A new gadolinium-based MRI zinc sensor

Article abstract of DOI:10.1021/ja901875v

The properties of a novel Gd³⁺-based MRI zinc sensor are reported. Unlike previously reported Gd³⁺-based MRI contrast agents, this agent (GdL) differs in that the agent alone binds only weakly with human serum albumin (HSA), while th

Full text of DOI:10.1021/ja901875v

Published on Web 07/24/2009
A New Gadolinium-Based MRI Zinc Sensor
Ana C. Esqueda,† Jorge A. Lo´pez,† Gabriel Andreu-de-Riquer,†
Jose´ C. Alvarado-Monzo´n,† James Ratnakar,‡ Angelo J. M. Lubag,‡
A. Dean Sherry,‡ and Luis M. De Leo´n-Rodr´ıguez*,†
Departamento de Qu´ımica, UniVersidad de Guanajuato, Cerro de la Venada s/n, Guanajuato,
Gto., C.P. 36040, Me´xico, and AdVanced Imaging Research Center, UniVersity of Texas
Southwestern Medical Center, 5323 Harry Hines BouleVard, Dallas, Texas 75390-9185
Received March 10, 2009; E-mail: lmdeleon@quijote.ugto.mx
Abstract: The properties of a novel Gd3+-based MRI zinc sensor are reported. Unlike previously reported
Gd3+-based MRI contrast agents, this agent (GdL) differs in that the agent alone binds only weakly with
human serum albumin (HSA), while the 1:2 GdL:Zn2+ ternary complex binds strongly to HSA resulting in
a substantial, 3-fold increase in water proton relaxivity. The GdL complex is shown to have a relatively
strong binding affinity for Zn2+ (KD ) 33.6 nM), similar to the affinity of the Zn2+ ion with HSA alone. The
agent detects as little as 30 µM Zn2+ in the presence of HSA by MRI in vitro, a value slightly more than the
total Zn2+ concentration in blood (∼20 µM). This combination of binding affinity constants and the high
relaxivity of the agent when bound to HSA suggests that this new agent may be useful for detection of free
Zn2+ ions in vivo without disrupting other important biological processes involving Zn2+
.
Introduction
in amyloid plaques. It is also known that pancreatic ꢀ-cells
release free chelatable Zn2+ simultaneously with release of
insulin such that the local concentration of Zn2+ surrounding
activated ꢀ-cells may be as high as 0.48 mM.9 Thus, monitoring
release of Zn2+ from ꢀ-cells in ViVo is an important goal in
understanding the etiology and treatment of diabetes.10
Zinc is the second most abundant trace element (after iron)
in mammalian tissues (the average adult human has ca. 3 g of
zinc).1 Zinc exists exclusively as a divalent ion (Zn2+) in biology
with most Zn2+ bound to proteins that play a pivotal role in
controlling gene transcription and metalloenzyme function.2
Today, there is broad awareness of Zn2+ signaling with a dozen
or more individual zinc-secreting cell types known.3 These
include cells in the submandibular salivary gland, the pancreatic
ꢀ-cells and pancreatic exocrine cells, the prostate epithelial cells,
Paneth cells in the intestine, mast cells, granulocytes, pituitary
cells and CNS neurons.4 Zn2+ is present in particularly high
concentrations in the mammalian prostate, pancreas and brain.
In addition to modulating neuronal transmission in brain, Zn2+
is reported to contribute to neuronal injury in certain acute
conditions, to suppress or induce apoptosis, and to induce the
formation of ꢀ-amyloid (ꢀA),5 which is reported to be related
to the etiology of Alzheimer’s disease.6,7 A considerable amount
of data now shows that Zn2+ and other metal ions (iron and
copper) contribute to formation of ꢀA deposits in the brain.8
Concentrations as high as 0.2-1 mM of Zn2+ have been found
Many disease conditions associated with either high or low
Zn2+ concentrations are still widely debated, and although it is
recognized that Zn2+ has many important cellular roles, its
physiological significance is not completely understood. Several
chemical Zn2+ sensors have been reported, most based on
fluorescence detection.11 While these have proven useful for
monitoring Zn2+ fluctuations in cells, their in ViVo application
is limited by light penetration in tissue, light scattering, and
toxic photobleached byproducts. To the best of our knowledge,
detection of free Zn2+ in ViVo has not been demonstrated to
date. Among alternatives to optical methods, magnetic resonance
imaging (MRI) is promising since this technique allows non-
invasive, high-resolution imaging of opaque bodies and moni-
toring of dynamic processes. There is considerable interest in
the development of MRI contrast agents (CAs) that respond to
biological events.12 Most MR contrast agents are based on
gadolinium (Gd3+) complexes that increase the relaxation rate
of water (r1) through exchange of a single Gd3+-bound water
molecule with bulk solvent. The design of responsive CAs is
typically based on changes in one or more factors that control
proton relaxation timesswater access to the metal, rotational
† Universidad de Guanajuato.
‡ University of Texas Southwestern Medical Center.
(1) Mills, C. F. Zinc in Human Biology; Springer-Verlag: New York, 1989.
(2) Auld, D. S. BioMetals 2001, 14, 271–313.
(3) Frederickson, C. J.; Perez-Clausell, J.; Danscher, G. J. Histochem.
Cytochem. 1987, 35, 579–583.
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B. P.; Suh, S. W.; Frederickson, C.; Fierke, C.; Herman, P. J. Neurosci.
Methods 2002, 118, 63–75.
(9) Kim, B. J.; Kim, Y. H.; Kim, S.; Kim, J. W.; Koh, J. Y.; Oh, S. H.;
Lee, M. K.; Kim, K. W.; Lee, M. S. Diabetes 2000, 49, 367–372.
(10) Chausmer, A. B. J. Am. Coll. Nutr. 1998, 17, 109–115.
(11) Kikuchi, K.; Komatsu, K.; Nagano, T. Curr. Opin. Chem. Biol. 2004,
8, 182–191.
(5) Mocchegiani, E.; Bertoni-Freddari, C.; Marcellini, F.; Malavolta, M.
Prog. Neurobiol. 2005, 75, 367–390.
(6) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353–356.
(7) Hardy, J. Curr. Alzheimer Res. 2006, 3, 71–73.
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6, 449–462.
(12) De Leon-Rodriguez, L. M.; Lubag, A. J. M.; Malloy, C. R.; Martinez,
G. V.; Gillies, R. J.; Sherry, A. D. Acc. Chem. Res. 2009, 42, 948–
957.
9
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Esqueda et al.
Scheme 1a
a (i) HBTU, DMF, (ii) TFA, DCM.
correlation times, metal bound water exchange ratessin re-
sponse to a specific stimulus (e.g., enzyme,13 protein,14 pH,15
metal,16 etc.). A few Zn2+-specific MRI CAs have been reported
previously. The first was reported by Nagano and co-workers17
who prepared a Gd3+ complex based on the diethylenetriamine-
pentaacetic acid (DTPA) ligand bifunctionalized with N,N-bis-
(2-pyridyl-methyl) ethylene diamine (BPEN), a moiety that has
shown to have a binding specificity for Zn2+ over other metals.
The relaxivity of this complex decreased from 6.1 to 4.0 mM-1
by Nagano forms a 1:2 complex with Zn2+, while the EuDOTA-
bisBPEN-dibutyl tetraamide PARACEST sensor (+3 charge)
only forms a 1:1 complex with Zn2+ 21 These differences must
.
reflect the differences in total charge on each Ln3+ complex.
Given these trends, a decision was made to investigate the Zn2+
binding properties of GdDOTA-bisBPEN diamide (net positive
charge), 3.
Conpound 3 was prepared as outlined in Scheme 1. Com-
pound 1 was first obtained by using a previous published
procedure following protection-derivatization-deprotection
schemes.22 Compound 1 was then coupled with 2 equiv of
BPEN (2) via activation of the free carboxylic acids with HBTU.
After product purification, the tert-butyl groups were removed
producing 3 in a 74% overall yield. The Gd3+ and Eu3+
complexes of 3 were prepared and further purified by HPLC.
s-1 (25 °C, pH 8, 7 T) in the presence of 1 equiv of Zn2+
,
presumably by restricting access of water to the Gd3+ coordina-
tion site. However, the relaxivity of this complex returned to
its initial value when a second Zn2+ was bound, so these features
make this system unattractive for testing in ViVo. The same
authors also reported a modified version of the DTPA derivative
wherein one of the pyridylmethyl groups was replaced by an
acetate moiety. This resulted in a complex that responded to
Zn2+ by changing its relaxivity from 4.8 to 3.5 mM-1 s-1 (25
The relaxivity of GdDOTA-diBPEN increased with addition
of Zn2+. In the absence of Zn2+, the relaxivity of the complex
was 5.0 ( 0.1 mM-1 s-1 (37 °C, pH 7.6, 0.1 M Tris buffer, 23
MHz) and this gradually increased to 6.0 ( 0.1 mM-1 s-1 with
addition of Zn2+ until 2 equiv had been added (Figure 1). This
shows that a 1:2 (Gd:Zn) complex is formed. These r1 values
are similar in magnitude to other GdDOTA-bis-amide complexes
previously reported.23 The relaxivity of GdDOTA-diBPEN
remained constant when dissolved in 0.1 M phosphate buffer
over a period of 24 h, indicating that the agent is stable in a
°C, pH 7.2, 7T) upon addition of 1 equiv of Zn2+ with no further
18
changes upon addition of a second equivalent of Zn2+
.
Although this was an improvement in one sense, both systems
result in an “on-off” response or image darkening upon Zn2+
bindingsclearly not an optimal situation. More recently, Meade
and co-workers19,20 reported an “off-on” Zn2+-responsive agent
derived from GdDO3A. In this agent, the metal water coordina-
tion site is occupied by a carboxylate group of a diaminoacetate
moiety attached through the fourth amino group of the macro-
cycle. When the diaminoacetate binds Zn2+, a water molecule
then coordinates to the Gd3+ which translates in an increase in
relaxivity from 2.3 to 5.1 mM-1 s-1 (37 °C, pH 7.4, 60 MHz),
a much more favorable 120% change. When dissolved in blood
serum, however, the change in relaxivity of this complex
changed much less in response to Zn2+, from 5.8 to 7.7 mM-1
s-1. Nevertheless, an in Vitro study showed that 100 µM Zn2+
could be detected by MRI by this agent in buffer and a Zn2+
binding constant of 240 µM was determined using a fluorescence
competition method. Given that one would like to detect Zn2+
at even lower levels in ViVo, continued development of agents
that respond to Zn2+ in ViVo under physiological conditions is
highly desirable.
(13) Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats,
R.; Jacobs, R. E.; Fraser, S. E.; Meade, T. J. Nat. Biotechnol. 2000,
18, 321–325.
(14) De Leon-Rodriguez, L. M.; Ortiz, A.; Weiner, A. L.; Zhang, S.;
Kovacs, Z.; Kodadek, T.; Sherry, A. D. J. Am. Chem. Soc. 2002, 124,
3514–3515.
(15) Zhang, S.; Wu, K.; Sherry, A. D. Angew. Chem., Int. Ed. 1999, 38,
3192–3194.
(16) Li, W. H.; Fraser, S. E.; Meade, T. J. J. Am. Chem. Soc. 1999, 121,
1413–1414.
(17) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Chem. Soc., Perkin
Trans. 2 2001, 1840–1843.
(18) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Narazaki, M.; Yokawa, T.;
Sakamoto, S.; Yamaguchi, K.; Nagano, T. Chem. Biol. 2002, 9, 1027–
1032.
(19) Major, J. L.; Parigi, G.; Luchinat, C.; Meade, T. J. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 13881–13886.
Results and Discussion
(20) Major, J. L.; Boiteau, R. M.; Meade, T. J. Inorg. Chem. 2008, 47,
10788–10795.
Gd3+-based MRI contrast agents derived from DOTA-like
macrocyclic ligands have advantages over their linear counter-
parts (DTPA) because of their higher thermodynamic stability
and kinetic inertness. Previous work has shown that the
GdDTPA-bisBPEN diamide agent (zero net charge) developed
(21) Trokowski, R.; Ren, J.; Kalman, F. K.; Sherry, A. D. Angew. Chem.,
Int. Ed. 2005, 44, 6920–6923.
(22) De Leon-Rodriguez, L. M.; Kovacs, Z.; Esqueda-Oliva, A. C.;
Miranda-Olvera, A. D. Tetrahedron Lett. 2006, 47, 6937–6940.
(23) Zhang, S.; Kovacs, Z.; Burgess, S.; Aime, S.; Terreno, E.; Sherry,
A. D. Chem.-Eur. J. 2001, 7, 288–296.
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Gadolinium-Based MRI Zinc Sensor
A R T I C L E S
Figure 2. Relaxivity of GdDOTA-diBPEN at 23 MHz and 37 °C in the
presence of MCl2, where M ) Zn2+, Cu2+, Ca2+, or Mg2+. All solutions
were prepared in 100 mM Tris buffer at pH 7.6 plus 600 µM human serum
albumin (HSA).
Figure 1. Relaxivity of GdDOTA-diBPEN at 23 MHz and 37 °C in the
presence of MCl2, where M ) Zn2+, Cu2+, Ca2+, or Mg2+. All solutions
were prepared in 100 mM Tris buffer at pH 7.6.
biological medium.24 The relaxivity of GdDOTA-diBPEN did
not change upon addition of Ca2+ or Mg2+ but did increase to
6.3 ( 0.1 mM-1 s-1 (37 °C, pH 7.6, Tris buffer, 23 MHz) with
addition of 2 equiv of Cu2+. Like Zn2+, the relaxivity did not
increase beyond this value with further addition of Cu2+. Given
that GdDOTA-bis-amide complexes are known to display slower
water exchange rates than required for optimal relaxivity, the
small increases in relaxivity promoted by binding of either Zn2+
or Cu2+ likely reflect an increase in water and/or proton
exchange rates, perhaps catalyzed by Zn2+-bound hydroxy
groups as previously found for the PARACEST-based Zn2+
sensor, EuDOTA-bisBPEN-dibutyl tetraamide21 or by creating
a more organized second sphere of water molecules (those bound
to Zn2+ or Cu2+) in close proximity to the single Gd3+-bound
water molecule of the complex. The mechanistic differences
that determine the relaxivity change in response to Zn2+ for
GdDOTA-diBPEN (altering water or proton exchange) as
compared to the DTPA derivative reported by Nagano and co-
workers17 (Gd3+ water coordination blockage when two BPEN
units coordinate one Zn2+ above the Gd3+) might be explained
by the difference in charges between these two Gd3+ complexes.
In the former, the complex has a net positive charge, while the
later Gd3+ complex is neutral so that a 1:1 Zn2+ complex
formation involving both BPEN units placing Zn2+ above the
Gd3+ is less electrostatically restricted compared to GdDOTA-
diBPEN. MALDI-TOF spectra of the GdDOTA-diBPEN-Zn2+
adduct showed two major peaks, m/z 1208 and 1108 assigned
to GdDOTA-Zn2(OH)4diBPEN and GdDOTA-Zn(OH)2diBPEN,
respectively (see Supporting Information Figure S1). The high
resolution 1H NMR spectrum of EuDOTA-diBPEN shows that
both possible coordination isomers are present in solution, the
square antiprismatic (SAP) and twisted square antiprismatic
(TSAP) in an approximate 3:2 ratio (see Supporting Information
Figure S2) and that this ratio does not change with addition of
Figure 3. Titration of GdDOTA-diBPEN 1 mM (triangles), and GdDOTA-
diBPEN 1 mM plus 2 mM of Zn2+ (squares) with HSA. All measurements
were made at 23 MHz and 37 °C in 100 mM Tris buffer at pH 7.6. The
solid curves represent the best fit to eq 3 in Supporting Information.
GdDOTA-Zn2diBPEN (per BPEN binding unit). As a reference,
a similar ligand, bis(6-pyridinylmethyl)amine has been reported
to form 1:1 complexes with Zn2+ with a KD of 26.9 nM.26
Given that GdDOTA-diBPEN has two aromatic rings per
BPEN unit, a further study was performed to investigate possible
interactions between the agent and human serum albumin
(HSA), the most abundant protein in blood and cerebral spinal
fluid. Since metal ion mediated binding of small molecules to
albumin has been reported previously,27 we also examined the
influence of Zn2+ on binding interactions between the agent and
albumin. A titration experiment performed by measuring the
relaxation rate of a solution of GdDOTA-diBPEN upon addition
of fatty acid-free HSA showed that the agent alone binds only
weakly with HSA possibly due to nonspecific electrostatic
interactions. Surprisingly, when 2 equiv of Zn2+ were added to
the agent prior to addition of HSA, a quite different result was
obtained (Figure 3). In this case, binding of the agent to HSA
was surprisingly strong and the relaxivity of GdDOTA-diBPEN
increased from 6.6 ( 0.1 to 17.4 ( 0.5 mM-1 s-1 when fully
bound to the protein (Figure 2), a 165% increase. A similar
increase in relaxivity was observed for GdDOTA-Cu2diBPEN
upon addition of HSA, but no change was observed for the
complex containing either Ca2+ or Mg2+. The increase in
relaxivity observed for GdDOTA-Zn2diBPEN when bound to
HSA likely reflects a slower rotational correlation time (τR) for
the complex when bound to the protein.
Zn2+
.
The apparent binding dissociation constant (KD) of GdDOTA-
Zn2diBPEN was determined by a competitive assay using the
commercially available fluorophore ZnAF-2F (see Supporting
Information and Figure S3). This dye was chosen since it shares
similar Zn2+ binding moieties as GdDOTA-diBPEN and binds
the metal with high affinity (KD) 5.5 nM).25 With the use of
the published dissociation constant for the dye, a binding
constant of 33.6 ( 0.4 nM was estimated for formation of
(24) Laurent, S.; Elst, L. V.; Copoix, F.; Muller, R. N. InVest. Radiol. 2001,
36, 115–122.
(26) Livieri, M.; Mancin, F.; Saielli, G.; Chin, J.; Tonellato, U. Chem.-
Eur. J. 2007, 13, 2246–2256.
(25) Hirano, T.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Am. Chem. Soc.
2002, 124, 6555–6562.
(27) Klotz, I. M.; Loh Ming, W.-C. J. Am. Chem. Soc. 1954, 76, 805–814.
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Esqueda et al.
Figure 5. T1-weighted phantom MR images. Images of a 100 µM solution
of GdDOTA-diBPEN in Tris buffer with 600 µM HSA at various
concentrations of ZnCl2. Spot A, 200 µM Zn2+; B, 100 µM Zn2+; C,
30 µM Zn2+; D, 0 µM Zn2+; E, 600 µM HSA but no contrast agent; F, Tris
buffer only (no HSA or contrast agent). Repetition time (TR) ) 200.0 ms;
echo time (TE) ) 8.3 ms; data matrix ) 128 × 128. FOV ) 15 × 15
mm2. Single scan, no averaging. A single slice of 5 mm was acquired
centered at the sample height (10 mm). Window level and image size has
been adjusted for printing purposes. Intensity scale is given in arbitrary
units. Images and T1 values were obtained at 400 MHz (9.4 T).
Figure 4. Displacement fluorescence plots for warfarin (squares, 5 µM)
and dansylglycine (circles, 5 µM) from equimolar HSA by GdDOTA-
Zn2diBPEN at 25 °C in 100 mM Tris buffer at pH 7.6. The solid curves
represent the best fit to the data.
To determine the KD of GdDOTA-Zn2diBPEN with HSA,
the proton relaxation enhancement, ε* (eq 2 in Supporting
Information) versus HSA concentration was then fitted to a
single site-specific binding model28 (eq 3 in Supporting
Information). The best fit to the model gave a KD of 42 ( 9
µM for GdDOTA-Zn2diBPEN binding to HSA.
Fluorescent displacement experiments were performed using
warfarin and dansylglycine as probes to determine whether
GdDOTA-Zn2diBPEN binds at site 1 or 2 of subdomain IIA or
IIIA of HSA.29 As shown in Figure 4, there is a decrease in
dansylglycine fluorescence when increasing amounts of Gd-
DOTA-Zn2diBPEN are added which suggests that the complex
binds HSA in site 2 of subdomain IIIA. The KD for GdDOTA-
Zn2diBPEN to HSA was determined from these displacement
results by assuming one site binding (eqs 4 and 5 in Supporting
Information).30 The fitting of the data gave a KD of 48 ( 6
µM, a value similar to that described above in the proton
enhancement titration experiment. Similar experiments were
performed using GdDOTA-diBPEN alone (no divalent metal
ion), but no displacement of either fluorescent probe was
observed.
Although nonmetal ion mediated, previous reports of posi-
tively charged GdDOTA complexes that bind HSA in site 2
can be found in the literature, but no definitive binding model
has been provided.31 To provide a model of how GdDOTA-
Zn2diBPEN might bind to HSA, we first performed a structural
analysis of site 2 using literature data. Site 2 comprises a largely
preformed hydrophobic cavity (∼16 Å deep and ∼8 Å wide
with a radius curvature ∼8.5 Å) with distinct polar features. It
contains a cationic group located close to one side of the
entrance of the binding pocket (R410) but also contains S489,
E382, and N386 located near the surface on the opposite side.
Y411 is centered inside the cavity (as a reference, site 1 contains
a cluster of four cationic residues at the entrance of the cavity
K195, K199, R218, R222). Unlike binding site 1 where the
binding entrance is enclosed by subdomains IIB and IIIA, the
entrance to site 2 is not encumbered and is exposed to solvent.
It is also known that binding to this site is favored with
compounds that are planar or near planar and that contain
aromatic rings.32 The fact that GdDOTA-diBPEN interacts only
weakly with HSA, while Zn2+ enhances this binding interaction,
considerably indicates that Zn2+ complexation “locks” the two
aromatic pyridine rings, making the ZnBPEN unit better
configured to fit into the binding site. Presumably, the agent
might first interact with S489, E382, and N386 at one side of
the entrance given its positive net charge, and once inside the
cavity, the hydroxyl group of Y411 may interact with the Zn2+
and thereby provide further binding stability (see Supporting
Information Figure S4 for a molecular model depicting the
binding of GdDOTA-Zn2diBPEN to HSA). With this model, it
is reasonable to consider that the presence of R410 on the
opposite side of the cavity restricts rotational freedom of the
bound agent through the methylene units connecting BPEN to
the GdDOTA moiety. This could explain the dramatic increase
in relaxivity that is observed for the agent bound to HSA, similar
to that previously reported for positively charged GdDOTA type
complexes.31
In Vitro MR images of GdDOTA-diBPEN at 9.4-T in the
presence of various Zn2+ concentrations demonstrates that Zn2+
can be detected at concentrations as low as 30 µM (Figure 5).
Unlike Zn2+, there were no discernible differences in image
intensity for samples of GdDOTA-diBPEN with added Ca2+
or Mg2+ (not shown).
To determine the effectiveness of GdDOTA-diBPEN as a
relaxation agent under physiological conditions, relaxivity
measurements were also performed in human blood serum. In
this case, the relaxivity increase upon addition of Zn2+ was
smaller, from 6.1 ( 0.1 to 8.6 ( 0.1 mM-1 s-1 (37 °C, pH 7.6,
Tris buffer, 23 MHz) than observed previously for the agent in
the presence of fatty acid free HSA. Nevertheless, this 40%
increase in relaxivity should still be enough to be detectable by
MRI in ViVo upon release of free Zn2+
.
Upon the basis of the binding equilibrium constants between
GdDOTA-diBPEN, Zn2+ and HSA (multiple equilibria) and the
relaxivities of GdDOTA-diBPEN, GdDOTA-Zn2diBPEN, and
ternary GdDOTA-Zn2diBPEN-HSA, one should be able to make
a reasonable prediction of the detection limits of the Zn2+ sensor
system in ViVo. The minimum requirement is that the agent should
be able to sense free, chelatable Zn2+ released in response to a
particular metabolic event or metabolic abnormality. The CA should
have a relatively strong binding affinity for Zn2+ but not so strong
(28) Mildvan, A. S.; Cohn, M. Biochemistry 1963, 2, 910–919.
(29) Krach-Hansen, U.; Chuang, V. T. G.; Otagiri, M. Biol. Pharm. Bull.
2002, 25, 695–704.
(30) Yung Chi, C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099–
3108.
(31) Montgomery, C. P.; New, E. J.; Parker, D.; Peacock, R. D. Chem.
Commun. 2008, 4261–4263.
(32) Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.; Otagiri,
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Figure 6. (A) Calculated plots of ∆R1 versus GdDOTAdiBPEN concentration for a 475 µM Zn2+ rise from physiological conditions at various dissociation
constants of the agent to Zn2+. [HSA] ) 600 µM and relaxivity values were used as determined in blood serum. (B) Calculated plots of the percent of total
GdDOTA-Zn2BPEN relative to the initial concentration of unbound agent (500 µM) versus the concentration of Zn2+ at various dissociation constants of the
agent with Zn2+. All other parameters were used as in panel A.
that it interferes with other endogenous metabolic processes. Given
that HSA (a main component in blood) has a binding affinity for
Zn2+ of 29.5 nM33 and if one assumes that the local extracellular
concentration of free chelatable Zn2+ near excitable cells rises to
at least 475 µM (as predicted for pancreatic ꢀ-cells),9 then one
can project the ideal tissue concentration of the Zn2+ sensor required
to detect such an increase. If one first assumes that a standard
clinical dose of CA will be injected (0.1 mmol/kg corresponds to
an average extracellular tissue concentration of ∼500 µM) and that
[HSA] in blood is ∼600 µM, then a multiple equilibrium model
(see Supporting Information) using the binding constants reported
here predicts a steady-state concentration of ternary GdDOTA-
Zn2diBPEN-HSA complex of ∼131 µM with 27% of the total
agent bound to Zn2+. Given the relaxivity values of the various
GdL species in human blood serum, this would correspond to a
relaxation rate change (∆R1) of 0.31 s-1 (∼10%) between a high
Zn2+ situation (475 µM) and normal physiology (∼20 µM Zn2+
in blood).34 This change in relaxation rate would be easily detected
in an image. Ideally, it would be preferable to use a much lower
concentration of sensor at a level where the agent is essentially
silent (no “background signal”) under normal physiological Zn2+
levels but then increases upon Zn2+ release from tissues. The lowest
concentration of agent required to detect 475 µM of Zn2+ can be
determined by extrapolating the previous calculation to a ∆R1 of
0.07 s-1, which is the predicted detection minimum of a targeted
GdL sensor reported recently.35 The calculated value corresponds
to 50 µM GdDOTA-diBPEN (Figure 6A) which coincides with
the actual “silent” sensor condition of 50 µM GdDOTA-diBPEN
in human blood serum. So, given the relaxivity and binding
characteristics of the Zn2+ sensor reported here, one would
anticipate being able to inject only minimal amounts of sensor (50
µM) and still detect increases in Zn2+ concentrations approaching
those expected for excitable cells. A second question that can be
addressed using this same model is how important is the binding
affinity between the sensor and Zn2+ for it to be useful in ViVo?
Figure 6B illustrates the estimated fractional amount of sensor-
Zn2+ complex formed with 500 µM sensor and the indicated
amount of total Zn2+ for four different binding constants. If one
again uses a ∆R1 of 0.07 s-1 as the lower limit of detection (this
corresponds to ∼6% of the total agent bound to Zn2+), then one
predicts that a sensor with a KDGdLZn ) 1 µM would only detect
changes in Zn2+ concentration approaching 500 µM, while a sensor
with a KDGdLZn ) 33.6 nM (the agent reported herein) should detect
changes in Zn2+ as low as ∼100 µM. This highlights the
importance of the Zn2+ binding constant in the in ViVo study.
Conclusions
A novel GdDOTA-based Zn2+ sensor that displays only a
modest relaxivity increase upon binding to 2 equiv of Zn2+ but
shows a substantially larger increase in relaxivity (165%) when
the ternary GdL-Zn2+ complex is bound to HSA is reported. The
Zn2+ sensor does not respond to either Ca2+ or Mg2+ but does
show a similar relaxation enhancement in the presence of Cu2+.
This competition, however, would not likely interfere in ViVo
because Cu2+ is typically found at much lower concentrations in
tissues compared to Zn2+. In Vitro imaging studies indicate that
the present agent can detect Zn2+ at concentrations as low as ∼30
µM in presence of HSA. Further analysis shows that the binding
dissociation constant of any CA to Zn2+ with relaxivities in serum
close to the agent presented herein should be at least 1 µM if the
goal is to apply such systems in ViVo.
Acknowledgment. The completion of this work was possible due
to the support of CONACYT, Mexico sabbatical fellowship 75381
and by NIH grant DK-058398.
Supporting Information Available: General experimental
details; full experimental details including synthetic and charac-
terization data for compound 3, and its Gd3+ and Eu3+ complexes;
full experimental details for fluorescence displacement experiments,
relaxivity measurements, data fitting, modeling programs and
mathematical derivations are provided. A proposed model figure
of the binding of the CA to HSA is also included. This material is
available free of charge via the Internet at http://pubs.acs.org.
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