the)bioorthogonal)clickreac:on)of)cyclobutenes)and)tetrazines) · synthesis...

Synthesis  The  rela(vely  new  field  of  bioorthogonal  or  copper-­‐free  “click”  chemistry  has  flourished  in  recent  years  because  of  the  poten(al  to  conduct  rapid  and  selec(ve  liga(ons  under  condi(ons  compa(ble  with  living  cells  (Bertozzi,  2003).  This  research  seeks  to  expand  on  the  narrow  set  of  cycloalkene  and  tetrazine  pairings  of  bioorthogonal  chemistry  that  are  suitable  for  inverse  electron-­‐demand  Diels-­‐Alder  4+2  cycloaddi(on  (the  “click”  mechanism)  with  the  introduc(on  and  characteriza(on  of  3-­‐subs(tuted  cyclobutene  as  a  viable  alterna(ve.        

Conclusions  Through  a  nine  step  synthe(c  pathway  of  our  cyclobutene  (ini(ally  established  by  Wantanee  SiOwong),  as  well  as  a  unique  tetrazine  synthe(c  method,  we  have  developed  a  model  system  with  which  to  explore  the  use  of  cyclobutene  in  inverse  electron-­‐demand  Diels-­‐Alder  4+2  reac(ons.  As  a  result  of  this  research,  we  hope  that  cyclobutene  garners  acceptance  as  a  new  and  effec(ve  bioorthogonal  molecule.  

Materials  &  Methods  

Background  Why  choose  click  chemistry?  

•  Wide  in  scope  •  Rapid  liga(on  rate  •  Selec(ve:  No  undesired  byproducts    •  Stable  and  irreversible  •  Effec(ve  means  of  immobilizing  or  linking  molecules  

A  recent  innova(on  •  Azide-­‐alkyne  Huisgen  1,3-­‐dipolar  cycloaddi(on  (2001)  •  Sharpless-­‐Fokin  Copper(I)-­‐catalyzed  Azide-­‐alkyne  cycloaddi(on  or  

CuAAC  (2002)  •  Bertozzi  Bioorthogonal  cycloaddi(on  (2003)  

The  popular  copper  catalyzed  click  reac:on  

The  Bioorthogonal  Click  Reac:on  of  Cyclobutenes  and  Tetrazines  William  Lambert1,  Wantanee  SiOwong1  &  Patrick  Dussault1,2  

Department  of  Chemistry1  and  The  Center  for  Nanohybrid  Func(onal  Materials2,  University  of  Nebraska–Lincoln  68588  

Purpose  

Work  on  the  synthesis  of  amphiphilic  cyclobutenes  developed  by  SiOwong,  et.  al.*      

Four  step  pathway  established  for  conversion  of  carboxylic  acid  func(onalized  tail  (SiOwong)  to  thiol  

Future  Work  •  SAM  forma(on  •  Click  reac(on  kine(cs  in  collabora(on  with  Prof.  Rebecca  Lai:  

Stability,  Selec(vity,  Liga(on  Rates  •  Improving  synthe(c  yields  and/or  shortening  pathways  •  Other  cyclobutene  func(onaliza(on  or  cons(tuents  •  Possible  biochemical  applica(ons  

I.  Chemical  sensors  II.  Imaging  proper(es  III.  Bioconjuga(ons  

Cyclobutene  Synthe:c  Pathway  

Tetrazine  Synthe:c  Pathway  q  Amphiphilic  cyclobutene  (hydrophobic  +  hydrophilic  ends)  for  coordinated  forma(on  of  self-­‐assembled  monolayer  (SAM)  

 q  Thiol  (-­‐SH)  func(onalized  tail  to  

take  advantage  of  strong  gold-­‐sulfur  affinity  

q  Forma(on  of  SAM  

q  1,2,4,5-­‐tetrazine  partner    

q  Incorporated  redox  indicator  for  quan(ta(ve  measurement  

q  The  bioorthogonal  click  reac(on  on  monolayer  surface    

1,2,4,5-­‐tetrazine  with  incorporated  redox  indicator  synthesized  in  two  part  pathway  followed  by  a  coupling  step    

Redox  Indicator  Synthesis  

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Bioorthogonal  Chemistry  Bioorthogonal  strain-­‐promoted  cycloaddi(ons  encompass  a  wider  variety  of  molecules  and  reac(on  condi(ons  than  azide-­‐alkyne  cycloaddi(ons.  Strained  cycloalkenes  are  paired  with  a  tetrazine  in  this  spontaneous  (uncatalyzed)  reac(on.  Three  types  are  typically  employed:                                                                                  Cyclooctenes                                                  Cyclopropenes                                                  Norbornenes                                                        1,2,4,5-­‐Tetrazine    

The  tetrazine  and  these  cycloalkenes  are  also  discrete  as  func(onal  groups  in  a  biological  system  and  do  not  perturb  regular  func(ons.  It  is  for  this  reason  the  bioorthogonal  click  reac(on  has  been  useful  in  biochemical  imaging  (fluorescent  and  PET).

Synthesis  

Acknowledgements    

Funding  for  this  research  was  provided  by  The  Center  for  Nanohybrid  Func(onal  Materials  (CNFM)  NSF  Grant  EPS-­‐1004094.    

*ChemMedChem  2014,  E-­‐pub.  ahead  of  print  June  5,  DOI:10.1002/cmdc.201402067