magnesium is the most 
 abundant divalent cation in the cell , with numerous roles that are 
 essential for cellular function . the estimated total concentration 
 of mg in mammalian cells varies between 14 and 20 mm , 
 the majority of it bound to atp and a lesser extent to proteins , phospholipids , 
 and various phosphometabolites . the concentration 
 of free cytosolic magnesium , [ mg]i , not associated with macromolecules , is 
 estimated to be in the sub - millimolar range . abnormal 
 levels of magnesium are associated with a number of age - related and 
 neuronal diseases ranging from hypertension to alzheimer s 
 disease . the mechanisms by which mg concentration is 
 regulated at the cellular level and the implications in human health , 
 however , remain poorly understood due to the scarcity of efficient 
 chemical tools for the study of this ion . most detection methods employed 
 thus far do not offer the combination of selectivity and spatiotemporal 
 resolution required to study magnesium in the complex chemical environment 
 offered by the cell . the use of fluorescent indicators has emerged 
 as a powerful approach to detect free magnesium in biological samples . most fluorescent indicators available commercially at this time 
 are based on o - aminophenol - n , n , o - triacetic acid ( aptra ) chelators ( figure 1 ) , which afford a rapid 
 response and dissociation constant in the low millimolar range , optimal 
 for measurements of free mg . nevertheless , the aptra binding group suffers from low selectivity 
 against ca and zn ( e.g. , kd , ca = 20 m and kd , zn = 11 nm for mag - fura-2 ) , which may give rise to artifacts in the detection 
 of mg in the cellular milieu . alternative chelators based 
 on -keto - acids and related -dicarbonyl bidentate binding 
 motifs have been incorporated recently into fluorescent probes for 
 the detection of mg . significantly , these chelators form magnesium complexes with typical 
 dissociation constants in the low millimolar range and exhibit dissociation 
 constants the same or 1 order of magnitude higher when complexed to 
 ca . as a result , they are practically insensitive to 
 biological concentrations of ca and show great promise 
 for the development of fluorescent probes with an improved metal selectivity 
 profile for biological applications . unlike the pentadentate aptra - based 
 sensors , -dicarbonyl species may occupy only two sites in the 
 coordination sphere of mg , leaving room for coordination 
 of further charged or neutral ligands and formation of ternary species . the formation of these species and its effect in the performance of 
 fluorescent sensors have been long overlooked and warrant further 
 investigation . in this paper , we provide evidence for the formation 
 of ternary complexes from interaction of bidentate fluorescent indicators 
 with mgatp , the most abundant form of bound mg in the 
 cell , and investigate its effect on the optical properties of the 
 indicators under conditions relevant to fluorescence imaging of biological 
 magnesium . magnesium - binding chelators o - aminophenol - n , n , o - triacetic acid ( aptra , 1 ) and 4-oxo-4h - quinolizine-3-carboxylic acid ( 2 ) employed in the design of fluorescent probes such as furaptra ( mag - fura-2 , 3 ) and kmg-301 ( 4 ) for imaging of mg . compounds kmg-301,2 , and 2a(15 ) were prepared according to reported 
 procedures , and their purity ( > 98% ) was verified by reverse phase 
 c-18 hplc . high - purity piperazine - n , n-bis(2-ethanesulfonic acid ) ( pipes ) , 37% hcl , 45% koh , and 
 99.999% anhydrous mgcl2 were purchased from aldrich . deuterium oxide ( d , 99.9% ) , sodium 
 deuteroxide ( d , 99.5% , 40% w / w in d2o ) , and deuterium chloride 
 ( d , 99.5% , 35% w / w in d2o ) were obtained from cambridge 
 isotope laboratories . all aqueous solutions for fluorescence experiments aqueous buffers were treated with chelex resin 
 ( bio - rad ) according to the manufacturer s protocol to remove 
 adventitious metal ions . fluorescence spectra were acquired on a quantamaster 40 photon technology 
 international spectrofluorometer equipped with xenon lamp source , 
 emission and excitation monochromators , excitation correction unit , 
 and pmt detector . all measurements were 
 conducted at 25.0  0.1 c controlled with quantum northwest 
 cuvette holders . fluorescence measurements at ph 7.0 were conducted 
 in aqueous buffer containing 50 mm pipes and 100 mm kcl . stock solutions 
 of compounds 2 and 2a , 1.0 mm , were prepared 
 in pipes buffer at ph 7.0 . all stock solutions for fluorescence measurements 
 were stored at 20 c in 20200 l aliquots 
 and thawed immediately before each experiment . excitation for compound 2 was provided at 385 nm and for compound 2a at 
 395 nm . excitation for kmg-301 was provided at 540 nm , and corrected 
 emission spectra were integrated in the 545750 nm range . apparent kd values for a 1:1 binding model ( probe / mg or probe / mgatp ) were obtained from nonlinear fit of integrated 
 florescence intensity or fluorescence ratio plots ( see supporting information for details ) . nmr spectroscopic data were acquired on a bruker 
 avance-400 nmr or bruker avance iii-600 nmr spectrometer equipped 
 with a 5 mm sample diameter inverse quadruple resonance probe . h nmr shifts were calibrated based on the protio impurity 
 of the solvent peak . changes of the chemical shift of the indicator s 
 protons as a function of total magnesium concentration were fitted 
 using a model including two sequential dissociation equilibria as 
 described in the supporting information . diffusion measurements were conducted on a bruker avance iii-600 
 nmr using the ledbpgp2s pulse program , with a longitudinal eddy current delay of 5 ms , diffusion time  
 = 100 ms , and effective gradient duration  = 3600 s . the gradient power of 
 the probe was calibrated from the diffusion coefficient of water . diffusion experiments were run with 16 , 32 , or 64 scans ; more scans 
 were used for samples with broad peaks ( concentrations of mgatp above 
 10 mm ) . linear gradient strength ramps from 8% to 50% were employed 
 for samples of sensors 2 and 2a with and 
 without mg . the intensities of non - overlapping 
 peaks were analyzed using the relaxation module in the bruker topspin 
 version 3.2 software ( supporting information ) . diffusion coefficients are reported as averages of the values 
 obtained from analysis of the different peaks corresponding to one 
 given compound . samples for nmr spectroscopy were prepared by 
 mixing appropriate amounts of na2atp and mgcl2 solutions in d2o with a sensor stock solution containing 
 tris buffer in d2o . the mixture 
 was adjusted to pd 7.40 with small amounts 
 of 37% dcl in d2o , and diluted to a final concentration 
 of 2.5 mm compound 2 in 10 mm tris or 5.0 mm compound 2a in 25 mm tris . na2atp was added from a 100 mm 
 stock solution in d2o , adjusted to pd 7.40 with naod . magnesium 
 was added from 100 mm or 1.00 m stock solutions prepared by dissolving 
 high purity anhydrous mgcl2 beads in d2o . crystals of 2mg(oh2)2 were obtained as follows : a 25 mm solution of 
 chelator 2 in dmso ( 50 l ) at 60 c was treated 
 with aqueous pipes buffer at ph 7.0 containing 50 mm mgcl2 ( 250 l ) , layered atop the first solution . the warm mixture 
 was allowed to cool slowly and diffuse over a period of 16 h protected 
 from light . single crystals suitable for x - ray analysis were coated 
 with paratone - n oil and mounted on fiber loops for analysis . diffraction 
 data were collected at 100(2 ) k on a bruker apexii ccd x - ray diffractometer 
 performing  scans using graphite - monochromated mo k 
 radiation . structure was solved by direct methods and standard difference 
 map techniques and was refined by full - matrix least - squares procedures 
 on f with shelxtl ( version 6.12 ) . crystallographic data collection and refinement parameters are summarized 
 in table s1 , supporting information . levy and co - workers developed a series 
 of 4-oxo-4h - quinolizine-3-carboxylic acid derivatives ( figure 1 ) that bind mg with dissociation constants 
 in the low millimolar range and show negligible response to ca . based on these chelators , 
 oka s group developed fluorescein and rosamine fluorescent 
 compounds kmg-104 and kmg-301 that perform as turn - on sensors with excitation 
 and emission in the visible range . in our study , we first employed 
 the most recently reported rosamine probe , kmg-301 , as a representative 
 example of a bidentate dicarbonyl - based chelator that would allow 
 us to investigate the formation of ternary complexes in conditions 
 akin to those employed in fluorescence bioimaging experiments . treatment of kmg-301 with increasing mg concentrations 
 in aqueous buffer at ph 7.0 leads to an increase in the fluorescence 
 emission ( figures 2 and s1 ) for an overall 24-fold turn on . nonlinear fit of the integrated fluorescence as a function 
 of magnesium concentration , obtained under our experimental conditions , 
 results in an apparent dissociation constant kd = 3.79  0.04 mm for the 1:1 complex of the chelator 
 with mg at 25 c . significantly , a titration employing 
 increasing concentrations of mg as a 1:1 complex with 
 atp ( figure 2b ) , leads to a 58% larger 
 increase in emission intensity compared to that observed in the titration 
 with mgcl2 over the same concentration range . the intensity 
 increase is accompanied by a slight red shift in the fluorescence 
 maximum . a control experiment conducted with the addition of atp , 
 in the absence of mg ( figure s1 , 
 supporting information ) , shows a similar red shift but only 
 a slight increase in the fluorescence intensity , thus supporting the 
 notion that the fluorescence turn on in each case involves magnesium 
 coordination . nonlinear fit of the binding isotherm for the titration 
 with mgatp yields an apparent dissociation constant kd = 14.2  0.3 mm at 25 c for a 1:1 binding 
 model . a similar value is obtained for a titration conducted in the 
 presence of a 1:2 ratio mg to atp ( data not shown ) . with 
 an apparent dissociation constant in the mid - micromolar range for 
 mgatp under physiological conditions , the quinolizine - based chelator is not expected to displace the atp 
 from the coordination sphere of magnesium . accordingly , the formation 
 of a ternary atpmgprobe species is the most 
 likely origin of the observed changes in fluorescence . fluorescence emission 
 of 1.0 m solutions of kmg-301 in response to increasing concentrations 
 of mg ( a ) or mgatp complex ( b ) . titrations were conducted 
 in 50 mm aqueous pipes buffer , 100 mm kcl , ph 7.0 , 25 c . insets correspond to the 
 integrated emission ( squares ) and nonlinear fit ( red line ) as a function 
 of [ mg ] or [ mgatp ] using a 1:1 binding model . we also investigated the interaction between mgatp 
 and the simpler 4-oxo-4h - quinolizine-3-carboxylate chelator 2 . unlike kmg-301 , compound 2 is fluorescent 
 in both mg - free and -bound states , which facilitates the study of the binding process and 
 allows for ratiometric determination of mg . treatment 
 of 2 with increasing mg concentrations in 
 aqueous buffer at ph 7.0 ( figure 3a ) led to 
 a blue shift in the fluorescence emission maximum , from 435 to 408 
 nm . the ratio of emission at these two wavelengths , f408/f435 , was employed to 
 calculate an apparent dissociation constant , which resulted in a value 
 of kd = 1.55  0.05 mm for the 1:1 
 complex of the chelator with mg under our experimental 
 conditions . treatment of 2 with increasing concentrations 
 of mgatp over the same range ( figure 3b ) also 
 led to a blue shift in the fluorescence emission maximum , from 435 
 to 412 nm . employing the ratio of emission at these two wavelengths , f412/f435 , an apparent 
 constant kd = 1.81  0.09 mm for 
 the dissociation of the fluorescent probe from a 1:1:1 atpmgprobe ternary complex was obtained . a control experiment 
 conducted with the addition of atp in the absence of mg only led to a slight decrease in the fluorescence of the sensor 
 with no significant blue shift ( data not shown ) , presumably due to 
 mild quenching effect of the purine base . therefore , the wavelength 
 shift observed in the presence of mgatp can be attributed to coordination 
 of the magnesium center to the bidentate chelator . fluorescence emission 
 of 1.0 m solutions of 4-oxo-4h - quinolizine-3-carboxylate , compound 2 , in response to increasing concentrations of mg ( a ) or mgatp complex ( b ) . all titrations were conducted in 50 mm 
 pipes buffer , 100 mm kcl , ph 7.0 , 25 c . insets 
 correspond to the ratio of fluorescence intensity at two wavelengths , f408/f435 ( f412/f435 for mgatp 
 titration , squares ) and nonlinear fit ( red line ) as a function of 
 added magnesium or mgatp using a 1:1 binding model . the apparent affinities of 2 for mg and mgatp are surprisingly similar and , in comparison to 
 those of the rosamine probe kmg-301 , indicate that the xanthene moiety 
 has an electronic effect on the apparent binding properties of the 
 chelator . with either sensor , however , the mgatp - induced optical changes 
 are expected to contribute significantly to the overall fluorescence 
 response obtained in cell or tissue imaging experiments . this contribution 
 can not be neglected in the interpretation of imaging studies of mg , as it does not allow for a clear distinction between the 
 detection of free mg from its nucleotide - bound forms 
 at typical physiological levels , based on steady state fluorescence 
 intensity or ratio measurements . in short , the -dicarbonyl - based 
 sensors tested herein do not report on levels of 
 free mg , as presumed in previous studies , but their optical 
 response may reflect changes in concentration of various forms of 
 mg ( e.g. free + nucleotide bound ) . this limitation may affect the performance of other low - denticity 
 chelators and must be considered carefully . however , the role of mg in mediating the interaction 
 is not clear in every case . the use of other metals such as zn and 
 cu has allowed for the isolation and structural characterization of 
 various ternary complexes in which the phosphate backbone of the nucleotide 
 and a second chelator share the coordination sphere of the metal center ; similar magnesium - containing ternary complexes with fluorescent 
 chelators are not unlikely . to gain further insight into the interaction 
 of the fluorescent probe with mgatp , we investigated the complex formation 
 by nmr spectroscopy ( figure 4 ) . for this purpose , 
 we employed a derivative of 2 , 1-(2,2-dicarboxyethyl)-4-oxo-4h - quinolizine-3-carboxylic 
 acid 2a , which displays similar fluorescence response 
 to both mg and mgatp ( figure s2 , 
 supporting information ) but offers increased solubility in 
 water compared to 2 . treatment 
 of a 5.0 mm solution of compound 2a in aqueous buffer 
 with increasing concentrations of mg reveals a gradual 
 shift and broadening of the aromatic signals , which sharpen at high 
 concentrations of mg ( figure 4a ) . analysis of the chemical shift as a function of total magnesium 
 concentration suggests at least two metal - binding steps , and a model 
 considering the formation of both 2:1 and 1:1 chelator / mg species provides the best fit ( figure 5 and supporting information ) . from this analysis , two apparent dissociation constants are obtained , one of kd1 = 12  1 mm for the chelator dissociation from 
 the 2:1 complex , and the second kd2 = 
 0.9  0.3 mm for the dissociation of the 1:1 species . h nmr spectra ( 600 mhz , d2o , 25 
 mm tris buffer , pd = 7.40 , 25 c ) of a 5.0 mm solution of chelator 2a after treatment with increasing concentrations of mgcl2 ( a ) or mgatp ( b ) . ( a ) changes in chemical shift of aromatic protons of chelator 2a ( 5.0 mm in d2o , 25 mm tris buffer , pd = 7.40 , 
 25 c ) as a function of total concentration of magnesium , obtained 
 from the h nmr spectroscopic titrations . ( b ) nonlinear 
 fit of the changes in chemical shift for ha in response 
 to increasing concentrations of magnesium , employing a model that 
 includes both the formation of complexes with 2:1 and 1:1 chelator - to - mg stoichiometry . the limited water solubility of 
 the simple chelator 2 at millimolar concentrations facilitated 
 the isolation and structural characterization of the complex 2mg(oh2)2 , as shown in 
 figure 6 . the solid - state structure exposes 
 a -[o2 ] coordination mode for two of 
 the quinolizine - derived fluorophores forming almost perpendicular 
 six - membered chelates around a pseudo - octahedral magnesium center . the concentration of this 2:1 chelator / mg species in 
 solution is negligible at the low chelator concentration and high 
 [ mg]/[chelator ] ratio involved in typical fluorescence 
 sensing experiments . its formation at high concentrations , however , 
 illustrates the ability of small low - denticity chelators to share 
 the coordination sphere with other multidentate ligands , such as another 
 molecule of 2 or potentially a biologically relevant 
 ligand such as atp . a second titration experiment 
 was conducted with a 5.0 mm solution of compound 2a in 
 aqueous buffer treated with increasing concentrations of mgatp and 
 followed by h nmr and p nmr spectroscopy 
 ( figures 4b and s10 and 
 s11 , supporting information ) . with the exception of the signal 
 corresponding to proton he , all the aromatic signals of 
 compound 2a shift upfield in the h nmr spectrum 
 indicating the formation of a complex that is electronically different 
 from that formed upon treatment of the chelator with mgcl2 alone . furthermore , these signals remain broad even at close - to - saturating 
 concentrations of mgatp , likely due to the effect of conformational 
 changes of the atp ligand in a ternary complex . further evidence 
 for the formation of ternary species upon interaction of the bidentate 
 fluorescent probes with mgatp was obtained through nmr diffusion studies . specifically , the translational self - diffusion coefficient of quinolizine - based 
 chelator 2a was determined via pulsed gradient spin  echo 
 ( pgse ) diffusion h nmr spectroscopy experiments in the absence and presence of near - saturating concentrations of 
 mg or mgatp ( table 1 ) . upon treatment 
 with mgcl2 , the diffusion coefficient of the chelator decreases , 
 thus reflecting an increase in the overall size of the molecule as 
 a result of metal complexation . the change in diffusion coefficient 
 is , however , more pronounced upon treatment with mgatp . at high concentration 
 of the nucleotide - bound magnesium source , the diffusion coefficient 
 of the chelator drops to a value lower than that obtained upon complexation 
 with magnesium and even lower than that of mgatp alone , thus consistent 
 with the formation of a larger species . additionally , a marked decrease 
 in diffusion coefficient was observed for chelator 2 in 
 the presence of mgatp ( table s2 , supporting information ) ; the low solubility of the compound in the presence of high concentration 
 of magnesium , however , prevented further studies . samples containing 5.0 mm of chelator 2a in d2o , with 
 25 mm tris buffer adjusted to pd = 7.40 , 25 c . sample containing 10 mm mgatp in tris 
 buffer adjusted to pd = 7.40 , 25 c . at 55 mm mgatp , the coefficient is , within error , the 
 same as the coefficient at 40 mm . much of our understanding of metal homeostasis and its implications 
 in health and disease stems from the use of fluorescent metal indicators 
 that enable optical tracking of ion accumulation and fluxes in the 
 complex matrix provided by the cell . sensor development endeavors 
 typically devote substantial effort to the fine - tuning of dissociation 
 constants as to provide maximum dynamic range , avoid competitive binding 
 of other metals , and prevent displacement of typical biological chelators , 
 thereby enabling the selective detection of the free or ionized forms of the cation under physiological 
 conditions . our results highlight the need for a deeper understanding 
 of the coordination properties of the chelator , and the consideration 
 of binding schemes beyond the simple formation of binary species , 
 to provide a more complete depiction of the  selectivity  
 of fluorescent metal chelators . we hereby provide spectroscopic 
 evidence for the formation of ternary complexes from -keto - acid 
 fluorescent chelators , namely , 4-oxo-4h - quinolizine-3-carboxylic acid 
 and kmg-301 , with mgatp , the most abundant bound form of biological 
 magnesium . the formation of such ternary species elicits comparable 
 or greater optical changes than those attributed to the formation 
 of binary complexes alone , with equilibrium constants that are similar 
 for the binding of the chelator to either free magnesium or its nucleotide 
 complex . as a result , these low - denticity chelators do not afford 
 a clear distinction between free ( ionized ) mg and mgatp 
 from simple ratio- or intensity - based steady - state fluorescence measurements . instead , they may co - report various magnesium - containing species , 
 thus posing challenges in the interpretation of results obtained from 
 fluorescence imaging of magnesium in nucleotide - rich biological samples . being a consequence of the mismatch between the typical coordination 
 number of mg and the denticity of the sensor , the formation 
 of ternary complexes is likely to influence the performance of other 
 low - denticity chelators and must be considered carefully .
fluorescent indicators based on -keto - acid 
 bidentate coordination motifs display superior metal selectivity profiles 
 compared to current o - aminophenol - n , n , o - triacetic acid ( aptra ) based 
 chelators for the study of biological magnesium . 
 these low denticity 
 chelators , however , may allow for the formation of ternary complexes 
 with mg2 + and common ligands present in the cellular milieu . 
 in this work , absorption , fluorescence , and nmr spectroscopy 
 were 
 employed to study the interaction of turn - on and ratiometric fluorescent 
 indicators based on 4-oxo-4h - quinolizine-3-carboxylic acid with mg2 + and atp , the most abundant chelator of biological magnesium , 
 thus revealing the formation of ternary complexes under conditions 
 relevant to fluorescence imaging . 
 the formation of ternary species 
 elicits comparable or greater optical changes than those attributed 
 to the formation of binary complexes alone . 
 dissociation of the fluorescent 
 indicators from both ternary and binary species have apparent equilibrium 
 constants in the low millimolar range at ph 7 and 25 c . 
 these 
 results suggest that these bidentate sensors are incapable of distinguishing 
 between free mg2 + and mgatp based on ratio or intensity - based 
 steady - state fluorescence measurements , thus posing challenges in 
 the interpretation of results from fluorescence imaging of magnesium 
 in nucleotide - rich biological samples .