unless otherwise noted , all reagents were purchased from sigma  aldrich ( st . louis , mo , usa ) and used without further purification .
boc - gly , merrifield resin and boc - lys - ome were purchased from bachem ( torrance , ca , usa ) .
boc - ala - oh , boc - lys - oh , boc - phe - oh and boc - val - oh were purchased from novabiochem ( merck kgaa , darmstadt , germany ) .
3-(4-phenylacetic acid)-1,2,4,5-tetrazine ( tz - cooh ) and ( e)-cyclooct-4-enyl 2,5-dioxopyrrolidin-1-yl carbonate ( tco - nhs ) were synthesized as described before .
,   hplc analyses were performed using a shimadzu hplc equipped with 2 lc-10at pumps , a spd - m10avp photodiode array detector , a flow count pin diode radiodetector from bioscan and a waters atlantis t3 column ( 6250  mm , 5  m ) .
a gradient of 95:50:100 h2o / ch3cn supplemented with 0.1  % tfa over 18  min at 1  ml  min was used . for preparative hplcs ,
a phenomenex jupiter ( 250100  mm , 5  m ) , and flowrates of 5  ml  min were used .
radioactivity measurements were performed with a capintec crc1243 dose calibrator ( capintec , ramsay , nj , usa ) . for tlc measurements with radioactive materials , a bioscan ar2000 ( bioscan , inc . , washington , dc , usa ) was used .
microwave irradiations were carried out using a biotage initiator microwave synthesizer ( biotage , llc , charlotte , nc , usa ) .
lrms were recorded with a waters acquity uplc with electrospray ionization sq detector ( esi ) .
hrms were recorded with a waters lct premier system ( esi ) . h  nmr spectra were recorded on a bruker aviii ( 500  mhz ) spectrometer .
chemical shifts for protons are reported in parts per million ( ppm ) and are referenced against the residual proton resonance of deuterated solvents ( cdcl3 : h , 7.26  ppm ; [ d4]meoh : h , 3.31  ppm ; [ d4]dmso : h , 2.50  ppm ) .
nmr data are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , m = multiplet ) , coupling constants ( hz ) and integration .
3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( tz - nhs ) : n - hydroxysuccinimide ( 163  mg , 1.42  mmol ) and et3n ( 495  l , 3.55  mmol ) were added to a mixture tz - cooh ( 150  mg , 0.71  mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edci , 544  mg , 2.84  mmol ) in ch2cl2 ( 15  ml ) , and the reaction mixture was stirred for 6  h at rt .
the mixture was extracted with acetic acid ( 1  m , 210  ml ) and h2o ( 210  ml ) , dried ( mgso4 ) , and volatiles were removed in vacuo .
the crude product was purified by column chromatography ( ch2cl2/meoh , 97.5:2.5 ) , yielding tz - nhs as a pink solid ( 67.3  mg , 0.22  mmol , 31  % ): h  nmr ( 500  mhz , [ d4]meoh ) : =10.60 ( s , 1  h ) , 8.51 ( d , j=8.3 , 2  h ) , 7.55 ( d , j=8.3 , 2  h ) , 7.20 ( d , j=7.7 , 1  h ) , 4.32 ( s , 2  h ) , 2.36 ( s , 4  h ) ; lc  ms ( esi ) : m / z ( % ) : 313.1 ( 100 ) [ mh ] , 312.1 ( 100 ) [ 2  mh ] .
n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysine ( boc--oh , 1 ) : tz - nhs ( 55.0  mg , 0.18  mmol ) was dissolved in a mixture of boc - l - lys - oh ( 53.2  mg , 0.36  mmol ) and et3n ( 63  l , 0.45  mmol ) in meoh ( 6  ml ) and stirred for 2  h. the reaction mixture was dried in  vacuo , and the resulting crude product purified by column chromatography ( 525  % meoh in ch2cl2 ) , yielding 1 as a pink film ( 40.3  mg , 0.09  mmol , 50  % ): h  nmr ( 500  mhz , [ d4]meoh ) : =10.31 ( s , 1  h ) , 8.55 ( d , j=8.4  hz , 2  h ) , 7.56 ( d , j=8.4  hz , 2  h ) , 4.05 ( dd , j=8.7 , 4.7  hz , 1  h ) , 3.22 ( t , j=6.9  hz , 2  h ) ,
1.861.61 ( m , 2  h ) , 1.581.38 ( m , 13  h ) ; lc  ms ( esi+ ) : m / z ( % ) : 467.3 ( 30 ) [ m+na ] , 911.6 ( 10 ) [ 2  m+na ] ; lc 
ms(esi ) : m / z ( % ) : 443.3 ( 10 ) [ mh ] , 887.6 ( 20 ) [ 2  mh ] ; hrms ( esi ) m / z [ m+na]calcd for c21h28n6o5na : 467.2005 , found : 467.2019 .
methyl n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysinate ( boc--ome , 2 ) : n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( 51.8  mg , 0.27  mol ) was added to a mixture of 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( 20  mg , 0.09  mmol ) ,
boc - l - lys - oh ( 28.6  mg , 0.11  mmol ) , et3n ( 63  l , 0.45  mmol ) in ch2cl2 ( 5  ml ) , and the reaction mixture was stirred over night at rt , before it was dried in vacuo .
the resulting crude product was purified by column chromatography ( 5  % meoh in dcm ) , yielding 2 as a pink film ( 20.8  mg , 0.05  mmol , 56  % ): h  nmr ( 500  mhz , [ d6]dmso ) : =10.57 ( s , 1  h ) , 8.44 ( d , j=8.2 , 2  h ) , 8.13 ( t , j=5.5  hz , 1  h ) , 7.55 ( d , j=8.2  hz , 2  h ) , 7.20 ( d , j=7.7  hz , 1  h ) , 3.933.88 ( m , 1  h ) , 3.61 ( s , 3  h ) , 3.55 ( s , 2  h ) , 3.05 ( q ,
j=6.5  hz , 2  h ) , 1.651.52 ( m , 2  h ) , 1.431.27 ( m , 13  h ) ; lc  ms ( esi+ ) m / z ( % ) : 359.3 ( 75 ) [ mboc+h ] , 459.3 ( 40 ) [ m+h ] , 917.7 ( 30 ) [ 2  m+h ] ; hrms ( esi ) : m / z [ m+h ] calcd for c22h30n6o5na : 481.2171 , found : 481.2175 .
n-(tetrazine)-l - lysine ( h--oh , 3 ) : boc--oh 1 ( 10  mg , 22.5  mol ) was dissolved in a mixture of tfa / ch2cl2 ( 1:1 ) and vigorously stirred for 1.5  h. volatiles were removed in vacuo , and the resulting crude material purified using hplc , yielding 3 as a pink solid ( 5.6  mg , 16.3  mol , 72  % ): h  nmr ( 500  mhz , [ d4]meoh ) : =10.32 ( s , 1  h ) , 8.55 ( d , j=8.3 , 2  h ) , 7.57 ( d , j=8.3 , 2  h ) , 3.92 ( t , j=6.3 , 2  h ) , 3.65 ( s , 2  h ) , 3.24 ( dt , j=6.3 , 3.2  hz , 2  h ) ,
2.001.84 ( m , 2  h ) , 1.621.42 ( m , 2  h ) ; lc  ms ( esi+ ) : m / z ( % ) : 345.2 ( 100 ) [ m+h ] , 367.2 ( 15 ) [ m+na ] ; hrms ( esi ) : m / z [ m+h ] calcd for c16h21n6o3 : 345.1668 , found : 345.1675 .
pentapeptide gafv ( 4 ) : the synthesis was carried out on a 0.010.02  mmol scale in a fritted syringe ( 3  ml volume ) , using a merrifield resin , which was preloaded with boc - gly - oh ( 20  mg , 0.51.0  mmol  g ) .
the resin was swollen by washing with dmf ( 22  ml ) and ch2cl2 ( 22  ml ) .
n-boc groups were removed by addition of a solution of tfa / ch2cl2 ( 1:1 ; 15  min , 2  ml and 120  min , 2  ml ) , followed by washing with ch2cl2 ( 22  ml ) and dmf ( 22  ml ) .
amino acids were coupled ( 1  h , rt ) without prior neutralization of the resin using a freshly prepared mixture of bop ( 0.04  mmol , 18  mg ) , hnigs base ( n , n-diisopropylethylamine , diea ; 0.12  mmol , 16  mg ) and amino acid ( 0.08  mmol ) in dmf ( 2.5  ml ) . after coupling ,
the resin was washed with dmf ( 32  ml ) and ch2cl2 ( 32  ml ) .
after completion of the deprotection / coupling sequence , the peptide was transferred to a microwave vessel , suspended in a solution of tfa / h2o ( 97.5:2.5 , 500  l ) , and microwaved ( 25  min , 50  hz ) .
the resin was removed by filtration and et2o ( 10  ml ) added to the supernatant .
the resulting crude precipitate was filtered and purified using hplc to yield 4 as a pink solid ( 0.9  mg , 1.3  mol , 7  % yield ) : lc  ms ( esi+ ) : m / z ( % ) : 719.5 ( 20 ) [ m+h ] ; lc  ms ( esi ) : m / z ( % ) : 717.5 ( 10 ) [ mh ] ; hrms ( esi ) : m / z calcd for [ m+h ] c35h47n10o7 : 719.3633 , found : 719.3629 .
trans - cyclooctene  desferrioxamine conjugate ( tco  dfo ) : tco - nhs ( 12  mg , 45  mol ) was added to a solution deferrioxamine mesylate salt ( 20  mg , 30  mol ) and et3n ( 21  l , 0.15  mmol ) in ch3cn / h2o ( 1:1 , 1  ml ) and stirred at rt for 90  min .
subsequently , volatiles were removed in vacuo , and the crude product was purified using hplc , yielding tco  dfo as a white solid ( 6.6  mg , 9  mol , 30  % ): h  nmr ( 500  mhz , [ d6]dmso ) : =9.689.54 ( m , 2  h ) , 7.787.74 ( m , 1  h ) , 6.936.88 ( m , 1  h ) , 5.605.40 ( m , 2  h ) , 4.214.17 ( m , 1  h ) , 3.483.41 ( m , 6  h ) , 3.032.87 ( m , 6  h ) , 2.612.53 ( m , 4  h ) ,
2.292.21 ( m , 7  h ) , 1.96 ( s , 3  h ) , 1.911.85 ( m , 4  h ) , 1.561.15 ( m , 22  h ) ; lc  ms ( esi+ ) : m / z ( % ) : 357.4 ( 10 ) [ m+2  h ] , 713.6 ( 100 ) [ m+h ] , 735.5 ( 70 ) [ m+na ] , 759.4 ( 10 ) [ m+hcoo ] ; hrms ( esi ) : m / z calcd for [ m+h ] c34h61n6o10 : 713.4474 , found : 713.4449 .
zr - trans - cyclooctene ( zr - tco , 5 ) : [ zr]zr - oxalate ( 59.274  mbq , 1.6002.000  ci ) in oxalic acid ( 1.0  m , 150  l ) was adjusted to ph  7.28.0 with na2co3 ( 1.0  m , 150  l ) .
after the evolution of co2(g ) stops , the zr was added to a solution of tco  dfo ( 1  mm , 100  l ) in pbs / dmso ( 9:1 ) .
the reaction solution was stirred at 37  c for 30  min , before the reaction progress was assayed using instant thin - layer chromatography ( itlc ) with an eluent of 1  m citric acid . in the itlc experiments ,
zr - tco remains at the baseline , while free zr ions and other [ zr]-citrate complexes elute with high rf values .
the crude reaction mixture was purified using hplc , and volatiles were removed in vacuo , yielding 5 in > 98  % radiochemical purity , 39  % uncorrected isolated radiochemical yield ( rcy ) , and a specific activity of > 5.59  mci  mol ( n=3 ) .
zr - gafv ( 6 ) : gafv ( 4 , 20  nmol , 20  l , 1  mm in dmso ) was added to zr - tco ( 56  ci , 10  l , 5.56  mci  ml in 1:1 ch3cn / h2o with 0.05  % tfa ) , and the mixture was agitated at rt for 20  min , before purification using hplc , yielding 6 in > 95  % purity , 65  % uncorrected isolated rcy , and a specific activity of > 3.00  mci  mol ( n=3 ) .
boc - deprotection conditions : tfa ( 760  l ) was added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 20  l , dmso ) and the resulting mixture stirred at room temperature . a control sample consisted of dmso ( 760  l ) , which was added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 20  l , dmso ) .
the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9  h using hplc analysis , and the results were compared to the control sample .
coupling conditions : solutions of ( benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate ( bop , 10  mm , 25  l , dmf ) and diea ( 100  mm , 25  l , dmf ) were added to a solution of 2 ( 5  mm , 25  l , dmf ) and coumarin ( 5  mm , 25  l , dmf ) . a control sample consisted of dmf ( 50  l ) , which was added to a solution of 2 ( 5  mm , 25  l , dmf ) and coumarin ( 5  mm , 25  l , dmf ) .
the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1  h , 2  h , 3  h , 4  h , 5  h , 6  h , 7  h , 8  h and 9  h using hplc analysis , and the results were compared to the control sample .
resin - cleavage conditions : tfa ( 950  l ) and deionized h2o ( 25  l ) were added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 5  l , dmso ) , and the resulting mixture was exposed to sequential microwave irradiation intervals ( 0112.5  min , 50  w ) . a control sample consisted of dmso ( 975  l ) , which was added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 5  l , dmso ) . at the end of each microwave irradiation interval ,
a sample was drawn , the amount of 3 relative to coumarin was measured using hplc analysis , and the results were compared to the control sample .
3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( tz - nhs ) : n - hydroxysuccinimide ( 163  mg , 1.42  mmol ) and et3n ( 495  l , 3.55  mmol ) were added to a mixture tz - cooh ( 150  mg , 0.71  mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edci , 544  mg , 2.84  mmol ) in ch2cl2 ( 15  ml ) , and the reaction mixture was stirred for 6  h at rt .
the mixture was extracted with acetic acid ( 1  m , 210  ml ) and h2o ( 210  ml ) , dried ( mgso4 ) , and volatiles were removed in vacuo .
the crude product was purified by column chromatography ( ch2cl2/meoh , 97.5:2.5 ) , yielding tz - nhs as a pink solid ( 67.3  mg , 0.22  mmol , 31  % ): h  nmr ( 500  mhz , [ d4]meoh ) : =10.60 ( s , 1  h ) , 8.51 ( d , j=8.3 , 2  h ) , 7.55 ( d , j=8.3 , 2  h ) , 7.20 ( d , j=7.7 , 1  h ) , 4.32 ( s , 2  h ) , 2.36 ( s , 4  h ) ; lc  ms ( esi ) : m / z ( % ) : 313.1 ( 100 ) [ mh ] , 312.1 ( 100 ) [ 2  mh ] .
n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysine ( boc--oh , 1 ) : tz - nhs ( 55.0  mg , 0.18  mmol ) was dissolved in a mixture of boc - l - lys - oh ( 53.2  mg , 0.36  mmol ) and et3n ( 63  l , 0.45  mmol ) in meoh ( 6  ml ) and stirred for 2  h. the reaction mixture was dried in  vacuo , and the resulting crude product purified by column chromatography ( 525  % meoh in ch2cl2 ) , yielding 1 as a pink film ( 40.3  mg , 0.09  mmol , 50  % ): h  nmr ( 500  mhz , [ d4]meoh ) : =10.31 ( s , 1  h ) , 8.55 ( d , j=8.4  hz , 2  h ) , 7.56 ( d , j=8.4  hz , 2  h ) , 4.05 ( dd , j=8.7 , 4.7  hz , 1  h ) , 3.22 ( t , j=6.9  hz , 2  h ) , 1.861.61 ( m , 2  h ) , 1.581.38 ( m , 13  h ) ; lc  ms ( esi+ ) : m / z ( % ) : 467.3 ( 30 ) [ m+na ] , 911.6 ( 10 ) [ 2  m+na ] ; lc 
ms(esi ) : m / z ( % ) : 443.3 ( 10 ) [ mh ] ,
887.6 ( 20 ) [ 2  mh ] ; hrms ( esi ) m / z [ m+na]calcd for c21h28n6o5na : 467.2005 , found : 467.2019 .
methyl n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysinate ( boc--ome , 2 ) : n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( 51.8  mg , 0.27  mol ) was added to a mixture of 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( 20  mg , 0.09  mmol ) , boc - l - lys - oh ( 28.6  mg , 0.11  mmol ) , et3n ( 63  l , 0.45  mmol ) in ch2cl2 ( 5  ml ) , and the reaction mixture was stirred over night at rt , before it was dried in vacuo .
the resulting crude product was purified by column chromatography ( 5  % meoh in dcm ) , yielding 2 as a pink film ( 20.8  mg , 0.05  mmol , 56  % ): h  nmr ( 500  mhz , [ d6]dmso ) : =10.57 ( s , 1  h ) , 8.44 ( d , j=8.2 , 2  h ) , 8.13 ( t , j=5.5  hz , 1  h ) , 7.55 ( d , j=8.2  hz , 2  h ) , 7.20 ( d , j=7.7  hz , 1  h ) , 3.933.88 ( m , 1  h ) , 3.61 ( s , 3  h ) , 3.55 ( s , 2  h ) , 3.05 ( q , j=6.5  hz , 2  h ) , 1.651.52 ( m , 2  h ) , 1.431.27 ( m , 13  h ) ; lc  ms ( esi+ ) m / z ( % ) : 359.3 ( 75 ) [ mboc+h ] , 459.3 ( 40 ) [ m+h ] , 917.7 ( 30 ) [ 2  m+h ] ; hrms ( esi ) : m / z [ m+h ] calcd for c22h30n6o5na : 481.2171 , found : 481.2175 .
n-(tetrazine)-l - lysine ( h--oh , 3 ) : boc--oh 1 ( 10  mg , 22.5  mol ) was dissolved in a mixture of tfa / ch2cl2 ( 1:1 ) and vigorously stirred for 1.5  h. volatiles were removed in vacuo , and the resulting crude material purified using hplc , yielding 3 as a pink solid ( 5.6  mg , 16.3  mol , 72  % ): h 
nmr ( 500  mhz , [ d4]meoh ) : =10.32 ( s , 1  h ) , 8.55 ( d , j=8.3 , 2  h ) , 7.57 ( d , j=8.3 , 2  h ) , 3.92 ( t , j=6.3 , 2  h ) , 3.65 ( s , 2  h ) , 3.24 ( dt , j=6.3 , 3.2  hz , 2  h ) , 2.001.84 ( m , 2  h ) , 1.621.42 ( m , 2  h ) ; lc  ms ( esi+ ) : m / z ( % ) : 345.2 ( 100 ) [ m+h ] , 367.2 ( 15 ) [ m+na ] ; hrms ( esi ) : m / z [ m+h ] calcd for c16h21n6o3 : 345.1668 , found : 345.1675 .
pentapeptide gafv ( 4 ) : the synthesis was carried out on a 0.010.02  mmol scale in a fritted syringe ( 3  ml volume ) , using a merrifield resin , which was preloaded with boc - gly - oh ( 20  mg , 0.51.0  mmol  g ) . the resin was swollen by washing with dmf ( 22  ml ) and ch2cl2 ( 22  ml ) .
n-boc groups were removed by addition of a solution of tfa / ch2cl2 ( 1:1 ; 15  min , 2  ml and 120  min , 2  ml ) , followed by washing with ch2cl2 ( 22  ml ) and dmf ( 22  ml ) .
amino acids were coupled ( 1  h , rt ) without prior neutralization of the resin using a freshly prepared mixture of bop ( 0.04  mmol , 18  mg ) , hnigs base ( n , n-diisopropylethylamine , diea ; 0.12  mmol , 16  mg ) and amino acid ( 0.08  mmol ) in dmf ( 2.5  ml ) .
after coupling , the resin was washed with dmf ( 32  ml ) and ch2cl2 ( 32  ml ) .
after completion of the deprotection / coupling sequence , the peptide was transferred to a microwave vessel , suspended in a solution of tfa / h2o ( 97.5:2.5 , 500  l ) , and microwaved ( 25  min , 50  hz ) .
the resin was removed by filtration and et2o ( 10  ml ) added to the supernatant .
the resulting crude precipitate was filtered and purified using hplc to yield 4 as a pink solid ( 0.9  mg , 1.3  mol , 7  % yield ) : lc  ms ( esi+ ) : m / z ( % ) : 719.5 ( 20 ) [ m+h ] ; lc  ms ( esi ) : m / z ( % ) : 717.5 ( 10 ) [ mh ] ; hrms ( esi ) : m / z calcd for [ m+h ] c35h47n10o7 : 719.3633 , found : 719.3629 .
trans - cyclooctene  desferrioxamine conjugate ( tco  dfo ) : tco - nhs ( 12  mg , 45  mol ) was added to a solution deferrioxamine mesylate salt ( 20  mg , 30  mol ) and et3n ( 21  l , 0.15  mmol ) in ch3cn / h2o ( 1:1 , 1  ml ) and stirred at rt for 90  min .
subsequently , volatiles were removed in vacuo , and the crude product was purified using hplc , yielding tco  dfo as a white solid ( 6.6  mg , 9  mol , 30  % ): h  nmr ( 500  mhz , [ d6]dmso ) : =9.689.54 ( m , 2  h ) , 7.787.74 ( m , 1  h ) , 6.936.88 ( m , 1  h ) , 5.605.40 ( m , 2  h ) , 4.214.17 ( m , 1  h ) , 3.483.41 ( m , 6  h ) , 3.032.87 ( m , 6  h ) , 2.612.53 ( m , 4  h ) , 2.292.21 ( m , 7  h ) , 1.96 ( s , 3  h ) , 1.911.85 ( m , 4  h ) , 1.561.15 ( m , 22  h ) ; lc  ms ( esi+ ) : m / z ( % ) : 357.4 ( 10 ) [ m+2  h ] , 713.6 ( 100 ) [ m+h ] , 735.5 ( 70 ) [ m+na ] , 759.4 ( 10 ) [ m+hcoo ] ; hrms ( esi ) : m / z calcd for [ m+h ] c34h61n6o10 : 713.4474 , found : 713.4449 . zr - trans - cyclooctene ( zr - tco , 5 ) : [ zr]zr - oxalate ( 59.274  mbq , 1.6002.000  ci ) in oxalic acid (
1.0  m , 150  l ) was adjusted to ph  7.28.0 with na2co3 ( 1.0  m , 150  l ) .
after the evolution of co2(g ) stops , the zr was added to a solution of tco  dfo ( 1  mm , 100  l ) in pbs / dmso ( 9:1 ) .
the reaction solution was stirred at 37  c for 30  min , before the reaction progress was assayed using instant thin - layer chromatography ( itlc ) with an eluent of 1  m citric acid . in the itlc experiments ,
zr - tco remains at the baseline , while free zr ions and other [ zr]-citrate complexes elute with high rf values .
the crude reaction mixture was purified using hplc , and volatiles were removed in vacuo , yielding 5 in > 98  % radiochemical purity , 39  % uncorrected isolated radiochemical yield ( rcy ) , and a specific activity of > 5.59  mci  mol ( n=3 ) .
zr - gafv ( 6 ) : gafv ( 4 , 20  nmol , 20  l , 1  mm in dmso ) was added to zr - tco ( 56  ci , 10  l , 5.56  mci  ml in 1:1 ch3cn / h2o with 0.05  % tfa ) , and the mixture was agitated at rt for 20  min , before purification using hplc , yielding 6 in > 95  % purity , 65  % uncorrected isolated rcy , and a specific activity of > 3.00  mci  mol ( n=3 ) .
boc - deprotection conditions : tfa ( 760  l ) was added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 20  l , dmso ) and the resulting mixture stirred at room temperature . a control sample consisted of dmso ( 760  l ) , which was added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 20  l , dmso ) .
the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9  h using hplc analysis , and the results were compared to the control sample .
coupling conditions : solutions of ( benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate ( bop , 10  mm , 25  l , dmf ) and diea ( 100  mm , 25  l , dmf ) were added to a solution of 2 ( 5  mm , 25  l , dmf ) and coumarin ( 5  mm , 25  l , dmf ) . a control sample consisted of dmf ( 50  l ) , which was added to a solution of 2 ( 5  mm , 25  l , dmf ) and coumarin ( 5  mm , 25  l , dmf ) .
the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1  h , 2  h , 3  h , 4  h , 5  h , 6  h , 7  h , 8  h and 9  h using hplc analysis , and the results were compared to the control sample .
resin - cleavage conditions : tfa ( 950  l ) and deionized h2o ( 25  l ) were added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 5  l , dmso ) , and the resulting mixture was exposed to sequential microwave irradiation intervals ( 0112.5  min , 50  w ) . a control sample consisted of dmso ( 975  l ) , which was added to a solution of 3 ( 5  mm , 20  l , dmso ) and coumarin ( 5  mm , 5  l , dmso ) . at the end of each microwave irradiation interval ,
a sample was drawn , the amount of 3 relative to coumarin was measured using hplc analysis , and the results were compared to the control sample .
 the need for post - synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents . in this regard , 
 the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far - reaching implications in both the laboratory and the clinic . 
 herein , we report the synthesis and development of a series of modular and versatile building blocks for inverse electron - demand diels  alder copper - free click chemistry : tetrazine - functionalized artificial amino acids . following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines 
 , we successfully demonstrated its effectiveness and applicability . 
 this versatile methodology has the potential to have a transformational impact , opening the door for the rapid , facile , and modular synthesis of bioorthogonally reactive peptide probes . 
Experimental Section
Synthesis
Tetrazine stability determination