Exofacial protein thiols as a route for the internalization of Gd(III)-based complexes for magnetic resonance imaging cell labeling

Article abstract of DOI:10.1021/jm901876r

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thio

Full text of DOI:10.1021/jm901876r

J. Med. Chem. 2010, 53, 4877–4890 4877
DOI: 10.1021/jm901876r
Exofacial Protein Thiols as a Route for the Internalization of Gd(III)-Based Complexes for Magnetic
Resonance Imaging Cell Labeling
Giuseppe Digilio,† Valeria Menchise,‡ Eliana Gianolio,§ Valeria Catanzaro,§ Carla Carrera,§ Roberta Napolitano,§
Franco Fedeli,§ and Silvio Aime*,§
†Department of Environmental and Life Sciences, University of Eastern Piedmont “A. Avogadro”, Viale T. Michel 11, I-15121 Alessandria, Italy,
‡Institute for Biostructures and Bioimages (CNR), c/o Molecular Biotechnology Center, University of Turin, Via Nizza 52, I-10125 Torino, Italy,
and §Department of Chemisty, IFM and Center for Molecular Imaging, University of Turin, Via Nizza 52, I-10125 Torino, Italy
Received December 20, 2009
Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties
on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a
large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for
suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or
DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs.
GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 ꢀ 1010 Gd(III) atoms/cell),
whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A
based compounds, that having the longest spacer (compound GdL1A) showed the best labeling effic-
acy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without
sequestration of the Gd(III) chelate within subcellular structures such as endosomes.
Introduction
agents (CA) for MRI detection to be possible. This makes it
mandatory to develop highly sensitive MRI labeling agents
and/or to optimize strategies to accumulate a large number of
these agents to cells for visualization.5 Labeling procedures
typically involve the internalization of the imaging reporters.
For instance, a veryhighMRI sensitivityhas beenobtained by
loading cells with nanosized superparamagnetic iron oxide
(IO) particles as T2-contrast agents (these agents produce
dark-spot image contrast, being henceforth also called nega-
tive agents).6,7 Efficient loading (up to (0.5-2) ꢀ 107 nano-
particles per cell) withIOparticles was achieved by coating the
particles with dextran to improve biocompatibility and by
further functionalization of the dextrane surface with a HIV-
tat derived peptide sequence as the membrane penetrating
vector.8 If positive image contrast is sought (bright spot image
contrast), Gd(III) complexes are best suited.5,9-11 The most
direct route for the internalization of Gd(III)-based CAs takes
advantage of pinocytosis, a physiological cellular process
characterized by the spontaneous invagination of the cell
membrane to form endosomal vesicles. During this process,
CAs added at high concentration in the culture medium
(50-100 mM) can be internalized to a significant extent. By
this procedure it has been possible to load several kinds of
cells, including stem cells, with suitable levels of GdHPDO3A
(a compound approved for clinical practice).12,13 As far as
contrast enhancement isconcerned, the internalization of CAs
within endosomal vesicles may be an issue; as the exchange
rate of water molecules across the endosomal membrane is
slow, an unwanted “quenching” of relaxivity is observed at
high concentrations of internalized Gd(III).14,15 To avoid
endosomal segregation, several procedures have been explo-
red, including electroporation14 or conjugation of the CAs
with polyarginine based peptides as membrane penetrating
The concept of tissue repair through transplantation or
grafting of suitable progenitor/stem cells is gaining more and
more strength as the forthcoming therapeutic answer to seve-
ral currently untreatable diseases. Cells can be “instructed” to
repair damaged tissues, and cells can find their way to the
tissue that has to be repaired. The development of cell-based
therapies will greatly benefit from any method allowing for a
noninvasive and repetitive monitoring of the delivery of repair-
ing cells to the microenvironment of interest. The possibility of
visualizing cell differentiation or response to biochemical sti-
muli would also be of immense value. “Cellular imaging” is a
relatively new and very rapidly evolving branch of in vivo
imaging techniques devoted to provide answers to these
needs.1-4 Among the armory of available in vivo imaging
techniques, MRIa is one of the most promising because of its
great spatial resolution (<100 μm with modern high field
equipment), lack of invasiveness (no ionizing radiation), and
ease of adaptability to the clinical practice. Cells to be visuali-
zed by MRI must be labeled with suitable compounds, often
referred to as “contrast agents” or “imaging probes”. Because
MRI suffers from low inherent sensitivity, cells to be tracked
must be heavily labeled with suitable paramagnetic contrast
*To whom correspondence should be addressed. Phone: þ 39 011
6706451. Fax: þ 39 011 6706487. E-mail: silvio.aime@unito.it.
a Abbreviations: EPTs, exofacial protein thiols; MRI, magnetic
resonance imaging; NMRD, nuclear magnetic relaxation dispersion;
CA, contrast agent; DO3A, 1,4,7,10-tetraazacyclododecane-1,4,7-tria-
cetic acid; DTPA, diethylenetriamine-N,N,N0,N0,N00-pentaacetic acid;
P2T, pyridine-2-thione; DTNB, 5,50-dithio-bis(2-nitrobenzoic acid);
NEM, N-ethylmaleimide; TFA, trifluoroacetic acid; DCC, N,N0-dicy-
clohexylcarbodiimide; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethane-
sulfonic acid; EBSS, Earl’s balanced salt solution.
r
2010 American Chemical Society
Published on Web 06/09/2010
pubs.acs.org/jmc

4878 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Digilio et al.
Scheme 1. Structure and Reactivity of the GdDO3A and the GdDTPA Based Probes for the Labeling of EPTs
vectors.16 An alternative concept to avoid the relaxivity
“quenching” associated with endosomal entrapment is that
of anchoring the imaging reporter on the extracellular side of
the cell membrane rather than pursuing its internalization.
This has been achieved, for instance, by forming a supramo-
lecular adduct between the cell, a cationic polyarginine pep-
tide, and the contrast agents under the form of negatively
charged micelles.17 Whatever the labeling strategy is, the
minimum amount of Gd(III) complexes to be delivered to
cells for MRI detection can be estimated by the following rule:
(r1)(N)>109, where N is the number of Gd(III) complexes per
cell and r1 is the actual relaxivity (mM-1 s-1) of the Gd(III)
complexes within the cellular environment.5
labeling method has finally been applied to cells other than
human myeloid leukemia K562 cells, initially chosen as a
model system, to test the broad range application of this
method.
Results
Synthesis of the Paramagnetic Probes. Ligand L1A has
been synthesized according to the route shown in Scheme 2.
The spacer designed to connect the activated disulfide
group with the DO3A-like chelating cage was synthesized
by reacting N-(benzyloxycarbonyl)-1,2-diamineethane with
6-bromohexanoyl chloride, and the product (compound 1)
was purified. Compound 1 was then added dropwise to
a solution of DO3A-tris-tert-butyl ester in acetonitrile to
obtain compound 2, which was purified by flash chroma-
tography on silica. The cleavage of the benzyloxycarbonyl
group has been achieved by hydrogenation with Pd/C at
atmospheric pressure and room temperature to yield the
free primary amino group, which was finally reacted with
3(2-pyridyldithio)propionic acid 4 in the presence of DCC.
After removal of the tert-butyl ester protecting groups by
treatment with trifluoroacetic acid, the compound was
carefully purified to >98% by liquid chromatography on
Amberlite XAD1600. Ligand L1B was synthesized starting
from N-(benzyloxycarbonyl)-2-bromoethylamine (13) (the
latter prepared from reaction of 2-bromoethylamine and
benzyl chloroformate in water at pH 7) and DO3A-tris-tert-
buthyl ester in acetonitrile. Hydrogenation to remove the
benzyloxycarbonyl group, coupling to 4, and deprotection
of the carboxyl groups on the DO3A chelating cage were
carried out essentially as for L1A. Ligand L2B, based on the
DTPA chelating cage, was synthesized according to the
route in Scheme 3. Compound 8 (BF3 salt) was synthesized
by reacting L-cysteine with aldrithiol in ethyl acetate in the
presence of BF3 ether ethylic complex. The primary amine
was then dialkylated with 9 to give 10, which was purified by
flash chromatography on silica. Compound 11 (i.e., ligand
L2B), obtained after deprotection of the carboxylic groups
with TFA, was, however, not stable in water. HPLC chro-
matograms done at increasing time lapses after dissolution
of L2B in water (at basic, neutral, and acidic pH) revealed
Recently we have undertaken a new method for labeling
cells withGd(III) based CAs exploiting the chemical reactivity
of protein thiols that are exposed on the outer surface of cell
membranes.18 The imaging probe we have first synthesized19
(GdL1A in Scheme 1, alias of Gd-DO3AS-Act in ref 18) is
composed of a Gd(III) chelate, a reactive moiety for the
recognition of thiols based upon the 2-pyridyldithio function,
and a flexible linker connecting them. This probe can bind to
exofacial protein thiols (EPTs) through a covalent disulfide
bridge, leading to a satisfactory level of labeling in human
myeloid leukemia K562 cell line. The proposed labeling
procedure can be thought as being quite general, as it is known
that several cell types express EPTs at sufficiently high levels:
for instance, the concentration of EPTs is in the range 9-62
nmol SH/106 cells for Chinese hamster ovary (CHO) cells,20
3
4-15 nmol SH/106 cells for lymphocytes,21 15-30 nmol SH/
3
3
106 cells for human fibrosarcoma (HT1080) cells.22 EPTs in
these cells are reactive toward several chemicals, forming
either reversible adducts (mixed disulfides) or irreversible
adducts (thio ethers by coupling with maleimide derivatives
or iodoacetamide derivatives).23,24
The purpose of this work is to clarify some aspects under-
lying the mechanism of EPT mediated cell labeling and assess
the molecular properties of the labeling probes that play key
roles for efficient labeling. Therefore, four probes based on
either the DO3A chelate or the DTPA chelate and differing in
the length of the spacer connecting the Gd(III) chelate with
the thiol reactive unit have been synthesized, characterized,
and evaluated for labeling efficacy (Scheme 1). The proposed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4879
Scheme 2. Synthesis of the DO3A Based Probes GdL1A and GdL1B
that the compound progressively released 2-pyridylmercap-
tane. For this reason the complexation of L2B with Gd(III)
was performed very quickly on a freshly prepared aque-
ous solution of the ligand (pH 7) by adding GdCl3 (0.95
equiv). The solution containing the complex was immedi-
ately freeze-dried. Unlike the free ligand, the GdL2B com-
plex was stable in water. HPLC analysis of a solution of
GdL2B in water (pH 7, room temperature) showed no
formation of 2-pyridylmercaptane within 2 weeks. The
DTPA-like L2A ligand was synthesized starting from 4
and 20, whose synthesis has been described elsewhere.25
The product 21 was purified by liquid chromatography on
silica and deprotected with TFA. Unlike L2B, ligand L1B is
stable in water. The complexation with Gd(III) of all ligands
has been done according to the method described in ref 11,
allowing a slight excess of the ligand (typically 2-3%) to
ensure that all Gd(III) is under the complexed form, as free
gadolinium is known to be readily taken up by cells.26 The
orange xylenol test27 confirmed complete complexation of
Gd(III).
1H and 17O NMR Relaxometric Characterization of the
Probes. The most relevant parameter summarizing the effi-
cacy of a Gd(III) complex as a contrast agent (CA) for MRI
applications is the millimolar relaxivity (r1mM, mM-1 s-1),
defined as the enhancement of the relaxation rate of sol-
vent water protons promoted by the paramagnetic complex
at 1 mM:
R1obs ¼ R1p þ R1w
ð1Þ
mM
R1p ¼ r1 ½CAꢁ
ð2Þ
In these formulas proton longitudinal relaxation rates R1
(s-1) are related to longitudinal relaxation times T1 (s) by the
relationship R1 = 1/T1. R1obs is the measured water proton
relaxation rate, R1p the paramagnetic enhancement to the
water proton relaxation rate, R1w the relaxation rate of pure
water, and [CA] the concentration of the Gd(III) complex in
mmol/L. The higher the millimolar relaxivity, the better the
ability of a given compound to enhance image contrast, other
conditions being equal. Although the physicochemical factors

4880 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Digilio et al.
Scheme 3. Synthesis of the DTPA Based Probes GdL2A and GdL2B
Table 1. Millimolar Relaxivities (mM-1 s-1, 25 °C) of the Probes in
Different Chemical Environments
contributing to the relaxivity of a given compound are
numerous and the theory governing them rather complex,28,29
the differences between the relaxivities of chemically homo-
geneous Gd(III) chelates can be explained at a first approxi-
mation in terms of three fundamental parameters, namely, (i)
the hydration number q, describing how many water mole-
cules are directly coordinated to the metal center (inner
HEPES buffer, pH 7.4,
with 20 mM lactate
HEPES buffer, pH 7.4
K562
cell pellets
300 MHz 300 MHz
20 MHz
300 MHz
20 MHz
DO3A
GdL1A
GdL1B
DTPA
GdL2A
GdL2B
6.0a
9.0
6.3
4.7b
6.7
6.9
5.4
7.4
5.9
4.0
5.8
5.7
3.5
3.0
3.1
2.2
sphere water molecules); (ii) the mean water residence life-
time τM, describing the exchange dynamics of inner sphere
water molecules with bulk water; and (iii) the reorientational
correlation time for molecular tumbling τR, describing the
overall rotational tumbling motions of the molecule.
2.6
2.5
2.0
2.3
Table 1 lists the millimolar relaxivities of the four com-
plexes in different environments, together with the relaxivity
of parent GdDO3A and GdDTPA.9 The relaxivities of the
DTPA-based complexes (GdL2A, GdL2B) are very similar
to each other and larger than that of parent GdDTPA be-
cause of the increased molecular size due to the introduction
of the functionalizing moieties (τR effect). These relaxivities
fall well within the range typical for low molecular weight
GdDTPA based structures and are well consistent with q=1
for both GdL2A and GdL2B. In comparison, the relaxivities
within the GdDO3A-based compounds (GdL1A, GdL1B,
and parent GdDO3A) show a less straightforward beha-
vior. Both GdL1A and GdL1B would be expected to have
a See also ref 31. b Taken from ref 9.
relaxivities higher than that of parent GdDO3A because of
their larger molecular size and because of the faster water
exchange kinetics typically observed for N-alkyl or N-aryl
derivatives of GdDO3A.30 However, only GdL1A shows
the expected relaxivity increase with respect to GdDO3A,
whereas the relaxivity of GdL1B is almost identical to that of
GdDO3A. Nuclear magnetic relaxation dispersion (NMRD)
profiles and 17O-R2p versus temperature profiles were
then acquired (Figure 1), as the multiparametric fitting of such
profiles according to the Salomon-Bloembergen-Morgan

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4881
Figure 1. (A) 1H NMRD profiles of GdL1A (solid squares) and GdL1B (open squares): probe concentration of 1.0 mM, HEPES buffer, pH
7.4, 25 °C. (B) 17O-R2p versus temperature profiles of GdL1A (solid squares) and GdL1B (open squares): probe concentration of 5 mM,
HEPES buffer, pH 7.4, 14.2 T. The solid lines represent best fit curves according to the Solomon-Bloembergen-Morgan theory with
parameters reported in Table 2.
Table 2. Relaxation Parameters for GdL1A and GdL1B Obtained after
Fitting of the NMRD and 17O-R2p versus T Profilesa
q
τm (ns)
τ
R (ps) τv (ps) Δ2 (s-2
)
ΔHm (kJ mol-1
)
GdL1A 2.0
GdL1B 1.1
28
13
113
110
22
29
6.2 ꢀ 1019
59
66
2.8 ꢀ 1019
a NMRD profiles were analyzed following Solomon-Bloembergen-
Morgan theory for the inner-sphere relaxivity and the Freed model for
the outer-sphere relaxivity (see text), fixing the diffusion coefficient (D)
at 2.24 ꢀ 105 cm2 s-1, the distance between Gd3þ and the inner sphere
˚
water protons (r) at 3.1 A, and the distance of closest approach of outer-
sphere water protons at 3.8 A. 17O-R2p versus T profiles were analyzed
˚
according to the Swift and Connick equation by fixing the distance
between Gd3þ and the inner sphere water oxygen (fixed to the values
determined from 17O-R2p versus T profiles of the complexes) at 2.5 A
˚
and the Gd-17O scalar coupling constant (A/p) at -3.8 ꢀ 106 rad s-1
.
theory of paramagnetic relaxation28 can give a more quanti-
tative picture of the contribution of each relaxation para-
meter to the observed relaxivity. The most relevant outcome
of such analysis (Table 2) is that, unlike GdL1A or GdDO3A
both having q = 2, GdL1B is characterized by q = 1. The
lower hydration can be explained by considering that the
spacer arm in GdL1B can fold to coordinate the Gd(III)
center, likely through the oxygen atom belonging to the
amide group. In doing so, one of the two inner sphere water
molecules that typically resides on the paramagnetic center
in DO3A based complexes is displaced,31 resulting into a
lower hydration. The quantitative analysis of the tem-
perature dependence of transverse relaxation rate (R2p) of
the metal bound 17O water resonance (Figure 1B) is consid-
ered the method of choice for the evaluation of the exchange
lifetime of the coordinated water molecule(s) (τM).32,33 In the
case of both the DO3A derivatives considered in this study,
very short τM values have been determined, namely, 28 and
13 ns for GdL1A and GdL1B, respectively. These values are
in the range of optimal values for the attainment of very high
relaxivity once the molecular tumbling of the system is
lengthened as a consequence of the formation of a macro-
molecular adduct.34 The difference in the amplitude of the
two profiles reported in Figure 1B (GdL1A shows 17O-R2p
values higher than those of GdL1B) is a consequence of the
higher hydration number of GdL1A with respect to GdL1B.
The Gd(III) ion in DO3A based structures can establish
coordinative bonds with anions (such as carboxylates) other
than those strictly belonging to the DO3A cage, leading to
the formation of ternary complexes characterized by their
Figure 2. Titration curves of GdL1A (solid squares) and GdL1B
(open squares) with lactate (0.47 T, HEPES buffer, pH 7.4, 25 °C).
The lines represent the best fitting of the curve according to the
model described in ref 37, from which the association constants (Ka)
and the millimolar relaxivities of the ternary complexes (r1bound) can
be extracted.
own relaxation properties.35,36 In this article it will become
clear that the formation of such a ternary complex has
important implications for the understanding of the EPT
dependent labeling mechanism. Therefore, we provide here
the characterization of the interaction between the DO3A
based probes and foreign carboxylates, such as lactate. The
0.5 mM solutions of either GdL1A or GdL1B have been
added with increasing amounts of lactate, which is known to
have a good affinity for GdDO3A and related compounds.37
The observed water proton relaxation rate R1obs decreases as
the concentration of lactate increases (Figure 2) because the
coordination of Gd(III) by lactate decreases the hydration at
the paramagnetic center. By fitting of the titration curve to
the model described in ref 37, the association constants (Ka)
and the relaxivity of the GdL1A/lactate and GdL1B/lactate
ternary complexes (r1bound) could be extracted. Association
constants of 1000 ( 60 M and 330 ( 40 M for GdL1A and
GdL1B, respectively, were found. The slightly higher affinity
of GdL1A with respect to GdL1B for lactate is likely due to
the fact that lactate binding in GdL1B partially competes
with intramolecular self-coordination by the functionalized
bound
arm. The relaxivities r1
of the ternary complexes were
2.3 and 2.4 mM-1 s-1 for GdL1A and GdL1B, respectively,

4882 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Digilio et al.
Figure 4. Amount of pyridine-2-thione (P2T) released during the
incubation of K562 cells (4 h, 37 °C) with the probes, expressed as
the number of released P2T molecules per single cell.
the incubation medium, pelleted, resuspended, and washed
to remove as much as possible the unreacted or aspecifically
bound probe and finally subjected to mineralization and
quantitative analysis of Gd(III) by a relaxometric assay.12,26
This experimental setup provides the measurement of the
total amount of Gd(III) taken up by cells, which is plotted in
Figure 3 as a function of the concentration of the probes in
the incubation medium. Parent GdDO3A and GdDTPA were
used as controls for GdL1A/GdL1B and GdL2A/GdL2B,
respectively, to evaluate the level of Gd(III) uptake unrelated
to the EPT chemistry. Among the DO3A-based probes,
GdL1A shows by far the highest levels of Gd(III) uptake
(as many as 1.2ꢀ1010 Gd(III) atoms/cell), while GdL1B has
a lower but still significant uptake (up to 2.2 ꢀ 109 Gd(III)
atoms/cell). The relationship between the total amount of
Gd(III) taken up by these two probes and the concentration
of the probe in the incubation medium is approximately
linear. No plateau is observed at the higher probe concentra-
tions, indicating that EPTs binding sites are not saturable.
On the other hand, the DTPA-based probe GdL2A showed a
barely detectable uptake, whereas GdL2B did not show any
significant uptake, neither in absolute terms nor in compari-
son with parent GdDTPA as the control (Figure 3B).
To gain further insight into the cause of the large differ-
ences in the uptake of the DO3A-based and DTPA-based
probes, the ability of each of the complexes to target EPTs
was evaluated by UV-vis spectrophotometry. When the
probes react with EPTs to form a disulfide bridge, pyridine-
2-thione (P2T) is released as a byproduct (Scheme 1) in the
incubation medium and it absorbs light at 343 nm. There-
fore, the increase in A343 from the start of the incubation to
the end of the incubation gives an estimate of the amount of
probe that has reacted with EPTs. Figure 4 shows that the
amount of P2T released increases linearly with the concen-
tration of the probe in the medium, with little (if any)
differences between the four probes, indicating that all
probes react with EPTs with similar reaction yields. Inter-
estingly enough, the release of P2T is uncorrelated with
Gd(III) uptake by cells. The DO3A-based probes react with
EPTs and are taken up by cells, whereas the DTPA-based
ones are poorly or not taken up at all while reacting with
EPTs to the same extent as the DO3A-based counterparts
do. Therefore, the different efficiency of cell labeling shown
by the two probe families cannot be explained on the basis of
a different reactivity toward EPTs. Finally we note that for
Figure 3. Uptake of Gd(III) chelates (expressed as Gd(III) atoms
per single cell) by K562 cells as a function of the concentration of the
probes in the incubation medium (4 h, 37 °C): (A) GdDO3A based
probes (GdL1A/GdL1B) with GdDO3A as the control; (B) GdDTPA
based probes (GdL2A/GdL2B) with Gd-DTPA as the control.
Note that the scaling of the y-axis in panels A and B differs by 2
orders of magnitude, pointing out the much higher labeling efficacy
of the GdDO3A based probes compared to the GdDTPA based
counterparts.
indicating that these complexes essentially behave as outer
sphere systems (i.e., they have no inner sphere water
molecules). This is due to the fact that lactate behaves as a
bidentate ligand, displacing both water molecules from the
Gd(III) center.37 In summary, GdL1A and GdL1B have q=
2 and q = 1, respectively, unless they interact with carboxy-
lates (or other coordinating anions) to form ternary com-
plexes characterized by the absence of inner sphere water
molecules. The interaction of GdDO3A-like complexes with
further anions is favored by the formal neutral net charge in
the complex and by the fact that the DO3A chelating cage is
heptadentate, leaving two easily accessible coordination sites
free on the Gd(III) center.
Labeling of K562 Cells. Preliminary to cell labeling with
Gd(III) chelates, the concentration of EPTs on K562 cells
has been assessed through the DTNB assay.18,20 The number
of DTNB-titrable EPTs is in the range (5.0-18.0) ꢀ 109
EPTs/cell, depending on the cell density in culture flasks (see
Supporting Information Figure S1). In typical cell labeling
experiments, 5 million K562 cells at a density of 1.5 ꢀ 106
cells/mL (displaying (5-7) ꢀ 109 EPTs/cell) were incubated
in a minimum medium (EBSS, Earl’s balanced salt solution
with HEPES buffer, thiol free) for 4 h at 37 °C in the presence
of the probe (0.5-3.5 mM). At the end of the incubation
period, cells showed a viability higher than 90%, indicating
an overall good tolerability. Cells were then separated from

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4883
Figure 5. Uptake of GdL1A (Gd atoms/cell) by K562 cells as a
function of the concentration of GdL1A in the incubation medium
(1 h incubation): white, labeling carried out at 37 °C; black, labeling
carried out at 4 °C.
GdL1A, showing the best labeling efficiency, the amount of
reacted probe per single cell is about 3 times larger than the
amount of Gd(III) units found in cells, indicating that only
one-third of the reacted probe is taken up by cells.
Figure 6. (Part A) T1-weighted spin-echo images (measured at
7 T) of agar phantoms containing K562 cell pellets: (A) control
(unlabeled) cells; (B, C) cells labeled with 1.0 and 2.0 mM GdL1A,
respectively; (D, E) cells labeled with 1.0 and 2.0 mM GdL2A,
respectively. (Part B) Effect of lactate added to the labeling medium
on cell labeling: (A) control cells; (B, C) cells labeled with GdL1A
(1.4 mM) without and with lactate 15 mM, respectively; (D, E) cells
labeled with GdL1B (1.4 mM) without and with lactate 15 mM,
respectively. See Supporting Information Figure S2 for MR images
of cells labeled with control GdDO3A.
To assess whether active cellular mechanisms were in-
volved in the labeling with GdL1A, labeling experiments
were carried out at 4 °C, where most of the active transmem-
brane transport systems are inhibited. The extent of label-
ing at 4 °C was then compared with that obtained in parallel
experiments carried out at 37 °C (Figure 5). In this set of
labeling experiments, the incubation time was shortened to
1 h to minimize cell suffering due to the low temperature. At
the lower temperature, the amount of gadolinium taken up
by cells is apparently constant within the probe concentra-
tion range, with an average value of around 6 ꢀ 108 Gd(III)
atoms/cell, and about 60% lower than the amount of gado-
linium that is taken up at 37 °C. Because of the short
incubation time (1 h), uptake of GdDO3A at both 37 and
4 °C is below the detection limit of the relaxometric assay,
estimated to be 1 ꢀ 108 Gd(III) atoms/cell for this set of ex-
periments. It must be emphasized that the difference in
Gd(III) uptake at the two temperatures cannot be ascribed
to temperature effects on the reactivity of GdL1A with EPTs
at 37 °C compared to that at 4 °C, as the release of P2T at
4 and 37 °C was comparable. These findings clearly indicate
that active cellular processes are at least partially involved in
EPTs-mediated cell labeling, likely yielding the internaliza-
tion of substantial amounts of the Gd(III) containing probes.
It must be emphasized that gadolinium in cells labeled with
GdL1A is present under the form of the intact complex and
that the dissociation of Gd(III) ions from the DO3A chelat-
ing cage to yield “free gadolinium” is negligible, as demon-
strated in a previous study.18
1 mM, respectively. For compound GdL2A, showing mini-
mal levels of uptake, a 2-fold signal enhancement becomes
detectable when labeling is carried out with a probe concen-
tration of 2 mM.
To assess the millimolar relaxivity r1
mM,cell
of the com-
plexes within the cellular environment, proton relaxation
rates were measured on a series of cell pellets labeled with
increasing concentration of the probes and the values nor-
malized by the total content of gadolinium in the cells (Gdcell
)
and by the volume of cells. To this purpose, 5 million cells
were labeled with increasing concentrations of GdL1A or
GdL1B (0.5-5 mM, 37 °C, 4 h), washed, and centrifuged, the
supernatant was carefully removed, and cells were dispersed
into agar phantoms. After the measurement of the water proton
relaxation rate R1obs by means of the saturation-recovery
spin-echo pulse sequence, pellets were recovered and ana-
lyzed for the total gadolinium content. These measurements
yielded millimolar relaxivities in cell of 2.0 and 2.3 mM-1 s-1
(at 7.0 T, 25 °C) for GdL1A and GdL1B, respectively
(Table 1). These quite similar values confirm that the differ-
ence in the signal enhancement between cells labeled with
GdL1A and GdL1B is due to the differential uptake of the
two probes rather than to differences between their relaxivi-
ties in the cellular environment.
The graph plotting R1obs versus the content of gadolinium
per cell (Gdcell, Gd(III) atoms/cell) can be further worked out
to gain insights into the cellular compartmentalization of the
Gd(III) complexes. This graph (Figure 7) also contains data
taken from the literature relative to the labeling of hepato-
carcinoma HTC cells with GdHPDO3A,14 a contrast agent
in clinical use. GdHPDO3A has been used to label cells
through the electroporation or the pinocytosis techniques.
GdHPDO3A is known to be internalized by both methods,
Relaxivity of the DO3A-Based Probes in the Cellular
Environment. The different degrees of cell labeling achievable
by the compounds considered in this work can be qualita-
tively appreciated by T1-weighted spin-echo MR images
(7.0 T) of labeled cell pellets. Figure 6A shows some repre-
sentative images, where cells labeled with GdL1A or GdL2A
have been dispersed into agar phantoms (representative
images with GdDO3A are given in Supporting Information
Figure S2). GdL1A produces a 7-fold or a 5-fold signal en-
hancement with respect to unlabeled cells, corresponding
to concentrations of the probe in the labeling medium of 2 or

4884 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Digilio et al.
(0.1-1 mM, 4 °C, 15 min) and then by titrating the amount
of residual EPTs by the DTNB spectrophotometric assay (i)
immediately after the treatment with NEM and (ii) after an
additional incubation time (recovery time) of 3 h at 37 °C in
minimal culture medium (data not shown). At the maximum
concentration of NEM (1 mM), cells showed a viability
larger than 80% (trypan blue test) in both circumstances.
The number of available EPTs measured immediately after
1.0 mM NEM treatment decreases by a factor of 5 with
respect to control, whereas the number of EPTs measured
after the recovery time showed only a 2-fold decrease of
EPTs, indicating that cells are able to partially restore the
functionality of EPTs. Next, true labeling experiments were
carried out by first pretreating cells with NEM (concen-
tration range 0.1-1 mM, 4 °C, 15 min) to increasingly mask
EPTs and then by incubating cells for 3 h at 37 °C with 2 mM
GdL1A. Although cell viability is not significantly affected
by any of the two single treatments (NEM at 4 °C or GdL1A
at 37 °C), the catenation of the two resulted in a severe
increase in cell death. Only pretreatment with the lower con-
centration of NEM (0.1 mM) resulted in a viability of >80%
at the end of the whole labeling experiment. The decrease of
Gd(III) uptake between control and cells pretreated with 0.1
mM NEM was ∼45% (data not shown). These results clearly
indicate a direct proportionality between the amount of
probe taken up and the concentration of available EPTs on
cells.
Figure 7. Observed water proton relaxation rates R1obs of cell
pellets (about 5 millions cells, 25 °C) that have taken up different
amounts of gadolinium complexes: (solid triangles) K562 cells (this
work) labeled with GdL1Ap; (open squares) HTC cell pellets
labeled with GdHPDO3A (taken from ref 14) through electropora-
tion, leading to cytosolic localization; (solid squares) HTC cell
pellets labeled with GdHPDO3A through pynocytosis, leading to
endosomal entrapment (taken from ref 14). Best fit straight lines are
shown for the open square and triangles data points. The curve
through the solid squares is drawn only to guide the eye and to
highlight the plateau effect that is typical of compartmentalization
of Gd(III) chelates into poorly water-permeable cell substructures.
but its distribution within the cell is different. If internalization is
achieved through the electroporation method, GdHPDO3A
resides mostly in the cytoplasm or, more strictly, it is not
compartmentalized within subcellular organelles delimited
by poorly water permeable membranes. Noncompartmenta-
lized internalization of GdHPDO3A results in a linear rela-
tionship between R1obs and the content of gadolinium per
cell, as there are no limitations due to slow water exchange
between intracellular compartments and the extracellular
space (Figure 7, open squares). On the other hand, if GdHP-
DO3A uptake occurs through pinocytosis, the complex is
compartmentalized into membrane-delimited vesicles that
limit the exchange of water between the intravesicle environ-
ment, the cytoplasm, and the extracellular space. A rather
complex three-site exchange model is required to describe
relaxation. In this case a relaxivity “quenching” effect is
observed, resulting in a plateau in the R1obs versus Gdcell plot at
a Gdcell of 2ꢀ109 Gd(III) atoms/cell (Figure 7, solid squares).
The functional form of these curves is then related to the
compartmentalization of Gd(III) complexes within the cell.
When cells are labeled with GdL1A, a linear plot is observed
up to a concentration of 3ꢀ1010 Gd(III) atoms per cell, and
no relaxivity quench is observed (Figure 7, triangles). This
suggests that after internalization the GdL1A complex is not
sequestered into subcellular organelles.
Effect of the Concentration of EPTs on Cell Surface on the
Uptake. GdL1A, showing the best labeling efficacy, has been
used to label K562 cells displaying different levels of EPTs to
gain further insights into the link between the number of
available EPTs and the Gd(III) uptake. To this purpose, cells
were treated with chemicals known to block EPTs prior to
the incubation with the labeling agents. NEM has been
chosen as the thiol blocking compound because it can form
irreversible thioether bonds with EPTs, it is relatively well
tolerated by cells, and it cannot cross cell membranes.24 The
efficacy of NEM in decreasing the number of available EPTs
has been evaluated by incubating K562 cells with NEM
Effect of Lactate on the Uptake of DO3A-Based Probes.
Since the DO3A-based probes are taken up by cells while the
DTPA-based ones are not, the characteristics of the metal
chelate must play a major role in the uptake mechanism. This
is quite unexpected, as the targeting to the EPTs in the
original design of the probes relied entirely on the thiol
reactive 2-pyridyldithio unit. The role of the metal chelate
might be related to the differences between GdDTPA and
GdDO3A-based complexes in terms of net charge and pos-
sibility to establish further coordination with foreign car-
boxylates or other anions. To assess whether the coordina-
tion chemistry at the Gd(III) center could have any effect on
the labeling efficacy, labeling experiments with the DO3A-
based probes have been carried in the presence of increasing
concentrations of lactate in the incubation medium. As stated
above, lactate can coordinate in a bidentate manner the Gd(III)
center in GdL1A or GdL1B complexes and can form ternary
complexes with dissociation constants in the millimolar
range. Figure 8 shows that the total amount of both GdL1A
and GdL1B (1.5 mM, 37 °C, 4 h) taken up by cells decreases
proportionally with the concentration of lactate added in the
incubation medium. In the case of GdL1A, the percentage
inhibition is particularly striking: as much as 73% when
lactate concentration is 15 mM (in these conditions, the
molar concentration of free GdL1A with respect to the
GdL1A/lactate ternary complex is about 5%). The millimo-
lar relaxivity of cell pellets labeled with GdL1A in the
presence or absence of lactate does not change appreciably.
The decrease of signal enhancement in cell pellets labeled in
the presence of lactate (Figure 6B) is entirely due to a lower
concentration of gadolinium taken up by the cells.
Labeling of C6 and B16 Cells. To probe the range of
applicability of the proposed labeling procedure, it has been
applied to rat glioma (C6) and murine melanoma (B16)
cultured cells. The latter cell types grow in adhesion to the
culture vessel and may offer a lower availability of EPTs to
the labeling probes compared to K562 cells, which grow as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4885
10-25% (depending on cell type) of these EPTs could be
labeled with Gd(III)-based probes having a millimolar relaxi-
vity of 2 mM-1 s-1, each cell would be covered with a number
of Gd(III) centers large enough to allow visualization by
MRI. The necessity of the formation of a covalent EPT-
probe adduct for labeling is well pointed out by the fact that
the chemical blockage of EPTs by NEM considerably de-
creases the extent of labeling. Moreover, the extent of labeling
is proportional to the concentration of EPTs available on
cells. On the other hand, the fact that all of the probes
considered do react with EPTs while showing widely different
labeling properties (maximum efficiency for GdL1A, no
labeling at all for GdL2B) clearly indicates that the formation
of the EPT-probe adduct is not sufficient by itself for labeling
cells to a suitable extent. Thus, the EPT-dependent labeling
mechanism goes beyond the simple anchorage of the probes
on the extracellular side of the plasma membrane, and the fate
of the EPTs-probe adduct must be taken into account. More
detailed labeling experiments performed with GdL1A, the
best performing probe, revealed that (i) the total amount of
Gd(III) taken up per single cell is significantly larger than the
number of EPTs available on cells; (ii) the relationship
between Gd(III) uptake and the concentration of the probe
in the incubation medium is linear up to 3.5 mM probe con-
centration, where the stoichiometric ratio between the probe
and the EPTs is largely in favor of the former (in these condi-
tions, a saturation of the EPTs and a plateau in the Gd(III)
uptakevsprobeconcentration graphwould be expected, butit
is not observed); (iii) labeling cells with GdL1A at 4 °C is
about 60% less effective than that achieved at 37 °C, indicat-
ing that active cellular transport mechanisms, which are
inhibited at 4 °C, must play a role and that some degree of
probe internalization is involved.
Figure 8. Uptake of GdL1A (Gd(III) atoms/cell) by K562 cells as
a function of the concentration of lactate in the incubation medium
(4 h of incubation, 37 °C, about 5 millions cells): white bars, cells
labeled with GdL1B; black bars, cells labeled with GdL1A.
Taken together, these findings provide evidence about the
internalization of a substantial amount of the probe, which
must take place upon cellular processing of the EPT-probe
adduct. It may be envisaged that after the formation of the
disulfide bridged EPT-probe adduct, the cell machinery
comes into play to restore the physiological thiol/disulfide
redox balance on the extracellular side of the plasma mem-
brane. This balance has been shown to be essential for proper
cell function, as exofacial protein thiols/disulfides may act as
redox switches involved in many functions,38 such as intra-
cellular signaling, cell adhesion, or integrin activity.23,39 As a
matter of fact, cells have been shown to have several ways to
buffer (at least partially) the exofacial protein thiol/disulfide
redox state.21,22,40 The cell response to the formation of the
EPT-probe mixed disulfide could ultimately result into the
cleavage of such an adduct (by reduction or by thiol/disulfide
exchange), which can lead to either the ejection of the probe in
the extracellular space or the internalization of the probe.
Dealingwithinternalization, itbecomes important toevaluate
the chemical form of Gd(III) and its intracellular localization.
Ina previous workit hasbeen proved that the GdL1A probe is
present in the cell environment as the integer GdDO3A like
complex and that the release of free Gd(III) from the DO3A
cage is negligible.18 Although we have no explicit data on the
cellular localization of the internalized probe, the analysis of
the plots linking the observed relaxivity of cell pellets as a
function of the cellular concentration of gadolinium may
provide some clues (Figure 7). It has been shown that the
relaxivity increases linearly (up to a total concentration of
gadolinium of 2 ꢀ 109 Gd(III) atoms/cell) when the inter-
nalized Gd(III) complex resides in the cytoplasm or, more
Figure 9. Extent of cell labeling (expressed as Gd(III) atoms/cell)
by GdL1A as a function of the concentration of GdL1A in the
labeling medium (about 5 ꢀ 106 cells, 37 °C, 4 h): rat glioma C6 (gray
bars); human myeloid leukemia K562 (white bars); murine mela-
noma B16 (black bars). See Supporting Information Figure S3 for
control uptake of GdDO3A.
suspended cells. The level of EPTs expressed by C6 and B16
cells (measured at a confluence of 80%) was (2-4)ꢀ109 and
(2-6) ꢀ 1010 EPTs/cell, respectively, by the DTNB spectro-
photometric assay, whereas the level of EPTs/cell is in the
range (5-7)ꢀ109 for K562 cells. For labeling, cells grown to
a confluence of 80% were subjected to a 4 h incubation with
2 mM GdL1A at 37 °C, washed three times, and detached
from the flasks by treatment with trypsin. As shown in
Figure 9, a good labeling could be obtained for all cell lines,
with a minimum labeling of 1ꢀ109 Gd(III) atoms/cell (data
for control GdDO3A in Supporting Information Figure S3).
The higher efficacy of the labeling on B16 cells with respect to
C6 and K562 correlates well with the higher level of EPTs
shown by the former.
Discussion
The original concept of cellular labeling through the bio-
conjugation of EPTs with a suitably activated paramagnetic
probe relied upon the fact that cells display a large number of
DTNB reactive EPTs (in the range of (2-60)ꢀ109 EPTs/cell
for those considered in this work) that can be exploited as
anchorage points for suitably activated probes. If as much as

4886 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Digilio et al.
generally speaking, when it is not sequestered in poorly water
permeable subcellular organelles. On the other hand, the
relaxivity goes to a plateau when the complex is sequestered
within cellular substructures such as the endosomes, where the
exchange rate of water molecules between the intraorganelle
and the cytoplasmatic compartments is so slow as to quench
the paramagnetic relaxivity enhancement.14 In the case under
study, we found a linear relationship between the observed
paramagnetic relaxivity and the total gadolinium content, up
to a Gdcell concentration of 3ꢀ 1010 Gd(III) atoms/cell. This
behavior suggests a noncompartmentalized cellular localiza-
tion of the probe.
Gd(III) chelate or the possibility of Gd(III) to form ternary
complexes within the EPT-probe adduct (for instance, with
exofacial protein carboxylates or membrane lipid phosphate
headgroups as donors). Anyway, the formal charge of the
complex and the coordination chemistry at the Gd(III) center
are molecularproperties that arestrongly linked toeach other.
Finally, it is worth noting that the length/flexibility of the
linker also has a role in determining the extent of labeling: the
longer is the spacer, the higher is the amount of gadolinium
taken up (compare GdL1A with GdL1B, Figure 3). In view
that the formation of a ternary complex between the EPT-
bound probe andsome coordinating groups on thecell surface
is important for internalization, a longer (and flexible) spacer
would be beneficial because a longer spacer would allow a
greater degree of freedom for the Gd(III) ion to probe the
surface of the membrane protein (or the protein/membrane
interface) in search of coordinating groups.
To assess the structural requirements favoring the inter-
nalization route of the probe, we compared the labeling
efficacy of four probes differing either in the length of the
spacer connecting the paramagnetic unit with the thiol bind-
ing unit or in the structure of the polyaminocarboxylic co-
ordination cage (DO3A-like versus DTPA-like structures).
These probes show wide differences in their cell uptake in the
order GdL1A (maximum uptake) . GdL1B > GdL2A .
GdL2B (no uptake). This trend highlights that (i) DO3A-
based probes are much more efficiently taken up than DTPA-
based ones and (ii) within each of these two classes of com-
pounds, uptake is favored by long, flexible spacers (GdL1A
more easily taken up than GdL1B). The structure of the che-
lating cage is more important than the spacer length in defining
the extent of uptake: the DO3A-based GdL1B, having a spacer
slightly shorter than that of the DTPA-based GdL2A, still
shows a better uptake. Although unplanned, the reporter
moiety of the probes appears to play an active role in the EPT
dependent labeling. A few remarks about the most important
structural differences between GdDO3A-based and GdDTPA-
based structures may help to shed light on such an unexpected
role. GdDO3A-like complexes have a neutral net charge and
typically two water molecules in the inner coordination
sphere. With the DO3A-based structures being heptadentate
and with their Gd(III) complexes being formally neutral, the
GdDO3A complex can approach foreign negatively charged
coordinating groups and make ternary complexes (for in-
stance, with lactate, amino acids, protein carboxylates19,30,37),
with the displacement of the inner sphere water molecules. On
the other hand, DTPA-based structures are octadentate and
their Gd(III) complexes have a doubly negative net charge,
preventing the formation of ternary complexes with foreign
coordinating groups. Typically, GdDTPA complexes have
one inner sphere water molecule. The analysis of NMRD and
17O-R2p versus temperature profiles of GdL1A and GdL1B
clearly shows that these compounds are characterized by q=2
and q=1, respectively, when they are in the free state, i.e., in
the absence of potential foreign coordinating groups. The
lower hydration of GdL1B is likely due to the further intra-
molecular coordination of the Gd(III) ion by the carbonylic
oxygen belonging to the amido group of the spacer closer to
the metal ion. In the presence of excess lactate, the hydration
of both the DO3A-based probes decreases because of the
formation of a ternary complex in which lactate acts as a
bidentate ligand leading formally to the displacement of both
inner sphere water molecules. Thus, in the presence of lactate,
the GdDO3A moiety acquires one negative net charge and a
lower hydration, quite resembling the GdDTPA structure.
Consistently, the uptake of GdL1A or GdL1B in the presence
of 15 mM lactate is strongly inhibited (by 73% and 56%,
respectively). Atpresent weareunabletostate whether the key
property for uptake is the formal neutral net charge of the
Conclusions
We have described a new method for labeling cells with
paramagnetic compounds based on the conjugation of
GdDO3A-based complexes with exofacial protein thiols fol-
lowed by the internalization of the probe. Suitable probes for
this labeling scheme are GdDO3A-like molecules functiona-
lized with the thiol reactive 2-pyridyldithio group through a
long, flexible spacer. The proposed method can be thought of
as being of quite general purpose, as reactive EPTs are present
in a wide variety of cell lines. All three cell lines considered in
this work could be successfully labeled for MRI visualization.
In comparison to other methods for cell labeling with low
molecular weight Gd(III) chelates the labeling proce-
dure appears faster or less invasive, the payload of Gd(III)
within cells being comparable. Segregation into intracellular
vesicles or endosomes leading to relaxivity “quenching” is
avoided.
Experimental Section
The purity of every compound synthesized was determined
through HPLC-UV and HPLC-ESI-MS by the ratio of the
integrated HPLC peak area for the compound of interest to the
integrated HPLC peak area for all peaks. For compounds
GdL1A, GdL1B, GdL2A, and GdL2B the purity was >95%.
Synthesis of GdL1A. N-(Benzyloxycarbonyl)-N0-6-bromo-
hexanoyl-1,2-diamineethane (1). A solution of 6-bromohexanoyl
chloride (14.0 g, 0.065 mol) in dichloromethane (100 mL) was added
to N-(benzyloxycarbonyl)-1,2-diamineethane (12.6 g, 0.065 mol)
and potassium carbonate (powder, 325 mesh,13.6 g, 0.10 mol) in
dichloromethane (200 mL) at 0-5 °C under vigorous stirring.
After 1 night at room temperature the solid was filtered and the
solution washed with water, HCl 0.1 N, and brine. The clear
solution was evaporated on a rotary evaporator to yield an oil
(24.3 g).
1-[5-[N-[2-[N-(Benzyloxycarbonyl)amine]ethyl]aminecarbonyl-
pentyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic(1,10-di-
methylethyl ester) (2). The free base of DO3A-tris-tert-butyl ester
(16) was obtained by elution of DO3A HBr with water/ethanol
3
on Amberlite IRA 410). Compound 1 (18.5 g, 0.050 mol) was
then added to a solution of DO3A-tris-tert-butyl ester (32.9 g,
0.064 mol) and N,N-diisopropylethylamine (8.3 g, 0.064 mol) in
anhydrous acetonitrile (160 mL). After 4 days, the solution was
evaporated on a rotary evaporator to yield an oil that was
purified by flash chromatography on silica with a dichloro-
methane/methanol 20:1 v/v to remove the impurities and with
pure methanol to recover the product (21.9 g).

Article
1-[5-[N-[2-Amineethyl]aminecarbonylpentyl]-1,4,7,10-tetra-
azacyclododecane-4,7,10-triacetic(1,10-dimethylethyl ester) (3).
Compound 2 (20.9 g, 0.026 mol) was dissolved in methanol
(60 mL), and 10% Pd/C (1.2 g) was added. Then the reaction was
carried out under hydrogen atmospheric pressure at room
temperature for 6 h. The mixture was then filtered and the
solvent evaporated to yield an oil (17.3 g).
3-(2-Pyridyldithio)propanoic Acid (4). To a solution of ald-
rithiol (4.4 g, 0.02 mol) in ethyl acetate (25 mL) was added
dropwise a solution of 3-thiopropionic acid (1.74 mL, 0.02 mol)
in ethyl acetate (25 mL). The solution turned immediately to
bright yellow after the addition of BF3 ether ethylic complex
(4 drops). After the mixture was stirred for 2 h at room tem-
perature, the formation of a yellow precipitate was observed.
After 24 h the suspension was filtered (the solid was 2-pyri-
dylmercaptane) and the solvent was removed by evaporation.
The oil was dissolved twice in cold ethyl acetate and filtered to
give at the end a yellow oil (4.0 g, purity <70%).
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4887
1-[N-(benzyloxycarbonyl)-2-aminoethyl]-1,4,7,10-tetraazacy-
clododecane-4,7,10-triacetic(1,1-dimethylethyl ester) (14). DO3A-
tris-tert-butyl ester (37.1 g, 0.072 mol, free base) was dissolved in
acetonitrile (150 mL), and K2CO3 (11.9 g, 0.086 mol) was added.
The mixture was kept under stirring at room temperature while a
solution of 13 (20.4 g, 0.079 mol) in acetonitrile (150 mL) was
added dropwise over a period of 1 h. The reaction mixture was
kept under stirring for 4 days at 50 °C. After filtration the solvent
was removed by evaporation under reduced pressure and the
residue treated with diethyl ether (100 mL) to give a white solid
corresponding to unreacted parent 16. After filtration the solution
was evaporated in vacuo and the residue purified by flash
chromatography on silica with a dichloromethane/methanol gra-
dient. Fractions containing the product were combined and
evaporated to give a yellow oil (34 g). 1H NMR (300 MHz,
CD3OD): δ 1.48 (s, 27H), 1.70-3.18 (b m, 26H), 5.18 (s, 2H), 7.38
(m, 5H). MS [M þ Hþ] calcd, 691.9; found, 692.83.
1-(2-Aminoethyl)-1,4,7,10-tetraazacyclododecane-4,7,10-tri-
acetic(1,1-dimethylethyl ester) (15). Compound 14 (34.0 g,
0.049 mol) was dissolved in methanol (200 mL), and 10% Pd/
C (2.0 g) was added. Then the reaction was carried out under
hydrogen atmospheric pressure at room temperature for 6 h.
The mixture was then filtered and the solvent evaporated (28.6 g).
1H NMR (300 MHz, CD3OD): δ 1.45 (s, 27H), 1.85-3.24 (b m,
26H). MS [M þ Hþ] calcd, 557.77; found, 558.65.
1-[6,11-Dioxo-7,10-diaza-14,15-dithio-15(2-pyridyl)pentadec-
anyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic(1,10-di-
methylethyl ester) (5). N,N0-Dicyclohexylcarbodiimide (DCC,
6.2 g, 0.030 mol) was added to a solution of 3 (15.9 g, 0.024 mol)
and 3(2-pyridyldithio)propionic acid 4 (5.4 g, 0.025 mol) in
dichloromethane (150 mL) under vigorous stirring. After 4 days,
the N,N0-dicyclohexylurea (DCU) was filtered and the solution
was washed with water and brine. The clear solution was
evaporated on a rotary evaporator to yield an oil (24.5 g).
1-[6,11-Dioxo-7,10-diaza-14,15-dithio-15(2-pyridyl)pentadec-
anyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic Acid (6).
Trifluoroacetic acid (TFA, 46 mL) was added to a solution of
5 (17.4 g, 0.020 mol) in dichloromethane (50 mL). The organic
solvent was evaporated on a rotary evaporator. Then a further
aliquot of trifluoroacetic acid (46 mL) and triisopropylsilane
(500 μL) was added. After 3 days diethyl ether (300 mL) was
added to obtain a white crystalline precipitate that was filtered,
washed with diethyl ether (10 mL), and dried. The solid was
dissolved in water and purified by liquid chromatography on
Amberlite XAD1600 700 mL with a water/methanol gradient
(from 0 to 100 in 6 CV). Fractions containing the product were
combined and evaporated to a white powder (2.25 g, global yield
from 1, 8.8%). 1H NMR (600 MHz, DMSO-d6): δ 1.24 (m, 2H),
1.53 (m, 4H), 2.10 (t, 2H), 2.54 (o, t, 2H), 2.73 (m, br, 2H),
2.91-3.01 (m, br, TAZA ring), 3.04 (o, t, 2H), 3.13 (m, br, 4H),
3.44 (s, 4H), 3.49 (s, 2H), 7.28 (m, 1H), 7.80 (m, 1H), 7.87 (m,
1-[N-[3(2-Pyridyldithio)propanoyl]2-aminoethyl]-1,4,7,10-tetra-
azacyclododecane-4,7,10-triacetic(1,1-dimethylethyl ester) (17).
A solution of 4 (4.0 g, 0.015 mol, purity of ∼70%) in ethyl
acetate (20 mL) was added to a solution of 15 (5.0 g, 0.009 mol)
in ethyl acetate (20 mL). Then triethylamine (1.4 mL, 0.01 mol)
and dicyclohexylcarbodiimide (3.7 g, 0.018 mol) were added and
the mixture was left stirring at room temperature for 4 days.
After dicyclohexylurea was filtered away, the organic phase was
washed with water, NaHCO3 1%, and brine. The solvent was
evaporated to give a yellow oil (11 g, purity of <60%). 1H NMR
(300 MHz, CD3OD): δ 1.49 (s, 27H), 2.58 (t, 2H), 3.17 (t, 2H),
7.24 (m, 1H), 7.73 (m, 2H), 8.42 (m, 1H). MS [M þ Hþ] calcd,
755.04; found, 755.66.
1-[N-[3(2-Pyridyldithio)propanoyl]2-aminoethyl]-1,4,7,10-tetra-
azacyclododecane-4,7,10-triacetic Acid (18). The compound
was obtained by a route similar to that of 11 and purified by
liquid chromatography on Amberchrom CG161 resin with a
water-methanol gradient. Fractions containing the product
were combined and evaporated to give a white solid (400 mg).
1H NMR (300 MHz, D2O): δ 2.46 (t, 2H), 2.87 (t, 2H),
2.91-3.75 (b m, 26H), 7.15 (t, 1H), 7.71 (m, 2H), 8.23 (t, 1H).
13C NMR (300 MHz, D2O): δ 33.9, 34.8, 36.3, 48.5, 48.8, 50.7,
51.2, 51.6, 54.0, 56.5, 121.9, 122.4, 140.0, 149.1, 158.9, 171.0,
174.8. MS [M þ Hþ] calcd, 557.77; found, 558.65.
1H), 8.49 (m, 1H), 8.45 (t, br, 1H, HN), 8.62 (t, br, 1H, NH). 13
C
NMR (150 MHz, DMSO-d6, from HMQC/HMBC): δ 25.6,
26.6, 35.2, 35.7, 36.0, 39.3, 54.4, 56.0, 56.6, 120.1, 122.0, 138.7,
150.6, 160.1, 170.7, 171.8, 172.0, 173.5. MS [M þ H]þ calcd for
C30H49N7O8S2, 699.88; found, 700.2.
1-[6,11-Dioxo-7,10-diaza-14,15-dithio-15(2-pyridyl)pentadecanyl]-
1,4,7,10-tetraazacyclo-dodecane-4,7,10-triacetate (3-) Gadoli-
nate (3þ) (1:1) (7). Gadolinium chloride hexahydrate (149 mg,
0.4 mmol) was added to an aqueous solution of 6 (280 mg,
0.4 mmol). The pH of the solution was slowly brought to
neutrality with NaOH, 2 N. Compound 6 (15 mg) was further
added in order to obtain an excess of the ligand (about 3%). The
solution was filtered on Millipore 0.22 μm and lyophilized.
Synthesis of GdL1B. N-(Benzyloxycarbonyl)-2-bromoethyl-
amine (13). A solution of 2-bromoethylamine hydrobromide
(71.7 g, 0.35 mol) in water (150 mL) and ethanol (150 mL) was
brought with NaOH 10 N at 10 °C to pH 7.1. Benzyl chlorofor-
mate (50.0 mL, 0.35 mol) in dimethoxyethane (100 mL) was
added to the mixture dropwise over 2 h at <20 °C, and the pH
was maintained at 7. After 16 h at room temperature the organic
phasewas removedby evaporationand the product was extracted
with dichloromethane (3ꢀ50 mL). The organic layer was washed
with water, HCl 0.1 N, water, brine, dried, and evaporated to give
a white solid (79.8 g). 1H NMR (300 MHz, CD3OD): δ 3.30 (t,
2H), 3.61 (t, 2H), 5.12 (s, 2H), 7.38 (m, 5H). 13C NMR(300 MHz,
CD3OD): δ 20.8, 42.7, 66.5, 127.9, 128.2, 128.6, 137.3, 157.8.
1-[N-[3(2-Pyridyldithio)propanoyl]2-aminoethyl]-1,4,7,10-tetra-
azacyclododecane-4,7,10-triacetate (3-) Gadolinate (3þ) (1:1)
(19). The complexation was performed with GdCl3 in aqueous
solution at pH 7 by the method of the addition of the ligand. An
equimolar amount of GdCl3 (127 mg, 0.34 mmol) solution in
water was added to the aqueous solution of ligand (200 mg, 0.34
mmol), maintaining the pH at 7 with NaOH 0.1 N. The mixture
was allowed to stir at room temperature until the pH remained
constant at 7. The solution was then lyophilized to give a white
solid (350 mg). The amount of residual free Gd3þ ion was
assessed by the orange xylenol spectrophotometric assay,27
and a suitable amount of ligand was added to have a slight
excess of the ligand (about 3%) and to avoid the presence of free
Gd(III) ions.
Synthesis of GdL2A. N,N0-Bis[2-[bis[2-(1,1-dimethylethoxy)-
2-oxoethyl]amino]ethyl]-L-N-[3-[(2-pyridyl)dithio]propionyl]lys-
ine(1,1-dimethylethyl ester) (21). A solution of N,N0-bis[2-[bis-
[2-(1,1-dimethylethoxy)-2-oxoethyl]-amino]ethyl]-L-lysine(1,1-
dimethylethyl ester) 20 (9.7 g, 0.013 mol) in dichloromethane
(30 mL) was added to a solution of 4 (3.64, 0.013 mol) in
dichloromethane (20 mL). Then dicyclohexylcarbodiimide

4888 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Digilio et al.
(3.20 g, 0.0156 mol) was added and the mixture was left under
stirring for 32 h at room temperature. After dicyclohexylurea
was filtered away, the organic phase was washed with NaCO3
5%, water, brine and dried with Na2SO4. After filtration, the
solution was concentrated to give an oil (12.8 g) that was
purified by flash chromatography on silica with a hexane/ethyl
acetate gradient. Fractions containing the product were com-
bined and evaporated to give a yellow oil (1.90 g). MS [M þ Hþ]
calcd, 942.28; found, 942.95.
complexation was performed very quickly by adding an equi-
molar amount of GdCl3 (83 mg, 0.2 mmol) in aqueous solution
at pH 7. The solution was then quickly lyophilized to give a pale-
yellow solid (350 mg). The concentration of the Gd was deter-
mined by 1H NMR T1 measurement of the mineralized complex
(in HCl 37% at 120 °C for 16 h), while the concentration of the
ligand was determined by UV-vis spectrophotometry.19
Cell Culture. The culture media DMEM F-12, DEME, RPMI
1640, and fetal bovine serum (FBS) were purchased from
Cambrex, East Rutherford, NJ. All other supplements/chemi-
cals were from Sigma Chemical Co., St Louis, MO. Human
myeloid leukemia (K562) cells were cultured in RPMI 1640
supplemented with 10% fetal bovine serum (FBS), 100 U/mL
penicillin, and 100 U/mL streptomycin. Cell were cultured in a
humidified 95:5 v/v air/CO2 atmosphere and serially passaged in
75 cm2 tissue culture flasks. For uptake experiments, 3 millions
cells were recovered and suspended in 2 mL of a incubation
medium in 6 cm Petri dishes. Rat glioma (C6) and murine
melanoma (B16) cells were cultured in DMEM F-12 and DEME
media, supplemented as described above with the exception
of FBS, which was 5%. C6 and B16 cells were detached with
a 0.25% trypsin-EDTA solution when confluence was about
80%. For uptake experiments, C6 and B16 cells were seeded in
6 cm Petri dishes at a density of 4 ꢀ 104 cell/cm2. Twenty-four
hours after seeding cells were ready for the uptake experiments.
The trypan blue exclusion test was used to assess cell viability.
An amount of 10 μL of each cell suspension was added to 10 μL
of 0.4% trypan blue solutions in PBS. The suspended cells, once
introduced in a counting chamber, appear round and bright
when viable and colored in deep blue when damaged.
N,N0-Bis[2-[bis[carboxymethyl]amino]ethyl]-L-N-[3-[(2-pyridyl)-
dithio]propionyl]lysine (22). Neat trifluoroacetic acid (0.71 mL,
0.0097 mol) was slowly added to a solution of 21 (1.40 g,
0.001 319 mol) in dichloromethane (5 mL) and the mixture stir-
red at room temperature for 1 h. Dichloromethane was evapo-
rated and the residue treated with trifluoroacetic acid (5.0 mL)
and triisopropylsilane (0.10 mL). The solution was stirred for
24 h. Then diethyl ether was slowly added (20 mL) to give a
precipitate which was filtered, washed with diethyl ether, and
1
dried (0.82 g). H NMR (300 MHz, CD3OD): δ 1.09 (t, 2 H),
1.42 (m, 3 H), 1.55 (m, 2 H), 1.76 (m, 2 H), 2.60 (m, 2 H), 3.09 (m,
10 H), 3.46 (m, 8 H), 7.70 (t, 1 H), 8.13 (d,1 H), 8.30 (t,1 H), 8.56
(d,1 H). 13C NMR (600 MHz, D2O): δ 14.4, 23.7, 28.0, 34.8,
39.4, 46.4, 53.2, 56.4, 63.6, 66.4, 115.7, 124.2, 125.6, 143.4, 145.5,
163.2, 170. MS [M þ Hþ] calcd, 661.64; found, 662.34.
N,N0-Bis[2-[bis[carboxymethyl]amino]ethyl]-L-N0-[3-[(2-pyri-
dyl)dithio]propionyl]lysinate (5-) Gadolinate (3þ) (1:1) Sodium
Salt (1:2) (23). An equimolar amount of aqueous GdCl3 (438 mg,
0.41 mmol) was added to the aqueous solution of ligand (107 mg,
0.41 mmol), maintaining the pH at 7 with NaOH 0.1 N. The
mixture was kept under stirring at room temperature until the pH
remained constant at 7. The solution was then lyophilized to give a
white solid (350 mg). The amount of residual free Gd3þ ion was
assessed by the orange xylenol spectrophotometric method, and a
suitable amount of ligand was added to have an excess of the
ligand of 2%. MS [M þ Hþ] calcd, 814.96; found, 815.45.
Synthesis of GdL2B. L-S-(2-Pyridylthio)cysteine BF3 Salt (8).
L-Cysteine (2.4 g, 0.02 mol) was added into a solution of
aldrithiol (4.4 g, 0.02 mol) in ethyl acetate (50 mL). Then BF3
ether ethylic complex (1.5 mL, 0.02 mol) was added dropwise to
the suspension, which immediately became bright yellow. After
4 days at room temperature, the suspension was filtered and the
yellow solid washed several times with cold ethyl acetate, filtered
again, and dried (3.8 g).
The growth curve of K562 cells was obtained by harvesting
cells from a 75 cm2 flask every 24 h for up to 13 days. After being
harvested, cells were stained with trypan blue and live cells
counted with a counting chamber. The RPMI 1640 culture
medium was renewed every 24 h.
Spectrophotometric Assay for EPTs. The amount of cell-
surface protein thiols was determined using the DTNB test.22
Cells were suspended in 1 mL of HEPES buffer, and DTNB was
added to a final concentration of 200 μM. After 30 min of incu-
bation at room temperature, cells were pelleted and the 2-nitro-
5-thiobenzoic acid content of the supernatant was evaluated by
measuring the absorbance at 412 nm (ε =14 150 M-1 cm-1).
Cell Labeling. The typical labeling procedure consisted of
incubating 3-5 million cells for 4 h at 37 °C in a minimum
medium (Earl’s balanced salt solution, EBSS, in which the
phosphate has been replaced by 1.2 mM HEPES sodium salt)
containing the Gd-based labeling agent in a typical concentra-
tion range of 0.5-4.0 mM. In experiments with lactate, lactate
was added up to 15 mM just before addition of the labeling
agent. In the experiments carried out at 37 °C, cells were in-
cubated for 4 h in CO2-air (5:95) atmosphere, while for
experiments at 4 °C cells were incubated for a maximum of
1 h in a sterile environment. After incubation the cells were
washed three times with 5 mL of HEPES buffer. For C6 and
B16 lines, cells were recovered mechanically by harvesting them
in 200 mL of HEPES with a scraper. After recovering from the
labeling medium, cells were washed and suspended in 200 mL
of HEPES and then sonicated for 10 s for a complete lysis. HCl
37% was added at the same volume, and the mixture was left at
120 °C overnight. Upon this treatment all Gd(III) was released
as the free aquo ion. By measurement of the water proton
relaxation rate of these solutions, it is possible to determine its
concentration.12,26 Relaxation rate measurements were per-
formed at 20 MHz and 25 °C on a Spinmaster spectrometer
(Stelar, Mede, Italy) by using a conventional inversion recovery
pulse sequence. The obtained R1obs data are related to the
concentration of the paramagnetic species according to the
formula
N,N0-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-
L-S-(2-pyridylthio)cysteine (10). A solution of 9 (9.9 g, 0.0268
mol) in acetonitrile (30 mL) was added to a suspension of 8 (3.8 g,
0.016 mol) and DIPEA (1.05 mL, 0.019 mol) in acetonitrile
(50 mL). After 24 h at room temperature the suspension was
filtered and the solution recovered and concentrated to give an
oil, which was washed with diethyl ether and then hexane. The
product was insoluble in hexane, and it was purified by flash
chromatography on silica by a diethyl ether/methanol gradient.
The fractions containing the product were combined and evapo-
rated to give a yellow oil (2.9 g). 1H NMR (300 MHz, CD3OD):
δ 1.51 (s, 27H), 2.45 (m, 1H), 2.56 (m, 1H), 2.98 (t, 4H), 3.15
(t, 4H), 4.05 (m, 1H), 7.32 (m, 1H), 7.71 (m, 2H), 7.96 (m, 1H),
8.47 (m, 1H). MS [M þ Hþ] calcd, 773.01; found, 774.26.
N,N0-Bis[2-[bis[carboxymethylen]amino]ethyl]-L-S-(2-pyridyl-
thio)cysteine (11). Neat trifluoroacetic acid (1.9 mL, 0.025 mol)
was slowly added to a solution of 10 (1.3 g, 0.0017 mol) in
dichloromethane (5 mL) and the mixture stirred at room
temperature for 1 h. Dichloromethane was evaporated and the
residue treated with trifluoroacetic acid (8.0 mL, 0.10 mol) and
triisopropylsilane (0.2 mL). The solution was stirred for 24 h.
Then diethyl ether was slowly added (20 mL) to give a slightly
yellow solid, which was filtered, washed with diethyl ether, and
dried (0.82 g).
[N,N0-Bis[2-[bis[carboxymethylen]amino]ethyl]-L-S-(2-pyridyl-
thio)cysteinate (5-)] Gadolinate (3þ) (1:1) Sodium Salt (1:2) (12).
Since ligand 11 (290 mg, 0.2 mmol) was not stable in water, the
GdðIIIÞ
R1obs ¼ R1W þ ½GdðIIIÞꢁr1p

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 4889
where R1W is the relaxation rate of pure water (0.38 s-1
)
(4) Pathak, A. P.; Gimi, B.; Glunde, K.; Ackerstaff, E.; Artemov, D.;
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and r1pGd(III) the millimolar relaxivity of the Gd(III) aquo ion
(13.5 mM-1 s-1 in 6 M HCl, 25 °C). This method was calibrated
using standard ICP solutions, and the accuracy was determined
to be within 1%. The moles of Gd(III) obtained in this way were
normalized against the weight (in milligrams) of cellular pro-
teins. The protein concentration of each sample was determined
from cell lysates by the Bradford method41 using bovine serum
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1H NMR and 17O NMR Relaxation Rate Measurements. The
longitudinal water proton relaxation rate was measured by
using a Stelar Spinmaster (Stelar, Mede, Pavia, Italy) spectro-
meter operating at 20 MHz by means of the standard inversion
recovery sequence. The temperature was controlled with a Stelar
VTC-91 air-flow heater equipped with a copper constantan
thermocouple (uncertainty of 0.1 °C). The proton 1/T1 NMRD
profiles were measured over a continuum of magnetic field
strength from 0.000 24 to 0.47 T (corresponding to 0.01-
20 MHz proton Larmor frequency) on a Stelar field-cycling
relaxometer. The relaxometer works under complete computer
control with an absolute uncertainty in 1/T1 of (1%. Data
points from 0.47 T (20 MHz) to 1.7 T (70 MHz) were collected
on a Stelar Spinmaster spectrometer working at variable field.
The concentrations of Gd complex solutions, for the relaxo-
metric characterization, were determined by the relaxometric
procedure as described above.
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For variable-temperature 17O NMR measurements, aqueous
solutions containing 2.6% 17O isotope (Yeda, Israel) were used.
Variable-temperature 17O NMR measurements were recorded
at 600 MHz on a Bruker spectrometer, equipped with a 5 mm
probe, using a D2O external lock. Experimental settings were
as follows: spectral width of 9000 Hz, 90° pulse for 14 μs,
acquisition time of 10 ms, 1024 scans, and no sample spinning.
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were transferred into glass capillaries that were centrifuged at
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were acquired on a Bruker Avance 300 spectrometer operating
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Napolitano, R.; Gringeri, C.; Aime, S. Targeting exofacial protein
thiols with Gd-III complexes. An efficient procedure for MRI cell
labeling. Chem. Commun. 2009, 893–895.
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F.; Longo, D.; Mortillaro, A.; Aime, S. Synthesis and character-
ization of a Gd(III) based contrast agent responsive to thiol
containing compounds. Dalton Trans. 2007, 4980–4987.
(20) Laragione, T.; Gianazza, E.; Tonelli, R.; Bigini, P.; Mennini, T.;
Casoni, F.; Massignan, T.; Bonetto, V.; Ghezzi, P. Regulation of
redox-sensitive exofacial protein thiols in CHO cells. Biol. Chem.
2006, 387, 1371–1376.
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Lymphocyte surface thiol levels. Proc. Natl. Acad. Sci. U.S.A.
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Acknowledgment. Economic and scientific support from
MIUR (PRIN 2005039914 projects), EC-FP6 projects DiMI
(Grant LSHB-CT-2005-512146), MEDITRANS (Targeted
Delivery of Nanomedicine: Grant NMP4-CT-2006-026668),
EU-FP7-HEALTH-2007-1.2-4 ENCITE (European Net-
work for Cell Imaging and Tracking Expertise), and Regione
Piemonte (POR-FESR Asse I I.1.1 “PIIMDMT”; Ricerca
Sanitaria Finalizzata, bando 2008bis; Bando Converging
Technologies BIO-THER) is gratefully acknowledged.
Supporting Information Available: Representative control
data for GdDO3A. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Gianazza, E.; Ghezzi, P. Redox regulation of surface protein thiols:
identification of integrin R-4 as a molecular target by using redox
proteomics. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14737–14741.
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Lorenzini, E.; Tongiani, R.; Comporti, M.; Pompella, A. Redox
modulation of cell surface protein thiols in U937 lymphoma cells: the
role of the γ-glutamyl transpeptidase dependent H2O2 production
and S-thiolation. Free Radical Biol. Med. 1999, 27, 623–635.
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