Serum albumin targeted, pH-Dependent Magnetic Resonance Relaxation Agents

Article abstract of DOI:10.1002/chem.201103344

The objective of this work was the synthesis of serum albumin targeted, Gd^III-based magnetic resonance imaging (MRI) contrast agents exhibiting a strong pH-dependent relaxivity. Two new complexes (Gd-glu and Gd-bbu) were synthesized based on the DO3A macrocycle modified with three carboxyalkyl substituents α to the three ring nitrogen atoms, and a biphenylsulfonamide arm. The sulfonamide nitrogen coordinates the Gd in a pH-dependent fashion, resulting in a decrease in the hydration state, q, as pH is increased and a resultant decrease in relaxivity (r₁). In the absence of human serum albumin (HSA), r₁ increases from 2.0 to 6.0 mM^-1 s^-1 for Gd-glu and from 2.4 to 9.0 mM^-1 s^-1 for Gd-bbu from pH 5 to 8.5 at 37 °C, 0.47 T, respectively. These complexes (0.2 mM) are bound (>98.9 %) to HSA (0.69 mM) over the pH range 5-8.5. Binding to albumin increases the rotational correlation time and results in higher relaxivity. The r₁ increased 120 % (pH 5) and 550 % (pH 8.5) for Gd-glu and 42 % (pH 5) and 260 % (pH 8.5) for Gd-bbu. The increases in r₁ at pH 5 were unexpectedly low for a putative slow tumbling q=2 complex. The Gd-bbu system was investigated further. At pH 5, it binds in a stepwise fashion to HSA with dissociation constants K _d1=0.65, K_d2=18, K_d3=1360 μM. The relaxivity at each binding site was constant. Luminescence lifetime titration experiments with the Eu^III analogue revealed that the inner-sphere water ligands are displaced when the complex binds to HSA resulting in lower than expected r₁ at pH 5. Variable pH and temperature nuclear magnetic relaxation dispersion (NMRD) studies showed that the increased r₁ of the albumin-bound q=0 complexes is due to the presence of a nearby water molecule with a long residency time (1-2 ns). The distance between this water molecule and the Gd ion changes with pH resulting in albumin-bound pH-dependent relaxivity. Copyright

Full text of DOI:10.1002/chem.201103344

FULL PAPER
DOI: 10.1002/chem.201103344
Serum Albumin Targeted, pH-Dependent Magnetic Resonance Relaxation
Agents
Loꢀck Moriggi,[a] Mohammad A. Yaseen,[a] Lothar Helm,[b] and Peter Caravan*[a]
Abstract: The objective of this work
was the synthesis of serum albumin tar-
geted, GdIII-based magnetic resonance
imaging (MRI) contrast agents exhibit-
ing a strong pH-dependent relaxivity.
Two new complexes (Gd-glu and Gd-
bbu) were synthesized based on the
DO3A macrocycle modified with three
carboxyalkyl substituents a to the three
ring nitrogen atoms, and a biphenylsul-
fonamide arm. The sulfonamide nitro-
gen coordinates the Gd in a pH-depen-
dent fashion, resulting in a decrease in
the hydration state, q, as pH is in-
creased and a resultant decrease in re-
laxivity (r1). In the absence of human
serum albumin (HSA), r1 increases
from 2.0 to 6.0 mmꢀ1 sꢀ1 for Gd-glu and
from 2.4 to 9.0 mmꢀ1 sꢀ1 for Gd-bbu
from pH 5 to 8.5 at 378C, 0.47 T, re-
spectively. These complexes (0.2 mm)
are bound (>98.9%) to HSA
(0.69 mm) over the pH range 5–8.5.
Binding to albumin increases the rota-
tional correlation time and results in
higher relaxivity. The r1 increased
120% (pH 5) and 550% (pH 8.5) for
Gd-glu and 42% (pH 5) and 260%
(pH 8.5) for Gd-bbu. The increases in
r1 at pH 5 were unexpectedly low for a
putative slow tumbling q=2 complex.
The Gd-bbu system was investigated
further. At pH 5, it binds in a stepwise
fashion to HSA with dissociation con-
stants Kd1 =0.65, Kd2 =18, Kd3 =
1360 mm. The relaxivity at each binding
site was constant. Luminescence life-
time titration experiments with the
EuIII analogue revealed that the inner-
sphere water ligands are displaced
when the complex binds to HSA result-
ing in lower than expected r1 at pH 5.
Variable pH and temperature nuclear
magnetic
relaxation
dispersion
(NMRD) studies showed that the in-
creased r1 of the albumin-bound q=0
complexes is due to the presence of a
nearby water molecule with a long resi-
dency time (1–2 ns). The distance be-
tween this water molecule and the Gd
ion changes with pH resulting in albu-
min-bound pH-dependent relaxivity.
Keywords: gadolinium · imaging ·
imaging agents · second-sphere re-
laxation · serum albumin
Introduction
MRI has better spatial resolution than MRS or PET, and
is not limited by light scattering and absorption like optical
imaging. pH-Dependent MRI requires a metal-based con-
trast agent, proton relaxation enhancing properties (relaxivi-
ty) of which change with pH. Water is imaged in MRI and
the contrast agent acts as a catalyst to enhance water proton
relaxation rates (1/T1, 1/T2) with relaxivity defined as the
change in relaxation rate normalized to agent concentration.
A change in pH can alter relaxivity by changing the hydra-
tion state of the inner- or second-coordination sphere,[4] by
affecting prototropic exchange,[5] or by changing the rota-
tional diffusion rate of the molecule.[6] For example, lower-
ing pH can result in protonation of a donor atom on a multi-
dentate ligand opening up a site(s) for a water ligand to co-
ordinate the metal ion and increase relaxivity.[4]
The desire to produce contrast agents that are activated by
changes in physiological pH is driven by the fact that the ex-
tracellular pH of solid tumors is more acidic than that of
healthy tissue.[1] Tissue ischemia, for example, in ischemic
heart disease and stroke, also results in a lower extracellular
pH.[2] Thus, pH can be a valuable biomarker for the identifi-
cation of tumors and ischemic tissue. There has been great
interest in mapping pH change and quantifying pH, in vivo,
by using a range of imaging techniques including positron
emission tomography (PET), magnetic resonance imaging
and spectroscopy (MRI and MRS) and optical imaging.[1a,3]
There are several examples of pH-dependent contrast
agents. However, quantification of pH by using MRI is chal-
lenging because the MR signal change depends on both the
relaxivity of the probe (pH-dependent) and its concentra-
tion. This challenge has been met through two techniques.
One approach is to use two very similar contrast agents, one
pH-independent and one pH-dependent. The MR signal in-
tensity versus time curve of the pH-independent contrast
agent is used to estimate time-dependent concentration of
the contrast agent, and it is assumed that the pH-dependent
agent will have the same concentration profile.[7] The limita-
[a] Dr. L. Moriggi, Dr. M. A. Yaseen, Prof. P. Caravan
A. A. Martinos Center for Biomedical Imaging
Department of Radiology
Massachusetts General Hospital and Harvard Medical School
Charlestown, MA 02129 (USA)
E-mail: caravan@nmr.mgh.harvard.edu
[b] Prof. L. Helm
Institut de Chimie Molꢀculaire et Biologique
Ecole Polytechnique Fꢀdꢀrale de Lausanne, EPFL-BCH
1015 Lausanne (Switzerland)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103344.
Chem. Eur. J. 2012, 18, 3675 – 3686
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3675

tion of this approach is the assumption of identical pharma-
cokinetics of two different compounds. Another approach is
to incorporate a marker that is independently quantifiable
within the pH-dependent contrast agent. For example, 19F
MR spectroscopy has been used to quantify concentration,[8]
but MR spectroscopy is limited to high concentrations and
lower spatial and temporal resolution compared to gadolini-
um enhanced MRI. Incorporation of a PET reporter like 18F
can provide quantification,[9] but this requires the use of a si-
multaneous MR–PET device.
We reasoned that a contrast agent with a large enough re-
laxivity change could identify ischemic and acidic tissue. The
pH range of physiological interest is between pH 6–7.5. The
ideal pH-dependent contrast agent would have a very high
relaxivity that is detectable at low concentrations, but more
importantly would have a large change in relaxivity, Dr1,
from pH 6.8 to 7.4. If the percentage change in relaxivity is
higher than the difference in concentration between ische-
mic and normal tissue, then low pH regions could be identi-
fied, at least qualitatively. Such a high relaxivity, high delta
relaxivity pH agent could also be adapted for quantitative
pH imaging by using the approaches described above.
These compounds had several favorable features. First, re-
laxivity increased significantly as pH was lowered. Second,
the pKa of the sulfonamide was in the appropriate range for
a physiological pH sensor, and this pKa could be modulated
by the choice of electronic substituent on the aromatic ring.
Third, the macrocyclic DO3A core should impart high ther-
modynamic stability and kinetic inertness with respect to
Gd decomplexation. Finally, the pendant carboxylate arms
overcame a major limitation of other GdACTHUNRTGNEUNG(DO3A) deriva-
tives, that of anion binding and quenching of relaxivity by
water displacement.
Here, we report two new ligands that build on the design
of Lowe et al.[4a] The GdIII complexes were prepared and
evaluated as pH-dependent contrast agents. The pH-depen-
dent relaxivity, serum albumin binding, and self-association
of these complexes was explored. In addition, the EuIII com-
plexes were synthesized to explore the effects of protein
binding and anion concentration on hydration number.
Results and Discussion
We looked to the literature to identify promising ap-
proaches. In ischemic and/or cancerous tissue, low pH is ob-
served in the extracellular interstitial space. A pH-depen-
dent contrast agent should be small enough to rapidly cross
the endothelial barrier into the extracellular space. This
rules out pH-dependent nanomaterials like liposomes,[10]
carbon nanotubes,[11] large dendrimers,[6b,c] and polymers.[6a,d]
Also, preferred would be contrast agents the relaxivity of
which increases with decreasing pH, such that pathology
would appear bright in an image. One class of compounds
Compound strategy and synthesis: We sought to build on
the successful molecular design of Lowe et al.[4a] Their sul-
AHCTUNGTREGfNNUN onamide derivatives showed an excellent relaxivity depend-
ence on pH brought about by a change in hydration number
from q=0 to q=2 when the sulfonamide nitrogen was pro-
tonated. Importantly, the pendant carboxylate arms ap-
peared to prevent anion coordination to the lanthanide
when q=2.[12] Anion coordination would have the effect of
displacing the inner-sphere water ligands and muting the
pH-dependent relaxivity effect. This was especially true for
the adipate derivative (n=2, Scheme 1).
that appeared particularly promising was GdACHTNUTRGNE(NUG DO3A) deriv-
atives with a pendant sulfonamide reported by Lowe et al.
(Scheme 1, in which R=CF3, OMe, or Me).[4a] At high pH,
the sulfonamide nitrogen is deprotonated and coordinates
the GdIII resulting in a complex with no inner-sphere water
ligands (q=0). At low pH, the sulfonamide is protonated
and does not coordinate, opening up two sites for water liga-
tion and concomitant higher relaxivity.
To increase relaxivity and the dynamic range even further
for this class of compounds, we sought to make them more
avid for serum albumin. Non-covalent protein binding is a
well-established technique for increasing the relaxivity of
paramagnetic complexes.[13] Slowing molecular tumbling in-
creases the efficiency of nuclear relaxation by the metal ion.
For q=1 complexes, the protein-bound relaxivity can be on
the order of 60 mmꢀ1 sꢀ1 at 0.5 T and 30 mmꢀ1 sꢀ1 at
1.5 T.[13a,14] Gianolio et al. showed that a q=2 complex can
have an albumin-bound relaxivity of 84 mmꢀ1 sꢀ1 at 0.5 T and
over 60 mmꢀ1 sꢀ1 at 1.5 T.[15] On the other hand, albumin-
bound q=0 complexes tend to have much lower relaxivities.
We reported q=0 complexes with albumin-bound relaxivi-
ties of 5–6 mmꢀ1 sꢀ1 at 1.5 T.[14c,16] Thus, in principal, it should
be possible to obtain a pH probe the relaxivity of which
varies by a factor of ten from low pH to high pH at the
common clinical field strength of 1.5 T.
With such a large change in relaxivity (6 to 60 mmꢀ1 sꢀ1)
even small changes in pH would be amplified. For instance,
by using a pKa of 6.7 from Lowe et al.,[4a] one predicts a re-
laxivity difference of 15 mmꢀ1 sꢀ1 between pH 7.4 (normal)
and pH 6.8 (ischemia, tumor); on a percentage basis this
would be a 100% change in relaxivity between these two
pH values.
Scheme 1. q-Modulated pH-dependent contrast agents.
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HSA Targeted pH-Dependent MRI Agents
FULL PAPER
Lowe et al. observed a modest twofold increase in relaxiv-
ity for their compounds in the presence of serum albumin,
but they did not report binding data for the albumin interac-
tion.[4a] Based on the literature, it is likely that their com-
pounds only exhibit a weak interaction with serum albumin;
for example, other hydrophilic chelates with a single aryl
group have dissociation constants in the millimolar range re-
sulting in only 10–15% of the complex being bound to pro-
tein under physiological conditions.[17] We have found that
biphenyl groups impart sufficient lipophilicity to enhance al-
bumin binding of gadolinium complexes such that >90% of
the complex is bound under physiological conditions
(ꢁ600 mm albumin).[13a,14c,18]
used benzyl-tert-butyl hydroxypentanedioate (g1) and con-
verted it to a mesylate for the alkylation. This intermediate
was available from prior studies in our laboratory.[19]
For the adipate derivative, we prepared (S)-dibenzyl-2-hy-
droxyhexanedioate (b1) using a similar procedure as Levy
et al.,[19a] and then converted this to a mesylate for alkyla-
tion of cyclen. The benzyl ester provided a convenient
UV/Vis handle for monitoring the reaction by HPLC. The
best conditions for the tris-alkylation were 5 equiv of the re-
spective mesylate, 1 equiv cyclen at 788C for 48 h in acetoni-
trile with potassium carbonate as a base. The major impurity
was the bis-alkylated product. The DO3A esters g2 and b2
were purified by preparative HPLC.
The biphenylsulfonamide moiety (a3) was prepared
through Suzuki coupling. For alkylation of the DO3A deriv-
atives, we converted the hydroxyl group to a mesylate (a4).
Following deprotection, the ligands (b4, g4) were chelated
with either GdCl3 or EuCl3. The final products were purified
by preparative HPLC.
Based on this reasoning, the target complexes for our
study were analogues of those reported by Lowe et al.[4a]
and denoted as Gd-glu and Gd-bbu (Scheme 2). The synthe-
sis strategy was similar to that reported previously but with
some modifications. We first prepared the DO3A esters (g2)
and (b2). Prior work utilized the dimethyl ester of 2-bromo-
glutarate or 2-bromoadipate. For the glutarate derivative we
Scheme 2. Synthetic scheme for Ln-bbu and Ln-glu.
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3677

P. Caravan et al.
Relaxivity: The relaxivities of Gd-glu (Figure 1a and Fig-
ure S2b in the Supporting Information) and Gd-bbu (Fig-
ure 1b and Figure S2d in the Supporting Information) were
was 260% at 20 MHz for Gd-bbu. Similar behavior was ob-
served for Gd-glu for which the relaxivity in the presence of
HSA was much lower than expected at pH 5. Relaxivities at
60 MHz (Figure S2a and c in the Supporting Information)
were slightly lower than at 20 MHz but the shape of the r1
versus pH curve was the same as for the 20 MHz data.
This anomalously low relaxivity at low pH could arise
from several possibilities. First, the affinity of the compound
for HSA could be lower at lower pH, either because of a
conformational change of the protein or because the phenol
moiety on the compound is protonated at lower pH. This
would result in fewer compounds bound at low pH and a
smaller observed relaxivity increase. An alternative explana-
tion is that the inner-sphere water molecules are displaced
when the complex binds to albumin. This has been seen
with other protein-bound GdACTHNUTRGENUGN(DO3A) derivatives, in which
presumably a protein side-chain coordinates the Gd and dis-
places the water ligands.[14c,16b,20] Lowe et al. found that the
presence of the three pendant carboxylates prevented coor-
dinating anions from displacing the inner-sphere water,[4a]
and we anticipated that these pendant carboxylates would
block protein side-chains from binding to the metal ion. A
third possibility is that the rate of inner-sphere water ex-
change is slow when the complex is protein bound. This is
not expected given the fast water exchange of q=2
GdACHTNUTRGNEUNG(DO3A) derivatives, but there is precedence for such an
effect.[21] Finally, we noted an aggregation/precipitation phe-
nomenon at lower pH values and this aggregation could in-
terfere with protein binding and/or hydration state. We per-
formed additional experiments to address these hypotheses.
Figure 1. Relaxivity versus pH at 378C and 20 MHz for: a) 0.22 mm
~
*
Gd-glu in the absence ( ) and presence of 0.79 mm HSA ( ); b) 0.21 mm
pH-Dependent aggregation: We repeated the pH titration
for Gd-bbu and performed dynamic light scattering (DLS)
at each pH value. At about pH 5.5 we began to see the ap-
pearance of large aggregates (diameter, d=700 nm) with
high polydispersity. Figure 2a shows the DLS results plotted
as relative scattering intensity versus pH (a relative scatter-
ing intensity of 1 indicates no aggregation). We then ex-
plored the effect of adding HSA to the aggregated Gd-bbu
at pH 5 (Figure 2b). Even at a low HSA:Gd-bbu ratio of
0.25, we see an immediate drop in scattering intensity. The
large 700 nm aggregate (Figure 2c–e) disappears with addi-
tion of HSA, suggesting that the HSA binding of discrete
Gd-bbu breaks up the aggregation. After addition of HSA,
the DLS analysis is the same as for HSA alone, with a
single species of diameter of 8 nm. This drop in scattering
intensity and loss of aggregation at low HSA:Gd-bbu ratios
is also consistent with the ability of HSA to bind several
copies of Gd-bbu, as will be shown below. The fact that the
free energy change for HSA binding is greater than that for
Gd-bbu self-association suggests that the lower-than-expect-
ed relaxivity at pH 5 in HSA solution is not due to some ag-
gregation phenomenon.
~
*
Gd-bbu in the absence ( ) and presence of 0.69 mm HSA ( ). Solid lines
indicate fits to determine the pKa of the sulfonamide moiety.
determined as a function of pH in the range 5 to 8.5
(20 MHz, 60 MHz, 310 K). In the absence of HSA, the pH-
dependent relaxivity of these two compounds was similar to
that reported by Lowe et al.[4a] Assuming that the change in
relaxivity is due to deprotonation of the sulfonamide nitro-
gen and concomitant change in q, the pKa for this deproto-
nation was determined to be 6.49ꢂ0.17 for Gd-bbu and
6.58ꢂ0.07 for Gd-glu.
We also noted that for Gd-bbu in the absence of protein,
the solution became turbid in the pH range 5.5 to 4.5 and
the onset of turbidity depended on the concentration of the
compound. In this pH range the observed relaxivity appears
to rise as a result of aggregation, but then decrease at
pH 4.5, which might be a result of microscopic precipitation.
Relaxivity increased at all pH values when this pH titra-
tion was repeated in the presence of excess HSA. However,
the relaxivity increase at low pH was much less than expect-
ed (42% at 20 MHz, pH 5). Instead of rising to
>60 mmꢀ1 sꢀ1 as expected for a protein-bound q=2 complex,
the relaxivity only approached 14 mmꢀ1 sꢀ1. At high pH, the
relaxivity was in the range expected for a q=0 complex
bound to albumin. The relaxivity enhancement at pH 8.5
We also examined whether increasing the ionic strength
would alter aggregation (Figure S3 in the Supporting Infor-
mation). Here, we titrated up to 30 equiv ammonium ace-
tate (NH4OAc) at either pH 5 (Figure S3a in the Supporting
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HSA Targeted pH-Dependent MRI Agents
FULL PAPER
does not have a measurable impact on protein binding. The
anomalously low relaxivity in HSA solution at pH 5 cannot
be attributed to a lack of protein binding at low pH.
We noted from the albumin titration experiments that the
protein appeared to bind multiple copies of Gd-bbu. We in-
vestigated this further by performing a full binding isotherm
at pH 5, 378C, and the results were plotted as [Gd-bbu]
bound per total [HSA] versus [Gd-bbu] unbound. Figure 3
Figure 2. a) Relative scattering intensity (RSI) compared to pure water as
a function of pH for Gd-bbu (0.32 mm) in the absence of HSA. b) Effect
of added HSA on relative scattering intensity at pH 5, Gd-bbu (0.20 mm).
Data in (a) and (b) are mean ꢂstandard deviation for triplicate experi-
ments; solid line indicates absence of aggregation. c) DLS data for
0.20 mm Gd-bbu at pH 4.7; d) DLS data for HSA in HEPES buffer at
pH 5; e) DLS data for 1.5:1 HAS/Gd-bbu at pH 5. All measurements
were performed at 378C.
Information) or pH 6.2 (Figure S3c in the Supporting Infor-
mation) and monitored the effect on DLS and relaxivity. At
pH 5, NH4OAc caused a reduction in the scattering intensity
but did not eliminate scattering. Ammonium acetate also
caused a dramatic reduction in relaxivity, dropping r1 to
2 mmꢀ1 sꢀ1 at high ammonium acetate ratios. This suggests
that aggregation is still present but the aggregate is in a dif-
ferent form. The very low relaxivity suggests q=0. Interest-
ingly, when we took this pH 5 solution with 30 equiv ammo-
nium acetate and added HSA, the relaxivity rose from 2 to
over 12 mmꢀ1 sꢀ1 when 0.5 equiv HSA per Gd-bbu were
added (Figure S3c in the Supporting Information). We re-
peated these experiments at pH 6.2 and there was no evi-
dence of aggregation. At pH 6.2, the addition of 30 equiv
NH4OAc had no effect on relaxivity. This latter result is con-
sistent with the work of Lowe et al. who found that the pres-
ence of acetate did not reduce the relaxivity of their com-
plexes.[4a]
Figure 3. Binding isotherm for Gd-bbu (378C, pH 5, 0.80 mm) to HSA
(pH 5, from 0.05 to 1.05 mm in HEPES buffer) from the ultrafiltration ex-
periment. Solid line is the best fit to a stoichiometric binding model.
shows a very strong initial binding event followed by addi-
tional binding. From the shape of the curve it is clear that
the binding does not saturate at 3 equiv Gd-bbu bound. The
data were fit to a classical stepwise thermodynamic binding
model and the binding constants are listed in Table 1 as dis-
Table 1. Stepwise stoichiometric binding constants, expressed as dissocia-
tion constants for Gd-bbu binding to HSA at 378C, pH 5.
Kd1 [mm]
Kd2 [mm]
Kd3 [mm]
0.65ꢂ0.29
18.3ꢂ8.4
1364ꢂ387
sociation constants (Kd =1/Ka). The initial binding constant
is in the low micromolar range, two orders of magnitude
higher affinity than the albumin-binding contrast agent,
MS-325.[13a] This albumin affinity is similar to that we report-
ed for other gadolinium complexes with biphenyl-based
binding protein moieties,[14c,18a] and is in the same range as
drugs like warfarin, naproxen, and ibuprofen.[22]
Binding to serum albumin: The non-covalent interaction be-
tween Gd-bbu or Gd-glu and HSA was explored over the
pH range 5 to 8.5. Protein complex binding was determined
by ultrafiltration across a membrane with a MW 5000 cutoff
under conditions of 0.2 mm Gd complex, 0.69 mm HSA,
378C (ratio [HSA]/ACHTUNGTRENNUNG[GdL]total =3.2).
The [Gd] in the filtrate was determined by ICP-MS and
this corresponded to the concentration of the unbound com-
plex. Under these conditions, both Gd-glu and Gd-bbu were
highly bound to HSA with the fraction bound ranging from
98.9 to 99.8%. Thus, for the relaxivity experiments, the ob-
served relaxivity equates to the relaxivity of the protein-
bound complex.
To account for the presence of aggregation in the un-
bound state, we ran the binding assay in the absence of pro-
tein. In the absence of protein, 65% of the complex concen-
tration was measured in the filtrate and presumably the re-
mainder was aggregated. We assume that this is constant
and we used this value to correct our data. Otherwise we
will underestimate the amount of unbound Gd-bbu and
therefore overestimate the affinity for albumin. It should be
noted that this correction only has an impact on the data at
Although the fluorophenol moiety on the sulfonamide
arm could undergo deprotonation over this pH range, it
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3679

P. Caravan et al.
high Gd-bbu:HSA ratios at which there is more unbound
Gd-bbu. Applying the correction has little effect on the first
binding event.
see the same relaxivity as for 1 equiv bound, indicating that
there is no site-dependent relaxivity.
We next prepared the EuIII analogue and performed lumi-
nescence lifetime studies. The difference in luminescence
decay rates when measured in H2O and D2O is proportional
to the number of inner-sphere water molecule ligands, q.[23]
The nature of the inner-sphere lanthanide coordination envi-
ronment at pH 5 was examined as a function of HSA con-
centration. We used a custom-designed multimodal confocal
imaging system built by Yaseen et al.[24] to measure the lumi-
nescence lifetime of the EuIII excited state. The microscopic
imaging device allowed us to measure EuIII luminescence
lifetimes on a pixel-by-pixel basis across the entire field of
view (~600 mm; Figure S6a, b and c in the Supporting Infor-
mation). The q values that we report in Figure 4b are aver-
ages of these local pixel-wise q values, and the standard de-
viations reported are those from the averaged q values. Fig-
ure 4b shows measured q as a function of HSA added at
pH 5. Prior to adding HSA and at the first addition of
0.22 equiv HSA we observed a wide range of q values. How-
ever, at the next addition of 0.33 equiv HSA and for subse-
quent additions (0.5–2.6 equiv) this heterogeneity was elimi-
nated and the q value settled at 0.2ꢂ0.1 over this range.
This spatially resolved q mapping was useful for measure-
ments in the absence of HSA when the aggregated Gd-bbu
is heterogeneous. Figure S4 in the Supporting Information
shows microscopic images of the slide prior to HSA addition
and indicates that parts of this large aggregate are starting
to precipitate. The first two experimental points on the left
Relaxivity and hydration number as a function of serum al-
bumin concentration: In our HSA binding studies at pH 5, it
was clear that Gd-bbu binds to more than one site on HSA.
Our initial relaxivity studies employed an excess of HSA in
order to favor protein binding. Together, the relaxivity and
binding data indicate that the initial high affinity binding
site is a low relaxivity site. We next sought to examine
whether subsequent binding sites also displayed this anoma-
lous low relaxivity or if relaxivity would increase as addi-
tional sites became occupied.
Figure 4 shows a plot of observed relaxivity versus the
number of equivalents of HSA added. These data corre-
spond to the light scattering data shown in Figure 2b. We
of Figure 4b ([HSA]/ACTHNUGRTENUNG[Eu-bbu]=0) show two values. The
higher value corresponds to q=1.2ꢂ0.8, and this was mea-
sured in the liquid (supernatant) surrounding the visible ag-
gregate. The lower point, with a value of q=0.6ꢂ0.8, corre-
sponds to the average of the solvation state within the ag-
gregate. The large standard deviations reflect the heteroge-
neous nature of the soluble and insoluble aggregates. After
the first addition of HSA, aggregation was still observed, ex-
plaining the low precision of the data. After the second ad-
dition of HSA, which brought the ratio of [HSA]/ACHTUNGTRENNUNG[Eu-bbu]
to 0.33, microscopic observation showed no precipitation,
and mean luminescent lifetimes throughout the sample
became highly precise with q=0.2ꢂ0.1. Additional aliquots
of HSA also resulted in precise measurements of q that
were unchanged. This behavior of aggregation followed by
solubilization with HSA is entirely consistent with the DLS
data shown for Gd-bbu. The unchanging relaxivity data with
Figure 4. a) Relaxivity at 378C, 0.47 T, pH 5, for Gd-bbu (0.42 mm) as a
function of added HSA. b) Hydration number q of Eu-bbu at pH 5 as a
~
function of added HSA. At [HSA]/
N
*
on a visible aggregate; : measurements made on the supernatant.
[HSA]:ACHTNUTRGNE[NUG Gd-bbu]>3 is consistent with luminescence lifetime
know from the light scattering data that when 0.5 equiv
HSA are added there is no aggregation in solution and es-
sentially all the Gd-bbu is bound to HSA. Addition of more
HSA results in a rise in relaxivity, which plateaus at 1 equiv
HSA. Figure 4 suggests that all binding sites offer low relax-
ivity at pH 5. This was confirmed in an additional study in
which we measured relaxivity and binding as a function of
pH for a 4:1 Gd-bbu/HSA solution and calculated the relax-
ivity due to the bound species (Figure S4 in the Supporting
Information). Even at 3 equiv Gd-bbu bound to HSA, we
data for Eu-bbu, when q is low and does not change over
this range. The q value estimation is based on an empirical
equation that has some uncertainty due to the quenching ef-
ꢀ
fects of other X H oscillators. Thus, a q value of 0.2 likely
indicates no inner-sphere water ligands.
To summarize the data so far, we prepared biphenyl ana-
logues of LnACTHNUGRTNEUNG(DO3A) type complexes previously reported by
Lowe et al.[4a] in order to promote albumin binding and en-
hance pH-dependent relaxivity. In the absence of albumin,
these compounds behaved as expected: low relaxivity at
3680
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Chem. Eur. J. 2012, 18, 3675 – 3686

HSA Targeted pH-Dependent MRI Agents
FULL PAPER
high pH due to coordination of sulfonamide arm and high
relaxivity at lower pH when sulfonamide is protonated and
q changes from 0 to 2. The biphenyl compounds show high
affinity for serum albumin over the pH range 5–8.5. At
pH 8.5, relaxivity increased by 260% upon albumin binding,
but the magnitude of relaxivity was in line with what we ex-
pected for a q=0 complex bound to albumin. At low pH,
the relaxivity of the albumin-bound complex increased but
was much, much lower than expected for a putative q=2, al-
bumin-bound complex. Luminescence studies on the EuIII
analogue revealed that the complex was q=0 at pH 5 when
bound to albumin, and this explained the low relaxivity.
The lack of inner-sphere water at low pH when protein-
bound was surprising. While most q=2 GdACTHUNRTGNEUNG(DO3A) deriva-
tives are well-known to bind coordinating anions resulting
in displacement of coordinated water and reduced relaxivi-
ty,[12,20,25] Lowe et al. showed that the presence of the pend-
ant carboxylate groups, and especially the adipate deriva-
tive, effectively suppressed anion binding.[4a] There is also
precedence for displacement of inner-sphere water from
GdACHTUNGTRENNUNG
(DO3A) derivatives by protein side-chains.[16b,20] We
were aware of these studies and hypothesized that the pend-
ant carboxylate groups would also suppress protein side-
chain coordination. In our case, it is not clear why the inner-
sphere waters are displaced when the complex binds to albu-
min. It could be that the high local concentration of gluta-
mate or aspartate carboxylate residues in the binding pocket
results in coordination to the GdIII. If side-chain coordina-
tion is occurring, one may expect that this might not occur
at different binding sites because there might not be a suita-
ble side-chain donor atom in close proximity to the GdIII.
However, we found that the relaxivity did not vary for the
first three binding sites. An alternate explanation is coordi-
nation of one of the pendant carboxylates to the GdIII ion.
This would involve an 8-membered chelate ring and would
not be expected to be very stable. In aqueous solution, the
solvation energy of these pendant carboxylates is very high
and coordination to GdIII is not energetically favored. In the
HSA binding site, the dielectric constant will be much lower
and the pendant carboxylates will be less well-solvated.[26]
These low dielectric conditions can favor coordination of a
pendant carboxylate to GdIII.
Figure 5. Nuclear magnetic relaxation dispersions (NMRD) of Gd-bbu:
a) 0.52 mm in the absence of HSA at pH 8.5 and 5, and 378C; b) 0.39 mm
at pH 8.5 with 1.4 mm HSA at 5 and 378C; c) 0.33 mm at pH 5 with
1.3 mm HSA at 5 and 378C. Solid lines in (b) and (c) are fits to the data;
see text for model and Table 2 for parameters.
range.[27] At pH 8.5, at which there is no evidence of aggre-
gation, we observe a typical sigmoidal shaped curve consis-
tent with a short correlation time.
Regardless of the mechanism, these complexes are q=0
when bound to HSA at either high or low pH, yet there is
still a pH-dependent relaxivity for the albumin-bound com-
plex. To investigate the nature of this pH dependence, we
studied the field-dependent relaxivity of Gd-bbu as a func-
tion of pH and temperature.
In the presence of HSA, there is an increase in relaxivity
at both pH values and temperatures (5 and 378C) and a
peak is observed in the NMRD profile in the 20–30 MHz
range. This peak is indicative of a long correlation time. We
hypothesize that the increase in relaxivity upon binding is
due to the presence of a relatively long-lived water mole-
cule(s) or exchangeable proton(s) in close proximity to the
GdIII ion, that is, a long-lived second-sphere water molecule.
The NMRD data were modeled to test this hypothesis.
Relaxivity can be factored into contributions arising from
different classes of exchangeable protons in the different co-
ordination spheres: inner-sphere, second-sphere, and outer-
sphere. Based on the luminescence data, there are no inner-
Nuclear magnetic relaxation dispersion (NMRD): Figure 5a
shows NMRD profiles of Gd-bbu in the absence of HSA at
pH 5 and 8.5 at 378C. These profiles are consistent with our
observations. At pH 5, Gd-bbu forms large aggregates and
this results in an increase in the rotational correlation time
of the Gd!H vector. It is well established from theory and
practice that such an increase in correlation time will result
in a maximum in the NMRD profile in the 20–40 MHz
Chem. Eur. J. 2012, 18, 3675 – 3686
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3681

P. Caravan et al.
sphere water ligands when Gd-bbu is bound to HSA. This
leaves second- and outer-sphere contributions to the ob-
served relaxivity. Relaxation due to outer-sphere water mol-
ecules and exchangeable protons (water molecules or NH or
OH protons from the protein) that undergo fast exchange
(>109 sꢀ1) will result in an NMRD profile that is sigmoidal
in shape and lacks the high-field peak. Relaxation due to a
nearby long-lived water molecule or exchangeable proton
(second-sphere residency time tꢂm >1 ns) will result in this
high-field peak in the 20–30 MHz range. We assumed that
the relaxivity of the former (fast exchanging protons) could
be approximated by the relaxivity of Gd-bbu at pH 8.5 in
the absence of protein. This is summarized in Equation (1),
we considered tv and D2 to be global parameters. We also as-
sumed that the rotational correlation time of the albumin-
bound complex would be much longer than the lifetime of
the long-lived second-sphere water molecule, that is, 1/tꢂm @
1/tR, and thus the relaxivity measurement would not be very
sensitive to tR. The NMRD data at two temperatures were
then simultaneously fit by iteratively varying six parameters:
tꢂm (378C), tꢂm (58C), q’/r6 (378C), q’/r6 (58C), tv and D2; tR
was fixed at 40 ns. After this initial analysis, we found that
the fitted NMRD data were rather insensitive to the value
of tR, so long as tR was long (>20 ns). We also found that tv
and D2 were strongly correlated and could not be independ-
ently determined. Ultimately, we were able to reproduce the
two NMRD curves (5 and 378C) at either pH by varying
only five parameters, tꢂm (378C), tꢂm (58C), q’/r6 (378C), q’/r6
(58C), and D2 (or tv), and the results of these fits are given
in Table 2. For electronic relaxation, we could identify lower
limits on tv and D2 and establish a linear relationship be-
tween these parameters; at pH 5, D2 ꢄ5ꢃ1018 sꢀ2 with tv =
9.0ꢃD2ꢀ19.9 (in ps). At pH 8.5, D2 ꢄ6ꢃ1018 sꢀ2 with tv =
5.3ꢃD2ꢀ9.4.
with which we define a relaxivity due to a long-lived water
LL
molecule (or other exchangeable proton(s)), r1
:
ꢀ
ꢁ
q0=½H2Oꢃ q0C
3tc
r1LL ¼ r1obs ꢀ r1pH8:5;HSAꢀ
¼
¼
ð1Þ
ð2Þ
T10 m
rG6 dH 1 þ w2Ht2c
ꢄ
ꢅ
ꢂ
ꢃ
2
2
m0
C ¼
g2Hg2em2BSðS þ 1Þ
15 4p
The NMRD of the slow exchanging proton(s) was modeled
by using Solomon–Bloembergen–Morgan (SBM) theory.[28]
Since SBM is a high-field theory, we only considered data
from 8–400 MHz; at lower fields the assumption that the
Zeeman energy is much greater than the static zero field
splitting breaks down and use of SBM is not appropriate.[16a]
Table 2. Molecular parameters obtained from fit of 1H NMRD data in
Figure 5.
Parameter
pH 5
pH 8.5
tR [ns]
>20[a]
>20[a]
tmꢂ (378C) [ns]
tmꢂ (58C) [ns]
rGd–H (378C) [ꢄ]
rGd–H (58C) [ꢄ]
1.8ꢂ0.5
1.1ꢂ0.2
2.5ꢂ0.6
1.5ꢂ0.2
4.19ꢂ0.14[b]
4.32ꢂ0.11[b]
At these higher fields, there is only one relevant spectral
density term to consider. Furthermore, the lifetime of the
long-lived water molecule is still much shorter than its relax-
ation time, tꢂm !T1m’, which is apparent from the fact that
relaxivity is much higher at 58C than at 378C (Figure S7 in
the Supporting Information, r1 vs. temperature for ratio 4:1
Gd-bbu/HSA). Together this results in a simple analytical
3.86ꢂ0.05[b]
4.18ꢂ0.07[b]
[a] Fixed in fitting at 40 ns; [b] assumes one long-lived water molecule,
that is, 2 equiv protons.
The NMRD analysis revealed that the lifetime of the
long-lived water molecule is similar at pH 8.5 and 5. The dif-
ference in relaxivity arises from a variation in the distance
between the GdIII ion and this water molecule, which is
shorter at pH 5. This difference in Gd–H distance might be
expected. First, at high pH, the sulfonamide group is coordi-
nated to the GdIII and this coordination will change the ori-
entation of the biphenyl HSA binding group with respect to
the gadolinium chelate compared to the situation at pH 5, at
which the sulfonamide is protonated and not coordinated. If
the HSA binding pocket is the same at both pH values, then
the Gd chelate will be oriented differently resulting in a dif-
ferent distance to the long-lived water. Changes in protein
structure could also give rise to this change in Gd–H dis-
tance. For instance, Qiu et al. reported studies on the solva-
tion dynamics and local rigidity in a series of reversible con-
formations of HSA under different pH conditions.[30] They
observed that HSA undergoes a series of reversible confor-
mational changes from acidic to basic conditions and even a
change by a factor of ten of the water exchange rate at the
surface. From acidic to neutral pH, structural changes occur
in domains II and III, changes in viscosity, solubility, and
a-helical content in the protein were observed. As pH in-
LL
expression for r1 that depends on the correlation time of
the long-lived water, tc, and the ratio of the number of long-
lived water molecules (q’) to the gadolinium–hydrogen dis-
tance, q’/r6, Equation (1); here C is a constant [Eq. (2)] the
symbols have their usual meanings.[27]. The correlation time
can have contributions from rotation of the complex (1/tR),
electronic relaxation (1/T1e), and the exchange of the long-
lived water (1/t’m) [Eq. (3)]. The shortest correlation time
will dominate. Electronic relaxation is field-dependent and
can be approximated by Equation (4):
1
1
1
1
¼
þ
þ
ð3Þ
ð4Þ
tC tR T1e t0m
ꢀ
ꢁ
1
T1e
D2t ½4SðS þ 1Þ ꢀ 3ꢃ
tv
4tv
¼
þ
25
1 þ ws2tv2 1 þ 4ws2tv2
We took a global analysis approach to fitting the data.
While data at each pH were considered independently, we
fit the 37 and 58C data together for each pH. Since, elec-
tronic relaxation of polyaminocarboxylato GdIII complexes
is not very temperature dependent over this range,[14c,16a,29]
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Chem. Eur. J. 2012, 18, 3675 – 3686

HSA Targeted pH-Dependent MRI Agents
FULL PAPER
creases, HSA shows an increased affinity for some ligands,
like warfarin.[31]
protons in close proximity to the GdIII ion. As the pH was
lowered, the distance between this water and the GdIII ion
decreased resulting in a pH-dependent relaxivity increase.
We used the simplest model to reproduce the NMRD
curves; no doubt, more complicated models could be used.
It is interesting to note that the data could be well-repro-
duced with a simple isotropic model. Most NMRD data in-
volving macromolecular systems require the Lipari–Szabo
modification to account for internal motion.[32] However, in
our case, the correlation time is dominated by the short
Experimental Section
Instrumentation: Ultrafiltration units (UFC3LCC00, regenerated cellu-
lose membrane of 5000 Da nominal molecular weight cutoff) were ob-
tained from Millipore Corp. (Bedford, MA). All gadolinium concentra-
tions were determined by ICP-MS on an Agilent 7500a system. 1H and
13C NMR spectra were recorded on a Varian 500 NMR system equipped
with a 5 mm broadband probe. Longitudinal relaxation times, T1, were
measured by using the inversion recovery method on Bruker Minis-
pecs mq20 (20 MHz) and mq60 (60 MHz). The 1/T1 NMRD profiles
were measured on a Stelar Spinmaster fast-field cycling NMR relaxome-
ter (2.35ꢃ10ꢀ4–0.47 T; 1H Larmor frequencies: 0.01–20 MHz) equipped
with a VTC90 temperature control unit, on Bruker Minispecs (0.71 T
[30 MHz], 0.94 T [40 MHz] and 1.41 T [60 MHz]) on a Bruker Avance-
200 console connected to 2.35 T (100 MHz) and 4.7 T (200 MHz) cryo-
magnets, and on a Bruker DRX-400 (9.4 T, 400 MHz). Microwave irradi-
ation was carried out by using a personal chemistry Emrys optimizer mi-
crowave synthesizer. Purifications were performed by using two methods.
Method A: normal phase chromatography on a Combiflash Companion/
TS (Teledyne ISCO, 120 g RediSep Rf silica cartridge) by using A:
hexane, B: ethyl acetate, flow-rate 85 mLminꢀ1 over 15 min. Method B:
preparative HPLC on a Rainin, Dynamax (column: 250 mm Kromasil
C18) by using A: 0.1% TFA in water, B: 0.1% TFA in MeOH, flow-rate
15 mLminꢀ1 over 15 min. HPLC purity analysis (both UV and MS detec-
tion) were carried out on an Agilent 1100 system (column: Phenomenex
Luna, C18(2) 100ꢃ2 mm) with UV detection at 220, 254 and 280 nm by
using a gradient of ammonium formate (20 mm, pH 6.8) with 5% (9:1
ACN/20 mm ammonium formate) to 95% (9:1 ACN/20 mm ammonium
formate), flow-rate 0.8 mLminꢀ1 over 15 min.
(ꢁ1 ns) lifetime of the long-lived water molecule and the re-
laxivity data are insensitive to rotational dynamics. The dis-
tances reported in Table 2 are based on one long-lived water
molecule with two protons, however, the adjustable parame-
ter is q’/r6. The NMRD data cannot distinguish between a
water molecule or a nearby exchangeable proton from the
protein.
The Gd-glu system showed similar relaxivity data when
bound to HSA. Although we did not perform either lumi-
nescence studies on the EuIII analogue or NMRD, it is
highly likely that the relatively small relaxivity observed at
low pH is due to displacement of inner-sphere water mole-
cules as well. Interestingly, the Gd-glu system had
a
U-shaped pH dependence when bound to HSA (Figure 1a).
HSA undergoes the normal-to-base form (N!B) conforma-
tional transition above pH 8.[33] It is likely that this results in
a change in the internuclear distance between the GdIII ion
and the long-lived water. However, the Gd-bbu relaxivity is
not sensitive to this N!B transition.
The majority of water molecules hydrating proteins have
residency times on the order of tens of picoseconds, but it is
well-established that some water molecules can have longer
residency times.[34] The 1–2 ns lifetime of the long-lived
water molecule observed here is in accord with a water mol-
ecule hydrogen bonded to the surface of a protein. Interest-
ingly, other albumin-bound q=0 complexes show similar be-
havior.[14c,16a] The combination of NMRD and q=0 com-
plexes could be used to probe the dynamics of protein hy-
dration.
Luminescence: Measurements were collected by using the confocal por-
tion of a custom-designed multimodal microscope.[21] Briefly, a continu-
ous-wave diode laser (l=532 nm, B&W Tek) provided excitation light
that was temporally gated by an electro-optical modulator (ConOptics,
Danbury, CT) with extinction ratio of approximately 200 at 532 nm. The
excitation beam passed through several conditioning optics, including a
beam expander with pinhole spatial filter, polarizer, shutter, dichroic
mirror, scan lens, and tube lens and a 20ꢃ magnification objective lens
(XLumPlan FL, Olympus, NA=0.95). With the use of a customized con-
trol software and galvanometric scanners (Cambridge Technology, Inc.
Lexington, MA) the excitation beam was guided to selected locations in
the approximately 600 mm field of view. The emitted luminescence was
descanned and collected by using an avalanche photodiode photon count-
ing module (APD, SPCM-AQRH-10, Perkin–Elmer, Waltham, MA) sam-
pled at 50 MHz with a high-speed DIO card (National Instruments,
Austin, TX). Data were processed by using custom-written software in C
and MATLAB (Mathworks, Natick, MA). Detected luminescent photons
were binned into 50 ms long bins, to yield time-dependent phosphores-
cence decay profiles. With the use of a nonlinear least squares fitting rou-
tine, the resulting time-courses were fit with a single-exponential func-
tion. A sampleꢂs luminescence lifetime is equal to its fitted profileꢂs cal-
culated time constant.
Conclusion
Two new pH-dependent MR contrast agents were synthe-
sized with high affinity for serum albumin. The agents were
designed to have no inner-sphere water ligands at high pH
(low relaxivity). Pendant carboxylate groups were included
to prevent anion binding to the complex at low pH, and it
was hoped, to prevent displacement of inner-sphere water li-
gands by protein side-chains. However, when the complex
was bound to HSA at pH 5, the water ligands were dis-
placed and the relaxivity was substantially lower than antici-
Chemicals: HSA (Fraction V powder 96–99% albumin, containing fatty
acids) was purchased from Sigma Chemical Co. (St. Louis, Mo.). The syn-
thesis of ligands was carried out as shown in Scheme 2. (4-(Benzyloxy)-3-
fluorophenyl)boronic acid was purchased from Frontier Scientific, Inc.,
and (S)-2-aminohexanedioic acid (98%) was purchased from Alfa Aesar
(A Johnson Matthey Company). Other reagents were supplied by Al-
drich Chemical Co., Inc., and were used without further purification. Sol-
vents (HPLC grade) were purchased from various commercial suppliers
and used as received.
pated. These results suggest that the [GdACTHNUTRGENNUG(DO3A)ACHTUNGTRENN(GUN H2O)2]
moiety is not suitable for use in protein targeted contrast
agents due to water displacement upon protein binding. De-
spite being q=0 while bound to HSA over the pH range 5–
8.5, a pH-dependent relaxivity was still observed. This was
due to a long-lived water molecule or other exchangeable
4-Bromo-N-(2-hydroxyethyl)benzenesulfonamide (a2): Chlorotrimethylsi-
lane (1.30 g, 12.0 mmol) was added to a stirred mixture of 2-aminoetha-
Chem. Eur. J. 2012, 18, 3675 – 3686
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3683

P. Caravan et al.
nol a1 (665 mg, 10.89 mmol) and Et3N (1.29 mg, 13.0 mmol) in CH2Cl2
(50 mL) cooled to 0–58C in an ice-bath and monitored by TLC (CH2Cl2/
MeOH/Et3N 92:8 stained with ninhydrin) for completeness. After 1 h,
the mixture was warmed to room temperature while being stirred and
was then concentrated, in vacuo. 4-Bromobenzene-1-sulfonyl chloride
(3.05 g, 12.0 mmol) was then added and Et3N (1.31 g, 13.0 mmol) in
CH3CN (50 mL) cooled to 0–58C in an ice-bath and monitored by TLC
(hexane/ethyl acetate 80:20) for completeness. After 1 h, the mixture was
warmed to room temperature while being stirred. After 2 h, the mixture
was concentrated, in vacuo, and purified by using normal phase chroma-
48 h, the reaction mixture was cooled to room temperature, filtered with
a 200 nm syringe filter to remove potassium salts and concentrated, in
vacuo. The mixture was then purified by preparative HPLC (method B,
gradient of 5 to 95% solvent B, monitoring at 220 nm). Fractions contain-
ing the product were concentrated to give 201 mg of b2 (0.18 mmol, 35%
1
conversion of cyclen). H NMR (CDCl3, 500 MHz, 303 K): d=7.3 (30H),
5.2 (12H), 4.4 (3H), 3.3 ppm (broad); 13C NMR (CDCl3, 125 MHz,
303 K): d=167, 164, 135, 128, 66.2, 45.7, 33.3, 30.1, 20.0 ppm; LC/MS
(ESI+): C68H80N4O12: m/z (%): calcd 1145.59 [MH+]; found 1146.4 (MH+
).
1
tography (method A) to give a2 (2.02 g, 7.19 mmol, yield 66%). H NMR
Hexabenzyl
2,2’,2’’-(10-(2-(4’-(benzyloxy)-3’-fluoro-[1,1’-biphenyl]-4-yl
(CDCl3, 500 MHz, 303 K): d=7.95 (4H), 7.75 (NH), 4.44 (OH), 3.85
(2H), 3.74 ppm (2H); 13C NMR (CDCl3, 125 MHz, 303 K): d=139.6,
133.2, 130.7, 127.9, 61.4, 48.4 ppm; LC/MS (ESI+): C8H10BrNO3S: m/z
(%): calcd 281.15 [MH+]; found: 281.1 (MH+).
sulfonamido)ethyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triyl)trihexane-
dioate (b3): Methanesulfonyl chloride (91.9 mg, 0.80 mmol) was added to
a stirred mixture of a5 (293 mg, 0.73 mmol) and Et3N (96 mg, 0.95 mmol)
in CH2Cl2 anhydrous (20 mL). After the addition was complete, the mix-
ture was warmed to room temperature while being stirred. After 1 h, the
mixture was concentrated, in vacuo, and purified by using normal phase
chromatography (method A) to give a4 (312 mg, 0.65 mmol). LC/MS
(ESI+): C22H22FNO6S2: m/z (%): calcd 480.10 [MH+]; found: 480.5
(MH+). The mesylate a4 (128 mg, 0.27 mmol), as a solution in anhydrous
4’-(Benzyloxy)-3’-fluoro-N-(2-hydroxyethyl)-[1,1’-biphenyl]-4-sulfona-
mide (a3): Compound a2 (48.1 mg, 0.17 mmol) and (4-(benzyloxy)-3-fluo-
rophenyl)boronic acid (49.85 mg, 0.202 mmol) were dissolved in ethanol
(5 mL) in a microwave vial. PdACTHNUTRGNE(UNG PPh3)2Cl2 (12 mg, 0.017 mmol) and Et3N
(347.5 mg, 3.44 mmol) were added, and the reaction mixture was irradiat-
ed in the microwave synthesizer at 1008C for 30 min. After the reaction
was cooled to room temperature, the product was filtered, the filtrate
was concentrated, and the crude mixture was purified by using normal
phase chromatography (method A) to give a5 (28.1 mg, 0.07 mmol, yield
42%). 1H NMR (CDCl3, 500 MHz, 303 K): d=7.75 (4H), 7.45 (NH),
7.40 (2H), 7.35 (2H), 7.22 (3H), 7.18 (1H), 5.21 (2H), 3.57 (OH),
2.98 ppm (4H); LC/MS (ESI+): C21H20FNO4S: m/z (%): calcd 402.12
[MH+]; found: 402.1 (MH+).
CH3CN (30 mL), was added to
a stirred mixture of b2 (254 mg,
0.22 mmol) and dry potassium carbonate (92 mg, 0.67 mmol) in CH3CN
(50 mL) preheated to 788C, and the reaction was monitored by HPLC
for completeness. After 12 h, the reaction mixture was cooled to room
temperature, filtered with a 200 nm syringe filter to remove potassium
salts and concentrated, in vacuo. The mixture was then purified by prepa-
rative HPLC (method B, gradient of 5 to 95% solvent B). Fractions con-
taining the product were concentrated to give 275 mg of b3 (0.18 mmol,
1
yield 82%). H NMR (CDCl3, 500 MHz, 303 K): d=7.5–7.92 (42H), 5.2–
(S)-Dibenzyl 2-hydroxyhexanedioate (b1): (S)-2-Aminohexanedioic acid
(1.00 g, 6.2 mmol) was dissolved in water (8 mL). Concentrated HCl
(0.977 mL, 12.1 mmol) was added. A solution of sodium nitrite (2.48 g,
35.98 mmol) dissolved in water (8 mL) was then added very slowly
(3 mLhꢀ1) at 0–58C. The solution was stirred, overnight. After acidifica-
tion (pH 2) the product was extracted with ethyl acetate and dried, in
vacuo, to give a mixture of 2-hydroxyhexanedioic acid and 5-carboxy-d-
lactone. LC/MS (ESI+): diacid C6H10O5: m/z (%): calcd 163.06 [MH+];
found: 163.1 (MH+); lactone C6H8O4: m/z (%): calcd 145.05 [MH+];
found: 145.1 (MH+). Then, a solution of 1N KOH (6.2 g, 6.2 mmol) was
added in a single portion to a stirred solution of the mixture dissolved in
THF (10 mL) and heated at 408C for 2 h. The reaction was then concen-
trated to a solid, in vacuo (558C, <30 Torr) dried under vacuum, over-
night, and acid was not purified and used crude in the next step. LC/MS
(ESI+): C6H10O5. m/z (%): calcd 163.06 [MH+]; found: 163.1 (MH+).
(S)-2-Hydroxyhexanedioic acid (613 mg, 3.78 mmol) was suspended and
stirred in DMF (8 mL) and benzyl bromide (1.29 g, 7.56 mmol) was
added at room temperature. After being stirred for 8 h, the mixture was
concentrated, in vacuo, and was purified by preparative HPLC (meth-
od B, gradient of 5 to 65% solvent B, monitoring at 220 nm). Fractions
containing the product were concentrated to give 230 mg of b1
(0.67 mmol, yield 11%). 1H NMR (CDCl3, 500 MHz, 303 K): d=7.3
(10H), 5.2 (2H), 5.1 (2H), 4.2 (1H), 2.4 (4H), 1.8 ppm (4H); 13C NMR
(CDCl3, 500 MHz, 303 K): d=174 (CO), 173 (CO), 135, 128 (aryl), 77.2,
67, 33.6, 20.3 ppm; LC/MS (ESI+): C20H22O5: m/z (%): calcd 343.15
[MH+]; found: 343.1 (MH+).
5.4 (12H), 3.21 (3H), 3.45 ppm (3H); 13C NMR (CDCl3, 125 MHz,
303 K): d=20.5, 21.9, 25.3, 29.1, 29.7, 36.1, 38.2, 41.4, 66.7, 71.4, 114.4,
115.1, 116.7, 129.2, 135.7, 173.4 ppm; LC/MS (ESI+): C89H98FN5O15S: m/z
(%): calcd 1528.68 [MH+]; found 1528.8 (MH+).
2,2’,2’’-(10-(2-(3’-Fluoro-4’-hydroxy-[1,1’-biphenyl]-4-ylsulfonamido)eth-
yl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triyl)trihexanedioic acid (b4):
Pd (10%) on carbon (3.00 g) was added to a methanol solution (20 mL)
of b3. The mixture was subjected to hydrogen bubbling and stirred for
12 h and monitored by HPLC for completeness. The mixture was then fil-
tered through a fine frit, and the filtrate was concentrated, in vacuo. LC/
MS (ESI+): C40H56FN5O15S: m/z (%): calcd 898.36 [MH+]; found 898.4
(MH+).
(2S,2’S,2’’S)-5-Tribenzyl-1-tri-tert-butyl-2,2’,2’’-(1,4,7,10-tetra-azacyclodo-
decane-1,4,7-triyl) tripentanedioate (g2): Methanesulfonyl chloride
(214 mg, 1.87 mmol) was added to a stirred mixture of g1 (500 mg,
1.70 mmol) and Et3N (192 mg, 1.90 mmol) in CH2Cl2 anhydrous (30 mL)
cooled to 0–58C in an ice-bath and monitored by analytical HPLC for
completeness. After the addition was complete, the mixture was warmed
to room temperature while being stirred. After 1 h, the mixture was con-
centrated, in vacuo. and purified by using normal phase chromatography
(method A) to give the mesylate form of g1 (525 mg, 1.41 mmol). LC/MS
(ESI+): C17H24O7S: m/z (%): calcd 373.13 [MH+]; found 373.4 (MH+).
The mesylate (360 mg, 0.964 mmol), as a solution in anhydrous CH3CN
(25 mL), was added under N2 to a stirred mixture of cyclen (1,4,7,10-
tetra-azacyclododecane; 42 mg, 0.24 mmol) and dry potassium carbonate
(268 mg, 1.93 mmol) in CH3CN (20 mL) preheated to 808C, and the reac-
tion was monitored by analytical HPLC for completeness. After 48 h, the
reaction mixture was cooled to room temperature, filtered with a 200 nm
syringe filter to remove potassium salts and concentrated, in vacuo. The
mixture was then purified by preparative HPLC (method B). Fractions
containing the product were concentrated to give 86.6 mg of g2
(2S,2’S,2’’S)-Hexabenzyl
2,2’,2’’-(1,4,7,10-tetra-azacyclododecane-1,4,7-
triyl) tri-hexane-dioate (b2): Methanesulfonyl chloride (184 mg,
1.60 mmol) was added to a stirred mixture of b1 (500 mg, 1.46 mmol) and
Et3N (177 mg, 1.75 mmol) in CH2Cl2 anhydrous (30 mL) cooled to 0–58C
in an ice-bath and monitored by HPLC for completeness. After the addi-
tion was complete, the mixture was warmed to room temperature while
being stirred. After 1 h, the mixture was concentrated, in vacuo, and puri-
fied by using normal phase chromatography (method A) to give b5
(580 mg, 1.38 mmol). LC/MS (ESI+): C21H24O7S: m/z (%): calcd 421.13
[MH+]; found: 421.1 (MH+). The mesylate b5 (840 mg, 2.0 mmol), as a
solution in anhydrous CH3CN (50 mL), was added to a stirred mixture of
cyclen (1,4,7,10-tetra-azacyclododecane; 86 mg, 0.5 mmol) and dry potas-
sium carbonate (553 mg, 4.0 mmol) in CH3CN (50 mL) preheated to
788C, and the reaction was monitored by HPLC for completeness. After
(0.08 mmol, 36% conversion of cyclen); LC/MS (ESI+): C56H80N4O12
:
m/z (%): calcd 1001.59 [MH+]; found 1002.3 (MH+).
(2S,2’S,2’’S)-2,2’,2’’-(1,4,7,10-Tetra-azacyclododecane-1,4,7-triyl)tripenta-
nedioic acid (g3): Compound a4 was prepared following the same condi-
tion described for the synthesis of b3. Compound a4 (90.0 mg,
0.19 mmol) as a solution in anhydrous CH3CN (15 mL) was added under
N2 to a stirred mixture of g3 (125 mg, 0.13 mmol) and dry potassium car-
bonate (52 mg, 0.38 mmol) in CH3CN (15 mL), preheated to 788C, and
3684
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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 3675 – 3686

HSA Targeted pH-Dependent MRI Agents
FULL PAPER
the reaction was monitored by HPLC for completeness. After 12 h, the
reaction mixture was cooled to room temperature, filtered with a 200 nm
syringe filter to remove potassium salts and concentrated, in vacuo. The
mixture was then purified by preparative HPLC (method B). Fractions
containing the product were concentrated to give 152 mg of g3
(0.11 mmol, 86%); LC/MS (ESI+): C77H98FN5O15S: m/z (%): calcd
1384.69 [MH+]; found 1384.7 (MH+).
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2,2’,2’’-(10-(2-(3’-Fluoro-4’-hydroxy-[1,1’-biphenyl]-4-ylsulfonamido)eth-
yl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triyl)tripentanedioic acid (g4):
Pd/C (10%, 3.00 g) was added to a methanol solution (20 mL) of g3
(115 mg, 0.08 mmol). The mixture was subjected to hydrogen bubbling
and stirred for 12 h. HPLC indicated the completion of the reaction. The
mixture was then filtered through a fine frit, and the filtrate was concen-
trated, in vacuo, and co-evaporated with acetonitrile in order to azeo-
trope out residual water and to remove any remaining methanol. Then a
cocktail of tri-isopropylsilane (5%), anisole (5%) and TFA (90%) was
added to the product, stirred for 4 h and monitored by analytical HPLC
for completeness; LC/MS (ESI+): C37H50FN5O15S: m/z (%): calcd 856.31
[MH+]; found 856.3 (MH+).
Ln-bbu and Ln-glu: Lanthanide complexes were prepared in aqueous so-
lution following the reaction for compound b4 and g4, respectively, with
hydrated LnCl3 at pH 6 (18 h, 508C) then raised to pH 9 (30 min). The
purification of Ln-bbu and Ln-glu was carried out through preparative
HPLC with neutral ammonium acetate buffer or pure water/acetonitrile
eluent. Each purification method gave a satisfactory LC/MS trace of the
final compounds Ln-bbu and Ln-glu with expected masses. LC/MS (ESI+
): C37H47FGdN5O15S (Gd-glu) : m/z (%): calcd 1011.21 [MH+]; found:
1011.0; C40H53FGdN5O15S (Gd-bbu) : m/z (%): calcd 1053.26 [MH+];
found: 1053.25; C40H53EuFN5O15S (Eu-bbu) : m/z (%): calcd 1048.26
[MH+]; found: 1048.25; see the Supporting Information for purity analy-
ses.
Preparation of 12.0% (w/v) HSA: Lyophilized HSA was dissolved in
HEPES (50 mm) buffer to generate the HSA solution (12.0%, w/v). A
molecular weight of 66 435 Da was used to estimate % (w/v) to a molar
concentration. The protein concentration [HSA] was determined by
measuring its absorbance at 280 nm for four dilutions. The linear regres-
sion on absorbance versus dilution gave a slope of [HSA]ꢃe, in which e
is the molar absorptivity of HSA (35700mꢀ1 cmꢀ1).
Ultrafiltration measurements of binding of Gd-bbu to HSA: Gd-bbu/
HSA samples ranging from 0.051 to 1.0 mm HSA in Gd-bbu (0.4 mm)
were made by combining appropriate amounts of 12.0% (w/v) HSA and
0.8 mm Gd-bbu. Aliquots (400 mL) of these samples were placed in 5 kDa
ultrafiltration units, incubated at 378C for 20 min, and then centrifuged
at 3500g for 7 min. The filtrates from these ultrafiltration units were used
to determine the free concentration of Gd-bbu of each of the samples.
Duplicate aliquots were processed for each concentration sample of Gd-
bbu in 4.5% (w/v) HSA. Concentrations of Gd-bbu/HSA samples and ul-
trafiltrates were determined by measuring the Gd concentration using
ICP-MS.
Acknowledgements
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L.M. acknowledges the Swiss National Science Foundation for a fellow-
ship (PBELP2-130907). Fabrice Yerly is thanked for providing the pro-
gram VISUALISEUR/OPTIMISEUR, version 2.3.7 (2010) for least-
squares fitting of the NMRD data and the pKa experiment. We thank
Zhaoda Zhang for helpful discussions regarding the synthesis. This work
was supported by the National Institute of Biomedical Imaging and Bio-
engineering (award numbers: R01EB009062 and R21EB009738) and the
National Institute of Neurological Disorders and Stroke (R01NS057476).
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Received: October 24, 2011
Published online: February 10, 2012
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Chem. Eur. J. 2012, 18, 3675 – 3686