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Applying aryltrifluoroborates as PET imaging agents Li, Ying 2012

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 APPLYING ARYLTRIFLUOROBORATES AS PET IMAGING AGENTS  by YING LI M.Sc., Nanjing University, 2006 B.Sc. (Honours), Nanjing University, 2003   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Chemistry)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    January 2012    © Ying Li, 2012  ii Abstract This dissertation is focused on applying aryltrifluoroborates (ArBF3s) as PET imaging agents. Several aspects of this new 18F-labeling technique are addressed. These include the hydrolytic stability of heteroaryltrifluoroborates (HetArBF3s), the fluoridation of arylboronic acids/esters and the radiosyntheses of several 18F-ArBF3 labeled biomolecules for potential PET imaging applications. The solvolysis of several N-HetArBF3s under physiological conditions was studied with 19F NMR spectroscopy in Chapter 2. All the N-HetArBF3s tested therein displayed excellent solvolytic stability under physiological conditions. It is expected that these HetArBF3s can be further applied as 18F-labeled PET imaging agents. In Chapter 3, a rapid fluoridation was carried out under conditions with low fluoride concentrations in a short reaction time (~ one hour). Via TLC-fluorescent densitometry, 19F NMR spectroscopy, and radio-HPLC, the fluoridation of different arylboronic acids/esters was investigated. It was found that the fluoridation occurs relatively rapidly in the presence of 3 to 5 equivalents of fluoride in acidic aqueous CH3CN at room temperature. Under such conditions, radiochemical yields of 20-30% can be achieved. It was also noticed that arylboronates with acid-sensitive protecting groups could undergo fluoridations rapidly comparable to the arylboronic acids. In Chapter 4, marimastat, an MMP inhibitor, was labeled with an 18F-ArBF3 to image breast cancer in mice. An unoptimized isolated radiochemical yield of ~ 1.5% and specific activities of 0.179 and 0.396 Ci/mol were obtained within two hours including packaging. The blocking experiment suggested that the tumor uptake of Mar-18F-ArBF3 was MMP specific. This one-step aqueous fluoridation was also applied to label a urea-based PSMA inhibitor (Chapter 5) and RGD-containing cyclopeptides (Chapter 8). Radiochemical yields ranging from 10% to 25% were obtained within one hour and good HPLC separation was achieved. In addition, a one-pot two-step labeling strategy was developed in Chapter 6 to label acid-sensitive biomolecules with 18F-ArBF3s. The copper(I) catalyzed 1,3-dipolar cycloaddition was successfully used to conjugate 18F-ArBF3s with biomolecules including oligonucleotides (Chapter 6), folate (Chapter 7), and a cyclic RGD-peptide (Chapter 8) with radiochemical yields of 20-30% over two steps in one hour.  iii Preface The projects presented in this dissertation are done in collaboration with many people. Therefore, “we” and “our” are used in many parts of this thesis. Throughout the whole dissertation, Dr. David M. Perrin and I designed most of the projects and set the goals of this thesis together. I have performed the majority of the work presented in this dissertation, and any contribution from other people is described in this section. Papers already published or manuscripts based on the work in this thesis were co-written by Dr. Perrin and me. Former students in the Perrin lab carried out a small number of the reactions presented as data in this thesis and their contribution will be stated in this part. The study presented in Chapter 2 was published in J. Fluorine Chem. in 2008 as “Hydrolytic Stability of Nitrogenous-Heteroaryltrifluoroborates under Aqueous Conditions at Neutral pH”. In this work, Dr. Ali Asadi prepared boronate ester 2.4. I synthesized all the other boronic acids/esters and prepared all the heteroaryltrifluoroborates (HetArBF3s). I also performed the 19F NMR experiments and data analysis. The work in Chapter 4 was published in Cancer Res. in 2010 as “Novel Matrix Metalloproteinase Inhibitor [18F]Marimastat-Aryltrifluoroborate as a Probe for in vivo Positron Emission Tomography Imaging in Cancer” and Med. Chem. Comm. in 2011 as “Towards kit-like 18F-labeling of marimastat, a noncovalent inhibitor drug for in vivo PET imaging cancer associated matrix metalloproteases” respectively in collaboration with Dr. Christopher M. Overall. Dr. Perrin and Dr. Overall initiated this collaboration in order to image breast cancer with marimastat-18F- aryltrifluoroborate (Mar-18F-ArBF3) 4.15. Dr. Curtis Harwig designed and accomplished the initial synthesis of marimastat-arylboronate 4.14 and the fluorescent marimastat-FITC 4.19. Dr. Richard Ting did the initial radiolabeling experiments. I also synthesized 4.14 and all the other hydroxamic acids tested in this chapter. I developed the HPLC method for the isolation of 4.15 and performed the ferroin test and acid stability studies. I also studied the radiolabeling conditions to prepare Mar-18F-ArBF3 4.15. Dr. David Perrin performed the radiolabeling experiments and formulation for the animal imaging experiments. Dr. Ulrich auf dem Keller, Dr. Caroline L. Bellac, Dr. Philipp F. Lange, and Dr. Reinhild Kappelhoff carried out all  iv the biological experiments including the inhibition assay and binding test; Dr. Yuanmei Lou and Dr. Shoukat Dedhar prepared the animal models including tumor cell implantations, while Drs. auf dem Keller and Bellac along with Ms. Siobhan McCormick carried out the tail vein injections and PET scan experiments. Dr. Francois Benard assisted in the imaging reconstruction; Dr. Tom J. Ruth, Dr. Mike Adam, and Dr. Paul Schaffer at TRIUMF provided supervision in the hot lab. Mr. Wade English and Ms. Linda Graham operated the cyclotron to generate 18F-fluoride from 18O-H2O. Dr. Mike Adam trapped and released 18F-fluoride from the anion exchange column and also concentrated the fluoride solution under helium flow. Mr. James A. H. Inkster performed the radio-HPLC purifications on the Waters system. Mr. Peter Tian, an undergraduate NSERC summer student whom I supervised, helped to synthesize some 4.14 for studies at the Center for Probe Development and Commercialization (CPDC). For other chapters, I performed all the experiments, including the syntheses of precursors, 19F NMR studies, and radiolabeling experiments. Ms. Shiqing Tang, an exchange student from Singapore whom I supervised, helped to synthesize BODIPY-boronate 3.6, the precursor 5.8 for the urea-based PSMA inhibitor, and some precursors for RGD-peptide synthesis. Ms. Angela Leung, an undergraduate 425 student whom I supervised, scaled up the synthesis of 3.6 and studied the fluoridation of 3.6 under various conditions by TLC-fluorescent densitometry. All the radiolabeling experiments carried out at the CPDC were in collaboration with Dr. John Valliant and Dr. Karin Stephenson. Dr. Ryan Simms collected and transported the 18F-fluoride solution from the cyclotron site to the 18F-hot lab and assisted me with the radiolabeling experiments and radio-UPLC analysis. For the radiolabeling experiments undertaken in collaboration with Dr. Tom Ruth, Dr. Paul Schaffer, and Dr. Mike Adam at TRIUMF, I performed all the experiments and Dr. Hua Yang helped with the set-up of the radio-HPLC system. All the high-resolution mass spectrometry was performed by Mr. David Wong and Mr. Marshall Lapawa. Mr. Marshall Lapawa also performed the MALDI-TOF experiments. Dr. Yun Ling and Mr. Derek Smith helped with the ESI-LCMS experiments. Dr. David Perrin and I co-wrote the manuscripts for J. Fluorine Chem. and Med. Chem. Comm.  v Table of contents Abstract ....................................................................................................................... ii Preface ........................................................................................................................ iii Table of contents ..........................................................................................................v List of tables ............................................................................................................. xiii List of figures............................................................................................................ xiv List of schemes ....................................................................................................... xviii List of abbreviations and symbols............................................................................xx Acknowledgements ................................................................................................ xxvi Dedication............................................................................................................... xxix Chapter 1 Introduction .........................................................................................1 1.1 Molecular imaging...........................................................................................1 1.2 Molecular imaging with PET...........................................................................4 1.2.1 How does PET work? ...............................................................................4 1.2.2 Choices of PET radionuclides ..................................................................6 1.3 18F-Labeling techniques...................................................................................8 1.3.1 Production of 18F-fluoride/fluorine...........................................................8 1.3.2 Radiosyntheses involving electrophilic 18F-F2 .........................................8 1.3.3 Radiosyntheses with nucleophilic 18F-fluoride.......................................10 1.3.3.1 To form the C-18F bond via nucleophilic reactions- the conventional technique for 18F-labeling .................................................................................10 1.3.3.2 Newly developed methods to prepare 18F-labeled molecules..........12 1.4 Applying ArBF3s as PET imaging agents......................................................16 1.4.1 Specific activity ......................................................................................17 1.4.2 Radiochemical yields and synthesis time ...............................................19 1.4.3 Solvolytic stability of ArBF3s.................................................................20 1.5 The goal of this dissertation...........................................................................22 Chapter 2 Hydrolytic defluoridation of N-HetArBF3s at neutral pH ............24 2.1 Introduction ...................................................................................................24 2.2 Results ...........................................................................................................25 2.2.1 Synthesis .................................................................................................25  vi 2.2.2 Solvolytic studies of N-HetArBF3s.........................................................27 2.3 Discussion......................................................................................................30 2.3.1 Synthesis .................................................................................................30 2.3.2 Solvolytic studies of N-HetArBF3s.........................................................30 2.4 Conclusion .....................................................................................................34 2.5 Materials and methods...................................................................................34 2.5.1 Preparation of several heteroarylboronic acids.......................................35 2.5.2 General protocols to prepare N-HetArBF3s............................................38 2.5.3 Kinetics of the hydrolytic defluoridation of N-HetArBF3s.....................39 Chapter 3 Fluoridation of arylboronates ..........................................................40 3.1 Introduction ...................................................................................................40 3.2 Results ...........................................................................................................43 3.2.1 Synthesis .................................................................................................44 3.2.2 Fluoridation studies based on BODIPY-boronate 3.6 ............................47 3.2.2.1 Test of organic solvents for the fluoridation....................................47 3.2.2.2 Examination on acids for the fluoridation of 3.6 .............................49 3.2.2.3 Fluoride concentrations for the fluoridation of 3.6..........................50 3.2.2.4 Reaction temperatures for the fluoridation of 3.6............................51 3.2.2.5 The content of the organic solvent for the fluoridation of 3.6 .........52 3.2.2.6 Acid concentrations for the fluoridation of 3.6 ...............................52 3.2.2.7 Effects of the ionic strength on the fluoridation of 3.6....................54 3.2.2.8 Kinetic studies on the fluoridation of 3.6 ........................................56 3.2.3 Fluoridation studies by 19F NMR spectroscopy .....................................56 3.2.4 The 18F-fluoridation of boronates ...........................................................59 3.3 Discussion......................................................................................................62 3.3.1 Synthesis .................................................................................................62 3.3.2 The fluoridation of BODIPY-boronate 3.6 .............................................63 3.3.3 Fluoridation studies by 19F NMR spectroscopy .....................................66 3.3.4 18F-Fluoridations of boronates................................................................66 3.4 Conclusion .....................................................................................................68 3.5 Materials and methods...................................................................................69 3.5.1 Synthesis .................................................................................................70  vii 3.5.2 General procedures of the fluoridation ...................................................80 Chapter 4 Radiosynthesis of matrix metalloproteinase inhibitor marimastat-18F-ArBF3 to image breast cancer .......................................................82 4.1 Introduction ...................................................................................................82 4.1.1 Matrix metalloproteinases ......................................................................82 4.1.2 MMP inhibitors.......................................................................................83 4.1.2.1 Endogenous MMP inhibitors ...........................................................83 4.1.2.2 Synthetic MMP inhibitors................................................................84 4.1.3 Imaging MMPs .......................................................................................86 4.1.4 Towards in vivo imaging of breast cancer with Mar-18F-ArBF3 targeting MMPs ................................................................................................................89 4.2 Results ...........................................................................................................90 4.2.1 Synthesis .................................................................................................90 4.2.2 The in vitro stability of MarArBF3 4.15..................................................92 4.2.3 The stability study of hydroxamic acids under acidic conditions...........93 4.2.4 In vitro enzyme inhibition assays of marimastat derivatives..................96 4.2.5 Fluorescent marimastat-FITC and in vitro cell imaging.........................99 4.2.6 Radiosyntheses of Mar-18F-ArBF3 4.15................................................100 4.2.6.1 The radiosynthesis of Mar-18F-ArBF3 4.15 ...................................100 4.2.6.2 The radiosynthesis to image MMPs in breast cancer ....................102 4.2.7 In vivo PET imaging of MMPs in murine breast carcinomas...............105 4.2.8 Several factors influencing the radiosynthesis of Mar-18F-ArBF3 4.15 ......  ..............................................................................................................106 4.3 Discussion....................................................................................................111 4.3.1 Synthesis ...............................................................................................111 4.3.2 The in vitro stability of MarArBF3 4.15................................................113 4.3.3 The acid stability of hydroxamic acids.................................................114 4.3.4 In vitro enzyme inhibitory activity of MarArBF3 4.15 .........................116 4.3.5 Cell binding assays with fluorescent marimastat-FITC........................117 4.3.6 In vivo PET imaging of MMPs in murine breast carcinomas...............118 4.3.7 Some factors related to the 18F-fluoridation of marimastat-boronate 4.14 .  ..............................................................................................................120 4.4 Conclusion ...................................................................................................122  viii 4.5 Materials and methods.................................................................................123 4.5.1 Synthesis ...............................................................................................124 4.5.2 Defluoridation study of MarArBF3 (4.15) ............................................135 4.5.3 Ferroin test ............................................................................................135 4.5.4 The acid stability of hydroxamic acids studied by NMR spectroscopy .....  ..............................................................................................................135 4.5.5 Enzyme inhibition assays .....................................................................135 4.5.6 Assays for fluorescent marimastat-FITC..............................................136 4.5.6.1 Binding assay of marimastat-FITC with MMPs............................136 4.5.6.2 Cell assay for MMP14 by marimastat-FITC .................................136 4.5.7 Radiolabeling marimastat-boronate......................................................137 4.5.8 MicroPET imaging ...............................................................................139 Chapter 5 Radiosynthesis of an 18F-ArBF3 labeled PSMA inhibitor for prostate cancer imaging ..........................................................................................140 5.1 Introduction .................................................................................................140 5.1.1 Prostate cancer ......................................................................................140 5.1.2 Biomarkers for prostate cancer.............................................................140 5.1.3 PSMA and molecular imaging of prostate cancer ................................142 5.1.4 Labeling the urea-based PSMA inhibitor with an 18F-ArBF3...............146 5.2 Results .........................................................................................................147 5.2.1 Synthesis ...............................................................................................147 5.2.2 HPLC conditions for urea-ArBF3 5.10 .................................................151 5.2.2.1 HPLC analysis of urea-ArBF3 5.10 ...............................................151 5.2.2.2 The fluoridation analyzed by ESI-LCMS......................................152 5.2.3 The radiosynthesis of urea-18F-ArBF3 5.10 ..........................................153 5.3 Discussion....................................................................................................155 5.3.1 Synthesis ...............................................................................................155 5.3.2 The HPLC analysis of urea-ArBF3 5.10 ...............................................159 5.3.3 Radiosyntheses of urea-18F-ArBF3 5.10................................................160 5.4 Conclusion and future perspectives .............................................................163 5.5 Materials and methods.................................................................................163 Chapter 6 Applying copper(I) catalyzed click chemistry to label oligonucleotides with 18F-ArBF3s ...........................................................................176  ix 6.1 Introduction .................................................................................................176 6.1.1 Click chemistry.....................................................................................176 6.1.2 The copper(I) catalyzed 1,3-dipolar cycloaddition applied in radiopharmaceutical chemistry...........................................................................182 6.1.3 Imaging oligonucleotides .....................................................................185 6.1.4 Radiolabeling oligonucleotides with 18F-ArBF3s via the copper(I) catalyzed 1,3-dipolar cycloaddition....................................................................189 6.2 Results .........................................................................................................192 6.2.1 The derivatization of oligonucleotides .................................................192 6.2.2 Synthesis of alkynes .............................................................................194 6.2.3 Click reactions between 5’-N3-31/32P-ONs and alkynes........................196 6.2.4 The 18F-labeling of oligonucleotides via click chemistry.....................203 6.2.5 Specific activity ....................................................................................209 6.3 Discussion....................................................................................................212 6.3.1 The derivatization of oligonucleotides .................................................212 6.3.2 Synthesis of alkynes .............................................................................214 6.3.3 Click reactions between 5’-N3-31/32P-ONs and alkynes........................216 6.3.4 The one-pot two-step 18F-labeling of oligonucleotides.........................218 6.3.5 Specific activity ....................................................................................221 6.4 Conclusion ...................................................................................................224 6.5 Materials and methods.................................................................................224 6.5.1 The derivatization of oligonucleotides .................................................226 6.5.2 Synthesis ...............................................................................................229 6.5.3 Click reactions between alkynylArBF3 6.2 and 5’-N3-31/32P-ONs .......232 6.5.4 The 18F-radiolabeling of boronic acid 3.11...........................................232 6.5.5 Click reactions on 5’-N3-31/32P-ONs with alkynyl-18F-ArBF3 6.2 ........233 Chapter 7 Labeling folate with 18F-ArBF3s ....................................................234 7.1 Introduction .................................................................................................234 7.1.1 Folate and folate receptors....................................................................234 7.1.2 Folate and molecular imaging ..............................................................238 7.1.3 Labeling folate with ArBF3s .................................................................241 7.2 Results .........................................................................................................243 7.2.1 Synthesis of Pte-Glu[(PEG)2N3]-OH 7.10............................................243  x 7.2.2 Click reactions between Pte-Glu[(PEG)2N3]-OH 7.10 and alkynes.....245 7.2.3 Radiolabeling folate with the 18F-ArBF3 via click chemistry...............247 7.3 Discussion....................................................................................................251 7.3.1 Synthesis of Pte-Glu[(PEG)2N3]-OH 7.10............................................251 7.3.2 Click reactions between Pte-Glu[(PEG)2N3]-OH 7.10 and alkynes.....252 7.3.3 Radiolabeling folate with the 18F-ArBF3 via click chemistry...............253 7.4 Conclusion and perspectives........................................................................254 7.5 Materials and methods.................................................................................255 Chapter 8 Preparation of 18F-ArBF3 labeled RGD-peptides for cancer imaging ...........................................................................................................262 8.1 Introduction .................................................................................................262 8.1.1 Integrin αvβ3 and RGD..........................................................................262 8.1.2 αvβ3 Antagonists based on RGD sequences ..........................................263 8.1.3 RGD-containing peptides as drug delivery vehicles ............................264 8.1.4 RGD-peptides in molecular imaging ....................................................266 8.1.5 Labeling RGD-containing peptides with ArBF3s .................................270 8.2 Results .........................................................................................................270 8.2.1 Preparation of the RGD-containing peptides........................................270 8.2.2 The one-step fluoridation of RGD-boronates .......................................276 8.2.2.1 The HPLC conditions for RGD-ArBF3s........................................276 8.2.2.2 The one-step radiolabeling of RGD-boronates..............................277 8.2.3 One-pot two-step syntheses of RGD-ArBF3s via click chemistry........277 8.2.3.1 The one-pot two-step synthesis via click chemistry to prepare an RGD-ArBF3.....................................................................................................278 8.2.3.2 The one-pot two-step 18F-labeling to prepare an RGD-ArBF3......279 8.3 Discussion....................................................................................................280 8.3.1 Synthesis ...............................................................................................280 8.3.2 The fluoridation to prepare RGD-ArBF3s ............................................282 8.3.2.1 The one-step fluoridation of RGD-boronates ................................282 8.3.2.2 The one-pot two-step labeling of an RGD-peptide with the ArBF3 ....  .......................................................................................................283 8.4 Conclusion and future perspectives .............................................................285 8.5 Methods and materials.................................................................................285  xi 8.5.1 Synthesis ...............................................................................................286 8.5.2 The fluoridation of RGD-peptides........................................................301 8.5.3 Preparation of the 18F-ArBF3 labeled RGD-peptides ...........................302 Chapter 9 Discussion and conclusion ..............................................................305 9.1 Discussion....................................................................................................305 9.1.1 Defluoridation and fluoridation: rapid preparation and slow decomposition of 18F-ArBF3s .............................................................................305 9.1.1.1 The stability of ArBF3s ..................................................................305 9.1.1.2 The fluoridation to prepare ArBF3s ...............................................306 9.1.2 One-step labeling or one-pot two-step labeling to prepare 18F-ArBF3s?....  ..............................................................................................................307 9.1.3 Specific activity of 18F-ArBF3s.............................................................309 9.2 Perspectives .................................................................................................311 9.2.1 Are 18F-alkyltrifluoroborates suitable for PET imaging? .....................311 9.2.2 Developing new protecting groups to facilitate the fluoridation of boronates.............................................................................................................312 9.2.3 Alternative one-pot two-step strategies ................................................313 9.2.4 Where is 19F-fluoride in the irradiated 18OH2 from? ............................314 9.3 Conclusion ...................................................................................................315 Bibliography.............................................................................................................317 Appendices ...............................................................................................................339 Appendix A. Preparation of various solutions ....................................................339 Appendix B. HPLC information .........................................................................340 Appendix B.1. HPLC systems .........................................................................340 Appendix B.2. HPLC C18 columns ................................................................340 Appendix B.3. HPLC programs ......................................................................340 Appendix C. HPLC chromatograms ...................................................................344 Appendix D. The kinetic study on the solvolysis of N-HetArBF3s by 19F NMR spectroscopy .......................................................................................................358 Appendix E. NMR spectra..................................................................................363 Appendix E.1. NMR spectra for Chapter 2 .....................................................363 Appendix E.2. NMR spectra for Chapter 3 .....................................................369 Appendix E.3. NMR spectra for Chapter 4 .....................................................392  xii Appendix E.4. NMR spectra for Chapter 5 .....................................................414 Appendix E.5. NMR spectra for Chapter 6 .....................................................430 Appendix E.6. NMR spectra for Chapter 7 .....................................................437 Appendix E.7. NMR spectra for Chapter 8 .....................................................446   xiii List of tables Table  1.1 A summary of imaging modalities.4, 10...........................................................4 Table  1.2 Some positron-emitting radionuclides for PET.20-22 ......................................6 Table  2.1 The kinetic data for the solvolysis of N-HetArBF3s ....................................29 Table  3.1 A check-list of the boronates studied for the fluoridation in this chapter. ...43 Table  4.1 18F-Radiosyntheses of Mar-18F-ArBF3 4.15 under various conditions. .....109 Table  6.1 The calculation for the specific activity of ON2-18F-ArBF3. ....................211 Table  7.1 A summary of the click reactions between 6.2 and 7.10 under radioactive conditions...................................................................................................................247 Table  9.1 A summary of the radiosyntheses of several bioligands in this dissertation. ...................................................................................................................................307       xiv List of figures Figure  1.1 An illustration of positron annihilation. .......................................................5 Figure  1.2 Hammett plot in the form of log(k) = σρ + log(k0).85.................................21 Figure  1.3 The structures of boronic acid/ester 3.1 and 3.8 that will be used in this dissertation...................................................................................................................22 Figure  2.1 The 19F NMR study of para-pyridinyltrifluoroborate (TFB-2.1) dissociation in 200 mM phosphate buffer (pH 6.89). ..................................................27 Figure  2.2 Proposed energy diagram of hydroxide-fluoride exchange for step 1 of mechanism A. ..............................................................................................................33 Figure  3.1 Screening organic solvents for the fluoridation of 3.6. ..............................47 Figure  3.2 The structure of the oxidatively-deboronated product from 3.6. ...............48 Figure  3.3 Examination on acids used in the fluoridation of 3.6.................................49 Figure  3.4 Effect of fluoride concentrations on the fluoridation of 3.6.......................50 Figure  3.5 Study on the temperature dependence of the fluoridation of 3.6. ..............51 Figure  3.6 Investigation of organic solvent content affecting the fluoridation of 3.6. 52 Figure  3.7 Concentrations of HCl used in the fluoridation of 3.6. ..............................53 Figure  3.8 Concentrations of TsOH used in the fluoridation of 3.6. ...........................53 Figure  3.9 Addition of concentrated acids to the fluoridation of 3.6...........................54 Figure  3.10 The study of salt effects on the fluoridation to prepare BODIPY-ArBF3 3.2. ...............................................................................................................................55 Figure  3.11 The kinetic study of the fluoridation of BODIPY-boronate 3.6 in CH3CN or NMP.........................................................................................................................56 Figure  3.12 The fluoridation of 3.7 studied by 19F NMR spectroscopy. .....................57 Figure  3.13 The 19F NMR study on the fluoridation of 3.10. ......................................58 Figure  3.14 The kinetic study on the 18F-fluoridation of 3.16.....................................60 Figure  3.15 The radio-HPLC chromatogram of the 18F-fluoridation of 3.11 ..............60 Figure  3.16 The radio-HPLC traces of the 18F-radiolabeling of 3.19, 3.20, 3.21 and 3.23. .............................................................................................................................61 Figure  4.1 Some synthetic MMPIs. .............................................................................85 Figure  4.2 Structures of some representative radiolabeled MMPIs. ...........................87 Figure  4.3 HPLC chromatograms of 4.14 and 4.15.....................................................92 Figure  4.4 The hydrolysis of MarArBF3 4.15 in 1 × PBS (pH 7.4) monitored by 19F NMR spectroscopy.......................................................................................................93  xv Figure  4.5 The study on the acid stability of 4.17 with the ferroin test analyzed by UV-vis spectroscopy. ...................................................................................................94 Figure  4.6 The acid stability of 4.17 studied by 1H NMR spectroscopy. ....................94 Figure  4.7 The acid stability of 4.18 studied by 1H NMR spectroscopy. ....................95 Figure  4.8 The determination of the concentration of MarArBF3 4.15 by the ferroin test................................................................................................................................97 Figure  4.9 The MMP2 inhibition assay with marimastat compounds.........................98 Figure  4.10 The specificity of marimastat-FITC 4.19 and the in vitro cell image of MMPs. .........................................................................................................................99 Figure  4.11 The autoradiographic TLC analysis of the 18F-fluoridation to prepare Mar-18F-ArBF3 4.15...................................................................................................101 Figure  4.12 The test of reductant additives to the 18F-fluoridation of marimastat-boronate 4.14. .........................................................................................101 Figure  4.13 Radio-HPLC chromatograms of the fluoridation to prepare Mar-18F-ArBF3 4.15. ..................................................................................................103 Figure  4.14 Radio-HPLC chromatograms of the 2nd synthesis of Mar-18F-ArBF3 4.15. ...................................................................................................................................104 Figure  4.15 In vivo PET imaging of murine breast carcinomas targeting MMPs. ....105 Figure  4.16 The radio-UPLC chromatogram of the 18F-radiolabeling reaction of marimastat- boronate 4.14. ........................................................................................107 Figure  4.17 Identification of the byproduct in the 18F-fluoridation to prepare Mar-18F-ArBF3 4.15. ..................................................................................................110 Figure  5.1 Examples for some synthesized PSMA inhibitors.232, 234, 236....................144 Figure  5.2 Several radiolabeled PSMA inhibitors reviewed in this chapter. .............145 Figure  5.3 NMR spectra of urea-ArBF3 5.10.............................................................150 Figure  5.4 The ESI-LCMS of urea-ArBF3 5.10. .......................................................151 Figure  5.5 The ESI-LCMS analysis of the fluoridation to prepare urea-ArBF3 5.10. ...................................................................................................................................152 Figure  5.6 The radio-HPLC chromatogram of the 18F-fluoridation of urea-boronate 5.9 in HCO2NH4/CH3CN solvent system. .................................................................153 Figure  5.7 The radio-HPLC chromatograms of the radiosynthesis of urea-18F-ArBF3 5.10. ...........................................................................................................................154 Figure  5.8 The HPLC traces of the deprotection of 5.8 and 5.20 with TFA..............157 Figure  5.9 Studies on the deprotection of 5.20 in neat TFA. .....................................158 Figure  5.10 The proposed mechanism of the fluoridation of the benzopinacol protected boronate. ....................................................................................................162 Figure  6.1 The 1,3-dipolar cycloadditions.................................................................178  xvi Figure  6.2 The proposed mechanism of the copper(I)-catalyzed alkyne-azide dipolar cycloaddition by Sharpless et al.261, 280......................................................................180 Figure  6.3 The autoradiographic gel image of the derivatization of 5’-31/32P-ON. ...193 Figure  6.4 The HPLC chromatograms at 229 nm of alkynylArBF3 6.2 (black) and the fluoridation of alkynylarylboronic acid 3.11 (red). ...................................................195 Figure  6.5 A radio-HPLC chromatogram of the 18F-fluoridation of alkynylarylboronic acid 3.11. ....................................................................................................................196 Figure  6.6 The autoradiographic gel image of the click reactions between the gel-purified 5’-N3-31/32P-ONs and alkynes. ...............................................................197 Figure  6.7 The study of the concentrations of Cu(II)-TBTA for the click reaction between 5’-N3-31/32P-ON2 and alkynylArBF3 6.2. ....................................................199 Figure  6.8 The kinetics of the click reaction between 5’-N3-31/32P-ON2 and alkynylArBF3 6.2. ......................................................................................................200 Figure  6.9 The click reaction between 5’-N3-31/32P-ON1 and alkynylArBF3 6.2......201 Figure  6.10 The one-pot two-step reaction to label oligonucleotide 5’-N3-31/32P-ON1. ...................................................................................................................................202 Figure  6.11 The autoradiographic gel images of the click reactions between alkynes and 5’-N3-31/32P-ON1.................................................................................................204 Figure  6.12 The resolution of various oligonucleotides in 10% polyacrylamide gel. ...................................................................................................................................205 Figure  6.13 The radio-HPLC chromatogram of the preparation of alkynyl-18F-ArBF3 6.2 and the autoradiographic gel images of click reactions with 5’-N3-31/32P-ON1..206 Figure  6.14 The HPLC chromatograms of the crude click reaction with 5’-N3-31P-ON2...........................................................................................................208 Figure  6.15 The UV absorption spectra of the sample collected from the radio-HPLC. ...................................................................................................................................210 Figure  6.16 The mechanism of the derivatization of oligonucleotides by Knorre’s protocol.351 .................................................................................................................213 Figure  6.17 The UV absorption of the decayed sample of the HPLC collected 5’-31P-ON2-18F-ArBF3 can be corrected by deconvolution of the UV spectrum......223 Figure  7.1 The folate receptor-mediated endocytosis................................................237 Figure  7.2 The structure of EC145.413-416 ..................................................................238 Figure  7.3 Several radiolabeled folate conjugates.302, 387, 425-427 ................................239 Figure  7.4 The preparation of Pte-Glu[(PEG)2N3]-OCH3 7.9 using different coupling reagents. .....................................................................................................................245 Figure  7.5 The click reaction between 6.4 and 7.10 monitored by HPLC. ...............246 Figure  7.6 The click reaction between 6.2 and 7.10 monitored by HPLC. ...............246  xvii Figure  7.7 The one-pot two-step radiolabeling of folate from alkynyl-18F-ArBF3 6.2 via click chemistry. ....................................................................................................248 Figure  7.8 The one-pot two-step radiosynthesis of folate-18F-ArBF3. ......................250 Figure  7.9 The radio-HPLC chromatograms of the one-pot two-step radiolabeling of folate with the 18F-ArBF3 via click chemistry. ..........................................................250 Figure  8.1 The macrolactamization of H2N-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)- Gly-OH (8.2a). ..........................................................................................................272 Figure  8.2 The test of different coupling reagents to conjugate boronate 3.1 to peptide 8.5. .............................................................................................................................273 Figure  8.3 The HPLC traces of the RGD-ArBF3s. ....................................................276 Figure  8.4 The radio-HPLC traces of the one-step 18F-fluoridation of RGD-boronates 8.7 and 8.13. ..............................................................................................................277 Figure  8.5 The HPLC chromatogram of the click reaction between 8.4 and 6.2. .....278 Figure  8.6 The LCMS chromatograms and ESI-spectra of the click reaction between 6.2 and 8.4. ................................................................................................................279 Figure  8.7 The radio-HPLC traces of the one-pot two-step labeling reaction to prepare RGD-18F-ArBF3.........................................................................................................280 Figure  9.1 The structures of possible pursued protecting groups for boronic acids..313   xviii List of schemes Scheme  1.1 The scheme of the nuclear reactions to produce 18F-fluorine/fluoride. .....8 Scheme  1.2 Radiosyntheses of 18F-FDG via electrophilic reactions.37, 40, 45 .................9 Scheme  1.3 Examples of fluorodestannylation to prepare functional 18F-labeled reagents.49, 50 ................................................................................................................10 Scheme  1.4 The radiosynthesis of 18F-FDG via a nucleophilic substitution.51 ...........11 Scheme  1.5 The nucleophilic aromatic substitution to prepare 18F-labeled compounds. .....................................................................................................................................11 Scheme  1.6 Reactions of diaryliodonium salts with 18F-fluoride.63-65.........................11 Scheme  1.7 The radiosynthesis of 18F-N,N,N’,N’-tetramethylphosphorodiamidic fluoride.66 .....................................................................................................................12 Scheme  1.8 18F-Labeled NOTA-octreotide with Al18F.69 ............................................13 Scheme  1.9 18F-Radiosyntheses of silicon-based building blocks.71-73, 76 ...................14 Scheme  1.10 The radiosynthesis of 18/19F-ArBF3s.78 ...................................................16 Scheme  2.1 The proposed kinetic scheme of the ArBF3 solvolysis. ...........................24 Scheme  2.2 Synthesis of several heteroarylboronic acids. ..........................................26 Scheme  2.3 Proposed mechanisms of the hydrolysis of HetArBF3s. ..........................32 Scheme  3.1 Proposed mechanism of the fluoridation of arylboronate esters..............40 Scheme  3.2 Synthetic scheme of BODIPY-boronate 3.6 and BODIPY-ArBF3 3.2. ...44 Scheme  3.3 Synthetic scheme of boronates protected with 1,8-diaminonaphthalene.45 Scheme  3.4 Synthesis of DiDiAN protected boronates...............................................45 Scheme  3.5 Synthesis of boronates containing a piperazine linker. ............................46 Scheme  4.1 Synthesis of marimastat-boronate 4.14 and MarArBF3 4.15. ..................91 Scheme  4.2 The proposed mechanism of the deboronation of marimastat-boronate 4.14. ...........................................................................................................................112 Scheme  4.3 The proposed mechanism of the solvolysis of MarArBF3 4.15. ............114 Scheme  4.4 Chelation between the hydroxamic acid and Fe3+.172 ............................115 Scheme  5.1 Synthesis of urea-borate 5.9 following the literature protocol...............147 Scheme  5.2 Modified synthetic route of urea-borate 5.9...........................................149 Scheme  5.3 The proposed deboronation mechanism of the benzopinacol protected boronate in neat TFA. ................................................................................................159 Scheme  6.1 Examples of some copper-free cycloaddition reactions.........................180 Scheme  6.2 The scheme of the one-pot two-step method to label biomolecules with  xix an 18F-ArBF3 via the copper(I) catalyzed alkyne-azide cycloaddition. .....................190 Scheme  6.3 The scheme of the oligonucleotide derivatization.341 ............................192 Scheme  6.4 Synthetic scheme of alkynes. .................................................................194 Scheme  7.1 The mechanism of the thymidylate synthesis.359 ...................................235 Scheme  7.2 Synthesis of Pte-Glu[(PEG)2N3]-OH 7.10. ............................................243 Scheme  7.3 The proposed mechanism of the synthesis of pteroic acid 7.8...............251 Scheme  8.1 Synthetic scheme of the cyclic RGD pentapeptides. .............................271 Scheme  8.2 Synthesis of cyclo[Arg-Gly-Asp-D-Phe-Lys(suc-piperazinyl-boronate)] 8.13 and the corresponding RGD-ArBF3 8.19...........................................................274 Scheme  8.3 Synthesis of the fluorescent RGD-peptides 8.20 and 8.21. ...................275 Scheme  9.1 Potential one-pot two-step labeling alternatives to label molecules with ArBF3s. ......................................................................................................................314    xx List of abbreviations and symbols  α α-particle, He2+ β+ positron γ gamma ray ε extinction coefficient  chemical shift  wavelength ρ reaction constant σ substituent value oC degrees centigrade (Boc)2O di-tert-butyl dicarbonate (PyS)2 dipyridyl disulfide 5,10-CH2-H4PteGlu 5,10-methylenetetrahydrofolate A260 nm absorbance at 260 nm A deoxyadenosine AcCl acetyl chloride Ala (A) L-alanine APS ammonium persulfate ArBF3 aryltrifluoroborate ArB(OH)2 arylboronic acid Ar argon Arg (R) L-arginine Asn (N) L-asparagine Asp (D) L-aspartic acid ATP adenosine-5'-triphosphate AU absorbance unit Av avidin Boc tert-butyloxycarbonyl BODIPY boron-dipyrromethene BOS beginning of synthesis br broad Bt biotin BTTES bis(tert-butyltriazolyl) triazolylethylene sulfonate BuLi butyllithium BuMgBr butylmagnesium bromide C deoxycytidine Cbz carbobenzyloxy Cbz-Osu N-(benzyloxycarbonyloxy) succinimide CDI carbonyldiimidazole Ci curie CPDC Center for Probe Development and Commercialization cpm count per minute  xxi CT computed tomography CTA cetyltrimethylammonium CTAB cetyltrimethylammonium bromide Cy cyanine dye Cys (C) L-cysteine d deuteron d doublet Dap 2,3-diaminopropionic acid DBU 1,8-diazabicycloundec-7-ene dC deoxyribocytosine DCC N,N’-dicyclohexylcarbodiimide DDQ 2,3-dichloro-5,6-dicyanobenzoquinone DEPC diethylpyrocarbonate DHFR dihydrofolate reductase DiDiAN 2,7-dimethoxy-1,8-diaminonaphthalene DIPEA diisopropylethylamine DMA N,N-dimethylacetamide DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DNA deoxynucleic acid DOPA 3,4-dihydroxylphenylalanine DOTA 1,4,7,12-tetraazacyclododecane D-Phe (f) D-phenylalanine DPPA diphenylphosphonic azide dTMP thymidine monophosphate DTPA diethylenetriamine pentaacetic acid DTT dithiothreitol dTTP thymidine triphosphate dUMP deoxyuridine monophosphate EC50 half maximal effective concentration ECM extracellular matrix EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA ethylenediaminetetraacetic acid EOS end of synthesis EPCA early prostate cancer antigen eq. equivalent ESI-HRMS electrospray ionization-high resolution mass spectrometry ESI-LRMS electrospray ionization-low resolution mass spectrometry ESI-MS electrospray ionization-mass spectrometry Et ethyl FACS fluorescence-activated cell-sorting FBA fluorobenzylamine FDA US Food & Drug Administration  xxii FDG 2-deoxy-2-fluoroglucose FITC fluorescein isothiocyanate Fmoc fluorenylmethyloxycarbonyl Fmoc-OSu fluorenylmethyloxycarbonyl succinimide FmocCl fluorenylmethyloxycarbonyl chloride FR folate receptor FR(-) FR negative FR(+) FR positive RFC reduced-folate carrier G deoxyguanosine G Gibbs free energy G4 generation 4 polyamidoamine dendrimer Gln (Q) L-glutamine Glu (E) L-glutamic acid Gly (G) L-glycine GRO G-rich oligonucleotide H2PteGlu dihydrofolate H4PteGlu tetrahydrofolate HBTU O-benzotriazole-N,N,N'N'-tetramethyl-uronium hexafluorophosphate HetArBF3 heteroaryltrifluoroborate HFIP hexafluoro-2-propanol His (H) L-histidine HOAc acetic acid HOBt N-hydroxylbenzotriazole HPLC high performance liquid chromatography HPMA N-(2-hydroxypropyl)methacrylamide hr hour HUVEC human umbilical vascular endothelial cells HYNIC 6-hydrazinonicotinic acid Hz Hertz IC50 half maximal inhibitory concentration J coupling constant Ile (I) L-isoleucine iso-Bu iso-butyl K2.2.2 Kryptofix 222 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]-hexacosane) KD dissociation constant Ki inhibitory constant kobs observed rate constatnt LCMS liquid chromatography mass spectrometry Leu (L) L-leucine Lys (K) L-lysine m multiplet M molar  xxiii mAb monoantibody MALDI-TOF matrix-assisted laser desorption time-of-flight MarArBF3 marimastat-aryltrifluoroborate mAU milli-absorbance unit Me methyl Met (M) L-methionine mg milligram MHz megahertz min minute mL milliliter mM millimolar MMP matrix metalloproteinase MMPI matrix metalloproteinase inhibitor mol mole Mp melting point MR magnetic resonance mV milliVolt MV MegaVolt L microliter n neutron ND not determined NHS N-hydroxysuccinimide NIRF near infrared fluorescence nm nanometer NMM N-methylmorpholine NMP N-methylpyrrolidone NMR nuclear magnetic resonance NOTA 1,4,7-triazacyclonane-1,4,7-triacetic acid ONs oligonucleotides p proton PAGE polyacrylamide gel electrophoresis Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PBS phosphate buffer saline PCA3DD3 prostate cancer gene 3 PCR polymerase chain reaction PEG polyethyleneglycol PET positron emission tomography Phe (F) L-phenylalanine PMB para-methoxybenzyl PNK polynucleotide kinase ppm parts per million Pro (P) L-proline PSA prostate-specific antigen PSMA prostate-specific membrane antigen  xxiv Pte pteroic PteGlu pteroyl-glutamic acid (folic acid) PTX paclitaxel Py pyridine PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) pz pyrazolyl QF24 (7-methoxylcoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)–L-2,3-diaminoproprionyl]-Ala-Arg-NH2 quant. quantitatively rC cytidine RCY radiochemical yield Rf retention factor RNA ribonucleic acid RNase ribonuclease RP-HPLC reverse phase high performance liquid chromatography rt room temperature s singlet SA specific activity SDS sodium dodecyl sulfate sec-BuLi sec-butyllithium SELEX systematic evolution of ligands by exponential enrichment Ser (S) L-serine SFB N-succinimidyl-4-fluorobenzoate siRNA short/small interfering RNA SN1 unimolecular nucleophilic substitution SN2 bimolecular nucleophilic substitution SPECT single proton emission computed tomography sst somatostatin T thymidine t time t triplet t1/2 half-life T4 PNK T4 polynucleotide kinase TBE Tris-borate-EDTA TBTA tris-[(1-benzyl-1H-1,2,3-triazol-yl)methyl]amine TBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate tBu tert-butyl tBuOH tert-butanol TEA triethylamine TEAA triethylammonium acetate Tf triflyly/triflate/trifluoromethanesulfonate TFA trifluoroacetic acid TFAA trifluoroacetic anhydride TFB trifluoroborate  xxv THF tetrahydrofuran Thr (T) L-threonine TIC the total ion current chromatography TIMP tissue-localized inhibitor of metalloproteinases TLC thin layer chromatography TMEDA tetramethylethylenediamine TMS trimethylsilyl Tol toluene tR retention time Tris tris(hydroxymethyl)aminomethane Trp (W) L-tryptophan Trt trityl TS transition state TsOH toluenesulfonic acid Tyr (Y) L-tyrosine UPLC ultra-performance liquid chromatography UV-vis ultraviolet-visible Val (V) L-valine VEGF vascular endothelial growth factor  xxvi Acknowledgements  It has been a long journey to reach this point. Thanks to all the help and support, personally and academically from many kind people around me, I can be here now. First of all, I would like to acknowledge my supervisor Dr. David M. Perrin. It would not have been possible for me to get involved in these great projects described in this dissertation without him. As a young scientist in the imaging field, Dave led me into this important and fascinating research area. His passion about great science always helps us out during times of difficulty. His persistence and enormous efforts are the major driving force to push everything forward. His critical comments and advice have been extremely helpful through the preparation of this thesis. Besides setting high scientific standards, he has also provided me a great deal of freedom to explore many aspects of the projects and offered me opportunities to present my work in many conferences, which have been great scientific experiences in these six years. In addition, I’d also like to acknowledge all members in my supervisory committee, Dr. Jennifer A. Love, Dr. Stephen G. Withers, and Dr. Chris Orvig, for their advice on the projects. Moreover, I would like to acknowledge my previous fellow graduate students, Dr. Curtis Lam, Dr. Christopher Hipolito, and Dr. David J. Dietrich for their efforts in proof-reading my thesis. Their advice clearly helped to improve this thesis. Besides, all of them have been very helpful and supportive during my stay in the Perrin lab. I really appreciate their friendship and support. Special thanks to several people who have been involved in this work. Dr. Richard Ting initiated this project in 2005 and thereafter has made numerous discoveries, which provided the foundation of this project. Richard has helped me a lot to understand many key things/concepts in this field since I took over the project. Dr. Curtis Harwig gave me useful advice for the synthesis, and discussion with him was always fruitful. Thank you very much. There were several undergraduate students having worked with me in this project. Ms. Shiqing Tang, Ms. Liqun Wang, Ms. Angela Leung, and Mr. Peter Tian have contributed to the project by making several precursors. It was quite enjoyable working with them. I must acknowledge our collaborators, to only some of whom it is possible to give  xxvii particular mention here. Dr. Christopher M. Overall, Dr. Ulrich auf dem Keller, Dr. Caroline L. Bellac, and Dr. Philipp F. Lange were our biological support for the marimastat project (Chapter 4). It was pleasant to work with them. And special thanks should go to Dr. Caroline L. Bellac, who I have harassed a lot during my thesis preparation for all those biological details. She is so nice and patient to respond to all my questions and requests. Thank you very much. I would like to thank all the people I have talked to and worked with at TRIUMF. It is impossible to have the work done without them. Dr. Tom Ruth, Dr. Mike Adam, Dr. Salma Jivan, and Dr. Paul Schaffer provided me training and guidance to work with radioactive materials and also helped to schedule our radiolabeling experiments. Mr. Wade English and Ms. Linda Graham have operated the cyclotron to produce 18F-fluoride for our experiments. I need to thank Dr. Hua Yang for her assistance on the radio-HPLC, frequently taking care of my radioactive waste and useful discussion on my radiolabeling work. I would like to thank Ms. Christine Takhar, Ms. Kathleen Genge, and (soon-to-be) Dr. James Inkster for their patience and help with my radiolabeling work undertaken at TRIUMF. On the other hand, we were fortunate to collaborate with Dr. John Valliant and Dr. Karin Stephenson at the CPDC. They provided us 18F-fluoride for about two weeks in 2010, which was a really productive period. I would like to especially thank Dr. Ryan Simms at the CPDC, who patiently assisted me with the radiolabeling work. I would also like to acknowledge Dr. Francois Benard from the BC Cancer Agency (BCCA) for his input to the imaging work of marimastat. I would like to thank Dr. Kuo-Shyan Lin, Dr. Jinhe Pan, Ms. Maral Pourghiasian, Ms. Jennifer Greene, and Mr. Jean-Pierre Appiah for their help on the labeling work at the BCCA. I would like to thank all my talented colleagues in the lab. It is because of them that my PhD life is more joyful. To Dr. Jason Thomas, Dr. Jonathan May, Dr. Marcel Hollenstein, Dr. Ali Asadi, Mr. Justin Lo, Mr. Jack Huang, (soon-to-be) Dr. Marie Willaing Johannsen, (soon-to-be) Dr. Erkai Liu, Dr. Liang Zhao, Mr. Henry Chen, Mr. Daniel Walker, Mr. Abid Hasan, Mr. Zhibo Liu, Mr. Wenbo Liu, Ms. Yajun Wang, Mr. Jerome Lozada, (soon-to-be) Dr. Antoine Blanc, Dr. Marleen Renders, and many undergraduate students ever working in the lab, thank you all. I would like to acknowledge the staff from the facilities in Chemistry Department, especially the staff in the NMR and Mass labs, who have helped a lot during my Ph.D study. Thank you to Dr. Paul Zhicheng Xia, Ms. Zorana Danilovic, Ms. Maria Ezhova,  xxviii and Mr. Jason Traer for their assistance with the NMR spectroscopy. Thank you to Dr. Yun Ling, Mr. Marshall Lapawa, Mr. David Wong, and Mr. Derek Smith for their advice and assistance with the mass spectrometry. I would also like to acknowledge the supporting staff in the department. They are Ms. Sheri Harbour, Ms. Lani Collins, Mr. Milan Coshizza, Mr. Des Lovrity, Ms. Jane Cua, Mr. Bojan Zimonjia, Mr. Xinhui Huang, Mr. John Ellis, Mr. Patrick Olsthoorn, Mr. Nate Kumar, Ms. Helen Bottriell, Ms. Bev Evans, Ms. Tram Nguyen, and Ms. Marjan Molouk-Zadeh. Also, I would like to take this opportunity to acknowledge my dear friends, physically in Vancouver or not. Qin, I guess I finally have accomplished both of our dreams. Huiying, Yuan (Li), Yan (Cai), Dan (Yu), Liang, Feng, Yuan (Ren), Xiaozhe, Arwen, Jian, Yan (Xu), Zhifeng, Qiao, Hui, Zhan, Xiufeng, Peng, Xuan, Yong, Kuan, Chengguang, Yan (Liu), and Dan (Wang), thank you all for your great friendship. Mostly, in the end, I am extremely grateful to my family. Without your support and understanding, I will not be able to pursue my Ph.D dreams. Thank you, Dad and Mum, for raising me up and educating me to be an honest, determined and strong person who is able to take the challenges and be persistent at tough times. Thank you, my parents-in-law, for your consideration and support during these years and your forgiveness to my childishness. Most of most, I would like to thank my husband, Yi, for his unequivocal support and encouragement throughout, as always, for which my mere expression of thanks likewise does not suffice. To everyone listed above and showing up in my life, thank you all. You made me!  xxix Dedication  This dissertation is dedicated to my beloved grandparents, who passed away during my graduate studies.              1 Chapter 1 Introduction While significant progress has been made in our understanding of various biological processes at the molecular levels in the past decades, the causes of many malignant diseases still remain unknown. The remarkable advances from various noninvasive imaging techniques that allow the direct visualization of in vivo processes, however, have provided better understanding about disease progression for suitable treatment, and significant in vivo information to evaluate tested drugs for improved pharmacokinetics, pharmacodynamics, specificity, and stability at a timely manner.1-4 Both drug development and therapies, therefore, have benefited from this newly emerging field: molecular imaging science. A brief review about molecular imaging will be presented in this chapter. 1.1 Molecular imaging Molecular imaging is a multi-disciplinary field, which has been defined as noninvasive, quantitative, and reproducible visualization of biological processes and targeted macromolecules in living organisms.1 It is now playing increasingly important roles in diagnosis, therapeutic evaluation, and drug development. There are two important elements for molecular imaging: i) the concentration and/or detectable properties of a molecular probe can be changed due to the biological process/distribution, and ii) a suitable technique is available to track the probe in vivo.1, 5 Molecular imaging is therefore expected to provide characteristic and quantitative information about biological processes at cellular and subcellular levels in intact living subjects.4 There are generally two categories of probes used in molecular imaging: direct and indirect probes, which target the biomarkers differently via either the concentration or the functionality of the biomarkers.1 In addition, based on different reporter modalities, there are many molecular imaging approaches extensively applied in animal studies or in clinical trials, such as optical imaging, ultrasound imaging, magnetic resonance imaging (MRI), X-ray computed tomography (CT), and radionuclide imaging including positron emission tomography (PET) and single photon emission computed tomography (SPECT).4-6  2 Optical imaging includes fluorescence imaging7 and bioluminescence imaging.8 Fluorescence imaging relies on the detection of fluorescent light that is emitted upon the excitation of fluorophores, which can be endogenously produced or exogenously administered.4, 6, 7 Bioluminescence imaging is based on the enzymatic reactions that release light, which can be detected to indicate aspects of a biological process.4, 8 Both of these optical imaging methods benefit from high sensitivity (up to 10-15 M), low instrumentation cost, general availability, and compatibility with high-throughput capabilities. They are quick and easy to perform. Nevertheless, both modalities suffer from high degrees of light scattering in vivo and absorption by overlying tissues, which limits their use in deep tissue detection.4, 6 Autofluorescence from normal tissues is another drawback of fluorescence imaging. Generally, optical imaging is most often used for preclinical studies in small animals. Ultrasound imaging examines the reflection of high-frequency sound waves from tissues to construct real-time ultrasound images to monitor the structure and movement of the body’s internal organs and to measure the blood flow.4, 6 It has been found that ultrasound imaging might be useful to study microcirculation, angiogenesis, and neovascularization of tumors.9 Ultrasound imaging is inexpensive, simple, and rapid to perform, but its poor tissue penetration and high dependence on operators’ skill and experience highly limit its application to intravascular structures. It is more regarded as a qualitative tool, since it has limited sensitivity to detect the contrast agents.6 Magnetic resonance imaging (MRI), based on the energy absorbed and emitted in the magnetic field, measures the rates of relaxation of hydrogen atoms or other atoms in high magnetic fields.6 As water is abundant in living beings, most MRI contrast agents based on paramagnetic metals have been designed to interact with water and perturb the magnetic properties of hydrogen atoms. Since the relaxation of hydrogen atoms bound to water is different in tissues that retain the targeted contrast agents, MRI is a powerful imaging technique that provides anatomical images with high resolution and is also able to map the contrast agents in vivo. However, its sensitivity is as low as 10-3 to 10-4 M; the acquisition time is long and the available contrast agents are limited.4, 10, 11 X-ray computed tomography (CT) is based on the differentiable absorption of X-rays  3 by different tissues,4, 6 and provides high resolution anatomical images. Since X-ray absorption is determined by the density of the tissues, CT is limited to providing information on soft tissues. In spite of its low detection sensitivity and lack of readily available targeted probes, CT is highly significant when used in combination with other imaging techniques such as MRI,12 SPECT,13 and PET.14 Single photon emission computed tomography (SPECT) detects γ-rays directly emitted by radioisotopes and reconstructs the localization of the contrast agents carrying the radioisotopes in vivo noninvasively.4, 6, 15 Since a collimator is installed between the detector and the imaging subject to allow only γ-rays of characteristic energies to pass through and be detected, photons with different energies can be differentiated and SPECT is thus able to provide the information of multiple radionuclides (2 ~ 3) simultaneously.4, 6 Notwithstanding the use of radionuclides, the sensitivity of SPECT is compromised by the collimator, which absorbs a large quantity of γ-rays emitted from the radionuclides. In turn, relatively high administered doses of the radiotracer and long scanning time are usually prerequisites for generating high quality SPECT images.4, 6, 10 Positron emission tomography (PET) detects γ-rays with the energy of 511 keV, which are released upon the annihilation of positrons. PET proceeds with high sensitivity (10-11 ~ 10-12 M) while small doses of radioactivity are required for the image acquisition, since nearly all the incidents can be directly measured by the detector.4, 16 PET imaging is also independent of the depth where the radioactive compound locates, and it is regarded as quantitative in that the signals are proportional to the concentration of the radioligand in the tissue. PET, however, has a functional limitation of spatial resolution, which relies on the size of the single detector component.4 Moreover, with the requirement of an expensive cyclotron to produce most of the PET-radionuclides and of PET scanners, PET imaging remains one of the most costly diagnostic techniques. The molecular imaging techniques have been briefly reviewed above. Several aspects including the advantages and disadvantages of these imaging modalities are listed in Table  1.1. Clearly, each technique carries its advantages and disadvantages. Currently, besides the application of the single imaging modality for diagnosis, the combination of different imaging techniques is a promising direction of molecular imaging to provide  4 more accurate measurement in monitoring in vivo processes. On the other hand, since the development of PET imaging agents is the goal of this dissertation, a more detailed review about PET imaging is given in the following section.  Table  1.1 A summary of imaging modalities.4, 10 Modality Resolution (mm) Spatial temporal Penetration depth Sensitivity (M) Advantages Disadvantages Fluorescence 2-3 Sec to min < 1 mm 10-9-10-12 Bioluminescence 3-5 Sec to min 1-2 mm 10-15-10-17 High-throughput screening High sensitivity Low penetration depth Limited clinical translation*  Ultrasound 0.05-0.5 Sec to min mm-cm - Clinical translation High spatial and temporal resolution Low cost Operator dependency Low penetration depth Lack of targeted probes Targeted imaging limited to vascular compartment CT 0.025-0.2 Min No limitation - High spatial resolution Unlimited depth penetration Clinical translation No target-specific imaging Use of X-radiation Poor soft-tissue contrast MRI 0.025-0.1 Min to hr No limitation 10-3-10-5 Superior spatial resolution Clinical translation Imaging time Costs SPECT 0.5-1.5 Min No limitation 10-10-10-11 High sensitivity Multiple isotope imaging Availability of tracers and instruments Attenuation-associated accuracy limit Size of reporter probes Limited spatial resolution PET 3-7 10 s to min No limitation 10-11-10-12 High sensitivity High throughput Attenuation correction Fundamentally limited spatial resolution Lack of clinical translation Costs * Clinical translation refers to availability of anatomical information.  1.2 Molecular imaging with PET 1.2.1 How does PET work? PET, as mentioned in the previous section, relies on the detection of γ-rays created during the annihilation of positrons. As shown in Figure  1.1, positrons, positively charged electrons, are released from the decay of positron-emitting radionuclides (PET-radionuclides).17 When positrons, which are antimatter particles, collide with negatively charged electrons, which are abundant in all matter, this collision results in the annihilation, in which both the positron and the electron disappear and two γ-ray photons are produced. These photons, with energy of 511 keV, correspond to the resting masses of  5 the positron and electron, following the laws of conservation of electric charge/linear and angular momentum/total energy. The two photons are released simultaneously in the 180o direction to each other and can be detected by the detector surrounding the subject. The positron might travel a short distance from where the decay of the radionuclide occurs, and the average net trajectory (positron range) is always determined by the positron energy in the emission, which is a characteristic decay property of the corresponding radionuclide. By recording many pairs of the coincident γ-ray photons, a spatial distribution of the radioactivity can be constructed through the use of computed tomography as a function of time to provide PET images. PET radionuclide positron electron γ gamma photon (511 keV) positron range γ γ Figure  1.1 An illustration of positron annihilation. A positron is emitted from the radionuclide during the decay process. Since the positron possesses momentum, it travels (shown as the black dot line) through elastic collision until it collides with an electron to release two γ-photons with 511 keV at two opposite directions.  The spatial resolution of a PET scan highly depends on the size of the detector and the positron range.18, 19 The detector size actually determines the intrinsic resolution of most scintillation detectors. In addition, the distance that a positron travels in the tissue, before it is annihilated via capturing an electron, also affects the resolution. The site of positron emission is always distant from the site of the annihilation as shown in Figure  1.1. This distance is defined as the positron range. Although the positron range is an average value, it is directly related to the positron energy in H2O or tissues. Higher positron energy always results in a longer positron range. As the detector actually records signals, which directly relate to the site of the annihilation, a longer positron range always results in poorer spatial resolution. Therefore, for a good image, PET-radionuclides with low positron energies are always preferred.  6 1.2.2 Choices of PET radionuclides The increased use of PET imaging depends on the development of new imaging probes. Every probe consists of both a functionally targeting moiety and a radionuclide. There are many positron-emitting radioisotopes, and several frequently used ones are listed in Table  1.2 with their radioactive properties presented. There are several criteria to consider when choosing a PET radioisotope for PET imaging studies:20 a), a reasonable half-life that allows the preparation of the radiotracers and the in vivo clearance of the radiotracer; b), suitable positron energy that affords high image resolution; c), acceptable radiation dosimetry to the subject; d) availability of the isotope in sufficient amounts and in high specific activities from a cyclotron or a generator; and e), one or more reliable and reproducible methods to incorporate the radionuclides into the bioligands with high radiochemical yields.  Table  1.2 Some positron-emitting radionuclides for PET.20-22 β+ energy (keV)** β + range in Water (mm) Nuclide t1/2 (min) SA* (Ci/mol) Decay (% β+) Max. Mean Max. Mean 11C 20.4 9220 99.77 960.1 385.6 4.1 1.1 15O 2.04 91730 100 1700 n/a 5.4 n/a 18F 109.8 1710 96.7 633.5 249.8 2.4 0.6 64Cu 768 245 17.87 652.9 278.1 2.9 0.64 68Ga 68.3 2766 87.7 1899.1 836 8.2 2.9 86Y 884 213 12.4 5.6 1253.5 1578 440 696 5.2 6.5 1.8 2.9 124I 6048 31 11.0 12.0 1532.3 2135 685.9 973.6 6.3 8.7 2.3 3.5 NOTE: The most characteristic emissions of positron for each radionuclide have been indicated. *SA is specific activity; **β+ is positron.  Among the radionuclides listed in Table  1.2, 11C-carbon is one of the radionuclides often used for PET studies and clinical trials. Although its short half-life of 20.3 minutes imposes a strict limitation on radiotracer preparation and imaging data acquisition, the radiation dose administered to the patients and the production chemists is intrinsically reduced. Additionally, as carbon is one of the key elements in various compounds, 11C-labeled radiotracers would retain largely the biological nature of the leading compounds by simply replacing 12C-carbon to 11C-carbon.23 The 11C-radiochemistry has  7 been expanded and developed to provide many new 11C-imaging compounds and some of the 11C-labeled radiotracers are currently under clinical evaluation.23-25 Another radionuclide that has received a large amount of attention is copper-64.26 The longer half-life allows its transport from one site to another (within hours of driving/flying etc.), and it is also useful to localize 64Cu-radiolabeled tracers, such as antibodies and macromolecules, the clearance of which requires longer time. The radionuclide 64Cu-copper has been incorporated into functional molecules via various chelating ligands and some 64Cu-labeled compounds have provided promising imaging results.26-28 Despite the fact that 64Cu is an emerging PET isotope, only 17.9% of its decay accounts for positron emission. Due to the low positron yield, longer acquisition times29 or higher administration doses17 may be required to accumulate enough data to achieve reasonable image quality. Gallium-68 (68Ga) has been recognized as an attractive alternative radionuclide in PET imaging.30 In addition to its moderate half-life (68.3 min), a high positron yield (87.7%), and the relatively low mean positron energy, its production from 68Ge in the commercially available generator accelerates the development of 68Ga-labeled radiotracers. By coordination with a variety of ligands, several 68Ga-labeled peptides such as [Tyr3]octreotide and bombesin have entered preclinical evaluations and encouraging results have been obtained.30 In addition, there are also many 18F-labeled PET imaging agents under investigation in the field. Some of them have exhibited high potential for clinical uses in cancer diagnosis.31 This is because of the optimal properties of fluorine-18, which conform to various requirements to achieve good qualities of PET images.17, 31 Fluorine-18 has a half-life of 109.8 minutes, weak positron energy, and a clean decay process (96.7%). It can be produced with high specific activities in a cyclotron efficiently. All these attributes make fluorine-18 one of the ideal isotopes for PET imaging development in terms of resolution and dosimetry.  8 1.3  18F-Labeling techniques 1.3.1 Production of 18F-fluoride/fluorine The radionuclide 18F-fluoride/fluorine can be produced in a cyclotron via two nuclear reactions as shown in Scheme  1.1.32 Fluorine-18 was first produced in the form of 18F-F2 gas from the bombardment of neon gas with 5 MV deuterons, described as the nuclear reaction of [20Ne(d,α)18F], by Snell and co-workers.33 Later, 18O-targeta was developed as a more widely used method for both 18F-F2 and 18F-fluoride via proton bombardment following [18O(p,n)18F].34-36 The 18O(p,n)18F nuclear reaction with 18O-enriched water is the most used nuclear reaction to produce 18F-fluoride with high specific activities.  Scheme  1.1 The scheme of the nuclear reactions to produce 18F-fluorine/fluoride. For 18O(p,n)18F, the nuclear reaction for the irradiation of 18O-enriched water is demonstrated.  Radiosyntheses, however, are constrained by radiation safety concerns, short half-lives of the radioisotopes, and the need for high specific activities. Short total radiosynthesis time and reasonable high radiochemical yields are necessary for the preparation of radiopharmaceuticals. To reduce the operation of radiation with regard to safety and production concerns, fewer steps, radioisotope incorporation at a later stage and simpler purification procedures are always favored.17 The two different forms of 18F-fluorine produced in the cyclotron demonstrate dramatically different chemical reactivities. Hence different chemical reactions have been employed to incorporate 18F-fluorine into molecules for PET imaging applications.16, 17 In the following part, chemistry involving 18F-fluorine incorporation is described. 1.3.2 Radiosyntheses involving electrophilic 18F-F2 Electrophilic 18F-fluorine has been applied to reactions involving electronically enriched molecules, such as alkenes and arenes. 18F-Fluorine gas (18F-F2),37-39 xenon  a 18O-Target is the target with oxygen-18.  9 difluoride (Xe18F2),40-44 and 18F-acetylhypofluoride (CH3CO218F)39, 45, 46 are used for the electrophilic fluorination.47 Using different electrophilic 18F-fluorine reagents via electrophilic addition reactions, the radiosyntheses of 2-deoxy-2-[18F]-fluoroglucose (18F-FDG), which is widely used in clinic to investigate various diseases by monitoring glucose mechanism, are summarized in Scheme  1.2.37, 40, 45 By the electrophilic addition of 18F-reagents to the double bond, an 18F-fluorinated intermediate is obtained. Following an acid treatment, the desired product 18F-FDG is prepared. In addition to the electrophilic addition reactions to introduce 18F-fluorine, electrophilic aromatic substitutions have also been developed.47-49 Particularly, the fluorodestannylation reaction has been applied to prepare useful imaging tracers such as 18F-fluoro-L-DOPA50 and 18F-fluoro-L-tyrosine49 regioselectively from trialkyltin derivatives, as indicated in Scheme  1.3. 18F-Fluoro-L-DOPA is known to target the cerebral dopamine metabolism for imaging neurodegenerative diseases, and 18F-fluoro-L-tyrosine images protein synthesis. O OAc OAc OAc 18 F- F 2 or Xe 18 F 2 CH3COO18F O OAc OAc OAcO OAc OAc OAc 18F 18F 18F 18F O OAc OAc OAc OAc 18F O OH OH OH OH 18F 18F-FDG H+ H+ 18F2 + CH3COONH4 CH3COOH CH3COO18F + NH418F  Scheme  1.2 Radiosyntheses of 18F-FDG via electrophilic reactions.37, 40, 45  Electrophilic addition using either Xe18F2 or 18F2 always provides stereometric intermediates, while at most 50% of 18F-fluorine can be incorporated to the final radiolabeled compounds. Moreover, 18F-fluorine production from the nuclear reaction always requires the addition of 19F-F2, which limits the specific activity to a few mCi/mol (< 20 mCi/mol).17, 51 However, as for the synthesis of 18F-fluoro-L-DOPA, the electrophilic fluorodestannylation remains the synthesis of choice due to its simplicity compared with the synthetic route involving 18F-fluoride nucleophilic  10 reactions.  Scheme  1.3 Examples of fluorodestannylation to prepare functional 18F-labeled reagents.49, 50  1.3.3 Radiosyntheses with nucleophilic 18F-fluoride In contrast to the production of 18F-F2, 18F-fluoride can be routinely prepared with very high specific activities (~ 5-14 Ci/mol).16, 17, 52 Although 18F-fluoride is highly hydrophilic and its hydrated form is quite inert, it can be converted to a very nucleophilic species by routine manipulations. Briefly, 18F-fluoride is activated by the removal of water and the addition of metal ion chelating agents such as Kryptofix-222 (K2.2.2).17, 22 In this section, the incorporation of 18F-fluoride will be reviewed. Two categories of labeling methods, based on the construction of C-18F and X-18F (X is any atom rather than carbon) bonds, will be described.16, 53 1.3.3.1 To form the C-18F bond via nucleophilic reactions- the conventional technique for 18F-labeling The majority of 18F-labeled radiopharmaceuticals are synthesized by nucleophilic substitutions on aliphatic substrates and aromatic compounds. Some of the 18F-labeled radiotracers have been routinely prepared by this method and used on a daily basis for diagnostics. The nucleophilicity of 18F-fluoride is usually activated by the addition of K2.2.2 in anhydrous solvent (DMSO or CH3CN) at elevated temperatures (120-150 oC).17 Most of the 18F-labeled aliphatic compounds are prepared from substrates with good  11 leaving groups such as halides and sulfonates (tosylate, mesylate or triflate).50 The best known example might be the radiosynthesis of 18F-FDG shown in Scheme  1.4.51 The precursor 1,3,4,6-tetra-O-acetyl-2-trifluoromethanesulfonyl-β-D-mannopyranose is first fluoridated in the presence of K2.2.2 at ~ 80 oC for about 5 minutes under basic anhydrous conditions. Then the additive K2.2.2 and salts are removed by passing through a C18 sep-pak cartridge. The removal of acetyl groups under acidic conditions gives 18F-FDG with high efficiency. By using a large quantity of precursors, the radiochemical yield of this reaction was determined to be 99% via radio-thin layer chromatography (TLC). Many 18F-labeling compounds have been prepared in a similar manner.54-57  Scheme  1.4 The radiosynthesis of 18F-FDG via a nucleophilic substitution.51  Aromatic nucleophilic substitutions, on the other hand, involve leaving groups (such as halides, nitro groups, and trimethylamine) and electron withdrawing groups (such as cyano, nitro, and acyl groups) at the para- or ortho-position to the leaving group on the aromatic ring are always required to activate the ring for the reaction.17, 58-60 For example, a simple 18F-prosthetic group 4-18F-fluorobenzaldehyde is frequently prepared following substitution as shown in Scheme  1.5. Once the 18F-synthon is prepared, it is rapidly incorporated to bioactive molecules via an oxime ether formation reaction.61, 62 N CHO K18F, K2.2.2 K2CO3,DMSO,120oC 18F CHO O H2N NH O R 18F O N N H O R 4-18F-fluorobenzaldehyde Scheme  1.5 The nucleophilic aromatic substitution to prepare 18F-labeled compounds.  I R2R1 18F R1 18F R2 or K18F, K2.2.2 K2CO3, solvent Scheme  1.6 Reactions of diaryliodonium salts with 18F-fluoride.63-65  In addition, diaryliodonium salts were reported as precursors for 18F-fluoroarenes  12 without electron withdrawing groups, as shown in Scheme  1.6.63 The fluoridation can be accomplished in one step with good yields. A recent study revealed that the regioselectivity in the reaction of unsymmetrical diaryliodoniums could be controlled by the ortho-substituents.64, 65 From the examples illustrated for the C-18F bond formation with nucleophilic 18F-fluoride, extremely anhydrous anionic 18F-fluoride, elevated temperatures, protecting-group chemistry, and additives such as K2.2.2 are almost always required. These often lead to tedious multistep syntheses, problems for purification, and incompatibility to many biomolecules. Ideally, a one-step labeling strategy to incorporate 18F-fluoride at room temperature under aqueous conditions is favored. 1.3.3.2 Newly developed methods to prepare 18F-labeled molecules Several newly developed methods have provided possible alternatives for the incorporation of 18F-fluoride, in the fashion dramatically different from the conventional C-18F bond formation. In this section, a brief introduction to the formation of a P-18F bond,66 Al-18F complexes,67-70 an Si-18F bond,71-77 and a B-18F bond78-82 will be presented. The P-18F bond formation was reported by Studenov and co-workers in 2005.66 It was described therein that a substitution reaction between 18F-fluoride and N,N,N’,N’- tetramethylphosphorodiamidic chloride in anhydrous CH3CN could yield the 18F-labeled phosphorodiamidic compound with a high radiochemical yield of 96%. Instead of K2.2.2, tetrabutylammonium carbonate was added to activate 18F-fluoride as a phase transfer agent (Scheme  1.7). Though the radiosynthesis ensued with a high radiochemical yield and efficiency, the 18F-labeled compound underwent relatively rapid defluoridation in aqueous conditions, as about 25% of the P-18F bond decomposed within 30 minutes. Unfortunately, ever since then, no further investigation on improving the stability of the phosphorofluoridates has been reported.  Scheme  1.7 The radiosynthesis of 18F-N,N,N’,N’-tetramethylphosphorodiamidic fluoride.66   13 18F-Fluoride-aluminum-chelates (Al-18F) have been developed in the McBride group to label thermostable peptides such as hapten-peptides67, 68, 70 and octreotide69 for in vivo imaging studies. On the basis of fluoride-metal interactions, McBride and colleagues first tested the stability of the Al18F complexes with various chelating groups.67 They found that peptides with the ligand 1,4,7-triazacyclonane-1,4,7-triacetic acid (NOTA) yielded Al18F complexes with the highest in vivo stability. It was also discovered in the same report that two of the carboxylic groups are critically required to stabilize the complex. With modifications of the NOTA ligands and optimization of the labeling conditions, both the radiochemical yield and labeling efficiency were improved.68 The radiosynthesis via this method is usually undertaken in aqueous sodium acetate solution (pH 4.5) at around 100 oC and is complete within 15 minutes in high yields. It is expected this labeling technique might be expanded to biomolecules with low thermostability via one-pot two-step radiosyntheses using useful prosthetic groups. The chelation of Al18F with NOTA-octreotide is shown in Scheme  1.8 as an example of this labeling method.69  Scheme  1.8 18F-Labeled NOTA-octreotide with Al18F.69  The Si-18F bond formation has received a great deal of attention recently. Synthetically, fluoride has frequently been used to remove silyl protecting groups, especially the sterically hindered ones.83 This might be due to the strong bond energy of the Si-F bond. In fact, dating back to 1985, the first preparation of 18F-trimethylsilylfluoride (18F-TMS-F) was reported by Rosenthal et al., who treated TMS-Cl with 18F-trimethylammonium fluoride in aqueous CH3CN to give 18F-TMS-F with a radiochemical yield of 80% (decay corrected).84 18F-TMS-F, however, decomposed quickly in vivo, resulting primarily in bone uptake as imaged in mice. It was not until recently that the formation of the Si-18F bond came back to the stage to provide potential 18F-labeled radiopharmaceuticals, with a better understanding of the stability of the 18F-fluorosilyl compounds.71-73 Schirrmacher and colleagues found that the hydrolytic  14 stability of 18F-fluorosilanes can be highly improved through sterics by introducing bulky substituents such as the tert-butyl group to the silicon atom.71 Consequently, the di-tert-butylphenyl-18F-fluorosilane exhibited high in vitro and in vivo stability. Moreover, the di-tert-butylphenyl-18F-fluorosilane can be prepared in the presence of K2.2.2 in anhydrous CH3CN at room temperature by either the isotopic exchange reaction or the substitution reaction of the related chlorosilane compound with 18F-fluoride, as indicated in Scheme  1.9. Since it is of great synthetic challenge to conjugate biomolecules to the hydrolytically labile chlorosilanes, Schirrmacher and co-workers have thereafter mainly focused on the development of an elegant isotopic exchange reaction to prepare Si-18F compounds, in spite of the fact that this represents a carrier-added experiment whereby the specific activity may be compromised.74, 75, 77 On the other hand, also indicated in Scheme  1.9, Ametamey and colleagues reported alternative ways to prepare the di-tert-butylphenyl fluorosilane from the corresponding silanol or silane.72, 73 The resulting di-tert-butylphenyl-18F-fluorosilane based bioconjugates, albeit with high preparative yields and good hydrolytic stability, are of high lipophilicity, which results predominantly in excretion by the liver. Modification of the silyl molecule will therefore be necessary to decrease its hydrophobicity for favored in vivo distribution and also improved clearance. Si tBu R tBu R' Si tBu 18F tBu R' R=Cl, 19F K18F, K2.2.2 CH3CN, rt K18F, K2.2.2 HOAc, DMSO 90 0C K18F, K2.2.2 DMSO, 70 0C R=H R=OH  Scheme  1.9 18F-Radiosyntheses of silicon-based building blocks.71-73, 76  The formation of the B-18F bond was reported as another fluoride capturing technique by Perrin and co-workers in 2005.78 By radiolabeling biotinylated p-aminophenylboronyl pinacolate under acidic conditions, Ting et al. were able to show 18F-fluoride  15 incorporation through the formation of an 18F-aryltrifluoroborate (18F-ArBF3) in the presence of 19F-fluoride using avidin magnetic particles to separate the 18F-labeled biotin from the unreacted 18F-fluoride.78 It was therein pointed out that the specific activity of the 18F-ArBF3 is three times that of the source 18F-fluoride. Via a systematic study on the in vitro solvolytic stability of a series of ArBF3s, it was recognized that the hydrolytic stability of ArBF3s can be controlled by different substituents on the aryl region and several potential ArBF3s with high hydrolytic stability were identified for further studies.80, 85 Furthermore, the in vivo stability of one 18F-ArBF3 was confirmed by an animal study on the clearance and biodistribution of the biotin-18F-ArBF3 conjugate.79 Recently, Tsien and colleagues reported the work in combination of 18F-PET and near infrared fluorescence (NIRF).81 Their “boron/optical multimodality beacon”, called 18F-BOMB, is based on the conjugation of the 18F-ArBF3/NIRF fluorophores to Lymphoseek to detect the distribution of the sentinel lymph node. We also have reported in vivo imaging work with an 18F-ArBF3 conjugated to marimastat, which is a broad-spectrum MMP inhibitor that might find use in breast cancer diagnosis.86 In addition to the efforts directed towards 18F-ArBF3s as PET imaging compounds, an isotopic exchange method has been reported to construct the B-18F bond for 18F-tetrafluoroborate (18F-BF4-) by Jauregui-Osoro et al. to detect the human sodium-iodide symporter.82, 87 They incubated 18F-fluoride with NaBF4 under acidic conditions at 120 oC for 20 minutes. The radiochemical yield (not decay corrected) of the isotopic exchange reaction was about 10%. Although both methods described here represent the radiosynthesis of boron compounds with the B-18F bond from carrier-added 18F-fluoride, 18F-ArBF3s provide extensive flexibility to label various functional molecules targeting different bioprocesses. The negative charge on 18F-ArBF3s may provide added advantage in that the labeled molecules have increased hydrophilicity. Moreover, the specific activity of 18F-ArBF3s is triple that of 18F-fluoride. This originates from the stoichiometric ratio (1:3) of 18F-ArBF3s to the bound fluorine atoms, which can compensate for any decrease in the specific activity of 18F-fluoride from the carrier addition. However, when considering the labeling conditions for 18F-ArBF3s, it is realized that the acidic conditions might be detrimental to various biomolecules, which might not survive low pHs. An alternative involving a one-pot two-step synthesis may  16 enable labeling the acid-sensitive molecules with 18F-ArBF3s.  Scheme  1.10 The radiosynthesis of 18/19F-ArBF3s.78  Although several 18F-incorporation techniques have been introduced above, there is still no perfect labeling technique so far to accommodate all the requirements for radiosyntheses, such as rapid labeling reactions, high radiochemical yields, high specific activities, and a one-step radiosynthesis to obtain the final product. Every method contains one or more drawbacks. Novel labeling methods thus are desired. Meanwhile current techniques should be optimized to achieve better radiosyntheses with high radiochemical yields and specific activities. 1.4 Applying ArBF3s as PET imaging agents In the previous section, it was mentioned that a good radiosynthetic scheme should minimize the number of radiosynthetic steps and shorten the synthesis time, while ensuring a reasonable radiochemical yield and a high specific activity. Moreover, the radiolabeled compounds need to possess good in vitro and most importantly in vivo stability, low lipophilicity for optimal clearance, and high in vivo target specificity.16, 17 As ArBF3s are anionic,78, 79 the ArBF3 labeled biomolecules would have higher hydrophilicity, which in turn should lead to rapid in vivo clearance of the 18F-ArBF3 labeled compounds. The moiety carrying 18F-atom should not impede the bioactivity of the biomolecules such as target specificity, and several 18F-ArBF3 labeled compounds have not been observed to influence the affinity of biomolecules for their targets.79, 81, 86 Nevertheless, the clearance and excretion of the labeled compound is likely to be enhanced due to the decreased lipophilicity derived from the negatively charged nature of ArBF3s. In this section, several aspects will be addressed in order to apply 18F-ArBF3s as potential PET imaging agents, including the specific activity, the radiochemical yield and solvolytic stability of 18F-ArBF3s.  17 1.4.1 Specific activity Specific activity, an important factor for imaging assays, is defined as the amount of radioactivity given by a certain amount of a radiolabeled compound.16, 88 Mathematically, it is the amount of radioactivity per micromole of the radiolabeled compound, as shown in Equation 1.1. Based on this definition, the theoretical specific activity of the carrier-free radionuclide can be calculated from the decay half-life of the radionuclide.  Equation  1.1  Practically, however, it is often impossible to obtain a carrier-free, 100% radiolabeled compound from a radiosynthesis, particularly in the case of 18F-fluoride. This is because the radionuclide is almost always contaminated with its stable isotope. Consequently, the highest practical specific activity is much lower than the theoretical number. Taking 18F-fluoride for example, the calculated theoretical specific activity is 1710 Ci/mol. In contrast, the specific activity of 18F-fluoride is usually measured to be < 40 Ci/mol, according to Equation 1.1.16, 17, 52 Usually 18F-labeled imaging agents are produced at much lower specific activities. Since most PET imaging agents are based on receptor binding, the radiotracer with a low specific activity would have to compete with the non-radiolabeled compounds (either the cold form or the decayed form), and less uptake of the radiolabeled compound will be expected. This is particularly true in cases with relatively low levels of receptors, in which case a significant percentage of receptors are occupied by the non-labeled compound. A significant amount of work has supported this hypothesis.89, 90 For instance, Frost and co-workers89 quantified the human opiate receptor in vivo via imaging experiments with high specific activities and low specific activities. The brain images clearly suggested that the radioactivity uptake by brain was highly reduced by the 11C-labeled ligand with low specific activities. Partial saturation of the receptors was regarded to account for the suppression. As for the case of any 18F-radiolabeling experiments introduced in the previous section, the specific activity of an 18F-labeled compound at any given time can be calculated if the specific activity of the source 18F-fluoride is known. This is due to at least two Specific activity Radioactivity (Ci) Amount of radiolabeled compound (mol)=  18 reasons. First of all, since 18F-fluoride has an insignificant kinetic isotopic effect compared to 19F-fluoride, it has the same physicochemical and biochemical properties as 19F-fluoride. In other words, 18F-fluoride and 19F-fluoride have the same opportunity to react in the same fashion in a radioreaction, and therefore the specific activity of 18F-fluoride can be directly transferred to that of the 18F-labeled compound. Secondly, 18F-fluoride and all the 18F-labeled species decay at the same rate, which implies that the specific activity at any given time could be calculated from the source 18F-fluoride via a first order decay function. For example, for the no-carrier-added nucleophilic substitution reaction to prepare 18F-FDG as shown in Scheme  1.4, if the 18F-fluoride solution is the only source of 19F-fluoride, and if we started with a radioactivity of 50 mCi at a specific activity of 2 Ci/mol (at t = 0 min), there is 25 nmol of 19F-fluoride present. After radiosynthesis and separation to give the pure 18F-FDG (containing both anomers) at t = 55 minutes (t1/2 for 18F is ~ 110 min), no matter what the radiochemical yield is, the specific activity of the purified 18F-labeled compound is 1.41 Ci/mol at t = 55 minutes, calculated via the first order decay kinetics.  Equation  1.2  When it comes to 18F-ArBF3s, Equation 1.2 briefly demonstrates the overall preparation of 18F-ArBF3s, though the reaction most likely proceeds stepwise. From the reaction, one boronic acid/ester molecule reacts with one HF molecule and one molecule of KHF2 to give one ArBF3 anion. The specific activity of the 18F-ArBF3 is three times that of 18F-fluoride, which can be derived as following: Equation  1.3  In 18F-fluoride generated from the 18O-H2O irradiation, trace amounts of 19F-fluoride are always present, but the amount of 19F-fluoride varies from one cyclotron to another.52 As a result, there is actually no good correlation to determine the specific activity for ArB(OR)2 + HF +KHF2 ArBF3 + K + + 2 ROH  19 18F-fluoride. Conventionally, the specific activity is determined by the incorporation of 18F-fluoride to afford an 18F-labeled compound, whose amount can be determined by methods such as UV absorption at a certain wavelength.91 Another possible method to estimate the specific activity is via the addition of a relatively large amount of carrier 19F-fluoride, which outweighs 19F-fluoride that normally contaminates the original 18F-fluoride solution. Generally, if a substantial amount of carrier 19F-fluoride is added to 18F-fluoride (no-carrier-added) and well mixed with 18F-fluoride, the specific activity of the carrier-added 18F-fluoride can be calculated as (the amount of the radioactivity of 18F-fluoride)/(the amount of carrier 19F-fluoride + the amount of 19F-fluoride present in the original 18F-fluoride solution + the amount of 18F-fluoride). Since a large amount of carrier 19F-fluoride is added, both the amount of 19F-fluoride from the original 18F-fluoride solution and that of 18F-fluoride itself become negligible. The specific activity of the carrier-added 18F-fluoride can be simplified to (the amount of the radioactivity of 18F-fluoride)/(the amount of carrier 19F-fluoride). For instance, if there is 200 mCi of 18F-fluoride from 18O-H2O irradiation and 800 nmol of 19F-fluoride is added (maybe in the form of KHF2) at t = 0 minute, the specific activity of 18F-fluoride (at t = 0 min) is 0.25 Ci/mol. Assuming that it takes 55 minutes for the radiosynthesis and purification to prepare an 18F-ArBF3, then at t = 55 minutes, the specific activity of 18F-fluoride is 0.177 Ci/mol. But for the specific activity of the 18F-ArBF3 at t = 55 minutes, it should be 0.531 Ci/mol. It is appreciated that the addition of carrier 19F-fluoride suppresses the specific activity of 18F-fluoride while the formation of 18F-ArBF3s compensates to some extent for the loss with a tripling of the specific activity. Thus, it is possible to adjust the specific activity by controlling for the amount of added carrier 19F-fluoride. Furthermore, optimal reaction conditions might favor the formation of 18F-ArBF3s in the presence of a smaller amount of 19F-fluoride or even under no-carrier-added conditions. 1.4.2 Radiochemical yields and synthesis time Though the yield is not always the top concern for radiosyntheses, it is important to be able to prepare radiolabeled compounds with “enough” radioactivity for animal imaging  20 experiments. Due to radiation safety, the radiosynthesis in the presence of lower initial radioactivity (< 200 mCi) is always favored. Therefore, a relatively good radiochemical yield is needed to provide enough radiolabeled compound for imaging applications. Most of the 18F-labeling experiments have been reported with radiochemical yields in the range of 5% to 95%.17, 53 In addition, it has also been addressed in the previous sections that radiosyntheses in a timely manner are preferred to prepare radiolabeled compounds and thus fewer steps are required.17 Although ArBF3s have been applied in synthetic chemistry for a long time, especially in transition-metal catalyzed cross-coupling reactions for the C-C bond formation, their preparation, particularly at relatively low concentrations of boronates and fluoride, is still not well understood. In part, this is because in the past a very large amount of fluoride has always been used to drive the reaction to completion. In contrast, in order to apply 18F-ArBF3s as PET imaging agents with a relatively high specific activity, the addition of a huge amount of carrier 19F-fluoride cannot be entertained without compromising the specific activity of the imaging agent. In addition, while a large excess of fluoride can favor the production of ArBF3s, the yield in terms of fluoride incorporation would drop abruptly. This means that not only the specific activity will be compromised by the addition of a large amount of carrier fluoride, but the radiochemical yield is also sacrificed. Therefore, a systematic study on the fluoridation of organoboronic acids/esters is extremely important for the further development and optimization of this labeling technique. 1.4.3 Solvolytic stability of ArBF3s Besides the importance of the radiolabeling technique to fulfill most of the requirements of radiosyntheses, the radiolabeled compounds must be sufficiently stable in vivo to be developed as useful imaging agents. To determine the factors that might influence the solvolytic stability of ArBF3s, Perrin and co-workers measured the hydrolytic rates for a series of ArBF3s with different substituents on the aromatic system by 19F NMR spectroscopy or 18F/19F TLC autoradiography.85 In the 19F NMR study, they did not observe any steady-state intermediate during the solvolysis for any of the ArBF3s investigated. More importantly, the kinetic data revealed a general trend of the influence  21 of different substituents on the solvolytic stability of ArBF3s. Specifically, electron withdrawing groups, at the para- and meta-positions to the trifluoroborate group, enhance the solvolytic stability of ArBF3s, while electron donating groups at the para-position accelerate the solvolysis of ArBF3s. Perrin and colleagues then were able to plot the kinetic data with the substituent constants (σ) to get a Hammett plot as shown in Figure  1.2. Among the ArBF3s studied therein, the ArBF3s from two boronates (3.1 and 3.8) were found to be especially stable and thus they have potential use for conjugation to biomolecules for PET imaging studies. The structures of the boronate synthons are shown in Figure  1.3. Via the carboxylic group, the boronate can be conjugated to various biomolecules. Hammett plot for the hydrolysis of ArBF3s m -.4 -.2 0.0 .2 .4 .6 .8 lo g( k o bs ) -2.5 -2.0 -1.5 -1.0 -.5 0.0 By 19F NMR spectroscopy By 18/19F TLC autoradiography  Figure  1.2 Hammett plot in the form of log(k) = σρ + log(k0).85 Data for the 18/19F exchange TLC experiment (○) and the 19F NMR fluoride dissociation experiment (●) were plotted against σ. The linear regression analysis of the 18/19F exchange TLC experiment (○) (black line) gave the reaction constant for trifluoroborate isotopic exchange, ρ = -1.20 ± 0.06 and R2 = 0.818. The linear regression analysis of the 19F NMR fluoride dissociation experiment (●) (red line) gave the reaction constant for trifluoroborate fluoride loss, ρ = -0.92 ± 0.07 and R2 = 0.807.  22 F F F B OO Ph Ph Ph Ph O OH B FF OHO HO OH 3.83.1 t1/2 (ArBF3) 781 min 410 min Figure  1.3 The structures of boronic acid/ester 3.1 and 3.8 that will be used in this dissertation. The t1/2 (the half-life of the defluoridation of the corresponding ArBF3s) was measured via 19F NMR spectroscopy in 192 mM phosphate buffer (pH 7).85  1.5 The goal of this dissertation Based on all the aspects discussed above for applying ArBF3s as PET imaging agents, this dissertation attempts to further understand the process of the fluoridation of boronates to prepare the radiolabeled 18F-ArBF3s in the presence of a low concentration and small amounts of fluoride. This thesis focuses on the conjugation of the arylboronic acid/ester to several biofunctional molecules for both radiolabeling and animal imaging studies. Based on the study from Ting et al.,85 the electronic properties on the aromatic system is significant for the solvolytic stability of ArBF3s. As electron withdrawing groups were found to stabilize ArBF3s against hydrolysis under physiologic conditions, the investigation of heteroaryltrifluoroborates (HetArBF3s) was expected to provide ArBF3s with higher stability since the π-deficient heteroaromatic systems provide high opportunities to further stabilize the corresponding HetArBF3s. As the aromatic structure containing –CH=N– unit(s) is considered to be π-deficient92 and the inductive effect from the endocyclic nitrogen is considerable, the hydrolytic stability of several N-HetArBF3s has been studied in this dissertation in order to discover more stable ArBF3s. This work will be presented in Chapter 2. A systematic fluoridation study based on boronates 3.1 and 3.8, including their derivatives, will be described in Chapter 3. In this chapter, we used TLC-fluorescent densitometry, 19F NMR spectroscopy, and radio-HPLC to analyze the fluoridation. Several reaction factors were investigated to achieve a reproducible, high yielding, and  23 low carrier-added radiosynthesis of 18F-ArBF3s. In this dissertation, boronate 3.1 has been conjugated to the matrix metalloproteinase (MMP) inhibitor marimastat (Chapter 4), a urea-based prostate-specific membrane antigen (PSMA) inhibitor (Chapter 5), and cyclic pentapeptides containing the Arg-Gly- Asp (RGD) sequence (Chapter 8). These boronates were all 18F-labeled under carrier-added conditions. The animal imaging work with marimastat-18F-ArBF3 4.15 is included in Chapter 4. To further explore this labeling technique to a broader application in terms of labeling biomolecules, an alkynyl prosthetic 18F-ArBF3 6.2 has been prepared for the subsequent copper(I) catalyzed click reaction to radiolabel biomolecules with 18F-ArBF3s. The radiolabeling of oligonucleotides (Chapter 6), folate (Chapter 7), and an RGD-containing cyclic pentapeptide (Chapter 8) has been undertaken using this newly developed one-pot two-step radiosynthesis.  24 Chapter 2 Hydrolytic defluoridation of N-HetArBF3s at neutral pH 2.1 Introduction Organotrifluoroborates are the more air-stable equivalents of boronic acids/esters.93, 94 These organotrifluoroborates have been increasingly used for synthetic reactions, including transition-metal catalyzed cross-coupling reactions to construct C-C bonds95-106 or C-X bonds.107 Moreover, aryltrifluoroborates (ArBF3s) have also been proposed to be useful as PET imaging agents.78 To understand better the solvolysis of ArBF3s, a systematic study was carried out and a Hammett plot was obtained to show that the rate constants of the solvolytic defluoridation of ArBF3s correlate with standard σ-values (the coefficient of correlation ρ is ~ -1) by Perrin and co-workers.85 This means that the solvolysis of ArBF3s can be retarded by introducing electron withdrawing substituents into the aromatic system yet enhanced in the presence of electron donating groups. Meanwhile, the solvolytic mechanism was proposed therein. It was believed that the reaction undergoes a stepwise process to lose the fluorine atoms following an SN1 mechanism, while the empty orbital on the boron is quickly occupied by the hydroxide to give intermediates as shown in Scheme  2.1.  Scheme  2.1 The proposed kinetic scheme of the ArBF3 solvolysis. The steps are all regarded reversible. a and a’ are the difluoro-species; b and b’ are the monofluoro-species.  In contrast, compared with the relatively large pool of reactions involving aryl-, alkenyl-, and alkyltrifluoroborates, there are fewer examples of the coupling reactions  25 with heteroaryltrifluoroborates (HetArBF3s).96-98, 100, 103-105, 107 This might be due to the less systematic understanding of the performance of HetArBF3s during the cross- coupling reactions. In fact, for the Pd-catalyzed cross-coupling reactions with ArBF3s, it is believed that defluoridation is necessary to provide a small amount of boronic acid or monofluoroboronate-species in order to facilitate transmetallation of the aryl moiety in H2O miscible solvents containing a small amount of H2O.98, 100, 103 Hence, the study of the solvolytic stability of these HetArBF3 synthons can provide important information to predict their synthetic reactivity in the transition-metal catalyzed cross-coupling reactions. In addition, HetArBF3s might possess the desired hydrolytic properties for PET imaging applications. The relatively low electron density, which is normally ascribed to heteroaromatic systems, is regarded as the favored property for stabilizing HetArBF3s. Therefore, in this chapter, we prepared some nitrogen-containing HetArBF3s and studied their stability under physiological conditions by 19F NMR spectroscopy. The results demonstrate that the N-heteroaromatic ring systems greatly retard the defluoridation of the HetArBF3s under buffered aqueous conditions at near neutral pH. Several N-HetArBF3s are found to display extraordinary hydrolytic resistance and therefore have very promising applications as PET imaging agents. 2.2 Results 2.2.1 Synthesis The heteroarylboronic acids/esters, which were converted to HetArBF3s, were either purchased or synthesized. The synthesis of three heteroarylboronic acids/esters has been summarized in Scheme  2.2. First, 2,6-dichloro-4-iodo-pyridine was treated with BuLi under a halo-lithium exchange reaction, the reaction mixture was quenched with B(OCH3)3; pinacol was added to protect the newly produced boronic acid to give 2.4 as one of the desired boronate esters.a In order to obtain N-methyl-4-pyridineboronic acid iodide 2.5, 4-pyridinylboronic acid was first protected with 2,3-dimethyl-1,3- propanediol, and N-methylated with MeI. The protecting group on boronate 2.5b was then removed to afford the desired product 2.5 with an overall yield of 74% over three  a Dr. Ali Asadi made this compound.  26 steps.   Scheme  2.2 Synthesis of several heteroarylboronic acids. (a), i. BuLi, Et2O, -78 oC, ii. B(OCH3)3, -78 oC, 2 hr, iii. pinacol and HOAc, 74%; (b), 2,2-dimethyl- 1,3-propanediol, 1,4-dioxane, molecular sieves, reflux, 18 hr, quant.; (c), MeI, CH3CN, reflux, overnight; (d), water/acetone, rt, 1 d, 74% over three steps; (e), hydrazine monohydrate, 1,4-dioxane, reflux, 89%; (f), 2,4-pentanedione, H2O, 70 °C, overnight, 80%; (g), NO/NO2, DMF, rt, 4 hr, 72%; (h), hydrazine monohydrate, CH3CN, reflux, 1.5 hr, 73%; (i), trichloroisocyanuric acid, CH3CN, 0 °C to rt, 1 hr, 46%; (j), i. BuLi, -78°C, 0.5 hr, ii. 2-isopropyloxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, -78 °C, 2 hr, iii. HCl in Et2O, -78 °C, 20 min, 37% over three steps; (k), xylenes, reflux, 24 hr, 14%.  Finally, a Diels-Alder reaction was used to prepare dichloropyridazinylboronate 2.7 from dichlorotetrazine 2.7e and alkynylboronate 2.7f. Hexyne was first treated with BuLi to give acetylide anions, which were quenched with B(OCH3)3. The boronate intermediate was further protected with pinacol to give 2.7f, which was purified via vacuum distillation. Meanwhile, 1,4-dichlorotetrazine 2.7e was synthesized from guanidinium chloride over five steps. Triaminoguanidinium chloride 2.7a, obtained from the hydrazinolysis of guanidinium chloride, was reacted with 2,4-pentanedione in H2O at 70 oC to afford 1,2-dihydro-1,2,4,5-tetrazine 2.7b, which was oxidized by NO/NO2 to  27 afford 1,2,4,5-tetrazine 2.7c. The dimethylpyrazole groups on 2.7c were replaced by hydrazine to provide 3,6-dihydrazino-1,2,4,5-tetrazine 2.7d. Tetrazine 2.7d underwent an electrophilic substitution with trichloroisocyanuric acid to give 1,4-dichlorotetrazine 2.7e. The Diels-Alder reaction between 2.7e and 2.7f was undertaken in xylenes at 140 oC for 24 hours to give 2.7. The overall yield to prepare 2.7 starting from guanidinium chloride was 2.4%. Then all the heteroarylboronic acids/esters were incubated with excess of KHF2 to prepare the corresponding N-HetArBF3s. The N-HetArBF3s were generally purified from free fluoride by flash chromatography with a small silica gel column using 5% NH4OH/EtOH as the elution buffer. Gratifyingly nearly all of the heteroarylboronic acids/esters showed very good conversions. 2.2.2 Solvolytic studies of N-HetArBF3s -80-60-40-200 3 min. 81 min. 190 min. 384 min. 1111 min. 1688 min. 2568 min. N B F F F F ppm    time (min) 0 500 1000 1500 2000 2500 Fr ac tio n of  T FB -2 .1  0.0 .2 .4 .6 .8 1.0  Figure  2.1 The 19F NMR study of para-pyridinyltrifluoroborate (TFB-2.1) dissociation in 200 mM phosphate buffer (pH 6.89). A, 19F NMR spectra of para-pyridinyltrifluoroborates TFB-2.1 dissociation; B, the exponential plot of trifluoroborate fraction against time as measured by 19F NMR spectroscopy with the 19F-signal of trifluoroacetic acid as the standard reference (0 ppm), kobs = (1.8  0.20)  10-3 min-1, R2 = 0.9940.  The solvolytic stability of various N-HetArBF3s in buffered aqueous solution at neutral pH was studied by 19F NMR spectroscopy. In general, it has been found that all the N-HetArBF3s studied herein have relatively long half-lives (t1/2  300 min) at room temperature in the phosphate buffer (pH ~ 7). An example of the 19F NMR spectroscopic A   28 assay for the solvolysis of para-pyridinyltrifluoroborate TFB-2.1 is shown in Figure  2.1. This assay monitors the loss of HetArBF3 peak (δ ~ -65 ppm, referenced to trifluoroacetic acid at 0 ppm) and simultaneously the increase of the free fluoride peak (δ ~ -42 ppm). The 19F NMR spectra showed that there were no other obvious fluorine-containing intermediates (monofluoro- or difluoro-species) in the reaction on the time scale of the 19F NMR data acquisition. The data analysis for the hydrolytic kinetics is therefore simplified due to the absence of any intermediate, since 19F-signals were used to construct a kinetic curve for the solvolysis. Typically, the percentage of the fluoride remaining on the N-HetArBF3 vs. time was best fitted to a standard first order rate process for all decomposition experiments, while the overall fluoride amounta was considered constant for each experiment. The data suggested a single rate-determining step, which is regarded to the loss of the first fluoride atom on the N-HetArBF3. This controls the overall rate of the decomposition. The study of the kinetics for the defluoridation of para-pyridinyltrifluoroborate (TFB-2.1) via 19F NMR spectroscopy, shown in Figure  2.1, exhibited a solvolytic decomposition half-life of 385 minutes at pH ~ 7. At higher pHs, the solvolytic rate of para-pyridinyltrifluoroborate TFB-2.1 was slightly enhanced, with half-lives of 210 minutes at pH 8 and 187 minutes at pH 9. These results encouraged us to extend the solvolytic studies at pH ~ 7 to several readily prepared N-HetArBF3s as summarized in Table  2.1. In brief, introducing one exocyclic halogen atom as fluorine to the pyridine ring, such as 2-fluoro-4-pyridinyltrifluoroborate (TFB-2.2), whose fluorine atom is at the meta-position to the BF3 group, showed little-to-no enhancement to the hydrolytic stability compared with that of TFB-2.1. However, when two halogen atoms, i.e. fluorine or chlorine, are introduced to 2- and 6-positions of the pyridine ring of the N-HetArBF3s (2,6-dihalopyridinyltrifluoroborates TFB-2.3 and TFB-2.4), the solvolysis was largely impeded. Since fluorine is more electronegative than chlorine, it is not surprising that the difluoropyridinyltrifluoroborate TFB-2.3 (t1/2 ~ 19 hr) is more stable than the dichloro-version TFB-2.4 (t1/2 ~ 14 hr). When the zwitterionic para-N-methylpyridinium trifluoroborate (TFB-2.5) was tested, the hydrolytic stability was further enhanced to give a half-life of approximately 4 days under the same conditions at room temperature.  a The overall fluoride only contains the fluoride in the form of ArBF3 or free fluoride.  29 Furthermore, pyridazinyltrifluoroborates TFB-2.6 and TFB-2.7, which contain two endocyclic nitrogen atoms and exocyclic chlorine atoms, displayed extraordinary stability against hydrolytic decomposition. Even at elevated temperatures (37 and 50 oC), the decomposition process was so slow that half-lives were measured in days.  Table  2.1 The kinetic data for the solvolysis of N-HetArBF3s Compound no. Structure Temperature k (min-1) t1/2 (min) 22 ± 2 oC (1.8 ± 0.20)  10-3 385 ± 43 22 ± 2 oC a (3.3 ± 0.20)  10-3 210 ± 12 22 ± 2 oC b (3.7 ± 0.10)  10-3 187 ± 5 TFB-2.1 N BF3  22 ± 2 oC c (1.9 ± 0.20)  10-3 365 ± 38 TFB-2.2 N BF3 F 22 ± 2 oC (1.9 ± 0.07)  10-3 364 ± 13 TFB-2.3 NF F BF3  22 ± 2 oC (0.6 ± 0.02) 10-3 1155 ± 31 TFB-2.4 N BF3 ClCl 22 ± 2 oC (8.0 ± 0.21)  10-4 866 ± 22 22 ± 2 oC (1.1 ± 0.03)  10-4 6177 ± 158 37 ± 2 oC (5.2 ± 0.20) 10-4 1329 ± 51 TFB-2.5  50 ± 2 oC (1.3 ± 0.06)  10-3 527 ± 24 22 ± 2 oC NDd ND 37 ± 2oC (3.7 ± 0.31)  10-5 18698 ± 1563 TFB-2.6  50 ± 2 oC (1.2 ± 0.05)  10-4 5996 ± 240 22 ± 2 oC (4.3 ± 0.11)  10-5 16187 ± 404 37 ± 2 oC (1.1 ± 0.03)  10-4 6478 ± 150  TFB-2.7  N N BF3 Bu Cl Cl  50 ± 2 oC (3.4 ± 0.07)  10-4 2069 ± 41 Note:  The hydrolytic study was undertaken in 200 mM phosphate buffer at pH 6.87 or otherwise noted; aThe hydrolytic study was undertaken in 200 mM phosphate buffer at pH 8.01; bThe hydrolytic study was undertaken in 200 mM phosphate buffer at pH 9.00; c The hydrolytic study was undertaken in 200 mM phosphate buffer at pH 6.87 in the presence of 10 mM KF; d ND stands for “not-determined” since the reaction rate was too slow to monitor.   30 More interestingly, when studied in the presence of free fluoride (10 mM), the hydrolytic defluoridation rate of para-pyridinyltrifluoroborate (TFB-2.1) was not significantly influenced. This may imply that at relatively high dilution these potential intermediates such as mono- and/or bis-fluoroborates tend to decompose rather than revert back to HetArBF3s under the solvolytic conditions at pH ~ 7. 2.3 Discussion 2.3.1 Synthesis Three heteroarylboronic acids/esters were prepared in this chapter for the corresponding N-HetArBF3s. Boronates 2.4 and 2.5 were synthesized with a reasonable yield of ~ 74%; however, the overall yield of the synthesis of 2.7 was only 2.4% over six steps from guanidium chloride. The very low yielding steps were the chlorination with trichloroisocyanuric acid for tetrazine 2.7e and the subsequent Diels-Alder reaction. Although the electrophilic substitution with trichloroisocyanuric acid was rapid, there seemed to be extensive loss during the work-up including the sublimation. For the Diels-Alder cycloaddition, it is possible that both the electron poor “diene” (tetrazine 2.7e) and the electron poor “dienophile” (alkynylboronate 2.7f) do not quite favor the electron demanding for the (inverse) Diels-Alder reactions. Even though the reaction was driven in terms of the production of N2 following the [2+4] cycloaddition, the first Diels-Alder reaction was slow and under the given conditions a relatively low yield was obtained. Since the preparation of the heteroarylboronic acids/esters was not the focus of the project, no further optimization was developed. Then heteroarylboronic acids/esters were treated with excess of KHF2 to prepare N-HetArBF3s, some of which required a bit longer time to achieve full conversions of the boronic acids/esters. 2.3.2 Solvolytic studies of N-HetArBF3s The results of the solvolytic study suggest that N-HetArBF3s are a class of compounds with high kinetic stability against hydrolytic defluoridation. Additional exocyclic electron-withdrawing groups on the heteroaromatic system can further improve the hydrolytic stability of N-HetArBF3s. The inductive effect of both the endocyclic heteroatoms and the exocyclic electron withdrawing substituents (e.g. fluorine and  31 chlorine) accounts for this increased stability. However, pyridinyltrifluoroborate TFB 2.2 with only one fluoride on 2-position did not demonstrate any significant enhancement to the hydrolytic stability compared with TFB-2.1, and there is no immediately forthcoming explanation for this. When comparing TFB-2.6 with TFB-2.7, they are very structurally similar but the position of the boron atom relative to the heteroatom might make a big difference for the ring electron properties. When the trifluoroborate group is next to the endocyclic nitrogen as for TFB-2.6, the defluoridation process is much slower than the other pyridazinyl compound TFB-2.7, which has two chlorine substituents. Even though the two chlorine substituents at 3- and 6-positions exert a very high inductive effect, TFB-2.7 with the trifluoroborate group at the 4-position hydrolyzed nearly three times as rapidly as TFB-2.6 under the same conditions. This suggests that the “in ring” nitrogen atoms greatly improve the stability of the trifluoroborate group at the 3-position. Moreover, although at elevated temperatures, the N-HetArBF3s demonstrated faster solvolysis than at room temperature, several N-HetArBF3s such as TFB-2.5, TFB-2.6, and TFB-2.7 still exhibited promising stability at higher temperatures under the same buffered conditions.a Among the N-HetArBF3s studied in this chapter, it was found that pyridazinyltrifluoroborates displayed extreme kinetic stability to solvolysis. These data suggest the potential use of these pyridazinyltrifluoroborates as 18F-PET imaging compounds but may also help to predict their poor reactivity to be used as substrates in transition-metal catalyzed cross-coupling reactions. It is possible to propose the mechanism of the hydrolytic defluoridation based on the 19F NMR studies. For all the data we have obtained, no fluorinated intermediate was ever observed in addition to the N-HetArBF3 and the product, namely free fluoride. This would imply very short half-lives of the possible intermediates, as shown in Scheme  2.1, on the NMR time scale. This leads us to hypothesize that the mechanism of N-HetArBF3 solvolysis involves the loss of the first fluoride as the rate-determining step, while the two subsequent fluorides are lost rapidly. This single rate-determining step is thus sufficient to explain the entire defluoridation process, consistent with the first order decay process observed with 19F NMR data. Based on this hypothesis, two possible  a Since pH changes due to temperatures, the same buffer was used for all the temperatures without pH corrections.  32 mechanisms are proposed in Scheme  2.3. Since no 19F-related intermediates were observed by 19F NMR spectroscopy for any compound studied herein, the second and third hydroxide-fluoride exchange processes should be very rapid. Moreover, when exogeneous fluoride source was added to the reaction, the hydrolytic process was not affected. This also suggests that loss of the second and third fluorine atoms is probably more kinetically favored under such conditions. N BF F F R OH F N BHO F F N B HO OH R R N B F F F R HO N B F F F R slow N BF F F R F N B F F R OH N BHO F F R N B HO OH R A B slow fast fast fast TS-A TS-B - - SN1 like SN2 like --  Scheme  2.3 Proposed mechanisms of the hydrolysis of HetArBF3s. A is the SN1 like mechanism and B is the SN2 like mechanism for the loss of the first fluoride; TS-X is the transition state for the slow step of either proposed mechanism.  Both the mechanisms shown in Scheme  2.3 propose a slow first step to lose the first fluoride, which is the rate-limiting step, and then fast subsequent steps, based on the observation. Mechanism A suggests the rate-determining B-F bond breakage is via an SN1-like mechanism, which is independent of the nucleophile concentration, where the nucleophile can be water or hydroxide. Alternatively, mechanism B is an SN2-like process, whose transition state involves a pentacoordinate boron, with the hydroxide ion attacking the boron atom and pushing away one of the fluoride ions. The kinetic data of para-pyridinyltrifluoroborate TFB-2.1 at various pHs, shows that the values of log(kobs)  33 for solvolysis are not dramatically changed. a  Data suggest that the reaction is pH-independent at slightly basic conditions, which corroborates the SN1 like mechanism A as the most possible mechanism. N BF F F R N R B F F N B F F F R   R=H R=EWG G Reaction Coordinate Figure  2.2 Proposed energy diagram of hydroxide-fluoride exchange for step 1 of mechanism A. The electron withdrawing groups (EWG) can largely stabilize ground states while the stabilizing effect is much weaker for transition states.  With regard to the overall reaction coordinate, the relative stability (free energies) of both the ground state and the transition state are important. The stabilizing effect from the electron withdrawing elements against hydrolysis increases the energy barrier between the ground state and the transition state. This might be interpreted by decreasing more of the free energy of the ground state or increasing more of the free energy of the transition state. The electron withdrawing groups discussed here exhibit strong inductive effects, which serve to decrease the -electron density available on the aromatic system and therefore to stabilize any negative charges in the system. Considering the experimental observations of the kinetic studies, it seems that the stabilizing effect of the ground state by the electron withdrawing groups is more than that of the transition state.  a The magnitudes of the rate constants among the conditions discussed are the same.  34 As at the ground state, the negative charge can be stabilized by the aromatic system via the inductive effect of the electron withdrawing substituents. The overall stabilization of the negative charge by the aromatic system at the transition state is less efficient than that at the ground state. As a result, a slightly less stabilizing effect with electron withdrawing groups should be expected for the transition state, as shown in Figure  2.2. Correspondingly, the energy gap between the ground state and the transition state is increased by the presence of electron withdrawing groups. As a result, the solvolysis is more obstructed. 2.4 Conclusion In this chapter, we have studied the hydrolytic stability of several N-HetArBF3s by 19F NMR spectroscopy. The N-HetArBF3s displayed very strong stability against hydrolytic decomposition. To develop new PET imaging compounds based on fluorine-18 chemistry, the N-HetArBF3s studied herein exhibit very favorable hydrolytic stability compared with the physical half-life of fluorine-18. In regard to the metal catalyzed coupling reactions, these N-HetArBF3s might not be good substrates. This hydrolytic study suggests that these HetArBF3s should be exceptional candidate components for in vivo PET imaging studies. 2.5 Materials and methods All chemicals were purchased from Sigma-Aldrich or Alfa-Aesar. Deuterated solvents were obtained from Cambridge Isotope Laboratories. Analytical thin layer chromatography was run on Silica Gel 60 F254 Glass TLC plates from EMD Chemicals, and SiliaFlash F60 from Silicycle was used for flash chromatography. Melting points were not corrected. All nuclear magnetic resonance (NMR) spectra were recorded at room temperature on a Bruker Avance 300 or 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm; all coupling constants (J) are reported in Hertz (Hz). Unless specified, 1H NMR spectra are referenced to the tetramethylsilane peak (δ = 0.00 ppm), 13C NMR spectra are referenced to the chloroform peak (δ = 77.23 ppm), and 19F NMR spectra are referenced to neat trifluoroacetic acid (δ = 0.00 ppm, -78.3 ppm relative to CFCl3). Due to the presence of 19F contamination in the NMR spectrometer probe,  35 baseline for samples less than 20 mM in 19F-fluoride concentration had to be adjusted by the multipoint linear baseline correction using MestReC 4.9.9.9. This correction did not affect the absolute chemical shifts or integration ratios of 19F signals. Low-resolution ESI mass spectrometry (ESI-LRMS) was performed on a Waters ZQ with a single quadruple detector, attached to a Waters-2695 HPLC. High-resolution ESI mass spectra (ESI-HRMS) were obtained on a Waters-Micromass LCT with a time-of-flight (TOF) detector. 2.5.1 Preparation of several heteroarylboronic acids 2,6-Dichloro-4-(4,4,5,5-tetramethyl-1,3,2 dioxaboryl)pyridine (2.4)108 Briefly, 2,6-dichloro-4-iodo-pyridine (1.4 g, 10 mmol) at -78 oC was added to a hexane solution of BuLi (8.0 mL, 15 mmol) in dry Et2O (30 mL). After 30 min at -78 °C, the mixture was treated with B(OCH3)3 (1.25 mL, 11.0 mmol) and the reaction was stirred for 1 hr. The temperature was then allowed to rise slowly over 2 hr up to rt, then pinacol (1.6 g, 13 mmol) was added and 10 min later AcOH (0.60 mL, 10.5 mmol) was added. The resulting mixture was filtered through Celite, which was then washed with ether, and the combined filtrates were evaporated under reduced pressure. The desired product was crystallized from cyclohexane. Yield: 74%. 1H NMR (300 MHz, CDCl3, rt): δ (ppm) 1.34 (s, 12 H), 8.06 (s, 2 H); 13C NMR (75.5 MHz, CDCl3, rt): δ (ppm) 24.8, 84.4, 111.9, 128.0, 149.0. N-Methyl-4-pyridineboronic acid iodide (2.5) This compound is prepared following a literature protocol.109 Briefly, 4-pyridinylboronic acid (0.5 g, 4 mmol) and 2,2-dimethyl-1,3-propanediol (0.4 g, 4 mmol) were dissolved in 1,4-dioxane (25 mL) in the presence of a few chips of 4Å molecular sieves. The mixture was refluxed for 18 hr. The reaction was then cooled down and filtered to remove the molecular sieves. The filtrate was concentrated under vacuum and the residue was dried over high vacuum to give 0.85 g (quantitatively) of a white solid, which was used directly without further purification. The white solid was dissolved in CH3CN (25 mL) and MeI (2.4 mL, 40.2 mmol) was added. The resulting solution was refluxed overnight, and concentrated under vacuum. To the yellowish residue, 1:1  36 water/acetone (30 mL) was added and the slurry was stirred at rt for 1 day. The mixture was then filtered and the filtrate was concentrated under reduced pressure. The product was precipitated from MeOH/Et2O to give a pale yellow powder as the desired product. Yield: 1.0 g, 74% over three steps. 1H NMR (400 MHz, d6-DMSO, rt): δ (ppm) 4.20 (s, 3 H), 7.54 (d, J = 6.16 Hz, 2 H), 8.55 (d, J = 6.24 Hz, 2 H); 13C NMR (100.6 MHz, d6-DMSO, rt): δ (ppm) 48.79, 50.05, 132.12, 144.30. Triaminoguanidine monohydrochloride(2.7a) This compound is prepared following a literature protocol with some modifications.110 Hydrazine monohydrate (3.21 g, 68.2 mmol) was added to a suspension of guanidine hydrochloride (1.91 g, 20.0 mmol) in 1,4-dioxane (10 mL) at rt. The mixture was then refluxed until ammonia was no longer releaseda. The reaction mixture was cooled to rt, filtered, washed with 1,4-dioxane and dried to give a white powder as the target molecule. Yield: 2.49 g, 89%. Mp: 215-216 oC (Lit. 230 oC); 13C NMR (75.5MHz, D2O, rt): δ (ppm) 159.78; ESI-LRMS: [M-Cl]+: 104.9 (100%). 3,6-Bis(3,5-dimethylpyrazol-1-yl)-1,2-dihydro-1,2,4,5-tetrazine(2.7b) This compound is prepared following a literature protocol with some modifications.110 2,4-Pentanedione (20.4 g, 0.2 mol) was dropwise added to triaminoguanidine monohydrochloride 2.7a (4.06 g, 0.1 mol) in H2O (100 mL) at rt. The mixture was stirred at 70 oC overnight. After the reaction was cooled down, the orange mixture was filtered. The solid was washed with water, and dried to provide pure 2.7b. Yield: 10.85 g, 80%. Mp: 130-131 oC (Lit. 150 oC); 1H NMR (300 MHz, d6-DMSO, rt): δ (ppm) 2.17 (s, 6 H), 2.39 (s, 6 H), 6.14 (s, 2 H), 8.82 (s, 2 H); 13C NMR (75.5 MHz, d6-DMSO, rt): δ (ppm) 12.90, 13.21, 109.39, 141.65, 145.24, 149.35; ESI-LRMS: [M+H]+, 273.2 (100%), 273.4 (15%). 3,6-Bis(3,5-dimethylpyrazol-1-yl)-1,2,4,5-tetrazine(2.7c) This compound is prepared following a literature protocol with some modifications.110 NO/NO2 was produced by adding 50 wt% sulfuric acid (78 mL, 0.55 mol)) dropwise to 0.6 N sodium nitrite (100 mL, 60 mmol); the resulting gas was bubbled into 2.7b (2.26g,  a The ammonia could be detected with a stripe of pH paper with moisture.  37 8.3 mmol) in DMF (40 mL) at rt for 4 hr. Then iced water (100 mL) was poured into the reaction to result in a purple precipitate. The reaction mixture was filtered, washed with cold water, and dried to give the desired product. Yield: 1.61 g, 72%. Mp: 216-218 oC (Lit. 223-224 oC); 1H NMR (300 MHz, d6-DMSO, rt): δ (ppm) 2.27 (s, 6 H), 2.58 (s, 6 H), 6.35 (s, 2 H); 13C NMR (75.5 MHz, d6-DMSO, rt): δ (ppm) 14.07, 14.27, 111.59, 143.71, 153.19, 159.38; ESI-LRMS: [M+Na]+: 293.2 (100%), 294.2 (15%). 3,6-Dihydrazino-1,2,4,5-tetrazine(2.7d) This compound is prepared following a literature protocol.111 Hydrazine monohydrate (1.3 g, 26 mmol) was added slowly to a slurry of 2.7c (3.2 g, 12 mmol) in CH3CN (30 mL). The resulting dark red solution was then heated to reflux for 1.5 hr. After the mixture was cooled down to rt, the slurry was filtered and the solid was washed with CH3CN to afford the desired product. Yield: 1.22 g, 73%. Mp: 137-138 oC. 13C NMR (75.5 MHz, d6-DMSO, rt): δ(ppm) 164.22; ESI-LRMS: [M+H]+: 143.1 (100%). 3,6-Dichloro-1,2,4,5-tetrazine (2.7e) To 3,6-dihydrazino-1,2,4,5-tetrazine 2.7d (1.28 g, 9.00 mmol) in CH3CN (35 mL) at 0 oC was added dropwise with CH3CN (25 mL) solution of trichloroisocyanuric acid (4.08 g, 18 mmol) for 30 min.112 The reaction mixture was then allowed to warm up to rt over 20 min. After filtration, the filtrate was concentrated in vacuum to give the crude product. Pure orange red crystals as the product were obtained via sublimation. Yield: 0.63 g, 46%. 13C NMR (75.5 MHz, CDCl3, rt): δ (ppm) 167.24; ESI-LRMS: [M+H]+: 149.8 (100%). 2-(1-Hexyn-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.7f) This compound is prepared following a literature protocol with some modifications.113 n-Hexyne (2 g, 24 mmol) in anhydrous Et2O (24 mL) was cooled to -78 oC and then 1.6 M BuLi (15.2 mL, 24.32 mmol) in hexane was added. The resulting slurry was stirred for 0.5 hr. 2-Isopropyloxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4.58 g, 24.6 mmol) in anhydrous Et2O (24 mL) was added quickly to the mixture and the reaction was stirred at -78 oC for another 2 hr. The reaction mixture was later wa