luminescent metal complexes 
 are increasingly employed as biomolecular and cellular probes due 
 to their unique photophysical properties that make them particularly 
 well suited to monitor biological processes .
examples include not only complexes of transition metals such as 
 pt , ir , and ru but also those that incorporate lanthanides , most commonly 
 eu and tb . in particular , the long luminescence lifetime 
 of the emitting metal mitigates the interference of background fluorescence 
 originating from the biological sample .
moreover , since the large 
 energy difference between the absorbing and emissive states of these 
 complexes removes self - absorption issues , their luminescence signal 
 intensity is proportional to their concentration over a wide range 
 of values . with this in mind ,
lanthanide - based molecular probes have 
 been applied to the detection of reactive oxygen species ( ros ) , redox - active metals , ph , and intracellular analytes , such 
 as atp .
the continued applications of 
 such metal - based luminescent probes , however , further rely on the 
 ability to limit their cellular association altogether for certain 
 tissue imaging experiments or , alternatively , to direct them to specific 
 cellular regions .
prior studies indicate that structural properties 
 of metal complexes , such as their hydrophobicity and size , influence 
 their cell viability and cellular association , including their membrane 
 permeability . although cell - penetrating 
 peptides have been applied to increase the cell uptake of lanthanide 
 complexes , the goal of this project is 
 to modify the intrinsic properties of the metal complex to control 
 cellular association . presented here
is a systematic study to evaluate 
 the effects of structural variations on a core structure common to 
 most luminescent lanthanide probes and an investigation of the practicality 
 of such probes for cellular and tissue applications requiring extracellular 
 imaging agents .
the most common platform of luminescent lanthanide 
 complexes features a chelating polyaminocarboxylate ligand substituted 
 with one or more carboxamide pendant arms from which additional moieties 
 are attached , notably the probe s antenna .
the laporte - forbidden 
 nature of the f  f transition dictates that , for practical applications 
 in biologically relevant settings , the probe must incorporate a sensitizing 
 antenna . in terms of the design of a responsive probe , the luminescence 
 intensity of the lanthanide complex
can then be modulated by altering , 
 among other parameters , the excited triplet state energy level of 
 the antenna and the antenna  lanthanide distance . for biological applications ,
it is thus important 
 to select an antenna with suitable energetic properties for lanthanide 
 sensitization that is also resistant to quenching by the medium . work 
 by parker and co - workers identified that the nature of the antenna , 
 specifically its point of attachment to the cyclen backbone of the 
 ligand , affects the subcellular localization of the complexes .
given the increasing use of phenanthridine and isophthalamide antennas 
 for the design of luminescent lanthanide probes , a 
 deeper understanding of the structural parameters influencing their 
 effect on cellular viability and cell association is necessary to 
 further optimize their structures for biological application both 
 extra- and intracellularly .
herein , two parameters were evaluated 
 for their effect on cell viability and association : the nature of 
 the antenna and that of the pendant amide arms .
the macrocyclic polyaminocarboxylate ligand provides kinetic 
 inertness and thus predicted lower toxicity on cell viability .
furthermore , the overall positively charged 
 lanthanide complexes of dota tetraamide ( dotam ) ligands are kinetically 
 more inert than the dota analogues as a result of the decreased basicity 
 of the nitrogen atoms . moreover , the 
 macrocyclic framework is less susceptible to enzymatic degradation .
the high kinetic inertness of macrocyclic lanthanide complexes and 
 their ability to resist degradation is critical for their use in vivo . 

three different antennas were investigated : the hydrophilic 2-hydroxyisophthalamide 
 ( iam ) , a reported sensitizer of both tb and eu , the closely related and synthetically more facile 2-methoxyisophthalamide 
 ( iam(ome ) ) , and the extended aromatic 6-methylphenanthridine ( phen ) 
 ( chart 1 ) . for each complex , the quantum yield , 
 cellular viability , and cell association were evaluated .
the effect 
 of charge and hydrophobicity on cell viability and association was 
 subsequently evaluated with complexes comprising the iam(ome ) antenna 
 functionalized with varying carboxamide arms ( chart 2 ) .
the isophthalamide complexes tb - dota - iam(ome ) ( [ tb-1 ] ) and tb - dota - iam ( [ tb-2 ] ) were 
 synthesized according to scheme 1 .
briefly , 
 the oxidation of bis(hydroxymethyl)-p - cresol with 
 mno2 and koh generated the diacid 11 .
subsequent 
 treatment with methyl iodide generated the methoxy - protected diester 12 , which was then converted to the diamide 13 with methylamine .
the aldehyde 14 was generated via 
 a riley oxidation of the aryl methyl group in naphthalene at 215 c . 

periodinane oxidation methods ( dess  martin and 2-iodoxybenzoic 
 acid ) , permananganates , and chromates ( jones reagent and pcc ) 
 were unsuccessful mediators of this transformation .
reaction of the 
 aldehyde 14 with hydroxylamine hydrochloride generated 
 the oxime 15 , which was crystallized by addition of diethyl 
 ether to an acidic solution of the crude reaction mixture in ethyl 
 acetate and methanol and then reduced to the amine form of the iam 
 antenna 16 .
the tris - boc - protected cyclen 17 was synthesized as reported and further 
 coupled to the amine 16 using hatu as the activating 
 agent .
boc deprotection in a hydrochloric acid / methanol mixture afforded 
 the iam(ome ) cyclen conjugate 19 , which served as a common 
 intermediate in the synthesis of complexes [ tb-1 ] , [ tb-2 ] , and [ tb-4][tb-10 ] .
reagents and conditions : ( a ) mno2 , ch3cl , 60 c , 20 h ; ( b ) koh ( s ) , 230 c , 
 1 h ; ( c ) ch3i , k2co3 , acetone , 56 
 c , 16 h ; ( d ) nh2ch3 , ch3oh , 
 65 c , 18 h ; ( e ) seo2 , naphthalene , 215 c , 2.5 
 h ; ( f ) hydroxylamine hydrochloride , pyridine , 22 c , 24 h ; ( g ) 
 pd / c ( 10% ) , hcl , ethanol / h2o , 5 bar , 16 h ; ( h ) hatu , dipea , 
 dmf , 22 c , 40 h ; ( i ) hcl , ch3oh , 22 c , 18 h ; 
 ( j ) cs2co3 , ch3cn , 40 c , 18 
 h ; ( k ) hcl , ch3oh , 22 c , 18 h ; ( l ) bbr3 , ch2cl2 , 25 c , 18 h ; ( m ) ch3oh , 65 c , 16 h ; ( n ) tbcl3 , h2o / ch3oh , ph 7 , 45 c , 48 h ; ( o ) 0.2 m koh , h2o , 
 22 c , 20 h. alkylation of the cyclen 
 derivative 19 with tert - butyl bromoacetate 
 yielded the protected ligand 20 ( scheme 1 ) .
deprotection in a hydrochloric acid / methanol mixture yielded 
 the free ligand dota - iam(ome ) ( 1 ) .
harsher deprotection 
 conditions with boron tribromide removed both the tert - butyl groups and the aryl methyl ether to yield dota - iam ( 2 ) . the final complexes [ tb-1 ] and [ tb-2 ] were obtained from their respective ligands , 1 and 2 , by heating with tbcl3 in 
 aqueous solutions at neutral to slightly basic ph for 2 days or more .
the phenathridine complex [ tb-3 ] was synthesized according to the published procedure . in order to evaluate the role of charge and hydrophobicity 
 of the lanthanide complex in its cellular compatibility , seven derivatives 
 of tb - dota - iam(ome ) ( [ tb-1 ] ) were synthesized ( chart 2 and scheme 1 ) .
the synthesis 
 was designed to take advantage of the common intermediate 19 , described above .
each bromoacetamide arm was synthesized using 
 a biphasic method in which bromoacetyl bromide in dichloromethane 
 and aqueous potassium carbonate were simultaneously added dropwise 
 to a solution of the corresponding amine in dichloromethane at 0 c . 

this method resulted in moderate to high yields ( 6585% ) of 
 the desired products in high purity .
the ligands were obtained upon 
 reaction of the bromoacetamide arms with the intermediate 19 .
the carboxylic acid derivatives [ tb-9 ] and [ tb-10 ] were obtained by saponification of their corresponding 
 esters [ tb-7 ] and [ tb-8 ] , respectively .
the nature of 
 the sensitizing antenna of a lanthanide complex can affect its function 
 in a biological environment . here , we compare [ tb-1 ] , 
 [ tb-2 ] , and [ tb-3 ] , three 
 complexes that feature either an isophthalamide- or phenanthridine - based 
 antenna for sensitizing the lanthanide emissions ( chart 1 ) .
all three complexes luminesce in the green region of the visible spectrum , 
 with maximum emission at 545 nm upon excitation at 350 nm ( figure 
 s1 ( supporting information ) ) .
these three 
 antenna platforms have different properties in terms of hydrophobicity 
 and interactions with biomolecules , including nucleic acids , reducing 
 agents , and proteins .
the hydrophilic isophthalamide and methoxyisophthalamide 
 are composed of a single aromatic ring and have no known interactions 
 with biomolecules , while the phenanthridine is an extended aromatic 
 system capable of base stacking with nucleotides and dna intercalation .
the quantum yield ,  , of the three tb complexes in pbs 
 was investigated by comparing the luminescence intensity of each complex 
 to that of a quinine sulfate standard in 0.1 m sulfuric acid ( r = 0.577 ) according to the optically 
 dilute method . at
ph 7.8 and 22 c , 
 the quantum yields of [ tb-1 ] and [ tb-3 ] 
 are statistically identical ( 4.6  1.2% vs 4.7  2.2% , respectively ) . 

the quantum yield of the phenathridine derivative [ tb-3 ] is comparable to that of similar complexes featuring either triamide- 
 or triphosphinate - substituted dota ligands .
however , [ tb-2 ] is a more efficient 
 emitter with an average quantum yield of 9.8  1.5% , indicating 
 that substituting the methoxy for the hydroxyl group on the isophthalamide 
 antenna substantially affects the quantum yield of the complex .
50% have been reported by raymond and co - workers 
 for tb complexes with tetra - iam - substituted ligands .
in contrast with the case for [ tb-2 ] , in the tetra - iam complex h(2,2)-iam the isophthalamide 
 directly coordinates the lanthanide , thereby enabling a dexter - type 
 energy transfer from the antenna to the tb that is much more efficient 
 than the frster - type , through - space mechanism likely taking 
 place in [ tb-2 ] . on the other hand , 
 [ tb-2 ]
has substantially higher water 
 solubility ( m range ) than the cryptand h(2,2)-iam ( m range ) 
 and enables the design of responsive probes .
nonetheless , the 
 quantum yields of [ tb-1 ] , [ tb-2 ] , and [ tb-3 ] are comparable to those of luminescent 
 d - block compounds used for cellular imaging . for example , the quantum 
 yields of ir complexes featuring 2-phenylpyridine ligands are between 
 4 and 8% in pbs buffer . not surprisingly , 
 the quantum yields of tb - centered luminescence are lower than those 
 of commonly used fluorescence dyes such as cy3 ( 4% in pbs ) , cy5 ( 30% 
 in pbs ) , alexa750 ( 12% in phosphate buffer ) , and texas red ( 30% in 
 water ) . however , in comparison with phosphorescent 
 metal complexes , organic dyes suffer from short fluorescence lifetimes 
 and small stokes shifts . as for 
 any luminescent or fluorescent probe ,
emission is susceptible to quenching 
 by reducing agents , proteins , and other components of biological media . 
 in the case of luminescent tb and eu complexes ,
the quenching pathways 
 can involve electron or charge transfer from the excited states to 
 intracellular reducing agents such as urate , ascorbate , and glutathione 
 that are present in mm concentrations in cells .
in addition , luminescence quenching by proteins , such as 
 human serum albumin , has been reported .
the degree of luminescence quenching was evaluated by measuring 
 the time - delayed luminescence intensity of [ tb-1 ] with 
 respect to its concentration in pbs and whole cell lysate at two different 
 protein concentrations .
in whole cell lysate containing 0.25 mg of 
 protein / ml , the luminescence intensity of [ tb-1 ] is quenched 
 by 22  2.8% ; at 0.5 mg of protein / ml the luminescence intensity 
 is reduced by 30  2.4% ( table 1 and figure 
 s2 ( supporting information ) ) . advantageously , 
 the isophthalamide antenna is less susceptible to quenching than complexes 
 containing electron - poor sensitizing moieties .
for instance , the metal - centered 
 emission of terbium azaxanthone complexes is quenched by 3060% 
 in the presence of 0.2 mm human serum albumin .
experimental conditions : [ tb-1 ] = 015 
 m , pbs , ph 7.8 , time delay 0.1 ms , excitation wavelength 345 
 nm , slit widths ( excitation and emission ) 5 nm , integrated emission 
 intensity from 470 to 635 nm , t = 20 c .
whole cell lysate was 
 generated from l6 myoblasts ; the protein concentration was determined 
 by a bca assay .
percent 
 quenching was calculated using the percent change in the slope of 
 the integrated luminescence intensity versus the [ tb-1 ] ( m ) plots ( figure s2 ( supporting information ) ) .
the effect of [ tb-1 ] , [ tb-2 ] , and [ tb-3 ] on the viability of l6 rat muscle myoblast cells was investigated 
 using an mtt assay .
it was hypothesized that the 2-methoxyisophthalamide 
 would have a reduced effect on cell viability due to the protecting 
 group on the phenol that could block nonspecific interactions with 
 the cell proteins and coordination of cellular metals , such as iron 
 and copper .
additionally , the phenathridine complex [ tb-3 ] was expected to have a greater effect on cell viability due to 
 its ability to intercalate dna and rna . following a 24 h incubation with 0300 m tb complex , 
 the phenanthridine derivative [ tb-3 ] decreased cell viability 
 to 74% at 100 m and to 63% at 300 m . on the other hand , 

the similar profiles indicate that the methoxy protecting group 
 on the isophthalamide does not significantly improve cell viability 
 at the doses studied here .
this is in contrast to a pair of rh probes 
 chelated by chrysenequinone diimine and n - functionalized 
 dipyridylamine ligands , for which small structural changes were shown 
 to have a substantial impact on cell viability . in that case , altering 
 the ethanol substituent to a propyl reduces the viability of hct116n 
 and hct116o cells from 80% to 60% after a 24 h incubation with 40 
 m complex .
obtained by an mtt assay after a 24 h incubation 
 with 100 m complex .
results are mean  sd ( n = 3 ) . determined by icp - ms 
 after a 4 h incubation with 50 m complex .
viability ( 24 h ) of l6 myoblasts treated with 0300 m 
 [ tb-1 ] ( solid squares ) , [ tb-2 ] ( open circles ) , and [ tb-3 ] ( solid triangles ) as determined 
 with an mtt assay .
overall , the complexes 
 in this study influence cell viability slightly less than complexes 
 containing tetraazatriphenylene or azaxanthone antennas with phenyl 
 or ethyl ester substituted carboxamide arms , which have ed50 values in the 100 m to > 240 m range .
they affect cell viability similarly 
 to ru(iii ) polypyridine complexes , which also have ed50 ( 72 h ) values 300 m in mcf-7 and ht-29 cell lines .
it is interesting to note that the europium 
 compound of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 
 containing no antenna moiety , [ eu - dota ] , has a 
 negligible effect on cell viability from 0 to 500 m with a 
 72 h incubation .
thus , with respect to 
 cell viability , [ tb-1 ] and [ tb-2 ] are preferred for cellular imaging applications compared to [ tb-3 ] .
the nature of the antenna was also expected to 
 affect the cellular association of the complexes . following a 4 h 
 incubation with 50 m of tb complex ,
the cellular accumulation 
 of the metal was determined by icp - ms and expressed relative to the 
 protein concentration as determined by a bca assay .
importantly , it 
 should be noted that the quantitative nature of measuring cellular 
 association of metal complexes by icp - ms is balanced by the inability 
 of the technique to distinguish between internalized or membrane - bound 
 complexes .
it was anticipated that the 
 extended aromatic antenna of [ tb-3 ] would facilitate 
 membrane permeability , as suggested by the increased cellular uptake 
 of more lipophilic ru complexes and phenanthridine - substituted cisplatin . however , the cell association 
 values of the hydrophilic tb - dota - iam(ome ) ( [ tb-1 ] ) and 
 tb - dota - iam ( [ tb-2 ] ) are statistically 
 not different from the values for the hydrophobic tb - dota - phen ( [ tb-3 ] ) ( table 2 ) .
small structural 
 changes to the ligands of metal complexes have been reported to exert 
 varied effects on the intracellular accumulation and distribution . 
 using a group of lanthanide complexes with tetraazatriphenylene- or 
 azaxanthone - based antennas
, parker identified that how the antenna 
 was linked to the cyclen ring affected membrane permeability and localization 
 more than small structural modifications on the antenna itself .
for the isophthalamide complexes , however , conversion 
 from the methoxy group in [ tb-1 ] to the phenolate of 
 [ tb-2 ] does affect cellular association . 

this observation is more in concert with the findings of barton , who 
 measured substantial differences in cell association between an alkyl - substituted 
 dipyridyl rh complex ( 705 ng of rh / mg of protein ) in comparison to 
 a complex that introduced a terminal alcohol on the alkyl group ( 165 
 ng of rh / mg of protein ) .
the cellular 
 association values measured for the hydroxyisophthalamide [ tb-2 ] are comparable to the values reported 
 for a tb complex with a tetraazatriphenylene antenna and phenyl - substituted 
 amide arms , which had a maximum accumulation of 10 mol / g of 
 protein into nih 3t3 cells after a 4 h incubation at 50 m . unfortunately , further comparisons with cellular 
 accumulation values reported in the literature are limited due to 
 differences in the method of data collection ( icp - ms vs flow cytometry ) , and data presentation of metal 
 per amount of protein or per cell . notable differences among the three metal complexes include 
 the increased effect of [ tb-3 ] on cell viability above 
 100 m in spite of its only modest cell association .
the synthesis 
 of [ tb-2 ] requires a synthetically 
 challenging boron tribromide deprotection to produce the 2-hydroxyisophthalamide 
 antenna , and the resulting complex has a cellular association only 
 slightly higher than that of its analogue [ tb-1 ] .
the 
 more facile synthesis of the 2-methoxyisophthalamide of [ tb-1 ] in comparison to [ tb-2 ] thus became 
 a significant factor in selecting an antenna for further studies evaluating 
 the effects of variable arms on the cellular compatibility of lanthanide 
 complexes .
the role 
 of charge and hydrophobicity of the polyaminocarboxylate arms on the 
 cell viability and association of the lanthanide probes was evaluated 
 with a library of derivatives of [ tb-1 ] .
complexes [ tb-4][tb-10 ] share a common cyclen backbone with a 2-methoxyisophthalamide antenna 
 but differ in the nature of the remaining substituents ( chart 2 ) .
the alkyl , benzyl , and trifluoro groups impart 
 increasing hydrophobicity to the complexes , whereas alanine and glutamine 
 amino acid based substituents in either an ethyl ester or carboxylic 
 acid form render greater hydrophilicity .
the resulting complexes also 
 differ in overall complex charge ; neutral carboxamide arms generate 
 the 3 + charged complexes [ tb-4][tb-8 ] , the addition of monoanionic arms forms the 
 zwitterionic complex [ tb-9 ] , and the dianionic glutamate 
 arm creates [ tb-10 ] bearing a net 
 3 charge . as with the previous complexes that differ 
 with respect to the antenna , the cell viability ( 24 h ) was measured 
 for the library of complexes featuring pendant arms with structural 
 and electronic variations . of the hydrophobic complexes , only [ tb-5 ]
decreases cell viability significantly to 
 60% , while the values for [ tb-4 ] and [ tb-6 ] are comparable to that of the parent complex 
 [ tb-1 ] , maintaining a viability above 80% in the concentration 
 range measured ( figure 2a and table 2 ) .
each of the amino acid derivatives [ tb-7 ] , [ tb-8 ] , [ tb-9 ] , and [ tb-10 ] also sustain viability 
 above 75% even in the presence of 300 m tb complex ( figure 2b and table 2 ) .
interestingly , 
 the notable reduction in cell viability upon addition of medium - length 
 alkyl chains matches the results of a previous study comparing a family 
 of ir(iii ) cyclometalated probes . in that study ,
the ir complexes 
 featuring the shortest ( c2 ) and longest ( c18 ) alkyl chains had comparably minimal effects on hela cell viability 
 ( ed50  15 m ) , while the median length alkyl 
 chain ( c10 ) was significantly more cytotoxic ( ed50 = 2 m ) .
the hexyl alkyl derivative 
 [ tb-5 ] excluded , the relatively low effect 
 on cell viability ( ed50 values > 300 m ) of this 
 entire class of tb complexes suggests they have only limited interactions 
 with cellular components and bodes well for their application .
viability ( 24 
 h ) of l6 myoblasts treated with 0300 m tb complexes 
 as determined with an mtt assay : ( a ) [ tb-1 ] ( solid squares ) , 
 [ tb-4 ] ( solid triangles ) , [ tb-5 ] ( open circles ) , and [ tb-6 ] ( open inverted triangles ) ; ( b ) [ tb-7 ] ( solid 
 squares ) , [ tb-8 ] ( solid triangles ) , [ tb-9 ] ( open circles ) , and [ tb-10 ] ( open inverted triangles ) .
the role of the pendant arms on the cellular association 
 of the tb complexes was also investigated by icp - ms .
it was hypothesized , 
 on the basis of the results of barton , that [ tb-4 ] , [ tb-5 ] , and [ tb-6 ] would penetrate cells more efficiently due 
 to their lipophilic substituents and cationic character that would 
 assist transport across the membrane .
indeed , higher tb accumulation is observed with the hydrophobic 
 hexyl - substituted [ tb-5 ] and trifluoro - containing 
 [ tb-6 ] than with the more hydrophilic amino 
 acid derivatives ( table 2 ) .
however , this conclusion 
 can not be universally applied to all hydrophobic arms , since the cellular 
 association of the benzyl - substituted [ tb-4 ] is less than that of the parent complex [ tb-1 ] .
the 
 alkyl - substituted ir complexes discussed previously also do not illustrate 
 a direct relationship between hydrophobicity and cellular association . 
 in that study , each complex investigated displayed measurable cellular 
 association by icp - ms , but the c10 version afforded the 
 greatest association , followed by the c2 and then the c18 derivative .
thus , it is clear 
 that the lipophilicity of the complex alone can not be used to predict 
 the cellular association of metal complexes . of the amino acid 
 derivatized complexes ,
the alanine - based [ tb-7 ] and [ tb-9 ] have higher cell association values than 
 the glutamine - based compounds [ tb-8 ] and 
 [ tb-10 ] .
positively charged complexes 
 containing esters are reported to have enhanced cell association in 
 comparison to carboxylic acid derivatives , and this is observed with the slightly higher cell association values 
 of the ethyl ester complex of alanine [ tb-7 ] in comparison to the neutral acid complex [ tb-9 ] .
this 
 reflects the finding that the cell association of [ eu - dotam ] is 5 times that of [ eu - dota ] , which showed negligible 
 cellular accumulation .
interestingly , 
 this trend is not repeated for the glutamine ethyl ester and acid 
 complexes , [ tb-8 ] and [ tb-10 ] , respectively , which both have low cell association 
 values despite their large differences in overall complex charge . 

this is in contrast with to lanthanide complexes studied by parker 
 in which the intracellular concentration of a 3 charged glutamate - functionalized 
 dota with a tetraazatriphenylene antenna was approximately 3 times 
 that of the neutral ln - dota - tetraazatriphenylene .
an important conclusion is that the parameters influencing 
 cellular association of lanthanide complexes are multifaceted and 
 include both structural features ( lipophilicity , nature of the antenna , 
 terminal functional groups ) and , to a lesser extent , overall complex 
 charge .
this study investigated the effect of structural modifications 
 of the ligand on cell association in the absence of targeting moieties ; 
 however , conjugating ln complexes to cell - penetrating peptides or receptor targeting groups has successfully 
 increased the cellular uptake .
progress in the field of cell - penetrating , 
 lanthanide - based contrast agents has been reviewed and 
 includes the use of polyarginine and 
 tat - based peptides .
this technique has 
 also been applied to luminescent metal complexes and dual imaging agents .
additional 
 approaches to increase cellular accumulation include coupling complexes 
 to moieties that facilitate interactions with cellular membrane receptors , transporters , or 
 special groups such as exofacial thiols .
the translation of these approaches to the luminescent macrocyclic 
 polyaminocarboxylate complexes described herein is being further investigated 
 by our group .
the cellular 
 association of the tb complexes was also investigated with epifluorescence 
 microscopy .
l6 myoblasts were treated with tb complex ( 200 m 
 for 4 h ) , washed with pbs at room temperature , and fixed with formaldehyde 
 prior to imaging .
results of representative cells indicate the presence 
 of weak cellular association for the trifluoro - substituted [ tb-6 ] , while the fluorescence intensity of phenanthridine - containing 
 [ tb-3 ] is comparable to that of the control cells ( figure 3 ) .
the remaining tb complexes exhibit similar weak 
 cellular fluorescence ( figure s3 ( supporting information ) ) . due to reports that different staining patterns can be observed 
 in experiments that differ only in whether cells were imaged in a 
 live or fixed state , the cellular association 
 of [ tb-6 ] was also examined in live cells 
 via fluorescence microscopy ( figure s4 ( supporting 
 information ) ) . in these images ,
an overall decrease in the 
 fluorescence to levels comparable to those for the untreated control 
 cells was observed .
fluorescence microscopy images of representative l6 myoblasts 
 treated with 200 m [ tb-3 ] and [ tb-6 ] for 4 h at 37 c : ( a ) fluorescence images , 60 objective , 
 325375 nm excitation filter , 470750 nm emission filter , 
 0.4 s exposure time ; ( b ) bright field images , 0.04 s exposure time . 

scale bars represent 10 m . the reduced punctate staining in live cells in comparison 
 to fixed cells has also been observed by belitsky .
however , unlike our 2-hydroxyisophthalamide complexes , 
 lanthanide complexes featuring tetraazatriphenylene antennas containing 
 phenyl , ester , or carboxylate substituents are taken up by cho or 
 nih 3t3 cells and can be visualized with fluorescence microscopy regardless 
 of the nature of their pendent arms . likewise , 
 bnzli observed lanthanide helicates ranging in overall charge 
 from 6 to 6 + localized to endosomes and lysosomes , consistent 
 with endocytotic uptake mechanisms irrespective of the polarity or 
 overall charge of the complex .
regardless 
 of the reasons , the low cell associations exhibited by this entire 
 class of complexes render them particularly useful for monitoring 
 extracellular species .
a family of luminescent 
 lanthanide complexes featuring different sensitizing antennas varying 
 in charge and hydrophobicity and pendant arms of variable structural 
 and electronic properties were synthesized . the cell viability and 
 association of this family of complexes
have been evaluated . with 
 respect to the antenna , the 2-methoxyisophthalamide of [ tb-1 ]
was selected as the ideal candidate for further studies due to 
 its synthetic ease ( in comparison to [ tb-2 ] ) , suitable quantum yield , little effect on cell viability , and modest 
 cell association .
surprisingly , the addition of hydrophobic moieties 
 ( benzyl , hexyl , and trifluoro ) did not increase cell association . 
 all of the complexes investigated minimally affect cell viability 
 and exhibit low cellular association , regardless of the overall complex 
 charge or relative hydrophobicity ; thus , it is presumed that it is 
 the hydrophilic nature of the 2-methoxyisophthalamide antenna that 
 confers the low membrane permeability of this class of compounds
. 
 thus , the probes based on the structural framework presented here 
 are well - suited for monitoring extracellular analytes such as group 
 i ions , polysaccharides , hormones , or other signaling molecules .
unless otherwise noted , starting materials were obtained from commercial 
 suppliers and used without further purification .
water was distilled 
 and further purified by a millipore cartridge system ( resistivity 
 18 m ) .
elemental analyses were performed by inductively coupled 
 plasma mass spectrometry on a thermo scientific xseries 2 icp - ms fitted 
 with an esi pc3 peltier cooled spray chamber , sc - fast injection loop , 
 and sc-4 autosampler at the department of geology at the university 
 of minnesota .
samples were diluted appropriately and analyzed in the 
 presence of a 20 ppb of in internal standard using the he / h2 collision - reaction 
 mode .
data were collected between 220 and 800 nm using 
 a quartz cell with a path length of 10 mm .
luminescence data were 
 recorded on a varian eclipse fluorescence spectrophotometer using 
 a quartz cell with a path length of 10 mm .
the absorbance and integrated luminescence 
 intensities of [ tb-1][tb-3 ] in pbs 
 ( ph 7.8 , n = 1.3345 ) were compared to the reference 
 fluorescence of quinine sulfate ( r = 0.577 ) in 0.1 m sulfuric acid ( n = 
 1.333 ) .
absorption spectra were collected on a cary 100 bio uv  visible 
 spectrophotometer ; emission spectra were recorded on a cary eclipse 
 fluorescence spectrophotometer . quantum yields (  ) were calculated 
 according to the optically dilute method using the equationwhere a is the absorbance at the excitation wavelength (  ) , n is the refractive index , and i is the 
 integrated intensity .
all data were collected 
 using the same spectrophotometer on the same day , resulting in a constant 
 intensity of the excitation light ( ex = 350 nm ) . 

measurements were obtained at 22 c in a quartz cell of 1 cm 
 path length in degassed solutions using an excitation wavelength of 
 350 nm and excitation and emission slit widths of 10 nm for both complexes . 

the fluorescence response of quinine sulfate was reported as the integrated 
 emission intensity from 365 to 665 nm ; the tb luminescence of each 
 complex was collected with a time delay of 0.1 ms and reported as 
 the integrated emission intensity from 470 to 688 nm .
rat muscle l6 myoblast cell line was obtained from atcc ( manassas , 
 va ) .
cells were cultured in 25 or 75 cm vented culture 
 flasks in dulbecco s modified eagle medium ( dmem ) supplemented 
 with 10% ( v / v ) bovine serum ( bs ) and 10 g of gentamicin ml at 37 c and 5% co2 .
the cells were 
 maintained by splitting every 34 days before reaching confluence ; 
 cells were rinsed with pbs , lifted using 0.25 g l typsin for 5 min , and diluted in fresh medium .
all cells were propagated 
 at low passage numbers ( < 20 ) to maintain their myoblastic character .
l6 cells were harvested 
 from two 75 cm culture flasks grown to 85% confluence 
 and centrifuged at 400 g for 10 min at 4 c . 

the resulting pellet was rinsed with ice - cold pbs ( 2 ml ) and centrifuged . 

the cells were then suspended in pbs ( 1 ml ) and transferred to a tight - fitting 
 homogenizer ( b clearance 0.00080.0022 in . ) .
approximately 
 90% of the cells were ruptured after 120 strokes , as determined by 
 cell counting with trypan blue and a hemocytometer .
the total protein 
 concentration of the whole cell lysate was determined with a bicinchoninic 
 acid ( bca ) assay ( bca protein assay kit , thermoscientific ) using bovine 
 serum albumin ( bsa ) protein standard solutions .
the whole cell lysate 
 was then diluted to a protein concentration of 1.0 mg / ml , and aliquots 
 ( 450 l ) were stored at 20 c for later use .
an aqueous suspension 
 of [ tb-1 ] ( 100 m ) in whole cell lysate diluted 
 in pbs ( 0.25 or 0.5 mg of protein / ml ) was titrated into an aqueous 
 solution of whole cell lysate in pbs ( 0.25 or 0.5 mg of protein / ml ) . 

the time - delayed emission profile was recorded in the presence of 
 015 m of tb complex .
the titration was repeated in 
 pure pbs buffer ( ph 7.8 ) without cell lysate .
measurements were recorded 
 with an excitation wavelength of 345 nm , a time delay of 0.1 ms , and 
 excitation and emission slit widths of 5 nm at a temperature of 20 c . 

the luminescence response was reported as the integrated emission 
 intensity from 470 to 640 nm .
the percent quenching of the luminescence 
 was calculated from the slopes of the integrated luminescence intensity 
 versus the concentration of tb complex ( m ) plots in pbs and 
 whole cell lysate at the two protein concentrations .
l6 myoblasts ( 225 l ) were plated at 4.0  10 cells ml in a 48-well cell culture plate . 

cells were allowed to recover 24 h , after which tb complexes in pbs 
 ( 25 l ) were added to the culture media ( final concentrations 
 of 0 , 1 , 3 , 10 , 30 , 100 , and 300 m ) . for the negative control , 
 pbs ( without tb complex ) was used .
the mtt assay was performed during 
 the last 3 h of the compound exposure time as follows .
mtt ( 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium 
 bromide , 25 l of 5 mg / ml stock solution , 0.5 mg / ml final concentration ) 
 was added to the media , and the plate was returned to the cell culture 
 incubator for 3 h. when a purple precipitate was clearly visible , 
 250 l of 0.04 m hcl in isopropyl alcohol was added to each 
 well .
after an incubation of 4 h at room temperature in the dark , 
 the samples were centrifuged at 10000 g for 5 min 
 to pellet cellular debris . for each sample ,
a portion of the supernatant 
 ( 200 l ) was transferred to a corresponding well of a 96-well 
 plate , and the absorbance at 570 and 690 nm was recorded using a microtiter 
 plate reader .
the cell viability was calculated according to the following 
 equation using the adjusted absorbance of the cells treated with tb 
 complex in comparison to the control cells .
the 
 cellular association of [ tb-1][tb-10 ] was investigated by seeding in triplicate l6 myoblasts ( 225 l ) 
 at 4.5  10 cells ml in a 48-well 
 plate ( 10000 cells ml ) .
cells were allowed 
 to recover for 48 h and had grown to 80% confluence , at which time 
 a concentrated solution of tb complex in pbs ( 25 l ) was added 
 to achieve a final concentration of 50 m in the cellular media
. 
 cells grown in tb complex free media ( addition of only pbs ) were included 
 as a negative control .
following 4 h of incubation , the media were 
 removed , and the wells were washed twice at room temperature by adding 
 pbs and rocking the culture plate back and forth several times .
the 
 addition of a lysis buffer ( 75 l of 10 mm tris , ph 7.5 , 100 
 mm nacl , 1 mm na2edta , and 1% triton x-100 ) followed by 
 a 20 min incubation at 4 c ruptured the cell membranes .
aliquots ( 3  8 l ) from each well 
 were removed for a bca assay to determine the protein concentration . 

samples ( 50 l ) were combined with concentrated nitric acid 
 ( 50 l ) and heated to 95 c for 18 h in flame - sealed glass 
 ampules prior to icp - ms analysis . the tb content ( ppb )
was determined 
 by icp - ms , and the cellular association of each tb complex ( ctb ) , expressed as mol of tb / g of protein , was calculated 
 according to the equationwhere cm is the measured metal concentration ( ppb ) of the cell 
 samples , m is the molecular mass of tb , and cp is the protein concentration ( mg / ml ) .
l6 myoblasts ( passage 9 or 10 ) were seeded in 
 a polylysine - coated 8-well glass - bottom plate and allowed to grow 
 for 24 h to 50% confluence at 37 c in 5% co2 .
then 
 tb complexes [ tb-2][tb-8 ] were added in pbs for a final concentration of 200 m . 

negative control wells containing cells grown in tb complex free media 
 ( addition of only pbs ) were included . following a 4 h incubation , 

the media were removed , and cells were washed three times with pbs 
 at room temperature by rocking the culture plate back and forth gently . 

cells were fixed with 4% formaldehyde in pbs at room temperature for 
 15 min .
additionally , live cell imaging of [ tb-6 ] was 
 performed in dmem phenol red free media .
epifluorescence images of 
 representative cells were acquired on an olympus ix81 inverted microscope 
 equipped with a 60 ( n.a .
excitation 
 was supplied by an exfo xcite 120 metal halide lamp source coupled 
 to the microscope via a liquid light guide , images were collected 
 with a c910001 ccd camera , and hcimage software was utilized 
 to control the microscope and camera and to process images .
an excitation 
 filter ( 325375 nm ) , dichoric filter ( 460 nm ) , and emission 
 filter ( 470750 nm ) were employed with exposure times of 0.4 
 s for fixed cells and 0.1 s for live cells .
bright field images of 
 cells were also collected to determine the position of cells ( exposure 
 time 0.04 s ) .
the contrast of bright field images was adjusted to 
 a contrast range of 0100 ( normal range 0255 ) using 
 hcimage software .
 a systematic 
 study of the effect of hydrophobicity and charge on the cell viability 
 and cell association of lanthanide metal complexes is presented . 
 the 
 terbium luminescent probes feature a macrocyclic polyaminocarboxylate 
 ligand ( dota ) in which the hydrophobicity of the antenna and that 
 of the carboxyamide pendant arms are independently varied . 
 three sensitizing 
 antennas were investigated in terms of their function in vitro : 2-methoxyisophthalamide 
 ( iam(ome ) ) , 2-hydroxyisophthalamide ( iam ) , and 6-methylphenanthridine 
 ( phen ) . 
 of these complexes , tb - dota - iam exhibited the highest quantum 
 yield , although the higher cell viability and more facile synthesis 
 of the structurally related tb - dota - iam(ome ) platform renders it more 
 attractive . 
 further modification of this latter core structure with 
 carboxyamide arms featuring hydrophobic benzyl , hexyl , and trifluoro 
 groups as well as hydrophilic amino acid based moieties generated 
 a family of complexes that exhibit high cell viability ( ed50 > 300 m ) regardless of the lipophilicity or the overall 
 complex charge . only the hexyl - substituted complex reduced cell viability 
 to 60% in the presence of 100 m complex . 
 additionally , cellular 
 association was investigated by icp - ms and fluorescence microscopy . 
 
 surprisingly , the hydrophobic moieties did not increase cell association 
 in comparison to the hydrophilic amino acid derivatives . 
 it is thus 
 postulated that the hydrophilic nature of the 2-methoxyisophthalamide 
 antenna ( iam(ome ) ) disfavors the cellular association of these complexes . 
 as such , responsive luminescent probes based on this scaffold would 
 be appropriate for the detection of extracellular species . 
Introduction
Results and Discussion
Conclusions
Experimental Section