probing the biological 
 functions of proteins and small molecules 
 is essential to our mechanistic understanding of cell biology .
recent 
 developments in bioorthogonal chemistry have provided a means to chemically tag biomolecules in live cells 
 without significantly perturbing their native functions .
these bioorthogonal 
 chemical tags provide unique handles that , after ligation with specific 
 reporters , enable visualization and/or identification of biological 
 targets .
bioorthogonal tags have been introduced into proteins in 
 a number of ways : reaction with clickable covalent inhibitors , ligand - directed affinity - based labeling with tosyl and other reactive groups , photoaffinity 
 labeling with clickable photoprobes , ligation using 
 enzymes ( such as lipoic acid ligase , protein farnesyltransferase or sortase ) , metabolic 
 labeling ( clickable glycans , amino acids , lipids , acetyl - coa and s - adenosyl - l - methionine analogues ) , and genetic 
 encoding of unnatural amino acids .
these techniques 
 are finding great utility in drug discovery efforts for target identification , 
 target - engagement , and target validation directly in living systems . with the widespread use of bioorthogonal labeling strategies , 
 there 
 is increasing effort to develop novel reactions for targeting cellular 
 biomolecules .
these reactions typically follow 
 second - order kinetics and have reaction rate constants ranging from 
 10 to 10 m s ; however , these rate constants are typically measured in vitro and 
 are not necessarily reflective of rates in complex biological systems . 

while many reactions have been evaluated in vitro or at the cell surface , 
 relatively few bioorthogonal reactions have been carried out in an 
 intracellular context .
in addition , with more than 20 unique reactions , selecting the appropriate chemistry for a given 
 application can be daunting .
we envisioned developing a system that 
 would enable the unbiased investigation of bioorthogonal reaction 
 progression in the complex intracellular biological mileu of living 
 cells , where issues such as reagent permeability , selectivity , and 
 cross - reactivity could be explored .
recently , methods have been 
 developed to label proteins with small - molecules 
 using genetically encoded fusion proteins with the covalent self - labeling 
 o - alkyl guanine - dna alkyltransferase ( agt ) known as snap - tag and an engineered haloalkane dehalogenase known as halotag .
these proteins covalently react with small molecule 
 substrates and allow selective labeling within living systems .
agt 
 fusion proteins can be covalently labeled with o - benzylguanine 
 derivatives and halotag fusion proteins covalently bind chloroalkane 
 ligands .
one advantage of these genetic fusion techniques is that 
 they can be designed to target selected organelles in live cells .
there are now a number of recombinant methods that can be used to 
 incorporate bioorthogonal groups into proteins , either via direct 
 encoding of unnatural amino acids , or through fusion of an acceptor peptide for ligase - mediated attachment 
 of a bioorthogonal tag .
several of these methods 
 have been applied to live - cell imaging . however , these techniques are not yet able to accept the full spectrum 
 of bioorthogonal tagging groups , many of which are sterically demanding 
 and hydrophobic . herein , we report the development 
 and validation of an organelle - targetable 
 model system that can be used to systematically evaluate bioorthogonal 
 reactions in live cells .
we adapted the halotag protein labeling technology by synthesizing chloroalkane derivatives incorporating various bioorthogonal 
 groups and covalently displaying these groups on halotag - fusion proteins . 

previously , this technology has been used to label halotag - fusion 
 proteins in live cells with chloroalkane - linked fluorescent molecules , photosensitizing chromophores , photoreactive 
 kinase inhibitors and hydrophobic tags .
halotag - protein fusions were selected to localize the halotag protein , 
 and hence the bioorthogonal reaction , to different subcellular compartments , 
 including the nucleus , cytosol , plasma membrane and endoplasmic reticulum 
 ( er ) .
because halotag labeling is broadly general , it can be used 
 to incorporate a wide range of bioorthogonal reaction partners thereby 
 enabling the comparative study of reaction efficiency and selectivity . 

moreover , the same halotag constructs can be used to directly incorporate 
 fluorophores , thereby providing a fluorescence readout for quantifying 
 the efficiency of bioorthogonal methods of attaching the same fluorophores . 

overall , this system enables a quantitative comparison between different 
 bioorthogonal ligations , as well as the participating chemical groups , 
 in the complex microenvironments within the confines of a living cell , 
 where true bioorthogonality can be evaluated . to validate this 
 system , we investigated two prominent bioorthogonal 
 chemistries that are amenable to live - cell labeling , the strain - promoted 
 azide  alkyne cycloaddition ( spaac ) between cyclooctynes and 
 azides and the inverse electron - demand diels  alder 
 ( iedda ) reaction of strained cycloalkenes and cyclooctynes with tetrazines .
we used bioorthogonal fluorophore 
 reporters to investigate reaction rates , reagent specificity , cellular 
 availability , stability , and optimal conditions for intracellular 
 labeling in living mammalian cells using in - gel fluorescence and fluorescence 
 microscopy ( scheme 1 ) .
this system provides a novel , live - cell platform for the unbiased 
 investigation of bioorthogonal chemistry in different subcellular 
 organelles and will be a useful tool to characterize novel reactions 
 and fluorophores in the context of a living cell .
we developed a model system based on halotag protein technology 
 to examine bioorthogonal reactions inside living cells and optimize 
 conditions for live - cell fluorescence imaging applications ( scheme 1 ) .
a suite of bioorthogonal 
 halotag ligands were synthesized ( figure 1a ) to investigate two prominent bioorthogonal 
 reactions that have become increasingly popular for live - cell labeling : 
 spaac and iedda ( see supporting information ( si ) for synthesis ) .
for spaac , several cyclooctynes with enhanced 
 reaction kinetics due to increased strain energy have been introduced , 
 including the dibenzocyclooctynes dibo , dbco ( also known as dibac ) and barac , and the bicyclononyne bcn .
we chose to prepare halotag ligands based on the commercially 
 available cyclooctynes dbco 1 and bcn 2 ( figure 1a ) and investigate 
 the spaac reaction with fluorophore - azides ( i.e. , 15 , 19 and 23 , figure 1b  d ) .
in addition , we examined the reverse bioorthogonal 
 group pairing , by preparing chloroalkane - azide halotag ligand 3 to tag the protein with an azide , followed by reaction with 
 dbco- and bcn - linked fluorescent reporters 16 and 17 , respectively .
for iedda , we prepared halotag ligands incorporating 
 seven different dienophiles that cover a broad range of reactivity , 
 including the parent trans - cyclooctene 4 , the conformationally strained trans - cyclooctenes known as stco ( 5 and 6 ) and dtco 7 , as well as derivatives of norbornene ( 8) and cyclopropene ( 9 ) ( figure 1a ) .
the iedda reaction was examined using fluorophore - tetrazines 
 ( i.e. , 13 , 14 , 2022 , figure 1b  d ) as the reaction partner .
we also examined the reaction 
 of tetrazine - chloroalkane 10 and methyl - tetrazine - chloroalkane 11(66,6971 ) with the bcn- 
 and tco - linked fluorophores ( 17 and 18 , 
 respectively ) to study the reverse pairing where the tetrazine was 
 the protein tag and the strained alkyne and alkene was incorporated 
 in the reporter molecule .
we chose to investigate the reactions with 
 clickable versions of three fluorophores , carboxytetramethylrhodamine 
 ( tamra ) , boron dipyrromethene ( bodipy ) , and fluorescein that have 
 been used extensively for live - cell imaging ( figure 1b  d ) .
structures of clickable ( a ) halotag chloroalkane 
 ligands and ( b ) 
 tamra- , ( c ) fluorescein- , and ( d ) bodipy - fluorophores .
halotag 
 fusion proteins were designed to contain three components : ( 1 ) an n - terminal halotag protein to react with bioorthogonal halotag 
 ligands , ( 2 ) an organelle - targeting protein sequence , and ( 3 ) a fluorescent 
 protein ( gfp or mcherry ) for confirmation of protein expression and 
 reaction colocalization by fluorescence microscopy . when expressed 
 in mammalian cells ,
the organelle - targeting sequence directs expression 
 of the halotag protein inside the lumen of the organelle of interest . 

localized halotag fusion protein can then be covalently labeled with 
 various clickable halotag ligands ( i.e. , 111 ) to tag the protein followed by reaction with the cognate 
 bioorthogonal fluorophore reporters ( scheme 1 ) .
expression and localization of fusion 
 proteins targeting the nucleus , endoplasmic reticulum ( er ) , cytosol , 
 and plasma membrane ( table 1 ) were confirmed by live - cell fluorescence microscopy ( figure s1 ) prior to analysis of the bioorthogonal 
 reactions .
we initially investigated the spaac and iedda 
 reactions in the nucleus of live hela cells using halo - h2b - gfp , which 
 has high protein expression levels and excellent nuclear localization . 

our initial investigations used tamra as the fluorophore reporter 
 as it exhibits low background fluorescence for live - cell imaging and 
 has a relatively uniform distribution within hela cells .
the capacity of halotag ligands 111 to label halo - h2b - gfp in hela cells was first examined using a competitive 
 pulse - chase assay with fluorescent tamra halotag ligand 12 .
halo - h2b - gfp expressing hela cells were labeled with halotag ligands 111 , and chased with tamra ligand 12 .
the ability of nonfluorescent ligands 111 to inhibit incorporation of 12 was determined 
 by in - gel fluorescence ( figure s2 ) .
while 
 we did not observe complete saturation of halotag protein under these 
 conditions , the incorporation of clickable halotag ligands 111 was similar ( 7079% incorporation ) , 
 enabling a semiquantitative comparison of subsequent spaac and iedda 
 reactions .
the spaac and iedda ligations were evaluated 
 in cells by reacting 
 the clickable halotag protein conjugates of 111 with the corresponding clickable tamra - based fluorescent 
 reporters 1318 .
the tamra - labeled 
 proteins were detected by in - gel fluorescence to quantify the click 
 chemistry reaction product as well as any nonspecific protein labeling . 

total halotag protein was measured by western blot to control for 
 protein loading . to account for data fluctuations , variability between 
 experiments , and the dynamic nature of a live - cell system ,
we included 
 tamra chloroalkane ligand 12 as a positive control to 
 determine the total amount of halotag protein that could be fluorescently 
 labeled per experiment ( see materials and methods for detailed procedures ) .
we first analyzed a dose response for 
 each reaction to determine the half - maximal effective concentration 
 ( ec50 ) , defined as the dose halfway between the baseline 
 and saturating level for each reaction .
the saturating level for each 
 reaction was based on the plateau from normalized curve fit data in 
 which we observed no further significant increase in fluorescence 
 intensity between the clickable halotag conjugate and bioorthogonal 
 tamra reporter .
an optimized dose was then selected for timecourse 
 studies to determine the reaction half - life ( t1/2 ) , defined as the time point halfway between the baseline 
 and saturating level for the indicated dose . in addition , we calculated 
 the efficiency for each reaction ( emax ) from timecourse data , which is defined as the saturating level 
 ( plateau from curve fit data ) for each reaction
the reaction rate 
 data reflect both the halotag labeling and the bioorthogonal reaction . 
 as the halotag labeling efficiency was consistent from experiment 
 to experiment ( figure s2 )
, differences 
 in these values mostly reflect differences between the bioorthogonal 
 reactions . from dose response 
 of tamra - azide 
 for 2 h. from timecourse 
 with 25 m 
 tamra - azide . for spaac ligations ,
hela cells expressing 
 halo - h2b - gfp were labeled with strained alkyne ligands dbco 1 or bcn 2 , followed by reaction with a dose 
 response of tamra - azide 15 for 2h ( figure 2a , b ) .
reactions were immediately quenched 
 with 500 m 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine 
 ( azide - amine 24 , figure s3a ) and processed as described in the materials 
 and methods section to determine the ec50 for each 
 reaction .
representative gels for the spaac reaction of tamra - azide 15 with the dbco 1- and bcn 2-halo - h2b - gfp 
 conjugates are shown in figure s4a and figure 2a , respectively and 
 the ec50 curves from quantitation of 36 replicates 
 for each reaction are shown in figure 2b .
the reactions were further investigated using a 
 timecourse with 25 m 15 to determine the t1/2 and emax values 
 ( figure 2c , d , table 2 ) .
representative 
 gels from the timecourse reaction of tamra - azide 15 with 
 dbco 1- ( figure s4b ) and bcn 2-halo - h2b - gfp conjugates ( figure 2c ) are shown .
quantification of the in - gel 
 fluorescence and graph of the one - phase association curves from 36 
 replicates are reported in figure 2d .
interestingly , we found significant differences 
 between ec50 , t1/2 , and emax values for dbco 1 compared 
 with bcn 2 when reacted with 15 ( table 2 ) .
the reaction between 1 and 15 was 10-fold faster and had a 
 20-fold lower ec50 .
consistent with our observations for 
 dbco in these live - cell studies , rate enhancements up to 400-fold 
 over in vitro reaction rates have been reported for more lipophilic 
 cyclooctynes in biological environments .
in addition , we observed a significant difference 
 in the saturating levels ( emax ) for spaac 
 ligations in the context of a living cell . here , 1 proceeded 
 with > 90% efficiency ( emax = 94% ) , 
 whereas 2 had an average emax of 67% ( table 2 ) . 

these findings 
 demonstrate the utility of our model system to directly compare bioorthogonal 
 reaction parameters in live cells .
furthermore , the reaction parameters 
 calculated herein are a reflection of multiple variables , including 
 the reaction kinetics , as well as the permeability and diffusion of 
 the reactants .
therefore , the differences in rates we observe can not 
 easily be predicted from in vitro models of reaction kinetics . taken 
 together
, our studies suggest that the combination of dbco with tamra - azide 
 is an efficient spaac reaction for live - cell nuclear labeling in mammalian 
 cells .
analysis of spaac reactions of dbco 1- and bcn 2-halotag conjugates with tamra - azide 15 in the 
 nucleus of live hela cells expressing halo - h2b - gfp .
( a ) cells were 
 treated with 10 m chloroalkane ligand bcn 2 for 
 30 min to generate the bcn 2-halo - h2b - gfp conjugate followed 
 by reaction with 50 nm-250 m tamra - azide 15 for 
 2 h at 37 c and analyzed by in - gel fluorescence ( top panel ) 
 and western blotting with anti - halotag antibody ( bottom panel ) .
as 
 a control , halo - h2b - gfp was treated with tamra ligand 12 to determine the maximum fluorescent labeling per experiment ( lane 
 1 ) .
( b ) dose  response curves from fluorescence intensity analysis 
 of gels from the spaac reaction of tamra - azide 15 with 
 dbco 1-halo ( orange ) and bcn 2-halo ( green ) 
 conjugates .
( c ) representative gel of spaac timecourse experiments 
 with bcn 2-halo and 25 m tamra - azide 15 analyzed by in - gel fluorescence ( top panel ) and western blotting 
 with anti - halotag antibody ( bottom panel ) .
( d ) one - phase association 
 curves from timecourse fluorescence intensity analysis of spaac reactions 
 between dbco 1-halo ( orange ) and bcn 2-halo 
 ( green ) reacting with 25 m tamra 
curves were generated in graphpad 
 prism from fluorescence intensity measurements of n = 36 independent replicates as described in the experimental section , and reported as the mean  
 sem .
from dose response of tamra - tz 13 for 1 h. from timecourse with 2 m 13 or 14 .
we next evaluated the iedda 
 reaction of tetrazines with four prominent dienophiles covering a 
 broad range of reactivity .
we calculated the ec50 , t1/2 , and emax values 
 as described above and in the materials and methods section . because of faster kinetics for iedda reactions compared 
 to spaac reactions , and the propensity of monosubstituted tetrazines 
 for nonspecific labeling at high concentrations ( vide infra , figure s6 , s8 and s13 ) , we used lower concentrations 
 of the fluorophore - tetrazine for the iedda dose response and timecourse 
 studies .
the strained alkyne bcn 2 , which has recently 
 been reported to undergo iedda reactions , and dbco 1 were also investigated 
 in these studies .
halo - h2b - gfp incorporating the tco 4 , bcn 2 , dbco 1 , nbor 8 and 
 cprop 9 halotag ligands were initially reacted with a 
 dose response of tamra - tz 13 for 1 h , and the reactions 
 were quenched with 100 m ( 4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine 
 ( tz - amine 27 , figure s3b ) 
 and processed as described .
figure 3a contains a representative gel for the reaction between 
 tco 4-halo - h2b - gfp conjugate and tamra - tz 13 , and a representative gel for the reaction of the bcn 2-halo - h2b - gfp conjugate with 13 is included in figure s5a . under these conditions ,
tco 4 had a slightly lower ec50 and the reaction proceeded 
 to a greater extent compared to bcn 2 ( figure 3b and table 3 ) .
in contrast , dbco 1 did not 
 react with tamra - tz 13 ( figure s6 ) .
this is consistent with previous reports , and is likely due to increased sterics present in the tricyclic 
 dibenzocyclooctyne ring system .
we next investigated the reaction 
 timecourse of bcn 2 and tco 4 with 2 m 
 tamra - tz 13 to determine the t1/2 and emax values .
representative gels 
 for the timecourse reaction of tamra - tz 13 with tco 4 or bcn 2 are shown in figure 3c and figure s5b , respectively . here , we observed much faster reaction rates than 
 the spaac ligations , consistent with the faster in vitro kinetics 
 reported for iedda ligations .
interestingly , bcn 2 was 
 found to undergo an iedda reaction with tamra - tz 13 almost 
 as fast as tco 4 in the nucleus of live mammalian cells , 
 and both reactions proceeded with similar efficiencies ( figure 3d and table 3 ) .
analysis of iedda reactions of bcn 2- and tco 4-halotag conjugates with tamra - tz 13 in the 
 nucleus of live hela cells expressing halo - h2b - gfp .
( a ) cells were 
 treated with 10 m chloroalkane ligand tco 4 for 
 30 min to generate the tco 4-halo - h2b - gfp conjugate followed 
 by reaction with 1 nm-20 m tamra - tz 13 for 1 h 
 at 37 c and analyzed by in - gel fluorescence ( top panel ) and 
 western blotting with anti - halotag antibody ( bottom panel ) . as a control , 

halo - h2b - gfp was treated with tamra - ligand 12 to determine 
 the maximum fluorescence labeling per experiment ( lane 1 ) .
response 
 curves from fluorescence intensity analysis of gels from the iedda 
 reaction of tamra - tz 13 with bcn 2-halo 
 ( black ) and tco 4-halo ( blue ) conjugates .
( c ) representative 
 gel of iedda timecourse reaction between 2 m tamra - tz 13 and tco 4-halo conjugate analyzed by in - gel 
 fluorescence ( top panel ) and western blotting with anti - halotag antibody 
 ( bottom panel ) .
( d ) one - phase association curves from timecourse fluorescence 
 intensity analysis of iedda reactions between bcn 2-halo 
 ( black ) or tco 4-halo ( blue ) with 2 m tamra - tz 13 for 0.5120 min at 37 c .
curves were generated 
 in graphpad prism from fluorescence intensity measurements of n = 36 independent replicates as described in the experimental section , and reported as the mean  
 sem .
as dbco was found to rapidly undergo 
 the spaac reaction with tamra - azide 15 , we were curious 
 to determine how the rate of this reaction 
 compared with iedda reactions of 2 and 4 with 13 . to provide a direct comparison between reaction 
 rates , we determined the half - life and efficiency of this reaction 
 under identical conditions as the iedda reactions . here , we observed 
 a half - life for this spaac reaction of 15 min , which was almost 
 as fast in live cells as the iedda reactions of 4 and 2 with 13 ( figure s7 ) .
this surprising rate enhancement for spaac reactions of dbco underscores 
 the importance of measuring relative reaction rates within a cellular 
 context , and not relying solely on in vitro rate data . under 
 the same conditions used in figure 3a ,
we did not observe significant reaction 
 with either the nbor 8 or cprop 9 halotag 
 conjugates ( data not shown ) , even though the halotag ligands were 
 effectively conjugated to the halotag protein ( figure s2 ) .
to more accurately assess the reactivity of nbor 8 and cprop 9 halotag ligands in our cellular 
 system , which display sluggish in vitro reaction rates compared to 
 other dienophiles , we evaluated higher concentrations of tamra - tz 13 over a longer 4 h reaction period . even under these modified 
 conditions , we were only able to detect 1020% reaction 
 ( figure s8 ) .
furthermore , we observed significant 
 background protein labeling by 13 at these high concentrations , 
 and background labeling dominated at 100 m .
these studies suggest 
 that 8 and 9 are either not stable in the 
 nucleus of mammalian cells or not sufficiently reactive under the 
 live - cell labeling conditions implemented here .
various tetrazines 
 have been synthesized for live - cell labeling , 
 and 3-methyl-6-aryltetrazine derivatives have been used for live - cell 
 imaging applications due to their improved stability .
therefore , we were curious to examine how this tetrazine variant 
 behaved in our model system . to this end , we determined the half - lives 
 and efficiency for the bcn 2-halo and tco 4-halo conjugates with tamra - tz - me 14 ( figure 1b ) .
consistent with published 
 in vitro kinetic data,14 reacts about 
 3 times more slowly than tamra - tz 13 under identical 
 parameters ( figure s9 and table 3 ) .
in addition , reaction with 
 tamra - tz - me 14 was also less efficient , reacting with 
 only 2530% efficiency when compared to our positive 
 tamra control within the 4 h timecourse .
overall , these studies 
 clearly demonstrate significant differences 
 in labeling rates and stability of reactants when placed in the context 
 of a living cell , and should help provide guidance when choosing the 
 reaction of choice and labeling conditions for a particular application .
our live - cell model system provided 
 interesting observations regarding spaac and iedda reactions when 
 either the strained alkyne or alkene was introduced into the cell 
 as a tag prior to reaction with the appropriate azide or tetrazine 
 reporter .
we also investigated the reverse situation where an azide 
 or tetrazine was introduced into the cell as a tag followed by reaction 
 with the appropriate strained alkyne or alkene reporter .
for the spaac 
 reactions , halo - h2b - gfp incorporating azide halotag ligand 3 was reacted with a timecourse of 25 m tamra - dbco 16 or tamra - bcn 17 ( figure s10 ) . here
, we observed an interesting disconnect where the azide tag / cycloalkyne 
 reporter combination did not perform as well and generally had decreased 
 reaction efficiency relative to the analogous cycloalkyne tag / azide 
 reporter systems . to better understand this disconnect , we determined 
 the passive permeability of the clickable fluorophore reporters 1318 in low - efflux mdckii cells ( table s1 ) .
we found 
 that tamra - azide 15 had moderate permeability , while 
 tamra - bcn 16 and tamra - dbco 17 had very 
 low permeability .
we also investigated the stability of 1318 in hela cells and found that tamra - azide 15 was completely stable over a 24 h period , while tamra - bcn 17 exhibited considerable instability with only 6% remaining 
 after 24 h ( table s2 ) .
therefore , the reduced 
 efficiency of the azide tag / cycloalkyne reporter combination can be 
 explained , in part , by the poorer permeability and stability of the 
 cycloalkyne reporters which leads to lower effective intracellular 
 concentrations of these reagents . in addition
, we observed a significant 
 amount of background protein labeling using 25 m 16 and 17 ( figure s12e , f ) that 
 was not observed with tamra - azide 15 ( figure s12a ) .
this is most likely due to nonspecific labeling 
 of cysteine thiol biomolecules which has been shown to be a limitation 
 of cyclooctynes including bcn in cell lysates and inside living cells .
these observations have implications for probe 
 design and argue for preferential placement of cycloalkyne bioorthogonal 
 tags directly on the probe or biological component to be labeled rather 
 than the reporter group .
( a ) h nmr spectra ( 600 mhz , cd3od ) of fresh stco 5 ( bottom ) and following a 3-day incubation 
 neat at 30 c ( top ) showing degradation and some trans - cis isomerization .
( b ) h nmr spectra 
 ( 600 mhz , cd3od ) of fresh ag - stco 6 ( bottom ) 
 and following a 3 day incubation neat at 30 c ( top ) showing 
 no degradation or trans - cis isomerization . 

( c ) stability of tco - derivatized halotag ligands 47 following formation of halo - h2b - gfp conjugates inside the 
 nucleus over 24 h. hela cells expressing halo - h2b - gfp were labeled 
 with 10 m chloroalkane ligands 47 for 30 min at 37 c .
unbound ligand was washed out 
 of the cells for 1 h , and the t = 0 time point was 
 labeled with a saturating dose of 2 m tamra - tz 13 for 1 h and analyzed by in - gel fluorescence .
all other time points 
 were labeled with tamra - tz 13 after the indicated incubation 
 times in media . for each time point , we labeled halo - h2b - gfp or halo - kdel 
 with tamra ligand 12 as a positive control to account 
 for halotag conjugate protein turnover during the course of the experiment . 

graphs represent fluorescence intensity measurements from 3 replicates , 
 and are reported as the mean  sem ( d ) stability of tco 47 halo - kdel conjugates inside the lumen 
 of the er over 24 h. hela cells expressing halo - kdel were labeled , 
 collected , and analyzed as in ( c ) . for iedda reactions , the efficiency of reaction between the 
 halotag 
 conjugate of tz 10 and 2 m tamra - tco 18 decreased by 50% ( figure s11 ) 
 compared to the corresponding reaction where tco 4 was 
 incorporated as the tag and tamra - tz 13 was used as the 
 reporter ( figure 3 ) . 

monosubstituted tetrazines have been 
 reported to have limited stability in serum upon prolonged exposure 
 and this could account for part of the reduced reaction efficiency . 

in addition , tamra - tz 13 displays nonspecific labeling 
 at high concentrations ( figure s8 and s12d ) so it is likely that a component of the instability is due to covalent 
 modification of biomolecules within the cell .
we also found that tamra - tco 18 was considerably more stable than tamra - bcn 17 in hela cells ( table s2 ) .
consistent 
 with the improved stability , very little background labeling was observed 
 for tamra - tco 18 under the conditions of our experiments 
 ( figure s12b ) .
even though tco has been 
 shown to undergo partial isomerization to the cis - isomer within the cell , this isomer 
 is inert and does not cause nonspecific labeling of background proteins . 

this isomerization may slightly lower the emax for the iedda reaction between 10 and 18 , but it is not a limitation because the selectivity is not affected .
analysis 
 of iedda reactions of stco 5- , ag - stco 6- , and dtco 7-halotag conjugates with tamra - tz 13 in the nucleus of live hela cells expressing halo - h2b - gfp . 

( a ) dose  response curves for the iedda reaction of tamra - tz 13 with the halotag conjugates of stco 5 ( red ) , 
 ag - stco 6 ( blue ) , and dtco 7 ( black ) chloroalkane 
 ligands .
( b ) one phase association plot of the iedda reaction timecourse 
 between 2 m tamra - tz 13 and the halotag conjugates 
 of chloroalkane ligands 57 .
curve 
 fits represent full 2 h timecourse , and are shown here up to 60 min 
 to highlight differences in the early part of the curves .
interestingly , the tz - me 11 halotag 
 conjugate retained 
 high efficiency when incubated with tamra - tco 18 ( figure s11 ) .
in addition , tamra - tz - me 14 was more stable in hela cells ( table s2 ) and displayed much less background labeling ( figure s12c ) compared to tamra - tz 13 ( figure s12d ) .
these observations are consistent 
 with prior reports that 3,6-disubstituted tetrazines are more stable 
 than monosubstituted tetrazines , and suggest that disubstituted 
 tetrazines may be useful as protein tagging agents for live - cell bioorthogonal 
 labeling in mammalian cells particularly when paired with more reactive 
 tco reporters , as is the case in bacteria .
recently , two conformationally strained trans - cyclooctene 
 derivatives have been described that display faster reactivity relative 
 to simpler tco derivatives .
these groups include the strained trans - cyclooctene stco , which 
 displays the fastest reactivity , and the cis - dioxolane - fused trans - cyclooctene dtco , which displays better stability 
 and solubility . to date , their use in 
 live cells has been limited to applications as reporter molecules , and efforts to genetically encode or to use ligases to selectively 
 introduce stco or dtco into proteins have thus far proved unsuccessful .
recent reports have shown that these conformationally strained derivatives 
 can undergo isomerization to the unreactive cis - isomer 
 in the presence of high thiol concentrations , presumbably via a free 
 radical mediated pathway .
using our model system , 
 we investigated both the stability and reactivity of these two highly 
 reactive tco derivatives to assess their utility as tags for bioorthogonal 
 labeling in mammalian cells .
we first examined the stability 
 of stco when heated neat at 30 c for 3 days and observed almost 
 complete decomposition with a trace of isomerization to the cis - isomer ( figure 4a ) .
we sought to find a way to stabilize the stco reagent 
 to permit prolonged storage .
the preparation of stco involves photoisomerization 
 of the cis - cyclooctene in a flow reactor where the trans - isomer is isolated through selective complexation 
 with agno3-impregnated silica gel .
the stco is then liberated by stirring the silica with nh4oh . we hypothesized that preparing the stco reagent as the silver 
 complex would provide long - term stability for storage , yet could be 
 applied directly to cell culture , which contains high concentrations 
 of nacl that could release the reactive stco in situ .
first we prepared 
 the ag - stco halotag ligand 6 ( figure 1a ) and demonstrated that it was stable when 
 heated at 30 c for 3 days ( figure 4b ) , unlike the parent stco ligand 5 ( figure 4a ) .
in addition , 
 we synthesized a halotag reagent derived from the newly reported dtco ( figure 1a , ligand 7 ) , which has increased stability compared 
 to stco while displaying similar reactivity .
we next examined 
 the relative stability of the tco series of halotag 
 ligands ( compounds 47 ) following 
 conjugation to halo - h2b - gfp in the nucleus or halo - kdel in the endoplasmic 
 reticulum ( er ) ( figure 4c , d ) .
hela cells were transfected with either halo - h2b - gfp or halo - kdel 
 and halotag ligands 47 were added 
 to make the corresponding clickable halotag conjugates and excess 
 unbound ligand was washed out of the cells .
tamra - tz 13 was added at the indicated time points over 24 h to evaluate how 
 much of the tco - halo conjugate was stable and could undergo the iedda 
 reaction .
the reaction of tco 4-halo - h2b - gfp with tamra - tz 13 progressed to the same extent over 24 h indicating that 
 tco 4 was stable in the nucleus under these conditions . 

in contrast , less tamra - tz 13 reacted over the 24 h period 
 with the conformationally strained tco derivatives presumably due 
 to isomerization to the unreactive cis - isomer with 
 prolonged incubation .
ag - stco 6 and stco 5 had the lowest stability , leading to significant loss of reactivity 
 after 24 h , while dtco 7 had intermediate stability . 

however , in the er , all the trans - cyclooctenes appeared 
 stable for up to 24 h ( figure 4d ) , suggesting that differences in subcellular microenvironments 
 can affect reagent stability .
the er provides a highly oxidizing environment 
 relative to the rest of the cell and 
 further study will be needed to determine if that contributes to the 
 enhanced stability of trans - cyclooctenes in the er 
 relative to the nucleus .
nonetheless , our data suggests that conformationally 
 strained tcos are useful as tagging molecules in the er , where some 
 enzyme - mediated protein labeling reactions are not compatible .
importantly , stco 5 and ag - stco 6 behaved almost identically demonstrating that the ag is 
 effectively decomplexed when 6 is added to cells .
confocal images 
 of the spaac and iedda reactions of clickable bcn 2- , 
 dbco 1- , and tco 4-halotag conjugates 
 with clickable tamra fluorophores in live cells expressing halo - h2b - gfp . 

halo - h2b - gfp was labeled with 10 m bcn 2 ( a ) , 
 dbco 1 ( b ) , or tco 4 ( c ) chloroalkane ligands 
 for 30 min , followed by reaction with a dose response of tamra - azide 15 ( spaac , a - b ) or tamra - tz 13 ( iedda , c ) for 
 30 min at 37 c and imaged live after a 2 h washout period .
total 
 nuclei were visualized with hoescht 33342 and are shown in blue ( column 
 1 on the left ) .
cells expressing halo - h2b - gfp are shown in green ( column 
 2 ) .
tamra - labeled nuclei from click chemistry are shown in red ( column 
 3 ) .
overlays were created between green and red channels to demonstrate 
 colocalization ( yellow ) between transfection and bioorthogonal chemistry 
 ( column 4 ) .
reaction rates were then examined between tamra - tz 13 and the more reactive dienophiles 57 in the nucleus to determine how these values compare to 
 tco 4 under the same reaction conditions .
consistent 
 with the 
 increased reactivity observed from in vitro kinetics data , we observed over 14-fold lower ec50 values for the stco 5 , ag - stco 6 and dtco 7 halo - h2b - gfp 
 conjugates when compared to tco 4 , with the lowest ec50  36 nm for ag - stco 6 ( figure 5a , table 3 ) .
furthermore , 
 a comparison of reaction half - life values demonstrates that ag - stco 6 ( half - life 1.4 min ) and stco 5 ( half - life 
 2.2 min ) react the fastest , followed by dtco 6 , and finally tco 4 ( figure 5b and table 3 ) .
in addition , the 
 reactions with conformationally strained tco derivatives 57 have the highest efficiencies with emax values in the 6786% range compared 
 to 47% for tco 4 .
overall , these data demonstrate that 
 the stco reagents ( 5 and 6 ) have the fastest 
 kinetics and highest efficiency for live - cell labeling , enabling reaction 
 saturation within minutes following addition of the tetrazine reporter . 

furthermore , consistent with the above stability data , stco was clearly 
 liberated from the silver complex 6 when added to the 
 cell culture since ag - stco 6 performed identically or 
 slightly better than the parent stco 5 .
in addition , 
 ag - stco 6 stock solutions remained stable up to 8 months 
 when stored at 20 c , whereas stco 5 stock 
 solutions , and even neat compound , required frequent preparation and/or 
 resynthesis of the ligand when stored at 20 c .
we also 
 tested stco 5 and ag - stco 6 in a standard 
 mtt cell viability assay in hela cells and found no cell toxicity 
 over a 24 h period ( figure s14 ) . given 
 that ag - stco has greatly improved stability for storage , we feel this 
 reagent will expand the utility of stco iedda chemistry in cells .
we next investigated the performance of the spaac and iedda reactions 
 in the endoplasmic reticulum and cytosol , where unique subcellular 
 microenvironments can affect the stability ( figure 4c , d ) and , potentially , reactivity of the 
 bioorthogonal groups investigated here .
we chose the fastest dienophiles , 
 ag - stco 6 and dtco 7 , to evaluate the iedda 
 reaction .
for spaac chemistry , we examined both the dbco 1 and bcn 2 chloroalkane ligands .
we restricted the bioorthogonal 
 reactions to the lumen of the er using halotag - kdel .
for cytosolic 
 localization , we designed a halotag - gap43-gfp construct which is present 
 throughout the cytosol , and excluded from the nucleus ( figure s1c ) .
hela cells were transfected with 
 either construct and treated under identical conditions used for evaluation 
 of the nuclear construct , halo - h2b - gfp .
overall , both the spaac and 
 iedda reactions proceeded with similar reaction rates in both the 
 cytosol and er ( figure s15 and table 4 ) .
the reaction of 
 ag - stco 6 and dtco 7 with tamra - tz 13 mirrored what was seen in the nucleus ( figure s15c , d and table 4 ) . as expected , ag - stco 6 was slightly 
 faster than dtco 7 , and both reactions proceeded with 
 similar efficiencies ( table 4 ) .
furthermore , the spaac reaction between tamra - azide 15 and dbco 1 was approximately 5-fold faster 
 and proceeded with greater efficiency than the reaction with bcn 2 ( figure s15a , b and table 4 ) .
these data suggest that both 
 spaac and iedda ligations are compatible with different subcellular 
 compartments .
confocal images of the spaac and iedda reactions between 
 clickable 
 halotag conjugates and clickable fluorescein derivatives in different 
 subcellular organelles .
( a ) confocal images of nuclei expressing halo - h2b - mcherry 
 labeled with bcn 2 followed by spaac reaction with 50 
 m fl - dipiv - azide 19 for 1 h under live ( top panel ) 
 and meoh fixed ( bottom panel ) conditions .
total nuclei were visualized 
 with hoescht 33342 and are shown in blue ( column 1 on left ) .
fluorophore - labeled 
 proteins from click chemistry are shown in green ( column 3 ) .
overlays 
 were created between red , green , and blue channels to demonstrate 
 colocalization ( yellow ) between transfection and bioorthogonal chemistry 
 ( column 4 ) .
( b ) confocal images of the er expressing halo - kdel - mcherry 
 labeled with bcn 2 followed by spaac reaction with 50 
 m fl - dipiv - azide 19 for 1 h under live ( top panel ) 
 and meoh fixed ( bottom panel ) conditions .
( c ) confocal images of nuclei 
 expressing halo - h2b - mcherry labeled with tco 4 followed 
 by iedda reaction with 1 m fl - diac - tz 20 for 1 
 h. ( d ) confocal images of the er expressing halo - kdel - mcherry labeled 
 with tco 4 followed by iedda reaction with 1 m 
 fl - diac - tz 20 for 1 h. ( e ) confocal images of the cytoplasm / nucleus 
 expressing halo - mcherry labeled with tco 4 followed by 
 iedda reaction with 1 m fl - diac - tz 20 for 1 h. 
 ( f ) confocal images of cell surface expressing halo - mcherry - pdgfr 
 labeled with tco 4 followed by iedda reaction with cell - impermeable 
 fl - tz 21 .
scale bar = 20 m . with 
 the systematic evaluation of the bioorthogonal reactions in hand , 

we were in a position to evaluate the spaac and iedda reactions using 
 our clickable reagents for the purpose of intracellular imaging in 
 live cells .
for the spaac reaction , hela cells expressing halo - h2b - gfp 
 were treated with either dbco ligand 1 or bcn ligand 2 , followed by reaction with a dose response of tamra - azide 15 .
representative 
 images for the reaction of bcn 2-halo - h2b - gfp ( figure 6a ) and dbco 1-halo - h2b - gfp ( figure 6b ) conjugates with tamra - azide 15 are shown . 

overall , we observed differences in the intensity of nuclear labeling 
 consistent with our reaction rate data in live cells ( table 2 ) .
the rate enhancement observed 
 with the halotag conjugate of dbco 1 enabled significant 
 reaction to be visualized with as little as 0.5 m tamra - azide 15 and the reaction becomes saturated with optimal signal - to - noise 
 levels with only 2 m tamra - azide 15 ( figure 6b ) .
in contrast , 
 10 m tamra - azide 15 is required to visualize the 
 reaction with bcn 2 , and 25 m 15 is 
 necessary to achieve adequate signal - to - noise levels ( figure 6a ) .
background fluorescence 
 becomes evident with 25 m tamra - azide 15 , and 
 dominates at 100 m 15 ( figure 6a , bottom panel ) .
these data suggest that 
 25 m is an upper limit to the amount of tamra - azide 15 that can be successfully used for these live - cell imaging studies . for iedda reactions , hela cells expressing halo - h2b - gfp
were treated 
 with chloroalkane tco ligand 4 followed by reaction with 
 a dose response of tamra - tz 13 ( figure 6c ) for 30 min , and imaged live after a 2 
 h washout . for iedda reaction with 13
, we observed a 
 direct correlation between our in - gel fluorescence analysis and imaging , 
 with significant signal observed using 0.5 m tamra - tz 13 and optimal signal - to - noise levels using 2 m tamra - tz 13 .
background labeling dominated when 10 m tamra - tz 13 was used ( figure 6c , bottom panel ) , suggesting lower concentrations ( 2 
 m ) should be used for live - cell imaging studies in mammalian 
 cells . here
, we also examined the reverse bioorthogonal group pairing 
 where tz ligand 10 or tz - me ligand 11 was 
 incorporated into halo - h2b - gfp as the tag , and tamra - tco 18 served as the fluorescent reporter ( figure s16 ) .
we observed labeling with a low dose of tamra - tco ( 0.1 m ) , 
 but the labeling did not increase in a dose proportionate fashion 
 when up to 25 m tamra - tco 18 was used on the tz 10 conjugate of halo - h2b - gfp ( figure s16a ) .
this data suggests that the monosubstituted tz 10 is not stable in the cells and a proportion decomposes before reacting 
 with tamra - tco 18 .
no reaction with 
 me - tz 11 was observed when 0.1 m tamra - tco 18 was used ; however , significant labeling was seen with 2 
 m 18 and the reaction appeared to reach saturation 
 when 10 m 18 was used with good colocalization 
 ( figure s16b ) .
this data is consistent 
 with the results from the in - gel fluorescence analysis where the reaction 
 of the tz - me 11 halotag conjugate retained high efficiency 
 when incubated with tamra - tco 18 , while the reaction 
 of the tz 10 conjugate was less efficient ( figure s11 ) .
it is also worthwhile to note that 
 very little background fluorescent labeling or nonspecific fluorophore 
 accumulation was observed with tamra - tco 18 , even up 
 to 25 m ( figure s16 ) .
this is also 
 consistent with the low background observed based on in - gel fluorescence 
 data ( figure s12b ) . these observations 
 highlight how tco - based reporters not only provide excellent reactivity , 
 but also label tetrazine - tagged proteins with high selectivity . from timecourse with 25 m 
 tamra - azide . from timecourse 
 with 2 m 
 tamra - tz .
we next explored the spaac and iedda ligations 
 in different organelles using two green fluorophores amenable to intracellular 
 imaging : bodipy - fl and fluorescein ( fl ) .
hela cells were transfected 
 with the intracellular organelle - targeting constructs for halotag 
 expression in the nucleus ( halo - h2b - mcherry ) , and er ( halo - kdel - mcherry ) . 
 for the spaac ligations , halotag protein was labeled with bcn ligand 2 followed by reaction with the green cell - permeable fluorophores , 
 fluorescein - dipivalate - azide ( fl - dipiv - azide ) 19 ( figure 7 ) and bodipy - fl - azide 23 ( figure s17 ) . here , we observed 
 significant background fluorescence for both 19 and 23 , even after a 2 h washout .
fl - dipiv - azide 19 signal appeared to be present diffusely throughout the cytosol and 
 nucleus of live cells ( figure 7a , b , top panels ) .
this background accumulation is likely due 
 to the high concentration of fluorophore needed to effectively promote 
 the spaac reactions , resulting in trapping of negatively charged fluorescein 
 once the pivaloyl groups have been removed by endogenous esterases 
 inside the cell .
this limits passive diffusion of unreacted fluorophore 
 out of the cell during the washout stage due to lower membrane permeability . 

fixing the cells in meoh completely removed background , and revealed 
 selective labeling in both the nucleus ( figure 7a , bottom panel ) and er ( figure 7b , bottom panel ) .
bodipy - fl - azide 23 displayed a different pattern of nonspecific accumulation , 
 with high concentrations of unreacted fluorophore that appeared to 
 accumulate in organelles ( figure s17a , b , top panels ) .
this background was not completely removed after fixation 
 ( figure s17a , b , bottom panels ) , suggesting 
 that bodipy - fl - azide 23 may not be as useful for intracellular 
 imaging .
we also evaluated iedda reactions in the nucleus ( halo - h2b - mcherry ) , 
 er ( halo - kdel - mcherry ) , cytosol ( halo - mcherry ) , and at the extracellular 
 face of the plasma membrane ( halo - mcherry - pdgfr ) .
hela cells expressing 
 the intracellular constructs were labeled with tco halotag ligand 4 , followed by reaction with 1 m fluorescein - diacetate - tz 
 ( fl - diac - tz ) 20 ( figure 7c  e ) or bodipy - fl - tz 22 ( figure s17c  f ) for 30 min .
tetrazines 
 have been reported to quench the fluorescence of some fluorophores , 
 resulting in turn - on fluorescence upon reaction with trans - cyclooctenes .
therefore , we initially 
 attempted to image subcellular compartments without a washout step 
 prior to imaging in live cells using these  turn - on  
 tetrazine - linked fluorescein- and bodipy - fl fluorophores . however , 
 the background fluorescence we observed was too high to distinguish 
 specific labeling ( data not shown ) .
this is in part due to stability 
 issues with the tetrazine which can lead to fluorescent impurities 
 ( i.e. , see table s2 for stability of tamra - tz 13 in hela cells ) .
fluorophore - tetrazines 
 with > 1000-fold fluorescence increases have recently been developed , 
 and these may provide better tools for intracellular iedda imaging 
 in live cells with no washout period . following 
 a 2 h washout , we were able to observe selective labeling with no 
 visible background for both fl - diac - tz 20 and bodipy - fl - tz 22 in the nucleus ( figure 7c and s17c ) , er ( figure 7d and s17d ) , and cytosol ( figure 7e and s17e ) . in 
 addition
, we demonstrated selective extracellular labeling with cell - impermeable 
 fluorescein - tz ( fl - tz ) 21 ( figure 7f ) , whereas cell - permeable bodipy - fl - tz 22 labeled both intracellular ( present in the secretory pathway ) 
 and extracellular halo - mcherry - pdgfr ( figure s17f ) , demonstrating that iedda reactions are successful for both intracellular 
 and extracellular labeling .
of note , fl - tz 21 is amenable 
 to selective extracellular labeling , even when the bioorthogonal reaction 
 partner is present intracellularly .
on the basis of these studies , 
 the cell - permeable iedda fluorophore - tetrazines 
 at the concentrations tested here exhibit excellent properties for 
 live - cell intracellular imaging experiments in all the organelles 
 tested , whereas the high concentrations of fluorophore - azides required 
 for efficient spaac labeling are not ideal for live - cell imaging and 
 cells need to be fixed to remove background fluorescence .
finally , 
 we evaluated the fastest iedda reactions observed in this work , for 
 rapid labeling in the nucleus at low fluorophore concentrations . here
, 
 we analyzed a dose response of tamra - tz 13 after 5 min 
 of incubation with the ag - stco 6-halo - h2b - gfp and dtco 7-halo - h2b - gfp conjugates compared to the tco 4-halo - h2b - gfp conjugate .
reactions were quenched for 5 min with 100 
 m tz - amine 27 ( figure s3 ) , and imaged after a 1 h washout period .
here , we observed some 
 signal for both ag - stco 6-halo ( figure 8a ) and dtco 7-halo ( figure 8b ) conjugates at 
 100 nm after 5 min ( top row ) , with significant labeling and excellent 
 signal - to - noise levels at 1 m ( third row ) .
furthermore , labeling 
 intensity increased with increasing fluorophore concentration , and 
 appeared to saturate with 15 m tamra - tz 13 within the 5 min incubation ( figure 8a and 8b ) .
in contrast , we needed 
 10-fold more fluorophore to see any signal with tco 4-halo ( figure 8c ) , further highlighting the superiority of the ag - stco and dtco 
 reagents over tco for rapid intracellular live - cell imaging .
halo - h2b - gfp expressing hela cells were labeled with halotag chloroalkane 
 ligands tco 4 , dtco 7 , or ag - stco 6 followed by reaction with a dose response of tamra - tz 13 for 5 min at 37 c .
reactions were quenched with tz - amine 27 followed by a 1 h washout prior to imaging .
cells were 
 imaged live under identical parameters and representative images are 
 shown for ag - stco 6-halo ( a ) , dtco 7-halo 
 ( b ) , and tco 4-halo ( c ) .
total nuclei were visualized 
 with hoescht 33342 and are shown in blue ( column 1 on left ) .
cells 
 expressing halo - h2b - gfp are shown in green ( column 2 ) .
tamra - labeled 
 nuclei from click chemistry are shown in red ( column 3 ) .
overlays 
 were created between red and green channels to demonstrate colocalization 
 ( yellow ) between transfection and bioorthogonal chemistry ( column 
 4 ) .
overall , our halotag model system provides 
 a unique and unbiased 
 method to systematically evaluate bioorthogonal labeling strategies 
 directly inside living mammalian cells , as well as in different subcellular 
 organelles . using this approach
, we were able to rapidly assess various 
 bioorthogonal groups for fast , efficient , and selective spaac and 
 iedda ligations for live - cell imaging applications .
while the spaac 
 reactions were slower overall , we were still able to identify conditions 
 for intracellular live - cell imaging studies using tamra fluorophores 
 for both bcn and dbco .
however , in many cases the high fluorophore 
 concentrations required for live - cell labeling studies limits the 
 fluorophore selection due to background fluorescence accumulation . 

unexpectedly , we discovered that the spaac reaction of dbco 1 when conjugated to halotag proteins was nearly as rapid 
 as the iedda reaction with slower dienophiles , tco and bcn , in different 
 subcellular organelles of live cells .
this rate acceleration allowed 
 the use of lower concentrations of the fluorophore - azide reporter 
 and suggests that this spaac reaction may have utility for intracellular 
 imaging applications where dbco is used as a tag .
when dbco was linked 
 to the reporter , we observed extensive background labeling at the 
 high concentrations required for the spaac reaction , limiting its 
 use in these instances .
for the first time , proteins have been 
 tagged in living mammalian 
 cells with stco and dtco , which when reacted with tetrazines constitutes 
 the fastest bioorthogonal coupling reaction known to date .
our studies 
 clearly demonstrate that iedda ligations are the fastest reactions 
 in multiple cellular compartments of live cells , enabling reaction 
 completion within minutes when selecting the fastest dienophiles , 
 stco and dtco .
previous attempts to tag proteins with stco or dtco 
 using genetically encoded amino acids or lipoic acid ligase have been 
 unsuccessful . here
, we demonstrate that protein 
 conjugates can be made in living mammalian cells using halotag ligands 
 of both stco ( 5 , 6 ) and dtco ( 7 ) .
the intracellular stability of stco and dtco was organelle dependent , 
 with significant loss of reactivity in the nucleus after 24 h , but 
 high stability in the er over 24 h. thus , while conformationally strained trans - cyclooctenes display remarkable kinetics , their utility 
 as tagging molecules is somewhat tempered by their organelle - dependent 
 stability . the impact is somewhat alleviated because the major side 
 reaction is isomerization to the unreactive cis - isomer 
 which appears to be 
in contrast , 
 cyclooctynes can undergo covalent modification with cysteine and other 
 thiol - containing biomolecules which can lead to issues associated 
 with nonspecific background labeling .
another advantage of iedda reactions 
 with trans - cyclooctenes is that efficient labeling 
 can be achieved using low concentrations of fluorophore - tetrazines , 
 thereby reducing the chance for nonspecific fluorophore accumulation 
 and labeling in live cells , an issue which is present with the high 
 fluorophore concentrations required for spaac reactions or iedda reactions 
 with less reactive dienophiles .
the modest shelf life of stco 
 has also limited its usefulness . 
 we observed degradation when stco was stored neat or as a dmso stock 
 solution .
in addition , freeze  thawing of stock solutions led 
 to decomposition / isomerization , and we found it was best to prepare 
 single - use frozen aliquots to prevent freeze  thaw decomposition . 

we addressed the shelf life issue by making a stable ag - stco complex 
 and demonstrated that it can be used directly in cells for reaction 
 with tetrazine - linked fluorophores .
this silver complex has been stored 
 as a stock solution in ethanol for 8 months at 20 c 
 without any observable loss of reactivity . to date
, most cell - labeling 
 studies involving iedda have focused 
 on use of tetrazines as reporter molecules with strained alkenes and 
 alkynes as the tags . in a  reversed  approach ,
our data 
 shows that methyl - tetrazine tag 14 could be used as the 
 protein tag in labeling studies with tamra - tco 18 as 
 the reporter . moving forward ,
we expect there will be considerable 
 advantages to utilizing stable tetrazine tags with the more reactive 
 conformationally strained stco or dtco based reporters .
we are presently 
 preparing ag - stco and ag - dtco fluorescent reporters for this purpose 
 and results will be disclosed in due course .
further expansion of 
 the suite of stable tetrazines with reduced size and good solubility 
 is also an important goal to enhance their utility as tagging molecules .
the selection of an appropriate bioorthogonal labeling strategy 
 for a given application is a difficult task , requiring multiple considerations 
 to minimally perturb the system of interest .
we hope these investigations 
 will help provide guidance for selecting the appropriate reaction 
 and reagents for various live - cell imaging applications .
we believe 
 it can also serve as a platform to evaluate new bioorthogonal reactions where one could easily prepare 
 halotag ligands of newly developed bioorthogonal groups to evaluate 
 the chemistry in live cells and within the various intracellular organelles . 

this platform is ideally suited 
 to help evaluate and optimize new fluorophores including fluorogenic 
 fluorophores for no - wash live - cell imaging as well as fluorophores for super - resolution 
 imaging .
synthesis 
 of chloroalkane halotag 
 ligands 111 and bioorthogonal fluorophores 
 are described in the supporting information .
n - terminal halotag 
 orf fusion protein plasmids in pfn21a mammalian expression vector 
 were purchased for targeted halotag expression in the lumen of the 
 endoplasmic reticulum ( human kdelr3 ; promega fhc05579 ) , nucleus ( human 
 hist1h2bk ; promega fhc05625 ) , and cytosol ( human gap43 ; promega fhc02950 ) . 

kdelr3 , hist1h2bk , and gap43 plasmids were cut with pmei / noti , and 
 ligated to a noti / smai fragment of either egfp ( pegfp - n1 , 
 clontech ) or mcherry ( pmcherry - n1 , clontech ) to create fluorescent 
 protein tags on each construct . the c - terminal 
 halotag orf phtn mammalian expression vector was purchased from promega 
 ( g772a ) .
a cytosolic / nuclear expressed halo - mcherry fusion construct 
 was generated by ligating an nhei / ecorv halo fragment into pmcherry - n1 
 ( clontech ) mammalian expression vector , cut with nhei / pspomi ( blunt 
 filled ) . for extracellular halotag protein expression ( halo - mcherry - pdgfr )
, 
 a construct was generated by ligating a synthetic , stop codon eliminated 
 halo - mcherry fusion to the transmembrane domain of the pdgfr in the 
 pdisplay vector ( invitrogen / lifetechnologies catalog # v66020 ) 
 at the restriction sites bglii / psti . a (
hela cells were 
 maintained in growth media containing dulbecco s modified eagle 
 medium ( dmem , life technologies ) supplemented with 10% ( v : v ) heat 
 inactivated fbs ( life technologies ) , 100 units / ml penicillin and streptomycin 
 ( life technologies ) , and 10 mm hepes ( life technologies ) in a humidified 
 incubator set at 37 c/5% co2 .
hela cells were transfected 
 with a 3:1 ratio of lipofectamine 2000 ( life technologies):dna according 
 to the manufacturer s protocol in antibiotic - free dmem/10% 
 fbs at 8090% confluence .
cells were incubated for 3 h at 37 
 c/5% co2 and media was changed into growth media 
 for 1620 h prior to halotag labeling experiments .
halotag ligands 111 were prepared as 10 mm stocks ( 1000 ) in anhydrous dmso within 
 1-week of nmr confirmation of ligand structure .
stocks were frozen 
 as single - use aliquots at 20 c , and kept for up to 6 
 months due to stability issues .
stco ligand 5 and cyclopropene 
 ligand 9 were especially unstable , and required constant 
 monitoring and resynthesis .
ag - stco ligand 6 was prepared 
 as a 10 mm stock in etoh , and maintained stability over at least 8 
 months when stored at 20 c .
fluorophore tetrazine derivatives 
 were prepared as 10 mm stocks in anhydrous dmso , and serially diluted 
 to 1000 working stocks .
solutions were frozen at 20 
 c and remained stable up to 3 freeze  thaw cycles . strained 
 alkyne and azide
fluorophores were prepared as 100 mm stock solutions 
 in anhydrous dmso , and frozen as 1000 working stocks at 20 
 c .
aliquots were used up to 3 times before disposal without 
 any loss of reactivity .
tco - tamra fluorophore was prepared as a 10 
 mm stock in anhydrous dmso , aliquoted , and stored at 20 c 
 as single - use aliquots .
hela cells expressing halotag constructs were treated in 6-well dishes 
 with 1 ml of 10 m halotag ligands 112 in growth media for 0.5 h at 37 c/5% co2 .
samples labeled with 12 served as a positive control 
 to determine the maximum amount of halotag protein labeling per experiment . 

cells were washed three times in dpbs and incubated in 2 ml new media 
 for 1 h with one media change to remove unbound halotag ligands .
for 
 spaac reactions , hela cells were labeled with chloroalkane ligands 13 followed by either a dose response 
 of 50 nm to 250 m fluorophores 1517 for 2 h , or a timecourse of 25 m fluorophore for 
 30 s to 4 h in growth media .
reactions were immediately quenched by 
 washing cells two times in 500 m azide - amine 24 , dbco - amine 26 ( click chemistry tools ) , or bcn - amine 25 in pbs ( spaac quench buffer ) .
for iedda , hela cells labeled 
 with chloroalkane ligands 2 , and 411 were treated with either a dose response from 1 nm-20 m 
 fluorophores 13 , 14 , 17 , and 18 for 1 h , or a timecourse of 2 m fluorophore for 
 10 s-2 h in growth media .
cells were quenched by washing two times 
 in 100 m tz - amine 27 ( click chemistry tools ) or 
 tco - amine 28 in pbs ( iedda quench buffer ) .
cells were 
 scraped in 1 ml quench buffer , spun at 2000 g for 
 3 min , the buffer was aspirated and cell pellets were immediately 
 frozen on dry ice . for spaac
, cell pellets were lysed by sonication 
 in 100 l 1% sds / spaac quench buffer .
for iedda , cell pellets 
 were lysed by sonication in 100 l 1% sds / iedda quench buffer . 

protein concentrations were determined with a bca protein assay ( thermo - fisher ) 
 and cell lysates were normalized by protein concentration .
samples 
 were prepared in 1 lds sample buffer ( life technologies ) , with 
 10 g protein loaded per well , and separated by sds - page on 
 nupage 412% bis - tris gels in mes running buffer ( life technologies ) . 
 tamra - fluorescence was analyzed on a typhoon variable mode imager 
 ( ge healthsciences ) using a tamra filter .
gels were then transferred 
 to nitrocellulose using iblot ( life technologies ) , blocked in odyssey 
 blocking buffer ( licor ) for 1 h at rt , and incubated in anti - halotag 
 pab ( promega , g9281 ) at 1:2000 overnight in tbst .
membranes were washed 
 3 times in tbst , and incubated in goat anti - rabbit irdye 800cw ( licor ) 
 at 1:10,000 in tbst for 1 h at rt .
membranes were washed three times 
 in tbst and imaged on the odyssey infrared imager ( licor ) .
fluorescence intensity measurements 
 were quantified in imagej 1.45 ( nih ) for both tamra fluorescence and 
 total halotag protein expression . in - gel fluorescence ( tamra ) signal 
 first was normalized to total halotag protein expression signal ( western 
 blot ) .
the positive tamra - control ( ligand 12 ) was set 
 at 100% for each experiment .
spaac and iedda data were normalized 
 to this value , and reported as a percent of control .
data from 3 to 
 6 independent replicates were quantified and plotted as the log[dose ] 
 vs response for generation of ec50 values .
response curve in graphpad 
 prism version 6.03 for windows , graphpad software , la jolla california 
 usa , www.graphpad.com .
for timecourse experiments , data 
 was plotted as the percent of control vs time and fit with pseudo - first 
 order association kinetics where y = y0 + ( plateau 
 exp(kx ) ) where 
 the plateau = emax and k = rate constant expressed as the reciprocal of x in units .
all data is reported as the mean  sem from 3 to 6 independent 
 replicates .
hela cells expressing halo - h2b - gfp were treated with 
 10 m halotag ligands 112 for 
 30 min in growth media at 37 c/5% co2 , washed three 
 times with pbs , and incubated for 1 h in growth media with one media 
 change to remove unbound halotag ligand .
samples were treated with 
 1 m halotag ligand 12 for 10 min at 37 c/5% 
 co2 , washed three times with pbs and processed as described 
 after lysis in 100 l 1% sds with sonication .
hela cells transfected with halo - h2b - gfp 
 ( nucleus ) or halo - kdel ( er ) were analyzed for dienophile stability . 

cells treated with 10 m halotag ligands 47 and 12 were incubated in dmem/10% fbs for 0.5 
 h followed by a 1 h washout period prior to reaction with tamra - tetrazine 13 at 37 c/5% co2 .
hela cells were incubated 
 with a saturating dose of 2 m 5-tamra - tetrazine 13 for 1 h at 37 c/5% co2 at the indicated time points . 

cells were washed with pbs , scraped in 1 ml pbs , pelleted at 2000 g for 3 min , and processed as described previously for in - gel 
 fluorescence .
each time point contained 12 as a control 
 to account for labeled halotag protein degradation during the course 
 of the experiment .
hela cells were plated on poly - lysine coated glass - bottom 
 dishes ( mattek , p35gc-1.514c ) , transfected , and treated as 
 described for in - gel fluorescence with the following modifications . 

following fluorophore incubation in culture media , cells were washed 
 3  1 ml pbs with the appropriate quench reagent , and quenched 
 for an additional 5 min in media .
cells were washed 3  1 ml 
 media to remove quench reagent and incubated in cell culture media 
 for 12 h prior to imaging .
cells were washed one time in phenol 
 red - free dmem ( life technologies)/10% fbs , and media was replaced 
 with phenol - red free dmem/10% fbs supplemented with 10 g / ml 
 hoescht 33342 ( life technologies , h3570 ) for nuclear labeling 5 min 
 prior to imaging live . for fixed - cell imaging ,
cells were fixed in 
 ice - cold meoh for 10 min , washed 3  1 ml pbs and incubated in 
 1 ml pbs overnight at 4 c .
pbs was aspirated and 100 l 
 vectashield ( vector laboratories ) cell mounting media containing dapi 
 ( vector laboratories , h-1200 ) was added prior to imaging .
live and fixed cells were imaged 
 on a zeiss axioobserver.z1 with a yokagawa csu - x1 m 5000 spinning disk 
 system using a zeiss planapochromatic 40/1.3 or 63/1.4 
 oil immersion objectives .
the imaging system was maintained in a 37 
 c heated incubation chamber along with humidified stage - top 
 incubation components set at 37 c/5% co2 for live - cell 
 imaging .
excitation of hoescht and dapi was carried out with a 405 
 nm laser and emission spectra were collected between 440 and 480 nm . 

excitation of fluorescein , bodipy - fl , and egfp was carried out using 
 a 488 nm laser and emission spectra were collected between 520 and 
 550 nm .
tamra and mcherry were excited with a 561 nm laser and emission 
 spectra were collected between 620 and 670 nm .
images were acquired 
 using a photometrics evolve 512 delta camera using the appropriate 
 filter conditions for the indicated fluorophores with the zen blue 
 2012 v. 8.1 software ( carl zeiss microscopy ) .
 bioorthogonal 
 reactions , including the strain - promoted azide  alkyne 
 cycloaddition ( spaac ) and inverse electron demand diels  alder 
 ( iedda ) reactions , have become increasingly popular for live - cell 
 imaging applications . 
 however , the stability and reactivity of reagents 
 has never been systematically explored in the context of a living 
 cell . here 
 we report a universal , organelle - targetable system based 
 on halotag protein technology for directly comparing bioorthogonal 
 reagent reactivity , specificity , and stability using clickable halotag 
 ligands in various subcellular compartments . 
 this system enabled a 
 detailed comparison of the bioorthogonal reactions in live cells and 
 informed the selection of optimal reagents and conditions for live - cell 
 imaging studies . 
 we found that the reaction of stco with monosubstituted 
 tetrazines is the fastest reaction in cells ; however , both reagents 
 have stability issues . 
 to address this , we introduced a new variant 
 of stco , ag - stco , which has much improved stability and can be used 
 directly in cells for rapid bioorthogonal reactions with tetrazines . 
 
 utilization of ag complexes of conformationally strained trans - cyclooctenes should greatly expand their usefulness especially when 
 paired with less reactive , more stable tetrazines . 
Introduction
Results and Discussion
Conclusions
Experimental Section