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Synthesis of ¹⁸F-radiolabeled LLP2A for use in PET imaging via aryltrifluoroborate formation Walker, Daniel 2012-12-31

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SYNTHESIS	
  OF	
  18F-­‐RADIOLABELED	
  LLP2A	
  FOR	
  USE	
  IN	
  PET	
  IMAGING	
   VIA	
  ARYLTRIFLUOROBORATE	
  FORMATION	
   	
   	
   	
   by	
   	
   	
   Daniel	
  Walker	
   	
   	
   B.Sc.	
  (Honours),	
  St.	
  Francis	
  Xavier	
  University,	
  2009	
   	
   	
   	
   A	
  THESIS	
  SUBMITTED	
  IN	
  PARTIAL	
  FULFILLMENT	
  OF	
   THE	
  REQUIREMENTS	
  FOR	
  THE	
  DEGREE	
  OF	
   	
   	
   MASTER	
  OF	
  SCIENCE	
   	
   	
   in	
   	
   	
   The	
  Faculty	
  of	
  Graduate	
  Studies	
   	
   (Chemistry)	
   	
   	
   	
   THE	
  UNIVERSITY	
  OF	
  BRITISH	
  COLUMBIA	
   	
   (Vancouver)	
   	
   	
   	
   July	
  2012	
   	
   	
   ©	
  Daniel	
  Walker,	
  2012	
    	
    Abstract	
   	
   Tumor-­‐specific	
   imaging	
   agents,	
   such	
   as	
   peptides,	
   allow	
   neoplasm	
   visualization	
   by	
   targeting	
  over-­‐expressed	
  extracellular	
  proteins	
  not	
  found	
  on	
  normal	
  cells.	
  	
  This	
  yields	
  not	
   only	
   diagnostic	
   information,	
   but	
   also	
   provide	
   insight	
   into	
   tumor	
   aggressiveness,	
   allowing	
   more	
  accurate	
  treatment	
  and	
  prognosis.	
  	
  LLP2A	
  has	
  been	
  recognized	
  as	
  a	
  strong	
  binder	
   of	
   the	
   α4β1	
  integrin.	
   	
   Its	
   binding	
   characteristics	
   along	
   with	
   its	
   proteolytic	
   resistance	
   make	
   it	
   an	
   excellent	
   candidate	
   for	
   PET	
   imaging.	
   	
   18F	
   is	
   widely	
   recognized	
   as	
   the	
   optimal	
   radionuclide	
   for	
   cancer	
   imaging,	
   although	
   its	
   unique	
   chemistry	
   presents	
   difficulties	
   in	
   biomolecule	
   incorporation.	
   	
   The	
   synthesis	
   of	
   18F-­‐aryltrifluoroborates	
   can	
   be	
   performed	
   under	
   mild	
   conditions,	
   while	
   also	
   creating	
   an	
   imaging	
   agent	
   with	
   triple	
   the	
   specific	
   activity	
   of	
   the	
   source	
   fluoride.	
   	
   Herein	
   is	
   described	
   the	
   synthesis	
   of	
   an	
   18F-­‐labeled	
   aryltrifluoroborate-­‐LLP2A	
   conjugate,	
   an	
   azide-­‐functionalized	
   LLP2A	
   for	
   use	
   in	
   click	
   radiolabeling,	
  and	
  cellular	
  binding	
  confirmation	
  of	
  the	
  aryltrifluoroborate-­‐LLP2A	
  peptide	
   to	
  α4β1	
  integrin	
  expressing	
  cells.	
    	
    	
    ii	
    Table	
  of	
  Contents	
   	
   Abstract	
  .......................................................................................................................	
  ii	
   Table	
  of	
  Contents	
  ........................................................................................................	
   iii	
   List	
  of	
  Tables	
  ................................................................................................................	
  vi	
   List	
  of	
  Figures	
  ..............................................................................................................	
  vii	
   List	
  of	
  Schemes	
  ............................................................................................................	
  ix	
   Abbreviations	
  ..............................................................................................................	
  xi	
   Acknowledgements	
  ....................................................................................................	
  xiii	
   Chapter	
  1	
  	
  Introduction	
  ................................................................................................	
  1	
   1.1	
   Cancer	
  and	
  its	
  Imaging	
  ...........................................................................................	
  1	
   1.1.1	
   A	
  Brief	
  Overview	
  of	
  Cancer	
  ..............................................................................	
  1	
   1.1.2	
   Cancer	
  Imaging	
  Technology	
  .............................................................................	
  2	
   1.1.3	
   PET	
  Isotopes	
  .....................................................................................................	
  4	
   1.1.4	
   18F	
  Production	
  ..................................................................................................	
  6	
   1.1.5	
   18FDG	
  ................................................................................................................	
  6	
   1.1.6	
   Tumor-­‐Specific	
  Imaging	
  ....................................................................................	
  7	
   1.1.7	
   Alternative	
  18F-­‐Labeling	
  Strategies	
  .................................................................	
  11	
   1.2	
   Arylborates	
  as	
  Fluoride	
  Captors	
  ...........................................................................	
  13	
   1.2.1	
  	
   Advantages	
  of	
  Trifluoroborates	
  .....................................................................	
  13	
   1.2.2	
   Stability	
  of	
  Aryltrifluoroborates	
  .....................................................................	
  14	
   1.2.3	
   Boronic	
  Acid	
  Protecting	
  Groups	
  .....................................................................	
  17	
   1.2.4	
   ArB18F3-­‐marimistat	
  .........................................................................................	
  19	
   1.3	
   LLP2A	
  ....................................................................................................................	
  20	
   1.3.1	
   The	
  α4β1	
  Integrin	
  ............................................................................................	
  20	
   1.3.2	
   High-­‐Affinity	
  α4β1	
  Ligand	
  Identification	
  ..........................................................	
  21	
   1.3.3	
   In	
  Vivo	
  Testing	
  of	
  LLP2A	
  .................................................................................	
  22	
   1.4	
  	
   ArB(OR)2-­‐LLP2A	
  Synthetic	
  Strategy	
  .......................................................................	
  23	
   1.4.1	
   One-­‐step	
  Strategy	
  ..........................................................................................	
  23	
   1.4.2	
   Two-­‐step	
  Strategy	
  ..........................................................................................	
  25	
   Chapter	
  2	
  	
  Results	
  and	
  Discussion	
  ..............................................................................	
  27	
   2.1	
   Peptide	
  Synthetic	
  Strategy	
  ...................................................................................	
  27	
   2.1.1	
   Solid	
  Phase	
  Peptide	
  Synthesis	
  (SPPS)	
  Overview	
  .............................................	
  27	
   2.1.2	
   Resin	
  and	
  Linker	
  Options	
  ................................................................................	
  28	
   2.1.3	
   Peptide	
  Coupling	
  Reagents	
  ............................................................................	
  30	
   2.2	
  	
   Solid	
  Phase	
  Peptide	
  Synthesis	
  of	
  LLP2A	
  ................................................................	
  32	
   2.3	
   Solution	
  Phase	
  Synthesis	
  and	
  Conjugation	
  to	
  LLP2A	
  ............................................	
  44	
   2.3.1	
   Synthesis	
  of	
  ArB(OR)2-­‐LLP2A	
  ..........................................................................	
  44	
   2.3.2	
   Synthesis	
  of	
  N3-­‐LLP2A	
  .....................................................................................	
  47	
   	
    iii	
    2.4	
   Cold	
  Fluorination	
  of	
  ArB(OR)2-­‐LLP2A	
  ....................................................................	
  49	
   2.4.1	
   Perspectives	
  on	
  ArBF3	
  Formation	
  ...................................................................	
  49	
   2.4.2	
   Slow	
  Fluorination	
  of	
  ArB(OR)2-­‐LLP2A	
  .............................................................	
  50	
   2.4.3	
   PET	
  Imaging	
  Scale	
  Fast	
  Fluorination	
  of	
  ArB(OR)2-­‐LLP2A	
  ................................	
  52	
   2.5	
   Biological	
  Activity	
  Confirmation	
  ...........................................................................	
  53	
   2.5.1	
   Synthesis	
  of	
  FITC-­‐LLP2A	
  ..................................................................................	
  53	
   2.5.2	
   Cell	
  Binding	
  Assay	
  ...........................................................................................	
  54	
   2.6	
   18F-­‐Radiolabeling	
  of	
  ArB(OR)2-­‐LLP2A	
  ....................................................................	
  58	
   2.6.1	
   One-­‐step	
  Radiolabeling	
  ..................................................................................	
  58	
   2.6.2	
   Two-­‐step	
  Radiolabeling	
  via	
  Click	
  Chemistry	
  ...................................................	
  60	
   Chapter	
  3	
  	
  Conclusions	
  and	
  Future	
  Directions	
   ............................................................	
  63	
   3.1	
   Conclusions	
  ...........................................................................................................	
  63	
   3.2	
   Future	
  Directions	
  ..................................................................................................	
  64	
   Chapter	
  4	
  	
  Experimental	
  .............................................................................................	
  65	
   4.1	
   Materials	
  ...............................................................................................................	
  65	
   4.2	
   Techniques	
  ...........................................................................................................	
  65	
   4.2.1	
  	
  	
   NMR	
  Spectroscopy	
  .........................................................................................	
  65	
   4.2.2	
   Mass	
  Spectrometry	
  ........................................................................................	
  66	
   4.2.3	
   Chromatography	
  ............................................................................................	
  66	
   4.2.4	
  	
  	
   UV-­‐Visible	
  Absorption	
  ....................................................................................	
  67	
   4.2.5	
   Cell	
  Proliferation	
  .............................................................................................	
  68	
   4.2.6	
   Fluorescence	
  Microscopy	
  ...............................................................................	
  68	
   4.3	
   Solid	
  Phase	
  Peptide	
  Synthesis	
  ..............................................................................	
  68	
   4.3.1	
   Resin	
  Techniques	
  ............................................................................................	
  68	
   4.3.2	
   Fmoc-­‐Ach	
  (resin	
  1)	
  .........................................................................................	
  69	
   4.3.3	
   Fmoc-­‐Aad(tBu)-­‐Ach	
  (resin	
  2)	
  ..........................................................................	
  70	
   4.3.4	
   Fmoc-­‐Lys(Dde)-­‐Aad(tBu)-­‐Ach	
  (resin	
  3)	
  ...........................................................	
  71	
   4.3.5	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl-­‐Lys(Dde)-­‐Aad(tBu)-­‐Ach	
  (resin	
  4)	
  ............	
  72	
   4.3.6	
   [2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl]-­‐Lys(3-­‐(3-­‐pyridyl)	
  acrylyl)-­‐Aad(tBu)-­‐Ach	
   (resin	
  5)	
  ......................................................................................................................	
  73	
   4.3.7	
   [2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl]-­‐Lys(3-­‐(3-­‐pyridyl)	
  acrylyl)-­‐Aad(tBu)-­‐Ach	
  	
   (LLP2A(tBu),	
  2)	
  ...........................................................................................................	
  74	
   4.4	
   Synthesis	
  in	
  Solution	
  .............................................................................................	
  75	
   4.4.1	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
  acid38	
  (1)	
  ................................................	
  75	
   4.4.2	
   2,4,6-­‐Trifluoro-­‐3-­‐(4,4,5,5-­‐tetraphenyl-­‐1,3,2-­‐dioxaborolan-­‐2-­‐yl)benzoic	
  acid	
  	
   (ArB(OR)2	
  ,	
  3)	
  ..............................................................................................................	
  76	
   4.4.3	
   ArB(OR)2-­‐LLP2A	
  (4)	
  .........................................................................................	
  78	
   4.4.4	
   5-­‐azido	
  pentanoic	
  acid46	
  (5)	
  ...........................................................................	
  80	
   4.4.5	
   N3-­‐LLP2A	
  (6)	
  ...................................................................................................	
  81	
   4.4.6	
   ArBF3-­‐LLP2A	
  (7)	
  ..............................................................................................	
  82	
   4.4.7	
   FITC-­‐LLP2A	
  (8)	
  ................................................................................................	
  85	
   4.5	
   Cell	
  Binding	
  Assays	
  ...............................................................................................	
  86	
   4.5.1	
   Fluorescence	
  Assay	
  ........................................................................................	
  86	
   	
    iv	
    4.5.2	
   Blocking	
  Assay	
  ................................................................................................	
  87	
   4.6	
   18F-­‐Radiolabeling	
  ..................................................................................................	
  87	
   4.6.1	
   One-­‐Step	
  One-­‐Pot	
  Synthesis	
  of	
  ArB18F3-­‐LLP2A	
  (9)	
  .........................................	
  87	
   4.6.2	
   One-­‐Pot	
  Two-­‐Step	
  Click	
  Synthesis	
  of	
  ArB18F3-­‐LLP2A	
  (10)	
  ...............................	
  88	
   References	
  .................................................................................................................	
  90	
   	
   	
   	
   	
    	
    v	
    List	
  of	
  Tables	
   	
   	
   Table	
  1.1	
  	
  	
  Properties	
  of	
  several	
  possible	
  isotopes	
  for	
  use	
  in	
  PET	
  imaging……………..	
    5	
    Table	
  2.1	
  	
  	
  Results	
  of	
  cell	
  binding	
  fluorescence	
  assay…………………………………………….	
    56	
    Table	
  2.2	
  	
  	
  Results	
  of	
  fluorescence	
  blocking	
  assay………………………………………………….	
    57	
    	
   	
    	
    	
    vi	
    List	
  of	
  Figures	
   	
   	
   Figure	
  1.1:	
  	
  Positron	
  (+e)	
  emission	
  by	
  18F………………………………………………………………….	
    4	
    Figure	
  1.2:	
  	
  The	
  structure	
  of	
  18FDG…………………………………………………………………….......	
    7	
    Figure	
  1.3:	
  	
  Select	
  peptides	
  currently	
  under	
  investigation	
  for	
  molecular	
  imaging	
   purposes………………………………………………………………………………………………………………….	
    8	
    Figure	
  1.4:	
  	
  RP-­‐HPLC	
  traces	
  of	
  the	
  one-­‐step	
  18F-­‐labeling	
  of	
  RGD	
  via	
  nucleophilic	
   aromatic	
  substitution	
  by	
  Chen	
  et	
  al…………………………………………………………………………	
    10	
    Figure	
  1.5:	
  	
  Charge	
  distribution	
  of	
  electron	
  donating	
  and	
  withdrawing	
  substituents	
   on	
  aryltrifluoroborates……………………………………………………………………………………………	
    16	
    Figure	
  1.6:	
  	
  Structure	
  of	
  the	
  boronic	
  acid	
  to	
  be	
  used	
  for	
  fluoride	
  labeling	
  studies……	
    17	
    Figure	
  1.7:	
  	
  Prevention	
  of	
  C-­‐B	
  bond	
  cleavage	
  by	
  base-­‐mediated	
  deboronation	
   through	
  use	
  of	
  a	
  sterically	
  encumbered	
  protecting	
  group……………………………………….	
    18	
    Figure	
  1.8:	
  	
  Structure	
  of	
  ArB18F3-­‐marimistat	
  (top)	
  and	
  in	
  vivo	
  imaging	
  results	
   (bottom)	
  of	
  murine	
  breast	
  carcinoma……………………………………………………………………..	
    20	
    Figure	
  1.9:	
  	
  The	
  structure	
  of	
  the	
  library	
  design	
  for	
  the	
  screening	
  of	
  α4β1	
  targeting	
   ligands……………………………………………………………………………………………………………………..	
    21	
    Figure	
  1.10:	
  	
  The	
  structure	
  of	
  LLP2A…………………………………………………………………………	
   22	
   Figure	
  1.11:	
  	
  Localization	
  of	
  Cy5.5-­‐LLP2A	
  in	
  α4β1	
  expressing	
  Molt-­‐4	
  cells…………………	
    23	
    Figure	
  1.12:	
  	
  Structure	
  of	
  the	
  target	
  molecule,	
  ArB(OR)2-­‐LLP2A,	
  and	
  its	
   retrosynthetic	
  analysis…………………………………………………………………………………………….	
    24	
    Figure	
  1.13:	
  	
  Retrosynthetic	
  analysis	
  of	
  18F-­‐labeling	
  of	
  N3-­‐LLP2A	
  via	
  click	
   chemistry…………………………………………………………………………………………………………………	
    26	
    Figure	
  2.1:	
  	
  O-­‐bis-­‐(aminoethyl)ethylene	
  glycol	
  trityl	
  resin………………………………………..	
    29	
    Figure	
  2.2:	
  	
  Structures	
  of	
  DCC,	
  DIC,	
  and	
  EDC	
  coupling	
  reagents……………………………….	
    30	
    Figure	
  2.3:	
  	
  Structures	
  of	
  coupling	
  reagents,	
  from	
  left	
  to	
  right:	
  	
  HBTU,	
  HATU,	
  and	
   HCTU..……………………………………………………………………………………………………………………..	
    31	
    Figure	
  2.4:	
  	
  RP-­‐HPLC	
  trace	
  of	
  LLP2A(tBu)	
  and	
  truncate…………………………………………….	
    43	
    Figure	
  2.5:	
  	
  RP-­‐HPLC	
  trace	
  of	
  purified	
  LLP2A(tBu)…………………………………………………….	
    44	
    	
    vii	
    Figure	
  2.6:	
  	
  Structure	
  of	
  deboronylated	
  side	
  product………………………………………………	
    50	
    Figure	
  2.7:	
  	
  RP-­‐HPLC	
  of	
  synthesis	
  of	
  ArBF3-­‐LLP2A	
  by	
  slow	
  fluorination	
  method……….	
    51	
    Figure	
  2.8:	
  	
  19F	
  NMR	
  of	
  ArB(OR)2	
  and	
  ArBF3-­‐LLP2A…………………………………………………..	
    51	
    Figure	
  2.9:	
  	
  RP-­‐HPLC	
  of	
  synthesis	
  of	
  ArBF3-­‐LLP2A	
  by	
  fast	
  fluorination	
  method…………	
    52	
    Figure	
  2.10:	
  	
  MOLT-­‐4	
  cells	
  exhibiting	
  fluorescence…………………………………………………..	
    56	
    Figure	
  2.11:	
  	
  MOLT-­‐4	
  cells	
  exhibiting	
  no	
  fluorescence……………………………………………..	
    58	
    Figure	
  2.12:	
  	
  Radiolabeled	
  product	
  ArB18F3-­‐LLP2A	
  9	
  and	
  RP-­‐HPLC	
  trace…………………..	
    59	
    Figure	
  2.13:	
  	
  RP-­‐HPLC	
  of	
  alkynyl-­‐ArB18F3......................................................................	
    61	
    Figure	
  2.14:	
  	
  RP-­‐HPLC	
  of	
  the	
  final	
  radiolabeled	
  ArB18F3-­‐LLP2A	
  10	
  and	
  alkynyl-­‐ ArB18F3	
  …………………………………………………………………………………………………………………….	
   62	
   Figure	
  4.1:	
  	
  RP-­‐HPLC	
  trace	
  of	
  LLP2A(tBu).	
  	
  Reproduced	
  from	
  page	
  44………………………	
    75	
    Figure	
  4.2:	
  	
  1H	
  NMR	
  of	
  1………………………………………………………………………………………….	
    76	
    Figure	
  4.3:	
  	
  1H	
  NMR	
  of	
  3..…………………………………………………………………………………………	
    77	
    Figure	
  4.4:	
  	
  19F	
  NMR	
  of	
  3………………………………………………………………………………………….	
    78	
    Figure	
  4.5:	
  	
  RP-­‐HPLC	
  trace	
  of	
  ArB(OR)2-­‐LLP2A………………………………………………………….	
    79	
    Figure	
  4.6:	
  	
  1H	
  NMR	
  of	
  5…………………………………………………………………………………………..	
    80	
    Figure	
  4.7:	
  	
  RP-­‐HPLC	
  trace	
  of	
  N3-­‐LLP2A.	
  …………………………………………………………………..	
   82	
   Figure	
  4.8:	
  	
  19F	
  NMR	
  of	
  ArBF3-­‐LLP2A…………………………………………………………………………	
   83	
   Figure	
  4.9:	
  	
  RP-­‐HPLC	
  trace	
  of	
  ArBF3-­‐LLP2A.	
  	
  Reproduced	
  from	
  page	
  50……………………	
    83	
    Figure	
  4.10:	
  	
  RP-­‐HPLC	
  of	
  ArBF3-­‐LLP2A.	
  	
  Reproduced	
  from	
  page	
  52…………………………..	
    84	
    Figure	
  4.11:	
  	
  UV-­‐visible	
  absorption	
  of	
  FITC-­‐LLP2A…………………………………………………….	
   86	
   Figure	
  4.12:	
  	
  RP-­‐HPLC	
  radio	
  trace	
  of	
  ArB18F3-­‐LLP2A	
  9.	
  	
  Reproduced	
  from	
  page	
  58……	
    88	
    Figure	
  4.13:	
  	
  RP-­‐HPLC	
  of	
  alkynyl-­‐ArB18F3.	
  	
  Reproduced	
  from	
  page	
  60………………………	
    89	
    Figure	
  4.14:	
  	
  RP-­‐HPLC	
  of	
  click	
  reaction	
  to	
  produce	
  ArB18F3-­‐LLP2A	
  10.	
  	
  Reproduced	
   from	
  page	
  61.	
  ………………………………………………………………………………………………………….	
   89	
   	
   	
    	
    	
    viii	
    List	
  of	
  Schemes	
   	
   	
   Scheme	
  1.1:	
  	
  One-­‐step	
  18F-­‐labeling	
  strategy	
  proposed	
  by	
  Chen	
  et	
  al…………………….	
    9	
    Scheme	
  1.2:	
  	
  A	
  multi-­‐step	
  18F-­‐labeling	
  scheme	
  employing	
  18F-­‐	
  in	
  a	
  nucleophilic	
   substitution	
  reaction……………………………………………………………………………………………..	
    11	
    Scheme	
  1.3:	
  	
  Two-­‐step	
  biomolecule	
  radiolabeling	
  via	
  click	
  conjugation………………..	
    12	
    Scheme	
  1.4:	
  	
  General	
  scheme	
  of	
  fluoride	
  capture	
  by	
  a	
  protected	
  arylborate	
  to	
   produce	
  an	
  aryltrifluoroborate	
  (ArBF3).	
  ……………………………………………………………….	
    14	
    Scheme	
  1.5:	
  	
  Mechanism	
  of	
  hydrolysis	
  of	
  an	
  aryltrifluoroborate………………………….	
    15	
    Scheme	
  2.1:	
  	
  General	
  SPPS	
  protocol…………………………………………..…………………………	
    27	
    Scheme	
  2.2:	
  	
  Deprotection	
  of	
  Fmoc	
  under	
  basic	
  conditions………………………………….	
    28	
    Scheme	
  2.3:	
  	
  Installment	
  of	
  Fmoc-­‐Ach-­‐OH	
  onto	
  the	
  resin……………………………………..	
    33	
    Scheme	
  2.4:	
  	
  Reaction	
  of	
  DBU	
  with	
  Fmoc	
  to	
  produce	
  DBF…………………………………….	
    34	
    Scheme	
  2.5:	
  	
  Removal	
  of	
  Fmoc	
  protecting	
  group	
  of	
  resin	
  1	
  and	
  coupling	
  of	
  Fmoc-­‐ Aad(tBu)-­‐OH	
  onto	
  Ach…………………………………………..………………………………………………	
    36	
    Scheme	
  2.6:	
  	
  Removal	
  of	
  Fmoc	
  protecting	
  group	
  of	
  resin	
  2	
  and	
  coupling	
  of	
  Fmoc-­‐ Lys(Dde)-­‐OH	
  onto	
  Aad…………………………………………..………………………………………………	
    38	
    Scheme	
  2.7:	
  	
  Synthesis	
  of	
  2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
  acid……………………….	
    39	
    Scheme	
  2.8:	
  	
  Removal	
  of	
  Fmoc	
  protecting	
  group	
  of	
  resin	
  3	
  and	
  coupling	
  of	
  2-­‐(4-­‐ (3-­‐o-­‐tolylureido)	
  phenyl)acetic	
  acid	
  onto	
  Lys.	
  ……………………………………………………….	
    40	
    Scheme	
  2.9:	
  	
  Removal	
  of	
  Dde	
  protecting	
  group	
  of	
  resin	
  4	
  and	
  coupling	
  of	
  3-­‐(3-­‐ pyridyl)acrylic	
  acid	
  onto	
  the	
  side	
  chain	
  of	
  Lys.	
  ………………………………………………………	
    41	
    Scheme	
  2.10:	
  	
  Cleavage	
  of	
  resin	
  5	
  to	
  produce	
  LLP2A(tBu)	
  …………………………………….	
    42	
    Scheme	
  2.11:	
  	
  Synthesis	
  of	
  ArB(OR)2…………………………………………..…………………………	
    45	
    Scheme	
  2.12:	
  	
  Synthesis	
  of	
  ArB(OR)2-­‐LLP2A…………………………………………………………..	
    46	
    Scheme	
  2.13:	
  	
  Synthesis	
  of	
  5-­‐azido	
  pentanoic	
  acid………………………………………………..	
    47	
    Scheme	
  2.14:	
  	
  Synthesis	
  of	
  N3-­‐LLP2A…………………………………………………………………….	
    48	
    Scheme	
  2.15:	
  	
  Synthesis	
  of	
  ArBF3-­‐LLP2A	
  7	
  by	
  slow	
  fluorination	
  method……………….	
    50	
    Scheme	
  2.16:	
  	
  Synthesis	
  of	
  FITC-­‐LLP2A………………………………………………………………….	
    54	
    	
    ix	
    Scheme	
  2.17:	
  	
  Synthesis	
  of	
  alkynyl-­‐ArB18F3……………………………………………………………	
    60	
    Scheme	
  2.18:	
  	
  Synthesis	
  of	
  the	
  final	
  product	
  of	
  the	
  two-­‐step	
  radiolabeling	
  of	
   LLP2A…………………………………………………………………………………………………………………….	
   	
   	
    	
    61	
    	
    x	
    Abbreviations	
   	
   	
   α	
   	
   	
   β	
   	
   	
   +	
   	
   	
   β δ	
   	
   	
   λ	
   	
   	
   AA	
   	
   	
   ACN	
   	
   	
   ArBF3	
   	
   	
   Asc	
   	
   	
   BOS	
   	
   	
   BuLi	
   	
   	
   D	
   	
   	
   DBU	
   	
   	
   DBF	
   	
   	
   DCC	
   	
   	
   DCM	
   	
   	
   Dde	
   	
   	
   DIC	
   	
   	
   DIPEA	
   	
   	
   DMF	
   	
   	
   DMSO	
   	
   	
   +	
   	
   	
   e -­‐ e 	
   	
   	
   EDC	
   	
   	
   EDG	
   	
   	
   EOS	
   	
   	
   ESI	
   	
   	
   EtOH	
   	
   	
   eV	
   	
   	
   EWG	
   	
   	
   18 FDG	
   	
   	
   FITC	
   	
   	
   Fmoc	
   	
   	
   Fmoc-­‐Aad(tBu)-­‐OH	
   Fmoc-­‐Ach-­‐OH	
   	
   G	
   	
   	
   HATU	
   	
   	
   	
   	
   	
   HBTU	
   	
   	
   	
   	
   	
    	
    alpha	
   beta	
   positron	
   delta	
   lambda	
   amino	
  acid	
   acetonitrile	
   aryltrifluoroborate	
   ascorbate	
   beginning	
  of	
  synthesis	
   butyl	
  lithium	
   aspartic	
  acid	
   1,8-­‐diazabicycloundec-­‐7-­‐ene	
   dibenzofulvene	
   N,N’-­‐dicyclohexylcarbodiimide	
   dichloromethane	
   2-­‐acetyldimedone	
   N,N’-­‐diisopropylcarbodiimide	
   N,N-­‐diisopropylethylamine	
   dimethylformamide	
   dimethyl	
  sulfoxide	
   positron	
   electron	
   1-­‐ethyl-­‐3-­‐(3-­‐dimethylaminopropyl)carbodiimide	
   electron	
  donating	
  group	
   end	
  of	
  synthesis	
   electrospray	
  ionization	
   ethanol	
   electronvolt	
   electron	
  withdrawing	
  group	
   18 F-­‐2-­‐fluorodeoxyglucose	
   fluorescein	
  isothiocyanate	
   fluorenylmethyloxycarbonyl	
   Fmoc-­‐2-­‐aminoadipic	
  acid	
   	
   1-­‐(Fmoc-­‐amino)cyclohexanecarboxylic	
  acid	
   glycine	
   O-­‐(7-­‐azaBenzotriazole-­‐1-­‐yl)-­‐1,1,3,3-­‐tetramethyluronium	
  	
   hexafluorophosphate	
   O-­‐(Benzotriazole-­‐1-­‐yl)-­‐1,1,3,3-­‐tetramethyluronium	
  	
   hexafluorophosphate	
    xi	
    HCTU	
   	
   	
   	
   HFIP	
   	
   HOBt	
   	
   HRMS	
   	
   IC50	
   	
   J	
   	
   L	
   	
   LRMS	
   	
   Lys	
   	
   m/z	
   	
   MMP	
   	
   MRI	
   	
   MS	
   	
   NHS	
   	
   NIR	
   	
   NIRF	
   	
   NMR	
   	
   Nu	
   	
   o	
   	
   PET	
   	
   PG	
   	
   ppm	
   	
   R	
   	
   Rf	
   	
   RBF	
   	
   RP-­‐HPLC	
   RT	
   	
   SA	
   	
   SPECT	
   	
   SPPS	
   	
   t1/2	
   	
   tR	
   	
   TBS	
   	
   tBu	
   	
   TFA	
   	
   THF	
   	
   TLC	
   	
   UV	
   	
   V	
   	
   	
   	
   	
    	
    	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
   	
    	
   	
   	
   	
   	
   	
   	
   	
    O-­‐(6-­‐chlorobenzotriazole-­‐1-­‐yl)-­‐1,1,3,3-­‐tetramethyluronium	
  	
   hexafluorophosphate	
   hexafluoro-­‐2-­‐propanol	
   hydroxybenzotriazole	
   high	
  resolution	
  mass	
  spectrometry	
   half	
  maximal	
  inhibitory	
  concentration	
   coupling	
  constant	
   leucine	
   low	
  resolution	
  mass	
  spectrometry	
   lysine	
   mass-­‐to-­‐charge	
  ratio	
   matrix	
  metalloproteinase	
   magnetic	
  resonance	
  imaging	
   mass	
  spectrometry	
   N-­‐Hydroxysuccinimide	
   near	
  infrared	
   near	
  infrared	
  fluorescence	
  	
   nuclear	
  magnetic	
  resonance	
   nucleophile	
   ortho	
   positron	
  emission	
  tomography	
   protecting	
  group	
   parts	
  per	
  million	
   arginine	
   retardation	
  factor	
   round-­‐bottomed	
  flask	
   reverse-­‐phase	
  high	
  performance	
  liquid	
  chromatography	
   room	
  temperature	
   specific	
  activity	
   single	
  photon	
  emission	
  tomography	
   solid	
  phase	
  peptide	
  synthesis	
   half-­‐life	
   retention	
  time	
   tris	
  buffered	
  saline	
   tert-­‐butyl	
   trifluoroacetic	
  acid	
   tetrahydrofuran	
   thin	
  layer	
  chromatography	
   ultraviolet	
   valine	
   	
    xii	
    Acknowledgements	
   	
   	
    First	
   and	
   foremost,	
   I	
   would	
   like	
   to	
   express	
   my	
   gratitude	
   to	
   my	
   supervisor,	
   Dr.	
    David	
  M.	
  Perrin.	
  	
  His	
  passion	
  for	
  science	
  and	
  work	
  ethic	
  are	
  qualities	
  I	
  will	
  continue	
  to	
   admire	
   for	
   years	
   to	
   come.	
   	
   I	
   would	
   like	
   to	
   thank	
   Dr.	
   Perrin	
   for	
   his	
   encouragement,	
   scientific	
  insight,	
  and	
  helpful	
  teachings	
  over	
  the	
  course	
  of	
  this	
  project.	
   	
    I	
   would	
   like	
   to	
   thank	
   Dr.	
   Ying	
   Li,	
   formerly	
   of	
   the	
   Perrin	
   group,	
   for	
   all	
   of	
   her	
    leadership	
   and	
   assistance	
   with	
   this	
   project.	
   	
   I	
   would	
   like	
   to	
   thank	
   all	
   my	
   fellow	
   group	
   members	
  for	
  making	
  my	
  research	
  an	
  enjoyable	
  experience	
  and	
  allowing	
  my	
  education	
  at	
   UBC	
  to	
  include	
  more	
  than	
  only	
  scientific	
  studies.	
   	
    Most	
   of	
   all,	
   I	
   would	
   like	
   to	
   thank	
   my	
   family	
   for	
   providing	
   me	
   with	
   the	
   love,	
    kindness,	
   support,	
   and	
   foundation	
   necessary	
   to	
   pursue	
   my	
   education.  	
    xiii	
    Chapter	
  1	
   	
    	
   1.1	
    Introduction	
   Cancer	
  and	
  its	
  Imaging	
    	
   1.1.1	
   A	
  Brief	
  Overview	
  of	
  Cancer	
   	
   	
   Cancer	
  can	
  be	
  defined	
  as	
  a	
  group	
  of	
  cells	
  that	
  exhibit	
  uncontrolled	
  division.	
  	
  These	
   cells	
  often	
  metastasize,	
  invading	
  neighboring	
  tissue	
  and	
  spreading	
  to	
  other	
  parts	
  of	
  the	
   body	
   via	
   the	
   lymphatic	
   system	
   and	
   the	
   bloodstream.	
   	
   The	
   malignancy	
   may	
   grow	
   and	
   destroys	
  healthy	
  tissue	
  until	
  necessary	
  organs	
  are	
  unable	
  to	
  function,	
  which	
  proves	
  fatal	
   to	
  the	
  patient.	
  	
  Cancer	
  is	
  among	
  the	
  leading	
  causes	
  of	
  death	
  in	
  the	
  developed	
  world	
  and	
   the	
  second	
  leading	
  cause	
  of	
  death	
  in	
  developing	
  countries.1	
  	
  In	
  Canada,	
  29	
  %	
  of	
  deaths	
   annually	
  are	
  related	
  to	
  cancer.	
  	
  The	
  incidence	
  and	
  mortality	
  rates	
  for	
  cancer	
  in	
  Canada	
   have	
  both	
  recently	
  been	
  on	
  the	
  rise,	
  increasing	
  by	
  1.6	
  and	
  1.2	
  %,	
  respectively,	
  from	
  2009	
   to	
   2010.2	
   	
   A	
   disease	
   with	
   the	
   global	
   reach	
   of	
   that	
   of	
   cancer	
   is	
   rare	
   and	
   its	
   increasing	
   prevalence	
  is	
  cause	
  for	
  concern.	
  	
  In	
  light	
  of	
  this,	
  research	
  investigating	
  all	
  aspects	
  of	
  this	
   broad	
   disease	
   has	
   also	
   been	
   growing.	
   	
   Increased	
   insight	
   into	
   cancer’s	
   epidemiology,	
   diagnosis,	
  and	
  treatment	
  will	
  hopefully	
  give	
  rise	
  to	
  a	
  diminished	
  incidence	
  of	
  cancer	
  and	
   advancements	
  in	
  patient	
  care	
  in	
  the	
  near	
  future.	
   	
    There	
   are	
   many	
   ways	
   that	
   cancer	
   can	
   be	
   diagnosed,	
   usually	
   beginning	
   with	
   the	
    patient	
   noticing	
   signs	
   and	
   symptoms.	
   	
   If	
   cancer	
   is	
   suspected,	
   several	
   tests	
   are	
   done	
   before	
  making	
  further	
  conclusions,	
  including	
  imaging	
  and	
  blood	
  screens.	
  	
  The	
  diagnosis	
    	
    1	
    may	
  then	
  be	
  confirmed	
  by	
  a	
  histological	
  examination	
  of	
  the	
  neoplastic	
  tissue	
  performed	
   by	
  a	
  pathologist.	
  	
  Cancer	
  can	
  be	
  treated	
  by	
  a	
  variety	
  of	
  methods	
  ranging	
  from	
  surgery	
  to	
   radiation	
  therapy	
  to	
  chemotherapy,	
  among	
  others.	
  	
  To	
  many	
  people,	
  the	
  word	
  cancer	
  is	
   synonymous	
  with	
  death,	
  although	
  mortality	
  rates	
  differ	
  widely	
  among	
  various	
  types	
  of	
   cancer.2	
  	
   	
   1.1.2	
   Cancer	
  Imaging	
  Technology	
   	
   	
   An	
   aging	
   population	
   coupled	
   with	
   an	
   increasing	
   incidence	
   of	
   cancer	
   means	
   that	
   novel	
  and	
  effective	
  methods	
  of	
  diagnosing	
  and	
  treating	
  cancer	
  are	
  more	
  essential	
  now	
   than	
   ever.	
   	
   One	
   way	
   to	
   monitor	
   the	
   progression	
   of	
   cancer	
   is	
   via	
   techniques	
   that	
   can	
   create	
   a	
   visual	
   image	
   of	
   the	
   neoplasm	
   itself.	
   	
   The	
   most	
   common	
   imaging	
   techniques	
   used	
   today	
   are	
   magnetic	
   resonance	
   imaging	
   (MRI),	
   single	
   photon	
   emission	
   computed	
   tomography	
  (SPECT)	
  and	
  positron	
  emission	
  tomography	
  (PET).	
   	
    MRI	
  is	
  used	
  to	
  image	
  bodily	
  structures	
  without	
  the	
  use	
  of	
  ionizing	
  radiation,	
  which	
    minimizes	
   the	
   risk	
   to	
   the	
   patient.	
   	
   Rather,	
   it	
   employs	
   the	
   nuclear	
   magnetic	
   resonance	
   properties	
   of	
   the	
   atoms	
   that	
   make	
   up	
   the	
   tissue	
   and	
   also	
   uses	
   a	
   contrast	
   agent	
   to	
   improve	
   internal	
   visibility.	
   	
   These	
   contrast	
   agents	
   are	
   most	
   often	
   gadolinium	
   based.	
  	
   Unfortunately,	
  the	
  sensitivity	
  to	
  these	
  contrast	
  agents	
  is	
  low	
  and	
  this	
  has	
  prevented	
  MRI	
   from	
   being	
   considered	
   among	
   the	
   best	
   techniques	
   for	
   molecular	
   imaging	
   of	
   localized	
   and	
  microscopic	
  disease.3	
  	
  This	
  technique	
  is	
  also	
  not	
  suited	
  for	
  patients	
  with	
  pacemakers	
   or	
  certain	
  metallic	
  implants	
  due	
  to	
  the	
  strong	
  magnetic	
  field	
  that	
  is	
  employed.	
   	
   	
    	
   2	
    	
    SPECT	
  produces	
  images	
  via	
  the	
  emission	
  of	
  a	
  single	
  gamma	
  ray	
  that	
  is	
  measured	
    directly	
   by	
   the	
   detector.	
   	
   The	
   emission	
   energy	
   of	
   these	
   gamma	
   rays	
   is	
   too	
   high	
   to	
   be	
   absorbed	
  by	
  body	
  tissues,	
  ergo	
  decreasing	
  the	
  radiation	
  absorption	
  by	
  the	
  patient.	
  	
  Long	
   half-­‐life	
   isotopes	
   may	
   be	
   used	
   for	
   SPECT,	
   enabling	
   more	
   synthetic	
   freedom	
   and	
   time	
   for	
   distribution	
   of	
   labeled	
   compounds.	
   	
   This	
   also	
   extends	
   timeframes	
   for	
   imaging,	
   permitting	
   physicians	
   to	
   observe	
   in	
   vivo	
   processes	
   within	
   hours	
   of	
   or	
   even	
   days	
   after	
   injection.4,	
  5	
  	
  The	
  need	
  for	
  a	
  second	
  dose	
  of	
  radiotracer	
  is	
  also	
  eliminated,	
  should	
  follow-­‐ up	
  scans	
  be	
  required	
  shortly	
  after	
  the	
  initial	
  imaging.	
  	
  The	
  main	
  limitation	
  of	
  SPECT	
  is	
  its	
   sensitivity;	
  i.e.	
  the	
  percentage	
  of	
  emitted	
  photons	
  that	
  are	
  actually	
  detected	
  and	
  used	
  to	
   construct	
   an	
   image.	
   	
   This	
   arises	
   because	
   a	
   collimator	
   is	
   required	
   to	
   absorb	
   photons	
   that	
   are	
   outside	
   of	
   the	
   angular	
   range	
   of	
   the	
   detector,	
   causing	
   only	
   a	
   small	
   number	
   of	
   photons	
   to	
   reach	
   the	
   detector.	
   	
   Due	
   to	
   this	
   necessity,	
   the	
   efficiency	
   of	
   SPECT	
   is	
   approximately	
  0.01	
  %.5	
   	
    PET	
   is	
   able	
   to	
   produce	
   high	
   resolution,	
   three-­‐dimensional	
   images	
   of	
   cancerous	
    tissue	
  using	
  nuclides	
  that	
  emit	
  positrons.	
  	
  When	
  a	
  positron	
  is	
  emitted,	
  it	
  travels	
  a	
  short	
   distance	
   (the	
   positron	
   range)	
   until	
   it	
   loses	
   enough	
   energy	
   that	
   it	
   can	
   interact	
   with	
   an	
   electron.	
   	
   At	
   this	
   point,	
   a	
   collision	
   occurs,	
   resulting	
   in	
   annihilation	
   and	
   producing	
   two	
   gamma	
  photons	
  of	
  511	
  keV	
  each.	
  	
  These	
  two	
  photons	
  are	
  emitted	
  at	
  180°	
  to	
  each	
  other	
   and	
  are	
  detected	
  coincidently,	
  increasing	
  the	
  acceptable	
  detection	
  angle	
  and	
  eliminating	
   the	
  need	
  for	
  a	
  collimators.	
  	
  This	
  results	
  in	
  a	
  sensitivity	
  of	
  about	
  1	
  %.5,	
  6	
  	
  The	
  resolution	
  of	
   the	
  image	
  depends	
  on	
  the	
  distance	
  travelled	
  by	
  the	
  positron	
  prior	
  to	
  annihilation,	
  which	
   is	
  directly	
  proportional	
  to	
  positron	
  energy.	
  	
  By	
  labeling	
  a	
  cancer	
  targeting	
  biomolecule	
    	
    3	
    with	
   a	
   positron-­‐emitting	
   isotope,	
   this	
   decay	
   can	
   be	
   localized	
   and	
   an	
   image	
   generated.	
  	
   Although	
   PET	
   is	
   able	
   to	
   produce	
   some	
   of	
   the	
   highest	
   quality	
   molecular	
   images,	
   it	
   is	
   handicapped	
   by	
   the	
   high	
   production	
   cost	
   of	
   imaging	
   agents	
   and	
   the	
   scanners	
   themselves.7,	
  8	
  	
   18F  PET Tracer  +e positron range  +e  +  -e  511 keV  	
    18  -­‐  Figure	
  1.1:	
  	
  Positron	
  ( e)	
  emission	
  by	
   F	
  followed	
  by	
  collision	
  with	
  an	
  electron	
  ( e),	
  resulting	
  in	
   annihilation	
  of	
  both	
  particles	
  and	
  producing	
  two	
  gamma	
  rays	
  of	
  511	
  keV	
  each.	
    	
   1.1.3	
   PET	
  Isotopes	
  	
  	
   	
   	
   The	
   choice	
   of	
   isotope	
   for	
   PET	
   imaging	
   depends	
   on	
   several	
   properties:9	
   	
   1)	
   a	
   low	
   positron	
   energy	
   that	
   will	
   yield	
   a	
   short	
   positron	
   range	
   and	
   therefore	
   good	
   image	
   resolution,	
   2)	
   a	
   moderate	
   half-­‐life	
   that	
   allows	
   enough	
   time	
   for	
   incorporation	
   into	
   a	
   biomolecule	
  without	
  significant	
  loss	
  of	
  specific	
  activity	
  but	
  not	
  so	
  long	
  that	
  the	
  patient	
   experiences	
   harmful	
   radiation	
   absorption	
   post-­‐imaging,	
   3)	
   a	
   clean	
   decay	
   process	
   consisting	
  of	
  mostly	
  positron	
  decay,	
  and	
  4)	
  the	
  ease	
  of	
  production	
  of	
  the	
  radioisotope	
   and	
   its	
   intrinsic	
   chemical	
   properties	
   allowing	
   for	
   simple	
   and	
   reproducible	
   methods	
   of	
   incorporation.	
   	
    4	
    	
    There	
   are	
   several	
   isotopes	
   that	
   are	
   potentially	
   suitable	
   for	
   use	
   in	
   PET	
   imaging;	
    selected	
  isotopes	
  are	
  outlined	
  in	
  Table	
  1.1.	
  	
   11C	
  would	
  be	
  an	
  excellent	
  isotope	
  for	
  PET	
   imaging	
   due	
   to	
   its	
   high	
   specific	
   activity	
   and	
   percentage	
   of	
   β+	
   decay,	
   however	
   its	
   half-­‐life	
   is	
  often	
  too	
  short	
  for	
  synthesis	
  and	
  distribution.9	
  	
   64Cu	
  and	
   68Ga	
  are	
  of	
  interest	
  because	
   these	
  can	
  be	
  easily	
  incorporated	
  into	
  biomolecules	
  through	
  the	
  use	
  of	
  chelators.	
  	
  Simply	
   mixing	
   the	
   imaging	
   compound	
   in	
   a	
   solution	
   of	
   the	
   metal	
   is	
   enough	
   to	
   almost	
   fully	
   incorporate	
  it.	
  	
  However,	
   64Cu	
  does	
  not	
  exhibit	
  a	
  clean	
  decay	
  process	
  which	
  results	
  in	
  a	
   low	
  percentage	
  of	
  β+	
  decay	
  and	
  thus	
  requires	
  longer	
  acquisition	
  times10.	
  	
  68Ga	
  has	
  a	
  high	
   positron	
   emission	
   energy,	
   consequently	
   decreasing	
   the	
   resolution	
   of	
   the	
   resulting	
   images.	
   	
   All	
   things	
   considered,	
   it	
   becomes	
   apparent	
   that	
   18F	
   is	
   the	
   ideal	
   isotope	
   to	
   be	
   used	
  in	
  PET	
  imaging	
  due	
  to	
  its	
  low	
  positron	
  energy,	
  clean	
  decay	
  process	
  and	
  moderate	
   half-­‐life.9,	
   11	
   	
   In	
   light	
   of	
   these	
   properties,	
   18F	
   has	
   become	
   the	
   most	
   commonly	
   used	
   isotope	
  for	
  PET	
  imaging,	
  employed	
  in	
  the	
  form	
  of	
  18F-­‐2-­‐fluorodeoxyglucose	
  (18FDG).	
   	
   	
   9  Table	
  1.1:	
  	
  Properties	
  of	
  several	
  possible	
  isotopes	
  for	
  use	
  in	
  PET	
  imaging. 	
    t1/2	
   (min)	
    SA	
   (Ci/μmol)	
    Decay	
   (%	
  β+)	
    Cu	
    768	
    245	
    Ga	
    68	
    Nuclide	
   64 68  11  C	
    18  	
    F	
    β+	
  energy	
  (MeV)	
    β	
  +	
  range	
  in	
  Water	
   (mm)	
    Max.	
    Mean	
    Max.	
    Mean	
    17.9	
    0.65	
    0.28	
    2.9	
    0.64	
    2766	
    87.7	
    1.90	
    0.84	
    8.2	
    2.9	
    20	
    9220	
    99.8	
    0.96	
    0.39	
    4.1	
    1.1	
    110	
    1710	
    96.7	
    0.63	
    0.25	
    2.4	
    0.6	
    5	
    1.1.4	
   18F	
  Production	
   	
   18 	
   F	
   can	
   be	
   produced	
   in	
   different	
   ways,	
   depending	
   on	
   whether	
   it	
   is	
   to	
   be	
   incorporated	
   into	
   the	
   biomolecule	
   as	
   an	
   electrophile	
   or	
   as	
   a	
   nucleophile.	
   	
   For	
   electrophilic	
   incorporation,	
   18O2	
   gas	
   is	
   converted	
   to	
   18F2	
   by	
   proton	
   bombardment.11	
  	
   Elemental	
   fluorine	
   is	
   highly	
   reactive,	
   providing	
   a	
   rapid	
   labeling	
   reaction	
   that	
   limits	
   radioactive	
  decay	
  and	
  loss	
  of	
  specific	
  activity.	
  	
  This	
  reactivity,	
  as	
  well	
  as	
  its	
  gaseous	
  state	
   makes	
   18F2	
  difficult	
  to	
  prepare	
  and	
  handle,	
  resulting	
  in	
  electrophilic	
  incorporation	
  being	
   rarely	
  used.	
  	
  In	
  addition,	
  electrophilic	
  fluorine	
  is	
  produced	
  at	
  low	
  specific	
  activity.	
  	
  The	
   preferred	
   method	
   is	
   nucleophilic	
   incorporation.	
   	
   Here,	
   H218O	
   is	
   bombarded	
   with	
   a	
   proton	
   beam	
   to	
   produce	
   18F-­‐.	
   	
   The	
   18F	
   anion	
   is	
   then	
   separated	
   out	
   using	
   an	
   anion	
   exchange	
   column	
   and	
   usually	
   stabilized	
   by	
   adding	
   a	
   crown	
   ether,	
   2,2,2-­‐cryptand,	
   and	
   eluted.11	
   	
   This	
   18F	
   anion	
   can	
   then	
   be	
   used	
   to	
   radiolabel	
   biomolecules	
   by	
   nucleophilic	
   displacement	
  reactions.	
   	
   1.1.5	
   	
   	
    18  18  FDG	
    FDG	
   is	
   an	
   analog	
   of	
   2-­‐deoxyglucose	
   on	
   which	
   an	
   18F	
   atom	
   is	
   installed	
   at	
   the	
   2’	
    position	
   instead	
   of	
   the	
   normal	
   hydroxyl	
   group	
   present	
   in	
   glucose.	
   	
   18FDG	
   is	
   injected	
   into	
   the	
   bloodstream	
   of	
   a	
   patient	
   and	
   is	
   then	
   imported	
   into	
   cells	
   via	
   specific	
   transporters	
   on	
   the	
   cell	
   membrane.	
   	
   It	
   then	
   undergoes	
   phosphorylation	
   within	
   the	
   cytosol	
   at	
   the	
   C-­‐6	
   position	
   by	
   the	
   enzyme	
   hexokinase.12	
   	
   There	
   are	
   two	
   reasons	
   why	
   this	
   is	
   useful	
   for	
   imaging	
  purposes.	
  	
  First,	
  cancer	
  cells	
  are	
  rapidly	
  growing	
  and	
  exhibit	
  accelerated	
  glucose	
   metabolism	
  compared	
  to	
  normal	
  cells,	
  causing	
   18FDG	
  to	
  become	
  more	
  concentrated	
  in	
   	
    6	
    cancerous	
   tissue.	
   	
   Second,	
   due	
   to	
   the	
   absent	
   2’	
   hydroxyl	
   group,	
   the	
   18FDG	
   cannot	
   be	
   metabolized	
   further.	
   	
   Following	
   phosphorylation	
   at	
   the	
   C-­‐6	
   position,	
   the	
   molecule	
   is	
   charged	
   and	
   must	
   remain	
   in	
   the	
   cell.7,	
  13	
   	
   With	
   the	
   18FDG	
   localized	
   and	
   trapped	
   within	
   the	
   cancer	
   cells,	
   an	
   image	
   may	
   be	
   produced	
   in	
   which	
   malignant	
   tissue	
   is	
   identified	
   by	
   locating	
  the	
  areas	
  of	
  concentrated	
  radioactive	
  decay.	
   OH HO HO  O 18F  OH 	
   18  18  Figure	
  1.2:	
  	
  The	
  structure	
  of	
   FDG.	
  	
  The	
  2’-­‐OH	
  of	
  glucose	
  is	
  replaced	
  with	
   F	
  to	
  allow	
  for	
  PET	
  imaging.	
    	
   1.1.6	
   Tumor-­‐Specific	
  Imaging	
   	
   	
   While	
   the	
   use	
   of	
   18FDG	
   to	
   image	
   cancer	
   is	
   an	
   extraordinary	
   breakthrough	
   in	
   diagnostic	
   nuclear	
   medicine,	
   it	
   has	
   several	
   drawbacks.	
   	
   It	
   is	
   often	
   difficult	
   to	
   visualize	
   malignant	
  tissue	
  contained	
  within	
  areas	
  of	
  the	
  body	
  that	
  have	
  a	
  naturally	
  high	
  glucose	
   uptake,	
  such	
  as	
  the	
  brain,	
  myocardium,	
  muscles,	
  and	
  urinary	
  tract.7,	
  12	
  	
  Therefore,	
  18FDG	
   is	
  only	
  useful	
  for	
  imaging	
  once	
  the	
  tumor	
  has	
  reached	
  a	
  minimum	
  size	
  to	
  allow	
  sufficient	
   18  FDG	
   accumulation,	
   permitting	
   suitable	
   differentiation	
   against	
   the	
   background.	
  	
    Perhaps	
  the	
  biggest	
  drawback	
  of	
   18FDG	
  is	
  that	
  it	
  is	
  rather	
  non-­‐specific;	
  it	
  does	
  not	
  help	
   elucidate	
  any	
  information	
  about	
  patient-­‐specific	
  tumor	
  biology.9	
  	
   	
    By	
  using	
  radiopharmaceuticals	
  that	
  are	
  specific	
  to	
  different	
  types	
  of	
  cancer,	
  more	
    specific	
   phenotypes	
   may	
   be	
   characterized.14	
   	
   This	
   allows	
   PET	
   imaging	
   to	
   not	
   only	
   be	
   useful	
   for	
   imaging	
   the	
   tumor,	
   but	
   for	
   choosing	
   the	
   appropriate	
   treatment	
   option	
   by	
   	
    7	
    offering	
   information	
   about	
   tumor	
   aggressiveness	
   and	
   proliferation.	
   	
   By	
   diagnosing	
   cancerous	
   tissue	
   earlier	
   and	
   more	
   specifically,	
   the	
   survival	
   rate	
   of	
   the	
   patient	
   is	
   increased	
   and	
   the	
   overall	
   cost	
   of	
   care	
   is	
   decreased.2	
   	
   In	
   order	
   to	
   provide	
   this	
   specific	
   imaging,	
   radiolabeled	
   biomolecules	
   such	
   as	
   peptides	
   or	
   oligonucleotides	
   that	
   exhibit	
   a	
   high	
   affinity	
   for	
   extracellular	
   markers	
   present	
   in	
   the	
   early	
   stages	
   of	
   specific	
   cancers	
   could	
  be	
  used.	
  	
   OH O  HO  O N H NH  HO  O HN  O NH O  HN  O  H N  N H  O  N H  O  O O N H  N  NH  N  N  NH2  NH NH H2N  N H  O  H N O  N H S S O  HO  H N O OH  N H  H N  NH O  O  HN H N O  NH2  OH  	
   15  Figure	
   1.3:	
  	
  Select	
  peptides	
  currently	
  under	
  investigation	
  for	
  molecular	
  imaging	
  purposes.	
  	
  RGD 	
  (top	
  left),	
   16  17  folic	
  acid 	
  (top	
  right),	
  and	
  octreotide 	
  (bottom)	
  are	
  shown.	
  	
  A	
  radioactive	
  imaging	
  moiety	
  would	
  be	
   attached	
  at	
  the	
  interrupted	
  bond	
  to	
  enable	
  PET	
  imaging.	
    	
   	
    A	
   covalent	
   bond	
   between	
   the	
   18F	
   atom	
   and	
   the	
   tumor-­‐targeting	
   biomolecule	
   is	
    necessary	
  for	
  PET	
  imaging	
  since	
  the	
  radioisotope	
  must	
  remain	
  anchored	
  to	
  the	
  imaging	
   agent	
   at	
   all	
   times	
   in	
   order	
   to	
   produce	
   a	
   clear,	
   high	
   resolution	
   image.	
   	
   Cleavage	
   of	
   the	
  	
    	
    8	
    18  F-­‐biomolecule	
   bond	
   will	
   result	
   in	
   the	
   free	
   radionuclide	
   itself	
   being	
   tracked	
   by	
   the	
    imaging	
   device,	
   as	
   opposed	
   to	
   nuclide-­‐biomolecule	
   conjugate.	
   	
   In	
   the	
   absence	
   of	
   the	
   biomolecule,	
  the	
  radionuclide	
  has	
  no	
  ability	
  to	
  localize	
  at	
  the	
  malignancy,	
  thus	
  defeating	
   the	
   purpose	
   of	
   the	
   imaging	
   scan.	
   	
   To	
   form	
   a	
   covalent	
   18F-­‐C	
   bond,	
   as	
   found	
   in	
   18FDG,	
   harsh	
  reaction	
  conditions	
  including	
  high	
  temperatures	
  and	
  aprotic,	
  organic	
  solvents	
  are	
   usually	
   required	
   in	
   order	
   to	
   increase	
   the	
   nucleophilicity	
   of	
   the	
   18F	
   anion.11,	
   18	
   	
   These	
   conditions	
   are	
   generally	
   not	
   compatible	
   with	
   biomolecules,	
   which	
   normally	
   must	
   be	
   stored	
   under	
   mild	
   physiological	
   conditions,	
   and	
   therefore	
   could	
   possibly	
   lead	
   to	
   an	
   alteration	
  of	
  their	
  biological	
  activity	
  and	
  loss	
  of	
  imaging	
  functionality.	
  	
  	
   	
    Despite	
   these	
   concerns,	
   studies	
   on	
   direct	
    18  F-­‐labeling	
   of	
   biomolecules	
   have	
    appeared	
   in	
   the	
   literature.19-­‐21	
   	
   Chen	
   et	
   al.	
   have	
   recently	
   reported	
   a	
   one-­‐step	
   18F-­‐ labeling	
   strategy	
   that	
   involves	
    18  F-­‐C	
   bond	
   formation	
   via	
   aromatic	
   nucleophilic	
    displacement.21	
   	
   Activation	
   of	
   a	
   nitro	
   group	
   by	
   an	
   ortho	
   trifluoromethyl	
   substituent	
   provides	
   sufficient	
   electron	
   withdrawing	
   properties	
   to	
   allow	
   nucleophilic	
   substitution	
   of	
   NO2	
  by	
  fluoride	
  at	
  high	
  temperature.	
   	
   	
   O2N H N  F3C  K[18F]-Kryptofix DMSO  Peptide  130 °C  18F  H N  F3C O  O  Peptide  	
    18  Scheme	
  1.1:	
  	
  One-­‐step	
   F-­‐labeling	
  strategy	
  proposed	
  by	
  Chen	
  et	
  al.	
    	
   	
   	
    9	
    	
    This	
   method	
   was	
   tested	
   by	
   incorporating	
   the	
   nitro-­‐arene	
   moiety	
   into	
   the	
   cyclic	
    RGD	
   peptide	
   (shown	
   in	
   Figure	
   1.3),	
   a	
   well-­‐known	
   ligand	
   for	
   the	
   α5β3	
   integrin.	
  	
   Predictably,	
   Chen	
   et	
   al.	
   observe	
   that	
   the	
   high	
   temperature	
   (130	
   °C)	
   of	
   the	
   labeling	
   reaction	
   resulted	
   in	
   decomposition	
   of	
   the	
   peptide,	
   as	
   is	
   evident	
   in	
   the	
   RP-­‐HPLC	
   traces	
   (Figure	
  1.4).	
  	
  Not	
  only	
  are	
  there	
  several	
   18F-­‐labeled	
  products,	
  unlabeled	
  peaks	
  are	
  also	
   observed	
  by	
  UV	
  detection	
  which	
  indicates	
  the	
  breakdown	
  of	
  the	
  peptide	
  at	
  a	
  number	
  of	
   positions.	
   	
   Significant	
   difficulty	
   in	
   separating	
   the	
   18F-­‐labeled	
   products	
   from	
   the	
   nitro	
   precursors	
   is	
   also	
   mentioned.	
   	
   This	
   method	
   is	
   an	
   interesting	
   study	
   of	
   a	
   one-­‐step	
   18F-­‐ labeling	
   for	
   use	
   in	
   PET	
   imaging,	
   but	
   the	
   aforementioned	
   drawbacks	
   demonstrate	
   the	
   necessity	
  of	
  an	
  alternate	
  route	
  for	
  viable	
  18F-­‐labeling	
  of	
  biomolecules. 	
   	
    	
   18  Figure	
   1.4:	
  	
  RP-­‐HPLC	
  traces	
  of	
  the	
  one-­‐step	
   F-­‐labeling	
  of	
  RGD	
  via	
  nucleophilic	
  aromatic	
  substitution	
  by	
   21  Chen	
  et	
  al. 	
  	
  The	
  arrow	
  indicates	
  the	
  desired	
  product.	
  	
  Reproduced	
  from	
  Chen	
  et	
  al.	
  w/o	
  permission.	
    	
   	
   	
    10	
    1.1.7	
   Alternative	
  18F-­‐Labeling	
  Strategies	
   	
   	
   Current	
   18F-­‐labeling	
  strategies	
  are	
  focused	
  on	
  first	
  labeling	
  a	
  synthon,	
  in	
  which	
  an	
   activated	
   group	
   is	
   exchanged	
   for	
   18F	
   via	
   nucleophilic	
   displacement,	
   then	
   undergoing	
   further	
   reaction	
   to	
   conjugate	
   the	
   snython	
   to	
   the	
   bioactive	
   moiety.	
   	
   This	
   allows	
   the	
   biomolecule	
   to	
   bypass	
   the	
   harsh	
   conditions	
   required	
   to	
   complete	
   a	
   nucleophilic	
   substitution	
   reaction	
   using	
   18F.	
   	
   An	
   example	
   of	
   this	
   strategy	
   for	
   labeling	
   antibodies	
   performed	
  by	
  Zalutsky22	
  is	
  shown	
  in	
  Scheme	
  1.2	
  below.	
   	
   18F  K[18F]-Kryptofix DMSO  N  150 °C 8 min  OH  150 °C 3 min  O  O  18F  KMnO4 NaOH  O NHS DCC  18F  H2N H N  18F  O O  25 °C 15 min  O 18  THF 25 °C 3 min  N  O O  	
    18 -­‐  Scheme	
  1.2:	
  	
  A	
  multi-­‐step	
   F-­‐labeling	
  scheme	
  employing	
   F 	
  in	
  a	
  nucleophilic	
  substitution	
  reaction,	
   followed	
  by	
  NHS	
  ester	
  formation	
  and	
  subsequent	
  antibody	
  coupling.	
    	
    	
    	
    The	
   main	
   drawback	
   with	
   this	
   method	
   is	
   the	
   number	
   of	
   steps,	
   and	
   ensuing	
    purifications	
   that	
   are	
   required.	
   The	
   purity	
   of	
   any	
   radio-­‐imaging	
   agent	
   is	
   of	
   the	
   utmost	
   importance,	
   meaning	
   purification	
   must	
   achieve	
   >95	
   %	
   purity.	
   	
   Multi-­‐step	
   reaction	
   schemes	
  and	
  lengthy	
  purification	
  to	
  prepare	
  a	
  sample	
  for	
  PET	
  imaging	
  causes	
  a	
  decrease	
    	
    11	
    in	
  specific	
  activity	
  due	
  to	
  the	
  radioactive	
  decay	
  occurring	
  simultaneously.	
  	
  This,	
  in	
  turn,	
   yields	
   a	
   diminished	
   image	
   quality	
   and	
   necessitates	
   a	
   higher	
   dosage	
   of	
   the	
   drug.	
   	
   For	
   these	
   reasons,	
   click	
   chemistry	
   has	
   recently	
   become	
   of	
   interest	
   to	
   the	
   PET	
   labeling	
   community23-­‐26	
   due	
   to	
   its	
   robust	
   nature,	
   mild	
   conditions	
   and	
   complete	
   conversion.27	
  	
   The	
   Huisgen	
   cycloaddition,	
   the	
   premier	
   click	
   reaction,	
   provides	
   a	
   facile	
   method	
   for	
   connecting	
  a	
  radiolabeled	
  synthon	
  with	
  a	
  biomolecule	
  through	
  the	
  formation	
  of	
  a	
  1,2,3-­‐ triazole	
   by	
   1,3-­‐dipolar	
   cycloaddition	
   between	
   an	
   alkyne	
   and	
   an	
   azide.	
   	
   A	
   typical	
   click	
   radiolabeling	
  strategy	
  is	
  shown	
  below.	
   	
   O K[18F]-Kryptofix  OTos  CH3CN 100 °C 10 min  18F  Peptide  O N3  CuSO4 Ascorbate  Peptide  N  N N 18F  	
    Scheme	
  1.3:	
  	
  Two-­‐step	
  biomolecule	
  radiolabeling	
  via	
  click	
  conjugation.	
    	
   	
    Although	
   the	
   click	
   strategy	
   increases	
   simplicity	
   by	
   minimizing	
   the	
   number	
   of	
   steps	
    and	
  purification,	
  there	
  remains	
  room	
  for	
  improvement.	
  	
  The	
  ideal	
  biomolecule	
  labeling	
   reaction	
   would	
   be	
   a	
   one-­‐step	
   reaction	
   that	
   takes	
   place	
   under	
   mild	
   conditions	
   and	
   affords	
   simple	
   purification	
   with	
   minimal	
   byproducts.	
   	
   A	
   method	
   able	
   to	
   fulfill	
   these	
   criteria	
   could	
   push	
   the	
   boundaries	
   of	
   PET	
   image	
   technology	
   further	
   than	
   previously	
   thought	
  possible.	
   	
   	
   	
    	
    12	
    1.2	
    Arylborates	
  as	
  Fluoride	
  Captors	
    	
   1.2.1	
  	
   Advantages	
  of	
  Trifluoroborates	
  	
   	
   	
   It	
   has	
   been	
   shown	
   that	
   trifluoroborates	
   can	
   be	
   synthesized	
   from	
   boronic	
   acids	
   under	
  mild	
  aqueous	
  conditions.28	
  	
  In	
  accord	
  with	
  these	
  findings,	
  Perrin	
  and	
  colleagues	
   have	
   hypothesized	
   that	
   fluoride	
   capture	
   by	
   boron	
   may	
   be	
   applicable	
   to	
   radiopharmaceuticals.29	
   	
   Perrin	
   et	
   al.	
   were	
   able	
   to	
   show	
   18F	
   incorporation	
   into	
   an	
   aryl	
   moiety	
   through	
   the	
   formation	
   of	
   an	
   18F-­‐aryltrifluoroborate.	
   The	
   advantages	
   to	
   this	
   strategy	
   are	
   threefold.	
   	
   By	
   labeling	
   a	
   biomolecule	
   in	
   one	
   step,	
   the	
   number	
   of	
   side	
   products	
  may	
  be	
  greatly	
  reduced,	
  decreasing	
  purification	
  times	
  and	
  thereby	
  increasing	
   yield.	
   	
   Furthermore,	
   a	
   rapid,	
   one-­‐step	
   reaction	
   favors	
   the	
   medium	
   length	
   half	
   life	
   of	
   18F,	
   allowing	
   bioconjugates	
   of	
   good	
   specific	
   activity	
   to	
   be	
   produced.	
   	
   Lastly,	
   three	
   fluoride	
   atoms	
  are	
  incorporated	
  in	
  a	
  trifluoroborate,	
  in	
  contrast	
  to	
  one	
  fluoride	
  seen	
  in	
  current	
   labeling	
   strategies.	
   	
   By	
   incorporating	
   three	
   fluoride	
   atoms	
   onto	
   a	
   single	
   molecule	
   the	
   specific	
   activity	
   is	
   tripled,	
   allowing	
   the	
   synthesis	
   of	
   compounds	
   attaining	
   previously	
   impossible	
  specific	
  activities	
  and	
  yielding	
  greatly	
  enhanced	
  images.	
  	
  	
   	
    Another	
  attraction	
  of	
  fluoride	
  capture	
  via	
  boron,	
  which	
  plays	
  to	
  the	
  economics	
  of	
    PET	
  imaging,	
  is	
  the	
  fact	
  that	
  these	
  boronate-­‐biomolecule	
  conjugates	
  could	
  be	
  premade	
   and	
   stored	
   on-­‐site	
   prior	
   to	
   labeling.	
   	
   One	
   could	
   envision	
   the	
   delivery	
   of	
   aqueous	
   18F	
   which	
   could	
   then	
   be	
   reacted	
   with	
   the	
   bioconjugate	
   using	
   a	
   simple	
   aqueous	
   wash-­‐in	
   immediately	
   prior	
   to	
   injection	
   into	
   the	
   patient.	
   	
   The	
   ability	
   to	
   produce	
   boron	
   bioconjugates	
  for	
  imaging	
  different	
  types	
  of	
  cancer	
  in	
  a	
  “kit-­‐like”	
  fashion	
  could	
  lead	
  to	
   decreased	
   operating	
   costs	
   for	
   hospitals	
   and	
   radiopharmaceutical	
   companies	
   alike.	
   	
   This,	
   	
    13	
    in	
   turn,	
   will	
   make	
   PET	
   imaging	
   technology	
   more	
   widely	
   available	
   to	
   those	
   who	
   might	
   otherwise	
  not	
  have	
  access	
  to	
  this	
  era’s	
  optimum	
  cancer	
  imaging	
  technique.	
   	
   PG O  R  PG B  O KH18F2/HCl  Biomolecule  	
    F  F B  F  R  Biomolecule  	
    ArBF3	
    Scheme	
  1.4:	
  	
  General	
  scheme	
  of	
  fluoride	
  capture	
  by	
  a	
  protected	
  arylborate	
  to	
  produce	
  an	
   aryltrifluoroborate	
  (ArBF3).	
    	
   1.2.2	
   Stability	
  of	
  Aryltrifluoroborates	
   	
   	
   As	
   mentioned	
   previously,	
   it	
   is	
   important	
   that	
   the	
   18F	
   atom	
   does	
   not	
   dissociate	
   from	
  the	
  biomolecule	
  on	
  the	
  imaging	
  timescale.	
  	
  For	
  this	
  reason,	
  the	
  stability	
  of	
  several	
   aryltrifluoroborates	
  was	
  studied	
  by	
  Dr.	
  Richard	
  Ting,	
  previously	
  of	
  the	
  Perrin	
  group.30,	
  31	
   It	
   is	
   proposed	
   that	
   decomposition	
   of	
   aryltrifluoroborates	
   under	
   aqueous	
   conditions	
   would	
   first	
   see	
   the	
   elimination	
   of	
   one	
   fluoride	
   atom,	
   bringing	
   the	
   boron	
   to	
   a	
   neutral	
   charge	
   with	
   sp2	
   hybridization.	
   	
   This	
   is	
   followed	
   by	
   nucleophilic	
   attack	
   of	
   a	
   water	
   molecule	
  at	
  the	
  vacant	
  p	
  orbital.	
  	
  Another	
  water	
  molecule	
  is	
  added	
  and	
  the	
  process	
  is	
   repeated,	
   concluding	
   in	
   the	
   formation	
   of	
   a	
   boronic	
   acid	
   and	
   complete	
   solvolytic	
   liberation	
  of	
  the	
  remaining	
  fluorides.	
   	
    	
    14	
    F  F B  F  F  B  F  H2O  +  R  HF  F  B  OH  H2O  HF  HO  B  OH  F  R  R  R  	
    	
   Scheme	
  1.5:	
  	
  Mechanism	
  of	
  hydrolysis	
  of	
  an	
  aryltrifluoroborate.	
  	
  R	
  group	
  represents	
  bioactive	
  moiety	
   such	
  as	
  a	
  peptide	
  or	
  antibody.	
    	
    	
    	
    The	
   effect	
   of	
   electron	
   withdrawing	
   groups	
   on	
   aryltrifluoroborate	
   hydrolysis	
   was	
    investigated	
   by	
   the	
   Perrin	
   group	
   using	
   a	
   Hammet	
   analysis.31	
   	
   The	
   results	
   of	
   the	
   experiments	
  suggested	
  that	
  electron	
  withdrawing	
  groups	
  (EWGs)	
  in	
  the	
  para	
  and	
  meta	
   position	
  to	
  the	
  trifluoroborate	
  group	
  slowed	
  the	
  defluorination	
  while	
  electron	
  donating	
   groups	
   (EDGs)	
   in	
   the	
   para	
   position	
   enhanced	
   the	
   rate	
   of	
   solvolysis	
   of	
   aryltrifluoroborates.	
   	
   Rationalization	
   of	
   this	
   can	
   be	
   achieved	
   through	
   observing	
   charge	
   localization	
   of	
   the	
   resonance	
   structures	
   of	
   aryl	
   rings	
   substituted	
   with	
   electron	
   withdrawing	
   and	
   electron	
   donating	
   groups.	
   	
   EWGs	
   are	
   able	
   to	
   stabilize	
   the	
   negatively	
   charged	
   boron	
   of	
   the	
   trifluoroborate	
   by	
   localizing	
   a	
   partial	
   positive	
   charge	
   on	
   the	
   adjacent	
  carbon,	
  resulting	
  in	
  slower	
  B-­‐F	
  solvolysis.	
   	
   	
   	
   	
   	
   	
   	
    15	
    stabilizing	
   F  O  F B  N  destabilizing	
    F  F  O  O  F B  N  F  O  F  F B  O  N  O  F B  F  O  O  destabilizing	
   F δ+ F B  F  F  stabilizing	
   F δ+ F B  F δ+ F B  N  O  O  O  F  B  F  O  	
  	
    	
   Figure	
  1.5:	
  	
  Charge	
  distribution	
  of	
  electron	
  donating	
  and	
  withdrawing	
  substituents	
  on	
  aryltrifluoroborates	
   leading	
  to	
  increased	
  or	
  decreased	
  rate	
  of	
  fluoride	
  dissociation.	
    	
    	
    	
    EDGs	
   have	
   the	
   opposite	
   effect.	
   	
   Accumulation	
   of	
   electron	
   density	
   on	
   the	
   carbon	
    adjacent	
   to	
   the	
   negatively	
   charged	
   trifluoroborate	
   destabilizes	
   the	
   trifluoroborate	
   and	
   enhances	
  the	
  rate	
  of	
  fluoride	
  dissociation.	
  	
  Loss	
  of	
  fluoride	
  from	
  the	
  aryltrifluoroborate	
   yields	
   an	
   aryldifluoroborate	
   in	
   which	
   the	
   boron	
   is	
   sp2	
  hybridized	
   with	
   an	
   empty	
   p	
   orbital	
   (see	
   Scheme	
   1.5).	
   	
   This	
   aryldifluoroborate	
   is	
   further	
   stabilized	
   by	
   EDGs,	
   which	
   can	
   distribute	
   electron	
   density	
   into	
   the	
   empty	
   p	
   orbital.	
   	
   EWGs	
   would	
   again	
   have	
   the	
   opposite	
   effect,	
   destabilizing	
   the	
   aryldifluoroborate	
   by	
   localizing	
   positive	
   charge	
   next	
   to	
   the	
   vacant	
   p	
   orbital	
   of	
   boron.	
   	
   Altogether,	
   EWGs	
   stabilize	
   the	
   aryltrifluoroborates	
   and	
   destabilize	
   the	
   transition	
   state	
   for	
   solvolysis	
   that	
   would	
   be	
   similar	
   to	
   the	
   aryldifluoroborate	
  ground	
  state.	
  	
  This	
  leads	
  to	
  slower	
  aryltrifluoroborate	
  solvolysis	
  and,	
   	
    16	
    importantly,	
   a	
   more	
   stable	
   imaging	
   agent.	
   	
   Out	
   of	
   several	
   commercially	
   available	
   aryl	
   boronic	
  acids,	
  it	
  was	
  found	
  that	
  ortho	
  and	
  para	
  fluorine-­‐substituted	
  aryltrifluoroborate	
   exhibited	
  a	
  significantly	
  decreased	
  rate	
  of	
  aryltrifluoroborate	
  solvolysis.	
   	
   	
    A	
   conjugatable	
   handle	
   is	
   also	
   required	
   to	
   link	
   the	
   aryltrifluoroborate	
   to	
   the	
    biomolecule.	
   	
   In	
   this	
   case,	
   a	
   carboxylic	
   acid	
   would	
   be	
   useful	
   due	
   to	
   its	
   facile	
   coupling	
   through	
   amide	
   bond	
   formation,	
   as	
   well	
   as	
   its	
   electron	
   withdrawing	
   properties	
   which	
   provide	
   further	
   stabilization	
   of	
   the	
   B-­‐F	
   bond.	
   	
   By	
   placing	
   each	
   of	
   these	
   electron	
   withdrawing	
   groups	
   on	
   the	
   aryl	
   ring	
   it	
   was	
   hypothesized	
   that	
   this	
   species	
   would	
   be	
   capable	
  of	
  stabilizing	
  the	
  negative	
  charge	
  of	
  a	
  trifluoroborate.	
   	
   HO  B  F  OH F OH  F  O  	
    Figure	
  1.6:	
  	
  Structure	
  of	
  the	
  boronic	
  acid	
  to	
  be	
  used	
  for	
  fluoride	
  labeling	
  studies.	
    	
   	
   1.2.3	
   Boronic	
  Acid	
  Protecting	
  Groups	
   	
   	
   In	
   order	
   to	
   prevent	
   unwanted	
   chemistry	
   from	
   occurring	
   at	
   the	
   boronic	
   acid	
   site	
   during	
  the	
  coupling	
  of	
  the	
  imaging	
  moiety	
  to	
  the	
  biomolecule,	
  a	
  protecting	
  group	
  is	
  also	
   needed.	
   	
   The	
   protecting	
   group	
   needs	
   to	
   be	
   acid-­‐labile	
   such	
   that	
   removal	
   occurs	
   simultaneously	
   with	
   the	
   radiolabeling,	
   while	
   also	
   preferably	
   protecting	
   both	
   hydroxyl	
   groups	
   on	
   the	
   boron.	
   	
   Taking	
   into	
   account	
   these	
   conditions,	
   the	
   Perrin	
   group	
   	
    17	
    investigated	
  pinacol	
  and	
  1,1,2,2-­‐tetraphenyl	
  pinacol	
  as	
  possible	
  protecting	
  groups.	
  	
  The	
   C-­‐B	
   bond	
   was	
   found	
   to	
   be	
   unstable	
   in	
   basic	
   conditions	
   when	
   pinacol	
   was	
   employed	
   as	
   a	
   protecting	
  group.	
  	
  It	
  was	
  hypothesized	
  that	
  base-­‐mediated	
  deboronation	
  occurred	
  when	
   the	
   boron	
   adopted	
   a	
   tetrahedral	
   geometry.	
   	
   In	
   order	
   to	
   counteract	
   this,	
   1,1,2,2-­‐ tetraphenyl	
   pinacol	
   was	
   used	
   to	
   add	
   steric	
   bulk,	
   inhibiting	
   the	
   formation	
   of	
   the	
   tetrahedral	
  borate	
  anion	
  and	
  increasing	
  the	
  stability	
  of	
  this	
  species.	
  	
  With	
  these	
  findings	
   in	
   mind,	
   a	
   synthetic	
   avenue	
   towards	
   2,4,6-­‐trifluoro-­‐3-­‐(4,4,5,5-­‐tetraphenyl-­‐1,3,2-­‐ dioxaborolan-­‐2-­‐yl)benzoic	
  acid	
  (henceforth	
  referred	
  to	
  as	
  ArB(OR)2)	
  was	
  devised.32	
   	
   F  F  O OH  F B  OH F  	
    F O  ✔	
    F  Nu  O  O  O  B  O  ArB(OR)2	
   F  O OH  F  F B Nu O O  	
    Nu O  B  F O  O  F  O  OH  + F  F  H-B  OH F  F  	
    	
   	
   Figure	
  1.7:	
  	
  Prevention	
  of	
  C-­‐B	
  bond	
  cleavage	
  by	
  base-­‐mediated	
  deboronation	
  through	
  use	
  of	
  a	
  sterically	
   encumbered	
  protecting	
  group. 	
    	
    18	
    1.2.4	
   ArB18F3-­‐marimistat	
   	
   	
   After	
  confirming	
  that	
  the	
  ArB18F3s	
  were	
  stable	
  in	
  vivo,32	
  the	
  first	
  neoplastic	
  tissue	
   PET	
   imaging	
   test	
   of	
   arylborates	
   as	
   fluoride	
   captors	
   was	
   done	
   through	
   conjugation	
   to	
   marimastat,	
   a	
   clinically	
   trialed	
   broad	
   spectrum	
   matrix	
   metalloproteinase	
   (MMP)	
   inhibitor.33	
  	
  The	
  arylborate	
  was	
  conjugated	
  through	
  amide	
  formation	
  via	
  a	
  small	
  linker	
   attached	
   to	
   the	
   marimastat.34	
   	
   This	
   compound	
   was	
   converted	
   to	
   the	
   radiolabeled	
   aryltrifluoroborate	
  and	
  imaged	
  in	
  vivo	
  in	
  mice	
  that	
  had	
  been	
  xenografted	
  with	
  murine	
   breast	
  carcinomas	
  (Figure	
  1.8).	
  	
  While	
  the	
  overall	
  results	
  of	
  this	
  imaging	
  were	
  positive,	
   there	
   was	
   room	
   for	
   improvement.	
   	
   Although	
   localization	
   around	
   the	
   malignant	
   tissue	
   was	
  observed,	
  the	
  quality	
  of	
  the	
  image	
  was	
  lacking.	
  	
  The	
  increase	
  in	
  contrast	
  required	
  to	
   make	
  the	
  image	
  viable	
  was	
  inconsistent	
  with	
  the	
  current	
  standard	
  for	
  PET	
  imaging.	
  	
  It	
   was	
  concluded	
  that	
  the	
  low	
  image	
  quality	
  was	
  a	
  product	
  of	
  the	
  broad	
  spectrum	
  nature	
   of	
  marimistat	
  itself.	
  	
  High	
  uptake	
  of	
  the	
  drug	
  was	
  observed	
  in	
  tissues	
  with	
  known	
  non-­‐ pathologic	
   MMP	
   expression	
   such	
   as	
   the	
   liver	
   as	
   well	
   as	
   the	
   blood,	
   yielding	
   a	
   poor	
   target/non-­‐target	
  contrast.34	
  	
  While	
  the	
  results	
  of	
  the	
  ArB18F3-­‐marimistat	
  imaging	
  were	
   encouraging	
   with	
   respect	
   to	
   the	
   aryltrifluoroborate	
   strategy,	
   a	
   new	
   bioactive	
   moiety	
   was	
  required	
  for	
  further	
  imaging	
  investigation.	
   	
   	
   	
    	
    19	
    HO  OH  H N O  N H  H N O  F  O O  O  N H F  F F  B F  F  	
    	
    Figure 4. In vivo PET imaging of MMPs in murine breast carcinomas. A, IVIS image of 67NR/CMV-Luciferase derived primary tumor ( 18  Figure	
  1.8:	
  	
  Structure	
  of	
  ArB (top)	
   nd	
   in	
  vivo	
  with imaging	
   esults	
   (bottom)	
   of	
  murine	
   breast	
   Control mouse injected with 440 μCi control-Ar sameFmouse imaged next dayaby MicroPET 50 μCi rof marimastat-ArBF 3-­‐marimistat	
   3 (middle). carcinoma.	
  	
    were reconstructed from a scan taken 50 to 80 min after tracer injection. B, MicroPET images of 67NR breast tumor mice with 100 μCi o (5) injected either in an unblocked tumor mouse (left) or in a tumor mouse preblocked with 300 nmol of marimastat (right). C, biologic B using tumors established on different dates and imaged on different days. D, time–activity curves of the primary tumor of unblocke marimastat-ArBF3 (5) injected 67NR mice, respectively.  	
    preferable, there is no universally accepted v C-11 dramatically reduces the imaging time window (19), minimal specific activity represents a thresho therefore reducing image quality compared with ligands laoften a value of ∼1 Ci/μmol is sufficient for beled with other PET isotopes (20). Our labeling technique tionally, radiosyntheses are performed un captures the advantages of a single-step labeling in the case added conditions to guarantee the highest of F-18 to afford ligands with specific activities that are 1.3	
   LLP2A	
   activities. A prevailing misconception in su potentially useful for imaging.7 no carrier–added fluoride has a specific activ As this approach is conceptually very different from other 	
   of carrier-free, that is, 1,720 Ci/μmol; in pr radiolabeling methods, it is essential to briefly address some the specific activity of “no carrier–added” [18F of the chemical attributes of an 18F-labeled ArBF3. The first is 1.3.1	
   The	
  α4β1	
  Integrin	
   the question of chemical purity. Although labeling must proally falls in the range of 3 to 10 Ci/μmol. As 	
   ceed through mono- and difluoronated intermediates en significant amount of carrier [19F]fluoride p to the labeled ArBF3,targets	
   Furthermore, the the mono-for	
   andcandidate	
   difluoroboranes/ 	
   Cellular	
   surface	
  route proteins	
   are	
   good	
   imaging	
  carrier–added agents	
   due	
  syntheses. to	
   companies multistep radiosyntheses of mid boronates are unstable at pH 7 (21), and as such, the ArBF3 is further reduces the final specific activities, w the only labeled species isolated. The second 35 concern relates their	
   over-­‐expression	
  toby	
   many	
   types	
   of	
   malignancies. 	
   	
   The	
   α4β1	
   integrin	
   has	
   been	
   fall in the range of 1 to 2 Ci/μmol, or less. Be specific activity. Although high specific activity is always ride ions condense with one arylboronate to 36 law of mass action ensures that the resul identified	
   as	
   a	
   regulator	
   of	
   tumor	
   growth,	
   metastasis	
   and	
   angiogenesis.the 	
   	
   Antibodies	
   decay-corrected specific activity that is th 7 18 Li Y, Ting R, Harwig C, et al. Kit-like F-labeling of small molecules with source fluoride; therefore, activities as high specific activities suitable for in vivo PET imaging: toward imaging cancer associated matrix metalloproteases. for publication. may to	
   be envisaged if no carrier–added fluorid against	
   this	
   integrin	
   have	
   been	
   effective	
  2010; in	
  submitted inhibiting	
   tumor	
   growth	
   due	
   its	
   over-­‐  	
    expression	
   during	
   these	
   phases.37	
   	
   The	
   α4β1	
   integrin	
   has	
   been	
   found	
   in	
   leukemias,	
   www.aacrjournals.org  lymphomas,	
   melanomas	
   and	
   sarcomas,	
   making	
   it	
   an	
   ideal	
   target	
   for	
   cancer	
   imaging	
   studies.	
    	
    Cancer Res; 70(19)  Downloaded from cancerres.aacrjournals.org on May 12, 2011 Copyright © 2010 American Association for Cancer Research  20	
    1.3.2	
   High-­‐Affinity	
  α4β1	
  Ligand	
  Identification	
   	
   	
   The	
   Kit	
   Lam	
   lab	
   at	
   UC	
   Davis	
   discovered	
   a	
   high-­‐affinity	
   and	
   high-­‐specificity	
   ligand	
   for	
   imaging	
   the	
   α4β1	
   integrin.38	
   	
   This	
   was	
   done	
   through	
   a	
   one-­‐bead-­‐one-­‐compound	
   combinatorial	
  library,39,	
  40	
  in	
  which	
  each	
  bead	
  contains	
  one	
  discrete	
  peptide.	
  	
  This	
  library	
   was	
   designed	
   around	
   the	
   LDV	
   motif41	
   and	
   the	
   2-­‐(4-­‐(3-­‐o-­‐toylureido)phenyl)acetic	
   acid	
   N-­‐ terminal	
   cap,42	
   which	
   greatly	
   enhances	
   LDV	
   interaction	
   and	
   decreases	
   proteolysis.	
  	
   Through	
   diversification	
   at	
   each	
   position,	
   X1-­‐X5,	
   the	
   binding	
   abilities	
   of	
   many	
   individual	
   peptides	
   were	
   screened.	
   	
   The	
   urea	
   terminal	
   cap	
   was	
   diversified	
   using	
   420	
   combinations	
   of	
   30	
   isocyanates	
   and	
   14	
   4-­‐amino	
   phenyl	
   acetic	
   acids	
   at	
   the	
   X1	
   and	
   X2	
   positions,	
   respectively.	
  	
  The	
  LDV	
  motif	
  was	
  diversified	
  using	
  6	
  leucine	
  and	
  20	
  lysine	
  analogs	
  at	
  X3,	
  3	
   aspartic	
  acid	
  analogs	
  at	
  X4,	
  and	
  18	
  valine	
  analogs	
  at	
  X5.	
  	
   	
   O R  N H X1  N H  X2 X3 X4 X5  L  D  V  	
   38  Figure	
  1.9:	
  	
  The	
  structure	
  of	
  the	
  library	
  design	
  for	
  the	
  screening	
  of	
  α4β1	
  targeting	
  ligands. 	
    	
   	
   	
    To	
  test	
  the	
  affinity	
  of	
  the	
  randomized	
  discrete	
  peptides,	
  the	
  beads	
  were	
  exposed	
    to	
   α4β1	
   expressing	
   Jurkat	
   cells.	
   	
   The	
   beads	
   which	
   localized	
   around	
   cells	
   exhibited	
   superior	
  binding	
  characteristics	
  and	
  their	
  peptide	
  sequences	
  were	
  elucidated.	
  	
  In	
  order	
   to	
  further	
  select	
  for	
  the	
  highest	
  affinity	
  ligands,	
  an	
  increasing	
  amount	
  of	
  a	
  known	
  α4β1	
   	
    21	
    antagonist42	
  was	
  added	
  into	
  the	
  screening	
  solution.	
  	
  This	
  competition	
  assay	
  between	
  the	
   antagonist	
   and	
   the	
   synthetic	
   ligand	
   was	
   able	
   to	
   isolate	
   one	
   high-­‐affinity	
   peptide	
   sequence,	
  henceforth	
  denoted	
  as	
  LLP2A	
  (Figure	
  1.10).	
  	
  LLP2A	
  was	
  determined	
  to	
  have	
   an	
  IC50	
  of	
  2.0	
  ±	
  1.4	
  pM.38	
  	
  A	
  low	
  IC50	
  value	
  improves	
  imaging	
  by	
  better	
  localizing	
  the	
  drug	
   and	
   limiting	
   the	
   amount	
   of	
   background	
   radiation	
   seen,	
   while	
   also	
   decreasing	
   the	
   amount	
  of	
  imaging	
  agent	
  required	
  to	
  obtain	
  a	
  viable,	
  high	
  quality	
  image.	
   	
   O HO O R  N H  O  H N O  N H  H N  O O  HN  N H  N H  N O  	
    Figure	
  1.10:	
  	
  The	
  structure	
  of	
  LLP2A,	
  a	
  high-­‐affinity	
  ligand	
  for	
  the	
  α4β1	
  integrin.	
  	
  R	
  represents	
  an	
  imaging	
   moiety	
  for	
  PET,	
  SPECT,	
  fluorescence	
  or	
  other.	
    	
   1.3.3	
   In	
  Vivo	
  Testing	
  of	
  LLP2A	
   	
   	
   To	
  continue	
  investigation	
  of	
  this	
  compound	
  and	
  demonstrate	
  its	
  targeting	
  abilities,	
   the	
  Lam	
  group	
  conjugated	
   LLP2A	
  to	
  Cy5.5,43	
  a	
  fluorophore	
  emitting	
  in	
  the	
  near	
  infrared	
   (NIR)	
  region.	
  	
  Molt-­‐4	
  leukemia	
  cells	
  expressing	
  the	
  α4β1	
  integrin	
  were	
  xenografted	
  into	
   mice	
   and	
   the	
   Cy5.5-­‐LLP2A	
   conjugate	
   was	
   injected	
   when	
   the	
   tumor	
   had	
   reached	
   approximately	
   0.8	
   cm	
   in	
   diameter.	
   	
   The	
   tumor	
   was	
   then	
   analyzed	
   both	
   in	
   vivo	
   and	
   ex	
   vivo	
  by	
  NIR	
  fluorescence	
  (NIRF);	
  the	
  results	
  of	
  both	
  showed	
  fluorescence	
  was	
  observed	
   	
    22	
    screening random and combinatorial libraries (35, 36). and proteomics, manybased potential rationally on therapeutic the structural information offocused natural ligimaging genomic of specific In the last few years, we have successfully used the one bead – targets have been identified. Discovery of novel imaging and targeted receptors, or they can be developed through With the advances in theseands one compound combinatorial library approach to identify agents against targets enables in vivo molecular screening libraries (35,ligands 36). (24, 35–38). We preseveral cancer-specific targeting assessment and, therefore,random facilitatesand the focused under- combinatorial otential target therapeutic viously reported that a one bead–one standing of their roles in disease progression and the In the last few years, we have successfully used the one bead –compound combinatoery of novel imaging rial library-derived a4h1 integrin targeting peptidomimetic development of targeted therapeutics. Targeting ligands one compound combinatorial library approach to to identify s in vivomust molecular (LLP2A), when conjugated fluorochrome-labeled streptahave the ability to reach the targets with sufficient 43 several cancer-specific targeting ligands (24, 35–38). We prein	
  the	
  malignant	
  tissue	
  with	
  low	
  uptake	
  in	
  other	
  organs	
  (Figure	
  1.11). 	
  	
  These	
  positive	
   vidin in a tetravalent form, could image lymphoma xenograft acilitates concentration the under-and retention time to be detectable in vivo. with high specificity (24). Here, we show An ideal molecular imaging agent should have the viously reported that a one bead–one compound combinato- that LLP2A, when progression and the findings,	
   along	
   with	
   its	
   low	
   IC50a4h1 	
   and	
   integrin proteolytic	
   resistance	
   due	
   to	
   its	
   unnatural	
   amino	
   rial library-derived targeting peptidomimetic cs. Targeting ligands (LLP2A), when conjugated to fluorochrome-labeled streptaargets with sufficient acid	
   composition,	
   LLP2A	
   as	
  could an	
   excellent	
   candidate	
   for	
   further	
   research	
   into	
   vidin in acemented	
   tetravalent form, image lymphoma xenograft be detectable in vivo. with high specificity (24). Here, we show that LLP2A, when nt shouldFigure have thefluorescence 3. NIR imaging of subcutaneous Molt-4 tumor- of	
  arylborates	
  as	
  fluoride	
  captors	
  for	
  PET	
  imaging.	
   the	
   a pplicability	
   bearing mice. The LLP2A-Cy5.5 was given at a dose of 2 nmol (A) or 0.5 nmol (B and C) per mouse via tail vein. All NIR fluorescence images were acquired with 30 s exposure time at different time points postinjection. A, in vivo fluorescence images of subcutaneous Molt-4 tumor-bearing mice received 2 nmol LLP2A-Cy5.5 conjugates. Fluorescence signals from Cy5.5 were pseudocolored. B, in vivo NIR fluorescence images of mice at 3 h after injection of 0.5 nmol LLP2A-Cy5.5 without blocking (left ) or with blocking (right ) by injecting 200 nmol LLP2A 30 min before probe administration. C, ex vivo images of excised tumors and organs 6 h after injection of 0.5 nmol LLP2A-Cy5.5.  	
    	
    Mol Cancer Ther 2008;7(2). February 2008  	
    Downloaded from mct.aacrjournals.org on May 1, 2012 Copyright © 2008 American Association for Cancer 43 Research Figure	
  1.11:	
  	
  Localization	
  of	
  Cy5.5-­‐LLP2A	
  in	
  α4β1	
  expressing	
  Molt-­‐4	
  cells. 	
  	
  a)	
  in	
  vivo	
  NIRF	
  image,	
  b)	
  	
  NIRF	
   image	
  of	
  excised	
  tumor	
  and	
  organs.	
  	
  Reproduced	
  from	
  Lam	
  et	
  al.	
  w/o	
  permission.	
    	
   	
    1.4	
  	
   ArB(OR)2-­‐LLP2A	
  Synthetic	
  Strategy	
   aded from mct.aacrjournals.org on May 1, 2012 	
   © 2008 American Association for Cancer Research  1.4.1	
   One-­‐step	
  Strategy	
   	
   	
   In	
   light	
   of	
   the	
   advantageous	
   properties	
   of	
   LLP2A,	
   a	
   synthetic	
   strategy	
   to	
   conjugate	
   this	
   peptide	
   with	
   an	
   arylborate	
   was	
   designed.	
   	
   If	
   both	
   LLP2A	
   and	
   the	
   arylborate	
   possess	
   carboxylic	
   acid	
   functional	
   groups,	
   these	
   two	
   could	
   be	
   joined	
   through	
   amide	
   formation	
   with	
   a	
   bis-­‐amino	
   terminally	
   functionalized	
   linker.	
   	
   The	
   final	
   project	
   target,	
   ArB(OR)2-­‐ LLP2A,	
  and	
  retrosynthetic	
  analysis	
  is	
  shown	
  in	
  Figure	
  1.12.	
   	
    23	
    	
   O HO F  F  O  H N  O B F  O  O  O  N H  O  O  H N O  N H  H N  O O  N H  ArB(OR)2-­‐LLP2A	
   HN  N H  N O  F  F OH  O B O  F  O  H2N  ArB(OR)2	
   	
   	
    O  O  NH2  O  linker	
    HO O HO  O  H N O  imaging	
  moiety	
   	
    N H  H N  O O  HN  N H N  O  	
    	
    N H  	
    LLP2A	
   	
    	
    peptide	
  moiety	
   	
    	
    	
   Figure	
  1.12:	
  	
  Structure	
  of	
  the	
  target	
  molecule,	
  ArB(OR) 2-­‐LLP2A,	
  and	
  its	
  retrosynthetic	
  analysis.	
    	
    	
    	
   	
    The	
   peptide	
   moiety	
   could	
   be	
   synthesized	
   by	
   standard	
   solid	
   phase	
   peptide	
    synthesis	
  using	
  Fmoc	
  protecting	
  group	
  chemistry.	
  	
  The	
  solid	
  phase	
  resin	
  was	
  purchased	
   preloaded	
   with	
   the	
   desired	
   linker,	
   allowing	
   synthesis	
   of	
   the	
   peptide	
   at	
   one	
   end	
   and	
    	
    24	
    subsequent	
   cleavage	
   from	
   the	
   resin	
   to	
   produce	
   the	
   terminal	
   amine.	
   	
   After	
   the	
   completion	
   of	
   the	
   peptide	
   moiety,	
   the	
   arylborate	
   would	
   be	
   conjugated	
   to	
   the	
   peptide	
   via	
   an	
   amide	
   forming	
   coupling	
   reaction.	
   	
   The	
   finished	
   molecule	
   could	
   then	
   be	
   fluorinated	
  and	
  its	
  biological	
  activity	
  confirmed	
  through	
  a	
  cellular	
  binding	
  assay.	
  	
  To	
  do	
   this,	
   a	
   fluorescent	
   version	
   of	
   the	
   peptide	
   would	
   need	
   to	
   be	
   created.	
   	
   Incubating	
   α4β1	
   expressing	
   cells	
   with	
   a	
   fluorescent	
   LLP2A	
   would	
   allow	
   for	
   visual	
   binding	
   confirmation	
   using	
  fluorescence	
  microscopy.	
  	
  A	
  positive	
  result	
  from	
  this	
  experiment	
  would	
  pave	
  the	
   way	
  for	
  radiolabeling	
  and	
  in	
  vivo	
  imaging	
  studies.	
   	
   1.4.2	
   Two-­‐step	
  Strategy	
   	
   	
   A	
   clickable	
   version	
   of	
   LLP2A	
   is	
   also	
   envisaged	
   to	
   allow	
   for	
   studies	
   of	
   a	
   two-­‐step	
   one-­‐pot	
   labeling	
   reaction	
   through	
   1,2,3-­‐triazole	
   formation.	
   	
   The	
   same	
   peptide	
   moiety	
   could	
   be	
   functionalized	
   with	
   an	
   azide	
   by	
   coupling	
   a	
   small,	
   straight-­‐chained	
   azido-­‐ carboxylic	
  acid.	
  	
  This	
  would	
  permit	
  the	
  radiolabeling	
  of	
  an	
  alkynyl	
  arylborate,	
  followed	
   by	
  click	
  conjugation	
  to	
  the	
  peptide	
  moiety	
  in	
  the	
  same	
  reaction	
  vessel.	
  	
  The	
  final	
  labeled	
   product	
  and	
  retrosynthesis	
  is	
  found	
  in	
  Figure	
  1.13.	
   	
    	
    25	
    O HO  O NH  F  F  N N  O  H N  N  O  O  O  H N  HN  O B  F  18F  H N  N H  O  O O  N H  F  N H  F HN  N O  O F  HO  O N H  F  O  H N  N3  O  F 18F  B F  O F  O  HN  O  H N O  N3-­‐LLP2A	
    N H  H N  O O  N H  HN  N H  N O  O F  O H2N  F O  B  HO O  N H F  OH  N3  O  O  O  O  H N  HN  O  O  N H  H N  O O  HN  N H  N H  N O  H2N  O  O  NH2  O HO O HO  O  H N O  N H  H N  O O  HN  N H  N H  N O  	
    	
  	
   18  Figure	
  1.13:	
  	
  Retrosynthetic	
  analysis	
  of	
   F-­‐labeling	
  of	
  N3-­‐LLP2A	
  via	
  click	
  chemistry.	
    	
   	
   	
    	
    26	
    Chapter	
  2	
   Results	
  and	
  Discussion	
   	
   	
    2.1	
    Peptide	
  Synthetic	
  Strategy	
    	
   2.1.1	
   Solid	
  Phase	
  Peptide	
  Synthesis	
  (SPPS)	
  Overview	
   	
   	
   The	
  synthesis	
  of	
  LLP2A	
  was	
  done	
  on	
  the	
  solid	
  phase	
  using	
  the	
  Fmoc	
  protecting	
   group	
  strategy,44	
  summarized	
  below	
  in	
  Scheme	
  2.1.	
  	
  Briefly,	
  the	
  first	
  amino	
  acid	
  (AA1)	
  in	
   the	
   sequence	
   is	
   installed	
   on	
   the	
   resin	
   via	
   amide	
   formation	
   utilizing	
   a	
   coupling	
   agent,	
   then	
  the	
  Fmoc	
  protecting	
  group	
  is	
  removed	
  by	
  washing	
  with	
  a	
  20	
  %	
  piperidine	
  in	
  DMF	
   solution.	
  	
  This	
  affords	
  a	
  free	
  terminal	
  amine,	
  at	
  which	
  point	
  the	
  next	
  amino	
  acid	
  (AA2)	
   can	
  be	
  added	
  using	
  the	
  same	
  conditions.	
  	
  Since	
  the	
  amine	
  of	
  the	
  AA2	
  is	
  Fmoc	
  protected,	
   this	
  ensures	
  that	
  only	
  one	
  coupling	
  reaction	
  between	
  the	
  free	
  amine	
  of	
  the	
  first	
  amino	
   acid	
   and	
   the	
   carboxylate	
   of	
   the	
   second	
   amino	
   acid	
   can	
   occur.	
   This	
   prevents	
   any	
   unwanted	
  elongation	
  and	
  allows	
  a	
  peptide	
  chain	
  of	
  specific	
  sequence	
  to	
  be	
  synthesized.	
    H N  O  O  H N  a  O  NH2  O  O  N H  H N  Fmoc  R1 b  H N  	
    O O  O  N H  R2  H N R1  O  N H  Fmoc  c  H N  O O  O  N H  NH2 R1  Scheme	
  2.1:	
  	
  General	
  SPPS	
  protocol.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  AA1,	
  HBTU,	
  DIPEA,	
  DMF,	
  shaken	
  1	
  h	
  at	
   RT.	
  	
  b)	
  20	
  %	
  piperidine	
  in	
  DMF,	
  7	
  min	
  x	
  3.	
  	
  c)	
  AA2,	
  HBTU,	
  DIPEA,	
  DMF,	
  shaken	
  1	
  h	
  at	
  RT.	
    	
    27	
    2.1.2	
   Resin	
  and	
  Linker	
  Options	
   	
   	
   Prior	
  to	
  beginning	
  the	
  synthesis	
  of	
  LLP2A,	
  one	
  must	
  choose	
  an	
  appropriate	
  solid	
   phase	
   resin.	
   	
   There	
   are	
   numerous	
   solid	
   phase	
   resins	
   available	
   to	
   meet	
   the	
   requirements	
   of	
   specialized	
   synthesis.	
   	
   For	
   this	
   project,	
   the	
   solid	
   phase	
   target	
   needs	
   to	
   be	
   cleaved	
   from	
  the	
  resin	
  without	
  the	
  use	
  of	
  strong	
  acid,	
  as	
  this	
  would	
  prematurely	
  remove	
  the	
  t-­‐ butyl	
  protecting	
  group	
  on	
  the	
  side-­‐chain	
  carboxylic	
  acid	
  of	
  the	
  2-­‐amino	
  adipic	
  acid	
  (Aad)	
   residue.	
   	
   This	
   would	
   permit	
   unwanted	
   chemistry	
   to	
   occur	
   at	
   this	
   position	
   during	
   the	
   conjugation	
  of	
  the	
  imaging	
  and	
  peptide	
  moieties.	
  	
  Self-­‐polymerization	
  and/or	
  cyclization	
   of	
  LLP2A	
  could	
  occur	
  between	
  the	
  free	
  side	
  chain	
  carboxylate	
  of	
  one	
  LLP2A	
  and	
  the	
  free	
   terminal	
   amine	
   of	
   the	
   same	
   or	
   another	
   molecule	
   of	
   LLP2A,	
   resulting	
   in	
   a	
   biologically	
   inactive	
  species.	
  	
  Therefore	
  the	
  t-­‐butyl	
  protecting	
  group	
  must	
  be	
  cleaved	
  only	
  once	
  the	
   imaging	
   moiety	
   has	
   been	
   conjugated	
   to	
   LLP2A.	
   	
   A	
   base	
   labile	
   resin	
   would	
   not	
   be	
   orthogonal	
   with	
   the	
   proposed	
   Fmoc	
   synthetic	
   strategy,	
   as	
   part	
   of	
   the	
   synthetic	
   strategy	
   employs	
   DIPEA	
   during	
   coupling	
   reactions	
   and	
   removal	
   of	
   Fmoc	
   with	
   piperidine	
   (Scheme	
   2.1),	
   both	
   of	
   which	
   could	
   inadvertently	
   cleave	
   the	
   peptide	
   from	
   a	
   base	
   labile	
   resin.	
  	
   Therefore,	
   a	
   resin	
   that	
   can	
   be	
   cleaved	
   though	
   use	
   of	
   mild	
   acid	
   would	
   be	
   appropriate	
   for	
   this	
  synthesis.	
    R  H N  CO2  O O  H  R  NH2  +  H N  	
   Scheme	
  2.2:	
  	
  Deprotection	
  of	
  Fmoc	
  under	
  basic	
  conditions	
  to	
  afford	
  a	
  free	
  amine.	
    	
    28	
    	
    SPPS	
   resins	
   are	
   often	
   available	
   with	
   a	
   linker	
   pre-­‐attached	
   since	
   many	
   peptide	
    projects	
   require	
   the	
   use	
   of	
   a	
   linker.	
   	
   For	
   the	
   purposes	
   of	
   this	
   project,	
   the	
   linker	
   must	
   fulfill	
   the	
   following	
   criteria:	
   	
   1)	
   allow	
   for	
   solubility	
   under	
   physiological	
   conditions	
   and	
   have	
  low	
  toxicity;	
  and	
  2)	
  be	
  a	
  sufficient	
  length	
  in	
  order	
  to	
  distance	
  the	
  imaging	
  moiety	
   from	
   the	
   peptide	
   so	
   that	
   it	
   does	
   not	
   affect	
   the	
   peptide’s	
   receptor-­‐binding	
   abilities.	
  	
   Many	
   commonly	
   used	
   linkers	
   in	
   bioconjugate	
   chemistry	
   are	
   composed	
   of	
   ethylene	
   glycol	
   units.	
   	
   Ethylene	
   glycol	
   affords	
   the	
   required	
   low	
   toxicity	
   and	
   the	
   oxygens	
   allow	
   increased	
  hydrophilicity	
  to	
  maximize	
  solubility	
  in	
  biological	
  systems.	
  	
  An	
  ethylene	
  glycol	
   unit	
  that	
  is	
  functionalized	
  at	
  both	
  ends	
  with	
  a	
  free	
  amine	
  would	
  enable	
  SPPS	
  at	
  one	
  end	
   and	
   facile	
   imaging	
   moiety	
   conjugation	
   at	
   the	
   other.	
   	
   As	
   such,	
   an	
   O-­‐bis-­‐ (aminoethyl)ethylene	
  glycol	
  linker	
  was	
  hypothesized	
  to	
  meet	
  the	
  criteria	
  outlined	
  above.	
  	
   	
    A	
   polystyrene-­‐based	
   trityl-­‐functionalized	
   resin	
   allows	
   cleavage	
   of	
   products	
    through	
   reaction	
   with	
   HFIP,	
   a	
   weak	
   acid.	
   	
   Exposure	
   to	
   HFIP	
   would	
   cleave	
   the	
   resin-­‐ peptide	
  bond,	
  yet	
  be	
  mild	
  enough	
  to	
  leave	
  any	
  other	
  acid-­‐labile	
  protecting	
  groups	
  intact.	
  	
   Combining	
  this	
  trityl	
  resin	
  with	
  the	
  aforementioned	
  linker	
  would	
  provide	
  a	
  starting	
  point	
   for	
  the	
  SPPS	
  of	
  LLP2A	
  (Figure	
  2.1).	
  	
  This	
  resin	
  was	
  purchased	
  preloaded	
  with	
  the	
  linker	
   moiety.	
    N H  O  O  NH2  	
   Figure	
  2.1:	
  	
  O-­‐bis-­‐(aminoethyl)ethylene	
  glycol	
  trityl	
  resin	
  to	
  be	
  used	
  in	
  the	
  SPPS	
  of	
  LLP2A.	
    	
   	
    29	
    2.1.3	
   Peptide	
  Coupling	
  Reagents	
   	
   	
   There	
   are	
   several	
   reagents	
   available	
   to	
   form	
   amide	
   bonds	
   from	
   amines	
   and	
   carboxylic	
   acids.	
   	
   The	
   unaided	
   nucleophilic	
   attack	
   of	
   the	
   amine	
   on	
   the	
   carbonyl	
   is	
   too	
   slow	
   to	
   be	
   of	
   any	
   synthetic	
   use,	
   thus	
   activating	
   groups	
   are	
   routinely	
   added	
   to	
   the	
   reaction	
   to	
   increase	
   the	
   rate.	
   	
   Carbodiimides	
   are	
   commonly	
   used	
   in	
   peptide	
   coupling;	
   these	
   react	
   with	
   the	
   carboxylate	
   of	
   the	
   amino	
   acid	
   to	
   create	
   a	
   good	
   leaving	
   group.	
   	
   First	
   a	
  base,	
  typically	
  DIPEA,	
  deprotonates	
  the	
  carboxylic	
  acid	
  of	
  the	
  amino	
  acid,	
  which	
  can	
   then	
   act	
   as	
   a	
   nucleophile	
   and	
   attack	
   the	
   central	
   carbon	
   of	
   the	
   carbodiimide.	
   	
   This	
   yields	
   an	
  O-­‐acyl	
  urea,	
  which	
  is	
  an	
  activated	
  ester.	
  	
  Subsequent	
  nucleophilic	
  attack	
  by	
  the	
  free	
   amine	
   of	
   the	
   other	
   amino	
   acid	
   on	
   the	
   O-­‐acyl	
   urea	
   yields	
   the	
   desired	
   peptide	
   bond,	
   as	
   well	
   as	
   the	
   urea	
   byproduct.	
   	
   The	
   most	
   popular	
   carbodiimide	
   coupling	
   reagents	
   are	
   DCC,	
   DIC,	
  and	
  EDC	
  (Figure	
  2.2).	
  	
  	
   	
    N C  N  N C  N  N C  N  N  	
   Figure	
  2.2:	
  	
  Structures	
  of	
  DCC,	
  DIC,	
  and	
  EDC	
  coupling	
  reagents.	
  	
    	
   	
    The	
   wide	
   variety	
   of	
   coupling	
   agents	
   that	
   accomplish	
   the	
   same	
   end	
   goal,	
   amide	
    bond	
   formation,	
   allows	
   the	
   user	
   to	
   choose	
   the	
   reagent	
   best	
   suited	
   for	
   the	
   task	
   at	
   hand.	
  	
    	
    30	
    DCC	
  is	
  most	
  useful	
  for	
  solution	
  phase	
  coupling,	
  as	
  the	
  urea	
  byproduct	
  is	
  highly	
  insoluble	
   in	
  nearly	
  all	
  organic	
  solvents.	
  	
  DIC	
  and	
  EDC	
  are	
  generally	
  preferred	
  for	
  SPPS,	
  since	
  their	
   urea	
   byproducts	
   are	
   soluble	
   in	
   a	
   variety	
   of	
   solvents,	
   allowing	
   facile	
   removal	
   of	
   these	
   species	
   by	
   filtration.	
   	
   The	
   high	
   reactivity	
   of	
   the	
   O-­‐acyl	
   urea	
   intermediate	
   can	
   sometimes	
   lead	
   to	
   racemization	
   and/or	
   transfer	
   of	
   the	
   acyl	
   group	
   to	
   an	
   adjacent	
   nitrogen	
   on	
   the	
   carbodiimide.	
   	
   In	
   order	
   to	
   limit	
   this,	
   HOBt	
   can	
   be	
   introduced	
   into	
   the	
   reaction.	
   	
   This	
   will	
   attack	
  the	
  O-­‐acyl	
  urea	
  before	
  the	
  free	
  amine	
  of	
  the	
  other	
  amino	
  acid,	
  forming	
  another	
   less	
  reactive	
  O-­‐acyl	
  urea	
  that	
  suppresses	
  racemization	
  and	
  eliminates	
  the	
  possibility	
  of	
   acyl	
  transfer.	
  	
  The	
  free	
  amine	
  then	
  attacks	
  this	
  species	
  and	
  the	
  amide	
  bond	
  is	
  formed,	
   ejecting	
  the	
  HOBt	
  molecule,	
  which	
  can	
  undergo	
  further	
  reaction.	
   	
    Ongoing	
  developments	
  in	
  peptide	
  synthesis	
  have	
  yielded	
  coupling	
  reagents	
  that	
    bypass	
  the	
  addition	
  of	
  a	
  carbodiimide.	
  	
  Here,	
  HBTU,	
  or	
  a	
  derivative	
  thereof,	
  is	
  added	
  as	
   the	
  salt	
  of	
  a	
  non-­‐nucleophilic	
  anion.	
  	
  Available	
  reagents	
  include	
  HBTU,	
  HATU,	
  and	
  HCTU,	
   pictured	
  below	
  in	
  Figure	
  2.3.	
  	
  For	
  the	
  purposes	
  of	
  this	
  project,	
  HBTU	
  was	
  the	
  coupling	
   reagent	
  of	
  choice	
  due	
  to	
  its	
  relatively	
  high	
  coupling	
  yield	
  and	
  cost	
  effectiveness.	
  	
   	
   	
   N  N  N N  N O  N  N N  N N  N O  PF6  Cl  N N  N O  PF6  N N  PF6  	
    Figure	
  2.3:	
  	
  Structures	
  of	
  coupling	
  reagents,	
  from	
  left	
  to	
  right:	
  	
  HBTU,	
  HATU,	
  and	
  HCTU.	
    	
   	
    31	
    2.2	
  	
   Solid	
  Phase	
  Peptide	
  Synthesis	
  of	
  LLP2A	
   	
   	
    The	
  beginning	
  of	
  the	
  SPPS	
  of	
  LLP2A	
  involves	
  the	
  loading	
  of	
  the	
  resin	
  with	
  Fmoc-­‐  Ach-­‐OH.	
  	
  The	
  coupling	
  of	
  the	
  first	
  amino	
  acid	
  to	
  the	
  resin	
  is	
  a	
  crucial	
  step	
  in	
  the	
  synthesis	
   of	
   any	
   peptide.	
   	
   Without	
   complete	
   loading,	
   the	
   second	
   amino	
   acid	
   in	
   the	
   sequence	
   may	
   be	
   inadvertently	
   attached	
   directly	
   to	
   the	
   resin	
   during	
   the	
   subsequent	
   coupling	
   reaction.	
  	
   This	
   results	
   in	
   the	
   formation	
   of	
   an	
   incomplete	
   peptide	
   that	
   will	
   likely	
   have	
   no	
   or	
   limited	
   biological	
  activity.	
  	
  In	
  order	
  to	
  maximize	
  yield	
  of	
  the	
  desired	
  peptide,	
  it	
  is	
  advantageous	
   to	
  ensure	
  complete	
  loading	
  of	
  the	
  first	
  amino	
  acid	
  prior	
  to	
  the	
  subsequent	
  reaction.	
  	
   	
    Resin	
  swelling	
  contributes	
  to	
  achieving	
  overall	
  resin	
  loading.	
  	
  Prior	
  to	
  use,	
  solid	
    phase	
   resins	
   must	
   be	
   swollen	
   in	
   the	
   appropriate	
   solvent	
   that	
   permits	
   the	
   resin	
   to	
   expand.	
   	
   Insufficient	
   swelling	
   can	
   cause	
   poor	
   reaction	
   site	
   accessibility	
   and	
   diminished	
   reaction	
   rates45	
   since	
   the	
   reactive	
   sites	
   are	
   not	
   in	
   an	
   available	
   position	
   to	
   undergo	
   reaction.	
   	
   Testing	
   revealed	
   that	
   swelling	
   for	
   two	
   hours	
   was	
   enough	
   to	
   allow	
   complete	
   loading,	
  with	
  additional	
  swelling	
  time	
  yielding	
  no	
  increase	
  in	
  loading.	
  	
  Thus,	
  in	
  the	
  SPPS	
   of	
  LLP2A,	
  the	
  resin	
  was	
  swollen	
  in	
  DCM	
  for	
  two	
  hours	
  with	
  occasional	
  stirring	
  prior	
  to	
   the	
  addition	
  of	
  any	
  reagents.	
   	
    After	
  swelling,	
  the	
  solvent	
  was	
  removed	
  from	
  the	
  resin	
  by	
  filtration.	
  	
  The	
  design	
    of	
  the	
  reaction	
  vessel,	
  a	
  10	
  mL	
  plastic	
  tube	
  (also	
  called	
  a	
  spin	
  column)	
  with	
  a	
  frit	
  at	
  the	
   bottom,	
   allows	
   for	
   simple	
   suction	
   filtration	
   enabling	
   the	
   resin	
   to	
   remain	
   in	
   the	
   same	
   vessel	
  for	
  the	
  duration	
  of	
  the	
  synthesis.	
  	
  The	
  reactants	
  were	
  then	
  added	
  to	
  the	
  vessel,	
   which	
   included	
   Fmoc-­‐Ach-­‐OH,	
   HBTU,	
   DIPEA	
   and	
   DMF	
   (Scheme	
   2.3).	
   	
   The	
   reaction	
   vessel	
   was	
  then	
  capped	
  at	
  both	
  ends	
  and	
  shaken	
  gently	
  for	
  one	
  hour.	
  	
  As	
  another	
  precaution	
    	
    32	
    to	
  ensure	
  complete	
  loading,	
  four	
  equivalents	
  of	
  the	
  amino	
  acid	
  and	
  coupling	
  reagent	
  are	
   added	
  to	
  the	
  resin	
  during	
  all	
  coupling	
  reactions.	
   	
    H N  O  O  H N  a NH2  O O  O  N H  H N  O O  	
    resin	
  1	
    	
    Scheme	
  2.3:	
  	
  Installment	
  of	
  Fmoc-­‐Ach-­‐OH	
  onto	
  the	
  resin.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  Fmoc-­‐Ach-­‐OH,	
   HBTU,	
  DIPEA,	
  DMF	
  shaken	
  1	
  h	
  at	
  RT.	
    	
   	
    The	
   time	
   of	
   reaction	
   is	
   also	
   important.	
   	
   If	
   the	
   reaction	
   time	
   is	
   too	
   short,	
    incomplete	
  loading	
  may	
  be	
  observed.	
  	
  However,	
  if	
  the	
  reaction	
  time	
  is	
  too	
  long,	
  there	
   may	
  be	
  removal	
  of	
  the	
  base-­‐labile	
  Fmoc	
  protecting	
  group	
  by	
  DIPEA	
  and	
  an	
  undesirable	
   double	
   coupling	
   reaction	
   at	
   the	
   deprotected	
   terminal	
   amine	
   site.	
   	
   Through	
   experimentation,	
   it	
   was	
   determined	
   that	
   a	
   coupling	
   reaction	
   time	
   of	
   one	
   hour	
   using	
   HBTU	
   provided	
   complete	
   coupling	
   without	
   elongation	
   and	
   this	
   method	
   was	
   used	
   throughout	
   the	
   SPPS	
   portion	
   of	
   this	
   project.	
   	
   After	
   the	
   reaction	
   was	
   complete,	
   the	
   solution	
   was	
   removed	
   by	
   filtration.	
   	
   The	
   resin	
   was	
   washed	
   thoroughly	
   with	
   DMF	
   and	
   DCM	
  in	
  order	
  to	
  remove	
  any	
  remaining	
  reactants	
  and	
  reagents.	
  	
  	
   	
    After	
    the	
    installment	
    of	
    the	
    first	
    amino	
    acid,	
    1-­‐(Fmoc-­‐  amino)cyclohexanecarboxylic	
   acid	
   (Fmoc-­‐Ach-­‐OH),	
   the	
   loading	
   of	
   the	
   resin	
   was	
   quantified	
  so	
  that	
  the	
  required	
  amount	
  of	
  amino	
  acid	
  for	
  subsequent	
  couplings	
  could	
  be	
   calculated.	
   	
   This	
   is	
   done	
   by	
   observing	
   the	
   amount	
   of	
   Fmoc	
   deprotection	
   product,	
   	
    33	
    dibenzofulvene	
  (DBF),	
  produced	
  from	
  a	
  known	
  mass	
  of	
  dry	
  resin.	
  	
  While	
  routine	
  Fmoc	
   deprotection	
   will	
   employ	
   piperidine	
   as	
   a	
   base,	
   1,8-­‐diazabicycloundec-­‐7-­‐ene	
   (DBU)	
   is	
   used	
  for	
  resin	
  loading	
  determination	
  (Scheme	
  2.4).	
  	
  Use	
  of	
  a	
  nucleophilic	
  base,	
  such	
  as	
   piperidine,	
  will	
  cause	
  deprotection	
  of	
  the	
  Fmoc	
  to	
  yield	
  not	
  only	
  DBF,	
  but	
  also	
  forming	
   the	
   DBF-­‐piperidine	
   adduct.	
   	
   This	
   may	
   cause	
   varying	
   UV	
   absorption,	
   depending	
   on	
   the	
   equilibrium	
   between	
   DBF	
   and	
   the	
   DBF-­‐piperidine	
   adduct.	
   	
   Thus,	
   the	
   non-­‐nucleophilic	
   sterically	
   encumbered	
   base	
   DBU	
   is	
   used	
   instead	
   since	
   it	
   will	
   not	
   form	
   an	
   adduct	
   with	
   DBF.	
   	
   The	
   amount	
   of	
   DBF	
   liberated	
   in	
   the	
   reaction	
   will	
   be	
   stoichiometric	
   with	
   the	
   amount	
  of	
  Fmoc	
  present	
  on	
  the	
  resin	
  and	
  permit	
  accurate	
  loading	
  quantification.	
   	
   R  H N  O  O  N H  O  +  R  H N  N N  DBU	
    NH2  +  O  DBF	
    λmax = 304 nm  	
    Scheme	
  2.4:	
  	
  Reaction	
  of	
  DBU	
  with	
  Fmoc	
  to	
  produce	
  DBF,	
  allowing	
  quantification	
  of	
  resin	
  loading.	
    	
   	
    A	
   small	
   amount	
   of	
   the	
   resin	
   at	
   the	
   tip	
   of	
   a	
   spatula,	
   approximately	
   15	
   mg,	
   was	
    removed	
   and	
   dried	
   under	
   vacuum	
   for	
   12	
   hours	
   to	
   remove	
   any	
   solvent	
   which	
   would	
   add	
   extra	
   mass	
   that	
   may	
   be	
   incorrectly	
   attributed	
   to	
   the	
   resin.	
   	
   Two	
   separate	
   vials	
   each	
   containing	
  a	
  precise	
  mass	
  (approximately	
  6	
  mg	
  each)	
  of	
  dried	
  resin	
  were	
  then	
  prepared.	
  	
   The	
   addition	
   of	
   2	
   mL	
   of	
   a	
   2	
   %	
   DBU	
   in	
   DMF	
   solution	
   allows	
   complete	
   cleavage	
   of	
   the	
   Fmoc	
   protecting	
   group	
   from	
   the	
   resin,	
   yielding	
   DBF.	
   	
   Two	
   serial	
   dilutions	
   are	
   then	
   made	
   with	
   ACN.	
   	
   A	
   blank	
   solution	
   is	
   prepared	
   in	
   the	
   same	
   way,	
   omitting	
   the	
   resin.	
   	
   The	
    	
    34	
    solutions	
   are	
   then	
   analyzed	
   with	
   UV-­‐Visible	
   spectroscopy	
   at	
   304	
   nm,	
   the	
   λmax	
   for	
   DBF.	
  	
   After	
  determining	
  the	
  absorbance,	
  the	
  molar	
  absorbtivity	
  of	
  DBF	
  (7624	
  M-­‐1cm-­‐1)	
  is	
  then	
   used	
   to	
   calculate	
   the	
   concentration.	
   	
   This	
   allows	
   for	
   a	
   final	
   measurement	
   of	
   Fmoc	
   quantity	
   in	
   mmol/g	
   resin,	
   thus	
   quantifying	
   the	
   amount	
   of	
   amino	
   acid	
   that	
   has	
   been	
   coupled	
  onto	
  the	
  resin.	
   	
    What	
  is	
  considered	
  to	
  be	
  “full	
  loading”	
  varies	
  by	
  resin,	
  and	
  also	
  differs	
  between	
    batches	
   of	
   the	
   same	
   resin.	
   	
   For	
   the	
   O-­‐bis-­‐(aminoethyl)ethylene	
   glycol	
   trityl	
   resin,	
   complete	
   loading	
   can	
   be	
   anywhere	
   from	
   0.3	
   –	
   1.0	
   mmol/g	
   according	
   to	
   the	
   manufacturer.	
   	
   After	
   installation	
   of	
   the	
   Fmoc-­‐Ach-­‐OH	
   residue,	
   the	
   loading	
   was	
   determined	
   to	
   be	
   0.47	
   mmol/g.	
   	
   This	
   is	
   a	
   reasonable	
   loading	
   value	
   and	
   a	
   Kaiser	
   test	
   can	
   be	
  used	
  to	
  check	
  for	
  free	
  amines	
  and	
  confirm	
  complete	
  loading.	
  	
  The	
  test	
  involves	
  the	
   addition	
   of	
   a	
   ninhydrin	
   solution	
   to	
   the	
   resin.	
   	
   If	
   any	
   free	
   amines	
   are	
   present	
   they	
   will	
   undergo	
  a	
  reaction	
  with	
  the	
  ninhydrin	
  molecules	
  to	
  yield	
  a	
  strong	
  blue	
  color.	
  	
  When	
  the	
   test	
  is	
  performed	
  and	
  no	
  blue	
  color	
  is	
  observed,	
  it	
  can	
  be	
  concluded	
  that	
  there	
  are	
  no	
   free	
  amines	
  and	
  complete	
  coupling	
  has	
  taken	
  place.	
  	
  A	
  Kaiser	
  test	
  was	
  used	
  to	
  confirm	
   there	
  were	
  no	
  free	
  amines	
  on	
  the	
  resin,	
  verifying	
  full	
  loading,	
  and	
  resin	
  1	
  (Scheme	
  2.3)	
   was	
  acquired.	
  	
  	
   	
    The	
   next	
   step	
   involves	
   the	
   coupling	
   of	
   Fmoc-­‐Aad(tBu)-­‐OH	
   (Scheme	
   2.5).	
   	
   The	
    resin	
   was	
   first	
   washed	
   with	
   a	
   piperidine	
   solution	
   (20	
   %	
   in	
   DMF)	
   for	
   10	
   minutes	
   to	
   deprotect	
  the	
  Fmoc	
  group.	
  	
  The	
  solution	
  was	
  then	
  drained	
  from	
  the	
  reaction	
  vessel,	
  and	
   this	
  process	
  repeated	
  twice	
  more.	
  	
  The	
  removal	
  of	
  the	
  Fmoc	
  group	
  could	
  be	
  followed	
  by	
   TLC,	
   as	
   DBF	
   is	
   strongly	
   UV	
   active	
   due	
   to	
   the	
   highly	
   conjugated	
   system.	
   	
   The	
   resin	
   was	
    	
    35	
    then	
   washed	
   thoroughly	
   with	
   DMF	
   to	
   remove	
   any	
   residual	
   piperidine.	
   	
   Lingering	
   piperidine	
   could	
   possibly	
   lead	
   to	
   deprotection	
   of	
   Fmoc-­‐Aad(tBu)-­‐OH	
   and	
   a	
   double	
   coupling	
   to	
   form	
   a	
   Fmoc-­‐Aad(tBu)-­‐Aad(tBu)-­‐OH	
   dipeptide	
   in	
   situ	
   which	
   could	
   link	
   with	
   the	
  Ach	
  residue.	
  	
  Mixed	
  in	
  a	
  vial	
  and	
  added	
  to	
  the	
  resin	
  were	
  Fmoc-­‐Aad(tBu)-­‐OH,	
  HBTU,	
   DIPEA	
   and	
   DMF.	
   	
   The	
   reaction	
   was	
   shaken	
   for	
   1	
   hour,	
   then	
   drained	
   and	
   washed	
   with	
   DMF	
  and	
  DCM.	
  	
  	
   	
    H N  O O  O  resin	
  1	
    N H  H N  O  O  O  a, b  H N  O O  O  O  N H  O  H N O  N H  O  	
    resin	
  2	
   	
   Scheme	
  2.5:	
  	
  Removal	
  of	
  Fmoc	
  protecting	
  group	
  of	
  resin	
  1	
  and	
  coupling	
  of	
  Fmoc-­‐Aad(tBu)-­‐OH	
  onto	
  Ach.	
   Reagents	
  and	
  conditions:	
  	
  a)	
  20	
  %	
  piperidine	
  in	
  DMF,	
  10	
  min	
  at	
  RT.	
  	
  Repeated	
  twice	
  more.	
  	
  b)	
  Fmoc-­‐ Aad(tBu)-­‐OH,	
  HBTU,	
  DIPEA,	
  DMF	
  shaken	
  1	
  h	
  at	
  RT.	
    	
   	
    Unfortunately,	
   it	
   was	
   at	
   this	
   point	
   in	
   the	
   synthesis	
   where	
   difficulties	
   were	
    encountered.	
  	
  Testing	
  showed	
  that	
  no	
  amino	
  acid	
  had	
  been	
  coupled	
  onto	
  the	
  free	
  amine	
   of	
   the	
   Ach	
   residue.	
   A	
   Kaiser	
   test	
   was	
   positive	
   for	
   free	
   amines.	
   	
   A	
   small	
   portion	
   of	
   the	
   resin	
   was	
   cleaved	
   and	
   investigated	
   by	
   mass	
   spectrometry,	
   which	
   did	
   not	
   indicate	
   the	
   presence	
   of	
   the	
   desired	
   product.	
   	
   Confused	
   by	
   the	
   failure	
   of	
   this	
   normally	
   robust	
   reaction,	
   the	
   starting	
   material	
   was	
   investigated.	
   	
   Analysis	
   of	
   Fmoc-­‐Aad(tBu)-­‐OH	
   by	
   mass	
    	
    36	
    spectrometry	
  and	
   1H	
  NMR	
  showed	
  neither	
  the	
  expected	
  mass	
  nor	
  any	
  expected	
  proton	
   signals.	
   	
   Thus,	
   it	
   was	
   found	
   the	
   starting	
   material	
   was	
   not	
   as	
   advertised	
   and	
   in	
   fact	
   appeared	
   to	
   be	
   some	
   type	
   of	
   inorganic	
   compound.	
   	
   Further	
   inquiry	
   with	
   the	
   vendor	
   could	
  not	
  explain	
  this	
  phenomenon,	
  and	
  new	
  product	
  was	
  shipped.	
   	
    The	
  new	
  product	
  was	
  tested	
  prior	
  to	
  use	
  and	
  the	
  results	
  were	
  in	
  agreement	
  with	
    the	
  data	
  provided	
  by	
  the	
  supplier.	
  	
  The	
  synthesis	
  was	
  started	
  anew,	
  with	
  the	
  installation	
   of	
   Fmoc-­‐Ach-­‐OH	
   on	
   fresh	
   resin.	
   	
   Subsequent	
   coupling	
   with	
   Fmoc-­‐Aad(tBu)-­‐OH	
   yielded	
   the	
   desired	
   product,	
   as	
   shown	
   by	
   mass	
   spectrometry.	
   	
   The	
   mass	
   spectrum	
   did	
   not	
   indicate	
   the	
   presence	
   of	
   a	
   truncate,	
   however,	
   a	
   Kaiser	
   test	
   indicated	
   the	
   presence	
   of	
   free	
   amines.	
   	
   It	
   was	
   hypothesized	
   that	
   although	
   the	
   new	
   batch	
   of	
   amino	
   acid	
   did	
   in	
   fact	
   contain	
   Fmoc-­‐Aad(tBu)-­‐OH,	
   it	
   was	
   perhaps	
   not	
   pure	
   by	
   mass.	
   	
   A	
   second	
   coupling	
   reaction	
  could	
  be	
  done,	
  however	
  this	
  risks	
  the	
  formation	
  of	
  an	
  unwanted	
  elongation	
  of	
   the	
  peptide.	
  	
  At	
  this	
  point,	
  due	
  to	
  time	
  constraints,	
  it	
  was	
  decided	
  to	
  move	
  forward	
  with	
   the	
  synthesis	
  and	
  deal	
  with	
  any	
  future	
  purification	
  that	
  may	
  be	
  necessary.	
   	
    After	
  removal	
  of	
  the	
  Fmoc	
  protecting	
  group	
  by	
  washing	
  with	
  a	
  20	
  %	
  solution	
  of	
    piperidine	
  in	
  DMF,	
  a	
  solution	
  of	
  Fmoc-­‐Lys(Dde)-­‐OH,	
  HBTU,	
  DIPEA	
  and	
  DMF	
  was	
  added	
  to	
   the	
   resin	
   (Scheme	
   2.6).	
   	
   The	
   reaction	
   was	
   shaken	
   gently	
   for	
   1	
   hour	
   and	
   the	
   solution	
   was	
   removed	
   by	
   filtration.	
   	
   Mass	
   spectrometry	
   analysis	
   showed	
   the	
   presence	
   of	
   the	
   product	
   and	
  a	
  Kaiser	
  test	
  was	
  negative	
  for	
  free	
  amines.	
   	
    	
    37	
    O O H N  O O  O  O  H N  N H  O  O  N H  resin	
  2	
    a, b O O H N  O O  O  O  H N  N H  O  N H  H N  O O  resin	
  3	
    HN O  O  	
   Scheme	
  2.6:	
  	
  Removal	
  of	
  Fmoc	
  protecting	
  group	
  of	
  resin	
  2	
  and	
  coupling	
  of	
  Fmoc-­‐Lys(Dde)-­‐OH	
  onto	
  Aad.	
  	
   Reagents	
  and	
  conditions:	
  	
  a)	
  20	
  %	
  piperidine	
  in	
  DMF,	
  10	
  min	
  at	
  RT.	
  	
  Repeated	
  twice	
  more.	
  	
  b)	
  Fmoc-­‐ Lys(Dde)-­‐OH,	
  HBTU,	
  DIPEA,	
  DMF	
  shaken	
  1	
  h	
  at	
  RT.	
    	
   	
    With	
   the	
   tripeptide	
   now	
   in	
   hand,	
   the	
   next	
   step	
   involves	
   the	
   coupling	
   of	
   the	
    terminal	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
   acid	
   1,	
   which	
   must	
   first	
   be	
   synthesized	
   in	
   solution	
  (Scheme	
  2.7).	
  	
  This	
  synthesis	
  involves	
  the	
  carbamide-­‐forming	
  condensation	
  of	
   4-­‐aminophenyl	
   acetic	
   acid	
   and	
   o-­‐tosyl	
   isocyanate.	
   	
   4-­‐Aminophenyl	
   acetic	
   acid	
   was	
   dissolved	
   in	
   DMF	
   and	
   added	
   dropwise	
   into	
   a	
   stirring	
   solution	
   of	
   o-­‐tosyl	
   isocyanate	
   in	
   DMF.	
  	
  After	
  stirring	
  for	
  2	
  hours,	
  the	
  solvent	
  volume	
  was	
  reduced	
  and	
  the	
  reaction	
  was	
   poured	
  onto	
  ethyl	
  acetate.	
  This	
  resulted	
  in	
  a	
  coffee	
  coloured	
  precipitate.	
  	
  The	
  mixture	
    	
    38	
    was	
   then	
   filtered	
   and	
   the	
   precipitate	
   was	
   characterized	
   by	
   1H	
   NMR	
   and	
   MS	
   with	
   no	
   further	
  purification	
  required.	
   	
   H2N N  a  O  OH  O  + C  OH O  N H  N H  O  1	
    	
    	
   Scheme	
  2.7:	
  	
  Synthesis	
  of	
  2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
  acid.	
  	
  Reactions	
  and	
  conditions:	
  	
  a)	
  DMF,	
   stirred	
  at	
  RT	
  for	
  2	
  hours.	
    	
   	
    With	
  this	
  product	
  in	
  hand,	
  the	
  Fmoc	
  protecting	
  group	
  was	
  removed	
  from	
  resin	
  3	
    by	
   washing	
   three	
   times	
   with	
   20	
   %	
   piperidine	
   in	
   DMF.	
   	
   A	
   solution	
   of	
   2-­‐(4-­‐(3-­‐o-­‐ tolylureido)phenyl)acetic	
   acid,	
   HBTU,	
   DIPEA	
   and	
   DMF	
   was	
   added	
   to	
   the	
   resin	
   and	
   the	
   vessel	
  was	
  shaken	
  for	
  1	
  hour	
  (Scheme	
  2.8).	
  	
  Another	
  coupling	
  reaction	
  was	
  performed	
   with	
  half	
  the	
  equivalents	
  used	
  previously	
  since	
  there	
  was	
  no	
  risk	
  of	
  peptide	
  elongation	
   due	
   to	
   the	
   absence	
   of	
   an	
   N-­‐terminus	
   on	
   the	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
   acid.	
  	
   After	
   filtration	
   to	
   remove	
   the	
   solvent,	
   the	
   resin	
   was	
   washed	
   with	
   DMF	
   and	
   DCM.	
  	
   Following	
  analysis	
  revealed	
  the	
  reaction	
  was	
  successful,	
  with	
  the	
  corresponding	
  product	
   peak	
  observed	
  in	
  the	
  mass	
  spectrum.	
  	
    	
    39	
    O O O  H N  O  O  O  H N  N H  H N  N H  O  O O  resin	
  3	
   a, b  HN O  O  O O H N  O O  O  N H  O  H N O  N H  H N  O O  N H  N H  HN  resin	
  4	
    O  O  	
   Scheme	
  2.8:	
  	
  Removal	
  of	
  Fmoc	
  protecting	
  group	
  of	
  resin	
  3	
  and	
  coupling	
  of	
  2-­‐(4-­‐(3-­‐o-­‐tolylureido)	
   phenyl)acetic	
  acid	
  onto	
  Lys.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  20	
  %	
  piperidine	
  in	
  DMF,	
  10	
  min	
  at	
  RT.	
  	
  Repeated	
   twice	
  more.	
  	
  b)	
  2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
  acid,	
  HBTU,	
  DIPEA,	
  DMF	
  shaken	
  1	
  h	
  at	
  RT.	
  	
    	
   	
    The	
  final	
  step	
  in	
  the	
  SPPS	
  of	
  LLP2A	
  is	
  the	
  coupling	
  of	
  3-­‐(3-­‐pyridyl)acrylic	
  acid	
  onto	
    the	
   side	
   chain	
   of	
   the	
   lysine	
   residue	
   (Scheme	
   2.9).	
   	
   Before	
   this	
   can	
   be	
   done,	
   the	
   Dde	
   group	
   must	
   be	
   deprotected.	
   	
   Dde	
   is	
   an	
   excellent	
   protecting	
   group	
   for	
   side	
   chain	
   amines	
   because	
   it	
   is	
   completely	
   orthogonal	
   with	
   both	
   acid	
   and	
   base	
   treatments.	
   	
   Dde	
   is	
   deprotected	
   by	
   washing	
   with	
   a	
   solution	
   of	
   2	
   %	
   hydrazine	
   in	
   DMF.	
   	
   Following	
   deprotection,	
  a	
  solution	
  of	
  3-­‐(3-­‐pyridyl)acrylic	
  acid,	
  HBTU,	
  DIPEA	
  and	
  DMF	
  was	
  added.	
  	
    	
    40	
    The	
  vessel	
  was	
  shaken	
  for	
  1	
  hour	
  and	
  the	
  solution	
  was	
  filtered	
  off	
  and	
  the	
  resin	
  washed	
   with	
  DMF.	
  	
  This	
  completes	
  the	
  SPPS	
  of	
  LLP2A.	
   O O H N  O O  O  O  H N  N H  O  N H  H N  O O  N H  resin	
  4	
   a, b  N H  HN O  O  O O H N  O O  O  N H  O  H N O  N H  resin	
  5	
    H N  O O  N H  HN  N H N  O  	
    Scheme	
  2.9:	
  	
  Removal	
  of	
  Dde	
  protecting	
  group	
  of	
  resin	
  4	
  and	
  coupling	
  of	
  3-­‐(3-­‐pyridyl)acrylic	
  acid	
  onto	
  the	
   side	
  chain	
  of	
  Lys.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  2	
  %	
  hydrazine	
  in	
  DMF,	
  4	
  min	
  at	
  RT.	
  	
  Repeated	
  twice	
  more.	
  	
   b)	
  3-­‐(3-­‐pyridyl)acrylic	
  acid,	
  HBTU,	
  DIPEA,	
  DMF	
  shaken	
  1	
  h	
  at	
  RT.	
    	
   	
    The	
  complete	
  peptide	
  could	
  now	
  be	
  cleaved	
  from	
  the	
  resin	
  (Scheme	
  2.10).	
  	
  After	
    placing	
   resin	
   5	
   in	
   a	
   round	
   bottom	
   flask,	
   a	
   solution	
   of	
   30	
   %	
   HFIP	
   in	
   DCM	
   was	
   added.	
  	
   Immediately,	
  the	
  resin	
  turned	
  a	
  bright	
  orange	
  colour,	
  indicating	
  the	
  presence	
  of	
  a	
  trityl	
   cation	
   and	
   that	
   the	
   product	
   had	
   been	
   liberated	
   from	
   the	
   resin.	
   	
   The	
   reaction	
   was	
   gently	
   stirred	
   for	
   30	
   minutes	
   to	
   ensure	
   that	
   all	
   peptide	
   had	
   been	
   cleaved;	
   the	
   orange	
   colour	
   faded	
   during	
   this	
   time.	
   	
   The	
   resin	
   was	
   filtered	
   off,	
   with	
   the	
   desired	
   product	
   now	
    	
    41	
    dissolved	
  the	
  filtrate.	
  	
  The	
  filtrate	
  volume	
  was	
  reduced	
  and	
  the	
  product,	
  LLP2A(tBu),	
  was	
   precipitated	
  as	
  a	
  fluffy,	
  white	
  solid	
  using	
  diethyl	
  ether.	
   O O H N  O O  O  O  H N  N H  N H  O  H N  O O  N H  resin	
  5	
   HN a, b  N H N  O  O O O H2N  O  O  N H  O  H N O  N H  LLP2A(tBu)	
  2	
    H N  O O  N H  HN  N H N  O  	
    Scheme	
  2.10:	
  	
  Cleavage	
  of	
  resin	
  5	
  to	
  produce	
  LLP2A(tBu)	
  2.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  30	
  %	
  HFIP	
  in	
   DCM,	
  30	
  min	
  at	
  RT.	
  	
  b)	
  diethyl	
  ether.	
    	
   	
    At	
   this	
   point,	
   the	
   precipitate	
   was	
   analyzed	
   for	
   purity.	
   	
   The	
   mass	
   spectrum	
    revealed	
   the	
   corresponding	
   [M+H+]	
   peak	
   of	
   999,	
   however,	
   a	
   peak	
   of	
   799	
   was	
   also	
   observed.	
   	
   This	
   undesired	
   peak	
   indicated	
   the	
   presence	
   of	
   a	
   truncate.	
   	
   As	
   a	
   result	
   of	
   previous	
   experimentation,	
   it	
   was	
   suspected	
   that	
   complete	
   coupling	
   of	
   the	
   Aad(tBu)	
   residue	
   may	
   not	
   have	
   occurred.	
   	
   The	
   corresponding	
   mass	
   of	
   Aad(tBu)	
   is	
   200	
   g/mol,	
   confirming	
   it	
   to	
   be	
   the	
   residue	
   absent	
   in	
   this	
   truncation.	
   	
   The	
   ratio	
   of	
   truncate	
   to	
    	
    42	
    LLP2A(tBu)	
   was	
   investigated	
   by	
   RP-­‐HPLC	
   (Figure	
   2.4).	
   	
   A	
   clean	
   trace	
   was	
   produced,	
   consisting	
  of	
  one	
  peak	
  at	
  8	
  minutes	
  and	
  one	
  at	
  12	
  minutes.	
  	
  These	
  peaks	
  were	
  collected	
   and	
   their	
   masses	
   were	
   analyzed,	
   indicating	
   that	
   the	
   second	
   peak	
   was	
   LLP2A(tBu),	
   and	
   the	
   first	
   peak	
   was	
   the	
   truncate.	
   	
   The	
   areas	
   of	
   these	
   peaks	
   suggest	
   that	
   the	
   truncate	
   was	
   approximately	
  20	
  %	
  of	
  the	
  product.	
  	
  	
   	
    LLP2A(tBu)	
    	
    truncate	
   	
    	
    Figure	
  2.4:	
  	
  RP-­‐HPLC	
  trace	
  of	
  LLP2A(tBu)	
  and	
  truncate.	
    	
   	
    Preparative	
  TLC	
  was	
  used	
  to	
  purify	
  the	
  desired	
  product	
  from	
  the	
  truncate.	
  	
  Using	
    a	
  10	
  %	
  NH4OH	
  in	
  EtOH	
  solution,	
  the	
  truncate	
  and	
  LLP2A(tBu)	
  could	
  be	
  resolved	
  on	
  TLC,	
   with	
   LLP2A(tBu)	
   running	
   slightly	
   higher	
   on	
   the	
   plate.	
   	
   The	
   strongly	
   absorbing	
   aromatic	
   residues	
   allowed	
   visualization	
   with	
   a	
   handheld	
   UV	
   lamp.	
   	
   The	
   corresponding	
   spot	
   was	
   scraped	
   off	
   of	
   the	
   glass	
   plate	
   and	
   MeOH	
   was	
   added	
   to	
   the	
   remove	
   the	
   product.	
   	
   The	
   mixture	
   was	
   vortexed	
   and	
   then	
   centrifuged.	
   	
   The	
   solvent	
   was	
   then	
   removed	
   and	
   this	
   process	
   was	
   repeated	
   twice	
   more.	
   	
   Complete	
   removal	
   of	
   the	
   product	
   could	
   be	
   followed	
   with	
   UV	
   lamp,	
   as	
   the	
   silica	
   would	
   not	
   exhibit	
   the	
   dark	
   colour	
   characteristic	
   of	
   a	
   UV	
    	
    43	
    absorbing	
   compound	
   once	
   the	
   entire	
   product	
   had	
   been	
   removed.	
   	
   The	
   volume	
   of	
   the	
   solution	
   was	
   then	
   reduced	
   under	
   vacuum	
   and	
   the	
   product	
   was	
   precipitated	
   via	
   addition	
   of	
  diethyl	
  ether.	
  	
  This	
  product	
  was	
  further	
  analyzed	
  with	
  RP-­‐HPLC	
  to	
  confirm	
  its	
  purity,	
   as	
  shown	
  in	
  Figure	
  2.5.	
   	
    LLP2A(tBu)	
    Figure	
  2.5:	
  	
  RP-­‐HPLC	
  trace	
  of	
  purified	
  LLP2A(tBu).	
    	
   	
    2.3	
    Solution	
  Phase	
  Synthesis	
  and	
  Conjugation	
  to	
  LLP2A	
    	
   2.3.1	
   Synthesis	
  of	
  ArB(OR)2-­‐LLP2A	
   	
   	
   	
   With	
   the	
   biologically	
   active	
   portion	
   of	
   the	
   target	
   compound	
   complete,	
   it	
   could	
   now	
   be	
   functionalized	
   to	
   allow	
   imaging	
   studies	
   to	
   take	
   place.	
   	
   This	
   is	
   conveniently	
   done	
   through	
   acylation	
   of	
   the	
   terminal	
   free	
   amine	
   of	
   the	
   LLP2A(tBu).	
   	
   Conjugation	
   to	
   a	
   compound	
  containing	
  a	
  protected	
  boronic	
  acid	
  will	
  enable	
  direct,	
  one-­‐step	
  radiolabeling	
   of	
   the	
   peptide.	
   	
   Alternatively,	
   functionalizing	
   the	
   LLP2A	
   with	
   an	
   azide	
   will	
   allow	
   for	
   conjugation	
  to	
  any	
  number	
  of	
  functional	
  groups	
  through	
  facile	
  click	
  chemistry.27	
   	
    44	
    	
    To	
    this	
    end,	
    2,4,6-­‐trifluoro-­‐3-­‐(4,4,5,5-­‐tetraphenyl-­‐1,3,2-­‐dioxaborolan-­‐2-­‐  yl)benzoic	
   acid	
   (referred	
   to	
   as	
   ArB(OR)2)	
   was	
   synthesized	
   (Scheme	
   2.11).	
   	
   The	
   starting	
   material,	
  2,4,6-­‐trifluorobenzoic	
  acid,	
  is	
  first	
  deprotonated	
  by	
  adding	
  two	
  equivalents	
  of	
   n-­‐butyl	
   lithium	
   while	
   at	
   -­‐78	
   °C.	
   	
   The	
   first	
   equivalent	
   deprotonates	
   the	
   carboxylic	
   acid,	
   while	
   the	
   second	
   equivalent	
   deprotonates	
   one	
   of	
   the	
   equivalent	
   aromatic	
   hydrogens	
   resulting	
   in	
   a	
   vivid	
   orange-­‐red	
   colour.	
   	
   At	
   this	
   point,	
   trimethyl	
   borate	
   is	
   added	
   to	
   the	
   reaction	
  and	
  there	
  is	
  nucleophilic	
  attack	
  by	
  the	
  carbanion	
  on	
  the	
  empty	
  p	
  orbital	
  of	
  the	
   boron.	
   	
   After	
   15	
   minutes,	
   the	
   reaction	
   was	
   quenched	
   using	
   HCl	
   and	
   allowed	
   to	
   warm	
   to	
   room	
   temperature.	
   	
   1,1,2,2-­‐Tetraphenyl	
   pinacol	
   was	
   then	
   added,	
   displacing	
   the	
   methoxy	
  groups	
  on	
  the	
  boron	
  and	
  producing	
  methanol.	
  	
  The	
  product	
  was	
  then	
  isolated	
   by	
  column	
  chromatography	
  using	
  an	
  ethyl	
  acetate	
  and	
  hexane	
  solvent	
  system.	
   F  O OH  F  O a, b, c OH  F  F  F O  B  O  F  	
   3	
    	
    Scheme	
  2.11:	
  	
  Synthesis	
  of	
  ArB(OR)2	
  3.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  2,4,6-­‐trifluorobenzoic	
  acid,	
  THF,	
  n-­‐ BuLi	
  at	
  -­‐78	
  °C	
  for	
  2	
  hours.	
  	
  b)	
  trimethyl	
  borate,	
  THF,	
  2	
  hours	
  at	
  RT.	
  	
  c)	
  HCl,	
  1,1,2,2-­‐tetraphenyl	
  pinacol,	
  THF,	
   2	
  hours	
  at	
  RT.	
    	
    	
    	
    45	
    	
    The	
  conjugation	
  of	
  the	
  ArB(OR)2	
  to	
  LLP2A(tBu)	
  was	
  done	
  in	
  similar	
  fashion	
  to	
  the	
    other	
  amide	
  bond	
  forming	
  reactions	
  used	
  during	
  the	
  SPPS	
  portion	
  of	
  the	
  project.	
  EDC,	
   HOBt,	
  pyridine	
  and	
  ArB(OR)2	
  were	
  added	
  to	
  a	
  solution	
  of	
  LLP2A(tBu)	
  and	
  DMF	
  and	
  the	
   reaction	
  was	
  allowed	
  to	
  continue	
  overnight	
  at	
  RT.	
  	
  The	
  addition	
  of	
  diethyl	
  ether	
  caused	
   a	
  white	
  precipitate	
  to	
  form,	
  which	
  was	
  then	
  be	
  isolated	
  from	
  the	
  solution.	
  	
  The	
  t-­‐butyl	
   protecting	
   group	
   on	
   the	
   side	
   chain	
   of	
   Aad	
   was	
   then	
   removed	
   by	
   adding	
   the	
   solid	
   to	
   a	
   50	
   %	
   solution	
   of	
   TFA	
   in	
   DCM	
   and	
   stirring	
   for	
   1	
   hour.	
   	
   The	
   solvent	
   volume	
   was	
   then	
   reduced	
  and	
  the	
  product	
  precipitated	
  with	
  diethyl	
  ether.	
  	
  The	
  product	
  was	
  isolated	
  by	
   chromatography	
  with	
  7	
  %	
  MeOH	
  in	
  DCM,	
  yielding	
  the	
  final	
  target	
  compound,	
  ArB(OR)2-­‐ LLP2A.	
   	
   O F  O  O OH O H2N  O  O  N H  O  H N O  N H  H N  F O O  	
  2	
    N H  HN  +  F O  B  O  N H N  O  	
  3	
    a, b O HO F  F H N  O B O  F  O  O O  O  N H  ArB(OR)2-­‐LLP2A	
  4	
    O  H N O  N H  H N  O O  HN  N H  N H  N O  	
    Scheme	
  2.12:	
  	
  Synthesis	
  of	
  ArB(OR)2-­‐LLP2A	
  4.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  ArB(OR)2,	
  LLP2A(tBu),	
  EDC,	
   HOBt,	
  pyridine,	
  THF	
  stirred	
  for	
  16	
  hour	
  at	
  RT.	
  	
  b)	
  TFA	
  in	
  DCM	
  stirred	
  for	
  1	
  hour	
  at	
  RT.	
    	
    46	
    2.3.2	
   Synthesis	
  of	
  N3-­‐LLP2A	
   	
   	
   In	
  order	
  to	
  prepare	
  LLP2A	
  for	
  click	
  chemistry,	
  the	
  peptide	
  must	
  be	
  functionalized	
   with	
  an	
  azide	
  to	
  allow	
  reaction	
  with	
  an	
  alkynyl-­‐modified	
  ArB(OR)2.	
  	
  These	
  two	
  molecules	
   may	
  then	
  be	
  conjugated	
  together	
  through	
  a	
  Huisgen	
  cycloaddition	
  click	
  reaction	
  yielding	
   a	
  1,2,3-­‐triazole,	
  which	
  is	
  catalyzed	
  by	
  copper(I).	
  	
  It	
  was	
  decided	
  that	
  5-­‐azido	
  pentanoic	
   acid46	
  would	
  provide	
  a	
  suitable	
  chain	
  length	
  and	
  would	
  allow	
  production	
  from	
  a	
  readily	
   available	
   starting	
   material,	
   ethyl	
   5-­‐bromo	
   pentanoate.	
   	
   This	
   material	
   was	
   dissolved	
   in	
   DMSO	
  and	
  NaN3	
  was	
  added,	
  causing	
  an	
  SN2	
  reaction	
  where	
  the	
  N3	
  anion	
  displaces	
  the	
   bromide.	
  	
  This	
  product,	
  ethyl	
  5-­‐azido	
  pentanoate,	
  was	
  then	
  extracted	
  into	
  diethyl	
  ether.	
  	
   The	
   ester	
   was	
   cleaved	
   by	
   addition	
   of	
   1N	
   NaOH	
   solution	
   at	
   RT,	
   stirring	
   overnight.	
   	
   The	
   solution	
  was	
  then	
  acidified	
  with	
  concentrated	
  HCl	
  and	
  the	
  product	
  was	
  again	
  extracted	
   into	
  diethyl	
  ether.	
  	
  After	
  removing	
  the	
  solvent,	
  a	
  yellow	
  oil	
  was	
  produced.	
  The	
  synthesis	
   could	
  be	
  followed	
  with	
   1H	
  NMR.	
  	
  The	
  shift	
  in	
  the	
  methylene	
  peaks	
  after	
  installation	
  of	
   the	
  N3	
  group,	
  as	
  well	
  as	
  the	
  absence	
  of	
  the	
  ethyl	
  peaks	
  following	
  ester	
  hydrolysis	
  served	
   as	
  good	
  indicators	
  of	
  successful	
  synthesis.	
   	
   	
   O Br 	
    O  a, b O  N3  OH 	
   5	
    Scheme	
  2.13:	
  	
  Synthesis	
  of	
  5-­‐azido	
  pentanoic	
  acid	
  5.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  Ethyl	
  5-­‐bromo	
   pentanoate,	
  sodium	
  azide,	
  DMSO	
  stirred	
  for	
  12	
  hours	
  at	
  RT.	
  	
  b)	
  NaOH,	
  stirred	
  for	
  12	
  hours	
  at	
  RT.	
    	
    47	
    	
    With	
  this	
  product	
  in	
  hand,	
  it	
  was	
  now	
  possible	
  to	
  acylate	
  the	
  LLP2A(tBu)	
  and	
    create	
  the	
  “clickable”	
  LLP2A	
  ligand.	
  	
  The	
  peptide	
  bond	
  between	
  5-­‐azido	
  pentanoic	
  acid	
   and	
  LLP2A(tBu)	
  was	
  formed	
  using	
  HBTU	
  and	
  DIPEA	
  in	
  DMF.	
  	
  The	
  reaction	
  proceeded	
  for	
   1	
  hour,	
  after	
  which	
  the	
  solvent	
  was	
  reduced	
  and	
  the	
  product	
  precipitated	
  with	
  diethyl	
   ether.	
  	
  The	
  precipitate	
  was	
  added	
  to	
  a	
  50	
  %	
  solution	
  of	
  TFA	
  in	
  DCM	
  to	
  remove	
  the	
  t-­‐ butyl	
  protecting	
  group	
  on	
  the	
  Aad	
  residue	
  since	
  the	
  carboxylate	
  cannot	
  participate	
  in	
   the	
  click	
  reaction	
  to	
  conjugate	
  an	
  alkynyl-­‐ArBF3.	
  	
  After	
  stirring	
  for	
  1	
  hour,	
  the	
  solvent	
   was	
  reduced	
  under	
  vacuum	
  and	
  the	
  product	
  precipitated	
  with	
  diethyl	
  ether,	
  yielding	
  a	
   white	
  solid	
  that	
  was	
  purified	
  by	
  RP-­‐HPLC.	
   	
   O O O H2N  O  O  N H  O  H N  N H  O  O  H N  O O  N H  N3  +  OH  5	
    N H  2	
   HN  N O a, b  O HO H N  N3  O O  O  HN  O  O  H N O  N H  H N  O O  N H  N3-­‐LLP2A	
  6	
   HN  N H  N O  	
    Scheme	
  2.14:	
  	
  Synthesis	
  of	
  N3-­‐LLP2A	
  6.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  5-­‐azido	
  pentanoic	
  acid	
  5,	
  LLP2A(tBu),	
   HBTU,	
  DIPEA,	
  THF	
  stirred	
  for	
  1	
  hour	
  at	
  RT.	
  	
  b)	
  TFA	
  in	
  DCM	
  stirred	
  for	
  1	
  hour	
  at	
  RT.	
    	
    	
    48	
    2.4	
   	
    Cold	
  Fluorination	
  of	
  ArB(OR)2-­‐LLP2A	
    2.4.1	
   Perspectives	
  on	
  ArBF3	
  Formation	
   	
   	
   Now	
   that	
   the	
   ArB(OR)2-­‐LLP2A	
   had	
   been	
   synthesized,	
   fluorination	
   experiments	
   could	
  be	
  conducted.	
  	
  Two	
  fluorination	
  methods	
  are	
  possible:	
  	
  1)	
  slow	
  fluorination	
  (~12	
   hours)	
  under	
  very	
  mildly	
  acidic	
  conditions	
  (pH	
  3-­‐4)	
  for	
  relatively	
  large-­‐scale	
  production	
   of	
   ArBF3-­‐LLP2A;	
   and	
   2)	
   fast	
   fluorination	
   (<	
   1	
   hour)	
   under	
   quite	
   acidic	
   (pH	
   <	
   2)	
   conditions	
   for	
  imaging-­‐scale	
  production.	
  	
  For	
  PET	
  imaging	
  purposes,	
  fluorination	
  needs	
  to	
  be	
  fast	
  in	
   order	
  to	
  minimize	
  the	
  amount	
  of	
  decay	
  that	
  occurs	
  during	
  labeling	
  and/or	
  prior	
  to	
  the	
   drug	
  reaching	
  the	
  patient.	
  	
  Unfortunately,	
  fast	
  fluorination	
  to	
  simulate	
  radiolabeling	
  of	
   ArB(OR)2s	
   requires	
   rather	
   acidic	
   conditions	
   to	
   increase	
   the	
   rate	
   of	
   removal	
   of	
   the	
   boronic	
   acid	
   protecting	
   group.	
   	
   This	
   low	
   pH	
   also	
   increases	
   the	
   possibility	
   that	
   the	
   peptide	
   may	
   be	
   altered	
   or	
   cleaved	
   in	
   some	
   way	
   during	
   the	
   fluorination	
   reaction.	
  	
   However,	
   slow	
   fluorination	
   necessitates	
   only	
   mildly	
   acidic	
   conditions,	
   allowing	
   for	
   significant	
   amounts	
   of	
   the	
   ArBF3	
   to	
   be	
   produced	
   without	
   regard	
   for	
   side	
   reactions.	
  	
   While	
   this	
   is	
   not	
   useful	
   for	
   imaging,	
   this	
   method	
   provides	
   an	
   opportunity	
   for	
   characterization	
   of	
   the	
   fluorinated	
   peptide	
   and	
   in	
   vitro	
   cell	
   binding	
   studies.	
   	
   If	
   the	
   products	
  of	
  the	
  slow	
  and	
  fast	
  fluorination	
  are	
  revealed	
  to	
  have	
  the	
  same	
  RP-­‐HPLC	
  profile	
   and	
   mass	
   spectrometry	
   analysis,	
   then	
   it	
   can	
   be	
   presumed	
   that	
   the	
   low	
   pH	
   of	
   the	
   fast	
   fluorination	
  is	
  not	
  altering	
  the	
  peptide	
  and	
  it	
  should	
  retain	
  its	
  imaging	
  abilities.	
   	
   	
   	
    	
    49	
    2.4.2	
   Slow	
  Fluorination	
  of	
  ArB(OR)2-­‐LLP2A	
   	
   	
   ArB(OR)2-­‐LLP2A	
  was	
  placed	
  in	
  an	
  Eppendorf	
  vial	
  with	
  THF	
  and	
  KHF2.	
  	
  This	
  mixture	
   was	
  vortexed	
  and	
  allowed	
  to	
  react	
  overnight	
  at	
  RT.	
  	
  The	
  product	
  was	
  precipitated	
  with	
   diethyl	
  ether.	
  	
  RP-­‐HPLC	
  and	
  mass	
  spectroscopy	
  analysis	
  of	
  the	
  precipitate	
  showed	
  a	
  high	
   conversion	
  to	
  the	
  ArBF3-­‐LLP2A	
  as	
  well	
  as	
  a	
  small	
  amount	
  of	
  deboronylated	
  side	
  product.	
   O HO F  F  O  H N  O B F  O  O  O  N H  O  O  H N  H N  N H  O  O O  N H  N H  4	
   HN  a O  N O  HO F  F  O  H N  F B F F  F  O  O  N H  O  O  H N O  ArBF3-­‐LLP2A	
  7	
    H N  N H  O O  N H  HN  N H  N  	
    O  Scheme	
  2.15:	
  	
  Synthesis	
  of	
  ArBF3-­‐LLP2A	
  7	
  by	
  slow	
  fluorination	
  method.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
   ArB(OR)2-­‐LLP2A,	
  KHF2,	
  THF	
  for	
  12	
  hours	
  at	
  RT.	
   	
   O HO F  F H N F  O  O O  O  N H  O  H N O  N H  H N  O O  HN  N H  N H  N O  	
    Figure	
  2.6:	
  	
  Structure	
  of	
  deboronylated	
  side	
  product.	
    	
    50	
    	
    ArBF3-­‐LLP2A	
    deboronylation	
    Figure	
  2.7:	
  	
  RP-­‐HPLC	
  of	
  synthesis	
  of	
  ArBF3-­‐LLP2A	
  by	
  slow	
  fluorination	
  method.	
    	
    	
    	
   	
    The	
  synthesis	
  of	
  ArBF3-­‐LLP2A	
  can	
  be	
  confirmed	
  through	
   19F	
  NMR.	
  	
  The	
  ArB(OR)2	
    will	
  have	
  three	
  peaks,	
  each	
  of	
  integration	
  1,	
  corresponding	
  to	
  the	
  fluorines	
  on	
  the	
  aryl	
   ring.	
   	
   After	
   fluorination,	
   three	
   peaks	
   of	
   integration	
   1	
   as	
   well	
   as	
   a	
   new	
   peak	
   of	
   an	
   integration	
   of	
   3,	
   representing	
   the	
   3	
   fluorides	
   captured	
   as	
   the	
   aryltrifluoroborate,	
   are	
   observed.	
   	
    	
   19  Figure	
  2.8:	
  	
   F	
  NMR	
  of	
  ArB(OR)2	
  (top)	
  and	
  ArBF3-­‐LLP2A	
  (bottom).	
    	
    51	
    2.4.3	
   PET	
  Imaging	
  Scale	
  Fast	
  Fluorination	
  of	
  ArB(OR)2-­‐LLP2A	
   	
   	
   Fast	
  fluorination	
  at	
  low	
  pH	
  using	
   19F	
  reflects	
  identical	
  conditions	
  that	
  would	
  be	
   used	
   during	
   radiolabeling	
   for	
   in	
   vivo	
   PET	
   imaging.	
   	
   Due	
   to	
   the	
   sub-­‐micromole	
   scale	
   of	
   this	
   reaction,	
   characterization	
   by	
   19F	
   NMR	
   is	
   not	
   an	
   option.	
   	
   However,	
   there	
   is	
   a	
   sufficient	
  amount	
  of	
  product	
  to	
  allow	
  investigation	
  by	
  mass	
  spectrometry	
  and	
  RP-­‐HPLC.	
   	
    100	
  Nmol	
  of	
  ArB(OR)2-­‐LLP2A	
  was	
  placed	
  in	
  a	
  small	
  Eppendorf	
  vial	
  and	
  dissolved	
    in	
  4	
  μL	
  of	
  THF.	
  	
  2	
  μL	
  of	
  0.125	
  M	
  KHF2(aq)	
  and	
  0.5	
  μL	
  HCl	
  were	
  added	
  and	
  the	
  mixture	
  was	
   vortexed	
   and	
   allowed	
   to	
   sit	
   for	
   1	
   hour.	
   	
   The	
   reaction	
   was	
   then	
   quenched	
   with	
   100	
   μL	
   of	
   a	
   solution	
   of	
   5:15:80	
   NH4OH:H2O:EtOH	
   and	
   then	
   analyzed	
   by	
   RP-­‐HPLC.	
   	
   As	
   in	
   the	
   RP-­‐ HPLC	
   of	
   the	
   slow	
   method	
   in	
   Figure	
   2.7,	
   two	
   major	
   peaks	
   were	
   present;	
   the	
   expected	
   ArBF3-­‐LLP2A	
  at	
  9.18	
  min	
  and	
  a	
  new	
  peak	
  at	
  13.17	
  min.	
  	
  These	
  peaks	
  were	
  collected	
  for	
   mass	
   spectrometry	
   analysis.	
   	
   The	
   identity	
   of	
   the	
   ArBF3-­‐LLP2A	
   was	
   confirmed,	
   and	
   the	
   corresponding	
  mass	
  of	
  the	
  other	
  peak	
  showed	
  it	
  to	
  again	
  be	
  the	
  deboronylation	
  product.	
  	
   The	
  other	
  peaks	
  were	
  not	
  examined	
  further.	
   	
    ArBF3-­‐LLP2A	
    	
    deboronylation	
    	
    Figure	
  2.9:	
  	
  RP-­‐HPLC	
  of	
  synthesis	
  of	
  ArBF3-­‐LLP2A	
  by	
  fast	
  fluorination	
  method.	
    	
    	
    52	
    	
    Although	
  the	
  fast	
  method	
  of	
  fluorination	
  also	
  leads	
  to	
  some	
  deboronylation,	
  this	
    byproduct	
   is	
   in	
   low	
   amount.	
   	
   Gratifyingly,	
   there	
   is	
   still	
   a	
   significant	
   amount	
   of	
   ArBF3-­‐ LLP2A	
   produced	
   by	
   the	
   fast	
   method,	
   suggesting	
   applicability	
   for	
   PET	
   imaging.	
   	
   It	
   is	
   important	
   to	
   note	
   that	
   the	
   ArBF3-­‐LLP2A	
   is	
   the	
   first	
   peak	
   to	
   elute	
   and	
   that	
   it	
   is	
   distanced	
   from	
   all	
   other	
   peaks,	
   allowing	
   easy	
   isolation	
   by	
   RP-­‐HPLC	
   in	
   a	
   timely	
   manner.	
   	
   In	
   a	
   radiolabeling	
   situation,	
   the	
   quenched	
   reaction	
   would	
   be	
   immediately	
   put	
   onto	
   an	
   RP-­‐ HPLC	
  and	
  the	
  pure	
  ArB18F3-­‐LLP2A	
  collected.	
  	
  A	
  sub-­‐ten	
  minute	
  elution	
  time	
  means	
  fast	
   purification	
  resulting	
  in	
  minimal	
  radioactive	
  decay	
  and	
  a	
  high	
  specific	
  activity	
  of	
  the	
  final	
   product.	
    	
   2.5	
    Biological	
  Activity	
  Confirmation	
    	
   2.5.1	
   Synthesis	
  of	
  FITC-­‐LLP2A	
   	
   	
   Before	
   the	
   ArB(OR)2-­‐LLP2A	
   can	
   be	
   radiolabeled	
   and	
   tested	
   in	
   vivo,	
   it	
   must	
   be	
   shown	
   that	
   the	
   compound	
   is	
   biologically	
   active	
   and	
   binds	
   to	
   cells	
   expressing	
   the	
   α4β1	
   integrin	
   receptor.	
   	
   To	
   perform	
   a	
   cellular	
   binding	
   assay	
   a	
   fluorescent	
   version	
   of	
   the	
   peptide	
   is	
   required.	
   	
   Of	
   the	
   many	
   fluorescent	
   labeling	
   compounds	
   available,	
   FITC	
   was	
   chosen	
   because	
   it	
   is	
   readily	
   available	
   as	
   the	
   isothiocyanate	
   which	
   makes	
   for	
   facile	
   conjugation	
  to	
  the	
  peptide	
  through	
  thiourea	
  formation.	
  	
  As	
  well,	
  it	
  provides	
  an	
  emission	
   wavelength	
  that	
  is	
  compatible	
  with	
  standard	
  fluorescent	
  microscopy	
  equipment	
  and	
  is	
   economically	
   responsible	
   compared	
   to	
   many	
   other	
   fluorescent	
   compounds.	
   	
   LLP2A(tBu)	
   was	
   added	
   to	
   a	
   solution	
   of	
   50:50	
   DMF:Na2CO3	
   buffer	
   and	
   FITC.	
   	
   The	
   reaction	
   was	
    	
    53	
    allowed	
   to	
   proceed	
   for	
   12	
   hours	
   at	
   4	
   °C	
   and	
   then	
   the	
   product	
   was	
   precipitated	
   with	
   diethyl	
  ether	
  and	
  purified.	
  	
  The	
  t-­‐butyl	
  protecting	
  group	
  was	
  then	
  removed	
  by	
  adding	
  it	
   to	
  a	
  solution	
  of	
  50	
  %	
  TFA	
  in	
  DCM	
  solution	
  and	
  stirring	
  for	
  1	
  hour.	
  	
  Addition	
  of	
  diethyl	
   ether	
  caused	
  a	
  yellow	
  solid	
  to	
  precipitate,	
  after	
  which	
  the	
  solvent	
  was	
  decanted	
  and	
  the	
   product	
  was	
  collected.	
  	
  This	
  compound	
  was	
  purified	
  by	
  TLC	
  prior	
  to	
  in	
  vitro	
  studies.	
   O HO  O O H2N  O  O  O  H N  N H  H N  N H  O  +  O O  N H  2	
    N C  O N H  S  O OH  HN  O  N a, b O O  H N  HO  HO C HN  O  O  S  O O  O  HN  O  H N O  OH  N H  H N  O O  N H  N H  FITC-­‐LLP2A	
  8	
   O  HN  N  	
    O  	
   Scheme	
  2.16:	
  	
  Synthesis	
  of	
  FITC-­‐LLP2A	
  8.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  FITC,	
  LLP2A(tBu),	
  50:50	
   DMF:Na2CO3	
  buffer	
  stirred	
  for	
  12	
  hours	
  at	
  4	
  °C.	
  	
  b)	
  TFA	
  in	
  DCM	
  stirred	
  for	
  1	
  hour	
  at	
  RT.	
    	
   	
   2.5.2	
   Cell	
  Binding	
  Assay	
   	
   	
   Two	
   experiments	
   were	
   used	
   to	
   confirm	
   the	
   compound’s	
   binding	
   activity.	
   FITC-­‐ LLP2A	
   binding	
   can	
   be	
   demonstrated	
   by	
   incubating	
   the	
   cells	
   in	
   a	
   buffer	
   containing	
   the	
    	
    54	
    compound.	
  The	
  buffer	
  is	
  removed	
  and	
  then	
  the	
  cells	
  are	
  observed	
  for	
  fluorescence.	
  	
  If	
   the	
  cells	
  exhibit	
  fluorescence,	
  the	
  peptide	
  is	
  bound	
  to	
  the	
  integrin.	
  	
  To	
  further	
  confirm	
   cellular	
  binding,	
  a	
  blocking	
  experiment	
  is	
  then	
  conducted.	
  	
  Fresh	
  cells	
  are	
  first	
  incubated	
   with	
   a	
   non-­‐fluorescent	
   analog	
   of	
   the	
   compound	
   that	
   would	
   be	
   used	
   in	
   vivo,	
   in	
   this	
   case,	
   ArBF3-­‐LLP2A.	
  	
  After	
  incubation	
  with	
  ArBF3-­‐LLP2A,	
  the	
  medium	
  is	
  removed	
  and	
  the	
  cells	
   are	
  washed	
  with	
  fresh	
  buffer.	
  	
  The	
  cells	
  are	
  then	
  incubated	
  with	
  FITC-­‐LLP2A	
  for	
  an	
  equal	
   amount	
   of	
   time.	
   	
   This	
   medium	
   is	
   washed	
   away	
   and	
   the	
   cells	
   are	
   examined	
   for	
   fluorescence.	
  	
  A	
  positive	
  binding	
  determination	
  is	
  made	
  if	
  no	
  fluorescence	
  is	
  observed,	
   as	
   the	
   binding	
   sites	
   should	
   be	
   occupied	
   by	
   ArBF3-­‐LLP2A	
   and	
   thus	
   prevent	
   the	
   binding	
   of	
   FITC-­‐LLP2A.	
  	
  With	
  positive	
  results	
  from	
  both	
  of	
  these	
  experiments,	
  the	
  biological	
  activity	
   of	
  the	
  product	
  would	
  be	
  confirmed.	
   	
    Human	
   MOLT-­‐4	
   leukemia	
   cells	
   expressing	
   the	
   α4β1	
   integrin	
   were	
   obtained	
   and	
    proliferated	
   in	
   medium	
   containing	
   10	
   mM	
   glucose.	
   	
   The	
   cells	
   were	
   passaged	
   10	
   times	
   before	
   being	
   used	
   for	
   binding	
   studies	
   to	
   ensure	
   continuing	
   viability.	
   	
   When	
   enough	
   cells	
   were	
  present,	
  they	
  were	
  split	
  into	
  several	
  batches	
  of	
  approximately	
  1	
  x	
  106	
  cells	
  each.	
  	
   One	
   aliquot	
   of	
   cells	
   was	
   incubated	
   in	
   TBS	
   buffer	
   containing	
   400	
   nM	
   FITC-­‐LLP2A	
   and	
   another	
   aliquot	
   of	
   cells	
   incubated	
   at	
   1	
   nM	
   FITC-­‐LLP2A.	
   	
   The	
   TBS	
   buffer	
   also	
   contained	
   1mM	
  Mn2+,	
  a	
  known	
  activator	
  of	
  the	
  α4β1	
  integrin.47,	
  48	
  	
  The	
  compound	
  was	
  allowed	
  to	
   bind	
   for	
   1	
   hour	
   after	
   which	
   time	
   the	
   cells	
   were	
   centrifuged	
   and	
   the	
   buffer	
   removed.	
  	
   Fresh	
   buffer	
   was	
   added	
   and	
   the	
   cells	
   centrifuged	
   and	
   fresh	
   buffer	
   added	
   once	
   more.	
  	
   This	
   process	
   was	
   repeated	
   again	
   to	
   ensure	
   that	
   no	
   fluorescent	
   compound	
   was	
   left	
   behind	
   that	
   could	
   cause	
   background	
   fluorescence.	
   	
   The	
   cells	
   were	
   then	
   spotted	
   on	
   a	
    	
    55	
    glass	
  plate	
  and	
  examined	
  for	
  fluorescence.	
  	
  Positive	
  results	
  for	
  fluorescence	
  were	
  seen	
   in	
  both	
  the	
  1	
  nM	
  and	
  400	
  nM	
  concentrations,	
  indicating	
  cellular	
  binding.	
  	
  The	
  results	
  are	
   summarized	
  in	
  Table	
  2.1	
  below.	
   	
   	
   Table	
  2.1:	
  	
  Results	
  of	
  cell	
  binding	
  fluorescence	
  assay.	
    	
    Conc.	
  FITC-­‐LLP2A	
  (nM)	
   Fluorescence	
  observed	
   1	
    Yes	
    400	
    Yes	
    	
   	
   	
    	
    a)	
    	
    	
    b)	
    	
    Figure	
  2.10:	
  	
  MOLT-­‐4	
  cells	
  exhibiting	
  fluorescence	
  after	
  being	
  treated	
  with	
  (a)	
  1	
  nM	
  FITC-­‐LLP2A	
  and	
  (b)	
   400	
  nM	
  FITC-­‐LLP2A.	
    	
    	
    	
    A	
  blocking	
  assay	
  was	
  then	
  preformed.	
  	
  The	
  cells	
  were	
  incubated	
  in	
  TBS	
  buffer	
  (1	
    mM	
  Mn2+)	
  containing	
  100	
  nM	
  ArBF3-­‐LLP2A.	
  	
  The	
  compound	
  was	
  allowed	
  to	
  bind	
  for	
  1	
    	
    56	
    hour	
  after	
  which	
  time	
  the	
  cells	
  were	
  centrifuged	
  and	
  the	
  buffer	
  removed.	
  	
  Fresh	
  buffer	
   containing	
   either	
   1	
   nM	
   or	
   400	
   nM	
   FITC-­‐LLP2A	
   was	
   then	
   added	
   and	
   the	
   cells	
   were	
   incubated	
  for	
  1	
  hour.	
  	
  The	
  buffer	
  was	
  then	
  washed	
  away	
  and	
  replaced	
  twice	
  to	
  ensure	
   removal	
   of	
   any	
   left	
   over	
   fluorescent	
   peptide,	
   after	
   which	
   the	
   cells	
   were	
   examined	
   for	
   fluorescence.	
   	
   In	
   this	
   case,	
   no	
   fluorescence	
   was	
   observed	
   for	
   both	
   samples.	
   	
   This	
   indicates	
   that	
   the	
   α4β1	
   integrin	
   binding	
   sites	
   were	
   occupied	
   during	
   the	
   first	
   incubation	
   with	
   ArBF3-­‐LLP2A,	
   preventing	
   the	
   binding	
   of	
   FITC-­‐LLP2A.	
   	
   The	
   results	
   of	
   the	
   two	
   cell	
   binding	
  assays	
  indicate	
  that	
  the	
  compound	
  of	
  interest,	
  ArBF3-­‐LLP2A,	
  is	
  biologically	
  active	
   and	
   will	
   bind	
   in	
   vivo	
   to	
   cancer	
   cells	
   expressing	
   the	
   α4β1	
   integrin.	
   	
   These	
   results	
   are	
   summarized	
  in	
  Table	
  2.2	
  below.	
   	
   	
   Table	
  2.2:	
  	
  Results	
  of	
  fluorescence	
  blocking	
  assay.	
    Conc.	
  ArBF3-­‐LLP2A	
  (nM)	
   Conc.	
  FITC-­‐LLP2A	
  (nM)	
   Fluorescence	
  observed	
   100	
    1	
    No	
    100	
    400	
    No	
    	
   	
   	
    	
    57	
    	
  	
  	
    a)	
    	
  	
  	
  	
    	
    b)	
    	
    Figure	
  2.11:	
  	
  MOLT-­‐4	
  cells	
  exhibiting	
  no	
  fluorescence	
  after	
  being	
  treated	
  with	
  100	
  nM	
  ArBF3-­‐LLP2A	
  and	
   subsequently	
  (a)	
  1	
  nM	
  FITC-­‐LLP2A	
  and	
  (b)	
  400	
  nM	
  FITC-­‐LLP2A.	
    	
   	
    2.6	
   	
    18  F-­‐Radiolabeling	
  of	
  ArB(OR)2-­‐LLP2A	
    2.6.1	
   One-­‐step	
  Radiolabeling	
   	
   	
   The	
   ArB(OR)2-­‐LLP2A	
   was	
   then	
   labeled	
   under	
   radioactive	
   or	
   “hot”	
   conditions	
   at	
   the	
  TRIUMF	
  facility	
  by	
  Dr.	
  Ying	
  Li	
  of	
  the	
  Perrin	
  group.	
  	
  Near	
  identical	
  conditions	
  to	
  those	
   listed	
   for	
   the	
   fast	
   fluorination	
   (section	
   2.4.3)	
   were	
   used,	
   except	
   now	
   using	
   a	
   18/19F	
   solution.	
  	
  The	
  reaction	
  was	
  allowed	
  to	
  proceed	
  for	
  1	
  hour	
  before	
  being	
  quenched	
  with	
   an	
   NH4OH	
   solution	
   and	
   analyzed	
   by	
   RP-­‐HPLC.	
   	
   The	
   detector	
   in	
   this	
   case	
   measures	
   radioactivity,	
  allowing	
  us	
  to	
  determine	
  if	
  more	
  than	
  one	
  radiolabeled	
  product	
  is	
  present.	
   The	
  radioactivity	
  detector	
  was	
  able	
  to	
  identify	
  only	
  one	
  radioactive	
  product	
  other	
  than	
   free	
  18F-­‐,	
  shown	
  below	
  in	
  the	
  RP-­‐HPLC	
  trace.	
    	
    58	
    O HO F F 18F  F  B F  O  H N F  O  O  N H  O  O  H N O  N H  H N  O O  N H  18  ArB F3-­‐LLP2A	
  9	
   HN  N H  N  	
    O  	
    18 -­‐	
    F  18  ArB F3-­‐LLP2A	
    	
   18  Figure	
  2.12:	
  	
  Radiolabeled	
  product	
  ArB F3-­‐LLP2A	
  9	
  (top)	
  and	
  RP-­‐HPLC	
  trace	
  (bottom)	
  of	
  radiolabeling	
   18  18 -­‐  showing	
  ArB F3-­‐LLP2A	
  9	
  (20	
  min)	
  and	
  free	
   F 	
  (4	
  min).	
    	
    	
    	
    The	
  reaction	
  was	
  done	
  using	
   18/19F	
  	
  with	
  a	
  specific	
  activity	
  of	
  1.96	
  mCi/µmol	
  at	
    the	
  beginning	
  of	
  synthesis	
  (BOS).	
  	
  At	
  the	
  end	
  of	
  synthesis	
  (EOS),	
  after	
  60	
  minutes,	
  the	
   specific	
  activity	
  was	
  measured	
  to	
  be	
  1.31	
  mCi/µmol.	
  	
  100	
  nmol	
  of	
  ArB(OR)2-­‐LLP2A	
  was	
   used	
   for	
   this	
   reaction,	
   with	
   each	
   molecule	
   capturing	
   three	
   fluorides	
   atoms,	
   therefore	
   tripling	
   the	
   specific	
   activity	
   of	
   the	
   imaging	
   compound,	
   yielding	
   a	
   radiotracer	
   with	
   a	
   specific	
  activity	
  of	
  3.93	
  mCi/µmol.	
   	
   	
    	
    59	
    2.6.2	
   Two-­‐step	
  Radiolabeling	
  via	
  Click	
  Chemistry	
   	
   	
   The	
   two-­‐step	
   click	
   labeling	
   of	
   N3-­‐LLP2A	
   was	
   also	
   investigated	
   by	
   Dr.	
   Li.	
   	
   During	
   the	
  course	
  of	
  this	
  project,	
  ongoing	
  study	
  concerning	
  the	
  fluorination	
  of	
  boronates	
  was	
   performed	
   by	
   the	
   Perrin	
   group.	
   	
   New	
   protecting	
   groups	
   for	
   boron	
   were	
   considered	
   in	
   hopes	
   of	
   identifying	
   a	
   more	
   acid	
   labile	
   protecting	
   group	
   that	
   would	
   afford	
   higher	
   fluorination	
   yields.	
   	
   One	
   of	
   the	
   more	
   promising	
   protecting	
   groups	
   was	
   diamino	
   naphthalene	
  (R’).	
  	
  An	
  alkynyl-­‐modified	
  ArB(OR’)2	
  that	
  uses	
  diamino	
  naphthalene	
  instead	
   of	
   1,1,2,2-­‐tetraphenyl	
   pinacol	
   (R)	
   as	
   a	
   protecting	
   group	
   was	
   used	
   during	
   the	
   click	
   labeling	
   of	
   N3-­‐LLP2A.	
   	
   The	
   alkynyl-­‐ArB(OR’)2	
   was	
   first	
   labeled	
   under	
   similar	
   conditions	
   to	
   those	
   used	
   in	
   the	
   one-­‐step	
   fluorination	
   described	
   above	
   in	
   section	
   2.6.1.	
   	
   The	
   specific	
   activity	
  at	
  the	
  BOS	
  was	
  3.68	
  mCi/µmol.	
   	
   	
   F  O F  N H F B  N H  a  F HN  O  F  NH  F 18F  B F  F  	
   18  Scheme	
  2.17:	
  	
  Synthesis	
  of	
  alkynyl-­‐ArB F3.	
  	
  Reagents	
  and	
  conditions:	
  	
  a)	
  alkynyl-­‐ArB(OR’)2,	
    18/19  F(aq),	
  HCl,	
    and	
  THF	
  reacted	
  for	
  22	
  minutes	
  at	
  RT.	
   	
   	
    	
    60	
    18  alkynyl-­‐ArB F3	
   18 -­‐	
    F  Figure	
  2.13:	
  	
  RP-­‐HPLC	
  of	
  alkynyl-­‐ArB F3.	
   18  	
    The	
   alkynyl-­‐ArB18F3	
   was	
   then	
   conjugated	
   to	
   N3-­‐LLP2A	
   via	
   click	
   chemistry	
   in	
   a	
    	
    solution	
   containing	
   CuSO4	
   and	
   sodium	
   ascorbate.	
   	
   The	
   specific	
   activity	
   at	
   the	
   EOS	
   was	
   2.04	
   mCi/µmol.	
   	
   When	
   taking	
   into	
   account	
   the	
   100	
   nmol	
   of	
   N3-­‐LLP2A	
   used	
   in	
   this	
   reaction,	
  the	
  specific	
  activity	
  of	
  the	
  labeled	
  compound	
  is	
  calculated	
  to	
  be	
  6.12	
  mCi/µmol.	
  	
   The	
  RP-­‐HPLC	
  analysis	
  of	
  the	
  labeling	
  reaction	
  and	
  the	
  click	
  conjugation	
  are	
  shown	
  below.	
   O HO H N  N3 F  O  +  O O  O  HN  O  H N  O  O  N H  H N  O O  N H  N H F  6	
    F 18F  B F  N H  HN  F  N  a  O O HO  O NH  F  F  N N  H N  N  O O  O  O F  B 18F  F  HN  O  H N O  N H  H N  O O  N H  18  ArB F3-­‐LLP2A	
  10	
    F  HN  N H  N O  Scheme	
  2.18:	
  	
  Synthesis	
  of	
  the	
  final	
  product	
  of	
  the	
  two-­‐step	
  radiolabeling	
  of	
  LLP2A.	
  	
  Reagents	
  and	
   conditions:	
  	
  a)	
  alkynyl-­‐ArB F3,	
  N3-­‐LLP2A,	
  NaAsc,	
  CuSO4,	
  EtOH(aq)	
  reacted	
  for	
  36	
  minutes	
  at	
  RT.	
   18  	
    61	
    18  ArB F3-­‐LLP2A	
    18 -­‐	
    F  18  alkynyl-­‐ArB F3	
    	
   18  18  Figure	
  2.14:	
  	
  RP-­‐HPLC	
  of	
  the	
  final	
  radiolabeled	
  ArB F3-­‐LLP2A	
  10	
  (19	
  min)	
  and	
  alkynyl-­‐ArB F3	
  (15	
  min).	
    	
    62	
    Chapter	
  3	
   Conclusions	
  and	
  Future	
  Directions	
   	
   3.1	
   	
    Conclusions	
    	
    Novel	
   methods	
   for	
   imaging	
   cancer	
   are	
   needed	
   in	
   the	
   face	
   of	
   an	
   increasingly	
    prevalent	
  disease	
  that	
  touches	
  all	
  corners	
  of	
  the	
  globe.	
  The	
  replacement	
  of	
   18FDG	
  with	
   tumor-­‐specific	
   imaging	
   agents	
   such	
   as	
   peptides	
   for	
   routine	
   PET	
   imaging	
   will	
   improve	
   both	
  the	
  diagnosing	
  and	
  prognosis	
  of	
  cancer	
  patients.9	
   	
    Perrin	
   and	
   colleagues	
   have	
   developed	
   an	
   innovative	
   method	
   of	
   18F-­‐labeling	
    through	
   aryltrifluoroborate	
   formation	
   under	
   conditions	
   that	
   are	
   compatible	
   with	
   biomolecules.29	
   	
   Aryltrifluoroborates,	
   having	
   been	
   earlier	
   found	
   to	
   be	
   stable	
   under	
   physiological	
   conditions,32	
   open	
   the	
   door	
   to	
   previously	
   unattainable	
   specific	
   activities	
   through	
  the	
  capture	
  of	
  three	
   18F	
  atoms	
  per	
  biomolecule,	
  effectively	
  tripling	
  the	
  specific	
   activity	
  of	
  the	
  source	
  fluoride.31	
   	
    The	
   α4β1	
   integrin-­‐targeting	
   compound	
   LLP2A38	
   was	
   synthesized	
   on	
   solid	
   phase	
    and	
  coupled	
  to	
  ArB(OR)2,	
  which	
  was	
  then	
  fluorinated	
  under	
  cold	
  conditions	
  to	
  produce	
   ArBF3-­‐LLP2A.	
  	
  This	
  compound,	
  along	
  with	
  the	
  fluorescent	
  FITC-­‐LLP2A,	
  was	
  tested	
  in	
  vitro	
   against	
  α4β1	
  expressing	
  cells	
  and	
  found	
  to	
  be	
  biologically	
  active.	
  	
  An	
  N3-­‐LLP2A	
  was	
  also	
   prepared	
   to	
   allow	
   for	
   two-­‐step	
   labeling	
   via	
   click	
   chemistry.	
   	
   ArB(OR)2-­‐LLP2A	
   and	
   N3-­‐ LLP2A	
   were	
   radiolabeled	
   by	
   one-­‐step	
   and	
   two-­‐step	
   methods,	
   respectively,	
   to	
   produce	
   ArB18F3-­‐LLP2A.	
  	
  These	
  compounds	
  are	
  now	
  ready	
  to	
  proceed	
  to	
  in	
  vivo	
  imaging.	
    	
    63	
    3.2	
    Future	
  Directions	
    	
   	
    With	
  the	
  successful	
  synthesis	
  of	
  ArB18F3-­‐LLP2A,	
  the	
  immediate	
  next	
  step	
  is	
  the	
  in	
    vivo	
   imaging	
   of	
   a	
   α4β1-­‐expressing	
   tumor.	
   	
   Discussion	
   on	
   collaborative	
   imaging	
   projects	
   with	
  research	
  groups	
  around	
  the	
  continent	
  is	
  underway.	
  	
  Continuing	
  investigation	
  into	
   LLP2A	
   and	
   the	
   aryltrifluoroborate	
   labeling	
   technology	
   allows	
   us	
   to	
   be	
   optimistic	
   that	
   compounds	
   based	
   on	
   these	
   structures	
   may	
   soon	
   find	
   their	
   way	
   into	
   clinical	
   research	
   trials.	
   	
    The	
   development	
   of	
   new	
   boronic	
   acid	
   protecting	
   groups	
   such	
   as	
    diaminonapthalene	
   grants	
   new	
   possibilities	
   for	
   one-­‐step	
   radiolabeling	
   of	
   a	
   ArB(OR)2-­‐ LLP2A.	
   	
   The	
   increased	
   acid-­‐lability	
   of	
   novel	
   protecting	
   group	
   may	
   provide	
   high	
   yield	
   fluorinations	
  under	
  milder	
  conditions.	
   	
    The	
   azido-­‐modified	
   LLP2A	
   presents	
   several	
   other	
   interesting	
   applications	
   for	
   this	
    bioligand.	
   	
   Facile	
   conjugation	
   under	
   mild	
   conditions	
   afforded	
   by	
   click	
   chemistry	
   have	
   potential	
  for	
  use	
  not	
  only	
  in	
  imaging,	
  but	
  also	
  therapeutics.	
  	
  The	
  high-­‐affinity	
  targeting	
   of	
  LLP2A	
  make	
  it	
  a	
  good	
  candidate	
  for	
  conjugation	
  with	
  a	
  high-­‐toxicity	
  drug.	
  	
  One	
  could	
   envision	
   a	
   LLP2A	
   ligand	
   functionalized	
   with	
   both	
   imaging	
   and	
   therapeutic	
   moieties,	
   allowing	
  simultaneous	
  visualization	
  and	
  treatment	
  of	
  a	
  tumor.	
  	
   	
   	
   	
   	
    	
    64	
    Chapter	
  4	
  	
   Experimental	
    	
   4.1	
    Materials	
    	
   	
    All	
  chemicals	
  and	
  solvents	
  were	
  purchased	
  from	
  Sigma-­‐Aldrich,	
  Alfa	
  Aesar,	
  Nova	
    Biochem	
   or	
   Chem-­‐Impex	
   International	
   and	
   used	
   without	
   further	
   purification,	
   unless	
   otherwise	
   noted.	
   	
   Thin	
   layer	
   chromatography,	
   both	
   analytical	
   and	
   preparative,	
   was	
   performed	
   on	
   either	
   aluminum	
   or	
   glass	
   backed	
   plates	
   coated	
   with	
   Silica	
   Gel	
   60	
   F254	
   which	
  had	
  been	
  purchased	
  from	
  EMD	
  Chemicals.	
  	
  Silica	
  Gel	
  60	
  for	
  chromatography	
  was	
   purchased	
   from	
   Silicycle.	
   	
   Spin	
   columns	
   for	
   SPPS	
   were	
   purchased	
   from	
   Thermo	
   Scientific.	
   	
   NMR	
   experiments	
   were	
   performed	
   in	
   deuterated	
   solvent	
   purchased	
   from	
   Cambridge	
   Isotope	
   Laboratories.	
   	
   Distillation	
   over	
   sodium	
   metal	
   under	
   nitrogen	
   atmosphere	
   was	
   used	
   to	
   produce	
   anhydrous	
   THF.	
   	
   All	
   other	
   solvents	
   were	
   used	
   as	
   received	
  from	
  the	
  vendor.	
  	
  Cell	
  lines	
  were	
  purchased	
  from	
  ATCC.	
  	
  Cell	
  growth	
  medium	
   was	
  purchased	
  from	
  Invitrogen.	
  	
    	
   4.2	
    Techniques	
    	
   4.2.1	
  	
  	
   NMR	
  Spectroscopy	
   	
   	
   All	
   NMR	
   experiments	
   were	
   performed	
   on	
   a	
   Bruker	
   AV-­‐300	
   (300	
   MHz)	
   spectrometer.	
  	
  All	
  NMR	
  samples	
  were	
  prepared	
  in	
  glass	
  vials	
  before	
  being	
  transferred	
  to	
   an	
   NMR	
   tube.	
   	
   The	
   solubility	
   of	
   each	
   sample	
   was	
   tested	
   in	
   non-­‐deuterated	
   solvent	
   prior	
    	
    65	
    to	
  sample	
  preparation	
  in	
  deuterated	
  solvent.	
  	
  NMR	
  tubes	
  from	
  Norell	
  were	
  used	
  for	
  all	
   experiments.	
  	
  All	
  NMR	
  spectra	
  are	
  referenced	
  to	
  the	
  residual	
  solvent	
  peak	
  in	
  accordance	
   with	
  the	
  values	
  listed	
  by	
  Gottlieb	
  et	
  al.49	
  	
  Peaks	
  for	
  all	
  spectra	
  are	
  reported	
  as	
  chemical	
   shifts,	
  δ	
  (ppm),	
  and	
  multiplicity	
  (s	
  =	
  singlet,	
  d	
  =	
  doublet,	
  t	
  =	
  triplet,	
  m	
  =	
  multiplet).	
  	
  All	
   data	
  was	
  processed	
  using	
  iNMR	
  or	
  Mnova	
  programs.	
  	
    	
   4.2.2	
   Mass	
  Spectrometry	
   	
   	
   Low	
   resolution	
   mass	
   spectra	
   were	
   acquired	
   using	
   a	
   Waters	
   ZQ	
   spectrometer.	
  	
   High	
  resolution	
  mass	
  spectra	
  were	
  acquired	
  by	
  staff	
  at	
  the	
  UBC	
  Mass	
  Spectrometry	
  lab	
   using	
  a	
  Waters/Micromass	
  LCT	
  time-­‐of-­‐flight	
  spectrometer.	
  	
  All	
  samples	
  were	
  prepared	
   in	
  either	
  methanol	
  or	
  a	
  mixture	
  of	
  acetonitrile	
  and	
  water.	
  	
  	
    	
   4.2.3	
   Chromatography	
   	
   	
   TLC	
  results	
  are	
  reported	
  as	
  Rf,	
  describing	
  the	
  ratio	
  of	
  the	
  distance	
  moved	
  by	
  the	
   analyte	
  to	
  the	
  distance	
  moved	
  by	
  solvent	
  front.	
  	
  Detection	
  of	
  Rf	
  was	
  performed	
  using	
  a	
   handheld	
  UV	
  lamp	
  operating	
  at	
  both	
  254	
  and	
  365	
  nm	
  wavelengths.	
  	
  In	
  the	
  absence	
  of	
   UV	
   absorbance,	
   or	
   for	
   further	
   analyte	
   identification,	
   ninhydrin	
   stain	
   was	
   also	
   used.	
  	
   Column	
  chromatography	
  was	
  conducted	
  using	
  Silica	
  Gel	
  60	
  packed	
  between	
  two	
  layers	
   of	
  sea	
  washed	
  sand	
  that	
  was	
  purchased	
  from	
  Fischer	
  Scientific.	
   	
    RP-­‐HPLC	
   analyses	
   are	
   reported	
   in	
   minutes	
   as	
   retention	
   time,	
   tR.	
   	
   An	
   Agilent	
   1100	
    HPLC	
   furnished	
   with	
   an	
   automatic	
   injection	
   unit	
   and	
   photodiode	
   array	
   detector	
   was	
    	
    66	
    used	
  to	
  perform	
  RP-­‐HPLC.	
  	
  Both	
  analytical	
  and	
  preparative	
  style	
  reverse	
  phase	
  columns	
   were	
  employed	
  for	
  purification:	
  	
  (1)	
  a	
  Phenomenex	
  Jupiter	
  C18	
  (250	
  x	
  4.60	
  mm)	
  and	
  (2)	
   an	
  Agilent	
  Eclipse	
  XDB-­‐C18	
  (250	
  x	
  9.4	
  mm).	
  	
  All	
  RP-­‐HPLC	
  was	
  performed	
  with	
  the	
  columns	
   maintained	
  at	
  50	
  °C.	
  	
  Three	
  different	
  programs,	
  each	
  run	
  at	
  1	
  mL/min,	
  were	
  used	
  for	
  RP-­‐ HPLC	
   analysis:	
   	
   Program	
   1,	
   Program	
   2,	
   and	
   Program	
   3.	
   	
   Program	
   1	
   consists	
   of	
   two	
   solvents:	
  	
  A)	
  	
  Water	
  +	
  0.1	
  %	
  TFA	
  and	
  B)	
  	
  ACN	
  +	
  0.05	
  %	
  TFA.	
  	
  Program	
  1	
  was	
  carried	
  out	
  as	
   follows	
  with	
  the	
  mobile	
  phase	
  composition	
  given	
  as	
  a	
  percentage	
  of	
  solvent	
  B:	
  	
  30	
  %	
  B	
   over	
  5	
  min;	
  30	
  %	
  to	
  90	
  %	
  B	
  over	
  15	
  min;	
  90	
  %	
  to	
  100	
  %	
  B	
  over	
  5	
  min;	
  100	
  %	
  B	
  over	
  3	
  min,	
   100	
  %	
  to	
  30	
  %	
  B	
  over	
  2	
  min;	
  30	
  %	
  B	
  over	
  2	
  min.	
  	
  Program	
  2	
  consisted	
  of	
  two	
  solvents:	
  	
  C)	
  	
   Water	
   and	
   D)	
   	
   MeOH.	
   	
   Program	
   2	
   was	
   carried	
   out	
   as	
   follows	
   with	
   the	
   mobile	
   phase	
   composition	
   given	
   as	
   a	
   percentage	
   of	
   solvent	
   D:	
   	
   15	
   %	
   to	
   35	
   %	
   D	
   over	
   5	
   min;	
   35	
   %	
   to	
   90	
   %	
  D	
  over	
  15	
  min;	
  90	
  %	
  to	
  100	
  %	
  D	
  over	
  5	
  min;	
  100	
  %	
  D	
  over	
  3	
  min;	
  100	
  %	
  to	
  50	
  %	
  D	
  over	
   2	
  min;	
  50	
  %	
  D	
  over	
  2	
  min.	
  	
  Program	
  3	
  consisted	
  of	
  two	
  solvents:	
  	
  E)	
  	
  0.04	
  M	
  HCO2NH4	
   and	
  F)	
  	
  ACN.	
  	
  Program	
  3	
  was	
  carried	
  out	
  as	
  follows	
  with	
  the	
  mobile	
  phase	
  composition	
   given	
  as	
  a	
  percentage	
  of	
  solvent	
  F:	
  	
  0	
  to	
  5	
  %	
  F	
  over	
  5	
  min;	
  5	
  %	
  to	
  20	
  %	
  F	
  over	
  5	
  min;	
  20	
  %	
   to	
  50	
  %	
  F	
  over	
  10	
  min;	
  50	
  %	
  to	
  100	
  %	
  F	
  over	
  5	
  min;	
  100	
  %	
  to	
  95	
  %	
  F	
  over	
  3	
  min;	
  95	
  %	
  to	
   5	
  %	
  F	
  over	
  2	
  min;	
  5	
  %	
  F	
  over	
  2	
  min.	
   	
   4.2.4	
  	
  	
   UV-­‐Visible	
  Absorption	
   	
   	
   A	
   Beckman	
   Coulter	
   DU800	
   spectrophotometer,	
   equipped	
   with	
   both	
   tungsten	
   and	
   deuterium	
   lamps,	
   was	
   used	
   to	
   record	
   all	
   UV-­‐Visible	
   absorbance	
   spectra.	
   	
   Samples	
   were	
   prepared	
   in	
   glass	
   vials	
   prior	
   to	
   transfer	
   to	
   quartz	
   cells	
   for	
   testing.	
   	
   Transfer	
   was	
   	
    67	
    accomplished	
   using	
   polyethylene	
   disposable	
   transfer	
   pipets	
   purchased	
   from	
   Fischer	
   Scientific.	
  	
  Quartz	
  cells	
  of	
  1	
  cm	
  path	
  length	
  were	
  purchased	
  from	
  Fisher	
  Scientific.	
   	
   4.2.5	
   Cell	
  Proliferation	
   	
   	
   Human	
  MOLT-­‐4	
  Leukemia	
  cells	
  were	
  incubated	
  at	
  37	
  °C	
  in	
  5%	
  CO2	
  atmosphere	
  in	
   RPMI-­‐1640	
  medium	
  containing	
  10%	
  fetal	
  bovine	
  serum.	
  	
  Cells	
  were	
  grown	
  in	
  Falcon	
   brand	
  T-­‐25	
  flasks.	
  	
   	
   4.2.6	
   Fluorescence	
  Microscopy	
   	
   	
   Fluorescence	
  microscopy	
  was	
  performed	
  on	
  an	
  Olympus	
  IX70	
  microscope.	
   	
    4.3	
    Solid	
  Phase	
  Peptide	
  Synthesis	
    	
   4.3.1	
   Resin	
  Techniques	
   4.3.1.1	
  Resin	
  Loading	
  Determination	
   	
   	
   Resin	
   loading	
   of	
   amino	
   acids	
   was	
   determined	
   by	
   DBF	
   quantification	
   resulting	
   from	
   Fmoc	
   cleavage.	
   	
   Briefly,	
   a	
   small	
   portion	
   of	
   resin	
   1	
   was	
   placed	
   in	
   a	
   glass	
   vial	
   and	
   dried	
   overnight	
   under	
   vacuum.	
   	
   This	
   was	
   done	
   in	
   order	
   to	
   remove	
   all	
   residual	
   solvent	
   that	
   could	
   be	
   incorrectly	
   attributed	
   to	
   resin	
   mass.	
   	
   The	
   dried	
   resin	
   was	
   weighed	
   and	
   then	
  swollen	
  in	
  1.980	
  mL	
  DMF	
  with	
  slow	
  stirring.	
  	
  After	
  20	
  minutes,	
  20	
  μL	
  of	
  DBU	
  was	
   added.	
  	
  This	
  reaction	
  was	
  allowed	
  to	
  proceed	
  for	
  approximately	
  20	
  minutes	
  with	
  slow	
   stirring.	
   	
   The	
   reaction	
   was	
   then	
   diluted	
   to	
   10	
   mL	
   with	
   ACN.	
   	
   One	
   mL	
   of	
   this	
   solution	
   was	
    	
    68	
    then	
   diluted	
   to	
   13	
   mL	
   with	
   ACN.	
   	
   A	
   blank	
   solution	
   was	
   prepared	
   in	
   parallel	
   to	
   the	
   procedure	
   described	
   above,	
   although	
   no	
   resin	
   was	
   used.	
   	
   The	
   absorbance	
   of	
   the	
   solution	
  was	
  measured	
  at	
  a	
  single	
  wavelength,	
  304	
  nm.	
  	
  The	
  molar	
  extinction	
  coefficient	
   of	
   DBF	
   is	
   ε	
   =	
   7624	
   M-­‐1cm-­‐1.	
   	
   The	
   loading	
   of	
   the	
   resin	
   was	
   then	
   calculated	
   to	
   yield	
   an	
   amount	
  in	
  mmol/g.	
   	
   4.3.1.2	
  Resin	
  Cleavage	
   	
   	
   All	
   products	
   were	
   cleaved	
   from	
   the	
   solid	
   phase	
   resin,	
   for	
   mass	
   spectrometry	
   analysis	
  or	
  product	
  collection,	
  using	
  HFIP.	
  	
  Briefly,	
  to	
  the	
  resin	
  to	
  be	
  cleaved	
  was	
  added	
  a	
   30	
  %	
  solution	
  of	
  HFIP	
  in	
  DCM.	
  	
  The	
  reaction	
  was	
  allowed	
  to	
  proceed	
  for	
  30	
  minutes.	
  	
  The	
   solution	
   was	
   separated	
   from	
   the	
   resin	
   by	
   gravity	
   filtration	
   and	
   the	
   resin	
   was	
   washed	
   with	
  DCM.	
  	
  The	
  volume	
  of	
  the	
  filtrate	
  was	
  decreased	
  using	
  a	
  rotary	
  evaporator	
  to	
  yield	
   the	
  product.	
   	
   	
   4.3.2	
   Fmoc-­‐Ach	
  (resin	
  1)	
   O H N  O  O  N H  H N  O O  	
    	
    O-­‐bis-­‐(aminoethyl)ethylene	
   glycol	
   trityl	
   resin	
   (0.642	
   g)	
   was	
   swollen	
   in	
   5	
   mL	
   of	
    DCM	
   for	
   2	
   hours	
   in	
   a	
   10	
   mL	
   RBF.	
   	
   The	
   resin	
   was	
   transferred	
   to	
   a	
   10	
   mL	
   plastic	
   spin	
   column	
  and	
  the	
  solvent	
  filtered	
  off.	
  	
  To	
  a	
  glass	
  vial	
  was	
  added	
  Fmoc-­‐Ach-­‐OH	
  (0.899	
  g,	
    	
    69	
    2.46	
   mmol),	
   HBTU	
   (0.958	
   g,	
   2.52	
   mmol),	
   DIPEA	
   (0.90	
   mL,	
   5.17	
   mmol),	
   and	
   4	
   mL	
   DMF.	
  	
   These	
   compounds	
   were	
   allowed	
   to	
   dissolve	
   and	
   this	
   solution	
   was	
   added	
   to	
   the	
   spin	
   column	
   containing	
   the	
   resin.	
   	
   The	
   resin	
   was	
   gently	
   shaken	
   for	
   1	
   hour,	
   after	
   which	
   the	
   solution	
  was	
  filtered	
  off.	
  	
  The	
  resin	
  was	
  washed	
  with	
  DMF	
  (3	
  x	
  5	
  mL)	
  and	
  DCM	
  (3	
  x	
  5	
  mL)	
   and	
  suction	
  dried	
  using	
  an	
  aspirator	
  for	
  20	
  minutes	
  to	
  afford	
  resin	
  1.	
  	
  A	
  sample	
  of	
  this	
   resin	
   was	
   removed	
   and	
   the	
   product	
   cleaved	
   off	
   and	
   dissolved	
   in	
   methanol	
   for	
   mass	
   spectrometry	
   analysis.	
   	
   The	
   loading	
   of	
   resin	
   1	
   was	
   determined	
   by	
   the	
   technique	
   described	
  in	
  section	
  4.3.1.1	
  to	
  be	
  0.47	
  mmol/g.	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C28H37N3O5	
  	
  [M+H+]:	
  	
  496.27,	
  found:	
  	
  496.5.	
   	
   	
   4.3.3	
   Fmoc-­‐Aad(tBu)-­‐Ach	
  (resin	
  2)	
   	
   O O O H N  O  O  N H  O H N  N H  O  O  	
   	
    Resin	
   1	
  (0.521	
  g)	
  was	
  shaken	
  with	
  7	
  mL	
  of	
  a	
  solution	
  of	
  20%	
  piperidine	
  in	
  DMF	
    for	
   10	
   minutes.	
   	
   The	
   solution	
   was	
   removed	
   from	
   the	
   resin	
   by	
   filtration	
   and	
   this	
   procedure	
  was	
  repeated	
  twice	
  more.	
  	
  The	
  resin	
  was	
  then	
  washed	
  thoroughly	
  with	
  DMF	
   to	
  afford	
  the	
  Fmoc-­‐deprotected	
  NH2-­‐Ach	
  resin	
   1.	
  	
  To	
  deprotected	
   resin	
   1	
  was	
  added	
  a	
   solution	
  of	
  Fmoc-­‐Aad(tBu)-­‐OH	
  (0.530	
  g,	
  1.20	
  mmol),	
  HBTU	
  (0.468	
  g,	
  1.23	
  mmol),	
  DIPEA	
   (0.43	
  mL,	
  2.46	
  mmol),	
  and	
  4	
  mL	
  DMF.	
  	
  The	
  mixture	
  was	
  gently	
  shaken	
  for	
  1	
  hour	
  and	
  the	
   	
    70	
    solution	
  was	
  removed	
  by	
  filtration.	
  	
  The	
  resin	
  was	
  washed	
  with	
  DMF	
  (3	
  x	
  5	
  mL)	
  and	
  DCM	
   (3	
  x	
  5	
  mL)	
  and	
  suction	
  dried	
  on	
  an	
  aspirator	
  for	
  20	
  minutes	
  to	
  afford	
  resin	
  2.	
  	
  A	
  sample	
   of	
   this	
   resin	
   was	
   removed	
   and	
   the	
   product	
   cleaved	
   off	
   and	
   dissolved	
   in	
   methanol	
   for	
   mass	
  spectrometry	
  analysis.	
  	
  	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C38H54N4O8	
  	
  [M+H+]:	
  	
  695.86,	
  found:	
  	
  695.7.	
   	
   	
   4.3.4	
   Fmoc-­‐Lys(Dde)-­‐Aad(tBu)-­‐Ach	
  (resin	
  3)	
   	
   O O O H N  O  O  N H  O H N  N H  H N  O  O O  HN O  O  	
   	
    Resin	
  2	
  (0.521	
  g)	
  was	
  shaken	
  with	
  7	
  mL	
  of	
  a	
  solution	
  of	
  20	
  %	
  piperidine	
  in	
  DMF	
    for	
   10	
   minutes.	
   	
   The	
   solution	
   was	
   removed	
   from	
   the	
   resin	
   by	
   filtration	
   and	
   this	
   procedure	
  was	
  repeated	
  twice	
  more.	
  	
  The	
  resin	
  was	
  then	
  washed	
  thoroughly	
  with	
  DMF	
   to	
  afford	
  the	
  Fmoc-­‐deprotected	
  NH2-­‐Aad(tBu)-­‐Ach	
  resin	
  2.	
  	
  To	
  deprotected	
  resin	
  2	
  was	
   added	
  a	
  solution	
  of	
  Fmoc-­‐Lys(Dde)-­‐OH	
  (0.642	
  g,	
  1.20	
  mmol),	
  HBTU	
  (0.477	
  g,	
  1.26	
  mmol),	
   DIPEA	
  (0.45	
  mL,	
  2.57	
  mmol),	
  and	
  4	
  mL	
  DMF.	
  	
  The	
  mixture	
  was	
  gently	
  shaken	
  for	
  1	
  hour	
   and	
  the	
  solution	
  was	
  then	
  removed	
  from	
  the	
  resin	
  by	
  filtration.	
  	
  The	
  resin	
  was	
  washed	
    	
    71	
    with	
   DMF	
   (3	
   x	
   5	
   mL)	
   and	
   DCM	
   (3	
   x	
   5	
   mL)	
   and	
   suction	
   dried	
   on	
   an	
   aspirator	
   for	
   20	
   minutes	
   to	
   afford	
   resin	
  3.	
   	
   A	
   sample	
   of	
   this	
   resin	
   was	
   removed	
   and	
   the	
   product	
   cleaved	
   off	
  and	
  dissolved	
  in	
  methanol	
  for	
  mass	
  spectrometry	
  analysis.	
  	
  	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C54H78N6O11	
  	
  [M+H+]:	
  	
  988.23,	
  found:	
  	
  988.0.	
   	
   	
   4.3.5	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl-­‐Lys(Dde)-­‐Aad(tBu)-­‐Ach	
  (resin	
  4)	
   	
    	
   	
    Resin	
  3	
  (0.521	
  g)	
  was	
  shaken	
  with	
  7	
  mL	
  of	
  a	
  solution	
  of	
  20%	
  piperidine	
  in	
  DMF	
    for	
   10	
   minutes.	
   	
   The	
   solution	
   was	
   removed	
   from	
   the	
   resin	
   by	
   filtration	
   and	
   this	
   procedure	
  was	
  repeated	
  twice	
  more.	
  	
  The	
  resin	
  was	
  then	
  washed	
  thoroughly	
  with	
  DMF	
   to	
   afford	
   the	
   Fmoc-­‐deprotected	
   NH2-­‐Lys(Dde)-­‐Aad(tBu)-­‐Ach	
   resin	
   3.	
   	
   To	
   deprotected	
   resin	
   3	
   was	
   added	
   a	
   solution	
   of	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
   acid	
   1	
   (0.362g,	
   1.27mmol),	
   HBTU	
   (0.492	
   g,	
   1.30	
   mmol),	
   DIPEA	
   (0.45	
   mL,	
   2.57	
   mmol),	
   and	
   4	
   mL	
   DMF.	
  	
   The	
  resin	
  was	
  gently	
  shaken	
  for	
  1	
  hour	
  and	
  the	
  solution	
  was	
  then	
  removed	
  by	
  filtration	
   and	
  the	
  resin	
  washed	
  with	
  DMF	
  and	
  DCM.	
  	
  The	
  reaction	
  was	
  repeated	
  using	
  0.178	
  g	
  1,	
    	
    72	
    0.251	
  g	
  HBTU,	
  and	
  0.25	
  mL	
  DIPEA	
  to	
  ensure	
  full	
  coupling.	
  	
  The	
  resin	
  was	
  washed	
  with	
   DMF	
   (3	
   x	
   5	
   mL)	
   and	
   DCM	
   (3	
   x	
   5	
   mL)	
   and	
   suction	
   dried	
   on	
   an	
   aspirator	
   for	
   20	
   minutes	
   to	
   afford	
   resin	
   4.	
   	
   A	
   sample	
   of	
   this	
   resin	
   was	
   removed	
   and	
   the	
   product	
   cleaved	
   and	
   dissolved	
  in	
  methanol	
  to	
  allow	
  mass	
  spectrometry	
  analysis.	
  	
  	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C55H82N8O11	
  	
  [M+H+]:	
  	
  1032.29,	
  found:	
  	
  1032.2.	
   	
   	
   4.3.6	
   [2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl]-­‐Lys(3-­‐(3-­‐pyridyl)	
  acrylyl)-­‐Aad(tBu)-­‐Ach	
   (resin	
  5)	
   	
    	
   	
    Resin	
  4	
   (0.521	
   g)	
   was	
   shaken	
   with	
   5	
   mL	
   of	
   a	
   solution	
   of	
   2%	
   hydrazine	
   in	
   DMF	
   for	
    4	
   minutes.	
   	
   The	
   solution	
   was	
   removed	
   from	
   the	
   resin	
   by	
   filtration	
   and	
   this	
   procedure	
   was	
  repeated	
  twice	
  more.	
  	
  The	
  resin	
  was	
  then	
  washed	
  thoroughly	
  with	
  DMF	
  to	
  afford	
   the	
  Dde-­‐deprotected	
  [2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl]-­‐Lys-­‐Aad(tBu)-­‐Ach	
  resin	
   4.	
  	
  To	
   deprotected	
   resin	
   4	
   was	
   added	
   a	
   solution	
   of	
   3-­‐(3-­‐pyridyl)acrylic	
   acid	
   (0.189g,	
   1.27mmol),	
   HBTU	
   (0.489	
   g,	
   1.29	
   mmol),	
   DIPEA	
   (0.45	
   mL,	
   2.57	
   mmol),	
   and	
   4	
   mL	
   DMF.	
  	
   The	
   resin	
   was	
   gently	
   shaken	
   for	
   1	
   hour	
   and	
   the	
   solution	
   was	
   then	
   removed	
   by	
   filtration.	
  	
   The	
   resin	
   was	
   washed	
   with	
   DMF	
   (3	
   x	
   5	
   mL)	
   and	
   DCM	
   (3	
   x	
   5	
   mL)	
   and	
   suction	
   dried	
   on	
   an	
    	
    73	
    aspirator	
   for	
   20	
   minutes	
   to	
   afford	
   resin	
   5.	
   	
   A	
   sample	
   of	
   this	
   resin	
   was	
   removed,	
   the	
   product	
  cleaved	
  and	
  dissolved	
  in	
  methanol	
  to	
  allow	
  mass	
  spectrometry	
  analysis.	
  	
  	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C53H75N9O10	
  	
  [M+H+]:	
  	
  999.2,	
  found:	
  	
  999.0.	
   	
   	
   4.3.7	
   [2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetyl]-­‐Lys(3-­‐(3-­‐pyridyl)	
  acrylyl)-­‐Aad(tBu)-­‐Ach	
  	
   (LLP2A(tBu),	
  2)	
   	
    	
   	
    Resin	
   5	
  (0.521	
  g)	
  was	
  placed	
  in	
  a	
  25	
  mL	
  RBF.	
  	
  9	
  mL	
  of	
  a	
  30%	
  HFIP	
  in	
  DCM	
  was	
    added	
  to	
  the	
  resin	
  and	
  the	
  mixture	
  was	
  stirred	
  for	
  30	
  minutes.	
  	
  The	
  resin	
  was	
  filtered	
  off	
   and	
  the	
  filtrate	
  volume	
  was	
  reduced	
  using	
  a	
  rotary	
  evaporator.	
  	
  To	
  the	
  resulting	
  yellow	
   solution	
  was	
  added	
  diethyl	
  ether,	
  precipitating	
  a	
  white	
  solid.	
  	
  The	
  solid	
  was	
  filtered	
  off	
   and	
   dried	
   in	
   vacuo.	
   	
   The	
   crude	
   product	
   was	
   purified	
   using	
   preparative	
   TLC	
   (1:9	
   NH4OH:EtOH)	
  to	
  afford	
  product	
  LLP2A(tBu)	
  2	
  (0.020	
  g,	
  7	
  %	
  yield	
  overall),	
  a	
  white	
  solid.	
  	
   The	
  analytical	
  HPLC	
  was	
  performed	
  using	
  Program	
  1.	
   HRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C53H75N9O10	
  [M+H+]:	
  	
  999.5708,	
  found:	
  	
  999.5715.	
   Rf	
  =	
  0.61	
  (1:9	
  NH4OH:EtOH)	
   tR	
  	
  =	
  12.16	
  min	
  (Program	
  1)	
    	
    74	
    LLP2A(tBu)	
    Figure	
  4.1:	
  	
  RP-­‐HPLC	
  trace	
  of	
  LLP2A(tBu).	
  	
  Reproduced	
  from	
  page	
  44.	
    	
    	
   	
    4.4	
    Synthesis	
  in	
  Solution	
    	
   4.4.1	
   2-­‐(4-­‐(3-­‐o-­‐tolylureido)phenyl)acetic	
  acid38	
  (1)	
   	
   OH  O N H  	
    N H  O  	
    To	
  a	
  solution	
  of	
  4-­‐aminophenyl	
  acetic	
  acid	
  (1.514	
  g,	
  10.0	
  mmol)	
  and	
  50	
  mL	
  DMF	
    in	
   a	
   250	
   mL	
   RBF	
   was	
   added	
   dropwise	
   o-­‐tosyl	
   isocyanate	
   (1.370	
   g,	
   10.3	
   mmol).	
   	
   This	
   mixture	
   was	
   stirred	
   for	
   2.5	
   hours	
   at	
   RT	
   and	
   the	
   volume	
   of	
   the	
   solvent	
   reduced	
   to	
   15	
   mL	
   using	
   a	
   rotary	
   evaporator.	
   	
   The	
   solution	
   was	
   poured	
   onto	
   ethyl	
   acetate	
   (100	
   ml)	
   with	
   stirring	
  which	
  resulted	
  in	
  the	
  formation	
  of	
  a	
  light	
  brown	
  precipitate.	
  	
  This	
  was	
  collected	
   by	
  filtration	
  and	
  washed	
  with	
  ethyl	
  acetate	
  (2	
  x	
  25	
  mL)	
  and	
  ACN	
  (3	
  x	
  25	
  mL).	
  	
  The	
  solid	
   was	
  dried	
  under	
  vacuum	
  to	
  afford	
  2.085	
  g	
  of	
  tan	
  coloured	
  solid	
  (73	
  %).	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C16H16N2O3	
  [M+Na+]:	
  	
  307.31,	
  found:	
  	
  307.3.	
    	
    75	
    1  H	
  NMR	
  (300	
  MHz,	
  DMSO	
  d6)	
  δ	
  (ppm):	
  	
  2.25	
  (s,	
  3H),	
  3.50	
  (s,	
  2H),	
  6.94	
  (t,	
  1H,	
  J=7.5Hz),	
    7.14-­‐7.20	
  (m,	
  4H),	
  7.40	
  (d,	
  2H,	
  J=8.5Hz),	
  7.84	
  (d,	
  1H,	
  J=7.9Hz),	
  7.90	
  (s,	
  1H),	
  8.99	
  (s,	
  1H).	
   	
   Water	
   DMSO	
    	
   	
    1  Figure	
  4.2:	
  	
   H	
  NMR	
  of	
  1.	
    	
   	
   4.4.2	
   2,4,6-­‐Trifluoro-­‐3-­‐(4,4,5,5-­‐tetraphenyl-­‐1,3,2-­‐dioxaborolan-­‐2-­‐yl)benzoic	
  acid	
  	
   (ArB(OR)2	
  ,	
  3)	
  	
   	
   F  O OH  F  F O  B  O  	
   	
    To	
  a	
  flame	
  dried	
  100	
  mL	
  RBF	
  was	
  added	
  2,4,6-­‐trifluorobenzoic	
  acid	
  (0.50	
  g,	
  2.9	
    mmol)	
  and	
  40	
  mL	
  of	
  dry	
  THF	
  which	
  had	
  been	
  distilled	
  over	
  sodium	
  metal.	
  	
  The	
  solution	
   was	
  then	
  cooled	
  to	
  -­‐78	
  °C	
  with	
  an	
  acetone/CO2(s)	
  bath	
  under	
  N2(g)	
  flow	
  and	
  stirred	
  for	
  30	
   minutes.	
  	
  Following	
  this,	
  4	
  mL	
  of	
  a	
  1.6	
  M	
  solution	
  of	
  n-­‐BuLi	
  in	
  hexanes	
  (6.4	
  mmol,	
  2.2	
   	
    76	
    eq.)	
  was	
  added	
  drop	
  wise	
  over	
  1	
  hour.	
  	
  The	
  solution	
  turned	
  orange	
  and	
  was	
  left	
  to	
  stir	
   for	
   15	
   minutes	
   after	
   addition	
   of	
   the	
   n-­‐BuLi.	
   	
   Next,	
   trimethylborate	
   (0.80	
   mL,	
   7	
   mmol)	
   was	
  added	
  via	
  a	
  syringe	
  and	
  the	
  solution	
  was	
  stirred	
  for	
  2	
  hours.	
  	
  The	
  reaction	
  was	
  then	
   quenched	
   with	
   4	
   mL	
   of	
   a	
   4.0	
   M	
   solution	
   of	
   HCl	
   in	
   dioxane.	
   	
   A	
   benzopinacol	
   solution	
   (1.6	
   g,	
  4.3	
  mmol	
  in	
  10	
  mL	
  THF)	
  was	
  then	
  added	
  to	
  the	
  reaction.	
  	
  The	
  acetone/CO2(s)	
  was	
  then	
   removed	
   and	
   the	
   solution	
   was	
   stirred	
   for	
   2	
   hours	
   as	
   it	
   warmed	
   to	
   RT.	
   	
   50	
   mL	
   of	
   toluene	
   was	
   added	
   to	
   the	
   reaction	
   and	
   it	
   was	
   then	
   concentrated.	
   	
   This	
   process	
   was	
   repeated	
   twice	
   more.	
   	
   The	
   resulting	
   solid	
   was	
   loaded	
   onto	
   a	
   2	
   cm	
   silica	
   column,	
   where	
   the	
   product	
   was	
   eluted	
   using	
   10	
   %	
   EtOAc	
   in	
   Hexanes.	
   	
   The	
   elution	
   was	
   monitored	
   by	
   TLC	
   and	
  the	
  appropriate	
  fractions	
  were	
  collected	
  to	
  yield	
  0.122	
  g	
  (8	
  %)	
  of	
  white	
  solid.	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C33H22BF3O4	
  [M+Na+]:	
  	
  573.33,	
  found:	
  	
  573.3.	
   1  H	
  NMR	
  (300	
  MHz,	
  CDCl3)	
  δ	
  (ppm):	
  	
  6.86	
  (t,	
  1H),	
  7.11	
  (m,	
  12H),	
  7.22	
  (m,	
  8H).	
  	
    	
  19F	
  NMR	
  (300	
  MHz,	
  CDCl3)	
  δ	
  (ppm):	
  	
  -­‐93.26	
  (1F),	
  -­‐96.10	
  (1F),	
  -­‐103.50	
  (1F).	
   Rf	
  =	
  0.35	
  (1:9	
  MeOH:DCM)	
   	
    grease	
    	
   1  Figure	
  4.3:	
  	
   H	
  NMR	
  of	
  3.	
    	
    	
    77	
    	
   19  Figure	
  4.4:	
  	
   F	
  NMR	
  of	
  3.	
    	
   	
   4.4.3	
   ArB(OR)2-­‐LLP2A	
  (4)	
   	
    	
   	
    To	
   a	
   5	
   mL	
   RBF	
   was	
   added	
   0.75	
   mL	
   DMF,	
   LLP2A(tBu)	
   (12.5	
   mg,	
   12.5	
   μmol),	
    ArB(OR)2	
  	
   (8.26	
  mg,	
  15.0	
  µmol),	
  EDCHCl	
  (3.1	
  mg,	
  16.1	
  µmol),	
  HOBtH2O	
  (2.5	
  mg,	
  16.3	
   µmol)	
  and	
  pyridine	
  (5.47	
  μL,	
  67.3	
  µmol).	
  	
  This	
  solution	
  was	
  allowed	
  to	
  react	
  over	
  night.	
  	
   The	
  solvent	
  was	
  then	
  removed	
  under	
  vacuum	
  and	
  the	
  resulting	
  oil	
  was	
  dissolved	
  in	
  1	
  mL	
   THF.	
  	
  The	
  addition	
  of	
  diethyl	
  ether	
  caused	
  a	
  white	
  precipitate	
  to	
  form.	
  	
  The	
  solution	
  was	
   centrifuged,	
  the	
  solvent	
  was	
  decanted	
  and	
  the	
  resulting	
  solid	
  was	
  dried	
  under	
  vacuum.	
  	
   To	
  this	
  solid	
  was	
  added	
  one	
  mL	
  of	
  a	
  50	
  %	
  solution	
  of	
  TFA	
  in	
  DCM	
  and	
  the	
  solution	
  was	
   stirred	
   for	
   one	
   hour	
   after	
   which	
   the	
   solvent	
   was	
   removed.	
   	
   The	
   resulting	
   oil	
   was	
   resuspended	
   in	
   0.5	
   mL	
   THF	
   and	
   diethyl	
   ether	
   was	
   added	
   to	
   precipitate	
   a	
   white	
   solid.	
  	
    	
    78	
    The	
  solvent	
  was	
  decanted	
  and	
  the	
  solid	
  dried	
  under	
  vacuum	
  to	
  afford	
  9.5	
  mg	
  of	
  crude	
   product.	
   	
   The	
   crude	
   was	
   dissolved	
   in	
   0.5	
   mL	
   DCM	
   and	
   loaded	
   on	
   to	
   a	
   1	
   cm	
   silica	
   column	
   and	
   eluted	
   with	
   7	
   %	
   MeOH	
   in	
   DCM.	
   	
   The	
   appropriate	
   fractions	
   were	
   pooled	
   together	
   and	
   the	
   volume	
   was	
   reduced.	
   	
   The	
   resulting	
   oil	
   was	
   dissolved	
   in	
   200	
   µL	
   THF	
   and	
   precipitated	
  with	
  diethyl	
  ether.	
  	
  This	
  solution	
  was	
  centrifuged,	
  the	
  solvent	
  decanted	
  and	
   the	
   left	
   over	
   white	
   solid	
   was	
   dried	
   under	
   vacuum	
   to	
   afford	
   6.0	
   mg	
   (32.6	
   %)	
   of	
   ArB(OR)2-­‐ LLP2A.	
  	
  	
   HRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C82H87BF3N9O13	
  [M+H+]:	
  	
  1474.6572,	
  found:	
  	
  1474.6547.	
   Rf	
  =	
  0.43	
  (1:9	
  MeOH:DCM)	
   tR	
  	
  =	
  18.90	
  min	
  (Program	
  1)	
   	
  	
   	
    ArB(OR)2-­‐LLP2A	
    Figure	
  4.5:	
  	
  RP-­‐HPLC	
  trace	
  of	
  ArB(OR)2-­‐LLP2A.	
    	
    	
   	
   	
   	
    	
    79	
    4.4.4	
   5-­‐azido	
  pentanoic	
  acid46	
  (5)	
   	
   O N3  	
    OH 	
    To	
  a	
  10	
  mL	
  RBF	
  was	
  added	
  5-­‐bromo	
  ethyl	
  pentanoate	
  (2.00	
  g,	
  9.53	
  mmol),	
  5	
  mL	
    DMSO	
  and	
  NaN3	
  (2.480	
  g,	
  38.5	
  mmol).	
  	
  This	
  mixture	
  was	
  stirred	
  for	
  24	
  hours	
  at	
  100	
  °C.	
  	
   Upon	
  cooling,	
  the	
  solution	
  turned	
  into	
  a	
  brown	
  solid,	
  which	
  was	
  then	
  dissolved	
  in	
  100	
   mL	
  water.	
  	
  This	
  solution	
  was	
  extracted	
  with	
  diethyl	
  ether	
  (4	
  x	
  50	
  mL).	
  	
  The	
  organic	
  layers	
   were	
  pooled	
  together.	
  	
  The	
  volume	
  was	
  reduced	
  to	
  30	
  mL	
  and	
  the	
  solution	
  diluted	
  to	
  60	
   mL	
   with	
   1N	
   NaOH.	
   	
   This	
   mixture	
   was	
   stirred	
   at	
   RT	
   overnight.	
   	
   The	
   solution	
   was	
   then	
   washed	
   with	
   ether	
   and	
   acidified	
   to	
   pH	
   1	
   with	
   concentrated	
   HCl.	
   	
   The	
   product	
   was	
   extracted	
  with	
  ether	
  (3	
  x	
  20	
  mL),	
  dried	
  with	
  Na2SO4,	
  and	
  the	
  solvent	
  was	
  removed	
  under	
   vacuum,	
  yielding	
  0.933	
  g	
  (68	
  %)	
  of	
  a	
  yellow	
  liquid.	
   1  H	
  NMR	
  (300	
  MHz,	
  CDCl3)	
  δ	
  (ppm):	
  	
  1.6-­‐1.8	
  (m,	
  4H),	
  2.41	
  (t,	
  2H),	
  3.31	
  (t,	
  2H).	
    CDCl3	
    	
   1  Figure	
  4.6:	
  	
   H	
  NMR	
  of	
  5.	
    	
   	
    	
    80	
    4.4.5	
   N3-­‐LLP2A	
  (6)	
   	
    	
   	
    To	
   a	
   5	
   ml	
   RBF	
   was	
   added	
   LLP2A(tBu)	
   (1.9	
   mg,	
   1.9	
   μmol),	
   HBTU	
   (4.0	
   mg,	
   10.5	
    μmol),	
  5-­‐azido	
  pentanoic	
  acid	
  (1.1	
  mg,	
  7.6	
  μmol),	
  and	
  DIPEA	
  (1.9	
  mg,	
  15	
  μmol)	
  and	
  2	
  mL	
   of	
  DMF.	
  	
  This	
  mixture	
  was	
  stirred	
  for	
  1	
  hour	
  and	
  the	
  solvent	
  was	
  then	
  reduced	
  using	
  a	
   rotary	
   evaporator.	
   	
   Addition	
   of	
   cold	
   diethyl	
   ether	
   caused	
   a	
   white	
   solid	
   to	
   precipitate.	
  	
   The	
   vial	
   was	
   centrifuged	
   and	
   the	
   solid	
   pellet	
   was	
   collected	
   and	
   dried	
   under	
   vacuum.	
  	
   This	
  product	
  was	
  added	
  to	
  a	
  5	
  mL	
  RBF	
  and	
  was	
  dissolved	
  in	
  2	
  mL	
  of	
  a	
  50%	
  TFA	
  in	
  DCM	
   solution.	
  	
  The	
  solution	
  was	
  stirred	
  for	
  1	
  hour,	
  after	
  which	
  the	
  solvent	
  was	
  removed	
  in	
   vacuo.	
   	
   Diethyl	
   ether	
   was	
   added	
   to	
   precipitate	
   a	
   white	
   solid,	
   which	
   was	
   separated	
   by	
   filtration	
   to	
   yield	
   a	
   white	
   solid.	
   	
   The	
   solid	
   was	
   dissolved	
   in	
   methanol	
   and	
   purified	
   by	
   RP-­‐ HPLC	
  (Program	
  1)	
  to	
  afford	
  1.6	
  mg	
  (89	
  %)	
  of	
  N3-­‐LLP2A.	
  	
  	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C54H74N12O11	
  [M+H+]:	
  	
  1068.24,	
  found:	
  	
  1068.0.	
   Rf	
  =	
  0.76	
  (5:95	
  NH4OH:EtOH)	
   tR	
  	
  =	
  11.42	
  min	
  (Program	
  1)	
   	
    	
    81	
    N3-­‐LLP2A	
    Figure	
  4.7:	
  	
  RP-­‐HPLC	
  trace	
  of	
  N3-­‐LLP2A.	
    	
    	
    	
    	
   4.4.6	
   ArBF3-­‐LLP2A	
  (7)	
   	
   O HO F  F  F B F F  O H N  F  O  O  O H N  N H  O  H N  N H  O  O  O N H  HN  N H  N O  	
    	
   	
    “Slow”	
  Method	
    	
    To	
   a	
   1	
   mL	
   eppendorf	
   vial	
   was	
   added	
   ArB(OR)2-­‐LLP2A	
   (3.3	
   mg,	
   2.24	
   μmol),	
   200	
   μL	
    THF	
   and	
   15	
   μL	
   (30	
   eq)	
   of	
   a	
   4.5	
   M	
   KHF2(aq)	
   solution.	
   	
   The	
   vial	
   was	
   vortexed	
   and	
   the	
   reaction	
   was	
   allowed	
   to	
   proceed	
   overnight.	
   	
   The	
   next	
   day,	
   a	
   solid	
   KHF2	
   crystal	
   was	
   removed	
  and	
  the	
  product	
  was	
  precipitated	
  upon	
  addition	
  of	
  diethyl	
  ether.	
  	
  The	
  solvent	
   was	
   decanted	
   and	
   the	
   product	
   redissolved	
   in	
   THF	
   and	
   precipitated	
   with	
   diethyl	
   ether	
    	
    82	
    again.	
  	
  The	
  solid	
  was	
  collected	
  and	
  dried	
  under	
  vacuum	
  to	
  give	
  1.6	
  mg	
  (57.6	
  %)	
  of	
  white	
   solid.	
  	
  The	
  HPLC	
  was	
  performed	
  using	
  Program	
  2.	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C56H67BF6N9O11	
  [M-­‐H-­‐F]:	
  	
  1146.98,	
  found:	
  	
  1147.0.	
   19  F	
  NMR	
  (300	
  MHz,	
  CDCl3)	
  δ	
  (ppm):	
  	
  -­‐99.12	
  (1F),	
  -­‐104.16	
  (1F),	
  -­‐117.91	
  (1F),	
  -­‐132.24	
  (3F)	
    Rf	
  =	
  0.25	
  (1:9	
  MeOH:DCM)	
    	
    tR	
  	
  =	
  9.18	
  min	
  (Program	
  2)	
    TFA	
    	
   19  Figure	
  4.8:	
  	
   F	
  NMR	
  of	
  ArBF3-­‐LLP2A.	
    	
   	
   ArBF3-­‐LLP2A	
    	
    Figure	
  4.9:	
  	
  RP-­‐HPLC	
  trace	
  of	
  ArBF3-­‐LLP2A.	
  	
  Reproduced	
  from	
  page	
  50.	
    	
    	
   	
    83	
    	
    “Fast”	
  Method	
    	
    To	
  a	
  0.5mL	
  eppendorf	
  vial	
  containing	
  100	
  nmol	
  of	
  ArB(OR)2-­‐LLP2A	
  was	
  added	
  2	
    μL	
  of	
  a	
  0.125	
  M	
  KHF2(aq)	
  solution,	
  4	
  μL	
  THF	
  and	
  0.5	
  μL	
  HCl.	
  	
  The	
  vial	
  was	
  vortexed	
  and	
  left	
   to	
  react	
  for	
  1	
  hour	
  at	
  RT.	
  	
  The	
  reaction	
  was	
  quenched	
  by	
  addition	
  of	
  100	
  μL	
  of	
  5:15:80	
   NH4OH:H2O:EtOH.	
   	
   The	
   resulting	
   solution	
   was	
   transferred	
   into	
   a	
   Waters	
   MS	
   vial	
   and	
   analyzed	
  by	
  RP-­‐HPLC	
  and	
  MS.	
  	
  The	
  HPLC	
  was	
  performed	
  using	
  Program	
  2.	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C56H67BF6N9O11	
  [M-­‐H-­‐F]:	
  	
  1146.98,	
  found:	
  	
  1147.1.	
   tR	
  	
  =	
  9.08	
  min	
  (Program	
  2)	
   	
   ArBF3-­‐LLP2A	
   	
    	
   Figure	
  4.10:	
  	
  RP-­‐HPLC	
  of	
  ArBF3-­‐LLP2A.	
  	
  Reproduced	
  from	
  page	
  52.	
    	
    	
   	
   	
   	
   	
   	
   	
   	
    84	
    4.4.7	
   FITC-­‐LLP2A	
  (8)	
   	
   O H N  HO  HO C  S  HN O  O  OH  O  O O  O  HN  O  H N O  N H  H N  O O  HN  N H  N H  N O  	
    	
    A	
  solution	
  of	
  FITC	
  (1.9	
  mg),	
  0.5	
  mL	
  DMF	
  and	
  0.5	
  mL	
  Na2CO3	
  buffer	
  (pH	
  8.4)	
  was	
    prepared.	
   	
   0.435	
   mL	
   of	
   this	
   solution	
   (2.12	
   μmol	
   FITC)	
   was	
   added	
   to	
   a	
   5	
   mL	
   RBF	
   containing	
   0.5	
   mL	
   DMF,	
   0.5	
   mL	
   Na2CO3	
   buffer	
   (pH	
   8.4)	
   and	
   LLP2A(tBu)	
   (2.1	
   mg,	
   2.1	
   μmol).	
  	
  This	
  mixture	
  was	
  allowed	
  to	
  react	
  for	
  12	
  hours	
  at	
  4	
  °C	
  after	
  which	
  the	
  solvent	
   was	
   reduced	
   under	
   vacuum.	
   	
   Addition	
   of	
   diethyl	
   ether	
   caused	
   a	
   yellow	
   solid	
   to	
   precipitate.	
   	
   The	
   vial	
   was	
   centrifuged	
   and	
   the	
   solid	
   pellet	
   was	
   collected.	
   	
   To	
   this	
   solid	
   was	
  added	
  2	
  mL	
  of	
  a	
  50	
  %	
  solution	
  of	
  TFA	
  in	
  DCM.	
  	
  This	
  mixture	
  was	
  allowed	
  to	
  react	
  for	
   1	
  h	
  at	
  RT	
  after	
  which	
  the	
  solvent	
  was	
  removed	
  under	
  vacuum.	
  	
  The	
  resulting	
  solid	
  was	
   redissolved	
  in	
  100	
  μL	
  THF	
  and	
  precipitated	
  with	
  diethyl	
  ether,	
  then	
  the	
  solvent	
  decanted	
   and	
  dried	
  under	
  vacuum	
  to	
  yield	
  a	
  yellow	
  solid.	
  	
  This	
  compound	
  was	
  dissolved	
  in	
  3.00	
   mL	
   THF	
   and	
   aliquoted	
   into	
   fractions	
   containing	
   60	
   µL	
   of	
   solution.	
   	
   Purification	
   of	
   the	
   compound	
   by	
   TLC	
   took	
   place	
   immediately	
   prior	
   to	
   use,	
   with	
   one	
   aliquot	
   providing	
   20	
   nmol	
  of	
  FITC-­‐LLP2A,	
  representing	
  an	
  overall	
  reaction	
  yield	
  of	
  47	
  %.	
   LRMS	
  (ESI)	
  m/z:	
  	
  calculated	
  for	
  C70H78N10O15S	
  [M+Na+]:	
  	
  1354.49,	
  found:	
  	
  1354.3.	
   Rf	
  =	
  0.60	
  (1:9	
  MeOH:DCM)	
   	
    	
    85	
    Figure	
  4.11:	
  	
  UV-­‐visible	
  absorption	
  of	
  FITC-­‐LLP2A.	
    	
    	
   	
    4.5	
    Cell	
  Binding	
  Assays	
    	
   4.5.1	
   Fluorescence	
  Assay	
   	
   	
   	
   TBS	
  buffer	
  solution	
  with	
  1	
  mM	
  Mn2+	
  (from	
  MnSO4)	
  and	
  containing	
  400	
  nM	
  FITC-­‐ LLP2A	
   was	
   prepared	
   by	
   diluting	
   20	
   nmol	
   of	
   FITC-­‐LLP2A	
   to	
   50	
   mL.	
   	
   1	
   nM	
   FITC-­‐LLP2A	
   solution	
  was	
  prepare	
  by	
  diluting	
  	
  120	
  μL	
  of	
  400	
  nM	
  FITC-­‐LLP2A	
  to	
  50	
  mL	
  using	
  TBS	
  buffer	
   solution	
   (1	
   mM	
   Mn2+).	
   	
   Aliquots	
   of	
   approximately	
   1	
   x	
   106	
   cells	
   each	
   were	
   prepared	
   in	
   TBS	
   buffer	
   containing	
   either	
   1	
   nM	
   FITC-­‐LLP2A	
   or	
   400	
   nM	
   FITC-­‐LLP2A.	
   	
   The	
   cells	
   were	
   allowed	
  incubate	
  for	
  1	
  hour	
  at	
  37	
  °C,	
  after	
  which	
  the	
  mixture	
  was	
  transferred	
  to	
  a	
  15	
  mL	
   Falcon	
  tube.	
  	
  The	
  mixture	
  was	
  centrifuged	
  and	
  the	
  buffer	
  was	
  removed.	
  	
  The	
  cell	
  pellet	
   was	
   resuspended	
   in	
   fresh	
   buffer	
   that	
   did	
   not	
   contain	
   FITC-­‐LLP2A,	
   centrifuged	
   and	
   the	
   buffer	
   removed.	
   	
   This	
   process	
   was	
   repeated	
   once	
   more.	
   	
   The	
   cell	
   pellet	
   was	
   the	
    	
    86	
    resuspended	
  in	
  2	
  mL	
  buffer	
  and	
  approximately	
  0.3	
  mL	
  was	
  spotted	
  on	
  a	
  glass	
  slide	
  and	
   observed	
  for	
  fluorescence.	
    4.5.2	
   Blocking	
  Assay	
   	
   	
   After	
  several	
  passages,	
  aliquots	
  of	
  1	
  x	
  106	
  cells	
  each	
  were	
  prepared	
  in	
  TBS	
  buffer	
   (1	
  mM	
  Mn2+)	
  containing	
  100	
  nM	
  ArBF3-­‐LLP2A.	
  	
  After	
  incubating	
  for	
  1	
  hour	
  at	
  37	
  °C,	
  the	
   buffer	
   was	
   removed	
   and	
   replaced	
   with	
   buffer	
   containing	
   either	
   1	
   nM	
   FITC-­‐LLP2A	
   or	
   400	
   nM	
  FITC-­‐LLP2A.	
  	
  The	
  cells	
  were	
  incubated	
  for	
  1	
  hour	
  at	
  37	
  °C,	
  after	
  which	
  each	
  mixture	
   was	
  transferred	
  to	
  a	
  15	
  mL	
  Falcon	
  tube.	
  	
  The	
  mixtures	
  were	
  centrifuged	
  and	
  the	
  buffer	
   was	
  removed.	
  	
  The	
  cell	
  pellet	
  was	
  resuspended	
  in	
  fresh	
  buffer	
  that	
  did	
  not	
  contain	
  FITC-­‐ LLP2A,	
  centrifuged	
  and	
  the	
  buffer	
  removed.	
  	
  This	
  process	
  was	
  repeated	
  once	
  more.	
  	
  The	
   cell	
  pellet	
  was	
  the	
  resuspended	
  in	
  2	
  mL	
  buffer	
  and	
  approximately	
  0.3	
  mL	
  was	
  spotted	
  on	
   a	
  glass	
  slide	
  and	
  observed	
  for	
  fluorescence.	
  	
    	
   	
   4.6	
   	
    	
    18  F-­‐Radiolabeling	
    The	
  following	
  procedures	
  were	
  performed	
  by	
  Dr.	
  Ying	
  Li	
  of	
  the	
  Perrin	
  group.	
    	
   4.6.1	
   One-­‐Step	
  One-­‐Pot	
  Synthesis	
  of	
  ArB18F3-­‐LLP2A	
  (9)	
   	
   	
   To	
  a	
  PCR	
  tube	
  loaded	
  with	
  ArB(OR)2-­‐LLP2A	
  (100	
  nmol),	
  HCl	
  (1	
  μL),	
  and	
  THF	
  (4	
  μL)	
   was	
  added	
  1	
  μL	
  of	
  [18/19F]-­‐fluoride	
  (507	
  nmol,	
  995	
  μCi	
  at	
  the	
  BOS).	
  	
  Following	
  incubation	
   	
    87	
    for	
  1	
  hour	
  at	
  RT,	
  the	
  reaction	
  was	
  quenched	
  with	
  100	
  μL	
  of	
  a	
  solution	
  of	
  5	
  %	
  NH4OH	
  in	
   50	
  %	
  aqueous	
  EtOH.	
  	
  Activity	
  was	
  664	
  μCi	
  at	
  the	
  EOS.	
  	
  A	
  sample	
  of	
  this	
  mixture	
  was	
  then	
   injected	
  into	
  the	
  RP-­‐HPLC	
  (Program	
  3).	
   	
   18 -­‐	
    F  18  ArB F3-­‐LLP2A	
    18  Figure	
  4.12:	
  	
  RP-­‐HPLC	
  radio	
  trace	
  of	
  ArB F3-­‐LLP2A	
  9.	
  	
  Reproduced	
  from	
  page	
  58.	
    	
    	
   	
   4.6.2	
   One-­‐Pot	
  Two-­‐Step	
  Click	
  Synthesis	
  of	
  ArB18F3-­‐LLP2A	
  (10)	
   	
   	
   To	
  a	
  PCR	
  tube	
  was	
  added	
  2	
  μL	
  [18/19F]-­‐fluoride	
  (500	
  nmol,	
  1.84	
  mCi	
  at	
  the	
  BOS),	
   alkynyl-­‐ArB(OR’)2	
   (100	
   nmol),	
   HCl	
   (0.5	
   µL)	
   and	
   4	
   µL	
   THF.	
   	
   The	
   reaction	
   was	
   allowed	
   to	
   proceed	
   at	
   RT	
   for	
   22	
   minutes,	
   after	
   which	
   the	
   reaction	
   was	
   quenched	
   with	
   10	
   µL	
   of	
   a	
   solution	
   of	
   5	
   %	
   NH4OH	
   in	
   50	
   %	
   aqueous	
   EtOH.	
   	
   A	
   sample	
   of	
   this	
   mixture	
   (2	
   µL)	
   was	
   diluted	
   to	
   100	
   µL	
   using	
   a	
   solution	
   of	
   5	
   %	
   NH4OH	
   in	
   50	
   %	
   aqueous	
   EtOH	
   for	
   RP-­‐HPLC	
   analysis.	
   	
   The	
   rest	
   of	
   the	
   quenched	
   reaction	
   was	
   then	
   added	
   to	
   another	
   PCR	
   tube	
   containing	
   N3-­‐LLP2A	
   (100	
   nmol),	
   after	
   which	
   0.6	
   M	
   sodium	
   ascorbate	
   (4	
   µL)	
   and	
   lastly	
   0.2	
  M	
  copper	
  sulfate	
  (2	
  µL)	
  were	
  also	
  added.	
  	
  This	
  reaction	
  was	
  allowed	
  to	
  proceed	
  for	
   36	
  minutes	
  at	
  RT.	
  	
  6	
  µL	
  of	
  the	
  reaction	
  was	
  then	
  diluted	
  to	
  100	
  µL	
  using	
  a	
  solution	
  of	
  5	
  %	
    	
    88	
    NH4OH	
  in	
  50	
  %	
  aqueous	
  EtOH.	
  	
  This	
  mixture	
  was	
  used	
  for	
  RP-­‐HPLC	
  analysis	
  (Program	
  3).	
  	
   Activity	
  was	
  1.02	
  mCi	
  at	
  EOS.	
  	
    18  Alkynyl-­‐ArB F3	
   F 	
    18 -­‐  18  Figure	
  4.13:	
  	
  RP-­‐HPLC	
  of	
  alkynyl-­‐ArB F3.	
  	
  Reproduced	
  from	
  page	
  60.	
    	
    ArB F3-­‐LLP2A	
   18  F 	
    18 -­‐  	
    alkynyl-­‐ArB F3	
   18  18  Figure	
  4.14:	
  	
  RP-­‐HPLC	
  of	
  click	
  reaction	
  to	
  produce	
  ArB F3-­‐LLP2A	
  10.	
  	
  Reproduced	
  from	
  page	
  61.	
    	
    	
    	
    	
    	
    89	
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