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Synthesis and characterization of H5decapa and related ligands Arane, Karen 2015

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  SYNTHESIS AND CHARACTERIZATION OF H5DECAPA  AND RELATED LIGANDS by KAREN ARANE Hons. B.Sc. McGill University, 2013   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2015  © Karen Arane, 2015ii  ABSTRACT Radiopharmaceuticals have provided a great breakthrough in tumor imaging and treatment, and the continued exploration in the field is required to make their use widespread. This vast potential lies in the variety of the radioisotopes, due to the different emission profiles and half-lives, and it is the chemist’s job to harness these isotopes into functional pharmaceuticals.  Bifunctional chelators (BFC), that incorporate a radiometal into a ligand scaffold that is functionalized to target a specific biological site, provide a mode to access many of these isotopes: α, β-, or auger electron emitters for therapy, β+ emitters for positron emission tomography (PET) imaging, and γ emitting isotopes for single photon emission computed tomography (SPECT) imaging.  The first requirement of a BFC is the thermodynamic stability and kinetically inert complex it forms with the isotope, especially in vivo.  The Orvig group has discovered the promise of the ligands H2dedpa for 67/68Ga and H4octapa for 111In, and has thus led to the idea of expanding this scaffold for larger radioisotopes.  An improved synthetic scheme for H5decapa, a decadentate ligand, allowed for thermodynamic testing and radiolabeling experiments to be performed with 86/90Y, 177Lu and 89Zr.  H5decapa showed promising serum stability over 5 days with 177Lu, and was able to quantitatively bind 89Zr after 30 minutes at room temperature.  A bifunctional version of H5decapa was synthesised, coupling para-nitroethylbenzene to the central nitrogen, for future conjugation to biomolecules.  As well, preliminary investigation into creating a version of H5decapa with hydroxamate groups catered to binding Zr4+was undertaken.     iii  PREFACE This dissertation is original, unpublished work by the author, K. Arane.  Chapter 2, Section 2.2.2 was done in collaboration with Dr. Eric Price at the Memorial Sloan Kettering Cancer Center in New York; and Section 2.2.4 was done in collaboration with Dr. Jacqueline Cawthray who performed the computational fittings.     iv  TABLE OF CONTENTS Abstract......................................................................................................................................................................... ii Preface ......................................................................................................................................................................... iii Table of Contents..........................................................................................................................................................iv List of Tables ............................................................................................................................................................. viii List of Figures ...............................................................................................................................................................ix List of Schemes ............................................................................................................................................................xi List of Abbreviations .................................................................................................................................................. xii Acknowledgements ..................................................................................................................................................... xv Chapter 1 : Introduction ................................................................................................................................................. 1 1.1 SPECT/PET Nuclear Imaging ............................................................................................................................. 1 1.2 Radiotherapy ........................................................................................................................................................ 3 1.3 Radiometal-Based Radiopharmaceuticals ........................................................................................................... 4 1.4 Radioisotopes 86/90Y, 177Lu, and 89Zr.................................................................................................................... 5 1.5 Current Radiopharmaceuticals ............................................................................................................................. 8 1.6 ‘Pa’ Family – A Versatile Acyclic Ligand System for Radiometals ................................................................... 9 1.7 Thesis Aims ....................................................................................................................................................... 11 Chapter 2 : H5decapa: An Acyclic Ligand for Radiopharmaceutical Applications ..................................................... 13 2.1 Introduction ....................................................................................................................................................... 13 2.2 Results and Discussion ...................................................................................................................................... 15 2.2.1 Synthesis and Characterization ................................................................................................................... 15 2.2.2 Radiolabeling Experiments ......................................................................................................................... 26 2.2.3 Serum Stability Studies ............................................................................................................................... 28 v  2.2.4 Thermodynamic Stability ........................................................................................................................... 31 2.2.5 Circular Dichroism ..................................................................................................................................... 33 2.2.6 Europium Fluorescence Studies .................................................................................................................. 37 2.2.7 Synthesis and Characterization of Bifunctional Decapa ............................................................................. 39 2.3 Conclusion ......................................................................................................................................................... 42 2.4  Experimental Methods ...................................................................................................................................... 44 2.4.1 Materials and Methods ............................................................................................................................... 44 2.4.2 2-Nitro-N,N-bis(2-(2-nitrophenylsulfonamido)ethyl)benzenesulfonamide (2.1) ....................................... 45 2.4.3 Dimethyl 6,6'-((((((2-nitrophenyl)sulfonyl)azanediyl)bis(ethane-2,1-diyl))bis(((2-nitrophenyl)sulfonyl)azanediyl))bis(methylene))dipicolinate (2.2) ..................................................................... 46 2.4.4  Dimethyl 6,6'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(methylene))dipicolinate (2.3) ............ 47 2.4.5 N,N’’-[[6-(Methoxycarbonyl)pyridin-2-yl]methylamino]-N,N’,N’’-[(tert- ............................................... 47 butoxycarbonyl)methyl]]diethylenetriamine (2.4) ............................................................................................... 47 2.4.6 H5decapa·6HCl·3H2O (2.5) ........................................................................................................................ 48 2.4.7 Na2[Y(decapa)] (2.6) .................................................................................................................................. 48 2.4.8 Na2[Lu(decapa)] (2.7) ................................................................................................................................. 49 2.4.9 Na[Zr(decapa)] (2.8) ................................................................................................................................... 49 2.4.10 Na2[Eu(decapa)] (2.9) ............................................................................................................................... 49 2.4.11 N,N”-(2-Nitrobenzensulfonamide)-1,2- triaminodiethane (2.10) ............................................................. 50 2.4.12 N,N'-(((4-Nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis(2 nitrobenzenesulfonamide) (2.11) .......... 50 2.4.13 Dimethyl 6,6'-(((((4-Nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis(((2-nitrophenyl)sulfonyl)azanediyl))bis(methylene))dipicolinate (2.12) ................................................................... 51 2.4.14 Dimethyl 6,6'-(((((4-Nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(methylene))dipicolinate (2.13) ...................................................................................... 52 vi  2.4.15 N,N’,N”-[(tert-Butoxycarbonyl)methyl-N.N”–[6-(methoxycarbonyl)pyridin-2-yl]methyl]-1,2,3-triaminodiethane (2.14)........................................................................................................................................ 52 2.4.16 BF·decapa (2.15) ....................................................................................................................................... 53 2.4.17 Zr(BF-decapa) (2.16) ................................................................................................................................ 53 2.4.18 Solution Thermodynamics ........................................................................................................................ 54 2.4.19 Purification of 86Y ..................................................................................................................................... 54 2.4.20 86Y Radiolabeling Studies ......................................................................................................................... 55 2.4.21 Ligand Stock Solutions and Radiolabeling Solutions ............................................................................... 55 2.4.22 89Zr Radiolabeling and Serum Stability53 ................................................................................................. 56 2.4.23 177Lu and 90Y Radiolabeling and Serum Stability ..................................................................................... 57 2.4.24 UV-vis and Circular Dichroism Experiments ........................................................................................... 58 2.4.25 Europium Fluorescence Study .................................................................................................................. 59 Chapter 3 : Attempted Synthesis: 89Zr-ligand .............................................................................................................. 60 3.1 Introduction ....................................................................................................................................................... 60 3.2 Results and Discussion ...................................................................................................................................... 61 3.2.1 Synthesis and Characterization ................................................................................................................... 61 3.2.2 Future Directions ........................................................................................................................................ 65 3.3 Experimental Methods ....................................................................................................................................... 68 3.3.1 Materials and Methods ............................................................................................................................... 68 3.3.2 N,N’-(Trifluoroacetamide)-1,2- triaminodiethane (3.1) ............................................................................. 68 3.3.3 N,N’-(Trifluoroacetamide)-1,2- triaminodiethane (3.2) ............................................................................. 68 3.3.4 N’-(2-Aminoethyl)-N’-(4-nitrophenethyl)ethane-1,2-diamine (3.3) .......................................................... 69 3.3.5 6-(Bromomethyl)picolinic acid (3.4) .......................................................................................................... 69 vii  3.3.6 N-(Benzyloxy)-6-(bromomethyl)picolinamide (3.5) .................................................................................. 69 3.3.7 N-(Benzyloxy)-6-methylpicolinamide (3.6) ............................................................................................... 70 3.3.8 N-(Benzyloxy)-N,6-dimethylpicolinamide (3.7) ......................................................................................... 71 Chapter 4 : Conclusions and Future Directions ........................................................................................................... 72 4.1 Conclusion ......................................................................................................................................................... 72 4.2 Future Directions ............................................................................................................................................... 73 References ................................................................................................................................................................... 75 Appendix ..................................................................................................................................................................... 79 Appendix A Supplementary Figures and Data .................................................................................................... 79   viii  LIST OF TABLES Table ‎1.1. Types of radiative emissions employed in radiotherapy (α, β and Auger) with corresponding linear energy transfer (LET), range in tissue, and selected example radiometals.9,10,11 ................................................... 3 Table ‎1.2. Biomolecules used in BFCs and their corresponding half-lives in humans. ................................................ 4 Table ‎1.3. Radiometal isotopes and their corresponding half-lives, decay modes and production methods.16 ............. 6 Table ‎2.1. Thermodynamic stability values (log KML and pM) for H4octapa, H5decapa, DTPA and DOTA with  In3+, Y3+, and Lu3+.1–3………………………………………………………………………......……………….32 Table ‎3.1. Reaction conditions, reagents and solvents tested for Scheme 3.2. ........................................................... 63 Table ‎3.2. Reaction conditions, solvents and reagents tested for Scheme 3.4. ........................................................... 65 Table A.1. 177Lu human serum competition via PD10 column - 1.5 and 24 hours, 37.0 ± 0.1°C at 550 rpm  agitation; background activity / mCi =0.5………………………………………………………………………80   ix  LIST OF FIGURES Figure ‎1.1. Depiction of clinically employed nuclear imaging techniques a) SPECT imaging and b) PET imaging, using the bifunctional chelate (BFC) method to specifically deliver the radioactive dose to the area of interest in vivo. ....................................................................................................................................................................... 1 Figure ‎1.2 Bifunctional chelator (BFC) ........................................................................................................................ 4 Figure ‎2.1. Periodic table of accessible radionuclides; γ – SPECT imaging, β+ - PET imaging, T – therapy (either α, β-, Auger electrons).31 .......................................................................................................................................... 14 Figure ‎2.2. 1H NMR spectra of (i) H5decapa (300 MHz) at ambient temperature, (ii) [Y(decapa)]2- at 25°C (400 MHz) and (iii) [Y(decapa)]2- at 85°C (400 MHz), showing the formation of multiple fluxional isomers upon Y3+ chelation in D2O. ........................................................................................................................................... 18 Figure ‎2.3. 1H-1H COSY NMR spectrum of [Y(decapa)]2- alkyl region (600 MHz, D2O, 25°C), red arrows mark diastereotopic splitting of dien backbone, pictured right. .................................................................................... 19 Figure ‎2.4. 1H-1H COSY NMR spectrum of [Y(decapa)]2- aromatic region (600 MHz, D2O, 25°C). ....................... 20 Figure ‎2.5. 1H-13C HSQC NMR spectrum of [Y(decapa)]2- (600 MHz, D2O, 25°C). ................................................. 21 Figure ‎2.6.  1H NMR spectra in D2O of (i) H5decapa (300 MHz) at ambient temperature, (ii) [Lu(decapa)]2- at 25°C (400 MHz) and (iii) [Lu(decapa)]2- at 85°C (400 MHz), showing the formation of multiple static isomers upon Lu3+ chelation. ..................................................................................................................................................... 22 Figure ‎2.7. 1H-1H COSY NMR spectrum of [Lu(decapa)]2- alkyl region (600 MHz, D2O, 25°C); red bracket depicting doublet of doublets of dien backbone, pictured right. .......................................................................... 23 Figure ‎2.8. 1H-13C HSQC NMR spectrum of [Lu(decapa)]2-, inset: zoom in on aromatic region (600 MHz, D2O, 25°C). .................................................................................................................................................................. 24 Figure ‎2.9. 1H NMR spectra at ambient temperature in D2O of (top) H5decapa (300 MHz) and (bottom) [Zr(decapa)]- (400 MHz) in D2O, showing the formation of one static isomer upon Zr4+ chelation. Correlation peaks determined from 2D COSY NMR (Appendix, Figure A.2)....................................................................... 25 Figure ‎2.10. 13C NMR spectra at ambient temperature of (top) H5decapa (400 MHz) and (bottom) [Zr(decapa)]- (400 MHz) in D2O, showing the formation of one static, asymmetric isomer upon Zr4+ chelation. ............................ 26 x  Figure ‎2.11. 90Y stability of transchelation by human serum proteins via CH3CN precipitation at 37°C with H5decapa and DOTA over 5 days. ....................................................................................................................... 29 Figure ‎2.12. 177Lu stability of transchelation by human serum proteins via CH3CN precipitation at 37°C with H5decapa, DOTA, and DTPA over 5 days. ......................................................................................................... 30 Figure ‎2.13. 89Zr stability of transchelation by human serum proteins via iTLC (50 mM EDTA) at 37°C with H5decapa, DFO and free 89Zr control over 3 days. .............................................................................................. 31 Figure ‎2.14. UV-vis spectrum of apo-transferrin with the addition of 1 and 2 equivalents of Lu3+. .......................... 34 Figure ‎2.15. CD spectra of apo-transferrin upon addition of metal-ligand complex [Lu(decapa)]2-. ......................... 35 Figure ‎2.16. CD spectra of apo-transferrin upon addition of metal (Lu3+) and ligand (H5decapa). ............................ 36 Figure ‎2.17. 1H NMR spectra at ambient temperature of [Eu(decapa)]2- (400 MHz) in D2O, showing the effect of binding the paramagnetic Eu3+ ion. ..................................................................................................................... 38 Figure ‎2.18. Eu3+ and [Eu(decapa)]2- emission spectrum (excitation wavelength = 280 nm). .................................... 39 Figure ‎2.19. 1H NMR spectra, 400 MHz at ambient temperature of (top) BF-decapa in D2O, (bottom) [Zr(BF-decapa)] in DMSO-d6, * - water suppression signal. ........................................................................................... 42 Figure ‎3.1. Potential ligands for Zr4+ based on the ‘pa’ family of ligands. ................................................................. 67 Figure A.1. [Lu(decapa)]2- 2D COSY NMR spectrum, aromatic region (600 Hz, in D2O)………………………....79 Figure A.2. [Zr(decapa)]- 2D COSY spectrum, 400 Hz, in D2O, aromatic region (left) alkyl region (right)...……...79  Figure A.3. Zr(BF-decapa) 2D COSY spectrum, 400 Hz, in DMSO-d6, aromatic region (left) alkyl region (right)..80  Figure A.4. iTLC-SA radioactivity distribution of [89Zr(decapa)]- labelled at ambient temperature, 30 minutes reaction time, >99% RCY (left) and after 72 hours, (free/serum bound 89Zr at ~60-100 mm solvent front)  ~17% stable (right)……………………………………………………………………………………………..80 Figure A.5. iTLC-SA radioactivity distribution of free 89Zr after 24h (free/serum bound 89Zr at ~100 mm solvent front), some sticks to baseline (~6%)…………………………………………………………………………..81 Figure A.6. iTLC-SA radioactivity distribution of 89Zr(DFO) in serum after 72 hours, (free/serum bound  89Zr at ~60-100 mm solvent front) >98% stable………………………………………………………………..81    xi  LIST OF SCHEMES Scheme ‎2.1. Improved synthetic scheme of H5decapa (2.1 – 2.5). ............................................................................. 16 Scheme ‎2.2. Previous synthetic scheme of H5decapa,27 highlighting the low yielding deprotection step. ................. 17 Scheme ‎2.3. Synthesis of BF-decapa (2.10 – 2.15). .................................................................................................... 41 Scheme ‎3.1. Synthesis of precursors 3.1, 3.2 and 3.3. ................................................................................................ 62 Scheme ‎3.2 Synthesis of precursors 3.4 and 3.5. ........................................................................................................ 64 Scheme ‎3.3. Synthesis of precursors 3.6 and 3.7. ....................................................................................................... 64 Scheme ‎3.4. Attempted reaction to methylate the hydroxamate nitrogen. .................................................................. 65 Scheme ‎3.5. Designed synthetic pathway for Zr-ligand. ............................................................................................. 66    xii  LIST OF ABBREVIATIONS ~     approximate  2D      two dimensional  3D     three dimensional  α     alpha particle  Å      Angstrom, 10 x 10-10 m  β−    beta particle  β+  positron  γ      gamma ray  δ      delta or chemical shift in parts per million (NMR)  Δ      heat  µ     micro (10-6) µM      micromolar (10-6 M)  AAS      atomic absorption spectroscopy  Ab  antibody BF  bifunctional BFC      bifunctional chelate, also means bifunctional ligand  biomolecule   vector, biovector, targeting vector, (e.g. antibody, peptide)  Bn      benzyl  br      broad (NMR) Bq  Becquerel °C      degrees Celsius calcd.     calculated  CD  circular dichroism CHX  cyclohexane/cyclohexyl Ci  Curie cm-1     wavenumber  CN      coordination number  COSY     correlation spectroscopy (1H-1H NMR)  CT     computed tomography  d      doublet (NMR)  Da  Dalton DCM     dichloromethane  DFO      desferrioxamine B  DFT      density functional theory (in silico calculations) Dien      diethylenetriamine  DGA  N,N,N’,N’,-tetra-n-octyl-diglycolamide DIPEA  diisopropylethylamine  DMF      dimethylformamide  DMSO     dimethyl sulfoxide  DOTA    1,4,7,10-tetraazacyclododecane -N,N',N",N'"-tetraacetic acid  DTPA     diethylenetetraaminepentaacetic acid  EA      elemental analysis    EDTA     ethylenediaminetetraacetic acid  equiv.     equivalent(s)  ESI-MS   electrospray ionization mass spectrometry  EtOAc    ethyl acetate  eV  electronvolt(s) FDA      Food and Drug Administration (USA)  xiii  g      gram  h  hours H2dedpa 1,2-[[6-carboxy-pyridin-2-yl]-methylamino]ethane H4octapa N,N’-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N’-diacetic acid H5dedpa 6,6'-(((((carboxymethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid  HPLC     high performance liquid chromatography HR  high resolution HSQC     heteronuclear single bond correlation/coherence (1H-13C NMR)   Hz      hertz  iTLC      instant thin layer chromatography (typically radioactive)  Ig  immunoglobulin IR      infrared  J      coupling constant (NMR)  k      kilo  KML      thermodynamic complex stability constant  L     litre or ligand  LET      linear energy transfer  m      milli- or multiplet (NMR) or meters M     molar (moles/ litre) or mega (106) or metal MeOH    methanol  min  minute(s) mol      mole  MRI      magnetic resonance imaging    MS      mass spectrometry n  nano (10-9), neutron NBS      N-bromosuccinimide  NMR     nuclear magnetic resonance  nM     nanomolar (10-9 M)  Nosyl     2-nitrobenzenesulfonamide (protecting group) p  proton or pico (10-12) p  para substituent  PBS      phosphate buffered saline  Pd/C  palladium on carbon (10% by weight) PET      positron emission tomography   pH      -log[H3O]+  pKa  protonation constant pM     -log[free metal], or picomolar (10-12 M)  ppm      parts per million  q      quartet (NMR)  quin  quintet (NMR) ®     trademark  RCY      radiochemical yield  Rf     retention factor  RGD  Arg-Gly-Asp cyclic peptide RP  reverse phase (column chromatography) rpm  rotations per minute RT      room temperature  Rt      retention time  s     singlet (NMR) or second (s) SPECT   single photon emission computed tomography  xiv  t      triplet (NMR)  tR      retention time (HPLC)  t1/2     half-life  TEA  triethylamine  TFA     trifluoroacetic acid  THF      tetrahydrofuran  TLC      thin layer chromatography  trastuzumab  HER2/neu targeting antibody  TRF  time resolved fluorescence  UV      ultraviolet  Vis  visible VT-NMR   variable temperature NMR    xv  ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor Professor Chris Orvig for his guidance and support throughout my degree. I would also like to thank all of the wonderful people I have had the chance to work with in the Orvig group over the past 2 years. In particular, I would like to thank Eric for the introduction into the ‘decapa’ project and making my transition into grad school a breeze, always being available for help and support, even since moving away for his post-doc. I would also like to thank Caterina for her constant support in a variety of realms including chemistry, social and physical endeavors. Thank you to Jacquie for her tireless efforts towards the physical and computational analysis included herein. And special thanks to Christina and Dorothee, for their contributions, as well as to Madeleine for her positive energy and stress relief in the lab. I would also like to thank the shops and services at UBC for their assistance, specifically Maria for her NMR expertise and Marshall for his MS knowledge, along with the staff in Bioservices and the SIF for the use of their facilities and training. The staff and researchers at TRIUMF provided great skills and assistance throughout my degree. I would like to thank my friends and family for their support and encouragement throughout the past 2 years. Finally, a special thanks to Kyle for his countless hours spent towards helping in chemistry, editing and overall support.   1  Chapter 1 : INTRODUCTION 1.1 SPECT/PET NUCLEAR IMAGING Computerized tomography (CT) scans and magnetic resonance imaging (MRI) both provide critical structural and anatomical information, and are the main detection methods for oncological tumours.  While significant research has greatly improved the detection limits of both these methods, there is still a major limit to the molecular and biochemical information accessible, which is needed for improved accuracy as well as early detection. Molecular imaging is the measurement and visualization of biological activities at the cellular and subcellular levels and provides a mode to characterize and phenotype diseases based on their biological information, to supplement the anatomical data.4  Nuclear imaging, a form of molecular imaging, relies on the radioactive emission of a nuclear agent (radiotracer) that specifically targets the area of interest. The radiative emissions of the nuclide are recorded by detector cameras which can convert the collected data into a 3D image visualising the accumulation of radioactivity in the body. In order to obtain high resolution and sensitive images, only very small amounts of radioactivity and minimal amounts of agent are needed (low mass technique), meaning the injected a) b) Figure ‎1.1. Depiction of clinically employed nuclear imaging techniques a) SPECT imaging and b) PET imaging, using the bifunctional chelate (BFC) method to specifically deliver the radioactive dose to the area of interest in vivo.  2  agent causes no physiological disturbance (tracer levels).    The two clinical nuclear imaging techniques are SPECT (single-photon emission computed tomography) and PET (positron-emission tomography).  SPECT (Figure 1.1 a) relies on the radionuclide to emit γ-rays that are then recorded by the detector cameras, allowing for resolution between 6-8 mm with a radiotracer concentration of ~10-6 M in vivo.5  Over 80% of diagnostic nuclear imaging scans today use SPECT due to the ease of production, wide-spread availability of SPECT cameras in hospitals and use of the γ-emitting radioisotope 99mTc.  Recent disruptions to the production and supply of the parent isotope, 99Mo, have spurred research into other potential isotopes, such as 67Ga, 111In, and 177Lu, that could potentially help fill this void.6   PET (Figure 1.1 b) involves the injected radiopharmaceutical emitting a positron (β+).  When the radionuclide decays, it releases a β+ that collides with an electron, releasing two 511 keV γ-rays that are 180° apart.5,7 The PET scanner then detects these coincident γ-rays with detector cameras.  PET is a more expensive method than SPECT, but provides higher resolution images (2-4 mm), and is much more sensitive (tracer concentrations on the order of 10-12 M) compared to SPECT imaging.  The main reason for the large difference in sensitivity is the use of lead-collimators in SPECT cameras, which filter out a large percentage of emitted gamma rays.  PET imaging agents are dominated by organic radioisotopes, particularly 18F, but also 15O, 13N and 11C, all of which have short half-lives (under 2 hours) and often involve multistep synthetic protocols to incorporate the radionuclide into the pharmaceutical.8  These drawbacks have limited the use of PET to effectively image biological processes that occur on the order of minutes.  Recent efforts have simultaneously been made to expand this library to include radiometals, such as 68Ga, 64Cu, 86Y and 89Zr, which possess varying half-lives and would normally require a less time-consuming synthetic protocol to prepare the radiopharmaceutical. These metals also provide the opportunity for different delivery methods to a known biological target using vectors such as peptides and antibodies that have longer biological half-lives and do not match well with the traditional short half-life non-metals.  3  1.2 RADIOTHERAPY A large array of radiometals may be found in the periodic table with varying emission profiles, allowing for a wide spectrum of uses.  Nuclear imaging, either via SPECT or PET, requires a γ or β+ emitting nuclide, respectively.  While nuclear imaging is crucial for diagnosis, monitoring, and management of many diseases, such as cancerous growths, the use of radioisotopes for therapeutic purposes provides an opportunity to eradicate these cancerous tumours (Table  1.1). Current radiotherapy focuses on beta particle (β-) emission, which is best suited for large and poorly vascularised tumours, as its emission is deposited over several milimeters.9,10 Conversely, α-particles have a path length ranging only over a few cell diameters, which translates to a strong linear energy transfer (LET) efficiency that makes α-emitters ideal for treating small tumors, if very well targeted.10  The challenge with incorporating an α-emitting radionuclide into a radiopharmaceutical is accounting for the daughter isotopes that are created upon decay of the parent isotope, and usually are radioactive themselves.  This leads to new radioisotopes being formed that do not necessarily conform with the desired radiopharmaceutical treatment, and most importantly are typically ejected from the radiopharmaceutical/chelator upon decay due to the high recoil energy of alpha emission.11 Auger electrons also emit radiation that can be used for therapeutic purposes; however, as the emitted electrons are of low energy, it is necessary for them to be located within the cell nucleus to be effective.9,12  Table ‎1.1. Types of radiative emissions employed in radiotherapy (α, β and Auger) with corresponding linear energy transfer (LET), range in tissue, and selected example radiometals.12,13,14 Emission  Energy LET Range Tumour size Isotopes α-particle high 5-8 MeV high 100 keV/μm 40-100 μm ~1-3 cell diameters small 212/213Bi, 225Ac β- particles high-medium 0.1-2.2 MeV low 0.2 keV/μm 0.5-10 mm 50-1000 cell diameters large 177Lu, 90Y Auger electron low ~1-10 keV high 1-20 μm <1 cell diameter (subcellular) small 67Ga, 111In,   4  1.3 RADIOMETAL-BASED RADIOPHARMACEUTICALS Central to the basic design principle for radiometal-based radiopharmaceuticals is the concept of a bifunctional chelator (BFC).  The BFC is designed to have binding properties catered towards a specific metal whilst also possessing a functional moiety that can be covalently coupled to a targeting vector (Figure  1.2).15    Figure ‎1.2 Bifunctional chelator (BFC)  These targeting vectors range from peptides and nucleotides to antibodies and nanoparticles (Table  1.2), all of which allow for site-specific delivery of the radioisotope via the BFC.  The linker connects the metal-chelate complex to the biovector, and usually takes advantage of the many primary amine sites located on the biomolecule.  This can be done via thiourea bond formation, which couples an isothiocynate (NCS) moiety on the chelator, to a primary amine.    Table ‎1.2. Biomolecules used in BFCs and their corresponding half-lives in humans. Biomolecule Biological Half-life  Antibody (Ab)/ Immunoglobulin (Ig) 3-4 weeks Peptide ~30-60 mins Antibody Fragment >10 hours Nanoparticle Variable  What gives the BFC radiopharmaceutical design so much specificity is that both the biomolecule and radiometal can be tuned such that the construct can be catered to treat or image a specific disease or tumour site.  There has been tremendous progress recently in identifying an array of over-expressed receptors (antigens) and their corresponding native binding ligands (biomolecules) that play a role in a variety of oncological tumours.16 Depending on the physicochemical characteristics of specific biomolecule linker chelator radiometal 5  biomolecules they will have different circulation times in the body; for example, large molecular weight antibodies often take several days to circulate the blood stream and localize at the tumor site, compared to smaller molecular weight peptides that have a very rapid clearance profile from blood and non-target tissue.  Consequently, this requires matching of the radiometal to the biomolecule, in order for the physical half-life of the radioisotope to match the biological half-life of the biovector.  This ensures high uptake of the radiometal at the tumour source, along with clearance of any unbound agent, in the time frame of the radioactive emission, to yield a good signal-to-noise ratio (also called tumor to background ratio).    When designing a new chelator for a specific radiometal, many factors must be accounted for:  good thermodynamic stability, kinetic inertness, and quick labeling kinetics at ambient temperatures.  All of these factors are essential to create a viable radiopharmaceutical, which possesses a very strong metal-chelate affinity and therefore does not allow the release of radiometal in vivo.  These properties can be assessed through experiments that are used to predict the effectiveness of the radiopharmaceutical metal-chelate complex. To assess the thermodynamic stability, potentiometric or spectrophotometric titrations can be performed to calculate log KML (KML = [ML]/[M][L]) and pM (-log[M]free), which quantify the binding affinity of the chelator to the specific metal.  Acceptable kinetic inertness can be accessed via in vitro competition assays with endogenous proteins and chelators that are found in the body and known to complex metals (eg. apo-transferrin, albumin).  While both of these methods attempt to mimic in vivo scenaria, in reality it is much more complicated to predict how a radiopharmaceutical complex will behave; hence, in vivo experiments and biodistribution studies tend to provide the most clear-cut results of the metal-ligand complex’s thermodynamic stability and kinetic inertness.   1.4 RADIOISOTOPES 86/90Y, 177LU, AND 89ZR Lutetium-177 emits β- particles that can be used for radiotherapy and has a half-life (t1/2) of 6.6 days.  As it decays, 177Lu also emits two γ-rays that can be used for SPECT imaging, making 177Lu an 6  extremely attractive radionuclide as it allows for simultaneous imaging and therapy to occur at the tumor site.  Due to its long half-life, 177Lu is best suited when conjugated to larger biomolecules, used for radionuclide targeting, with comparably longer biological half-lives.  Lu3+ has an ionic radius ranging between 86-109 pm (CN = 6-9),17 and commonly exists in an oxidation state of 3+ with a coordination number of 9.13 177Lu can be produced through a few different methods, such as in a medium flux reactor by irradiating 176Lu(176Lu(n,γ)177Lu). Currently there are late phase-II clinical trials investigating the therapeutic potential of 177Lu-DOTA-TATE (Lutetium-177 octreotate), a radiolabeled peptide targeting neuroendocrine tumors.18  Table ‎1.3. Radiometal isotopes and their corresponding half-lives, decay modes and production methods.19 Isotope t1/2 (h) Decay mode Production method 67Ga 78.2 EC (100%) cyclotron, 68Zr(p,2n)67Ga 68Ga 1.1 β+ (90%) EC (10%) 68Ge/68Ga generator 86Y 14.7 β+ (33%) EC (66%) cyclotron, 86Sr(p,n)86Y 90Y 64.1 β- (100%) 90Zr(n,p)90Y 89Zr 78.5 β+ (23%) EC (77%) cyclotron, 89Y(p,n)89Zr 111In 67.2 EC (100%) cyclotron, 111Cd(p,n,)111m,gIn 177Lu 159.4 β- (100%) 176Lu(n,γ)177Lu 225Ac 240 α (100%) 226Ra(p,2n)225Ac, n-Capture of 232Th  223U 225Ac EC = electron capture  Yttrium possesses two useful radioisotopes, 86Y and 90Y, the first possessing suitable properties for PET imaging and the latter suitable for radiotherapy.  86Y decays via high-energy β+ particle emission and has a half-life of 14.7 hours. It can be produced using a small biomedical cyclotron via the 86Sr(p,n)86Y nuclear reaction.5 90Y, which decays solely via β- emission, has a half-life of 64 hours and is an ideal radioisotope for β- radiotherapy.  The general production of 90Y is through the use of a 90Zr(n,p)90Y reaction in a nuclear reactor.5  Yttrium prefers an oxidation state of 3+ and has an ionic radius between 90-108 pm (CN = 6-9)12 allowing for coordination numbers ranging from 6 to 9. It is 7  considered to behave as a hard acidic cation, showing preference for harder donor atoms like oxygen and nitrogen.   Because both yttrium isotopes have the same binding preferences, 86Y can be used as an imaging surrogate for 90Y, to perform dosimetry prior to injection of the therapeutic formulation using 90Y. There is currently an FDA approved 90Y labeled monoclonal antibody, Zevalin®, that is targeted for β- therapy towards a form of B-cell non-Hodgkin’s lymphoma.20  Zirconium-89 is a β+ emitter that can be used for PET imaging and has a half-life of 78.4 hours, which makes it a suitable match for antibodies, due to their longer biological half-lives.  Zirconium prefers an oxidation state of 4+ which makes it an extremely acidic cation with an ionic radius of 84-89 pm (CN = 8-9)19 and consequently prefers to form 8-9 coordinate complexes. Because of its very hard nature, it is difficult to radiolabel, as it tends to form insoluble polynuclear species under non-acidic conditions.21  It preferentially binds with hard donor molecules that are rich in anionic oxygen atoms and functional groups such as hydroxamates, carboxylates, carbonyls and hydroxypyridones.22  The most suitable chelator to date for Zr(IV) is desferrioxamine (DFO), and 89Zr-DFO complexes are currently in clinical trials.23  Presently, radioactive 89Zr is produced using the (p,n) reaction of 89Y using a cyclotron.5 Due to its markedly long half-life in comparison to other β+ emitters, there is much interest in using 89Zr as a PET imaging nuclide; however, its challenging chemical properties has made finding a suitable stable chelator more difficult.  Although DFO is a sufficient chelator for 89Zr, some 89Zr can be observed to leach out of the radiopharmaceutical and into the bone over time.22  This observation has spurred a recent surge in interest for developing new 89Zr chelators with superior in vivo stability to DFO.   8  1.5 CURRENT RADIOPHARMACEUTICALS   Macrocyclic chelators form kinetically inert metal complexes and are thermodynamically favourable due to their prearranged structure, known as the macrocycle effect.24  Conversely, they normally require elevated temperatures and longer reaction times to bind the radiometal in quantitative yields, which is much less desirable.  Longer reaction times lead to loss of the radioactive half-life, and once the BFC is conjugated to a biovector, the elevated temperatures that are required for radiolabeling can lead to denaturing of the peptide or antibody.  Nevertheless, because they offer such strong metal-chelate complexes, the current ‘gold-standards’ for many radioisotopes are macrocyclic ligands.  DOTA (1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid), an N4O4 macrocyclic chelator, is the leading chelator for a number of radiometals including 111In, 177Lu, 86/90Y, and 225Ac.  It has a coordination number of 8, and although it has slow radiolabeling kinetics and requires heating, the versatility and thermodynamic stability of its resultant complexes make it the leading chelator candidate.  Many BFCs have been designed by using one of the carboxylic acid appendages of DOTA to conjugate to a biovector, or by functionalization of the carbon-skeleton, to create derivatives such as p-SCN-Bn-DOTA (C-DOTA).                On the other hand, acyclic chelators often provide very fast labeling kinetics with minimal to no heating required, but are overall less thermodynamically favourable due to the loss in entropy ensued by the amount of rearrangement necessary to form a metal-chelate complex.  The most widespread used acyclic chelator is DTPA (diethylenetriaminepentaacetic acid), an N3O5 donor, which also has a coordination number of 8.  DTPA can bind many radiometals, such as 111In, 177Lu, 86/90Y, and 44/47Sc, at 9  room temperature within minutes.  However, further testing has shown DTPA to be unstable in vivo with many radioisotopes.25 The next generation chelator CHX-A”-DTPA, incorporates a cyclohexyl backbone linkage that provides increased rigidity to the ligand and leads to a greater degree of preorganization of the chelate structure, reducing the thermodynamic barrier and improving the kinetic inertness of the resultant metal-complexes.19 However, this modification also slows the radiolabeling kinetics and CHX-A”-DTPA has failed to demonstrate improved stability over DOTA.26   1.6 ‘PA’ FAMILY – A VERSATILE ACYCLIC LIGAND SYSTEM FOR RADIOMETALS The Orvig group has recently investigated a new set of acyclic chelators based on the picolinic acid (pa) moiety for radiolabeling a variety of radiometals, referred to as the ‘pa’ family.  The first member of this family, hexadentate H2dedpa (N4O2), showed a remarkable ability to complex 67/68Ga/Ga3+, and exhibited quantitative radiolabeling yields (RCY > 99%) after 10 minutes at room temperature.27 This ligand was then functionalized to create the BFC p-SCN-Bn-H2dedpa that was subsequently conjugated to a cyclic targeting peptide RGD for in vivo PET imaging.28,29  Further work on this specific backbone has included the addition of a cyclohexyl group onto the ethylenediamine backbone to improve rigidity and pre-organization; concurrent modifications through addition of lipophilic moieties to the backbone nitrogen atoms were explored towards use in cardiac perfusion imaging.28 The ‘pa’ family was then expanded to include larger ligands, the next being H4octapa (N4O4), an octadentate chelator. H4octapa shows very promising radiochemistry with both 111In and 177Lu demonstrating quantitative radiolabeling at room temperature in less than 10 minutes.1  A bifunctional 10  derivative of this ligand was synthesized creating the BFC p-SCN-Bn-H4octapa that was subsequently conjugated to the Trastuzumab antibody and tested on mice with ovarian cancer.3 Both the 111In and 177Lu H4octapa complexes have shown comparable, if not improved, in vivo stability compared to the gold standard DOTA analogues.3    Other derivatives based on this family have also since been synthesized and tested.  H2azapa (N6O2) uses the H2dedpa scaffold with the addition of two triazole rings (containing lipophilic benzyl “place-holders”), which stands as a model for click-based bifunctional chelating agents.  H2azapa was radiolabeled with 64Cu, which showed quantitative labeling at ambient temperature; however in vivo testing revealed high uptake of 64Cu in the liver and the gut, demonstrating that the metal-chelate complex was not stable in vivo.30 H6phospa is a methylene-phosphonate derivative of H4octapa with the bifunctional derivative p-SCN-Bn-H6phospa, and was evaluated in vivo with radioisotopes 111In, 177Lu and 89Zr when conjugated to the antibody Trastuzumab.  Although none of the conjugates achieved quantitative radiochemical yields (90% to 8% respectively), H6phospa demonstrated a scaffold with potential for future 89Zr chelators as it was the only known chelate to show any binding with 89Zr after DFO.31  a) b) c) d) 11  The final ligand in this series is the largest, decadentate, chelator H5decapa (N5O5).  Limited research into its radiolabeling potential has been undertaken, besides the poor labeling demonstrated with 111In.1 This is most likely due to the big difference in coordination preference between the metal and the chelator, where H5decapa has 10 potential binding sites, and a large binding cavity, and In3+ only has an ionic radius of 62-92 pm (CN of 4-8).  The limitations in the synthesis of H5decapa were what had been restricting further testing of this ligand.  This was in conjunction with the lack of a synthetic route to create the bifunctional version of the ligand, which is needed for the eventual in vivo testing and attachment of a biovector.  1.7 THESIS AIMS Because many of the potentially clinically useful radiometals (such as 177Lu and 89/90Y) are relatively large in size and prefer to form complexes of high denticity (CN >7), we hypothesized that the previously studied decadentate chelator H5decapa may be of interest for its coordination properties with these radiometals. The following chapter will discuss a new synthetic scheme that has been developed to synthesize H5decapa, and the initial characterization and radiolabeling studies performed.  This ligand was designed with the intended purpose to chelate larger radiometals in a more stable fashion, as the need for an expanded ligand library that can cater to a different variety of radiometals is growing.  The complexes of H5decapa with the metals Lu3+, Y3+ and Zr4+ were analysed in both their ‘cold’ (non-radioactive) chemistry characteristics, via NMR and thermodynamic studies, as well as their ‘hot’ (radioactive) chemistry labeling efficiency and serum stability.  In order to utilise H5decapa as part of a fully functional radiopharmaceutical, the chelate must bear a reactive linker group which can be used for conjugation to biomolecules (i.e. a BFC version of H5decapa), yet no bifunctional H5decapa analogues have been previously prepared. Herein, a novel synthetic route was developed which resulted in the preparation of a bifunctional H5decapa analogue for the first time, to allow for future in vivo testing.   12  Translating this ligand design to suit the hard radiometal 89Zr will also be discussed.  New synthetic pathways, based on the ‘pa’ ligand design, were tested that attempt to cater to the preferences of the Zr4+ ion.   13  Chapter 2 : H5DECAPA: AN ACYCLIC LIGAND FOR RADIOPHARMACEUTICAL APPLICATIONS 2.1 INTRODUCTION  As discussed in Chapter 1, the need to continuously grow and expand the current library of bifunctional chelators is omnipresent, requiring new ligand designs in order to match a wider range of clinically relevant radiometals. The current commercially available ligand standards (such as DOTA and DTPA (vide supra)) are used extensively with a wide range of radiometals, despite fundamental differences in the coordination properties of each metal. Consequently, one must often make a compromise between fast and mild radiolabeling conditions, and forming a kinetically inert radiometal-chelate complex. For example, acyclic chelators that can be quantitatively radiolabeled under ambient temperatures provide an advantage that their macrocyclic counter parts cannot match; however, macrocyclic chelators tend to form kinetically inert complexes, due to their prearranged structure, that prevent the radioisotope from being released from the chelate in vivo. Therefore, expansion of the ligand library to amalgamate the fast, labile chelation with very strong, stable binding, into new and improved ligands for radiopharmaceuticals is required.  14   Figure ‎2.1. Periodic table of accessible radionuclides; γ‎– SPECT imaging, β+ - PET imaging, T – therapy (either α, β-, Auger electrons).32   As seen in Figure 2.1, metals that have potentially attractive decay properties for imaging, therapy, or both span the length of the periodic table. Each of the radiometal isotopes exhibits different decay profiles while possessing dissimilar coordination chemistry.  Many of these radiometals contain large ionic radii and reside with higher oxidation states of 3-4, meaning they prefer to form complexes of high denticity (CN 8-10) in order to form a stable complex. Consequently there is need for new chelators that can accommodate these larger radiometals.  Due to the great potential of the isotopes Y3+ and Lu3+, coupled with their similar coordination chemistry and ionic radii (as discussed in Chapter 1), a ligand scaffold that can form kinetically inert complexes while also retaining the ability to facilitate fast and efficient radiolabeling chemistries would be very attractive.   Based on the previous success of the ligands H2dedpa and H4octapa, which form stable complexes with Ga3+ and In3+ respectively, the synthesis and metal complexation properties of the largest ligand in the ‘pa’ family H5decapa was explored herein. It was hypothesized that the decadentate N5O5 chelating ligand would satisfactorily saturate the coordination sphere of larger radiometals (i.e. Y3+, Lu3+) 15  due to its elongated dien (diethylenetriamine) backbone and larger binding cavity. In this chapter, an improved synthesis of the chelator was designed, followed by the derivatization to create a bifunctional version of the ligand.  The unfunctionalized ligand was analysed for its thermodynamic stability via potentiometric titrations and serum stability assays to help predict the metal-chelates’ in vivo kinetic inertness.  UV-vis spectroscopy and circular dichroism were used as an alternative method for predicting the in vivo stability of the M-decapa complexes.  2.2 RESULTS AND DISCUSSION 2.2.1 Synthesis and Characterization  Although the synthesis and characterization of H5decapa has been previously reported,1 herein an upgraded synthetic approach has been developed, improving the previous 5-step synthetic yield of ~2.5% to a yield of ~16.7% (Scheme  2.1).  H5decapa was synthesized from dien beginning with N-nosyl (nosyl = 2-nitrobenzene sulfonamide) protection to yield 2.1, followed by selective N-alkylation using 2 equivalents of bromo-methyl picolinate to give 2.2. Nosyl deprotection was accomplished via addition of thiophenol to yield 2.3, followed by a second N-alkylation reaction using t-butyl bromoacetate to give the fully protected pro-ligand 2.4. Complete methyl ester and butyl ester deprotection was accomplished in a two-step one-pot reaction, whereby LiOH was added to remove the methyl esters and refluxing HCl removed the remaining t-butyl ester protecting groups.    16  Scheme ‎2.1. Improved synthetic scheme of H5decapa (2.1 – 2.5).    Previous methods to synthesize H5decapa involved N-benzyl protection, where the vigorous hydrogenation conditions required for deprotection consequently resulted in a major loss of yield (Scheme 2.2). Furthermore, the previous scheme involved the addition of tert-butylacetate prior to N-alkylation of the bromopicolinates, as deprotection of the benzyl protecting group with the picolinate moieties resulted in an unwanted cleavage.  The hydrogenation of the benzyl protecting group also resulted in cleavage of the backbone, drastically reducing the overall yield, an unexpected result that was not observed using analogous benzyl-protection schemes during the synthesis of H4octapa.1 The above synthetic pathway alleviates the aforementioned synthetic pitfalls, also observed with H4octapa,3 as the nosyl protecting group’s deprotection is carried out using milder conditions that do not affect the surrounding functional groups.33         17  Scheme ‎2.2. Previous synthetic scheme of H5decapa,1 highlighting the low yielding deprotection step.    Following the successful preparation of the ligand H5decapa, metal complexation experiments with ‘cold’ non-radioactive metal solutions were performed to assess the coordination chemistry of decapa with Y3+ and Lu3+. The Y3+ complex of H5decapa, [Y(decapa)]2- was synthesized by mixing H5decapa with a small excess of YCl3·6H2O in deionized water to ensure quantitative complexation.  The pH of the solution was adjusted using NaOH (0.1 M) to ~4-5 and stirred at ambient temperature for 1 hour.  The formation of the expected coordination complex was confirmed by both low and high resolution mass spectroscopy and by NMR techniques (Figure 2.2).  18   Figure ‎2.2. 1H NMR spectra of (i) H5decapa (300 MHz) at ambient temperature, (ii) [Y(decapa)]2- at 25°C (400 MHz) and (iii) [Y(decapa)]2- at 85°C (400 MHz), showing the formation of multiple fluxional isomers upon Y3+ chelation in D2O.   Generally, metal-chelate complexes that exhibit minimal isomerization in solution are preferred as this is thought to imply higher stabilities,34 although no definitive proof of this trend has been established yet.  Comparing the 1H-NMR spectrum of H5decapa to [Y(decapa)]2- (Figure  2.2), very large broad peaks can be observed upon metal complexation, suggesting that there are multiple fluxional isomers existing in solution. Because Y3+ prefers a coordination sphere of 8-9, and H5decapa has 10 available coordination sites, it is possible that one of the acetate arms is not strongly bound and this structure may fluctuate in solution. This is different in comparison to the [Y(octapa)]- 1H-NMR spectra, where sharp, distinct peaks were seen, presumably representing multiple, well-defined, static isomers in solution.2   D2O (i) (ii) (iii) 19  To further probe the coordination structure and isomerization of [Y(decapa)]2-, variable-temperature NMR (VT-NMR) and 2D-COSY/HSQC experiments were performed.  As the temperature of the NMR sample (in D2O) was increased from 25°C to 85°C, the peaks began to coalesce, most notably in the aromatic region, where the resonances of the picolinate moieties resemble those of the metal-free ligand, potentially alluding to a single major isomer (Figure 2.2).  The VT-NMR experiments suggest that multiple fluxional isomers are present under aqueous conditions at ambient temperature.  The fluctuation between isomers of [Y(decapa)]2- is originally on the order of the NMR timescale (relatively slow), which is why the 1H spectrum contains such broad peaks as the signals are all being averaged out.  As the temperature is increased, the rate of these fluctuations increases, and the signals of the different isomers coalesce into sharper peaks.  This can especially be observed in the aromatic region at 85°C, where the original broad peaks have coalesced into three sharp peaks, a result of the weighted average of all the individual conformations.     Figure ‎2.3. 1H-1H COSY NMR spectrum of [Y(decapa)]2- alkyl region (600 MHz, D2O, 25°C), red arrows mark diastereotopic splitting of dien backbone, pictured right.  A 2D-COSY experiment was performed to probe the 1H-1H through-bond correlations and to help determine if distinct isomers could be observed at ambient temperature, supplementing the VT-NMR   20  results (Figure 2.3). The many off-diagonal correlation peaks displayed in the spectrum suggest that there are many different isomers present in solution.  Although the exact number of isomers cannot be ascertained, certain other pieces of information can be extracted from this spectrum.  When H5decapa coordinates with a metal, a chiral complex most likely is formed; the complex can either be symmetric or asymmetric, and in this case, 2D NMR spectroscopy can provide a better understanding of the complex behaviour in solution.  Although the resonances are poorly defined and are very broad, implying that conformations are changing in solution, there appears to be a 1H signal (at ~2.81 ppm) that correlates to three other 1H signals (2.57 ppm, 3.22 ppm, 3.38 ppm) (red brackets in Figure 2.3).  This pattern would be expected for an asymmetric, chiral complex where the hydrogen atoms on the ethylene backbone are diastereotopic, confirming that the ligand is indeed bound to the metal.  The other correlations seen in the COSY spectrum may correspond to other conformational isomers present in solution.   Further investigation of the aromatic region (Figure 2.4) of the [Y(decapa)]2- 2D COSY spectra appears to provide additional support towards the suspected appearance of one major conformational isomer. Off-diagonal correlations of the red indicated signals corroborate these suspicions, while blue highlighted peaks appear to be a less abundant fluxional isomer (Figure 2.4).     Figure ‎2.4. 1H-1H COSY NMR spectrum of [Y(decapa)]2- aromatic region (600 MHz, D2O, 25°C).   21  The highly fluxional nature of the metal-ligand complex [Y(decapa)]2- resulted in no observable signal in 13C NMR experiment after 12 hours at 151 MHz.  A 2D HSQC (1H-13C) correlation NMR was performed to help elucidate some of the 13C NMR signals.  Four peaks were observed in the aromatic region (Figure  2.5), supporting that an asymmetric complex is formed as only three aromatic peaks would be expected for a symmetric isomer.  However, not many other correlations could be observed, once again indicating that the [Y(decapa)]2- complex is very fluxional in solution, which foreshadows the weakness in stability of this metal-ligand complex.     Figure ‎2.5. 1H-13C HSQC NMR spectrum of [Y(decapa)]2- (600 MHz, D2O, 25°C).  The [Lu(decapa)]2- complex was synthesized as above, whereby the lutetium nitrate [Lu(NO3)3·6H2O] salt was mixed with H5decapa to form the desired coordination complex (quantitative).  Comparing the 1H-NMR spectrum of H5decapa to [Lu(decapa)]2-, upon complexation many distinct sharp peaks are observed, suggesting that multiple static isomers are formed in solution (Figure 2.6).  This is in stark contrast to what was previously described for the [Y(decapa)]2- complex, where many broad, 22  undefined peaks were seen. The appearance of multiple, static isomers was similarly observed for the [Lu(octapa)]- complex, which demonstrated high kinetic inertness in vivo.1    Figure ‎2.6.  1H NMR spectra in D2O of (i) H5decapa (300 MHz) at ambient temperature, (ii) [Lu(decapa)]2- at 25°C (400 MHz) and (iii) [Lu(decapa)]2- at 85°C (400 MHz), showing the formation of multiple static isomers upon Lu3+ chelation.  In order to gain more insight into the type of isomerization occurring, 2D COSY NMR was performed to assess the 1H-1H correlations (Figure  2.7).  Although there are quite a few correlations, it can be noted that there appears to be a doublet of doublets (~2.59 - 2.79 ppm) which most likely corresponds to the hydrogens on the diethylenetriamine backbone, as this is the splitting pattern you would expect for a symmetric chiral molecule (red bracket, Figure  2.7).   D2O (i) (ii) (iii) 23   Figure ‎2.7. 1H-1H COSY NMR spectrum of [Lu(decapa)]2- alkyl region (600 MHz, D2O, 25°C); red bracket depicting doublet of doublets of dien backbone, pictured right.  The aromatic region of the COSY is less informative since many correlations were observed (Appendix, Figure A.1), and is similar to the HSQC spectrum (Figure  2.8) where there are multiple static isomers all showing their own individual correlations.  This is in contrast to the [Y(decapa)]2- spectrum, where the isomers are not static and result in very few 13C NMR correlations.  The [Lu(decapa)]2- spectrum has many broad HSQC correlations due to multiple isomer signals overlapping with each other. As seen in the HSQC [Lu(decapa)]2- spectrum, there are a few sharp peaks in the 13C NMR spectrum that correlate to many different peaks in the 1H NMR spectrum (see inset in Figure  2.8).  This implies that the many isomers present in the 1H NMR spectrum represent very similar structures, as their corresponding carbon signals are at the same frequency.  The fact that there are static isomers in solution gives promise that the metal-chelate complex will exhibit stronger thermodynamic stability.     24   Figure ‎2.8. 1H-13C HSQC NMR spectrum of [Lu(decapa)]2-, inset: zoom in on aromatic region (600 MHz, D2O, 25°C).  Finally, to gain more insight into the solution chemistry of the [Lu(decapa)]2- complex, VT 1H NMR experiments were performed.  At 85 ºC, the maximum temperature available for D2O solutions, only slight coalescence and peak broadening can be observed (Figure  2.6). This implies that for the [Lu(decapa)]2- complex while it forms many isomers, these isomers are relatively static and not exchanging in solution, even at high temperatures.    H5decapa was also complexed with Zr(IV), despite the fact that the ligand was not specifically designed for Zr chemistry.  Zr(IV) ions tend to precipitate quickly, forming aggregates and polynuclear hydroxo species, especially in mildly acidic or neutral environments.  Due to its appropriate denticity and binding cavity size, the Zr-decapa complex was synthesized and subsequently analysed.  H5decapa was mixed with ZrCl4 and complexation was confirmed by mass spectrometry.  The 1H NMR of [Zr(decapa)]- is very different than those of the previous M-decapa complexes, showing sharp well defined resonances 25  (Figure  2.9).  The splitting pattern of the 1H signals depict an asymmetric complex and interrogating the aromatic region, one can see clear splitting of the two picolinic acid moieties, which was subsequently confirmed by 2D COSY NMR (Appendix, Figure A.2).   Figure ‎2.9. 1H NMR spectra at ambient temperature in D2O of (top) H5decapa (300 MHz) and (bottom) [Zr(decapa)]- (400 MHz) in D2O, showing the formation of one static isomer upon Zr4+ chelation. Correlation peaks determined from 2D COSY NMR (Appendix, Figure A.2).   Due to the static nature of this metal-ligand complex, a clear 13C NMR could be obtained and shows 24 distinct peaks compared to the 12 peaks in the free ligand spectra (Figure  2.10).  These results confirm that [Zr(decapa)]- forms one asymmetric isomer in solution.  This could mean that the stable complex that forms causes each binding arm of H5decapa to be in a different conformation.  It is also possible that one of the picolinic arms is not bound to the Zr ion, as Zr4+ can bind with a CN of 8.  While it is very promising that the NMR experiments show one static isomer for [Zr(decapa)]-, if one of the arms is not bound to the metal center this could provide instability in vivo.  Not only would it provide undistributed charge on the complex, as was seen in the [In(decapa)]2- DFT studies,1 but it would also provide an unwanted available site for in vivo serum proteins to bind to the Zr(IV).   D2O 26   Figure ‎2.10. 13C NMR spectra at ambient temperature of (top) H5decapa (400 MHz) and (bottom) [Zr(decapa)]- (400 MHz) in D2O, showing the formation of one static, asymmetric isomer upon Zr4+ chelation.  2.2.2 Radiolabeling Experiments To determine the ability of H5decapa to label lutetium and yttrium isotopes, and to evaluate the metal-chelate kinetic inertness, radiolabeling and competition experiments using blood serum were performed.  Initial radiolabeling experiments with H5decapa showed less than promising results with 86/90Y and 177Lu.  Due to the lack of availability of many radiometals, the majority of the radiolabeling experiments were conducted in collaboration with Dr. Eric Price at the Memorial Sloan Kettering Cancer Center (MSKCC) in New York. However, initial 86Y radiolabeling experiments were performed at TRIUMF using the salty target purification method.  This involves the irradiation of a Sr(NO)3 solution on the target (13 MeV TR13 cyclotron).  This solution was then purified with a DGA resin that selectively retains Y(III) while the remaining Sr(II) is eluted under acidic conditions.35  Successive washes with nitric and hydrochloric acids remove the residual cold Sr(II), followed by elution of the desired radioactive 86Y collected in 1 mL fractions of water.   These aliquots were then used to immediately radiolabel both H5decapa and DOTA, which is used as a control as it is a ‘gold standard’ chelator for Y(III).  Incubation of purified 86Y with H5decapa for 10 minutes at room temperature and subsequently 40 minutes at 80°C failed to yield any detectable  27  metal-ligand complex. In contrast, mixing of DOTA with 86Y at 80ºC resulted in a single sharp peak on the HPLC radiotrace at tR = 5.4 min, compared to the free 86Y peak at tR = 3.1 min.  The poor 86Y labeling efficiency with H5decapa was further corroborated by studies done at MSKCC, that also found no radiolabeling under various incubation conditions with H5decapa, yet DOTA showed quantitative labeling with 86Y, as has been previously reported.34 One potential explanation for the low observed results may be due to the production method of 86Y. Even following purification, there is often excess cold strontium remaining in the 86Y solution, which may interfere with the binding of H5decapa, if the ligand shows similar or stronger affinity for Sr(II) versus Y(III). Radiolabeling with 90Y, that can be produced without other side products by Perkin Elmer’s patented process was also tested. These binding experiments were conducted with H5decapa, DOTA and DTPA in ammonium acetate buffer and were each heated to 70°C for 30 minutes, to promote quantitative binding. Radiolabeling experiments with 177Lu, also obtained from Perkin Elmer, were performed following the same method. Radiochemical yield measurements were attempted via instant thin-layer chromatography using acidified silica-embedded paper strips (iTLC-SA) and by HPLC.  Unfortunately, useful data could not be obtained by iTLC as the metal-complexes [177Lu(decapa)]2- and [90Y(decapa)]2- could not be separated from the free-metal (177Lu and 90Y), both eluting with the solvent front.  Due to the high polar nature of the metal-ligand complexes, HPLC separation was not useful either as the free metal solutions along with the metal-chelate solutions all eluted in the void-volume of the column (< 4 min).  No method was found to completely separate the metal-complexes from the free-metal, preventing the quantification of binding.  Serum stability studies were performed to help gauge the stability as well as the percent binding of these complexes as separation of large serum proteins (60-100 kDa) from small chelates is typically efficient. Radiolabeling 89Zr using synthetic chelators has proven to be very challenging over the past several years and few complexes have achieved similar results to the gold standard DFO.  Our group has searched extensively for a potential ligand that would show strong binding with 89Zr. Although H4octapa was seen to be an excellent ligand for both 111In and 177Lu radiometals, no measurable quantity of its 89Zr complex could be detected under a vast array of conditions.36  To that end, H6phospa was designed, where 28  the carboxylic acids of H4octapa were substituted with methylene phosphonates to better suit zirconium’s oxophilic preferences. The new ligand, H6phospa was seen to incorporate 89Zr with a maximum yield of 12 % (18 hours, 37°C).31  Due to the well resolved NMR data of [Zr(decapa)]-, appearing as a single static isomer, there was hope that H5decapa may be even more successful.  After incubation of 89Zr with H5decapa for 30 minutes at 37°C, quantitative binding (>99%) was shown by a single sharp peak in the radiotrace on the HPLC, tR = 6.3 min, (Appendix, Figure A.4) compared to the absence of signal corresponding to free 89Zr (tR = 3.8 min); this was further confirmed via iTLC-SA strips (Appendix, Figure A.5).  These results were compared with 89Zr(DFO) which also demonstrated quantitative binding, as seen by both methods (Appendix, Figure A.6).    2.2.3 Serum Stability Studies In vivo, kinetic inertness is an imperative parameter in determining the stability of a complex; likewise, competition experiments using native biological ligands such as apo-transferrin and albumin are useful methods of predicting the in vivo stability and kinetic inertness of radiometal ion complexes.  Competition experiments using [90Y(decapa)]2- against human blood serum were conducted over a span of 5 days (Figure  2.11). Serum stability was assessed by precipitation with CH3CN, where the addition of cold CH3CN causes the serum proteins to precipitate out of solution, allowing for the separation and quantification of supernatant (ligand + metal) versus precipitate (protein + metal).  Measurement of the radioactivity for each, in comparison to control experiments with free 90Y, provided percentages of intact metal-chelate complex after 1, 3 and 5 days. The caveat to this method of serum stability analysis is that the metal-ligand complex must be soluble in a mixture of water and acetonitrile, as precipitation of the metal-ligand complex along with serum proteins would provide inaccurate data.  Additionally, this method cannot distinguish between radiometal that has been transchelated from chelator to serum proteins and intact chelate-radiometal complexes that are merely adsorbed or occluded with the precipitated serum proteins. The 90Y serum stability experiments were tested with three ligands, to compare H5decapa with 29  the well-characterized ligands DOTA and DTPA, the gold standards in the field of 90Y chelation. The serum stability results for H5decapa show less than 30% of the H5decapa-90Y complex remained intact after 24 hours. However, this result may reflect the poor radiolabeling yield for [90Y(decapa)]2-, as the bound metal-ligand levels remain relatively constant over the 5 day period. This implies that the 90Y that is bound to H5decapa remains chelated, however much of the radiometal is left unbound.  A low radiolabeling yield could also explain why no labeling was seen during the 86Y experiments, especially if the solution also had some Sr(II) contaminations. The serum stability experiment supports previously reported data that DOTA is a stable ligand for 90Y whereas DTPA suffers from stability issues in vivo.37 Furthermore, this supports the obtained NMR data that showed the [Y(decapa)]2- complex to be fluxional in solution, ultimately determining that H5decapa is not an ideal ligand for Y(III) chelation.    Figure ‎2.11. 90Y stability of transchelation by human serum proteins via CH3CN precipitation at 37°C with H5decapa and DOTA over 5 days.  Analogous metal-ligand serum stability tests were performed with [177Lu(decapa)]2- yielding more promising results than were observed with 90Y (Figure  2.12). [177Lu(decapa)]2- showed comparable binding abilities to 177Lu(DOTA) and 177Lu(DTPA) after 24 hours (69.5 ± 1.7%, 76.9 ± 1.3% and 74.7 ± 1.0% respectively).  However, [177Lu(decapa)]2- demonstrated improved kinetic inertness over the 5 day 01020304050607080901001 d 3 d 5 dStability of 90Y(chelate) Complex (%) 90Y(decapa)90Y(DOTA)90Y(DTPA) 390 ( a) 90 ( ) 90Y(DTPA)  30  period, showing a minimal change in stability (3%), whereas 177Lu(DTPA) decreased stability 15% over the same time frame.  These findings are comparable to previous results obtained via PD10 column stability experiments, where [177Lu(decapa)]2- showed improved stability over 177Lu(DTPA), but slightly lower values compared to 177Lu(DOTA) and [177Lu(octapa)]- (Appendix, Table A.1). While DOTA remains the best known chelator for 177Lu, H5decapa demonstrates comparable results and promising kinetic inertness.  These results also confirm that 177Lu was effectively bound by H5decapa, despite iTLC and HPLC methods being unable to confirm radiochemical yields.    Figure ‎2.12. 177Lu stability of transchelation by human serum proteins via CH3CN precipitation at 37°C with H5decapa, DOTA, and DTPA over 5 days.    Serum stability experiments were also performed with human blood serum and both the 89Zr(DFO) and 89Zr(decapa) complexes compared to a control of neutralized free 89Zr (Figure  2.13).  The results from the serum stability tests were analysed by the CH3CN precipitation method, described previously for 90Y and 177Lu, as well as by iTLC-SA.  Unfortunately, the [89Zr(decapa)]- complex was found to be unstable in serum, and shows almost complete transchelation to serum proteins after just 1 day.  Nevertheless, the difficulty of labeling 89Zr in any quantity, as seen for most ligands, makes these preliminary results very promising.  In light of the extreme preference of Zr(IV) for hard oxygen donor atoms, it was very surprising that the ligand H5decapa could achieve such high radiochemical yields with 01020304050607080901001 d 3 d 5 dStability of 177Lu(chelate) Complex (%) 177Lu(decapa)177Lu(DOTA)177Lu(DTPA)177Lu(decapa) 177Lu(DOTA) 177Lu(DTPA) 31  89Zr. These results will inspire potential modifications to the H5decapa scaffold which can be explored in an attempt to further stabilize the Zr-chelate complex.   Figure ‎2.13. 89Zr stability of transchelation by human serum proteins via iTLC (50 mM EDTA) at 37°C with H5decapa, DFO and free 89Zr control over 3 days.   2.2.4 Thermodynamic Stability  Stability constants (log KML) are well-established methods for determining and comparing relative thermodynamic stabilities for metal-ligand complexes; however, a more accurate measurement is the pM value (-log[Mn+free]), which indicates the metal-scavenging ability of the ligand, and simultaneously provides a more accurate figure for predicting the in vivo thermodynamic stability of metal-ligand chelates under relevant physiological conditions.  pM values are calculated under specific conditions (usually 10 μM total ligand, 1 μM total [M3+], pH 7.4, 25°C) that account for ligand basicity (ligand pKa values), free metal concentration, ligand-to-metal ratios, pH, variable denticity, and metal hydroxide formation.  The thermodynamic stability of [Y(decapa)]2- was determined by potentiometric titrations to be log KML = 24.5 (1) (pM = 20.0), which is slightly higher, and therefore better, than the other ‘gold standard’ ligands tested with Y3+.  This aptly illustrates how thermodynamic stability does not always predict the stability of the metal-chelate complex in vivo.  While [Y(decapa)]2-  has a comparable log KML 01020304050607080901000 24 72% Serum Stability by iTLC elution Time (hours) 89Zr(decapa)89Zr(DFO)89Zr control89Zr(decapa) 89Zr(DFO) 89Zr control 32  value in comparison to DOTA, the Y(DOTA) complex is significantly more kinetically inert than the H5decapa complex.  This was also seen with In3+ and H5decapa, which has a high log KML value, but is shown not to label with very high yields.1  Conversely, [Lu(decapa)]2- was found to have much lower thermodynamic stability values, log KML = 20.4 (pM = 16.2), compared to [Y(decapa)]2- and [In(decapa)]2-; however, the [Lu(decapa)]2- complex obtained the highest radiolabeling yields and serum stability over the 5 day experiment between the three compounds.        Table ‎2.1. Thermodynamic stability values (log KML and pM) for  H4octapa, H5decapa, DTPA and DOTA with In3+, Y3+, and Lu3+.1–3  Ligand log KML pMa In3+ octapa4- 26.8(1) 26.5 decapa5- 27.56(5) 23.1 DTPA4- 29.0 25.7 DOTA4- 23.9(1) 18.8 Y3+ octapa4- 18.3(1) 18.1 decapa5- 24.5(1) 20.0 DTPA4- 21.2-22.5 17.6-18.3 DOTA4- 24.3-24.9 19.3-19.8 Lu3+ octapa4- 20.08(9) 19.8 decapa5- 20.4(1) 16.2 DTPA4- 22.6 19.1 DOTA4- 21.6(1) 17.1 a Calculated for 10 μM total ligand and 1 μM total [M3+] at pH 7.4 and 25°C.   While thermodynamic stability values (log KML and pM) can provide a good gauge of stability of a metal-chelate complex, facilitating comparisons to other known ligands, discrepancies between predicted and actual stabilities in vivo have been clearly shown.38 Therefore, this serves to demonstrate that thermodynamic parameters are not the only factors contributing to the biological stability of the metal-ligand complexes: kinetic parameters such as ligand-metal on/off rates are far more important in determining biological stability.    33  2.2.5 Circular Dichroism   Circular dichroism (CD) measures the difference in absorption of left and right circularly polarized light, which makes it a good probe for chiral molecules such as proteins.  The CD spectrum provides information regarding the bonds and structures that lead to this chirality in the solution phase.39  CD is extensively used to help elucidate protein secondary structure; the number of α-helices, β-sheets and random coils can all be deduced from the difference in absorption in the UV range (250-350 nm) of the CD spectra.40 Moreover, it can be used to study changes in structural formation during the binding of metals, ligands or to probe protein-protein interactions.   While the synthetic chelator may efficiently bind a desired metal isotope that can be confirmed and characterized by NMR studies and thermodynamic stability constants, it does not provide a measure of how the anticipated complex will behave when exposed to other metal-seeking chelators, which are omnipresent in vivo. Human serum albumin (HSA) is the constitutive protein of blood plasma and is essential in distribution and transport of transition metals in the body.41  HSA has four distinct metal binding sites and can bind a plethora of metal ions including Cu2+, Zn2+, Fe3+ and VO4+.41 Another widely distributed metal-binding protein, apo-transferrin, serves principally to transport Fe3+ ions in the body, yet it can also bind a number of other metal ions in vivo.42 Previously it has been shown that Lu3+ can bind both apo-transferrin and albumin,43,44 and that Y3+ can also bind albumin, through various spectroscopic methods.45  UV-vis spectroscopy and CD were used in an attempt to assess the stability of [Lu(decapa)]2- upon exposure to apo-transferrin.  For the UV-vis experiments, apo-transferrin solutions were made up in phosphate buffer (pH 7.4, 10 mM) and their spectra were recorded.  Upon the addition of 1 and 2 equivalents of Lu3+, one can see the decrease in the absorbance at the 280 nm peak, along with the increase in the peak around 230 nm, (Figure  2.14) which have been previously characterized.44   34   Figure ‎2.14. UV-vis spectrum of apo-transferrin with the addition of 1 and 2 equivalents of Lu3+.   A set of experiments were designed using CD to evaluate the stability of the preformed metal-chelate complex upon exposure to human metal binding proteins.  Initial experiments were performed with Lu3+ and apo-transferrin as the protein has been proven to bind the metal isotope, and Lu3+ has been demonstrated to form a stable complex with H5decapa.  Circular dichroism spectra can be measured over a large range of wavelengths.  The intrinsic region, in the far UV region (190-245 nm), depicts major changes in the secondary structure of the protein, and would only cause a change in the CD spectrum if the binding of the metal isotope caused significant conformational changes to the α-helices and β-pleated sheets.46,47 The aromatic region, in the near-UV region (245-320 nm), probes the absorption of the aromatic amino acids residues (Trp, Phe, Tyr), and is the region where native apo-transferrin has a characteristic absorbance spectrum.  The aromatic region will report changes to the tertiary folding of the polypeptide chain that induces alterations to the chiral environment of the aromatic side group chromophores.48  The final region is the visible region (320-600 nm), where there is no notable absorption 00.511.522.533.544.55230 240 250 260 270 280 290 300 310Absorbance (AU) Wavelength (nm) -  - -   apo-transferrin  apo-transferrin + 1 eq Lu3+ apo-transferrin + 2 eq Lu3+  0.60.70.80.9270 275 280 285 29035  for apo-transferrin.  Induced CD spectra can be observed in this region if the whole metal-ligand complex binds the transferrin together, due to d  d electronic transitions.49,50    The CD spectrum of apo-transferrin was recorded in phosphate buffer (pH 7.4, 10 mM) in the aromatic region, and then subsequently monitored upon incubation with [Lu(decapa)]2- over 2 hours (Figure  2.15).  As only slight spectral changes were observed over the 2 hours from the initial apo-transferrin curve, it appears as if the Lu3+ is remaining bound to the ligand and is not being released to the apo-transferrin.    Figure ‎2.15. CD spectra of apo-transferrin upon addition of metal-ligand complex [Lu(decapa)]2-.   Next, apo-transferrin was exposed to equimolar amounts of Lu(III) (Figure  2.16).  Larger changes were observed between the 2 spectra, suggesting that Lu3+ is bound to apo-transferrin.  It is known that Fe3+ interacts with two tyrosine residues within the apo-transferrin binding site and that coordination induces conformational changes that can be observed via CD.48  If Lu3+ is binding in the Fe3+ binding sites, similar conformational changes to the aromatic amino acid residues should be observed.  The local minimum at 288 nm is characteristic of tyrosine (Tyr) and tryptophan (Trp) absorption, and changes in intensity on binding Lu3+ would indicate that these amino acids are involved.46  The Lu-transferrin -60000-50000-40000-30000-20000-10000010000250 260 270 280 290 300 310 320Elipticity (ε) Wavelength (nm) -  - -   apo-transferrin  apo-transferrin + [Lu(decapa)]2- apo-transferrin + [Lu(decapa)]2- + 2 hrs 36  solution was subsequently exposed to free ligand (H5decapa) to observe potential conformational changes towards the original apo-transferrin spectrum; such results would indicate that H5decapa preferentially binds Lu3+ in the presence of apo-transferrin.  Although, many slight changes can be observed that are similar to the native protein, the most notable region is at 288 nm where the two spectra (green & blue) overlap, suggesting that the tyrosine and tryptophan residues have returned to their original orientation.     Figure ‎2.16. CD spectra of apo-transferrin upon addition of metal (Lu3+) and ligand (H5decapa).   Although a more exhaustive set of experiments are required to draw any conclusive results from these experiments, the initial findings provide insight into a potentially unique method of measuring competitive metal binding between ligand and human serum proteins.  Classically, these experiments are conducted with radioactive isotopes so that the metal isotope can be easily traced.  However, experiments based on UV-vis and CD would allow for some preliminary in vitro competition assays to be performed using the ‘cold’ metal complexes, avoiding the cost and safety considerations that encumber the use of radioisotopes.    -60000-50000-40000-30000-20000-1000001000020000250 260 270 280 290 300 310 320Elipticity (ε) Wavelength (nm) -  - -   apo-transferrin  apo-transferrin + 1 eq Lu3+ apo-transferrin + 1 eq Lu3+ + H5decapa 37  2.2.6 Europium Fluorescence Studies There are major differences between nuclear imaging and optical imaging; however, each mode provides unique attributes that can be combined together to provide a wider breadth of application. Optical imaging provides resolution down to the micrometer scale with real time imaging, but is limited by its penetration depth in tissue, which is only a few millimeters; however, there are a variety of situations where optical imaging is critical.  Coupling nuclear imaging with optical imaging can help narrow the gap between the vast depth but poor detail of the former, compared with the poor depth but great detailing of the later.  One potential widespread need could be for tumor imaging and removal, for instance, a patient could be injected with a BFC radiolabeled with an isotope for SPECT or PET imaging.  Once the tumour has been localized, a fluorescent BFC could be injected to assist clinicians with removing the cancerous cells by illuminating the tagged cells using fluorescence techniques.  Even before the clinical stages, where fluorescent BFCs could be of great use, they could also be used to help understand and elucidate pathways in many preclinical assessments.  Often, little is known about antigen-biomolecule interactions, especially whether or not they are uptaken into the cell.  Fluorescent techniques allow for real-time imaging and are also widely used in luminescent assays to evaluate receptor ligand interactions, critical to the field of drug discovery and development.    Previously, organic fluorescent reagents had been widely used as tags or labels; unfortunately, the high cellular auto-fluorescence decreased the signal-to-noise ratio, reducing the dynamic range and sensitivity of the tags.51   Lanthanide complexes, on the other hand, are efficient fluorescent labels due to their unique properties such as excitation in the UV region (310 - 340 nm) and long lifetimes (100 μs - 1 ms). These characteristics led to lanthanide detection by time-resolved fluorescence (TRF) which yields a specific signal with very low background.51  The luminescent properties of lanthanides are dominated by their low extinction coefficients; however, unbound in solution, they tend to lack significant fluorescent properties and often require a covalently attached organic chromophore (antenna effect).  Europium is a luminescent lanthanide which prefers the oxidation state of 3+ and has an ionic radius of 95-112 pm (CN 6-10).52  Since water molecules quench the lanthanide fluorescence, an ideal chelator would saturate the 38  coordination sphere of the ion. Because of its large binding cavity, H5decapa was projected as a potential chelator for Eu3+ to be used for TRF in cell studies.    To form the metal-ligand complex, H5decapa was mixed with Eu(NO3)3·6H2O and the product was detected by mass spectrometry, confirming the synthesis of the cold metal complex as Na2[Eu(decapa)].  The 1H NMR spectrum of [Eu(decapa)]2- is very different than the previous metal complexes due to europium being a paramagnetic isotope (Figure  2.17).   While integrations, peak splitting and chemical shifts do not unambiguously show the coordination chemistry of the metal-chelate complex, the fact that clear peaks can be detected indicates that one isomer is formed in solution.  Fifteen peaks can be distinguished in the spectrum, corresponding to the number of protons expected for H5decapa if an asymmetric complex is formed.    This suggests that [Eu(decapa)]2- forms a stable complex in solution and could be suitable for in vivo experiments.   Figure ‎2.17. 1H NMR spectra at ambient temperature of [Eu(decapa)]2- (400 MHz) in D2O, showing the effect of binding the paramagnetic Eu3+ ion.   The major benefit of lanthanide-based labels is the characteristically long lifetime of the excited state, which allows for separation of the specific signal from the surrounding non-specific signals.  The typical cellular background fluorescence is emitted on the order of picoseconds, a cell can be excited and the emission signal recorded until the background signals have completely decayed, leaving only the lanthanide fluorescence.  Europium, when bound to a chelator, exhibits a strong, sharp emission at 615  39  nm (when excited at 340 nm), which can be seen in Figure  2.18.  This provides confirmatory evidence that Eu3+ forms a stable chelate with H5decapa, as no quenching is observed, and the known shift in fluorescent emission from the free-metal to the bound-metal is observed.     Figure ‎2.18. Eu3+ and [Eu(decapa)]2- emission spectrum (excitation wavelength = 280 nm).  Multicellular spheroids, which are 3-dimensional structures of cancerous cells that act as in vitro tumor models, were grown to measure the uptake of the [Eu(decapa)]2- complex.  Unfortunately, access to a time-resolved fluorescence microscope prevented this experiment from being completed.  The emission spectrum shown in Figure  2.18 suggests that the [Eu(decapa)]2- complex should be further investigated in the future when access to an appropriate microscope is obtained.    2.2.7 Synthesis and Characterization of Bifunctional Decapa While it is important to first assess the metal-ligand stability and binding characteristics of a ligand scaffold before increasing the synthetic complexity, until the system is bifunctional no truly informative in vivo experiments can be done.  Furthermore, it is critical that once a metal-ligand system shows promise, the bifunctional derivative must retain the radiometal complex stability and properties -20020406080100120450 500 550 600 650 700Intensity (AU) Wavelength (nm) -  -  Eu3+  [Eu(decapa)]2- 40  upon the addition of a linking moiety.  Previous attempts to synthesize a bifunctional version of H5decapa stemming from the diethylenetriamine backbone, in a similar manner to what was accomplished for H2dedpa and H4octapa,1,27 proved to be unsuccessful. The larger backbone of H5decapa led to greater asymmetry and instability when one of the bridging ethyl moieties was functionalized, rendering the complex unstable.  The synthesis of a linker moiety from the central nitrogen in exchange for an acetate arm was attempted.  This maintained the symmetry of the compound, while removing a single potential binding group.  Because H5decapa is decadentate and many of the metal isotopes in consideration do not require all 10 binding sites, changing one arm may not affect the binding of the ligand too much.  The chosen linker used in this case was an ethyl-nitrobenzene sidechain.  The ethyl linkage should provide distance and flexibility from the metal binding atoms in the chelate so that it does not sterically hinder their binding properties.  The nitrobenzene group has been the linker of choice for most of the bifunctional ‘pa’ family ligands as it is a stable functional group that can be chemically modified to the phenyl-isothiocyanate, and this isothiocyanate can subsequently be coupled to a suitable primary amine on a chosen biomolecule, forming a thiourea linkage under mildly basic conditions.    The synthesis of the bifunctional complex (BF-decapa) followed a similar synthesis to the original ligand, differing only in the addition of the linking arm. BF-decapa was synthesized beginning with N-nosyl protection (2.10), then N-alkylation with ethyl-nitrobenzene (2.11), N-alkylation with an alkyl halide (2.12), nosyl deprotection via thiophenol (2.13), a second alkyl halide N-alkylation (2.14), and finally deprotection using boiling HCl (2.15) (Scheme  2.3).  The nosyl protection was performed under milder conditions then was done for the synthesis of H5decapa to prevent the protection of the more hindered central nitrogen.  Since the bromo-nitrobenzene was quite unreactive, there was no major concern of over alkylating the other nitrogens as they were more sterically hindered.  This allowed for the subsequent alkylation to occur with the methyl-picolinate before deprotecting with thiophenol.      41  Scheme ‎2.3. Synthesis of BF-decapa (2.10 – 2.15).      The Zr4+ complex of BF-decapa, Zr(BF-decapa), was synthesized by mixing the BF-decapa salt (2.15) with ZrCl4 in deionized water, adjusting the pH with NaOH (0.1M) to ~4-5 and stirring at room temperature for 1 hour.  The formation of the coordination complex was confirmed by mass spectroscopy and the product was studied by NMR spectroscopy.  The resulting metal complex was highly insoluble, most likely due to the neutral charge of the resulting complex compared to [Zr(decapa)]-, which led to difficulties in studying the NMR spectra.  The 1H-NMR spectrum of Zr(BF-decapa) in DMSO-d6 was analyzed on the 600 Hz spectrometer using a suppressed water measurement (Figure  2.19).     42   Figure ‎2.19. 1H NMR spectra, 400 MHz at ambient temperature of (top) BF-decapa in D2O, (bottom) [Zr(BF-decapa)] in DMSO-d6, * - water suppression signal.   The 1H NMR spectrum of Zr(BF-decapa) appears to depict multiple isomers in solution upon complexation, when compared to the bare ligand spectrum.  However, there are sharp peaks which perhaps imply that there is at least one static isomer along with some broader peaks, which could be depicting a fluxional isomer.  The 2D COSY spectrum (Appendix, Figure A.3) has six correlation peaks in the aromatic region, which hints that the major isomer is asymmetric, as only three correlations would be observed for a symmetric isomer.  Nonetheless, further NMR studies, along with thermodynamic testing, radiolabeling and serum stability experiments, are required in order to gain more insight and understanding on the binding abilities and stabilities between BF-decapa and select radioisotopes.    2.3 CONCLUSION Preliminary investigations into the decadentate ligand H5decapa and its bifunctional derivative BF-decapa show this ligand scaffold to be a promising candidate for radiopharmaceutical applications.  While H5decapa did not show promising radiolabeling kinetics with 86Y/90Y, the radiolabeling results with H2O DMSO MeOD 43  177Lu and thermodynamic stability values obtained for Lu3+ demonstrate that H5decapa is a suitable candidate for further testing of the metal-chelate complex.  These experiments also demonstrate how finely tuned a chelate must be designed, as even though Y3+ and Lu3+ bear similar coordination preferences, a ligand may not bind each in a stable manor.  Quantitative radiolabeling was observed for 89Zr with H5decapa, and although the complex was not stable over time, the quick labeling at room temperature was unique for a non-hydroxamate ligand, and provides a basis from which to build future Zr4+ chelates.   Initial investigation into alternative methods for in vitro testing of the metal-chelate stability via spectroscopic experiments was performed.  CD shows promise as a technique to monitor competition experiments between endogenous metal binding proteins found in the body with the ‘cold’ metal-chelate complex.  A preliminary investigation on the use of Eu3+ with a radioisotope chelator to expand the scope for clinical use in imaging and removing tumours was explored.  Cell studies would give a better understanding of the chelate in vivo and would also benefit from the fluorescent chelate. Following the synthesis of the bifunctional derivative of H5decapa, which can now be bound to an appropriate biomolecule, the metal-ligand binding properties must be reassessed. These tests will help determine whether the newly added ethyl nitrobenzyl moiety modifies the binding properties relative to the native ligand.     44  2.4  EXPERIMENTAL METHODS 2.4.1 Materials and Methods  All solvents and reagents were purchased from commercial suppliers (Sigma Aldrich, TCI America, Fischer Scientific, Alfa Aesar) and were used as received. DOTA/DTPA was purchased from Macrocyclics, STREM chemicals, Acros Organics. The analytical thin-layer chromatography (TLC) plates used were aluminum-baked ultra-pure silica gel 60 Å, 250 µm thickness. 1H and 13C NMR spectra were recorded at ambient temperature unless otherwise noted on Bruker AV300, AV400, or AV600 instruments; the NMR spectra are expressed on the δ scale and were referenced to residual solvent peaks.  Low-resolution mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia.  Microanalysis for C, H, and N was performed by UBC MS staff on a Carlo Erba Elemental Analyzer EA 1108.  The HPLC system used for analysis and purification of cold compounds consisted of a Waters 600 controller, Waters 2487 dual wavelength absorbance detector, and a Waters delta 600 pump.  Phenomenex Synergi Hydro-RP 80 Å columns (250 x 4.6 mm analytical and 250 x 21.2 mm semi-preparative) were used for purification of the deprotected ligands.  Analysis of radiolabeled complexes was carried out on a Phenomenex Synergi Hydro-RP 80 Å analytical column (250 x 4.6 mm) using a Waters Alliance HT 2795 separation module equipped with a Raytest Gabi Star NaI (Tl) detector and a Waters 996 photodiode array (PDA). DGA (N,N,N’,N’,-tetra-n-octyl-diglycolamide) resin (Eichrom) was used for the purification of 86Y.   DMSO used for chelator stock solutions was of molecular biology grade (>99.9%: Sigma, D8418). Desferrioxamine mesylate (DFO) was purchased from Sigma Aldrich (>92.5%). Water used was ultrapure (18.2 MΩ cm−1 at 25°C, Milli-Q, Millipore). 90Y and 177Lu radiolabeling reactions were performed in ammonium acetate buffer (pH 4.5, 200 mM, made from ammonium acetate >99.9995% TraceSELECT®, Fluka), and 89Zr radiolabeling was performed in phosphate buffered saline (PBS, 45  Sigma). 177Lu-(chelate) and 90Y-(chelate) analysis was performed using an HPLC system comprised of a Shimadzu SPD-20A prominence UV−vis, LC-20AB prominence LC, a Bioscan flow-count radiation detector, and a C18 reverse phase column (Phenomenex Luna Analytical 250 × 4.6 mm). 177Lu was procured from Perkin-Elmer (Perkin-Elmer Life and Analytical Sciences, carrier free) as 177LuCl3 in 0.05 M HCl. 90Y was procured from Perkin-Elmer (Perkin-Elmer Life and Analytical Sciences, carrier free) as 90YCl3 in 0.05 M HCl. 89Zr was produced at Memorial Sloan-Kettering Cancer Center on an EBCO TR19/9 variable-beam energy cyclotron (Ebco Industries Inc.) via the 89Y(p,n)89Zr reaction and purified in accordance with previously reported methods to yield 89Zr with a specific activity of 5.28-13.43 mCi/μg (195-497 MBq/μg).53 Labeling reactions were monitored using acidic silica-gel impregnated glass-microfiber instant thin layer chromatography paper (iTLC-SA, Varian) and analyzed on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio- TLC software (Bioscan Inc.). All radio-labeling chemistry was performed with ultrapure water (>18.2 MΩ cm−1 at 25°C, Milli-Q, Millipore) that had been treated by stirring with Chelex resin (~1.5 g per liter of water for 24 hours, BioRad Laboratories) for 24 hours, followed by filtration with a 0.22 μm nylon media filter (Nalgene). Human blood serum (Sigma, Sera, human, aseptically filled, S7023−100 mL) competition solutions were agitated at 550 rpm and held at 37°C using an Eppendorf Thermomixer. Radioactivity in samples was measured using a Capintec CRC-15R dose calibrator (Capintec). Centrifugation of small Eppendorf tubes was performed with an Eppendorf 5424 centrifuge, and large samples with an Eppendorf 5810R centrifuge.  The UV-vis system used was a Cary 5000 UV-Vis-NIR spectrophotometer.  The CD system was a Jasco J-815 CD spectrophotometer operated with Spectra Manager software.  The fluorescence spectra were recorded on the Agilent Cary Eclipse Fluorescence Spectrophotometer.  2.4.2 2-Nitro-N,N-bis(2-(2-nitrophenylsulfonamido)ethyl)benzenesulfonamide (2.1) Diethylenetriamine (0.76 mL, 7.00 mmol) was dissolved in dichloromethane (DCM) (50 mL) with trimethylamine (TEA) (1.65 mL, 11.82 mmol, 1.7 equiv).  2-Nitrobenzene sulfonyl chloride (5.00 g, 22.56 mmol, 3.2 equiv) was dissolved in DCM (50 mL) and slowly added to the first solution, which was 46  allowed to stir overnight at room temperature. The solvent was then removed in vacuo and the orange solution was redissolved in DCM (100 mL).  The organic layer was washed with saturated NaHCO3, and washed with H2O (3 x 50 mL).  The organic layer was then dried with MgSO4 and the solvent was removed under reduced pressure to yield an orange fluffy solid.  This solution was dissolved in a minimal amount of DCM/CDCl3 (9:1) and recrystalized to give an off white solid (83%, 3.80 g). 1H NMR (300 MHz, Acetone-d6, 25oC) δ: 8.16-8.08 (m, 3H) 7.98-7.88 (m, 6H), 3.63-3.58 (m, 4H), 3.39-3.35 (m, 4H). 13C NMR (101 MHz, Acetone-d6, 25oC) δ: 149.45, 149.37, 135.76, 135.41, 134.26, 134.13, 133.55, 133.14, 131.78, 131.66, 126.27, 125.69, 49.79, 43.32. HR-ESI-MS calcd. for [C22H22N6O12S3+Na]+: 681.0356; found: 681.0349 [M+Na]+, PPM = -1.0.   2.4.3 Dimethyl 6,6'-((((((2-nitrophenyl)sulfonyl)azanediyl)bis(ethane-2,1-diyl))bis(((2-nitrophenyl)sulfonyl)azanediyl))bis(methylene))dipicolinate (2.2) To a solution of 2.1 (1.05 g, 1.59 mmol) in dimethylformamide (DMF) (10 mL, dried over molecular sieves 4 Å) was added methyl-6-bromomethyl picolinate1 (0.84 g, 3.66 mmol, 2.3 equiv) and Na2CO3 (0.96 g, 9.11 mmol, 6.0 equiv). The faint yellow reaction mixture was stirred at 60oC overnight, filtered to remove sodium carbonate, and concentrated in vacuo. The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 80 g HP silica; A: Hexanes, B: Ethyl Acetate, 100% A to 100% B gradient) to yield the product 2.2 as a yellow/white fluffy solid (65%, 0.99 g) (Rf : 0.6, TLC in ethyl acetate). 1H NMR (300 MHz, Acetone-d6, 25oC) δ: 8.21 (d, J = 7.7 Hz, 2H), 8.02 (d, J = 7.7 Hz, 1H), 7.93-7.79 (m, 13 H), 7.57 (t, J = 5.1 Hz, 2H), 4.80 (s, 4H), 3.85 (s, 6H), 3.74 (s, 8H). 13C NMR (75 MHz, Acetone-d6, 25oC) δ: 165.86, 157.42, 149.13, 149.01, 148.46, 139.00, 135.36, 135.09, 132.78, 131.51, 131.33, 126.71, 125.34, 125.14, 124.72, 53.87, 52.81, 48.57, 48.08. HR-ESI-MS calcd. for [C38H37N8O16S3]+: 957.1490; found: 957.1511, [M+H]+, PPM = 2.2.  47  2.4.4  Dimethyl 6,6'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(methylene))dipicolinate (2.3) To a solution of 2.2 (0.62 g, 0.65 mmol) in tetrahydrofuran (THF) (~8 mL) was added thiophenol (0.22 mL, 2.14 mmol, 3.3 equiv) and potassium carbonate (excess, ~1 g). The reaction mixture was stirred at 50oC for 48 hours, during which time the solution turned a bright yellow colour.  The solution was filtered through a medium frit glass filter funnel and evaporated in vacuo.  The resulting crude yellow oil was purified by neutral alumina chromatography (CombiFlash Rf automated column system; 2 x 24 g neutral alumina; A: dichloromethane, B: methanol, 100% A to 20% B gradient) to yield the product 2.3 as a yellow oil (79%, 0.20 g). 1H NMR (300 MHz, CDCl3, 25oC) δ: 7.97 (d, J = 7.7 Hz, 2H), 7.61 (t, J = 7.7 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H), 4.80 (s, 4H), 3.99 (s, 4H), 3.96 (s, 6H), 2.76 (s, 8H). 13C NMR (75 MHz, CDCl3, 25oC) δ: 165.82, 160.93, 147.46, 137.44, 125.64, 123.51, 55.13, 52.87, 49.36, 49.09. HR-ESI-MS calcd. for [C20H27N5O4+H]+: 402.2141; found: 402.2141, [M+H]+, PPM = -2.0.  2.4.5 N,N’’-[[6-(Methoxycarbonyl)pyridin-2-yl]methylamino]-N,N’,N’’-[(tert- butoxycarbonyl)methyl]]diethylenetriamine (2.4) To a solution of 2.3 (0.17 g, 0.43 mmol) in acetonitrile (10 mL) was added tert-butylbromoacetate (0.21 mL, 1.41 mmol, 3.3 equiv) and sodium carbonate (0.15 g, 1.37 mmol, 3.3 equiv). The reaction mixture was stirred at 60oC overnight, filtered to remove sodium carbonate, and concentrated in vacuo. The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 40 g HP silica; A: dichloromethane, B: methanol, 100% A to 20% B gradient) to yield the product 2.4 as an off-white solid (52%, ~0.18 g).  1H NMR (300 MHz, CDCl3, 25oC) δ: 7.94-7.91 (m, 2H), 7.80-7.71 (m, 4H), 3.95 (s, 4H), 3.83 (s, 6H), 3.26 (s, 6H), 2.69 (s, 8H), 1.38 (s, 27H). 13C NMR (75 MHz, CDCl3, 25oC) δ: 170.66, 166.02, 161.17, 147.28, 137.51, 123.23, 123.67, 27.04, 60.74, 56.57, 52.94, 52.90, 28.28). HR-ESI-MS calcd. for [C38H57N5O10 + H]+: 744.4184; found: 744.4174, [M+H]+, PPM = -1.3. 48   2.4.6 H5decapa·6HCl·3H2O (2.5) Compound 2.4 (0.14 g, 0.18 mmol) was dissolved in 3:1 THF/dH2O (15 mL), lithium hydroxide (0.026 g, 1.09 mmol, 6.0 equiv) was added, and the mixture stirred for one hour at room temperature.  The reaction was then concentrated in vacuo and redissolved in HCl (15 mL, 6M) and heated with a heat gun for 5-10 minutes.  The reaction mixture was again concentrated in vacuo and then purified via semi-prep reverse-phase HPLC (10 mL/min, gradient A: 0.1% TFA (trifluoroacetic acid) in deionized water, B: CH3CN. 0 to 95% B linear gradient 30 min. tR = 12.3 min, broad). Product fractions were pooled, concentrated in vacuo, dissolved in 6 M HCl, and concentrated in vacuo. The HCl salt H5decapa·6HCl·3H2O (2.5) was obtained as an off-white powder (49% yield, 0.079 g, using the molecular weight of the HCl salt as determined by elemental analysis). 1H NMR (300 MHz, D2O) δ: 8.25-8.13 (m, 4H), 7.78-7.76 (m, 2H), 4.62 (s, 4H), 4.04 (s, 4H), 3.73 (s, 2H), 3.50 (s, 4H), 3.32 (s, 4H).  13C NMR (75 MHz, D2O) δ: 172.71, 169.94, 166.16, 150.71, 146.03, 141.82, 128.44, 125.99, 57.68, 54.76, 53.99, 51.87, 50.48. Elemental analysis: calcd. for H5decapa·6HCl·3H2O (C24H29N5O10 ·6HCl·3H2O = 820.3279): C 35.14, H 5.04, N 8.53; found: C 35.01, H 5.09, N 8.29. HR-ESI-MS calcd. for [C24H29N5O10 + H]+: 548.1993; found: 548.1989, [M+H]+, PPM = -0.7.  2.4.7 Na2[Y(decapa)] (2.6) H5decapa·6HCl·3H2O (2.5) (15.00 mg, 0.019 mmol) was dissolved in deionized water (1 mL) and YCl3·6H2O (7.44 mg, 0.025 mmol, 1.3 equiv) was added.  The pH was adjusted to 4-4.5 using 0.1 M NaOH and the solution was stirred at 60°C overnight.  After confirmation of the product via mass spectrometry, the solvent was removed in vacuo to yield detectable Na2[Y(decapa)] (2.6) and excess salts. HR-ESI-MS calcd. for [C24H24N5O1089Y + H]-: 632.0660; found: 632.0660, [M+H]-, PPM = 0.0. Multiple isomers in solution were observed; NMR spectra can be found in Figure  2.2.    49  2.4.8 Na2[Lu(decapa)] (2.7) H5decapa·6HCl·3H2O (2.5) (15.00 mg, 0.019 mmol) was dissolved in deionized water (1 mL) and Lu(NO3)3·6H2O (11.50 mg, 0.025 mmol, 1.3 eq.) was added.  The pH was adjusted to 4-4.5 using 0.1 M NaOH and the solution was stirred at 60°C for 3 hours and then left stirring at room temperature for 72 hours.  After confirmation of the product via mass spectrometry, the solvent was removed in vacuo to yield detectable Na2[Lu(decapa)] and excess salts. HR-ESI-MS calcd. for [C24H24N5O10175Lu + H]-: 718.1009; found: 718.1011, [M+H]-, PPM = 0.3. Multiple isomers in solution were observed; NMR spectra can be found in Figure 2.6.   2.4.9 Na[Zr(decapa)] (2.8) H5decapa·6HCl·3H2O (2.5) (3.00 mg, 0.005 mmol) was dissolved in deionized water (1 mL) and ZrCl4 (1.66 mg, 0.0071 mmol, 1.3 equiv) was added.  The pH was adjusted to 4 using 0.1 M NaOH and the solution was heated to 60°C and left stirring overnight.  After confirmation of the product via mass spectrometry, the solvent was removed in vacuo to yield detectable Na[Zr(decapa)] (2.8) and excess salts. 1H NMR (600 Hz, D2O, 25oC) δ: 8.25 (t,  J = 7.8 Hz, 1H), 8.21 (t,  J = 7.8 Hz, 1H),  8.00 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 4.67 (m, 2H), 4.33 (d, J = 17.0 Hz, 1H), 3.92-3.73(m, 6H), 3.65 (q, J = 13.4, 2H), 3.27 (d, J = 13.7 Hz, 1H), 3.08-3.02 (quin, 2H), 2.97 (d, J = 12.3 Hz, 1H), 2.90 (d, J = 13.5 Hz, 1H). 13C NMR (151 Hz, D2O, 25oC) δ: 179.26, 179.11, 171.50, 171.48, 170.90, 155.87, 155.05, 148.93, 146.63, 142.16, 142.04, 126.24, 126.20, 124.31, 123.84, 123.37, 123.33, 65.69, 65.24, 60.63, 60.24, 57.71, 56.80, 56.41, 54.33, 54.22. HR-ESI-MS calcd. for [C24H25N5O10 90Zr + H]+: 634.0727; found: 634.0721, [M+H]+, PPM = -0.9.  2.4.10 Na2[Eu(decapa)] (2.9) H5decapa·6HCl·3H2O (2.5) (3.00 mg, 0.005 mmol) was dissolved in deionized water (1 mL) and Eu(NO3)3·6H2O (3.18 mg, 0.0071 mmol, 1.3 equiv) was added.  The pH was adjusted to 4 using 0.1 M 50  NaOH and the solution was left stirring overnight at room temperature.  After confirmation of the product via mass spectrometry, the solvent was removed in vacuo to yield detectable Na2[Eu(decapa)] (2.9) and excess salts. 1H NMR (400 Hz, D2O, 25oC) δ: 25.50, 21.87, 11.01, 10.54, 7.91, 7.36, 6.53, -0.97, -1.90, -4.24, -7.30, -8.73, -9.61, -11.17, -12.76. HR-ESI-MS calcd. for [C24H25N5O10 151Eu + H]+: 694.0800; found: 694.0797, [M+H]+, PPM = -0.4.  2.4.11 N,N”-(2-Nitrobenzensulfonamide)-1,2- triaminodiethane (2.10) Diethylenetriamine (0.50 mL, 4.60 mmol) was dissolved in THF (30 mL) in a round bottom flask (50 ml) that was placed in an ice bath. Sodium bicarbonate (1.17 g, 11.05 mmol, 2.4 equiv) was then added, followed by slow addition of 2-nitrobenzensulfonyl chloride (2.45 g, 11.05 mmol, 2.4 equiv), causing the reaction mixture to turn a pale yellow.  The reaction mixture was allowed to warm to ambient temperature and stirred overnight.  The off-white mixture was filtered to remove sodium bicarbonate and rotary evaporated. The crude product was purified by silica chromatography (CombiFlash Rf automated column system 80 g HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield the product 2.10 as an orange solid (63%, 1.37 g). 1H NMR (300 MHz, Acetone, 25oC) δ: 8.14-8.11 (m, 2H), 7.97-7.88 (m, 6H), 3.11 (t, J = 4.5 Hz, 4H), 2.79 (d, J = 10.2 Hz, 4H), 2.68 (t, J = 4.4 Hz, 4H). 13C NMR (75 MHz, Acetone, 25oC) δ: 148.42, 134.17, 133.24, 132.83, 130.73, 125.10, 47.88, 42.75. HR-ESI-MS calcd. for [C16H19N5O8S2+H]+: 474.0753; found: 474.0749, [M+H]+, PPM = -0.8.  2.4.12 N,N'-(((4-Nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis(2 nitrobenzenesulfonamide) (2.11) To a solution of 2.10 (1.00 g, 2.11 mmol) in DMF (5 mL) was added Na2CO3 (0.38 g, 2.75 mmol, 1.3 equiv) and 4-(2-bromoethyl)nitrobenzene (0.63 g, 2.75 mmol, 1.3 equiv). The reaction mixture was heated to reflux and left stirring for 4 days.  After 4 days the reaction mixture was cooled to room temperature, filtered, and dried in vacuo.  The crude product was purified by silica chromatography 51  (CombiFlash Rf  automated column system; 40 g HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield the product 2.11 as a dark orange oil (52%, 0.18 g). 1H NMR (400 MHz, CDCl3, 25oC) δ: 8.07-8.01 (m, 2H), 7.74-7.72 (m, 4H), 7.29 (d, J = 8.6 Hz, 2H), 3.04-3.03 (m, 4H), 2.81 (m, 2H), 2.71 (d, J = 7.8 Hz, 2H), 2.67 (t, J = 6.0 Hz, 4H). 13C NMR (101 MHz, CDCl3, 25oC) δ: 158.01, 157.64, 147.47, 146.81, 129.57, 129.52, 123.99, 117.30, 114.44, 54.96, 52.53, 37.55, 33.53. HR-ESI-MS calcd. for [C24H26N6O10S2 +H]+: 623.1230; found: 623.1237, [M+H]+, PPM = 1.1.  2.4.13 Dimethyl 6,6'-(((((4-Nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis(((2-nitrophenyl)sulfonyl)azanediyl))bis(methylene))dipicolinate (2.12) To a solution of 2.11 (0.35 g, 0.56 mmol) in DMF (8 mL, dried over molecular sieves 4 Å) was added methyl-6-bromomethyl picolinate (0.30 g, 1.29 mmol, 2.3 equiv) and sodium carbonate (0.14 g, 1.29 mmol, 2.3 equiv). The bright orange reaction mixture was stirred at 60oC overnight, filtered to remove sodium carbonate, and concentrated in vacuo. The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 80 g HP silica; A: Hexanes, B: Ethyl Acetate, 100% A to 100% B gradient) to yield the product 2.12 as an orange/brown oil (53%, 0.28 g). 1H NMR (300 MHz, CDCl3, 25oC) δ: 8.02-8.00 (m, 4H), 7.96 (d, J = 7.7 Hz, 2H), 7.78 (t, J = 7.8 Hz, 2H), 7.67-7.60 (m, 6H), 7.54 (d, J = 7.8 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 4.67 (s, 4H), 3.89 (s, 6H), 3.29 (t, J = 6.8 Hz, 4H), 2.58-2.51 (m, 8H). 13C NMR (101 MHz, CDCl3, 25oC) δ: 165.28, 156.98, 148.13, 147.55, 146.43, 138.14, 132.05, 129.74, 125.97, 125.93, 124.39, 124.38, 123.53, 55.31, 53.90, 52.91, 52.73, 46.87, 33.37. HR-ESI-MS calcd. for [C40H40N8O14S2 +H]+: 921.2184; found: 921.2184, [M+H]+, PPM = 0.    52  2.4.14 Dimethyl 6,6'-(((((4-Nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(methylene))dipicolinate (2.13) To a solution of 2.12 (0.28 g, 0.30 mmol) in THF (~8 mL) was added thiophenol (0.070 mL, 0.68 mmol, 2.3 equiv) and potassium carbonate (0.14 g, 0.98 mmol, 3.3 equiv). The reaction mixture was stirred at 50oC for 48 hours, becoming a dark orange colour.  The excess salts were removed by centrifugation (5 min, 4000 rpm) and the filtrate was concentrated in vacuo.  The resulting crude orange oil was purified by neutral alumina chromatography (CombiFlash Rf automated column system; 2 x 24 g neutral alumina; A: dichloromethane, B: methanol, 100% A to 20% B gradient) to yield the product 2.13 as a yellow/orange oil (79%, 0.20 g). 1H NMR (400 MHz, CDCl3, 25oC) δ: 8.01 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 7.73 (t, J = 7.8 Hz, 2 H), 7.45 (d, J = 7.7 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 3.99 (s, 4H), 3.91 (s, 6H), 2.79-2.72 (m, 12H). 13C NMR (101 MHz, CDCl3, 25oC) δ: 165.62, 158.88, 148.61, 147.39, 164.44, 137.76, 129.68, 126.00, 123.87, 123.67, 55.94, 54.02, 52.96, 52.69, 46.99, 33.27. HR-ESI-MS calcd. for [C28H34N6O6+H]+: 551.2618; found: 551.2617, [M+H]+, PPM = -0.2.  2.4.15 N,N’,N”-[(tert-Butoxycarbonyl)methyl-N.N”–[6-(methoxycarbonyl)pyridin-2-yl]methyl]-1,2,3-triaminodiethane (2.14) To a solution of 2.3 (0.04 g, 0.07 mmol) in acetonitrile (5 mL) was added tert-butylbromoacetate (0.025 μL, 0.17 mmol, 2.3 equiv) and sodium carbonate (0.02 g, 0.17 mmol, 2.3 equiv). The reaction mixture was stirred at 60oC overnight, filtered to remove sodium carbonate, and concentrated in vacuo. The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 24 g HP silica; A: dichloromethane, B: methanol, 100% A to 20% B gradient) to yield the product 2.14 as an orange oil (52%, 0.18 g). 1H NMR (400 MHz, CDCl3, 25oC) δ:  8.11 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 7.7 Hz, 2H), 7.89 (t, J = 7.7 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 4.18 (s, 4H), 3.96 (s, 6H), 3.90 (s, 4H), 3.73 (m, 2H), 3.54 (s, 4H), 3.45 (br s, 4H), 3.24 (m, 2H), 1.38 (s, 18H). 13C NMR (75 MHz, CDCl3, 25oC) δ: 168.74, 165.14, 156.74, 147.32, 147.25, 143.78, 139.10, 130.05, 127.49, 124.95, 53  124.02, 83.20, 57.47, 55.99, 54.47, 53.26, 50.42, 48.83, 29.83, 28.01. HR-ESI-MS calcd. for [C40H54N6O10 + H]+: 779.3980; found: 779.3973, [M+H]+, PPM = -0.9.  2.4.16 BF·decapa (2.15) Compound 2.14 (0.009 g, 0.01 mmol) was dissolved in 6 M HCl (2.5 mL), heated to reflux and left stirring overnight.  The reaction mixture was concentrated in vacuo and then purified via semi-prep reverse-phase HPLC (10 mL/min, gradient A: 0.1% TFA (trifluoroacetic acid) in deionized water, B: CH3CN. 5 to 100% B linear gradient 25 min. tR = 15.4 min, broad). Product fractions were pooled and then concentrated in vacuo.  BF·decapa (2.15) was obtained as an off white powder (41% yield, 0.003 g). 1H NMR (400 MHz, CDCl3, 25oC) δ: 8.13 (d, J = 7.6 Hz, 2H), 8.02 (d, J = 7.0 Hz, 2H), 7.93 (t, J = 7.7 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 4.06 (s, 4H), 3.70-3.65 (m, 6H), 3.51 (s, 4H), 3.51 (s, 4H), 3.24-3.17 (m, 2H). 13C NMR (101 MHz, MeOD-d4, 25ºC) δ: 30.93, 50.21, 52.55, 55.93, 56.01, 59.09, 124.91, 125.51, 128.33, 131.32, 140.09, 145.89, 148.70, 149.03, 160.20, 167.59, 174.30. HR-ESI-MS calcd. for [C30H34N6O10  + H]+: 639.2415; found: 639.2417, [M+H]+, PPM = 0.3.   2.4.17 Zr(BF-decapa) (2.16) BF-decapa (2.15) (5.00 mg, 0.005 mmol) was dissolved in deionized water (1 mL) and ZrCl4 (2.17 mg, 0.0093 mmol, 1.3 equiv) was added.  The pH was adjusted to 4 using 0.1 M NaOH and the solution was left stirring at room temperature for 4 hours. After confirmation of the product via mass spectrometry, the solvent was removed in vacuo to yield detectable Zr(BF-decapa) and excess salts. HR-ESI-MS calcd. for [C30H30N6O10 90Zr + Na]+: 747.0968; found: 747.0987, [M+Na]+, PPM = 2.5. Multiple isomers in solution were observed; NMR spectra can be found in Figure  2.19.    54  2.4.18 Solution Thermodynamics As a result of the strength of the binding of Y3+ and Lu3+ to H5decapa, the complex formation constants with this ligand could not be determined directly and the ligand-ligand competition method using the known competitor Na2H2EDTA was used by me. Potentiometric titrations were performed using a Metrohm Titrando 809 equipped with a Ross combination pH electrode and a Metrohm Dosino 800.  Data was collected in triplicate using PC Control (Version 6.0.91, Metrohm). The titration apparatus consisted of a water-jacketed glass vessel maintained at 25.0 (± 0.1°C, Julabo water bath). Prior to and during the course of the titration, a blanket of nitrogen (passed through 10% NaOH to exclude any CO2) was maintained over the sample solution.  Lutetium and yttrium ion solutions were prepared by dilution of the appropriate atomic absorption standard (AAS) solution.  The precise amount of acid present in each metal standard was determined by titration of an equimolar solution of Y3+/Lu3+ and Na2H2EDTA.  The amount of acid present in the metal standard solutions was determined by Gran’s method.54 Calibration of the electrode was performed prior to each measurement by titrating a known amount of HCl with 0.1 M NaOH.  Calibration data was analyzed by standard computer treatment provided within the program GLEE55 to obtain the calibration parameters E0 and pKw. Equilibration times for titrations were 10 minutes for pKa and 15 minutes for metal complex titrations.  Concentrations of ligand and metal were in the range of 0.75-1.0 mM for potentiometric titrations.  The data were analysed by Dr. Jacqueline Cawthray using the program Hyperquad2008.56 The proton dissociation constants corresponding to the hydrolysis of Y3+(aq)/Lu3+(aq) ions were taken from Baes and Mesmer.57 The KML value for yttrium-EDTA and lutetium-EDTA complexes were taken from Martell.38  All values and errors represent the average of at least two independent experiments.    2.4.19 Purification of 86Y Purification of 86Y followed the procedure by E. Oehlke et al.35 The liquid target solution was prepared by dissolving Sr(NO3)2 (14 g) in H2O (22.7 mL ) and adding concentrated HNO3 (4 M, 2.3 mL). 55  The solution was irradiated on TRIUMF’s TR13 cyclotron, a 13 MeV self-shielded, negative hydrogen ion cyclotron.  The irradiated solution was diluted with ultrapure H2O (8 mL) and conc. HNO3 (4 M, 4 mL).  This solution was loaded onto a 50 mg prewashed (5 mL H2O and 5 mL of 4 M HNO3) DGA resin column.  The column was then washed with HNO3 (4 M, 5 mL), HNO3 (3 M, 5 mL), and HCl (6M, 5 mL).  This was followed by aliquots of H2O (1 mL) that were collected individually and measured for radioactivity.  The 86Y was collected in1 mL vials between 4-8 mL of H2O (1.59 mCi).  The pH of the 86Y solutions were adjusted to 3 with NaOH (s) before radiolabeling experiments.    2.4.20 86Y Radiolabeling Studies The ligands H5decapa (10 mg/mL, ~10-2 M) and DOTA (1 mg/mL, ~10-3 M) were made up as stock solutions in pH 5.5 NaOAc (100 mM).  A 10 μL (H5decapa) or 100 μL (DOTA) aliquot of stock solution was transferred to a screw-cap mass spectrometry vial and diluted with pH 5.5 NaOAc (100 mM) and 86Y (~0.3-0.5 mCi) was added to the vials containing the ligand and heated to 70°C for 40 minutes to allow for radiolabeling.  The complexes were then analyzed by RP-HPLC to confirm radiolabeling and to calculate yields.  Areas under the peaks observed in the HPLC radiotrace were integrated to determine radiolabeling yields.  Elution conditions used for RP-HPLC analysis were gradient: A: 0.1% TFA in H2O, B: CH3CN; 0 to 100% B linear gradient 20 min, 1mL/min).  Free 86Y (tR = 3.073), [86Y(DOTA)] (tR = 5.420).  2.4.21 Ligand Stock Solutions and Radiolabeling Solutions The following radiolabeling and serum stability experiments were performed by Dr. Eric Price at the Memorial Sloan Kettering Cancer Center, Department of Radiology. The chelators DTPA, DOTA, and H5decapa were dissolved in DMSO (>99.9%, molecular biology grade) to make stock solutions at concentrations of 10 mM. For radiolabeling reactions, 5 μL of these stock solutions (10 mM, DMSO) were transferred to 495 μL of labeling buffer (ammonium acetate, 200 mM, pH 4.5 for 90Y and 177Lu, and 56  PBS pH 7.4 for 89Zr) to make 100 μM ligand solutions for radiolabeling (50 nmol total ligand each sample).   2.4.22 89Zr Radiolabeling and Serum Stability53 Radiolabeling solutions (500 μL, 100 μM, 50 nmol ligand, PBS pH 7.4) were mixed with ~300 μCi of 89Zr (neutralized to pH ~7 with Na2CO3, 1 M) and reacted for 30 minutes (550 rpm agitation, 37°C, thermomixer). Radiolabeling reactions were evaluated by iTLC–SA (regular non-acidic iTLC-SG did not separate free from bound radiometal) using an EDTA mobile phase (50 mM, pH 5), and by HPLC (5%-100% CH3CN (B) in 25 minutes, A = H2O with 0.1% TFA). “Free” radiometal eluted to the solvent front of the iTLC-SA strips, where 89Zr(decapa) and 89Zr(DFO) remained near the baseline. iTLC-SA strips were scanned using a Bioscan AR-2000 iTLC plate reader, and the area under the peaks was used to determine radiolabeling yields. After 30 minutes, both H5decapa and DFO achieve >99% radiochemical yields with 89Zr. Using RP-HPLC the 89Zr(decapa) complex had a retention time of 6.3 minutes by radiotrace, where “free” neutralized 89Zr had a retention time of 3.8 minutes. 89Zr(decapa) showed no free 89Zr at 3.8 minutes on the radiotrace. These methods of analysis confirmed quantitative 89Zr radiolabeling with both DFO and H5decapa after 30 minutes at 37°C.  Aliquots (~100 μCi, ~150 μL, n = 3) of 89Zr(DFO), 89Zr(decapa), and neutralized “free” 89Zr (control) were added to human blood serum (750 μL), and placed on a thermomixer (550 rpm, 37°C). Serum stability was evaluated by iTLC-SA as described above, or by precipitation with CH3CN. For the precipitation method, 300 μL aliquots of the serum competition mixture were transferred to Eppendorf tubes (1.5 mL) and 700 μL of cold CH3CN was added to precipitate serum proteins. Precipitated serum mixtures were centrifuged for 10 minutes (10,000 rpm), and the supernatant was decanted to a new Eppendorf tube. The radioactivity present in the serum protein tube (serum protein bound 89Zr) and the radioactivity present in the supernatant (ligand bound) were counted with a dose calibrator, and the percentage of radioactivity in the supernatant was calculated to be the percentage intact radiometal 57  complex at each time point, [(μCi supernatant) / ((μCi supernatant) + (μCi serum proteins))] X 100 %. Control experiments with “free” 89Zr showed 99.5 ± 0.1% to be serum protein bound after 24 hours by the precipitation method, and 88.1 ± 1.3 % by iTLC-SA, suggesting the CH3CN precipitation method was most accurate.   2.4.23 177Lu and 90Y Radiolabeling and Serum Stability 177LuCl3 (0.05 M HCl) and 90YCl3 (0.05 M HCl) were transferred (~300-500 μCi, ~2-5 μL) to separate DOTA, DTPA, and H5decapa radiolabeling solutions (50 nmol ligand each as described above, ammonium acetate buffer, 200 mM, pH 4.5), and heated at 70°C for 30 minutes to ensure quantitative radiolabeling (550 rpm agitation, thermomixer). Radiolabeling reactions were evaluated by iTLC–SA using an EDTA mobile phase (50 mM, pH 5), and by HPLC (5%-100% CH3CN (B) in 25 minutes, A = 0.1% TFA in H2O) as described above in the 89Zr section. iTLC-SA elution was not successful for these samples, as 177Lu(decapa) and 90Y(decapa)  could not be separated from free 177Lu/90Y (both eluted near the solvent front). Standard aluminum-backed silica TLC plates and RP-TLC (C18) (eluted with 50:50 methanol:water) also could not separate free from bound radiometal. Due to the high polarity of these radiometal-ligand complexes, HPLC was also not ideal for evaluation, as the 177Lu/90Y(chelate) complexes as well as the free 177Lu/90Y radiometal eluted at the same retention time at the void-volume of the column (~3.9-4.0 minutes). The retention times for 177Lu/90Y (free), 177Lu/90Y(decapa), and 177Lu/90Y(DOTA) were all 3.9-4.0 minutes; however, the 177Lu/90Y(decapa) samples had more intense UV absorption at 254 nm than the free 177Lu/90Y injections due to the presence of the picolinic acid rings in decapa. The free 177Lu/90Y injections had UV absorption values at 254 nm of ~4-5 mAU, where 177Lu/90Y(decapa) had absorption values of ~80 mAU. Although not ideal for accurate radiochemical yield determination, no method could be found to provide a clean separation between free 177Lu/90Y and the H5decapa radiolabeled radiometals. Serum stability results for 177Lu(decapa) (vide infra) suggest that 177Lu was indeed bound by H5decapa as it showed resistance to binding by serum proteins. In contrast to 58  the serum stability of 177Lu(decapa), control samples of free 177Lu showed complete binding to serum proteins after 24 h.   Aliquots (~100 μCi, 150 μL, n = 3) of 177Lu/90Y (free, control), 177Lu/90Y(decapa), 177Lu/90Y(DOTA), and 177Lu/90Y(DTPA) were added to human blood serum (750 μL), and placed on a thermomixer (550 rpm, 37°C). Serum stability could not be evaluated by iTLC-SA as described above for 89Zr, due to poor separation between free and chelate bound radiometal. Precipitation of serum proteins with cold CH3CN was performed as described above in the 89Zr section, by transfer of 300 μL aliquots of the serum competition mixture to Eppendorf tubes (1.5 mL) and addition of 700 μL of cold CH3CN. 90Y samples were also counted in a dose calibrator using the 90Y calibration setting, and were not counted using scintillation fluid on a scintillation counter. The readings for 90Y using the dose calibrator were approximate, as this method produces a lot of drift in the reading and is very sensitive to geometry. In order to minimize inaccuracy from sensitivity to geometry in the dose calibrator, each sample was affixed to the same physical location in the dose calibrator well by using a piece of sticky tape, and readings were allowed to stabilize for several minutes. The percent stability was determined in the same manner as described for 89Zr above, and control samples of free 90Y and 177Lu were 98.1 ± 0.1 % and 99.6 ± 0.1 % serum protein bound after 24 hours, respectively.   2.4.24 UV-vis and Circular Dichroism Experiments   The following stock solutions were prepared in phosphate buffer (pH 7.4, 10 mM): NaHCO3 (0.17 g in 5 mL, 0.4 M), human apo-transferrin (4 mg in 5 mL, 10-5 M), lutetium atomic absorption standard solution (350 μL in 2 mL, 10-3 M), H5decapa (0.80 mg in 1 mL, 10-3 M).  Aliquots of H5decapa (500 μL) and Lu3+ (500 μL) buffered solutions were mixed together and the pH was adjusted to 7.4.  Baselines on both the UV-vis and CD spectrometers were taken of phosphate buffer (2.5 mL) and NaHCO3 (25 μL). UV-vis spectra were taken of apo-transferrin (2.5 mL) and NaHCO3 (25 μL) in a quartz cuvette to measure the exact concentration of protein (at 278 nm, ε = 93, 000) using Beer’s Law (𝐴 =𝜀𝑐ℓ).39 Both UV-vis and CD spectra were recorded after the addition of [Lu(decapa)]2- (50 μL, 1 equiv) 59  and measured over time (until 2 hours) to monitor changes.  UV-vis and CD spectra were also recorded after the addition Lu3+ (25 μL, 1 equiv) followed by the addition of H5decapa (25 μL, 1 equiv). The CD spectra were converted from mdeg to ellipticity using the concentration of protein obtained from the UV-vis spectrum.     2.4.25 Europium Fluorescence Study  The following stock solutions were prepared in acetate buffer (pH 5, 50 mM): H5decapa (3.97 mg in 5 mL, 10-3 M) and Eu3+ AAS (3 μL in 5 mL, 10-3 M).  H5decapa solution (5 μL) was combined with Eu3+ solution (5 μL) in buffer (5 mL).  Each solution was measured by UV-vis initially to record absorption wavelengths; H5decapa solution absorbs at 280 nm.  Fluorescence spectra were measured for each, recording the emission profiles for Eu3+, H5decapa and [Eu(decapa)]2- solutions individually excited at 280 nm.   60  Chapter 3 : ATTEMPTED SYNTHESIS: 89ZR-LIGAND 3.1 INTRODUCTION  The positron-emitting radiometal zirconium-89 (89Zr) has recently garnered a lot of attention for its use in PET imaging.  Unlike the current, commonly used PET imaging agents (see Chapter 1), 89Zr boasts a long half-life of 78.41 hours (3.27 days). This is much longer than most β+ emitting radionuclides, whose half-lives are rarely longer than 1 or 2 hours.  The longer half-life allows for a radioimmunoconjugate to circulate throughout the body and accumulate at the target site, while any unbound tracer is cleared through the normal pathways, greatly improving the image contrast and tumor-to-background activity.22  Furthermore, the lengthier half-life perfectly matches the biological lifetime of antibodies, subsequently expanding the scope of PET imaging’s capabilities. In addition to its ideal half-life, 89Zr also exhibits low-energy positron decay which yields high resolution images, while ultimately being relatively simple and low cost in its production.58  Although 89Zr exhibits many ideal properties for improved PET imaging, one major factor has limited its extensive use: zirconium’s coordination properties.  Unlike most radiometals, where chelators are designed to accommodate the unique size and coordination preferences of the desired metal, additional factors need to be deliberated when considering Zr4+ chelation.  The Zr4+ cation is a highly charged Lewis acid, that has a relatively large radius (84 pm) and prefers to form complexes with a high coordination number (8-9).21  Under acidic to neutral conditions, Zr4+ tends to form polynuclear hydroxo-species, subsequently precipitating out of solution, making it a difficult ion to chelate.  Due to its hard acid characteristics, it prefers hard anionic donors, like oxygen, which should therefore be incorporated into any potential ligand scaffolds.31   The current gold standard (and it is a poor one) for 89Zr chelation is DFO, desferrioxamine B, an acyclic bacterial siderophore (see Chapter 1).  DFO contains three hydroxamate groups that provide six oxygen donors for coordination of the metal, as well as a pendent amine that allows for derivatization 61  towards creating bifunctional conjugates to desired biomolecules.  While DFO has been shown to radiolabel 89Zr under mild conditions (30-60 minutes, room temperature), in vivo studies have shown that the complex lacks kinetic inertness, as significant uptake of 89Zr can be seen in the bones, due to the osteophilic nature of the free metal ion.22  This major shortcoming in the clinical potential of 89Zr has increased the urgency to design and characterize a more suitable chelator for this hard metal ion Zr4+.   Subsequently, these factors all contributed to the design of a novel chelator with potential for Zr(IV) chelation, for use as a radiopharmaceutical.  This design amalgamates aspects of the ’pa’ ligand family with the functional binding groups of DFO. The ‘pa’ family of ligands has shown promise with a variety of radioisotopes and has a relatively robust synthesis.  Due to the large ionic radius of Zr4+, H5decapa appears to be the logical chelate size, and this was proven in Chapter 2.  Therefore, the scaffold of the new ligand is based on H5decapa’s dien backbone; however, to make a kinetically inert zirconium chelator, specific atoms should be used to cater to its hard acid preferences.  Hydroxamic acids are bidentate ligands that are known to have a high binding affinity to a range of transition metals, including Zr4+.58,59 Because of DFO’s success as a zirconium chelator, many new ligands have attempted to incorporate the hydroxamate functionality, the metal-binding moiety of DFO.58,60 Herein, the synthesis of a zirconium ligand was attempted, combining facets of the H5decapa binding scaffold with the DFO hydroxamate binding moiety.     3.2 RESULTS AND DISCUSSION 3.2.1 Synthesis and Characterization The synthesis of this new chelator was deconstructed into two goals, the synthesis of the backbone and the synthesis of the binding arms.  To increase efficiency, the synthetic design of the backbone revolved around incorporating a bifunctional handle right from the start.  To do this, the central nitrogen of the dien was selected to be the site of attaching the linker, as was done in the case of BF-decapa. To allow for functionalization on the secondary nitrogen of the diethylenetriamine, the two outer 62  primary amines need first be protected.  The trifluoroacetyl (TFA) moiety was chosen as a protecting group (Scheme  3.1) as it offers selective primary amine acylation in the presence of secondary amines, due to the difference in steric hindrance of primary and secondary amines during the aminolysis of esters.61 Although several synthetic procedures have been reported,61,62,63 the reaction that was the most successful was carried out in cold diethyl ether, and the desired product precipitated from solution (3.1).64   Scheme ‎3.1. Synthesis of precursors 3.1, 3.2 and 3.3.    A nitro-benzene group was chosen as the linker moiety, as it can later be transformed to an isothiocyanate that would provide a site for conjugation to biomolecules.  The addition of the ethyl nitrobenzene proved to be challenging due to the steric hindrance surrounding the central nitrogen and the electron withdrawing characteristics of the para-nitro group.  Following the procedure by  Ramaswamy et al.,65 the coupling was first attempted in a polar aprotic solvent (acetonitrile) with an inorganic base (NaHCO3).  The reaction was carried out both at room temperature and then subjected to heating (60°C) yet mass spectroscopy analysis over the course of three days displayed no change from the starting material.  As these conditions failed to produce detectable amounts of the coupled product, reaction optimization was performed as the temperature, solvent and base choices were explored (Table  3.1).  Prior to changing solvent, the use of a soluble organic base and a halide salt were tested.  N, N-diisopropylethylamine (DIPEA) was chosen as the base as it has a similar pKa to sodium bicarbonate (10.7 vs 10.3), but there is no issue of solubility, which increases the reactivity.  When this was unsuccessful, the addition of potassium iodide that can act as a nucleophilic catalyst for alkylations with 63  bromides (Finkelstein reaction) was added; nevertheless, no change to the starting material was observed.  Finally, changing solvents to DMF, which has a slightly higher dielectric constant, ultimately helped the reaction proceed.  The best results were achieved using DMF and a stronger inorganic base (K2CO3); however, it still required elevated temperatures and time (3.2).    Table ‎3.1. Reaction conditions, reagents and solvents tested for Scheme 3.2.  Solvent Base Temperature Duration Result Trial 1: Acetonitrile NaHCO3 RT, 60°C 3 days SM Trial 2: Acetonitrile DIPEA 60°C, 80°C reflux 4 days SM Trial 3: Acetonitrile DIPEA, KI 80°C, reflux 4 days SM Trial 4: DMF NaHCO3 80°C 4 days SM + P Trial 5: DMF K2CO3 80°C 4 days P DIPEA= N,N-diisopropylethylamine; SM = starting material; P = product  Another benefit of using TFA as a protecting group for the dien backbone is the ease with which it can be removed.   The cleavage of the trifluoroacetamide only requires mild basic conditions to deprotect,66 and was achieved using 1M sodium hydroxide (3.3).  Having successfully synthesized the functionalized backbone, the next task was to design pendant arms with suitable chemical properties for binding zirconium.  Due to the previous success of the picolinic acid moieties as chelating arms for a range of radioisotopes, this scaffold was thought to be a suitable starting point for terminal amine functionalization. Furthermore, following the binding interactions of DFO, the carboxylates were to be converted to the subsequent hydroxamate, that had been best shown to suit zirconium.60  The first approach to this synthesis began with saponification of the 6-(bromomethyl)picolinic acid obtained from dimethyl pyridine-2,6-carboxylate, to yield the desired methylated bromo-picolinic acid (3.4) (Scheme  3.2).  This acid could then be condensed with the benzyl protected hydroxylamine, via the mixed anhydride, to give the o-protected pyridine hydroxamate (3.5).67 This step involved the formation of the activated mixed anhydride intermediate using ethylchloroformate; however, N-64  methylation in the subsequent step was hindered by the inability to properly purify the desired hydroxamate product from the crude mixture.      Scheme ‎3.2 Synthesis of precursors 3.4 and 3.5.    Due to the aforementioned issues during purification and methylation observed in the previous approach, a different method towards creating the desired o-protected 6-(bromomethyl)-N-hydroxypicolinamide (3.5) was attempted (Scheme  3.3).  In order to avoid some issues previously observed with the labile terminal bromine, this synthetic scheme began instead with the 6-methylpicolinic acid, which was then similarly transformed into the benzyl-protected hydroxamate product.  This reaction occurred without any observable side product formation, which allowed for facile isolation and purification of the N-(benzyloxy)-6-methylpicolinamide (3.6). In order to perform N-methylation on the hydroxamate moiety, relatively harsh conditions were employed.68  Sodium hydride was chosen as the base, as it is very capable of deprotonating secondary amines, allowing for nucleophilic attack of the alkyl halide.  The alkylating agent chosen for this case was methyl iodide, a common reagent used for alkylating a wide variety of compounds, including amines (3.7).    Scheme ‎3.3. Synthesis of precursors 3.6 and 3.7.    65  In order to easily alkylate the diethylenetriamine backbone with the 6-methyl-pyridine hydroxamate, a good leaving group was required on the terminal alkane to facilitate coupling.  Halogens, such as bromine, are often employed as a suitable leaving group. The most common way to add a bromine to an alkyl chain is via NBS bromination (Scheme  3.4). This radical reaction requires a radical initiator, such as a peroxide, and an external initiator such as heat or light.  Although this type of bromination reaction has previously provided an efficient method to brominating the protected 6-methyl picolinic acid,2 in this case the hydroxamate moiety appears unstable towards radical conditions.  Reactions conditions were varied in an attempt to optimize the bromination (Table 3.2); however, fragmentation of the N-O bond was most often observed.   Scheme ‎3.4. Attempted reaction to methylate the hydroxamate nitrogen.     Table ‎3.2. Reaction conditions, solvents and reagents tested for Scheme 3.4. Trial NBS (equiv) ‘Base’ Solvent Conditions: Result: 1 0.7 + (24 hrs) 0.5 Benzoyl peroxide CCl4 -refluxing 60°C,2  SM 2 0.7 Benzoyl peroxide CCl4 Reflux, IR lamp fragmented 3 1.169 Benzoyl peroxide CCl4 Reflux, IR lamp fragmented 4 1.1 (new bottle) AIBN69,70  CCl4 Reflux, IR lamp fragmented  IR lamp = 150 W Halogen lamp, 5 cm from reactor – heat is used to reflux 71  3.2.2 Future Directions Due to time constraints, the synthetic approaches were not exhaustive towards the synthesis of the potential bifunctional Zr chelator.  Many alternative synthetic pathways could be attempted in order to make the activated picolinic hydroxamate. First, a non-radical initiated bromination reaction could be attempted in place of the analogous reaction in Scheme 3.4. To do so, the methyl 6-66  (hydroxymethyl)picolinate would serve as the starting material (the first step in the synthesis of the methyl 6-(bromomethyl)picolinate). Once the methylated hydroxamate was formed, the bromination could ensue by substituting the terminal alcohol with bromine, using phosphorous tribromide, a milder reaction. Second, methylation of the o-benzylhydroxylamine68,72 prior to coupling with bromo-picolinic acid would eliminate the aforementioned troublesome methylation step and potentially allow for the final pendant arm to be synthesized.   Alternatively, purchasing the N,O-dimethylhydroxylamine hydrochloride for the coupling reaction with the picolinic acid may prove to be more successful.   Scheme ‎3.5. Designed synthetic pathway for Zr-ligand.   Ideally, once the o-protected 6-(bromomethyl)-N-hydroxy-N-methylpicolinamide is successfully synthesized, coupling to form the desired ligand can be accomplished (Scheme  3.5). To perform the required N-alkylation to the previously functionalized backbone, a strong base and aprotic solvent could be used. Reduction with H2/Pd could deprotect the benzylated arms and simultaneously reduce the aromatic nitro group to the corresponding primary amine.  The prepared ligand could then be bound with Zr4+, characterized and tested rigorously for thermodynamic stability, radiolabeling yield, and serum stability, as was done for H5decapa.  The success of the hydroxylamine pendant arm would uncover the possibility of synthesizing a range of new ligands based off the ‘pa’ family scaffold that could be investigated towards Zr4+ chelation (Figure  3.1).  67   Figure ‎3.1. Potential ligands for Zr4+ based on the‎‘pa’‎family‎of‎ligands.   68  3.3 EXPERIMENTAL METHODS 3.3.1 Materials and Methods   General materials and methods are the same as those listed in Chapter  2.  3.3.2‎N,N’-(Trifluoroacetamide)-1,2- triaminodiethane (3.1)  The synthesis was adapted from a literature preparation.64 To a solution of diethylenetriamine (0.70 ml, 6.45 mmol) in 10 mL of diethyl ether on ice was added ethyl trifluoroacetate (1.76 mL, 14.8 mmol, 2.3 equiv). The reaction mixture was sealed and stirred at room temperature for 6 hours and placed in the freezer overnight.  The crude product was rinsed with cold ether and dried in vacuo to yield 3.1 as a white solid (68%, 1.3 g). 1H NMR (400 MHz, CDCl3, 25oC) δ: 6.97 (br s, 2H), 3.44 (s, 4H), 2.86 (t, J = 7.6 Hz, 4H). 13C NMR (101 MHz, CDCl3, 25oC) δ: 158.00, 157.63, 120.28, 117.42, 114.56, 111.70, 47.52, 39.46. 19F NMR (282 MHz, CDCl3, 25oC) δ: 76.44. HR-ESI-MS calcd. for [C8H11N3O2F6 + H]+: 296.0834; found: 296.0832, [M+H]+, PPM = -0.7.  3.3.3‎N,N’-(Trifluoroacetamide)-1,2- triaminodiethane (3.2)   The synthesis was adapted from a literature preparation.65 To a solution of 3.1 (0.50 g, 1.69 mmol) in 5 mL DMF was added K2CO3 (0.30 g, 2.20 mmol, 1.3 equiv) and 4-(2-bromoethyl)nitrobenzene (0.50 g, 2.20 mmol, 1.3 equiv). The reaction mixture was heated to reflux and left stirring for 4 days.  After 4 days it was cooled to room temperature, filtered, and dried in vacuo.  The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 40 g HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield the product 3.2 as a red/orange oil (52%, 0.18 g).  1H NMR (300 MHz, CDCl3, 25oC) δ: 8.16 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 8.7, 2H), 6.67 (br s, 1H), 3.42 (q, J = 5.8 Hz, 2H), 2.81-2.80 (m, 2H), 2.74 (t, J = 6.1 Hz, 2H). 13C NMR (101 MHz, CDCl3, 25oC) δ: 158.01, 157.64, 147.47, 146.81, 129.57, 129.52, 123.99, 117.30, 114.44, 54.96, 52.53, 37.55, 33.53. 19F NMR (282 MHz, CDCl3, 25oC) δ: 75.90. HR-ESI-MS calcd. for [C16H18N4O4F6 +H]+: 445.1310; found: 69  445.1309, [M+H]+, PPM = -0.2.  3.3.4 N’-(2-Aminoethyl)-N’-(4-nitrophenethyl)ethane-1,2-diamine (3.3)  The synthesis was adapted from a literature preparation.73 Compound 3.2 (0.20 g, 0.45 mmol) was dissolved in MeOH/H2O (3:1, 12 mL). 1M NaOH was added drop wise and the reaction progression was monitored by TLC. When the pH reached 10, the product was extracted with DCM (4 x 30 mL). The organic layer was dried with MgSO4 and the solvent was evaporated in vacuo to yield 3.3 as a brown oil (80%, 0.090g). 1H NMR (300 MHz, MeOD-d4, 25oC) δ:  8.53 (s, 3H), 8.15 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 2.90 (d, J = 7.4 Hz, 2H), 2.79 (d, J = 5.7 Hz, 2H), 2.64 (d, J = 5.3 Hz, 2H), 2.59 (d, J = 5.5 Hz, 2H). 13C NMR (75 MHz, MeOD-d4, 25oC) δ: 150.57, 147.66, 131.03, 124.46, 57.40, 56.55, 40.03, 34.20. HR-ESI-MS calcd. for [C12H20N4O2 +H]+: 253.1665; found: 253.1666, [M+H]+, PPM = 0.4.  3.3.5 6-(Bromomethyl)picolinic acid (3.4)  The synthesis was adapted from a literature preparation.74 Methyl 6-(bromomethyl)picolinate1 (1.00 g, 4.35 mmol) was dissolved in THF/H2O (2:1, 12 mL). LiOH (0.26 g, 10.87 mmol, 2.5 equiv) was added and the reaction mixture was stirred at ambient temperature for 1 hr. The pH was adjusted to 2 with 1M HCl and the aqueous layer was extracted with ethyl acetate (5 x 60 mL). The combined organic phases were dried with MgSO4 and concentrated in vacuo yielding 3.4 as a white solid (83%, 0.78 g). 1H NMR (300 MHz, CDCl3, 25oC) δ:  8.15 (d, J = 7.7 Hz, 1H), 7.95 (t, J = 7.7 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 4.58 (s, 2H). 13C NMR (101 MHz, CDCl3, 25oC) δ: 164.01, 156.35, 146.04, 139.77, 127.98, 123.33, 32.02. HR-ESI-MS calcd. for [C7H6NO2Br +Na]+: 237.9480; found: 237.9482, [M+Na]+, PPM = 0.8. HR-ESI-MS calcd. for [C7H6NO2Cl +Na]+: 193.9985; found: 193.9982, [M+Na]+, PPM = -1.5.  3.3.6 N-(Benzyloxy)-6-(bromomethyl)picolinamide (3.5)  The synthesis was adapted from a literature preparation.71 Compound 3.4 (0.098 g, 0.45 mmol) 70  was dissolved in 5 mL of THF at 0°C. TEA (0.19 mL, 1.36 mmol, 3.0 equiv) and ethyl chloroformate (0.064 mL, 0.68 mmol, 1.5 equiv) were added to the vial.  After 1 hour the reaction mixture had turned an opaque yellow/white colour and the o-benzyl hydroxylamine (0.073 g, 0.45 mmol) was added.  After 4 hours the reaction mixture was quenched with saturated sodium bicarbonate, and the organic layer was separated with EtOAc. The organic layer was then separated with 0.1 M HCl, and washed with a brine solution.  The organic layer was dried with MgSO4, filtered, and dried in vacuo.  The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 24 g HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield the product 3.5 (16%, 0.018 g). 1H NMR (300 MHz, CDCl3, 25oC) δ:  8.15 (d, J = 7.7 Hz, 1H), 7.93 (t, J = 7.8 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.41 (m, 2H), 7.44-7.41 (m, 4H), 5.10 (s, 2H), 4.63 (s, 2H). 13C NMR (75 MHz, CDCl3, 25oC) δ: 161.60, 155.66, 148.78, 135.28, 129.45, 128.98, 128.79, 126.03, 121.87, 78.83, 24.21. HR-ESI-MS calcd. for [C14H13N2O2Cl +H]+: 277.0744; found: 277.0752, [M+Na]+, PPM = 2.9.  3.3.7 N-(Benzyloxy)-6-methylpicolinamide (3.6)  The synthesis was adapted from a literature preparation.71 6-Methylpicolinic acid (0.12 g, 0.86 mmol) was dissolved in 5 mL of THF at 0°C. TEA (0.36 ml, 2.59 mmol, 3.0 equiv) and ethyl chloroformate (0.12 mL, 1.30 mmol, 1.5 equiv) was added to the vial.  After 1 hour the reaction mixture had turned an opaque yellow/white colour, and the o-benzylhydroxylamine (0.14 g, 0.86 mmol, 1.2 equiv) was added.  After 4 hours the reaction mixture was quenched with saturated sodium bicarbonate, and the organic layer was separated with EtOAc. The organic layer was then separated with 0.1 M HCl, and washed with a brine solution.  The organic layer was dried with MgSO4, filtered, and dried in vacuo.  The crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 24 g HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield the product 3.6 (21%, 0.044 g). 1H NMR (300 MHz, CDCl3, 25oC) δ:  8.02 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.51-7.49 (m, 2H), 7.41-7.30 (m, 3H), 7.30 (d, J = 7.7 Hz, 2H), 5.08 (s, 2H), 2.51 (s, 3H). 13C NMR (75 MHz, CDCl3, 25oC) δ: 162.32, 157.50, 148.50, 137.58, 135.43, 128.79, 126.54, 119.49, 78.67, 24.21. HR-ESI-MS 71  calcd. for [C14H14N2O2 + Na]+: 265.0653; found: 265.0952, [M+Na]+, PPM = -0.4.  3.3.8 N-(Benzyloxy)-N,6-dimethylpicolinamide (3.7)  The synthesis was adapted from a literature preparation.68 Compound 3.6 (0.11 g, 0.45 mmol) was added to a three headed round bottom with NaH (0.042 g, 0.59 mmol, 1.3 equiv) and placed under argon.  Dry DMF (5 mL) was added to the solution before adding the MeI (0.042 ml, 0.68 mmol, 1.5 equiv).  The reaction mixture was heated to 50°C under reflux and an inert atmosphere and left stirring overnight.  The solvent was removed in vacuo and redissolved in EtOAc and separated with H2O.  The organic layer was dried with MgSO4 and the crude product was purified by silica chromatography (CombiFlash Rf  automated column system; 40 g HP silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield the product 3.7 (59%, 0.068 g). 1H NMR (300 MHz, CDCl3, 25oC) δ:  10.28 (s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.73 (t, J = 7.7 Hz, 1H), 7.51-7.49 (m, 2H) 7.41-7.39 (m, 3H), 7.30 (d, J = 7.7 Hz, 1H), 5.08 (s, 2H), 2.51 (s, 3H). 13C NMR (75 MHz, CDCl3, 25oC) δ: 157.57, 152.79, 136.85, 134.79, 128.52, 124.46, 120.33, 77.58, 34.13*, 24.44 (*from HSQC data). HR-ESI-MS calcd. for [C15H16N2O2 +H]+: 257.1290 found: 257.1291, [M+H]+, PPM = 0.4.   72  Chapter 4 : CONCLUSIONS AND FUTURE DIRECTIONS 4.1 CONCLUSION The studies performed throughout this thesis focused on designing syntheses of ligands for larger radioisotopes based on the ‘pa’ family scaffold.  The excellent results demonstrated by the original ligand H2dedpa with 68Ga/Ga3+, as well as the second generation H4octapa with 111In/In3+, gave promise for matches between other radiometals and future generation ligands.  H5decapa had previously been synthesized and tested with 111In; however, poor matching of the radioisotope and chelate size lead to poor observed coordination stability between the two in vivo.  The synthetic difficulties of synthesizing H5decapa previously limited the ability of research to test the ligand on other radiometals.  A new and improved synthetic strategy was developed to make H5decapa in considerably higher yield than described.  This allowed for tests to be performed with other radioisotopes in an attempt to expand the library of available radiopharmaceuticals for PET/SPECT imaging and therapy.  Preliminary experiments were performed between H5decapa and 90Y, 177Lu and 89Zr to measure the radiolabeling efficiently as well as the serum stability.  While H5decapa had overall poor stability with both 90Y and 89Zr, [177Lu(decapa)]2- appeared to be relatively stable upon exposure to serum over the span of 5 days.  Other spectroscopic methods to assess various parameters of the ‘cold’ metal-ligand complexes were undertaken.  Although these were very preliminary experiments, there appears to be potential in using CD as a method to determine serum stability of a metal-ligand complex.  The potential to use lanthanide fluorescence to perform cell studies and monitor the BFC in vitro was also explored, with the europium complex of H5decapa showing promise.  In order for H5decapa to be a viable radiopharmaceutical, a bifunctional derivative had to be designed.  Functionalization of the backbone was originally attempted to mimic the previous ‘pa’ family ligands; however, it proved to be synthetically challenging.  Instead, the central nitrogen of the dien was used as a point of functionalization, removing one of the binding arms.  Since this could potentially 73  change the binding preferences compared to H5decapa, many more binding and characterization experiments are required to compare its function to the unfunctionalized H5decapa.   Although H5decapa showed very poor long-term stability over several days with 89Zr, the fact that it was able to bind 89Zr quantitatively after 30 minutes at room temperature is very promising, as this is relatively rare amongst standard chelators.  In order to help retain the Zr4+ isotope within the chelator, adjustments to the binding arms were designed to cater to zirconium’s observed binding preferences.  The addition of a hydroxamate moiety to the picolinic acid arms was attempted through various synthetic pathways, but synthesis was incomplete at the time of writing and the desired product has not been obtained to date.  Further work, as outlined above, will be done to overcome these synthetic barriers and allow for assessment of the new and improved ligands towards Zr4+ chelation.  4.2 FUTURE DIRECTIONS  The new synthesis for H5decapa, along with that of the bifunctional derivative, will help expand the ‘pa’ family library that has been extensively studied by our group over the past several years. While H5decapa did not show any improvements to current “gold standard” ligands, there is still a great deal of potential that lies ahead.  These preliminary results illustrate the complexity required in designing ligands that cater to specific radiometals; while Y3+ and Lu3+ both share similar chemical properties, ionic radii, coordination numbers and charge, they clearly have differing binding preferences, as demonstrated by the NMR studies and radiolabeling experiments performed.  The viability of a potential radiopharmaceutical can only be truly assessed by in vivo experiments that can measure the biodistribution and clearance of the radioactivity, although physical and in vitro tests aid in directing the choice of ligand to study.  The bifunctional derivative of H5decapa will allow for these experiments to be performed by providing a site to couple a biological targeting vector.  Although imaging of cancerous tumors is critical to diagnosis, the attraction that these isotopes provide as potential therapeutics is undeniable.  The need to harness α-emitting isotopes via a more accessible means is growing, so that their properties can be harnessed for 74  radiopharmaceutical testing.  While there still lie many challenges with the incorporation of an α-emitting isotope into a practicable radiopharmaceutical, once the major challenge of production is overcome, rigorous testing can proceed to find an appropriate match.  Due to the large size of many of these potentially accessible α-emitters, H5decapa provides a good basis towards chelation of these larger isotopes.     75  REFERENCES  (1)  Price, E. W.; Cawthray, J. F.; Bailey, G. A.; Ferreira, C. L.; Boros, E.; Adam, M. J.; Orvig, C. J. Am. Chem. Soc. 2012, 134, 8670–7683. (2)  Price, E. W.; Cawthray, J. F.; Adam, M. J.; Orvig, C. Dalton Trans. 2014, 43, 7176–7190. (3)  Price, E. W.; Zeglis, B. M.; Cawthray, J. F.; Ramogida, C. F.; Ramos, N.; Lewis, J. S.; Adam, M. J.; Orvig, C. J. Am. Chem. Soc. 2013, 135, 12707–12721. (4)  Hoffman, J. M.; Gambhir, S. 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[Lu(decapa)]2- 2D COSY NMR spectrum, aromatic region (600 Hz, in D2O).    Figure A.2. [Zr(decapa)]- 2D COSY spectrum, 400 Hz, in D2O, aromatic region (left) alkyl region (right).     80  Table A.1. 177Lu human serum competition via PD10 column - 1.5 and 24 hours,  37.0 ± 0.1°C at 550 rpm agitation; background activity / mCi = 0.5. Chelator Radiolabeling  conditions Average % chelate intact 1.5 hr Average % chelate intact 24 hr DTPA 10 min, RT 77.4 ± 1.2% 81.6 ± 2.3% DOTA 90°C, 1 hour 87.7 ± 0.7% 87.4 ± 2.1 % H4octapa 10 min, RT 88.1 ± 1.2% 86.2 ± 0.7% H5decapa 10 min, RT 81.2 ± 1.0% 79.7 ± 1.9% Experiments performed by Dr. Eric Price at MSKCC.1    Figure A.3. Zr(BF-decapa) 2D COSY spectrum, 400 Hz, in DMSO-d6, aromatic region (left) alkyl region (right).    . Figure A.4. iTLC-SA radioactivity distribution of [89Zr(decapa)]- labelled at ambient temperature, 30 minutes reaction time, >99% RCY (left) and after 72 hours, (free/serum bound 89Zr at ~60-100 mm solvent front) ~17% stable (right).  05001000150020002500300035004000-6 37 80 122 165 208Radioactivity (counts) Distance traveled on iTLC-SA strip (mm) 0100200300400500600700800-6 37 80 122 165 208Radioactivity (counts) Distance traveled on iTLC-SA strip (mm) 81   Figure A.5. iTLC-SA radioactivity distribution of free 89Zr after 24h (free/serum bound 89Zr at ~100 mm solvent front), some sticks to baseline (~6%).     Figure A.6. iTLC-SA radioactivity distribution of 89Zr(DFO) in serum after 72 hours, (free/serum bound 89Zr at ~60-100 mm solvent front) >98% stable.       0100200300400500600700800-6 37 80 122 165 208Radioactivity (counts) Distance traveled on iTLC-SA strip (mm) 0500100015002000250030003500-6 37 80 122 165 208Radioactivity (counts) Distance traveled on iTLC-SA strip (mm) 

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