magnetic resonance 
 imaging ( mri ) is a powerful noninvasive imaging technique , providing 
 high - resolution anatomical images with excellent soft - tissue contrast . 

a large fraction of mri studies utilize a contrast agent , e.g. , more 
 than half the clinical studies performed at our institution make use 
 of a gadolinium(iii)-based contrast agent . these contrast agents are 
 simple , water - soluble , ternary gadolinium(iii ) complexes with gadolinium(iii ) 
 coordinated by an octadentate poly(aminocarboxylato ) ligand and an 
 aqua coligand .
gadolinium(iii ) is advantageous 
 for use in mri contrast agents because of its large electron spin 
 number ( s = /2 ) and long electronic 
 relaxation time , which result in efficient nuclear relaxation of nearby 
 solvent water molecules .
contrast agents are characterized by their 
 relaxivity , defined as the change in the solvent relaxation rate ( 1/t1 or 1/t2 ) normalized 
 to the concentration of the contrast agent ( units of mm s ) .
first - generation clinical contrast agents 
 are monomeric gadolinium(iii ) complexes based on a [ gd(dota)(h2o ) ] or [ gd(dtpa)(h2o ) ] ( dtpa = diethylenetriaminepentaacetic acid ) framework and have relaxivities 
 in the range 35 mm s. second - generation contrast agents such as gadofosveset 
 ( ms-325 , ablavar ) used reversible protein binding to increase the 
 relaxivity . protein binding slows the 
 rotational tumbling rate of the complex , and as this rate approaches 
 the proton larmor frequency , nuclear relaxation is enhanced .
specific protein binding also serves to direct the contrast agent 
 to specific pathologies , e.g. , thrombosis , as well as enhance the 
 relaxivity .
however , the increase in the relaxivity 
 due to protein binding is greatest by far at low magnetic fields ( 1.5 
 t ) and falls off precipitously at higher fields .
clinically , 3 t mri 
 is becoming common , and the major equipment vendors all sell 7 t scanners . 
 for small - animal imaging ,
even higher field strengths are common . 
 at high fields ( 3 t ) ,
the proton larmor frequency is much 
 faster than the tumbling rate of protein - bound complexes but still 
 much slower than the tumbling rate of discrete monomeric complexes . 

we and others have reported multimeric contrast agents with increased high - field 
 relaxivity due to a tumbling rate intermediate between that of protein - bound 
 agents and small molecules .
multiple gd(dotala ) 
 moieties could be incorporated into oligopeptides of defined structure 
 and size , resulting in relaxivities tuned for high - field applications . 

the modular nature of gd(dotala ) also lends itself to the design of 
 serum albumin - targeted contrast agents . here
, we report on the synthesis and properties of a modified 
 derivative of the dotala ligand , dotalap .
we sought to improve the 
 properties of the dotala ligand by replacing one of the acetate moieties 
 by methylenephosphonate .
the increased charge of the phosphonate is 
 expected to increase the solubility of the resultant gadolinium(iii ) 
 complexes .
gadolinium(iii ) complexes with phosphonate - containing ligands 
 often yield higher relaxivities than complexes of the analogous carboxylate 
 ligand due to an effect on water in the second coordination sphere , 
 and this second - sphere effect persists at high fields .
we also expected that inner - sphere water exchange 
 should be faster for the phosphonate - substituted ligand . for gadolinium(iii ) 

complexes undergoing water exchange via a dissociative mechanism , 
 substitution of a carboxylate with the bulkier phosphonate donor group 
 results in faster water exchange as the energy difference between 
 the 8-coordinate transition state and the 9-coordinate ground state 
 becomes smaller . on the other hand
, this steric crowding can also 
 result in an 8-coordinate ground state with an inner - sphere water 
 ligand eliminated , which would result in lower relaxivity . in 
 this study
, we sought to address the following questions : ( i ) how 
 does substitution of one of the acetate donors in dotala for methylenephosphonate 
 impact the hydration number and water exchange kinetics and , in turn , 
 the relaxivity ?
( ii ) how does phosphonate substitution affect kinetic 
 inertness with respect to gadolinium(iii ) dissociation ? ( iii ) what 
 is the effect of amine versus carboxylate functionalization of the 
 propionamine arm of dotalap on the hydration number , water exchange 
 kinetics , relaxivity , and gadolinium(iii ) dissociation kinetics ?
( iv ) 
 are any of these properties altered when a gd(dotalap ) derivative 
 is bound to serum albumin ?
( v ) does phosphonate substitution confer 
 enhanced high - field relaxivity properties and , if so , do these result 
 in an increased in vivo mri signal ? to address these questions , 
 we synthesized the dotalap ligand by altering the previously established 
 dotala synthesis . additionally , we prepared 
 four dotalap derivatives substituted at either the amine or carboxylate 
 of the propionamine arm .
the gadolinium(iii ) complexes were prepared 
 and characterized with respect to variable - field , variable - temperature 
 relaxivity , water exchange kinetics , albumin binding studies , and 
 gadolinium(iii ) dissociation kinetics .
europium(iii ) complexes were 
 also synthesized , and luminescence lifetime measurements were performed 
 to quantify the hydration number of these complexes .
benzyl 2-[[(benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate 
 represents an intermediate common to both dotala and dotalap , so we 
 used the previously established synthetic steps to furnish this first intermediate ( scheme 1 ) . in order to introduce a protected phophonate arm onto the 
 monoderivatized cyclen macrocycle
, tri - tert - butyl 
 phosphite was synthesized according to ref ( 22 ) and appended using a mannich - type reaction . 

the reaction was monitored for disappearance of the monofunctionalized 
 macrocycle using liquid chromatography  mass spectrometry ( lc  ms ) , 
 followed by filtration and aqueous workup of the filtrate . the introduction 
 of these two chelator arms at this stage is key : because of their 
 steric hindrance , it is not possible to incorporate them efficiently 
 at a later stage .
the crude intermediate containing the difunctionalized 
 macrocycle was then redissolved in acetonitrile and subjected to alkylation 
 with tert - butyl bromoacetate , followed by preparative 
 high - performance liquid chromatography ( hplc ) to purify the protected 
 intermediate compound 1 .
orthogonal protection group 
 chemistry provided us with a free and ready - to - couple carboxylate 
 and primary amine on compound 2 after hydrogenation - mediated 
 removal of the carboxybenzyl ( cbz ) and benzyl protecting groups of 1 .
the overall number of steps starting from cbz - nh - ser - oh - obn 
 is 5 , and compound 2 is obtained with an approximate 
 10% overall yield based on the protected serine .
the lower yield is 
 due to the formation of a greater number of side products compared 
 to the dotala synthesis .
one single phosphonate species ( 1,7-functionalized 
 with respect to the aminopropanoate arm ) is obtained and isolated , 
 as indicated to us by h nmr .
compound 2 also 
 represents the last common intermediate for the synthesis of compounds 35b , which all are furnished by activation 
 of the carboxylate with 1-[bis(dimethylamino)methylene]-1h-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate 
 ( hatu ) in the presence of diisopropylethylamine ( dipea ) as a base , 
 followed by overnight amide coupling and subsequent hplc purification . 

the coupled , purified products still carry tert - butyl 
 ester protective groups on the acetate groups and the phosphonate . 
 these esters
are then removed using an acid - catalyzed deprotection 
 reaction with trifluoroacetic acid in dichloromethane , yielding the 
 final ligands without the formation of side products .
complexes were formed under standard conditions 
 by mixing the ligand with an aqueous solution of the lanthanide trichloride 
 salt .
the ph of the solution was adjusted to 7 by the dropwise addition 
 of a 0.1 m solution of naoh .
complexation was followed by hplc in 
 order to confirm that > 95% of ligand was complexed .
we also used 
 the xylenol orange test to further confirm that no free lanthanide 
 was present . according to our hypothesis , 

gd(dotalap ) derivatives should display water exchange rates that exceed 
 the previously reported water exchange rates of gd(dotala ) derivatives . 

we determined the water exchange rates of the gadolinium(iii ) complexes 
 by variable - temperature measurement of the transverse relaxation time 
 of h2o in the presence and absence of each 
 gadolinium(iii ) complex at 11.7 t. in the case of gd(4a ) , gd(4b ) , and gd(5b ) , we observed only 
 minor changes in the transverse relaxation rate when the complexes 
 were added , indicating a lack of an inner - sphere water ligand for 
 these complexes .
for instance , the maximum o relaxivity , r2 , was 2.5 mm s for these three complexes , while the maximum r2 is typically > 15 mm s for q = 1 complexes at this 
 field strength .
on the other hand , gd(3 ) and gd(5a ) had much higher maximum r2 values consistent with an inner - sphere 
 water ligand ( 18 and 16 mm s , respectively ) .
figure 1 shows the reduced 
 transverse relaxation rate r2r as a function 
 of the reciprocal temperature .
the crossover to exchange - limited r2r occurs at a low temperature for both complexes , 
 which indicates the predicted , very fast water exchange kinetics . 

we used a four - parameter model described previously to fit the data and obtained water residency times at 37 
 c of m = 8.1 ns for gd(3 ) and m = 6.4 ns for gd(5a ) ; see table 1 . while these water 
 residency times are considerably shorter than those seen for [ gd(dota)(h2o ) ] and [ gd(dotala)(h2o ) ] , they are similar to the water residency time for 
 [ gd(do3ap)(h2o ) ] , a close structural 
 analogue of dotalap ( table 1 ) . the coordination 
 spheres of gd(3 ) and gd(5a ) differ in that 
 gd(3 ) contains an anionic propionate oxygen donor and 
 gd(5a ) contains a neutral propionamide oxygen donor but 
 their water exchange kinetics are similar .
the propionate to propionamide 
 substitution does not result in different water exchange kinetics 
 in other gadolinium(iii ) complexes .
this is in 
 contrast to the acetate to acetamide substitution , which is well established 
 to reduce the water exchange rate .
interestingly , 
 substitution at the propionamine [ gd(4a ) and gd(4b ) ] gives a ligand with the same donor set as 3 , but these peripheral modifications now result in a gadolinium complex 
 with no inner - sphere water ligand .
similarly , changing the amide substituent 
 from gd(5a ) to gd(5b ) results in the latter 
 complex having no inner - sphere water ligand .
for complexes with exceedingly 
 fast water exchange kinetics , these results indicate that seemingly 
 minor modifications to the periphery of the ligand can alter the hydration 
 number of the complex .
temperature dependence of the o nmr ( 11.7 
 t ) reduced transverse relaxation rates of gd(3 ) ( 6.73 
 mm , left ) and gd(5a ) in pbs ( 4.16 mm , right ) .
the solid 
 line represents a fit to the data to determine the mean water residency 
 time m .
determination of the relaxivity of the gadolinium(iii ) 
 complexes also gives further indication of their hydration state . 

the longitudinal proton relaxivity ( r1 ) was measured in ph 7.4 pbs at 37 c at two larmor frequencies , 
 20 and 60 mhz . for small molecules with q
> 0 , 
 the relaxivity is dominated by the short rotational correlation time 
 at these frequencies and the hydration state .
additionally , phosphonate - containing gadolinium(iii ) chelates have 
 been reported to result in increased second - sphere relaxivity .
if all of the complexes were q = 1 , then we would 
 expect the relaxivity to increase proportionally with the molecular 
 weight , ranking them as follows : gd(3 ) < gd(4a )  gd(5a ) < gd(4b ) 
  gd(5b ) .
in fact , only gd(3 ) and 
 gd(5a ) behave as expected with a relatively high , but 
 for q = 1 phosphonato complexes typical , relaxivity .
on the other hand , gd(4a ) , gd(4b ) , and gd(5b ) show relaxivities 
 below 4 mm s , which is more 
 typical for q = 0 complexes with elevated outer - sphere 
 relaxivity ( table 2 ) .
these relaxivity measurements are consistent with the o nmr measurements , which suggests the lack of an inner - sphere water 
 ligand for gd(4a ) , gd(4b ) , and gd(5b ) .
the hydration number of a gadolinium(iii ) 
 complex can be estimated using a surrogate complex with the luminescent 
 lanthanide ions eu or tb .
this is done by 
 measuring the rate constants for luminescence decay in d2o and h2o solutions .
an empirical expression originally 
 reported by horrocks and sudnick relates these rate constants to q ( table 3 ) .
it is then assumed that gadolinium(iii ) has the same hydration number 
 based on the similar ionic radii of these ions and their propensity 
 for forming isostructural complexes . however , application of this 
 method to phosphonate - containing complexes typically results in an 
 overestimation of q by 0.30.6 . with this consideration in mind , we find that the q values obtained mirror the relaxometric behavior of the corresponding 
 gadolinium complexes .
the hydration numbers calculated are proportional 
 to the obtained relaxivity values in pbs at 20 and 60 mhz , with eu(3 ) and eu(5a ) providing q values 
 above 1 , while the other complexes show lower q values . 

figure s1 ( supporting information ) shows 
 the correlation of the 20 mhz relaxivity values of the gadolinium(iii ) 
 complex and the apparent hydration number of the corresponding europium(iii ) 
 complex .
nd = not determined . because of the decreased solubility 
 of eu(5b ) compared to the other europium complexes described 
 here , we were not able to generate a solution with high enough complex 
 concentration required for this measurement .
we had previously conjugated ibuprofen to dotala 
 and found that a 0.1 mm solution of its gadolinium(iii ) complex [ compound 
 gd(6 ) , figure 2 ] was 70% bound 
 to 4.5% ( w / v ) hsa , and this complex also displayed high relaxivity 
 ( at low fields ) in the presence of hsa . to understand 
 how the phosphonate modification affects protein binding and relaxivity 
 for a slow tumbling system ,
we synthesized the ibuprofen conjugates 
 gd(4b ) and gd(5b ) shown in figure 2 .
structures of ibuprofen conjugates gd(4b ) 
 and gd(5b ) reported here , as well as previously reported 
 compound gd(6 ) .
compared to gd(6 ) , the affinity of gd(4b ) for hsa was increased 3-fold ( kd = 260 and 80 m , respectively ) .
the reason for the higher 
 percentage of bound complex in the case of gd(4b ) is 
 likely the double negative charge , which may provide a stronger interaction 
 with the lysine - rich , positively charged binding pocket of hsa . on 
 the other hand , the zwitterionic gd(5b )
we also measured 
 the relaxivity for these complexes in a 4.5% hsa solution ( table 4 ) .
at 20 mhz , the relaxivity of gd(4b ) increased over 5-fold when hsa was added , whereas for 
 gd(5b ) , the increase was less than 3-fold . such a difference 

could not be accounted for by the difference in the fraction bound 
 to albumin between the two complexes . in the absence of albumin , the 
 relaxivity of gd(4b ) is about 25% higher than that of 
 gd(5b ) , but in the presence of albumin , the relaxivity 
 of gd(4b ) is 120% and 80% higher than that of gd(5b ) at 20 and 60 mhz , respectively ( table 4 ) . in order to better understand this phenomenon , we performed 
 additional r1 measurements at different 
 temperatures and additional field strengths .
we acquired 
 relaxivity data for gd(4b ) and gd(5b ) in 
 pbs and 4.5% ( w / v ) hsa at 20 , 30 , 60 , 100 , 200 , and 400 mhz and at 
 10 , 25 , 37 , and 50 c ( tables s1s4 in the supporting information , si ) .
we also measured 
 the fraction ( fb ) of gd(4b ) and gd(5b ) bound to hsa at each of these temperatures 
 ( table s5 in the si ) . because we know the 
 fraction bound to hsa for each complex and we measure the relaxivity 
 of the unbound complex independently , we can calculate the relaxivity 
 due to the hsa - bound species ( r1bound ) for both gd(4b ) and gd(5b ) ( eq 1).1 the magnitude and field dependences of these hsa - bound relaxivities 
 are shown in figure 3 . the high relaxivity 
 of gd(4b )
could not be well described by a pure second - sphere 
 model , which assumes some adjustable number of long - lived water molecules 
 in close proximity to the metal ion .
instead , we reasoned that the 
 difference in the hsa - bound relaxivities between gd(4b ) and gd(5b ) was due to the presence of an inner - sphere 
 water ligand on some fraction of the gd(4b ) complexes 
 ( eq 2 ) .
we assumed that the difference in the 
 hsa - bound relaxivity between gd(4b ) and gd(5b ) was the contribution from this inner - sphere water ( eq 3 ) .
we then calculated this difference at each temperature 
 and field and simultaneously fit the resultant data set using solomon  bloembergen 
morgan 
 theory ( figure 3 and eqs 28 ) . 
 while the hsa - bound relaxivity of gd(4b ) was higher than 
 expected ,
it was not as high as one would anticipate for a complex 
 with one coordinated water , and so we left the hydration number q as a temperature - dependent adjustable parameter defined 
 by an equilibrium constant .
we found that this entire data set could 
 be reasonably well described ( figure 3 ) by 
 five adjustable parameters : q ( mole 
 fraction of q = 1 species at 310 k ) , h ( enthalpy change for equilibrium between q = 0 and 1 species ) , m , 
 h ( activation energy for 
 water exchange ) , and  [ square of the trace of zero - field - splitting 
 ( zfs ) tensor ; eqs 38].2345678we found that the data were relatively insensitive to the rotational 
 correlation time as long as this correlation time was large .
this 
 is reasonable because the very short water residency time should dominate 
 the correlation time for relaxation .
in addition , the data were insensitive 
 to v , the correlation time for electronic relaxation , 
 and so we fixed this to 20 ps , in line with other studies of similar 
 complexes .
the quality of the fit could 
 likely be improved by the addition of more adjustable parameters , 
 e.g. , lipari  szabo - type treatment , but the conclusions 
 are unlikely to change .
relaxivity of hsa - bound gd(5b ) 
 ( filled circles ) and gd(4b ) ( open circles ) at ph 7.4 , 
 at ( a ) 10 c , ( b ) 25 c , ( c ) 37 c , and ( d ) 50 c . 
 the difference between the solid and open circles is the inner - sphere 
 contribution to the gd(4b ) relaxivity , and the solid 
 line is the fit to the data described in the text .
the analysis shows that there is a 15% fraction 
 of gd(4b ) that exists as a q = 1 complex 
 with a short residence time for the first sphere water molecule .
for 
 the equilibrium in eq 2 , the two reactions are 
 the formation and dissociation of the q = 1 complex . 
 in principle , there can also be water exchange on the q = 1 complex .
because we only observe free water , we can not distinguish 
 between these two reactions ( dissociation to a stable q = 0 complex versus water exchange at the q = 1 
 complex ) .
therefore , we likely are measuring the formation 
 and dissociation of water to the gadolinium complex ; i.e. , m , the residence time in the bound state , corresponds to 1/kd .
the relatively 
 high relaxivity of gd(4b ) in the presence of hsa , especially 
 at high fields , prompted us to further explore the kinetic inertness 
 of this complex with respect to gadolinium(iii ) dissociation .
acetate 
 to methylenephosphonate substitution in lanthanide(iii ) polyaminocarboxylates 
 typically results in a more thermodynamically stable complex ( because 
 of the increased basicity of the phosphonate ) , but at the same time , 
 the complex can become more kinetically labile .
we measured the half - life of gd(4b ) under 
 different forcing conditions and compared it directly to the acetate 
 analogue gd(6 ) measured under the same conditions . in 
 the first experiment , we challenged each complex with 1 equiv of ms-325 
 ligand [ a dtpa derivative of higher thermodynamic stability than gd(dotala ) 
 derivatives ] at ph 3 and 37 c and monitored transchelation by 
 hplc . under these conditions
, we found no measurable decomplexation 
 of gd(4b ) , while gd(6 ) underwent complete 
 transchelation to ms-325 with a half - life of 115 h. this result suggests 
 that gd(4b ) is much more thermodynamically stable than 
 ms-325 . in a second experiment , we challenged each complex with a 
 100-fold excess of citrate at ph 3 and 37 c . with a citrate 
 challenge
, we found the opposite behavior : transchelation proceeded 
 to completion in both cases but over 1 order of magnitude more rapidly 
 in the case of gd(4b ) ( t1/2 = 9.5 h ) than gd(6 ) ( t1/2 = 110 h ) ( figure 4 ) .
dechelation of gd(4b ) ( filled circles ) and gd(6 ) ( open circles ) 
 at ph 3 and 37 c in the presence of 10 mm citrate .
this is in agreement with the 
 previously reported kinetic data obtained for acyclic phosphonate 
 complexes compared to their acetate analogues .
we hypothesize that the phosphonate arm promotes the formation 
 of ternary complexes with citrate molecules , which facilitates decomplexation 
 in the case of gd(4b ) .
although gd(4b ) was 
 more labile than gd(6 ) , we note that acyclic complexes 
 like [ gd(dtpa)(h2o ) ] or its derivatives , 
 such as [ gd(bopta)(h2o ) ] ( table s5 
 in the si ) , are much more labile under 
 these conditions , e.g. , reaching transchelation equilibrium with ms-325 
 ligand in under 10 min . because the high field relaxivity of 
 gd(4b ) was 50% higher than that of the approved contrast 
 agent ms-325 at 4.7 t , we decided to assess its relaxation properties 
 in vivo . in addition , both gd(4b ) and ms-325 showed equivalent 
 affinity to hsa , making this comparison more direct .
a catheter was placed in the tail vein so the mouse could be injected 
 while positioned in the scanner .
we performed 3d t1-weighted imaging before and immediately after injection 
 of each compound ( 0.1 mmol kg ) . immediately after 
 injection , the blood vessel enhancement observed with gd(4b ) was 38  2% higher than that observed with ms-325 ( figure 5 ) consistent with the higher relaxivity of the former .
preinjection , 
 as well as 1 min postinjection , coronal images obtained with ms-325 
 ( second from left ) and gd(4b ) ( right ) .
gd(4b ) shows visibly better contrast in the vena cava , which can be quantified 
 as 38  2% better contrast ( vs muscle ) .
the dose of agent , time 
 point , and image windowing was the same for both studies .
kidney ( ki ) 
 and vena cava ( vc ) are denoted in the far right image .
we have synthesized and 
 evaluated the properties of five different derivatives of a new phosphonate 
 analogue ( dotalap ) of the single amino acid chelate dotala . on the 
 basis of literature examples , we anticipated and observed an increased 
 relaxivity due to the established second - sphere effect of the phosphonate . 

however , for [ gd(dotala)(h2o ) ] and gd(dotala)-propionamide 
 derivatives , water exchange is already very fast ( m = 9 and 17 ns , respectively ) . for the gd(dotalap ) complexes 
 investigated here , we found that further acceleration of water exchange 
 through crowding of the coordination sphere may instead lead to stabilization 
 of the q = 0 , 8-coordinate state .
the five complexes 
 investigated here comprised two that existed primarily as 9-coordinate q = 1 complexes , while the other three appear to be primarily q = 0 , presumably 8-coordinate complexes .
these conclusions 
 are supported by o nmr and h relaxivity data 
 on the gadolinium(iii ) complexes and by luminescence lifetime measurements 
 on the europium(iii ) complexes .
the energy difference between the 
 8- and 9-coordinate states is quite small , and peripheral changes 
 to the macrocyclic ligand outside the inner coordination sphere can 
 alter the coordination number in a nonpredictable manner . this delicate 
 equilibrium between q = 0 and rapidly exchanging q = 1 underlines a challenge in the design of gadolinium(iii ) 
 complexes with extremely fast water exchange rates .
two of the 
 investigated derivatives , gd(4b ) and gd(5b ) , were designed to bind hsa .
the relaxivities of these compounds 
 in the absence of hsa were similar and low , although r1 for gd(4b ) was about 25% higher than r1 for gd(5b ) .
this small increase 
 in the relaxivity could be due to the presence of a small amount of q = 1 species for gd(4b ) .
however , in the presence 
 of hsa , the relaxivity difference between the complexes is magnified , 
 and this increased difference seen when the molecular tumbling rate 
 is decreased is indicative of a small fraction of q = 1 species .
variable - temperature nmrd allowed us to estimate that 
 about 15% of gd(4b ) exists as a q = 
 1 species at 37 c , at least while it is bound to albumin .
the 
 presence of this q = 1 complex whose coordinated 
 water has an extremely short residency time results in remarkably 
 high relaxivities at high field for a complex bound to hsa .
for instance , 
 the relaxivity at 4.7 t was 50% higher than that for the albumin - targeted 
 approved agent ms-325 .
the nmrd results have profound consequences 
 for the development of slow tumbling contrast agents for high - field 
 applications .
the variable - temperature data clearly indicate that 
 the water residency time , m , is the dominant correlation 
 time for modulating the relaxivity in this system .
figure 6 shows data at two temperatures and two fields to 
 illustrate this point . at 0.47 t ( 20 mhz ) ,
m at 2.4 ns is too short for optimal relaxivity , but as the 
 temperature is lowered , m increases and the relaxivity 
 goes up . on the other hand , at higher fields ( > 2 t ) ,
the optimal 
 correlation time is in the 13 ns range . at 37 c ,
a m of 2.4 ns is ideal 
 for relaxivity at 2.4 t ( 100 mhz ) .
however , as the temperature is 
 lowered and m increases , the correlation time becomes 
 too long for optimizing high - field relaxivity .
the very short m provides increased relaxivity at high fields and offers a 
 strategy to increase the relaxivity of slow tumbling entities at high 
 magnetic fields .
we note that the data in figure 6 are for a complex that is only 25% ( at 10 c ) or 15% 
 ( at 37 c ) for q = 1 .
thus , the relaxivity gains 
 for a single q = 1 species are 46-fold higher .
inner - sphere 
 relaxivity of gd(4b ) bound to hsa at two different temperatures 
 ( 10 and 37 c ) and larmor frequencies .
because the relaxivity of q = 0.15 gd(4b ) was still clearly enhanced compared to q = 1 ms-325 at higher fields , we considered its evaluation for high - field 
 in vivo imaging .
for this , we first tested the kinetic inertness of 
 gd(4b ) under forcing conditions .
we found the somewhat 
 decreased kinetic inertness , when compared with the all - acetato analogue 
 gd(6 ) , is a typical reflection of the results previously 
 reported in conjunction of phosphonato compounds .
this encouraged the 
 in vivo evaluation and cross - comparison with the approved hsa binding 
 compound ms-325 .
the subsequent in vivo imaging experiment at 4.7 
 t was found to mirror the in vitro results well because we were able 
 to obtain better angiographic contrast for gd(4b ) compared 
 to ms-325 .
an increased blood signal is clearly found in the increased 
 relaxivity for this compound at intermediate magnetic fields .
we consider 
 this outcome a promising result that encourages us to further investigate 
 m acceleration as a way to enhance the relaxivity 
 at higher fields because an entirely q = 1 complex 
 with such a fast exchange rate would have 5 times the relaxivity observed 
 with gd(4b ) .
however , the somewhat unpredictable nature 
 of the compound behavior in terms of the inner - sphere hydration number 
 is a clear challenge .
in summary , we have 
 successfully synthesized a monophosphonate analogue of the single 
 amino acid chelator dotala , which we term dotalap .
the gd(dotalap ) 
 derivatives show enhanced second - sphere relaxivity compared to gd(dotala ) . 
 in two derivatives , we found increased water exchange kinetics , and 
 this combination of fast water exchange and increased second - sphere 
 relaxivity results in improved relaxometric behavior
. we also found , 
 however , that functionalization of this single amino acid chelate 
 of either the nitrogen or carbon terminus with different r groups 
 can sometimes yield gadolinium complexes with no inner - sphere water 
 ( q = 0 ) or a mixture of q = 0 and 
 1 complexes .
this decrease in the hydration number appears as a side 
 effect of accelerated water exchange and does not correlate with a 
 specific r group or location of functionalization . for one derivative , 
 gd(4b ) , where r was chosen such that the corresponding 
 gadolinium complex had affinity to hsa , we found that the hydration 
 number was 0.15 in the presence of hsa , i.e. , a 6:1 mixture of q = 0 to q = 1 .
the corresponding mean 
 water residency time obtained for the albumin - bound complex was shorter 
 than the rotational correlation time , which , in this case , leads to 
 m becoming the dominant correlation time for relaxation . 

because m is considerably shorter than r , higher inner - sphere relaxivity was achieved for this complex 
 at intermediate and high magnetic fields than what can be obtained 
 with the clinically approved hsa targeting agent ms-325 .
we have demonstrated 
 the effect of higher relaxivity also in vivo with higher blood vessel 
 to muscle contrast for gd(4b ) when compared to ms-325 . 

future endeavors will include the identification of fast exchanging 
 complexes that are exclusively q = 1 in order to 
 maximize the relaxivity gains at high fields .
h and c nmr spectra were recorded on a varian 11.7 t nmr system 
 equipped with a 5-mm broad - band probe .
hplc purification of intermediates 
 was performed on a rainin , dynamax ( phenomenex c18 column : 250 mm 
  21.2 mm , 10 m ) using method a : 0.1% trifluoroacetic 
 acid ( tfa ) in water with a gradient of 595% ( 0.1% tfa in mecn ) 
 over 20 min with a 15 ml min flow rate .
hplc purity 
 analyses ( both uv and ms detection ) were carried out on an agilent 
 1100 system ( phenomenex luna c18(2 ) column : 100 mm  2 mm , 0.8 
 ml min flow rate ) with uv detection at 220 , 254 , 
 and 280 nm and positive - mode electrospray ionization ( esi ) using the following methods .
method b : solvent a = water , solvent 
 b = mecn ; 260% b over 15 min .
method c : solvent a = 10 mm 
 nh4oac , solvent b = 10% 10 mm nh4oac , 90% mecn ; 
 595% b over 15 min .
method d : solvent a = 10 mm nh4oac , solvent b = 10% 10 mm nh4oac , 90% mecn ; 535% 
 b over 15 min .
chemicals were supplied by aldrich 
 chemical co. , inc . , and were used without further purification .
solvents 
 ( hplc grade ) were purchased from various commercial suppliers and 
 used as received . the monoalkylated cyclen precursor to compound 1
tri - tert - butyl phosphite was synthesized 
 according to a procedure reported by manning et al .
amide coupling followed by deprotection 
 of the tert - butyl protective groups was done by activation 
 of the carboxylate with hatu ( 1.2 equiv ) and dipea ( 1.2 equiv ) for 
 5 min in n , n - dimethylformamide ( dmf ) , 
 followed by the addition of amine ( 1 equiv ) dissolved in dmf and stirring 
 at room temperature for 18 h. after confirmation of the presence of 
 the amide - coupling product by lc  ms ( method b ) , the intermediate 
 was isolated by preparative hplc ( method a ) , eluting between 11 and 
 13 min .
the clean fractions containing the desired product were pooled 
 and lyophilized to afford the intermediate as an off - white powder 
 in 1535% yield .
redissolution 
 of intermediates described above in a 1:1 mixture of dichloromethane 
 and tfa , followed by stirring for 18 h , afforded the final ligand 
 with quantitative yield .
an amount of a stock solution 
 containing lncl36h2o ( 0.95 equiv based 
 on the ligand weight ) is added to the ligand solution under monitoring 
 of the ph .
the 
 lightly cloudy solution is filtered and lyophilized to afford the 
 corresponding lanthanide complex as an off - white powder .
ms ; the presence of free 
 lanthanides was excluded using the xylenol orange test .
2-[[(benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate 
 ( 1.475 g , 3.05 mmol , 1 equiv ) and paraformaldehyde ( 0.34 g , 11.45 
 mmol , 3.75 equiv ) were dissolved in tetrahydrofuran and vigorously 
 stirred under a dinitrogen atmosphere for 0.5 h. tri - tert - butyl phosphite ( 1.145 g , 4.5 mmol , 1.5 equiv ) was then added , and 
 the reaction was stirred for 16 h. reaction monitoring by lc  ms 
 showed the product as [ m + h ] ( m / z 690.4 ) .
the reaction mixture was then filtered , concentrated 
 in vacuo , and redissolved in etoac ( 200 ml ) .
the organic layer is 
 washed with 50 ml of saturated na2co3 and 50 
 ml of brine , dried with sodium sulfate , and concentrated to afford 
 the dialkylated intermediate ( 1.73 g , 2.5 mmol , 82% ) , which was used 
 in the subsequent step without further characterization .
k2co3 ( 0.68 g , 
 4.9 mmol , 2 equiv ) was added , and the mixture was stirred vigorously 
 after the addition of tert - butyl bromoacetate ( 0.766 
 g , 0.575 ml , 3.94 mmol , 1.6 equiv ) .
after 16 h , water ( 70 ml ) was 
 added and most of the mecn was removed in vacuo .
the oily residue 
 was taken up in etoac ( 80 ml ) and washed with water and brine ( 100 
 ml each ) .
the organic layer was separated , dried with sodium sulfate , 
 and concentrated to afford the crude product , which was redissolved 
 in mecn ( 5 ml ) and purified using preparative hplc ( method a ) .
fractions containing the product are pooled 
 and lyophilized to afford the product as a white powder ( 0.43 g , 0.46 
 mmol , 20% yield ) .
h nmr ( cdcl3 , 500 mhz , ppm ) : 
 9.9 ( s , br , 2h ) , 7.367.34 ( m , 10h ) , 5.255.16 ( m , 4h ) , 
 4.3 ( m , 1h ) , 3.352.68 ( m , 21h ) , 2.04 ( s , 4h ) , 1.511.43 
 ( m , 36h ) .
c nmr ( cdcl3 , 125.7 mhz , ppm ) : 175.4 , 
 170.6 , 170.3 , 135.0 , 131.1 , 129.4 , 128.6127.9 , 81.4 , 81.0 , 
 67.7 , 57.347.4 , 30.6 , 28.2 , 22.1 .
calcd for c47h76n5o11p4 : m / z 917.5 . found : m / z 918.6 ( [ m + h ] ) .
compound 1 ( 0.43 g , 0.47 mmol ) was 
 dissolved in etoh ( 40 ml ) .
pd / c [ 10% ( w / v ) , 0.215 g ] was added , and 
 the reaction mixture was purged first with dinitrogen and then charged 
 with dihydrogen ( 1 atm ) .
the mixture was then stirred under a dihydrogen 
 atmosphere for 3 h , after which the reaction was found to be complete . 

the pd / c was removed by filtration , and the solvent was removed in 
 vacuo to afford the product as a colorless oil ( 0.32 g , 0.46 mmol , 
 quantitative conversion ) .
h nmr ( cd3od , 500 
 mhz , ppm ) : 5.15 ( s , br , 1h ) , 4.462.48 ( m , 24h ) , 1.631.47 
 ( m , 36h ) .
c nmr ( cd3od , 125.7 mhz , ppm ) : 167.9 , 
 160.5 , 86.6 , 82.6 , 55.248.5 , 30.6 , 29.2 , 26.9 .
calcd for c32h64n5o9p : m / z 693.4 . found : m / z 694.7 ( [ m + h ] ) .
h nmr ( cd3od , 
 500 mhz , ppm ) : 5.15 ( s , br , 2h ) , 4.262.46 ( m , 25h ) .
c nmr ( cd3od , 125.7 mhz , ppm ) : 175.1 , 169.6 , 55.448.9 .
calcd for c16h32n5o9p : m / z 469.2 found : m / z 470.1 ( [ m + h ] ) .
h nmr ( cd3od , 
 500 mhz , ppm ) : 7.387.23 ( m , 5h ) , 5.15 ( s , br , 1h ) , 4.76 ( m , 
 1h ) , 3.982.86 ( m , 24h ) , 1.47 ( t , 3h ) .
c nmr ( cd3od , 125.7 mhz , ppm ) : 159.4 , 140.9 , 128.5126.8 , 116.7 , 
 114.4 , 53.3 , 49.5 , 46.1 , 17.1 .
calcd for c25h40n5o10p : m / z 601.2 . found : m / z 602.4 
 ( [ m + h ] ) .
h nmr ( cd3od , 
 500 mhz , ppm ) : 7.28 ( d , 2h ) , 7.20 ( d , 2h ) , 4.73 ( s , br , 1h ) , 3.912.87 
 ( m , 24h ) , 2.45 ( m , 2h ) , 1.84 ( m , 1h ) , 1.481.29 ( m , 5h ) , 0.89 
 ( d , 6h ) .
c nmr ( cd3od , 125.7 mhz , ppm ) : 172.8 , 
 169.6 , 140.5 , 138.2 , 129.5 , 127.0 , 54.748.2 , 30.0 , 21.3 , 17.1 .
calcd for c28h48n5o9p : m / z 629.3 . found : m / z 630.5 ( [ m + h ] ) .
h nmr ( cd3od , 
 500 mhz , ppm ) : 7.397.26 ( m , 5h ) , 5.02 ( s , br , 1h ) , 4.152.46 
 ( m , 24h ) , 1.641.46 ( m , 3h ) .
c nmr ( cd3od , 125.7 mhz , ppm ) : 169.7 , 166.3 , 160.4 , 142.7 , 128.5125.8 , 
 119.6 , 117.3 , 114.9 , 112.7 , 53.349.5 , 27.2 , 20.85 .
calcd for c24h41n6o8p : m / z 573.3 . found : m / z 574.6 ( [ m + h ] ) .
h nmr ( cd3od , 
 500 mhz , ppm ) : 7.24 ( d , 2h ) , 7.10 ( d , 2h ) , 4.212.44 ( m , 30h ) , 
 1.941.29 ( m , 6h ) , 0.89 ( d , 6h ) .
c nmr ( cd3od , 125.7 mhz , ppm ) : 175.4 , 169.8 , 167.4 , 140.2 , 138.8 , 128.9 , 
 126.7 , 54.648.1 , 38.2 , 30.1 , 21.3 , 17.1 .
p nmr 
 ( cd3od , 202.4 mhz , ppm ) : 6.1 . lc 
calcd for 
 c31h54n7o9p : m / z 699.3 . found : m / z 700.6 ( [ m + h ] ) .
calcd for c16h28gdn5o9p [ gd(3 ) ] : m / z 623.1 . 
 found : m / z 625.1 ( [ m + 2h ] ) .
. 
 found : m / z 620.1 ( [ m + 2h ] ) .
. 
 found : m / z 757.2 ( [ m + 2h ] ) .
calcd for c24h36eun5o9p [ eu(4a ) ] : m / z 750.1 . 
 found : m / z 752.1 ( [ m + 2h ] ) .
calcd for c29h44gdn5o10p [ gd(4b ) ] : m / z 811.2 . 
 found : m / z 813.4 ( [ m + 2h ] ) .
calcd for c29h44eun5o10p [ eu(4b ) ] : m / z 806.2 . 
 found : m / z 808.4 ( [ m + 2h ] ) .
calcd for c29h44tbn5o10p [ tb(4b ) ] : 812.2 . found : m / z 814.4 ( [ m + 2h ] ) .
calcd for c24h38gdn6o8p [ gd(5a ) ] : m / z 727.2 . found : m / z 728.2 ( [ m + h ] ) .
calcd for c24h38eun6o8p [ eu(5a ) ] : m / z 722.2 . found : m / z 723.3 ( [ m + h ] ) .
calcd for c31h51gdn7o9p [ gd(5b ) ] : m / z 852.3 . found : m / z 853.5 ( [ m + h ] ) .
calcd for c31h51eun7o9p [ eu(5b ) ] : m / z 849.3 . found : m / z 850.4 ( [ m + h ] ) .
luminescence 
 lifetime measurements of europium complexes in h2o and 
 d2o were performed on a hitachi f-4500 fluorescence spectrophotometer . 

for the measurements 
 in d2o , the complexes were first dissolved in d2o ( 99.98% d ) , lyophilized , and dissolved in d2o again 
 to reduce the amount of residual hdo .
measurements were taken with 
 the following settings : excitation at 396 nm ( eu ) emission at 616 
 nm ( eu ) 30 replicates , 0.04 ms temporal resolution ( 020 ms ) , 
 pmt voltage = 400 v. lifetimes were obtained from monoexponential 
 fits of the data using igor pro software ( version 6.0 , wavemetrics , 
 lake oswego , or ) .
o nmr 
 measurements of solutions were performed at 11.7 t on 350 l 
 samples contained in 5 mm standard nmr tubes on a varian spectrometer . 

the temperature was regulated by an air flow controlled by a varian 
 vt unit .
the h2o transverse relaxation times 
 of a > 4 mm solution of all gadolinium complexes ( ph 7.4 , 10 mm 
 pbs buffer ) were measured using a cpmg sequence . the exact concentration 
 of the sample was determined by inductively coupled plasma mass spectrometry 
 ( icp - ms ) . reduced relaxation rates , 1/t2r , were calculated from the difference of 1/t2 between the sample and water blank and then divided by the 
 mole fraction of coordinated water , assuming q = 
 1 .
the temperature dependence of 1/t2r was fit to a four - parameter model as previously described . the gd 
o hyperfine coupling constant , a/ , was assumed to be 3.8  10 rad 
 s. ultrafiltration measurements 
 of protein binding solutions of a 0.1 mm gadolinium compound and 4.5% 
 hsa ( 200 l total ) were added to a 5000 mw ultrafiltration unit 
 ( regenerated cellulose membrane , millipore , bedford , ma ) .
the samples 
 were incubated at a set temperature for 30 min and subsequently centrifuged 
 to filter off the nonprotein - bound components .
the concentration 
 of bound complex in the protein samples was determined by subtracting 
 the gadolinium concentration ( unbound complex ) found in the filtrate 
 from the gadolinium concentration of the initial solution .
stock solutions of ms-325-l ( the ligand of the 
 ms-325 complex ) , gd(4b ) , 
 and gd(6 ) were prepared . for experiment 1 , ms-325-l was 
 added to solutions of the gadolinium complexes in ph 3 water and incubated 
 at 37 c .
a 10 l aliquot was removed for hplc 
 analysis and analyzed at time points 1 , 20 , 50 , 72 , 80 , 96 , 120,168 , 
 192 , 216 , 240 , 288 , 312 , 336 , 360 , and 480 h , while the remainder 
 of the solution was incubated at 37 c . for experiment 2 , the 
 gadolinium complexes
were added to a 10 mm ph 3 citrate buffer , affording 
 a 0.1 mm complex concentration , and incubated at 37 c .
a 10 
 l aliquot was removed and analyzed at 0 , 0.75 , 1.5 , 2 , 3 , 4 , 
 5 , 6 , 24 , 72 , 96 , 120 , 168 , 192 , 216 , 240 , 288 312 , 336 , and 408 h. nu / nu mice 
 were anesthetized with isoflurane ( 12% ) and placed in a specially 
 designed cradle with the body temperature maintained at 37 c . 

the tail vein was cannulated for intravenous delivery of the contrast 
 agent while the animal was positioned in the scanner .
imaging was 
 performed at 4.7 t , using a custom - built volume coil to acquire coronal t1-weighted images with a field of view that 
 covered the major organs of the thorax and abdomen ( heart , stomach , 
 liver , intestines , and kidney ) .
t1-weighted images 
 were acquired using a 3d fast low angle shot ( flash ) sequence : tr / te / flip 
 angle = 15.3 ms/1.54 ms/40 , fov = 4.8  2.4  2.4 
 cm , matrix = 192  96  96 , 1 average .
 here , 
 we describe the synthesis of the single amino acid chelator dotalap 
 and four of its derivatives . 
 the corresponding gadolinium(iii ) complexes 
 were investigated for their kinetic inertness , relaxometric properties 
 at a range of fields and temperatures , water exchange rate , and interaction 
 with human serum albumin ( hsa ) . 
 derivatives with one inner - sphere 
 water ( q = 1 ) were determined to have a mean water 
 residency time between 8 and 6 ns in phoshate - buffered saline at 37 
 c . 
 the corresponding europium complexes were also formed and 
 used to obtain information on the hydration number of the corresponding 
 coordination complexes . 
 two complexes capable of binding hsa were 
 also synthesized , of which one , gd(5b ) , contains no inner - sphere 
 water , while the other derivative , gd(4b ) , is a mixture 
 of ca . 
 15% q = 1 and 85% q = 0 . in 
 the presence of hsa 
 , the latter displayed a very short mean water 
 residency time ( m310 = 2.4 ns ) and enhanced 
 relaxivity at intermediate and high fields . 
 the kinetic inertness 
 of gd(4b ) with respect to complex dissociation was decreased 
 compared to its dotala analogue but still 100-fold more inert than 
 [ gd(bopta)(h2o)]2. magnetic resonance 
 imaging in mice showed that gd(4b ) was able to provide 
 38% better vessel to muscle contrast compared to the clinically used 
 hsa binding agent ms-325 . 
Introduction
Results
Discussion
Conclusions
Experimental
Procedures