053.053.063.073.083.603.783.783.793.793.803.807.047.047.057.057.067.067.267.277.287.287.287.287.297.297.307.307.317.317.317.327.328.128.148.388.4128321,210,1411,1317,18,19416,20 21512N345S6O78O91011121314151617181920 21N+22O23O-2425O26O27 CH32829O30O31CH332-100102030405060708090100110120130140150160170180f1 (ppm)0100002000030000400005000060000700008000090000ZT04-56-1-CNMRZT04-56-10.0032.4644.4045.2150.6651.9152.8765.1676.7277.0377.35124.23127.65127.72128.77128.96136.40144.46150.20171.41171.6310,1417,1911,131816,204 51212823215252981212N345S6O78O91011121314151617181920 21N+22O23O-2425O26O27 CH32829O30O31CH332N CO2CH3Ph CO2CH3Ns71aCDCl3APPENDIX B 159 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-100010020030040050060070080090010001100120013001400150016001700ZT04-50-1-HNMRZT04-50-19.481.081.141.093.122.830.971.001.903.240.001.261.532.062.082.102.122.262.292.312.337.137.147.157.267.297.317.327.347.3614,15,16281121,22,2320,242 4 6' 6''3N123456O78O9O10 CH311O1213CH314CH315CH31617O18192021222324 25O26O27CH328-100102030405060708090100110120130140150160170180190f1 (ppm)-4000-200002000400060008000100001200014000160001800020000220002400026000280003000032000340003600038000ZT04-50-1-CNMRZT04-50-10.0027.9434.4436.9551.4351.8952.8762.0076.6977.0177.3384.20127.91128.82129.53133.20149.51170.89171.33171.94N123456O78O9O10 CH311O1213CH314CH315CH31617O18192021222324 25O26O27CH328N CO2CH3Ph CO2CH3BocO72bCDCl3APPENDIX B 160 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)-50050100150200250300350400450500550600ZT04-57-1-HNMRZT04-57-11.041.011.041.063.151.692.800.862.212.22-0.002.042.062.352.372.392.412.482.502.532.552.952.972.993.013.293.293.303.353.683.703.713.723.753.784.204.227.268.068.088.358.37 292511,1310,14241 5 18''18'12N345S67O8O91011 12131415O16OH1718N+19O20O-2122O23O24CH32526O27O28CH3292.352.402.452.502.55f1 (ppm)0501.041.012.352.372.392.412.482.502.532.55-100102030405060708090100110120130140150160170180190f1 (ppm)-100000100002000030000400005000060000700008000090000100000ZT04-57-1-CNMRZT04-57-10.0032.5642.1345.5149.4252.2152.9964.6876.7077.0277.2277.34124.21128.84143.59150.31171.00171.29175.8811,1310,14292512422261857 211512N345S67O8O91011 12131415O16OH1718N+19O20O-2122O23O24CH32526O27O28CH329N CO2CH3HO2C CO2CH3Ns72aCDCl3APPENDIX B 161 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)-2000200400600800100012001400160018002000220024002600280030003200ZT04-58-1-HNMRZT04-57-16.304.352.292.331.040.991.161.861.216.353.040.993.660.890.931.061.081.071.981.941.010.97-0.001.231.251.261.261.341.351.391.411.431.581.601.611.652.252.282.292.392.412.452.983.003.003.023.063.083.083.103.273.273.293.293.413.423.433.443.443.453.473.493.653.673.693.703.743.764.214.226.246.516.526.656.656.676.687.277.477.498.058.078.368.388.688.83502',5133,3431,32,3926 10,1411,1341,422225 4402''4324 18'4418''45513712 N345S67O8O91011 1213141516O171819202122232425 262728O29N303132CH333CH334O3536NH37O38394041424344NH45O46O47O48O49CH350CH351N+52O53O-540102030405060708090100110120130140150160170180190f1 (ppm)-20000-100000100002000030000400005000060000700008000090000100000110000120000130000140000150000160000170000180000190000200000210000220000ZT04-58-1-CNMRZT04-58-1-0.0012.4325.5725.6628.7729.3032.2938.7238.7442.8045.0945.7649.9652.0252.8565.2796.50108.37110.04124.17128.95131.23143.38148.10150.19152.62162.87163.31169.27171.30171.9710,1433,3411,1331,322316 50 401939441 524 451414225 22261528364321 2018122712 N345S67O8O91011 1213141516O171819202122232425 262728O29N303132CH333CH334O3536NH37O38394041424344NH45O46O47O48O49CH350CH351N+52O53O-54O OEt2NOHNNCO2CH3CO2CH3NsNHO73CDCl3APPENDIX C 162 162 Appendix C 1D NOE spectra of compounds in Chapter 4 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)12ZT06-01-1-NOESY1DZT06-01-1ZT06-01-1-HNMRZT06-01-14 2'',16' 15'16'' 15''12'512N345S6O7O8910111213141516OH17N+18O19 O-2021O22O23 CH32425O26O27 CH328N CO2CH3CO2CH3NsH HHNOEHO6%2%85CDCl3APPENDIX C 163 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)12ZT06-04-1-NOESY1DZT06-04-1ZT06-04-1-HNMRZT06-04-113,1512,161848'2''8''2' 1 512N345S67 8O910O111213141516O17H1819O20O21 CH322N+23O24O-2526O27O28CH329N CO2CH3CO2CH3NsH HHNOEHO5%2%86aCDCl3APPENDIX C 164 N CO2CH3CO2CH3NsH HHNOE3%1%86aCDCl3-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)12ZT06-05-1-NOESY1D_002ZT06-05-1ZT06-05-1-HNMRZT06-05-1212812,1613,15174 2'2''8' 8''5112N345S67 8O910O111213141516CH1718O19O20 CH321N+22O23O-2425O26O27CH328APPENDIX C 165 NMR spectra of compounds in Chapter 4 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)ZT06-01-1-HNMRZT06-01-10.981.060.870.900.981.941.033.042.780.881.942.002.392.412.432.452.552.552.572.582.592.602.602.612.632.652.662.682.882.892.902.912.912.922.932.943.423.433.443.463.503.503.513.523.523.533.543.553.573.593.603.623.643.703.744.304.317.268.078.088.088.088.098.108.108.118.358.368.368.388.388.388.394 2'',16' 15'16'' 15''2'1512N345S6O7O8910111213141516OH17N+18O19 O-2021O22O23 CH32425O26O27 CH328-100102030405060708090100110120130140150160170180190200f1 (ppm)ZT06-01-1-CNMRZT06-01-10.0032.3541.6542.1349.5052.1552.7860.4465.5876.7077.0277.2277.34124.17128.81144.49150.17171.40172.121541516212 N345S6O7O891011 1213141516OH17N+18O19O-2021O22O23CH32425O26O27CH328N CO2CH3CO2CH3NsHO85CDCl3APPENDIX C 166 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)ZT06-04-1-HNMRZT06-04-10.951.000.961.091.273.112.910.860.832.262.150.73-0.002.362.382.402.422.482.502.522.543.103.123.133.133.453.463.473.483.503.503.533.573.583.593.603.613.623.633.663.673.673.683.683.693.693.693.703.703.713.713.713.723.733.733.743.753.753.753.763.773.783.783.793.823.843.853.864.244.257.268.048.078.078.388.409.629.639.639.6312,1613,1518 48'2''8''2'1 52.352.402.452.502.55f1 (ppm)0.951.002.362.382.402.422.482.502.522.544.204.214.224.234.244.254.264.274.28f1 (ppm)0.834.244.2512N345S67 8O910O111213141516O17H1819O20O21 CH322N+23O24O-2526O27O28CH329-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT06-04-1-CNMRZT06-04-1-0.0332.3941.5046.4551.3752.2152.9865.2076.7177.0377.35124.30128.72143.80150.29170.74171.18198.29N CO2CH3OHC CO2CH3Ns86aCDCl3APPENDIX C 167 N CO2CH3OHC CO2CH3Ns86a 86b2.3 ғ1N CO2CH3OHC CO2CH3NsmixtureCDCl3-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)ZT06-25-11.000.900.280.67-0.002.362.382.402.422.482.502.522.542.562.582.622.643.123.473.483.593.613.623.633.663.663.683.693.693.703.703.703.713.713.713.723.733.733.743.743.753.753.753.773.773.823.843.853.864.114.134.244.254.334.347.278.048.068.078.098.378.388.388.398.408.409.589.589.629.639.639.63trans isomercis isomerAPPENDIX C 168 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)ZT06-05-1-HNMRZT06-05-10.831.001.040.930.981.212.881.322.770.902.061.950.001.921.932.432.452.472.492.702.722.742.762.792.802.812.812.822.822.832.842.843.293.303.313.313.323.333.333.333.613.623.633.643.693.703.713.723.723.733.743.794.074.097.268.058.068.068.078.088.088.098.358.368.368.378.388.39212812,1613,15174 2'2''8' 8''5112N345S67 8O910O111213141516CH1718O19O20 CH321N+22O23O-2425O26O27CH328-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT06-05-1-CNMRZT06-05-10.0032.8533.5843.3352.0752.9753.2164.0974.0776.7076.7677.0277.2277.3479.01124.20128.94143.76150.28170.98171.3212,1613,155 117 4821228182571014N CO2CH3CO2CH3Ns87aCDCl3APPENDIX C 169 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)ZT06-26-1-HNMRZT06-26-10.981.142.201.271.250.613.630.303.271.031.002.582.502.172.172.572.592.612.632.632.692.702.712.722.722.732.742.752.762.762.772.782.793.313.343.363.683.733.923.933.943.964.314.338.078.098.378.391110 6,7'2''2'7''912N345H6 78H9H10CH1112 O13O14CH31516 O17O18 CH319202122232425N+26O27 O-28S29 30O31-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT06-26-1-CNMRZT06-26-1-0.0034.3434.7546.7351.9652.2652.7764.4572.5676.5376.6976.9277.0177.2177.3379.52124.29128.76144.58150.26170.79170.85N CO2CH3CO2CH3Ns87bCDCl3APPENDIX C 170 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)ZT06-08-1-HNMRZT06-08-10.961.300.831.000.980.931.201.023.142.802.192.202.612.612.622.632.642.652.662.662.662.662.672.672.682.682.692.692.692.702.702.722.722.742.802.802.812.812.832.852.872.872.872.883.073.083.093.103.173.183.183.193.193.203.203.213.213.223.313.323.333.353.353.563.583.703.757.26 121682'7''57'4 2''10102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT06-08-1-CNMRZT06-08-10.0034.7335.2043.2051.7552.4052.8463.7972.7776.7176.8577.0277.2277.3482.30172.54174.377181224 516691312NH3456 7CH89O10O11 CH31213O14O15CH316NHCO2CH3CO2CH388CDCl3APPENDIX C 171 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)ZT06-09-1-HNMRZT06-09-10.902.101.151.200.941.101.002.722.732.772.772.802.813.513.513.523.533.533.543.543.553.563.573.603.623.633.653.853.874.795,82''4 7''2' 7'112NH3456 7CH89O10OH1112O13OH14-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT06-09-1-CNMRZT06-09-133.7337.2843.3250.2963.5375.4779.06173.02179.417524 1812 9612NH3456 7CH89O10OH1112O13OH14NHCO2HCO2H89D2OAPPENDIX C 172 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)data_s2pul_001ZT06-32-14.2011.693.762.931.051.921.443.040.450.890.923.082.001.761.001.844.602.051.221.251.271.271.291.301.311.311.341.371.401.431.451.461.491.581.601.611.651.661.681.811.821.841.861.911.931.952.102.122.142.162.192.202.222.652.662.692.712.993.013.033.133.203.603.623.643.783.803.813.833.994.014.034.044.114.114.134.314.334.354.796.266.286.296.316.556.586.627.347.367.767.817.837.847.877.938.038.078.108.2610,11,26,27283,6,19,22,504013,15294339 37 33,46'2,18 3231303812,1614 41'' 41' 46''44123456N789CH310CH3111213 1415 16171819202122N+232425CH326CH327CH328293031323334O35NH363738394041NH42434445 46N47N48N4950S51O52O53OH54S55O56O57O-5859O60OH6162O63OH641.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f2 (ppm)1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)data_gCOSY_001ZT06-32-12,183,6,19,22,5013,15 14 12,16 39 432941''41' 402844 37 46'' 33,46' 383010,11,26,2731322,183,6,19,22,5013,151412,163943 2941''41'4028443746''33,46'38 3010,11,26,2731 32NN ONHKO3S-O3SNHCO2HCO2HNNN90D2OAPPENDIX C 173 173 APPENDIX D 174 174 Appendix D NMR spectra of photolysis Figure S5–1. 1H NMR spectra for progressive photolysis of DECM-Glu by 465 nm photoreactor Figure S5–2. Photolysis rate of DECM-Glu under 465 nm photoreactore 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.50204060801001301601902202502903303704104705306507709501190Chemical shift (ppm)Time (s)0 400 800 1200020406080100Time (s)Conversion (%)0 400 800 1200012Time (s)Ln[Caged Glu]a bAPPENDIX C 175 Figure S5–3. 1H NMR spectra for progressive photolysis of DECM-Glu by 445 nm photoreactor -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.501020304050607080100120140160Time (s)Chemical shift (ppm)APPENDIX C 176 Figure S5–4. 1H NMR spectra for progressive photolysis of DECM-PhKA by 445 nm photoreactor 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.50102030406080100125150175200225240260280310350Chemical shift (ppm)Time (s)APPENDIX C 177 1D NOE spectra of compounds in Chapter 5 NH CO2HCO2HH HHNOE4%1%124NNOH2ND2O1.31.41.51.61.71.81.92.02.12.22.32.42.52.62.72.82.93.03.13.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.7f1 (ppm)12ZT05-03-1-NOESY1DZT05-03-1ZT05-03-1-HNMR152' 2''13 6' 6''1412NH34567N8N910O11NH212H13 H14H1518O19OH2021O22OH23APPENDIX C 178 NMR spectra of compounds in Chapter 5 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)ZT04-91-1-HNMRZT04-91-12.062.172.051.003.382.061.081.242.162.182.202.212.762.782.803.093.113.133.783.793.813.954.796.916.937.817.831.52.02.53.03.54.04.55.05.56.06.57.07.5f2 (ppm)1.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)ZT04-91-1-gCOSYZT04-91-1NOMeO CO2HNH2NO2·TFA101D2OAPPENDIX C 179 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)05001000150020002500300035004000ZT04-110-1-HNMR5.924.150.940.920.981.020.960.990.001.181.201.212.993.373.393.413.425.215.245.856.486.496.546.546.566.577.217.247.277.527.54-100102030405060708090100110120130140150160170180190f1 (ppm)-4000-200002000400060008000100001200014000160001800020000220002400026000280003000032000ZT04-110-1-CNMRZT04-110-10.0012.5244.6576.7277.0477.3687.4493.3598.10107.91108.12124.83146.62150.13152.41156.35163.55O OEt2NNMe2111CDCl3APPENDIX C 180 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)ZT04-119-1-HNMRZT04-119-16.444.430.911.001.030.940.76-0.001.201.221.243.413.423.443.466.456.526.536.626.626.646.657.278.308.3210.03O OONCH3CH3-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT04-119-1-CNMRZT04-119-10.0012.4544.8176.7277.0377.3597.62103.71109.52117.36127.04143.92151.04157.40161.86192.54O OONCH3CH3O OEt2NO112CDCl3APPENDIX C 181 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)ZT04-120-1-HNMR5.964.281.870.900.981.000.99-0.001.171.181.203.363.373.393.414.826.286.456.536.567.277.297.31O OOHNCH3CH3-100102030405060708090100110120130140150160170180190f1 (ppm)ZT04-120-1-CNMR0.0012.4544.7060.7776.7677.0877.3997.63105.19106.37108.66124.42150.50155.45156.05163.06O OEt2NOH113CDCl3APPENDIX C 182 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)ZT05-35-1-HNMRZT05-35-16.214.221.920.861.001.031.111.891.870.001.201.221.243.403.423.443.465.405.416.236.546.546.596.606.626.627.267.307.337.417.448.308.32O OOOONCH3CH3 N+OO--100102030405060708090100110120130140150160170180190f1 (ppm)ZT05-35-1-CNMRZT05-35-10.0012.4344.8165.7876.7077.0277.3397.96105.59107.00108.79121.74124.30125.43147.64150.89152.17156.42161.55O OOOONCH3CH3 N+OO-OEt2NOOONO2114CDCl3APPENDIX C 183 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)0100200300400500600700800900ZT05-07-1-HNMR5.8618.340.910.951.843.890.740.701.940.820.931.001.010.001.191.211.211.231.261.281.321.401.401.441.481.481.491.491.511.561.601.631.901.921.931.961.992.052.182.212.232.262.452.472.472.492.512.522.532.552.552.572.592.592.612.643.393.413.433.444.244.265.015.095.115.235.306.126.516.526.576.576.596.607.277.287.30123 456O78910O1112 O131415 1617O1819O20N21CH32223CH32425 O2627CH328CH329CH330NH31O3233CH334 CH335CH33637 O38-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT05-07-1-CNMR-0.0212.4127.9928.1228.3030.0644.7453.1961.5776.6977.0177.3379.8882.3797.83106.01106.64108.66124.43149.21150.66156.27161.78171.20172.13123 456O78910O1112 O131415 1617O1819O20N21CH32223CH32425 O2627CH328CH329CH330NH31O3233CH334 CH335CH33637 O38OOEt2N OOtBuONHBocO116CDCl3APPENDIX C 184 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)-5000500100015002000250030003500400045005000550060006500ZT05-33-1-HNMR5.941.991.804.100.761.920.950.981.011.001.191.211.223.303.303.303.313.313.783.803.814.855.345.345.366.066.546.556.736.736.756.767.467.4821,2322',24'12'491015'177 16'O1 2O3456789101112O1314151617O18NH219N20CH32122CH3232425O26OH27-100102030405060708090100110120130140150160170180190200f1 (ppm)-2000-10000100020003000400050006000700080009000100001100012000130001400015000160001700018000190002000021000220002300024000ZT05-33-1-CNMR11.2825.6026.7129.2444.1946.9347.1447.2347.3647.3847.4747.5747.6447.7847.8548.0048.0748.2161.3996.86104.74105.71109.04124.76151.11151.22156.13OOEt2N OOHONH2O102·TFACD3ODAPPENDIX C 185 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)-50050100150200250300350400450500550600ZT05-14-1-HNMR6.731.000.900.964.632.613.480.860.880.811.821.101.171.241.683.62-0.001.191.211.221.752.052.062.072.102.122.132.252.272.282.302.332.353.063.083.093.113.123.143.413.423.623.633.773.823.893.913.923.933.953.964.044.064.084.114.234.254.265.305.315.336.076.166.516.566.586.606.627.077.097.277.297.317.317.327.327.33-100102030405060708090100110120130140150160170180f1 (ppm)-50000500010000150002000025000300003500040000450005000055000ZT05-14-1-CNMR12.4233.1233.1543.4444.1544.5744.7344.7645.1150.4550.6951.7951.8652.6752.7162.6763.2063.6765.3376.7177.0377.3597.7697.81105.73105.83105.85105.91105.94106.87108.68108.72108.83115.72124.30124.33124.44126.05127.46127.50127.63128.94137.62137.74148.30149.88150.02150.64150.67150.79153.29153.83154.23156.19156.31161.75161.96162.06171.79171.90171.93172.08CO2CH3Ph CO2CH3OONEt2OON117CDCl3APPENDIX C 186 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)ZT05-51-1-HNMR6.491.070.941.004.430.972.070.882.060.991.061.092.101.112.281.181.181.181.201.201.211.221.231.241.941.992.012.373.073.303.313.313.313.323.443.463.463.473.483.493.503.513.773.883.913.923.964.024.044.114.234.865.315.355.385.395.405.436.126.166.556.556.706.716.736.736.746.746.766.777.127.147.247.267.287.317.317.337.337.357.357.477.497.5235,3736',38'827,2926,3011281714 5''26 3 5'4112 34567891011O12131415O1617O1819O20N2122O23OH24252627282930 31O32OH33N34CH33536CH337 38-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT05-51-1-CNMRZT05-51-111.3044.1946.9346.9546.9847.0347.0447.0547.0647.0747.0847.0947.1147.1247.1547.1847.2247.2347.2447.2547.2547.2647.2747.2847.2847.2947.3047.3147.3147.3547.3647.4347.4747.4947.5047.5247.5747.5947.6147.6447.6947.7247.7347.7347.7447.7547.7848.0048.0348.0348.0648.0748.1348.2162.5296.83109.05109.08124.65124.81126.90127.46128.48128.50151.06151.07156.05156.1436,3835,3717214 4 512 34567891011O12131415O1617O1819O20N2122O23OH24252627282930 31O32OH33N34CH33536CH337 38CO2HPh CO2HOONEt2OON100CD3ODAPPENDIX C 187 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)-50050100150200250300350400450500550600650700750ZT04-81-1-HNMR3.203.182.082.061.000.003.823.844.606.476.496.497.187.207.26123456 7N8O9O10CH31112S13CH314-100102030405060708090100110120130140150160170180f1 (ppm)-20000200040006000800010000120001400016000180002000022000240002600028000ZT04-81-1-CNMR0.0044.0955.4555.4976.6977.0177.3398.61104.13115.08129.43157.88161.27123456 7N8O9O10CH31112S13CH314H3CO OCH3NCS119CDCl3APPENDIX C 188 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)-50005001000150020002500300035004000450050005500ZT04-82-1-HNMRZT04-82-12.902.891.931.930.970.991.000.970.942.492.502.502.502.513.333.743.804.494.564.586.456.456.476.476.556.567.077.097.948.700102030405060708090100110120130140150160170180190200f1 (ppm)-500005000100001500020000250003000035000400004500050000ZT04-82-1-CNMRZT04-82-138.8939.1039.3139.5239.7339.9440.1541.6255.2255.4598.25104.24119.01128.99157.78159.76181.25H3CO OCH3NHNHSNH2120DMSO-d6APPENDIX C 189 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)-10001002003004005006007008009001000110012001300140015001600170018001900ZT04-83-1-HNMRZT04-83-11.001.031.043.208.173.942.940.960.950.970.971.931.92-0.002.322.342.372.382.582.602.632.653.033.053.073.083.103.603.803.864.274.284.304.324.334.344.354.374.385.276.446.446.466.466.486.497.197.217.277.907.928.248.271,2'',30,38292',4010,1411,1336 4,24333517''17'52312N345S67O8O910111213141516 1718N19N2021O22NH2324O25O26O27O28CH329CH330313233343536O37CH338O39CH340N+41O42O-43-100102030405060708090100110120130140150160170180190200210f1 (ppm)01000002000003000004000005000006000007000008000009000001000000ZT04-83-1-CNMRZT04-83-10.0032.0337.8142.6443.5250.7252.0353.0255.4355.4664.4976.7377.0577.3698.77103.83117.88124.31128.39130.65143.31150.31156.46158.64161.09163.49171.04171.2910,1411,13384030233 4 53618153524116 3431 1721 322971212N345S67O8O910111213141516 1718N19N2021O22NH2324O25O26O27O28CH329CH330313233343536O37CH338O39CH340N+41O42O-43NNsCO2CH3CO2CH3NNOHNH3COOCH3121CDCl3APPENDIX C 190 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)ZT04-88-1-HNMRZT04-88-10.940.961.003.174.760.920.901.671.812.010.002.372.392.422.432.562.582.602.623.643.813.873.873.883.903.903.923.934.334.355.277.268.008.028.358.37-100102030405060708090100110120130140150160170180190200f1 (ppm)ZT04-88-1-CNMRZT04-88-10.0032.1337.7642.6750.5452.1253.0764.5176.7077.0277.33124.31128.59143.46150.36170.95171.16NNsCO2CH3CO2CH3NNOH2N122CDCl3APPENDIX C 191 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)ZT04-118-1-HNMRZT04-118-11.001.090.850.931.020.942.770.980.882.651.74-0.002.452.482.502.522.622.632.662.672.692.772.942.952.962.962.972.972.982.992.993.003.013.293.303.323.333.413.433.443.453.653.663.673.673.683.693.693.703.713.733.765.515.517.27 2017412'12''11 2''2'15312NH3456N7N89O10NH211121314O15O16 CH317O18O19CH320-100102030405060708090100110120130140150160170180190f1 (ppm)ZT04-118-1-CNMRZT04-118-134.0239.8243.4650.1151.8852.5464.0076.7177.0377.35159.89163.22172.07174.14201251417124136912NH3456N7N89O10NH211121314O15O16 CH317O18O19CH320NHCO2CH3CO2CH3NNOH2N123CDCl3APPENDIX C 192 0.00.51.01.52.02.53.03.54.04.55.05.5f1 (ppm)ZT05-03-1-HNMR1.001.011.201.031.050.971.132.062.092.102.132.662.672.702.723.043.063.063.073.083.093.113.123.353.753.773.783.803.853.873.893.903.923.944.014.024.034.044.044.064.79152' 2''13 6' 6''1412NH34567N8N910O11NH212H13 H14H1518O19OH2021O22OH23-100102030405060708090100110120130140150160170180190200f1 (ppm)ZT05-03-1-CNMRZT05-03-136.3237.6843.1446.5264.37158.16164.69172.37178.0212NH34567N8N910O11NH212H13 H14H1518O19OH2021O22OH23NHCO2HCO2HNNOH2N124D2OAPPENDIX C 193 -101234567891011121314f1 (ppm)ZT04-93-2-HNMRZT04-93-22.984.022.000.772.492.502.502.502.513.347.607.617.617.627.637.637.647.647.927.937.937.947.947.947.957.957.967.967.977.987.988.138.148.148.168.168.1713.240102030405060708090100110120130140150160170180190200210f1 (ppm)ZT04-93-2-CNMRZT04-93-238.8939.1039.3139.5239.7339.9440.15122.57122.84129.60130.65132.22132.86151.91154.26166.70NNOHO126DMSO-d6APPENDIX C 194 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.513.0f1 (ppm)ZT04-100-1-HNMRZT04-100-11.001.922.000.432.042.042.052.062.062.092.847.537.557.557.577.687.687.707.708.1712.04-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT04-100-1-CNMRZT04-100-129.2629.4629.6529.8430.0330.2230.42127.38127.93130.87131.40132.15133.27147.82151.26164.74206.14NNOHOClClClCl127C3D6OAPPENDIX C 195 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)ZT05-56-1-HNMR2.000.800.303.560.004.717.237.257.257.267.277.447.46NNOHClClClCl-100102030405060708090100110120130140150160170180190f1 (ppm)ZT05-56-1-CNMR-0.0063.3876.7277.0377.35127.14127.21127.54129.38129.60143.53146.33147.71123456 N7 N89101112131415OH16Cl17Cl18Cl19Cl20NNOHClClClCl128CDCl3APPENDIX C 196 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)ZT05-58-1-HNMR2.001.193.520.004.407.247.247.267.267.267.267.267.287.287.427.437.437.437.457.457.477.477.470102030405060708090100110120130140150160170180190200f1 (ppm)ZT05-58-1-CNMRZT05-58-10.0053.1876.7177.0277.34127.32127.78128.61129.44129.74137.92147.16147.61NNN3ClClClCl129CDCl3APPENDIX C 197 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)ZT05-63-1-HNMR2.152.000.171.250.243.18-0.001.663.853.937.127.147.147.167.237.237.237.247.247.257.257.267.267.267.277.437.447.447.457.457.457.467.480102030405060708090100110120130140150160170180f1 (ppm)ZT05-63-1-CNMRZT05-63-10.0044.9445.2276.7077.0277.34126.00126.02127.18127.59127.70127.97128.01129.14129.26129.33129.36129.39129.45145.82145.96146.56147.89148.24NNNH2ClClClCl130CDCl3APPENDIX C 198 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)ZT05-61-1-HNMRZT05-61-12.000.360.953.820.004.784.784.787.257.267.267.277.277.297.437.437.437.467.467.487.48-100102030405060708090100110120130140150160170180190200f1 (ppm)ZT05-61-1-CNMRZT05-61-10.0047.4376.7077.0277.33127.38127.61127.98129.47129.82136.44147.37147.56NNNCSClClClCl131CDCl3APPENDIX C 199 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)-1000100200300400500600700800900100011001200130014001500ZT05-25-1-HNMR1.862.071.001.781.670.770.792.483.324.594.754.777.487.507.527.587.687.708.598.90-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)-100001000200030004000500060007000800090001000011000120001300014000150001600017000180001900020000ZT05-25-1-CNMRZT05-25-139.3439.5539.7639.9640.1740.3840.5945.45126.30126.52128.84130.34131.53145.13147.18NNNHClClClClNHSNH2132DMSO-d6APPENDIX C 200 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)-200-100010020030040050060070080090010001100120013001400150016001700180019002000210022002300ZT04-106-1-HNMR0.941.001.072.862.870.851.790.810.821.642.101.551.771.951.961.961.961.961.971.971.981.992.282.302.332.352.432.452.472.493.103.123.133.153.173.543.723.743.753.753.773.783.883.893.904.314.327.417.437.437.457.467.597.617.988.008.258.308.33494624,2617,1939,4138,42251,2'42''58'8''12N345S6789N10N1112O13141516 171819N20N212223242526 2728NH29O30Cl31Cl32Cl33Cl3435O36O37383940414243O44O45CH346O47O48CH349N+50O51O-52-100102030405060708090100110120130140150160170180190f1 (ppm)-100000100002000030000400005000060000700008000090000100000110000120000130000140000150000160000170000180000190000ZT04-106-1-CNMRZT04-106-10.700.911.111.321.531.731.9433.1239.0543.3950.8752.5753.4565.81118.30125.47127.28127.79129.51130.84130.88131.84144.43147.99149.83151.30171.88172.0924,2617,1938,4239,4123,27464914,1674340 5 142225 835 21812N345S6789N10N1112O13141516 171819N20N212223242526 2728NH29O30Cl31Cl32Cl33Cl3435O36O37383940414243O44O45CH346O47O48CH349N+50O51O-52NNsCO2CH3CO2CH3NNOHNONNClClClCl133CDCl3APPENDIX C 201 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)ZT05-68-1-HNMR1.081.001.113.113.132.941.040.301.630.150.840.550.743.522.112.082.352.372.392.412.502.522.552.573.073.083.093.103.613.613.783.813.823.833.843.843.853.864.334.354.364.364.374.504.525.417.247.257.257.257.267.277.277.277.287.457.457.467.467.477.478.018.018.028.028.028.038.038.048.048.048.048.058.338.348.348.358.358.358.368.368.378.378.37475124,28,33,351,211,1310,1434422 16''16'21 512N345S67O8O91011 1213141516N17N1819O20NH21222324252627 28N29N303132333435 36Cl37Cl38Cl39Cl40N+41O42O-4344O45O46CH34748O49O50CH351-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)ZT05-68-1-CNMRZT05-68-1-0.0032.4137.9342.6946.1550.2052.1453.0464.6276.7077.0277.33124.31127.31127.82128.37128.58128.61129.24129.45129.75139.62143.83147.08147.61150.36157.32163.06170.95171.1233,353424,2810,1411,132625,2751 54471623444822 121273132,36151912N345S67O8O91011 1213141516N17N1819O20NH21222324252627 28N29N303132333435 36Cl37Cl38Cl39Cl40N+41O42O-4344O45O46CH34748O49O50CH351NNsCO2CH3CO2CH3NNONNHNClClClCl135CDCl3APPENDIX C 202 -0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)ZT05-99-1-HNMR0.881.191.352.001.932.202.382.131.450.102.242.262.282.282.302.312.322.342.372.382.392.402.432.872.892.902.922.932.952.962.982.993.113.133.133.143.153.163.303.313.313.313.323.753.763.783.793.803.833.984.004.014.034.054.064.074.084.094.104.114.114.134.144.304.344.384.424.444.534.867.327.337.347.357.367.367.367.377.387.397.397.417.427.437.437.447.477.497.507.547.567.587.5913'13''2,5'' 4,5'6''3 6'NH1234567N8N910O11NH1213141516171819N20N21222324252627Cl28Cl29Cl30Cl3132O33OH3435O36OH370102030405060708090100110120130140150160170180190f1 (ppm)ZT05-96-1-CNMRZT05-96-132.4140.6945.3546.9447.0547.1547.3647.5847.7948.0048.2161.58127.00128.03128.23128.96129.09129.32129.56135.51168.52168.70172.52172.77NHCO2HCO2HNNONNHNClClClCl136CD3OD 204 References (1) Armstrong, N.; Sun, Y.; Chen, G.-Q.; Gouaux, E. 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Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Synthesis of kainic acid derivatives and their application as kainate receptors probes
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