the term  click chemistry  describes a collection of organic reactions that proceed rapidly and selectively under mild conditions to covalently link molecular components.(1 ) among the many click reactions described to date , the huisgen 1,3-dipolar cycloaddition of azides and alkynes(2 ) has received the most attention .
the reaction is highly exergonic ( g  61 kcal / mol),(3 ) the starting materials are easily prepared , the 1,2,3-triazole products are exceptionally stable , and the reaction occurs readily in both organic and aqueous solvents .
however , elevated temperatures or pressures are necessary to accelerate the reaction when simple alkynes and azides are employed , since the activation energy for the cycloaddition is high ( g  + 26 kcal / mol).(3 ) in the past decade , several strategies have been pursued in order to lower the activation barrier .
sharpless and co - workers(4 ) and meldal and co - workers(5 ) first reported that cu(i ) catalysis dramatically accelerates the reaction of terminal alkynes and azides , regioselectively forming the 1,4-disubstituted triazoles .
more recently , ruthenium - based catalysts have been employed to produce the 1,5-disubstituted isomer.(6 ) these metal - catalyzed variants of the huisgen [ 3 + 2 ] cycloaddition are now central tools for combinatorial library synthesis,(7 ) construction of peptidomimetics and glycomimetics,(8 ) supramolecular synthesis,(9 ) and labeling of biomolecules in vitro or in fixed samples .
however , the reliance of these chemistries on cytotoxic transition metals has largely precluded their use for applications in vivo , despite parallel growth in the use of azides as chemical reporters in biological systems.(15 ) as an alternative approach to lower the activation barrier for [ 3 + 2 ] cycloaddition , we have employed the intrinsic ring strain of cyclooctynes , following the precedent of wittig and krebs . these highly strained alkynes ( 18 kcal / mol of ring strain(18 ) ) react selectively with azides to form regioisomeric mixtures of triazoles at ambient temperatures and pressures without the need for metal catalysis and with no apparent cytotoxicity ( eq 1 ) : we further enhanced the cycloaddition rate by installing propargylic fluorine atoms intended to lower the lumo , thereby increasing its interaction energy with the homo of the azide,(19 ) an effect that was recently studied using density functional theory calculations.(20 ) the difluorinated cyclooctyne we termed  difo  ( compound 1 in figure 1 ) reacted with azides on intact proteins at a rate comparable to that of cu - catalyzed click chemistry and has since been employed for dynamic imaging of cell - surface glycans in live cells(19 ) and in developing zebrafish embryos.(21 ) other groups have reported the use of strained oxanorbornadiene(22 ) and dibenzocyclooctyne(23 ) reagents as substrates for [ 3 + 2 ] cycloaddition with azides , suggesting a rich future for cu - free click chemistry .
terminal alkynes can be installed in biomolecules using simple building blocks , such as commercial alkynoic acids .
in contrast , the synthesis of difo comprised 12 steps and an overall yield of 1%.(19 ) the final step of the sequence , elimination of a vinyl triflate to form the cyclooctyne , suffered from significant decomposition and low yield .
thus , synthetically tractable cyclooctynes with the capabilities of difo are needed in order to expand the use of this cu - free click reagent in biological settings . here
we report the design , synthesis , and biological evaluation of the second - generation difo reagents 2 and 3 ( figure 1 ) , which retain the difluorinated cyclooctyne core but possess a cc bond to a linker substituent at c4 rather than a co bond at c6 as in 1 ( figure 1 ) .
this structural change dramatically simplified the synthesis without impact on the kinetics or bioorthogonality of [ 3 + 2 ] cycloaddition with azides .
these synthetically tractable second - generation difo reagents should facilitate further applications of cu - free click chemistry .
all of the chemical reagents were analytical grade , obtained from commercial suppliers , and used without further purification , unless otherwise noted .
flash chromatography was carried out with merck 60 230400 mesh silica gel according to the procedure described by still et al.(24 ) when necessary , deactivated silica gel was prepared by rinsing silica gel thoroughly with a solution of 1% et3n in the starting solvent mixture used for flash chromatography .
reactions and chromatography fractions were analyzed with analtech 250 m silica gel g plates and visualized by staining with ceric ammonium molybdate , anisaldehyde , vanillin , or 2,4-dinitrophenylhydrazine or by absorbance of uv light at 245 nm .
solvents were removed using a rotary evaporator at reduced pressure ( 20 torr ) . unless otherwise noted , h , c{h } , f , and p{h } nmr spectra were obtained with 300 , 400 , or 500 mhz bruker spectrometers .
chemical shifts (  ) are reported in parts per million referenced to the solvent peaks for h and c , to cfcl3 for f , and to h3po4 for p. coupling constants ( j ) are reported in hertz .
low - resolution and high - resolution ( hr ) fast atom bombardment ( fab ) , electron impact ( ei ) , and electrospray ionization ( esi ) mass spectra were obtained at the uc berkeley mass spectrometry facility .
reversed - phase hplc was performed using a rainin dynamax sd-200 hplc system with 210 nm detection on a microsorb c18 analytical or preparative column .
dulbecco s phosphate - buffered saline ( pbs ) and fetal bovine serum ( fbs ) were purchased from hyclone laboratory , and rpmi-1640 and f12 media were obtained from invitrogen life technologies , inc .
fluorescein isothiocyanate ( fitc)-labeled avidin was purchased from sigma - aldrich . flow cytometry analysis was performed on a bd facscalibur flow cytometer using a 488 nm argon laser .
cell viability was ascertained on the basis of forward scatter ( to sort by size ) and side scatter ( to sort by granularity ) .
the average fluorescence intensity was calculated from each of three replicate experiments in order to obtain a representative value in arbitrary units .
for all of the flow cytometry experiments , data points were collected in triplicate and represent at least two separate experiments .
fluorescence microscopy was performed on a zeiss 200 m epifluorescence microscope , and the images were deconvolved using the nearest - neighbor algorithm of slidebook 4.2 ( intelligent imaging innovations , inc . ) .
a flame - dried round - bottom flask was charged with 1,3-cyclooctanedione ( 5.10 g , 36.4 mmol ) and mecn ( 260 ml ) . cs2co3 ( 24.3 g , 74.6 mmol ) was added , and the reaction mixture was stirred at room temperature ( rt ) for 30 min .
the reaction mixture was cooled to 0 c , and selectfluor ( 31.0 g , 87.4 mmol ) was added , after which the mixture was stirred for an additional 15 min .
the system was allowed to warm to rt , stirred for 1.5 h , concentrated under reduced pressure , diluted with 1 m hcl ( 200 ml ) , and extracted with diethyl ether ( 4  200 ml ) .
the combined organic layers were washed with brine ( 2  100 ml ) , dried over mgso4 , and filtered through a glass frit .
the solution was concentrated under reduced pressure and purified by flash chromatography ( 9:1 hexanes / etoac ) to yield a white solid ( 4.70 g , 73% ) .
h nmr ( 500 mhz , cdcl3 ) :  2.67 ( m , 4h ) , 1.81 ( m , 4h ) , 1.63 ( m , 2h ) .
c nmr ( 125 mhz , cdcl3 ) :  197.9 ( t , j = 25.1 hz ) , 109.5 ( t , j = 261.5 hz ) , 38.7 , 26.2 , 24.7 .
ir ( thin film , cm ) : 3468 , 2946 , 2867 , 1732 .
hrms ( ei ) : calcd for c8h10o2f2 , 176.0649 ; found , 176.0646 . to a flame - dried round - bottom flask
were added diketone 4 ( 1.57 g , 8.92 mmol ) , phosphonium bromide 5 ( 4.60 g , 9.37 mmol ) , and thf ( 200 ml ) .
the system was cooled to 0 c , and dbu ( 1.34 ml , 8.92 mmol ) was added , after which the reaction mixture was stirred for 20 min at 0 c . after the reaction mixture was warmed to rt , it was stirred for an additional 48 h ; the reaction was quenched with acoh ( 1.5 ml ) , and the mixture was diluted with meoh ( 20 ml ) , concentrated under reduced pressure , and purified by flash chromatography ( 05% etoac in a 2:1 mixture of hexanes / toluene ) to yield a white solid ( 2.63 g , 96% ) .
h nmr ( 400 mhz , cdcl3 ) :  8.05 ( d , 2h , j = 8.4 hz ) , 7.38 ( d , 2h , j = 8.2 hz ) , 7.23 ( s , 1h ) , 3.92 ( s , 3h ) , 2.70 ( t , 2h , j = 6.6 hz ) , 2.52 ( app t , 2h , j = 6.2 hz ) , 1.86 ( m , 2h ) , 1.53 ( m , 4h ) .
c nmr ( 125 mhz , cdcl3 ) :  202.1 ( t , j = 28.9 hz ) , 166.8 , 140.0 , 134.6 ( t , j = 19.6 hz ) , 131.2 ( t , j = 10.3 hz ) , 130.0 , 129.7 , 129.0 , 115.2 ( t , j = 253.4 hz ) , 52.4 , 37.5 , 27.3 , 26.0 , 25.7 , 25.3 ( t , j = 2.5 hz ) .
hrms ( fab ) : calcd for c17h18o3f2 [ m + h ] , 309.1302 ; found , 309.1302 . to a round - bottom flask
were added olefin 6 ( 2.63 g , 8.53 mmol ) and meoh ( 100 ml ) .
the system was flushed with n2 , and a catalytic amount of pd / c was added .
the system was again flushed with n2 followed by h2 , and the reaction mixture was stirred under an h2 atmosphere ( using a balloon ) for 24 h. the system was then flushed thoroughly with n2 , after which the reaction mixture was diluted with ch2cl2 ( 100 ml ) , filtered through celite , and concentrated under reduced pressure .
the crude product was purified by flash chromatography ( 02% etoac in a 2:1 mixture of hexanes / toluene ) to yield a white solid ( 2.47 g , 93% ) .
h nmr ( 400 mhz , cdcl3 ) :  7.99 ( dm , 2h , j = 8.3 hz ) , 7.27 ( d , 2h , j = 8.14 hz ) , 3.92 ( s , 3h ) , 3.31 ( dd , 1h , j = 13.6 , 2.9 hz ) , 2.822.73 ( m , 1h ) , 2.662.48 ( m , 2h ) , 2.452.28 ( m , 1h ) , 2.172.02 ( m , 1h ) , 1.971.84 ( m , 1h ) , 1.661.42 ( m , 4h ) , 1.411.29 ( m , 1h ) , 1.291.11 ( m , 1h ) .
c nmr ( 125 mhz , cdcl3 ) :  205.7 ( dd , j = 30.4 , 25.1 hz ) , 167.1 , 144.7 , 130.0 , 129.4 , 128.6 , 119.4 ( dd , j = 258.0 , 250.7 hz ) , 52.2 , 46.4 ( t , j = 21.6 ) , 39.1 , 33.8 ( t , j = 4.8 hz ) , 27.2 , 24.3 ( d , j = 6.9 hz ) , 24.1 ( d , j = 3.4 hz ) , 22.9 .
f nmr ( 376 mhz , cdcl3 ) :  102.72 ( d , 1f , j = 245.5 hz ) , 122.67 ( d , 1f , j = 251.7 hz ) .
hrms ( fab ) : calcd for c17h20o3f2 [ m + h ] , 311.1459 ; found , 311.1467 . to a flame - dried round - bottom flask was added thf ( 175 ml ) followed by khmds ( 14.81 ml of a 0.5 m solution in toluene , 7.40 mmol ) .
the reaction mixture was cooled to 78 c with stirring , and ketone 7 ( 2.19 g , 7.05 mmol ) in thf ( 70 ml ) was added dropwise over 20 min .
the reaction mixture was stirred for an additional 40 min , and then a solution of tf2nph ( 2.65 g , 7.40 mmol ) in thf ( 70 ml ) was added via syringe .
the system was warmed to rt with stirring over 21 h , and the reaction was then quenched with meoh ( 10 ml ) .
the reaction mixture was concentrated under reduced pressure and purified by flash chromatography ( 05% etoac in 4:1 hexanes / toluene with 1% et3n ) to yield a pale - yellow oil ( 2.74 g , 88% ) .
h nmr ( 400 mhz , cd3cn ) :  7.93 ( d , 2h , j = 8.2 hz ) , 7.36 ( d , 2h , j = 8.2 hz ) , 6.25 ( t , 1h , j = 9.4 hz ) , 3.85 ( s , 3h ) , 3.24 ( dd , 1h , j = 13.5 , 3.8 hz ) , 2.882.70 ( m , 1h ) , 2.61 ( dd , 1h , j = 13.5 , 10.2 hz ) , 2.562.42 ( m , 1h ) , 2.412.30 ( m , 1h ) , 1.681.52 ( m , 3h ) , 1.511.44 ( m , 2h ) , 1.431.34 ( m , 1h ) .
c nmr ( 125 mhz , cd3cn ) :  167.6 , 145.8 , 143.6 ( t , j = 29.4 hz ) , 130.5 , 130.4 , 129.5 , 129.1 ( t , j = 4.0 hz ) , 120.5 ( dd , j = 246.2 , 243.1 hz ) , 119.5 ( q , j = 319.1 hz ) , 52.7 , 46.8 ( app t , j = 22.0 hz ) , 35.2 ( app d , j = 5.4 hz ) , 27.0 , 26.2 , 23.0 , 21.8 .
f nmr ( 376 mhz , cd3cn ) :  74.45 ( s , 3f ) , 93.93 ( d , 1f , j = 272.2 hz ) , 105.18 ( d , 1f , j = 266.9 hz ) .
hrms ( fab ) : calcd for c18h19o5f5s [ m + h ] , 443.0952 ; found , 443.0960 . to a flame - dried round - bottom flask
was added vinyl triflate 8 ( 2.74 g , 6.20 mmol ) in thf ( 160 ml ) .
the mixture was cooled to 20 c with stirring . in a separate flame - dried round - bottom flask ,
a 0.199 m solution of lda was made by adding n - butyllithium ( 8.46 ml of a 2.5 m solution in hexanes , 21.2 mmol ) dropwise to a solution of diisopropylamine ( 3.59 ml , 25.4 mmol ) in thf ( 93.8 ml ) while stirring at 78 c . a portion of the lda solution ( 37.4 ml , 7.44 mmol ) was added dropwise to the first mixture over 1 h using a syringe pump at 20 c .
the reaction mixture was then brought to rt over 20 min , and the reaction was quenched with meoh ( 10 ml ) ; the mixture was then concentrated under reduced pressure and purified by flash chromatography ( 03% etoac in 2:1 hexanes / toluene ) to yield a white solid ( 1.58 g , 87% ) .
h nmr ( 400 mhz , cdcl3 ) :  7.99 ( d , 2h , j = 8.1 hz ) , 7.27 ( d , 2h , j = 7.9 hz ) , 3.92 ( s , 3h ) , 3.16 ( app d , 1h , j = 11.2 hz ) , 2.602.43 ( m , 2h ) , 2.412.24 ( m , 2h ) , 2.101.88 ( m , 2h ) , 1.871.68 ( m , 2h ) , 1.621.44 ( m , 1h ) , 1.211.08 ( m , 1h ) .
c nmr ( 125 mhz , cdcl3 ) :  167.2 , 145.5 , 130.0 , 129.4 , 128.4 , 119.5 ( t , j = 238.6 hz ) , 109.9 ( t , j = 11.1 hz ) , 85.1 ( dd , j = 47.2 , 41.6 hz ) , 58.2 ( t , j = 24.3 hz ) , 52.2 , 34.5 ( d , j = 4.7 hz ) , 32.6 , 30.8 ( d , j = 4.4 hz ) , 28.0 , 20.4 .
f nmr ( 376 mhz , cdcl3 ) :  94.32 ( d , 1f , j = 260.2 hz ) , 101.36 ( dm , 1f , j = 259.8 hz ) .
hrms ( fab ) : calcd for c17h18o2f2 [ m + h ] , 293.1353 ; found , 293.1357 . to a round - bottom flask fitted with a reflux condenser were added cyclooctyne methyl ester 9 ( 1.58 g , 5.42 mmol ) , lioh ( 2.59 g , 108 mmol ) , water ( 6 ml ) , and dioxane ( 24 ml ) under an air atmosphere .
the reaction mixture was heated to 55 c and stirred for 3 h. the mixture was then cooled to rt and diluted with 1 m hcl until the ph of the solution was < 2 .
the combined organic layers were then washed ( 1:1 1 m hcl / brine , 50 ml ) , dried over mgso4 , filtered through a glass frit , and concentrated under reduced pressure .
the crude product was then purified by flash chromatography ( 9:1 to 3:1 hexanes / etoac with 12% acoh ) to yield a white solid ( 1.08 g , 72% ) .
rf = 0.45 ( 4:1 hexanes / etoac with 1% acoh ) ; mp 181.0182.0 ( dec ) .
h nmr ( 400 mhz , cd3cn ) :  10.108.80 ( br s , 1h ) , 7.94 ( d , 2h , j = 8.2 hz ) , 7.35 ( d , 2h , j = 8.1 hz ) , 3.10 ( d , 1h , j = 10.9 hz ) , 2.702.50 ( m , 2h ) , 2.432.24 ( m , 2h ) , 2.041.84 ( m , 2h ) 1.831.67 ( m , 2h ) , 1.551.41 ( m , 1h ) , 1.221.11 ( m , 1h ) .
c nmr ( 125 mhz , acetone - d6 ) :  167.6 , 146.2 , 130.8 , 130.2 , 129.6 , 120.2 ( dd , j = 239.1 , 235.4 hz ) , 111.6 ( app t , j = 11.3 hz ) , 85.5 ( dd , j = 46.7 , 41.9 hz ) , 58.6 ( t , j = 24.2 hz ) , 34.9 ( d , j = 4.9 hz ) , 33.3 ( d , j = 2.1 hz ) , 31.6 ( d , j = 4.7 hz ) , 28.4 , 20.5 . f nmr ( 376 mhz , cd3cn ) :  93.81 ( d , 1f , j = 258.7 hz ) , 101.32 ( ddt , 1f , j = 258.8 , 20.3 , 7.0 hz ) .
hrms ( fab ) : calcd for c16h16o2f2 [ m + h ] , 279.1197 ; found , 279.1190 . to a flame - dried round - bottom flask were added dcc ( 3.66 g , 17.7 mmol ) , dmap ( 98.6 mg , 0.807 mmol ) , 3-methyl-3-oxetanemethanol ( 1.59 ml , 16.1 mmol ) , and ch2cl2 ( 20 ml ) . the mixture was cooled to 0 c with stirring . a solution of iodoacetic acid ( 3.00 g , 16.1 mmol ) in ch2cl2 ( 30 ml )
the reaction mixture was stirred for 1 h at 0 c and allowed to warm to rt over an additional 1.5 h ; the reaction was quenched with acetic acid ( 1 ml ) , and the mixture was stirred for an additional 30 min .
the filtrate ( 300 ml total ) was then washed with water ( 200 ml ) , saturated nahco3 ( 2  200 ml ) , and brine ( 200 ml ) .
the organic layer was then dried over mgso4 , filtered through a glass frit , and concentrated under reduced pressure .
the crude product was diluted with ch2cl2 and again filtered through celite to remove any residual dicyclohexylurea that had precipitated .
this material was then transferred to a new round - bottom flask and dissolved in thf ( 100 ml ) .
triphenylphosphine ( 4.65 g , 17.7 mmol ) was added , and the reaction mixture was stirred under n2 at rt for 40 h and then diluted with diethyl ether ( 100 ml ) and filtered through a glass frit to isolate the precipitated product .
residual solvent was removed under reduced pressure to yield a pale - yellow solid ( 7.93 g , 92% over two steps ) .
h nmr ( 400 mhz , cdcl3 ) :  7.937.84 ( m , 6h ) , 7.847.78 ( m , 3h ) , 7.737.65 ( m , 6h ) , 5.51 ( d , 2h , j = 13.5 hz ) 4.26 ( s , 4h ) , 4.15 ( s , 2h ) , 1.22 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  164.3 ( d , j = 3.5 hz ) , 135.4 ( d , j = 3.1 hz ) , 133.9 ( d , j = 10.8 hz ) , 130.4 ( d , j = 13.2 hz ) , 117.4 ( d , j = 89.2 hz ) , 79.1 , 71.1 , 38.8 , 33.6 ( d , j = 56.6 hz ) , 21.0 .
hrms ( esi ) : calcd for c25h26o3p , 405.1620 ; found , 405.1631 . to a flame - dried round - bottom flask were added phosphonium iodide 10 ( 5.66 g , 10.6 mmol ) , ch2cl2 ( 75 ml ) , and dbu ( 1.51 ml , 10.1 mmol ) , and the reaction mixture was stirred for 20 min . in a separate flame - dried round - bottom flask , difluoroketone 4 ( 1.78 g , 10.1 mmol ) was dissolved in ch2cl2 ( 125 ml ) , and this solution was then added to the phosphonium iodide solution .
the reaction mixture was allowed to stir for 48 h , concentrated under reduced pressure , and purified by flash chromatography ( 10:1 to 4:1 hexanes / etoac ) , yielding a white solid ( 2.87 g , 94% ) .
h nmr ( 500 mhz , cdcl3 ) :  6.49 ( s , 1h ) , 4.51 ( d , 2h , j = 6.0 hz ) , 4.40 ( d , 2h , j = 6.0 hz ) , 4.25 ( s , 2h ) , 2.81 ( t , 2h , j = 6.6 hz ) , 2.66 ( t , 2h , j = 6.7 hz ) , 1.82 ( m , 2h ) , 1.72 ( m , 2h ) , 1.52 ( m , 2h ) , 1.35 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  200.2 ( t , j = 28.1 hz ) , 165.1 , 150.4 ( t , j = 20.0 hz ) , 121.1 ( t , j = 9.6 hz ) , 114.2 ( t , j = 254.6 hz ) , 79.7 , 69.3 , 39.2 , 37.5 , 26.7 , 26.6 , 26.0 , 25.5 , 21.3 . f nmr ( 376 mhz , cd3cn ) :  113.03 ( s ) .
hrms ( fab ) : calcd for c15h20o4f2 [ m + h ] , 303.1408 ; found , 303.1404 . to a round - bottom flask
were added compound 11 ( 804 mg , 2.66 mmol ) and meoh ( 30 ml ) .
the system was flushed with n2 , and a catalytic amount of pd / c was added .
the system was then flushed again with n2 followed by h2 , and the reaction mixture was stirred under an h2 atmosphere for 24 h. the system was flushed thoroughly with n2 , and the reaction mixture was diluted with ch2cl2 ( 30 ml ) and filtered through celite to remove the catalyst .
the crude material was dissolved in ch2cl2 ( 17 ml ) and transferred via syringe to a new flame - dried round - bottom flask , which was under a n2 atmosphere and contained activated 4  molecular sieves . the mixture
was then cooled to 0 c with stirring . in a separate flame - dried conical flask ,
a 0.20 m solution of bf3oet2 ( 100 l , 0.780 mmol ) in ch2cl2 ( 3.9 ml ) was prepared , and a portion of this solution ( 1.0 ml , 0.20 mmol ) was added to the reaction mixture via syringe .
the reaction mixture was warmed to rt and stirred for an additional 20 h before the reaction was quenched with et3n ( 0.5 ml ) .
the reaction mixture was then concentrated under reduced pressure and purified by flash chromatography ( 20:1 hexanes / etoac with 1% et3n over deactivated silica gel ) to yield a white solid ( 731 mg , 90% over two steps ) . rf = 0.70 ( 2:1 hexanes / etoac ) ; mp 80.383.3 c .
h nmr ( 500 mhz , cdcl3 ) :  3.89 ( s , 6h ) , 2.65 ( m , 1h ) , 2.622.46 ( m , 2h ) , 2.21 ( d , 1h , j = 14.6 hz ) , 2.101.94 ( m , 2h ) , 1.90 ( br s , 1h ) , 1.60 ( m , 2h ) , 1.561.42 ( m , 2h ) , 1.36 ( m , 2h ) , 0.80 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  205.8 ( dd , j = 29.7 , 25.7 hz ) , 119.3 ( dd , j = 257.6 , 250.2 hz ) , 109.0 , 72.8 , 39.2 ( t , j = 21.2 hz ) , 38.9 , 34.6 ( t , j = 4.7 hz ) , 30.5 , 27.0 , 26.3 ( d , j = 7.2 hz ) , 24.8 ( d , j = 2.7 hz ) , 23.3 , 14.7 .
f nmr ( 376 mhz , cd3cn ) :  103.10 ( d , 1f , j = 246.4 hz ) , 124.20 ( dm , 1f , j = 249.0 hz ) .
hrms ( fab ) : calcd for c15h22o4f2 [ m + h ] , 305.1564 ; found , 305.1558 . to a flame - dried round - bottom flask was added thf ( 30 ml ) followed by khmds ( 2.66 ml of a 0.5 m solution in toluene , 1.33 mmol ) .
the reaction mixture was cooled to 78 c with stirring , and ketone 12 ( 354 mg , 1.16 mmol ) in thf ( 15 ml ) was added dropwise via syringe over 15 min .
the reaction mixture was stirred for an additional 3 h , and then a solution of tf2nph ( 457 mg , 1.28 mmol ) in thf ( 15 ml ) was added via syringe .
the system was allowed to slowly warm to rt with stirring over 19 h. the reaction was then quenched with deactivated silica gel , and the mixture was concentrated under reduced pressure and purified by flash chromatography ( 20:1 to 15:1 hexanes / etoac with 1% et3n over deactivated silica gel ) to yield a white solid ( 407 mg , 80% ) .
h nmr ( 400 mhz , cdcl3 ) :  6.05 ( t , 1h , j = 9.6 hz ) , 3.89 ( s , 6h ) , 2.742.56 ( m , 1h ) , 2.512.31 ( m , 2h ) , 2.20 ( dd , 1h , j = 14.5 , 1.7 hz ) , 2.001.87 ( m , 1h ) , 1.721.49 ( m , 6h ) , 0.80 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 )  143.2 ( t , j = 30.2 hz ) , 126.9 , 119.2 ( app t , j = 246.5 hz ) , 118.6 ( q , j = 320.0 hz ) , 108.9 , 72.8 , 40.9 ( app t , j = 22.3 hz ) , 35.1 , 30.5 , 27.2 , 26.6 , 22.6 , 21.8 , 14.6 .
f nmr ( 376 mhz , cdcl3 ) :  74.52 ( s , 3f ) , 93.80 ( d , 1f , j = 269.8 hz ) , 104.75 ( dm , 1f , j = 278.0 hz ) .
hrms ( fab ) : calcd for c16h21o6f5s [ m + h ] , 437.1057 ; found , 437.1050 . in a round - bottom flask , vinyl triflate 13 ( 407 mg , 0.932 mmol ) was dissolved in toluene ( 20 ml ) and concentrated under reduced pressure to remove trace moisture .
the material was then dissolved in thf ( 20 ml ) , and the solution was cooled to 20 c with stirring . in a separate flame - dried round - bottom flask , a 0.20 m solution of lda was made by adding n - butyllithium ( 1.12 ml of a 2.5 m solution in hexanes , 2.80 mmol ) dropwise to a solution of diisopropylamine ( 475 l , 3.36 mmol ) in thf ( 12.4 ml ) at 78 c .
a portion of the lda solution ( 5.6 ml , 1.12 mmol ) was added dropwise via syringe pump to the first mixture over 1 h. the reaction mixture was then brought to rt over 30 min , and the reaction was quenched with deactivated silica gel ; the mixture was then concentrated under reduced pressure and purified by flash chromatography ( 20:1 to 15:1 hexanes / etoac with 1% et3n over deactivated silica gel ) to yield a white solid ( 159 mg , 59% ) .
h nmr ( 500 mhz , cdcl3 ) :  3.90 ( s , 6h ) , 2.54 ( app dq , 1h , j = 23.5 , 9.1 hz ) , 2.402.25 ( m , 2h ) , 2.252.11 ( m , 2h ) , 2.102.02 ( m , 2h ) , 1.811.71 ( m , 1h ) , 1.65 ( dd , 1h , j = 14.6 , 10.3 hz ) , 1.50 ( app quint , 1h , j = 7.5 hz ) , 1.33 ( m , 1h ) , 0.81 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  120.0 ( t , j = 238.3 hz ) , 109.8 ( t , j = 11.2 hz ) , 85.4 ( dd , 47.1 , 41.8 hz ) , 72.8 , 51.9 ( t , j = 23.8 hz ) , 35.2 ( dd , j = 4.5 , 1.6 hz ) , 32.8 ( d , j = 4.8 hz ) , 32.7 ( d , j = 2.1 hz ) , 30.5 , 29.9 , 28.2 , 20.5 , 14.8 .
f nmr ( 376 mhz , cd3cn ) :  94.82 ( d , 1f , j = 258.7 hz ) , 101.81 ( ddt , 1f , j = 259.3 , 24.1 , 7.2 hz ) .
hrms ( fab ) : calcd for c15h20o3f2 [ m + h ] , 287.1459 ; found , 287.1461 . to a scintillation vial under an air atmosphere were added cyclooctyne orthoester 14 ( 90.4 mg , 0.316 mmol ) , meoh ( 4.5 ml ) , water ( 450 l ) , and ppts ( 159 mg , 0.632 mmol ) .
the reaction mixture was stirred at rt for 24 h , after which the reaction was quenched with saturated nahco3 ( 2 ml ) and the mixture concentrated under reduced pressure .
the crude product was diluted with brine ( 8 ml ) and extracted with etoac ( 3  10 ml ) .
the combined organic layers were washed with brine ( 10 ml ) and a hcl / brine solution ( 1:1 1 m hcl / brine , 2  10 ml ) , dried over mgso4 , and filtered through a glass frit .
the filtrate was concentrated under reduced pressure to yield a white solid ( 106.8 mg ) .
a portion of this material ( 57.8 mg ) was transferred to a round - bottom flask , where it was dissolved in dioxane ( 1 ml ) and water ( 200 l ) . to this
was added lioh ( 91 mg , 3.8 mmol ) , and the reaction mixture was stirred at rt for 3 h under an air atmosphere , after which the reaction was quenched with 1 m hcl ( 5 ml ) .
the reaction mixture was further diluted with brine ( 3 ml ) and extracted with etoac ( 4  10 ml ) .
the combined organic layers were washed ( 1:1 1 m hcl / brine , 10 ml ) , dried over mgso4 , and filtered through a glass frit .
the filtrate was concentrated and purified by flash chromatography ( 20:1 hexanes / etoac with 2% acoh ) to give a white solid ( 33 mg , 96% over two steps ) .
rf = 0.66 ( 1:1 hexanes / etoac with 1% acoh ) ; mp 87.488.9 c .
h nmr ( 400 mhz , cdcl3 ) :  11.9010.60 ( br s , 1h ) , 2.842.69 ( m , 2h ) , 2.462.28 ( m , 3h ) , 2.212.05 ( m , 2h ) , 1.901.74 ( m , 2h ) , 1.67 ( m , 1h ) , 1.41 ( m , 1h ) .
c nmr ( 125 mhz , cdcl3 ) :  177.8 , 119.1 ( t , j = 238.6 hz ) , 110.7 ( t , j = 11.2 hz ) , 84.7 ( dd , j = 47.0 , 41.6 hz ) , 52.6 ( t , j = 24.4 hz ) , 33.7 ( app d , j = 4.4 hz ) , 32.9 ( d , j = 4.6 hz ) , 32.7 ( d , j = 2.0 hz ) , 27.9 , 20.5 .
f nmr ( 376 mhz , cdcl3 )  94.64 ( d , 1f , j = 260.0 hz ) , 100.82 ( ddt , 1f , j = 260.2 , 21.1 , 6.8 hz ) . hrms ( esi ) : calcd for c10h11o2f2 [ m ] , 201.0722 ; found , 201.0729 .
to a flame - dried round - bottom flask were added cyclooctyne 2 ( 10.5 mg , 0.0377 mmol ) , ch2cl2 ( 0.5 ml ) , and diisopropylethylamine ( 16.4 l , 0.0943 mmol ) , and the mixture was cooled to 0 c with stirring .
pentafluorophenyl trifluoroacetate ( 7.1 l , 0.042 mmol ) was then added , and after 10 min , the system was warmed to rt .
the reaction mixture was stirred an additional 1.5 h , concentrated , filtered through a plug of silica gel eluting with hexanes , and then concentrated to yield a white solid .
this material was transferred to a new round - bottom flask and dissolved in anhydrous dmf ( 0.5 ml ) .
biotinylated amine 16(25 ) ( 12.0 mg , 0.0268 mmol ) was then added , followed by diisopropylethylamine ( 7.0 l , 0.040 mmol ) .
the reaction mixture was stirred overnight , concentrated , and purified twice by flash chromatography ( 1% et3n in ch2cl2 , then 5% meoh in ch2cl2 ) to yield a clear oil ( 9.7 mg , 36% over two steps ) .
h nmr ( 400 mhz , cd3od ) :  8.41 ( m , 1h ) , 7.93 ( m , 1h ) , 7.77 ( d , 2h , j = 8.2 hz ) , 7.32 ( d , 2h , j = 8.2 hz ) , 4.48 ( dd , 1h , j = 7.8 , 4.7 hz ) , 4.29 ( dd , 1h , j = 7.9 , 4.5 hz ) , 3.683.53 ( m , 10h ) , 3.48 ( q , 4h , j = 6.0 hz ) , 3.283.16 ( m , 4h ) , 3.10 ( d , 1h , j = 10.8 hz ) , 2.92 ( dd , 1h , j = 12.7 , 5.0 hz ) , 2.70 ( d , 1h , j = 12.7 hz ) , 2.632.47 ( m , 2h ) , 2.442.27 ( m , 2h ) , 2.19 ( t , 2h , j = 7.4 hz ) , 2.081.99 ( m , 1h ) , 1.981.79 ( m , 4h ) , 1.791.69 ( m , 4h ) , 1.691.55 ( m , 3h ) , 1.51 ( dd , 1h , j = 15.9 , 8.1 hz ) , 1.471.37 ( m , 2h ) , 1.31 ( t , 1h , j = 7.3 hz ) , 1.231.12 ( m , 1h ) .
c nmr ( 125 mhz , cd3od ) :  176.1 , 170.1 , 166.3 , 145.2 , 134.0 , 130.5 , 128.7 , 121.0 ( dd , j = 238.9 , 236.0 hz ) , 111.2 ( t , j = 11.2 hz ) , 86.0 ( dd , j = 46.8 , 41.8 hz ) , 71.7 , 71.7 , 71.4 , 71.4 , 70.4 , 70.1 , 63.5 , 61.8 , 59.5 ( t , j = 24.3 hz ) , 57.2 , 41.2 , 38.9 , 38.0 , 37.0 , 35.2 ( d , j = 4.0 hz ) , 33.8 ( d , j = 1.5 hz ) , 32.0 ( d , j = 4.6 hz ) , 30.6 ( d , j = 3.8 hz ) , 30.0 , 29.7 , 29.1 , 27.0 , 20.8 , 9.4 .
f nmr ( 376 mhz , cd3od ) :  95.40 ( d , 1h , j = 259.9 hz ) , 102.76 ( ddt , 1h , j = 259.9 , 20.1 , 6.8 hz ) .
hrms ( fab ) : calcd for c36h52n4o6f2s [ m + li ] , 713.3736 ; found , 713.3736 . to a flame - dried round - bottom flask
were added cyclooctyne 3 ( 26.3 mg , 0.130 mmol ) , ch2cl2 ( 2.0 ml ) , and diisopropylethylamine ( 57.0 l , 0.325 mmol ) , and the mixture was cooled to 0 c with stirring .
pentafluorophenyl trifluoroacetate ( 25.0 l , 0.143 mmol ) was added , and after 10 min , the system was warmed to rt .
the reaction mixture was stirred for an additional 1 h , concentrated , and filtered through a plug of silica gel using 1% etoac in hexanes as the eluent to yield a white solid ( 41.4 mg ) . a portion of this material ( 6.5 mg , 0.018 mmol ) was then transferred to a flame - dried conical flask and dissolved in anhydrous dmf ( 0.5 ml ) . biotinylated amine 16(25 ) ( 7.9 mg , 0.018 mmol ) was added , followed by diisopropylethylamine ( 5.0 l , 0.027 mmol ) .
the reaction mixture was stirred for 4 h , concentrated , and purified twice by flash chromatography ( 010% meoh in ch2cl2 ) to yield a clear oil .
the oil was further purified by reversed - phase hplc ( gradient of 570% ch3cn in h2o over 40 min , eluting at 27 min ) to yield a clear oil ( 6.4 mg , 50% over two steps ) .
h nmr ( 400 mhz , cd3od ) :  4.49 ( dd , 1h , j = 7.9 , 4.3 hz ) , 4.30 ( dd , 1h , j = 7.9 , 4.5 hz ) , 3.683.55 ( m , 8h ) , 3.52 ( dt , 4h , j = 6.2 , 1.4 hz ) , 3.27 ( m , 4h ) , 3.21 ( m , 1h ) , 2.93 ( dd , 1h , j = 12.8 , 5.0 hz ) , 2.822.66 ( m , 2h ) , 2.49 ( dd , 1h , j = 14.7 , 3.9 hz ) , 2.462.28 ( m , 2h ) , 2.20 ( t , 2h , j = 7.4 hz ) , 2.182.00 ( m , 3h ) , 1.851.70 ( m , 7h ) , 1.701.52 ( m , 4h ) , 1.501.33 ( m , 3h ) .
c nmr ( 125 mhz , cd3od ) :  176.1 , 173.8 , 166.3 , 120.9 ( app t , j = 237.4 hz ) , 111.8 ( app t , j = 11.0 hz ) , 85.7 ( dd , j = 46.6 , 42.0 hz ) , 71.7 , 71.7 , 71.4 , 71.4 , 70.1 , 70.0 , 63.5 , 61.8 , 57.2 , 54.6 ( t , j = 24.3 hz ) , 41.2 , 38.1 , 38.0 , 37.0 , 36.3 ( d , j = 4.0 hz ) , 33.9 ( d , j = 1.9 hz ) , 33.5 ( d , j = 4.5 hz ) , 30.6 , 30.0 , 29.7 , 29.0 , 27.1 , 20.8 , 14.6 . f nmr ( 376 mhz , cd3od ) :  95.57 ( d , 1f , j = 259.0 hz ) , 102.08 ( ddt , 1f , j = 259.4 , 20.9 , 6.8 hz ) .
hrms ( fab ) : calcd for c30h48n4o6f2s [ m + h ] , 631.3341 ; found , 631.3324 .
cyclooctyne 2 or 3 was mixed at a 1:1 molar ratio ( 15 mm ) with benzyl azide in either ( a ) cd3cn or ( b ) a 7:3 mixture of cd3cn and 25 mm potassium phosphate in d2o ( ph 7 ) , and the reaction was monitored by h nmr .
the kinetic data were derived by following the change in integration of resonances corresponding to the benzylic protons in benzyl azide (  4.4 ) compared to those of the benzylic protons in the triazole products (  5.55.7 ) .
the second - order rate constants for the reaction were determined by plotting 1/[azide ] versus time and subsequently using analysis by linear regression .
the second - order rate constant k ( ms ) corresponds to the determined slope .
jurkat or chinese hamster ovary ( cho ) cells were maintained in a 5% co2 , water - saturated atmosphere and grown in rpmi-1640 ( jurkat ) or f12 ( cho ) media supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 0.1 mg / ml ) .
cell densities were maintained between 1  10 and 1.6  10 cells / ml .
jurkat cells were incubated for 3 days in media containing 25 m ac4mannaz or no sugar .
the cells were distributed into a 96-well v - bottom tissue culture plate and washed three times by sequential concentration by centrifugation ( 2500 g , 3 min , 4 c ) and resuspension in 200 l of labeling buffer [ pbs ( ph 7.4 ) containing 1% fbs ] .
the cells were then incubated at rt with 100 l of 010 m 15ac in labeling buffer for 060 min , with dilutions made from a 2.5 mm stock solution in 7:3 pbs / dmf .
after incubation , cells were washed three times and resuspended in 100 l of fitc - labeled avidin ( 1:200 dilution in labeling buffer of a 1 mg / ml stock solution ) .
after a 15 min incubation in the dark at 4 c , the cells were washed once and then incubated with fitc - labeled avidin for an additional 15 min at 4 c .
the cells were washed three times and then diluted to a volume of 400 l for flow cytometry analysis .
annexin v - pe staining was performed according to instructions from the manufacturer immediately prior to flow cytometry analysis .
cho cells were incubated for 3 days in media containing 25 m ac4mannaz or no sugar in an eight - well labtek ii chambered coverglass ( nunc ) .
the cells were washed three times by sequential gentle aspiration of the media and addition of 500 l of media .
the cells were then treated with 100 l of a 10 m solution of 15b or 15c ( 1:250 dilution in media from a 2.5 mm stock solution in 7:3 pbs / dmf ) for 60 min at rt .
the cells were washed three times , stained with fitc - labeled avidin ( 100 l of a 1:200 dilution in media from a 1 mg / ml stock solution ) for 10 min at rt , washed three times , treated with hoechst 33342 dye ( 100 l of a 1:1000 dilution in media from a 1 mg / ml dmso stock solution ) for 2 min at rt to stain the nuclei , washed twice , and imaged by epifluorescence microscopy .
the synthesis of cyclooctyne 2 ( scheme 1 ) began with 1,3-cyclooctanedione , which was prepared in 67% yield as previously described.(26 ) difluorination of 1,3-dicarbonyl compounds with selectfluor has been reported to occur sluggishly under neutral conditions(27 ) and more rapidly using enamine intermediates(28 ) or microwave - assisted strategies.(29 ) we found that simply treating the diketone with selectfluor and cs2co3 at 0 c produced 2,2-difluoro-1,3-cyclooctanedione ( 4 ) in 73% yield . in order to install a linker with a protected carboxylic acid
fortuitously , we observed exclusive formation of monosubstituted product 6 even when excess amounts of 5 and base were used , thus enabling efficient desymmetrization of the diketone .
the observed chemoselectivity is likely due to the high electrophilicity of the first ketone equivalent compounded with the low nucleophilicity of the stabilized ylide .
hydrogenation of the olefin to saturated compound 7 and subsequent conversion of the ketone to a vinyl triflate ( 8) proceeded in good yield ( 82% over two steps ) .
cyclooctyne 9 was formed by lda - mediated elimination , and the methyl ester was saponified to yield difo reagent 2 in a total of six steps in 36% overall yield from 1,3-cyclooctanedione .
this yield is 25-fold higher than that for the previously reported synthesis of 1(19 ) and was achieved in half as many steps .
a lesson from previous studies is that the hydrophobicity of difo reagents can contribute to nonspecific protein and cell binding .
thus , we also synthesized a second - generation difo analogue lacking the nonessential phenyl moiety in the linker .
the target , compound 3 ( figure 1 ) , possessed a carboxymethyl group to which probes were later attached .
a key protecting - group strategy masked the carboxymethyl group as an oxabicycloortho ( obo ) ester in order to avoid acidic protons that would later interfere with vinyl triflate formation and elimination .
as shown in scheme 2 , 2,2-difluoro-1,3-cyclooctanedione ( 4 ) was reacted under basic conditions with phosphonium salt 10 , which bears an oxetane ester , an obo ester precursor.(30 ) again , we observed selective monosubstitution to yield olefin 11 in 85% yield .
hydrogenation of the olefin and conversion of the oxetane ester to the orthoester using bf3oet2(31 ) yielded ketone 12 in 90% yield .
formation of vinyl triflate 13 and elimination to form cyclooctyne 14 proceeded in 80% and 59% yields , respectively .
two - step deprotection of the obo ester to carboxylic acid 3 was accomplished by acidic deprotection using ppts to form a simple ester and subsequent saponification using lioh ( 86% yield).(32 ) the synthesis of 3 was accomplished with an overall yield of 28% from 1,3-cyclooctanedione . with these reagents in hand
, we used h nmr to measure the kinetics of the copper - free click reaction with the model compound benzyl azide .
for the reactions with 2 and 3 performed in cd3cn , we obtained second - order rate constants of ( 4.2  0.1 )  10 and ( 5.2  0.2 )  10 m s , respectively ; these values are comparable to that for the reaction of 1 with benzyl azide under identical conditions ( 7.6  10 ms).(19 ) to evaluate the kinetics of the [ 3 + 2 ] cycloaddition in an aqueous environment , we performed reactions of 2 and 3 with benzyl azide in a 7:3 mixture of cd3cn and 25 mm potassium phosphate in d2o ( ph 7 ) and obtained k values of ( 9.0  0.3 )  10 and ( 8.6  0.9 )  10 m s , respectively .
all three reagents are considerably more reactive than cyclooctynes lacking the difluoromethylene group.(33 ) additionally , we investigated the stabilities of compounds 2 and 3 with respect to biologically relevant nucleophiles .
we dissolved each cyclooctyne ( 20 mm ) in a 7:3 mixture of cd3cn and 25 mm potassium phosphate in d2o ( ph 7 ) and then added either 2-mercaptoethanol or 2-aminoethanol ( 20 mm ) .
we observed no reaction after 24 h , as monitored by h and f nmr , suggesting that compounds 2 and 3 are stable to water , thiols , amines , and alcohols at physiological ph .
finally , compounds 2 and 3 were found to be stable for many months when stored at 20 c .
an important application of copper - free click chemistry is the nontoxic and rapid detection of azides within living systems , either for molecular imaging or for subsequent affinity capture .
difo reagent 1 and its derivatives ( e.g. , the biotinylated derivative 15a(19 ) ) have proven useful for bioorthogonal labeling of cell - surface glycans bearing azides , and we set out to evaluate the second - generation difo reagents in this context .
we first derivatized carboxylic acids 2 and 3 as the biotinylated reagents 15b and 15c , respectively , via formation of the corresponding activated pentafluorophenyl esters and subsequent conjugation to an amine - derivatized biotin reagent ( scheme 3 ) .
jurkat cells were incubated with 25 m peracetylated n - azidoacetylmannosamine ( ac4mannaz ) for 3 days , resulting in the metabolic labeling of cell - surface glycans with azido sialic acid ( sianaz ) residues ( figure 2a).(34 ) the cells were washed and labeled with biotinylated reagents 15b and 15c either at various concentrations ( 010 m ) for 60 min ( figure 2b ) or for various reaction times ( 060 min ) at 10 m ( figure 2c ) . in all cases ,
the cells were subsequently stained with fitc - labeled avidin and analyzed by flow cytometry , as described previously.(35 ) both of the second - generation difo reagents displayed concentration- and time - dependent reaction profiles with cell - surface - associated azides .
we were pleased to observe that both reagents displayed very little background fluorescence labeling , though the more hydrophobic 15b resulted in slightly higher background fluorescence than 15c .
cells were first incubated with ac4mannaz , which is metabolically converted to cell - surface sianaz residues , and subsequently reacted with difluorinated cyclooctyne probes for visualization .
( be ) jurkat cells were metabolically labeled with 25 m ac4mannaz ( + az ) or no sugar ( az ) for 3 days .
the cells were labeled with 15b or 15c either ( b ) for 60 min at 0 , 1 , 2 , 5 , or 10 m or ( c ) for 0 , 15 , 25 , 35 , or 65 min at 10 m .
the cells were then stained with fitc - labeled avidin and analyzed by flow cytometry .
( de ) the cells were labeled with 15ac or no reagent ( no rgt ) for 60 min at 10 m and then sequentially stained with fitc - labeled avidin and annexin v - pe , followed by flow cytometry analysis .
the error bars indicate the standard deviation of three replicate samples . shown in ( d ) is the mean fluorescence intensity ( mfi ) in arbitrary units ( au ) . shown in ( e ) is the percentage of cells in each sample belonging to the population that stains highly with annexin v - pe .
we then compared the efficacy of cell - surface azide labeling of 15b and 15c to the first - generation reagent 15a in a 60 min reaction at 10 m ( figure 2d ) .
the observed labeling with 15b and 15c was approximately half of that with 15a , consistent with their relative rate constants .
these labeling efficiencies are all significantly higher than those previously reported for mono- or nonfluorinated cyclooctyne reagents or triarylphosphines capable of staudinger ligation.(33 ) finally , we tested the toxicity of probes 15ac by incubation of cells labeled as above ( 60 min , 10 m ) with phycoerythrin - conjugated annexin v , a marker of apoptosis .
as shown in figure 2e , none of the reagents caused a significant change in the percentage of annexin v - positive cells , indicating that difo reagents 15ac are not toxic to jurkat cells .
finally , we applied these novel cyclooctynes to image cell - surface glycans in live cho cells .
the cells were incubated for 3 days with 25 m ac4mannaz or with no sugar as a negative control and labeled with 10 m 15b or 15c for 60 min .
subsequently , the cells were treated with fitc - labeled avidin and hoechst 33342 , a live - cell nuclear stain , and imaged by epifluorescence microscopy .
we observed clear azide - dependent labeling at the cell surface in the ac4mannaz - treated cells and minimal background fluorescence in the negative control for both 15b and 15c ( figure 3 ) , indicating that these second - generation difo reagents can be utilized for live - cell imaging applications .
live - cell imaging of cell - surface glycans using second - generation difluorinated cyclooctynes .
cho cells were metabolically labeled with ( a , b ) 25 m ac4mannaz or ( c , d ) no sugar for 3 days .
the cells were labeled for 60 min with 10 m ( a , c ) 15b or ( b , d ) 15c , washed , stained with fitc - labeled avidin and hoechst 33342 , and imaged by epifluorescence microscopy .
images were deconvolved using the nearest - neighbor algorithm of slidebook 4.2 and are shown as maximum intensity z - projection fluorescence images over 7.5 m .
we have developed synthetically tractable second - generation difluorinated cyclooctyne reagents for copper - free click chemistry .
the synthesis of these reagents involves a novel method for one - step difluorination of 1,3-diketones , a selective wittig reaction to install the linkers , and the use of an orthoester as a cyclooctyne - compatible protecting group .
furthermore , these reagents can be prepared efficiently , with overall yields that are more than 20-fold higher than that of the first - generation reagent .
critically , we determined that these novel difo reagents are nontoxic to cells and can selectively tag azide - labeled biomolecules in living systems with efficiencies approaching that of the parent compound .
we envision that these reagents will enable widespread use of copper - free click chemistry in the context of in vivo imaging and ex vivo labeling of biomolecules and in the generation of novel biocompatible materials.(36 )
 the 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site - selective labeling of biomolecules in vitro and in vivo . 
 while copper catalysis has been widely employed to activate terminal alkynes for [ 3 + 2 ] cycloaddition , this method , often termed  click chemistry  
 , is currently incompatible with living systems because of the toxicity of the metal . 
 we recently reported a difluorinated cyclooctyne ( difo ) reagent that rapidly reacts with azides in living cells without the need for copper catalysis . here 
 we report a novel class of difo reagents for copper - free click chemistry that are considerably more synthetically tractable . 
 the new analogues maintained the same elevated rates of [ 3 + 2 ] cycloaddition as the parent compound and were used for imaging glycans on live cells . 
 these second - generation difo reagents should expand the use of copper - free click chemistry in the hands of biologists . 
Introduction
Experimental Section
Results and Discussion
Conclusion