A paramagnetic chemical exchange-based MRI probe metabolized by cathepsin D: Design, synthesis and cellular uptake studies

Article abstract of DOI:10.1039/b926639a

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimer's disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE). The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b926639a

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A paramagnetic chemical exchange-based MRI probe metabolized by
cathepsin D: design, synthesis and cellular uptake studies†
Mojm´ır Suchy´,a,b Robert Ta,b Alex X. Li,b Filip Wojciechowski,a Stephen H. Pasternak,c Robert Barthab and
Robert H. E. Hudson*a
Received 22nd December 2009, Accepted 9th March 2010
First published as an Advance Article on the web 26th March 2010
DOI: 10.1039/b926639a
Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimer’s
disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe
for the potential detection of localized cathepsin D activity has been synthesized. The probe design
includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin
D cleavable sequence. This design results in the selective intracellular deposition (determined
fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D.
The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic
chemical exchange effects (OPARACHEE).
(containing metals other than gadolinium) have been found
Introduction
capable of inducing large hyperfine shifts of coordinated water
Cathepsin D is a member of the mammalian lysosomal aspartyl
protons as well as other exchangeable protons present in close
proximity to the metal center.16 These new MRI contrast agents
are referred to as PARACEST MRI CAs.17 Among other ap-
plications PARACEST MRI CAs have been successfully applied
towards the detection of enzymatic activities; for example b-
galactosidase,18 caspase-3,19 or urokinase plasminogen activator.20
The PARACEST phenomenon exploits slowly exchanging water
or protons in the ligand framework whereas agents with faster
exchange rates can be used to generate contrast using on-resonance
effects.
Within the framework of our interest in the development of
MRI contrast agents capable of detecting important physiological
parameters21–24 we have designed and synthesized a probe for
the MRI detection of localized Cat D enzymatic activity. The
design adopts features of Tsien’s earlier work which exploited a
cell penetrating peptide (CPP) conjugated to a cleavable peptide
domain and imaging agent cargo.25 Probe 1 possesses four impor-
tant structural features including a DOTA-like metal chelator, an
optical tag for detection by fluorescent microscopy (to facilitate
in cultureo cellular uptake studies), and a 23 amino acid containing
peptide sequence possessing the TAT CPP sequence26 and a
protease-cleavable site (1, Fig. 1). A variety of passenger cargos
have been shown to be transported intracellularly by TAT or other
cell-penetrating peptides, such DNA, polymers, nanoparticles,
liposomes, and small molecule based drugs and imaging agents.26
The protease-cleavable site is expected to give selectivity for Cat D
as it was designed on literature precedent and examination of the
MEROPS peptidase database.27
protease family found in all cells. However, cathepsin D (Cat
D) is overexpressed in some cancers and in Alzheimer’s disease
(AD), which makes it a potentially useful marker for these
diseases. Specifically, Cat D is upregulated in breast cancer,1
ovarian cancer,2 endometrial cancer3 and prostate cancer,4 and has
been associated with an increased risk of metastasis.5 Cat D may
be involved in processes of early tumour progression including
the stimulation of cell proliferation, fibroblast outgrowth and
angiogenesis.6,7 Therefore overexpression of this protease may have
prognostic value8 and help to identify subjects that require more
aggressive treatment, making it an excellent target as an imaging
biomarker. With respect to Alzheimer’s disease (AD), a prevalent
neurodegenerative brain disorder, it has been well documented that
increased levels of lysosomal hydrolases in normal appearing neu-
rons precede the earliest known histopathological features of AD.9
Overexpression (up to 4 fold) of Cat D,10–13 in Alzheimer’s disease
patients also makes this protease a viable imaging biomarker for
nascent AD detection since very early clinical diagnosis remains
difficult.14
A new method of generating contrast in magnetic resonance
imaging (MRI) has been recently introduced based on chemical
exchange saturation transfer (CEST).15 Subsequently a large
number of paramagnetic ion-containing lanthanide(III) complexes
aDepartment of Chemistry, The University of Western Ontario, London,
Ontario, Canada, N6A 5B7. E-mail: robert.hudson@uwo.ca; Fax: 1 519-
661-3022; Tel: 1 519-661-2111 ext. 86349
bDepartment of Medical Biophysics and the Imaging Research Group,
Robarts Research Institute, 100 Perth Drive, The University of Western
Ontario, London, Ontario, Canada, N6A 5K8
The western half of probe 1 is designed to provide magnetic
resonance imaging (MRI) contrast. The DOTA-like cage has been
introduced in order to chelate Tm3+ (thulium ion), which will
produce contrast using the mechanism of paramagnetic chemical
exchange effects.16a,28 The Tm3+ cation effectively produces contrast
using the on-resonance paramagnetic chemical exchange effect
(OPARACHEE),29 which requires less radiofrequency energy
cDepartment of Physiology and Pharmacology, and the Molecular Brain
Research Group, Robarts Research Institute, 100 Perth Drive, The University
of Western Ontario, London, Ontario, Canada, N6A 5K8
† Electronic supplementary information (ESI) available: High resolution
mass spectral data for 1, 1cleaved and data for the metabolism of 1 by Cat D.
See DOI: 10.1039/b926639a
2560 | Org. Biomol. Chem., 2010, 8, 2560–2566
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Scheme 1 The cathepsin D digestion of probe 1 initially gives the fragment
1cleaved which possesses the MR-active group and the Oregon GreenꢀR
fluorophore.
Fig. 1 Structure of probe 1 incorporating the fluorescent tag derived from
Oregon GreenꢀR maleimide. The dominant Cat D cleavage site between
phenylalanine (F) residues is indicated by an arrowhead with asterisk.
Tm = thulium.
Results and discussion
Synthesis of DO3A terminal monomer
than off-resonance PARACEST detection and therefore may be
advantageous for in vivo imaging. Since the chemical exchange be-
tween bulk and bound water associated with the Tm3+ compounds
is fast, the Z-spectrum of such compounds do not typically show
a peak at the chemical shift of the exchangeable pool. Therefore,
off-resonance excitation of the bound pool would be ineffective
at the low power levels mandated by in vivo imaging applications.
It is also possible to envision this moiety metalated with other
lanthanide(III) cations (e.g. Gd3+) in order to generate contrast by
other effects.
The optical reporter was introduced for visualization of cellular
uptake in cell culture or for future histological post mortem animal
studies. It was attached to the cysteine primary sulfanyl group via a
Michael addition to the commercially available maleimide dye—
Oregon Green.30 The bifunctional peptide sequence possesses a
CPP to facilitate the potential transport of probe 1 across the
blood brain barrier and promote cellular uptake, while the central
portion of the peptide sequence (G to L) is a cleavage domain
designed to be recognized by Cat D.31
Monoalkylation33 of DO3A tert-butyl ester (2) with N-
bromoacetyl Gly-OBn (3)34 followed by reductive removal (H2,
Pd/C, in EtOAc) of the benzyl group (Scheme 2) has been used to
prepare DO3A terminal monomer 4 (in 94% overall yield) suitable
for the conjugation with the peptide sequence. Purification of 4 by
normal phase column chromatography (CH2Cl2–MeOH, 9 : 1) was
found to be somewhat complicated by unexpected esterification of
the glycine carboxylic group. Replacement of MeOH with MeCN
resulted in the retention of the material on the column, even using
higher proportions of MeCN. Although analytical samples of 4
have been obtained by careful chromatographic purification using
an original solvent mixture, it was later found that purification
of compound 4 was unnecessary as it can be subjected for the
conjugation chemistry with the protected peptide attached to the
resin without any significant yield loss.
As reported in the literature,31,32 the Cat D-promoted enzymatic
hydrolysis of peptides takes place between two lipophilic amino
acid units, wherein two phenylalanine units were shown to be a
superior substrate. In addition to the fluorophore-labelled MRI
probe 1, we have also prepared the expected major product of
the Cat D-promoted enzymatic hydrolysis of 1 (1cleaved, Scheme
1). Fragment 1cleaved is expected to be formed upon the cleav-
age of the dual probe 1 within an intracellular compartment
and represents the moiety that would be the origin of MRI
contrast.
Probe 1 and the enzymatic hydrolysis product 1cleaved were
synthesized and it was experimentally determined that probe
1 was metabolized by Cat D as predicted (see ESI†). Cellular
uptake studies by fluorescence microscopy revealed selective
accumulation of the probe within cells overexpressing Cat D and
minimal accumulation in native cells. Furthermore, the expected
probe metabolism product 1cleaved was detected in vitro by MRI
using the OPARACHEE contrast mechanism. Together, these
results provide the foundation for studies directed toward the
localized detection of cathepsin D activity in vivo.
Scheme 2 Synthesis of DO3A terminal monomer 4.
Org. Biomol. Chem., 2010, 8, 2560–2566 | 2561
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Conjugation of DO3A terminal monomer with the peptides,
labeling with Oregon Green and metalation with TmCl3·6H2O
a short time, it was possible to observe both the unmetabolized
probe 1 and a new signal that corresponded to the mass of
metabolized probe 1cleaved simultaneously (Scheme 1, see ESI†,
Fig. 4, Rt = 2.42 min). In order to confirm the molecular
identity of the species that gave the signal at Rt = 2.42 min,
we independently synthesized 1cleaved and analyzed it by the same
UPLC-MS technique. Happily, it possessed the same retention
time as the product observed in the mixture resulting from the
digestion of probe 1 by Cat D. Longer incubation times did not
increase the proportion of 1cleaved as this product itself is likely
a substrate for further cleavage because it possesses lipophilic
residues. However, we were unable to detect any other products of
digestion.
Standard Fmoc peptide chemistry protocols35 have been used to
conjugate the DO3A terminal monomer 4 with fully protected
(protecting groups are listed in the Experimental section) 22-
mer peptides 5a or 5b attached to the resin (Scheme 3). Crude
DO3A conjugated peptides 6a or 6b were cleaved off the resin
(5% TES in wet TFA) and the residues obtained after drying were
purified (see ESI†) by semi-preparative HPLC (details given in
the Experimental section). Conjugate 6a was obtained in 14%,
whereas conjugate 6b was obtained in 28% yield. Structures of both
6a and 6b were confirmed by mass spectrometry (high resolution
ESI-TOF MS).
Cell uptake studies
Cell uptake studies of probe 1, possessing a cell penetrating
peptide, were performed using SN56 cells because they pos-
sess neuronal properties which are relevant to our interest in
Alzheimer’s disease and because they were easily transfected.
Cells were transfected with a DNA construct that induces high
expression of human Cat D fused to mCherry (a red fluorescent
protein). The cells were treated with 50 mM of 1 for 30 min at
37 ◦C and then the live cells were imaged using laser scanning
confocal microscopy, and representative images are shown (Fig.
2). Panels B–D and F–H (Fig. 2) show laser scanning confocal
epifluorescence images of Cat D (red) and 1 (green) with matching
brightfield differential interference contrast (DIC) images (A and
E). Cells that overexpressed Cat D–mCherry also took up and
retained probe 1 (for example panel C) whereas cells that did
not overexpress Cat D did not show intracellular accumulation
of 1 (panel G). Cells were scored as containing or not containing
contrast agent by an observer blinded to their transfection status.
In 4 independent experiments, 17% (56/326) of cells took up and
expressed the DNA construct encoding CatD–mRFP. Of these
cells, 68.4 4.9% (SEM) of transfected cells took up the contrast
agent, showing bright green signal that was clearly intracellular
(n = 56) whereas only 4.7 1.3% (SEM) of untransfected cells
(n = 270) took up the agent. Small amounts of the agent are
sometimes seen outside of the cell bodies. Closer examination
of cells overexpressing Cat D after incubation with probe 1 by
fluorescence microscopy showed extensive punctuate distribution
(data not shown).
Scheme 3 Solid phase conjugation of DO3A terminal monomer 4 to fully
protected peptides 5a and 5b, labeling with Oregon Green maleimide and
subsequent metalation with TmCl3·6H2O.
The resulting dual probe 1 was obtained in 78% yield, based on
6a, and was purified by size exclusion chromatography. The Cat D-
promoted cleavage product 1cleaved was obtained in 41% yield, based
on 6b. Absence of free Tm3+ in both 1 and 1cleaved was confirmed
by xylenol orange test.36 The structures of 1 (Fig. 1) and 1cleaved
(Scheme 1) were confirmed by mass spectrometry (HR ESI-TOF,
see ESI†). The protocol described above allows for the preparation
of 10–20 mg of the dual probe 1 in one synthetic sequence.
MRI studies
MRI detection of fragment 1cleaved was demonstrated at 9.4 Tesla
using the OPARACHEE method. To attribute a decrease in image
intensity to chemical exchange, two images were acquired. The
first image served as a control for altered T1 and T2 relaxation
time constants induced by the paramagnetic lanthanide within the
agent. The second image, acquired following application of an on-
resonance WALTZ16 preparation pulse includes additional signal
loss due to direct saturation and chemical exchange. The signal
intensity difference between these images can be compared to the
signal intensity difference observed in a sample without contrast
agent to determine the on-resonance paramagnetic chemical
exchange effect (OPARACHEE).
Metabolism of the dual probe by cathepsin D
The enzymatic activity of Cat D toward probe 1 has been verified
as depicted in Scheme 1 and described in the Experimental section.
Cat D avidly digested probe 1; thus, in order to observe the
initial product of cleavage it was necessary to carefully determine
the experimental conditions. It was also found necessary to use
Pepstatin A, a well known inhibitor of aspartyl proteases,37 to
control the course of the reaction. By quenching the digestion after
2562 | Org. Biomol. Chem., 2010, 8, 2560–2566
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Fig. 2 SN56 cells overexpressing Cat D–mCherry internalize probe 1. Images are of representative cells that expressed (A–D) or did not express (E–H)
cathepsin-D–mCherry. Panels show brightfield microscopy (DIC) images (A and E), red fluorescent image channel (B and F), green fluorescent contrast
agent image channel (C and G) and the green and red channels overlaid (D and H). Note that in typical cells transfected with CatD–mcherry, bright
green fluorescence was observed clearly inside the cells. Small amounts of the agent were sometimes observed outside of cells (D–H). Scale bars are 5 mm
(A–D) and 10 mm (E–H).
In addition to the 1cleaved molecular fragment, the conjugate 6b
containing neither Tm3+ nor the Oregon Green label was used as
a control (control 2, Fig. 3), as well as a blank solvent mixture
(8 : 2 H2O–MeCN, control 1, Fig. 3). The relative decrease in
signal intensity (% change) induced by the WALTZ-16 pulse
was calculated for each sample and the resultant OPARACHEE
contrast was determined (Fig. 3, panel C).
accumulation and MR active group for molecular imaging
applications.
Experimental
General experimental procedures
The average relative decrease in signal intensity (% change) for
sample 1 was 41%, while it was only 19% and 17% for samples 2 and
3 respectively. Therefore, relative to the solvent mixture (control 1),
All reagents were commercially available. All solvents were HPLC
grade and used as such, except for water which was deionized
(18.2 MX cm-1). Organic extracts were dried with Na2SO4 and
solvents were removed under reduced pressure in a rotary evapo-
rator. Flash column chromatography (FCC) was carried out using
1cleaved produced a contrast of 24%, while 6b (control 2) produced a
contrast of 2%. The signal change observed in the control samples
1 and 2 was due to the direct saturation of the bulk water, while the
change induced by the sample containing 1cleaved included the addi-
tional chemical exchange contrast induced by the lanthanide metal
Tm3+. These experiments confirm that the origin of the contrast is
from the chelated metal and not due to the fluorophore or peptide
fragment.
˚
silica gel, mesh size 230–400 A. Thin layer chromatography (TLC)
was carried out on Al backed silica gel plates, compounds were
visualized by UV light or I2 vapors. Size exclusion chromatography
was carried out on SEPHADEX G-25, 50–150 mm mesh resin
(5 g, per 7.5 mmol of compound 1). Ten fractions (3 mL each)
were collected. Fractions containing the desired product were
identified by fluorescence under UV illumination (l = 366 nm).
HPLC analysis and purification was carried out using a radial
Conclusions
˚
compression C18 300 A column (particle size 15 mm; 8 ¥ 100 mm)
A dual (fluorescent microscopy/MRI) probe (1) designed to be
metabolized by cathepsin D which contains a DOTA-derived
metal chelator (metalated with Tm3+), a fluorescent label (Ore-
gon Green) and a peptide sequence (TAT peptide + Cat D
cleavage site) is reported. Enzymatic degradation studies re-
vealed that 1 was a substrate for Cat D, where mass spec-
trometry studies confirmed cleavage between the two pheny-
lalanine residues. Fluorescence-based studies showed that the
probe exhibited selective intracellular accumulation in cells that
over-expressed cathepsin D. It is speculated that cleavage of
the protease domain traps the probe within the cell. In vitro
MRI contrast was realized using the OPARACHEE method.
Further studies are aimed at exploiting the selective cellular
using a gradient elution. The mobile phases: Method A—90%
H2O–10% MeCN → 70% H2O–30% MeCN over 30 min then
100% MeCN over 5 min; Method B—90% H2O–10% MeCN →
40% H2O–60% MeCN over 15 min then 100% MeCN over 5 min;
Method C—90% H2O–10% MeCN → 40% H2O–60% MeCN
over 20 min then 100% MeCN over 5 min; linear gradients, flow
rates 3 mL min-1. UPLC analysis was carried out using dual UV
and mass (ESI-TOF) detection, with the separation performed
on a C18 column (particle size 1.7 mm; 2.1 id ¥ 50 mm). Mobile
phase: Method D—100% H2O → 25% H2O–75% MeCN over
3 min, linear gradient, flow rate 0.25 mL min-1. NMR spectra were
recorded on 400 MHz spectrometer; for 1H (400 MHz), d values
were referenced as follows CDCl3 (7.26 ppm); for 13C (100 MHz)
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2560–2566 | 2563
©

View Article Online
approximately 25% of its original volume and then was diluted
with EtOAc (30 mL). The resulting mixture was washed with water
(30 mL); the aqueous phase was extracted with EtOAc (20 mL).
The combined organic extract was dried and the solvent was
evaporated to leave a colorless oil of sufficient purity for the next
reaction. This material was dissolved in EtOAc (6 mL) to which
10% Pd/C (140 mg) was added and the mixture was vigorously
stirred under an atmosphere of H2 at room temperature for 18
h. The catalyst was filtered off using a CELITE pad, the filter
was washed with MeOH and the filtrate was concentrated to
afford 230 mg (94%, based on 3) of DO3A terminal monomer
4. Analytical samples of 4 were obtained by FCC on 20 g SiO2
(CH2Cl2–MeOH, 9 : 1). 1H NMR d 6.99 (m, D2O exch., 1H); 3.75
(br s, 2H); 3.36 (br s, 4H); 2.89-2.15 (br m, 20 H); 1.44 (m, 27H);
13C NMR (CDCl3) d 172.5, 172.3, 170.3, 169.3, 82.2, 82.0, 56.4,
55.7, 55.6, 44.3, 28.0, 27.9. HRMS (ESI) m/z: found 630.4073
[M+H]+ (630.4078 calcd for C30H56N5O9).
Conjugation of DO3A terminal monomer 4 with protected peptides
5a and 5b
In a separate peptide synthesis vessels, resins containing the
protected (R-Pbf, C-Trt, Q-Trt, K-Boc, Y-tBu, ‘N’ terminal-
Fmoc) peptides 5a (N-GCG-KPI-LFF-RLY-GRK-KRR-QRR-
R-C, 2.66 ¥ 10-5 mol, 100 mg of the resin) or 5b (N-GCG-KPI-
LF-C, 3.00 ¥ 10-5 mol, 50 mg of the resin) were treated with 20%
piperidine in DMF (2 mL, repeated twice) for 5 min. The resins
were consecutively washed with dry DMF and CH2Cl2 (ca. 2 ¥
10 mL each, repeated twice). In a separate vials DIPEA (32 mL,
1.85 ¥ 10-4 mol, to conjugate with 5a; 37 mL, 2.10 ¥ 10-4 mol,
to conjugate with 5b) and HBTU (32 mg, 8.28 ¥ 10-5 mol, to
conjugate with 5a; 36 mg, 9.45 ¥ 10-5 mol, to conjugate with
5b) were added to a cooled (0 ◦C) solutions of DO3A terminal
monomer 4 (60 mg, 9.31 ¥ 10-5 mol, to conjugate with 5a; 66 mg,
1.05 ¥ 10-4 mol, to conjugate with 5b) in DMF (700 mL). The
resulting mixtures were added (after 10 min at 0 ◦C) to the washed
resins in the separated peptide vessels followed by agitation for
1.5 h at rt. The resins were washed as described above and were
treated with cleavage cocktail (5 mL, for 2 h at rt) prepared by
dissolving water (300 mL) and TES (200 mL) in TFA (10 mL).
The obtained TFA solutions were concentrated to ca◦. 10% of
Fig. 3 PARACEST contrast agent MRI detection sensitivity. Samples
vials are identified by labels, left to right: 1cleaved; control 2: 6b; control 1:
80% water–20% acetonitrile.
CDCl3 (77.0 ppm). Mass spectra (MS) were obtained by electron
spray ionization (ESI) time-of-flight (TOF) methods.
Preparation of N-bromoacetyl Gly-OBn (3)
◦
A solution of bromoacetyl bromide (7.0 mL, 80 mmol) in CH2Cl2
(25 mL), was added dropwise over 1 h to a suspension of H-Gly-
OBn·p-TsOH (25.3 g, 75 mmol) and DIPEA (26.5 mL, 150 mmol)
their original volumes, were cooled to 0 C, cold (-20 C) Et2O
(20 mL) was added and the mixtures were set aside for 1 h at -20
◦C. The resulting mixtures were centrifuged (3 min at 1000 rpm);
the supernatants were decanted and discarded and the recovered
pellets were dissolved in water (3 mL). The resulting solutions were
lyophilized to afford the crude peptide conjugates 6a (46 mg) or 6b
(28 mg). These materials were purified by semi-preparative HPLC
as described in the General experimental procedures. Conjugate
6a (Method A, 12.1 mg, 14% based on 4); colorless solid; HPLC: tR
25.1 min; HRMS (ESI) m/z: found 3235.8900 [M+H]+ (3235.8578
calcd for C142H241N52O33S). Conjugate 6b (Method B, 10.8 mg, 28%
based on 4); colorless solid; HPLC: tR 8.3 min; HRMS (ESI) m/z:
found 1277.6594 [M+H]+ (1277.6564 calcd for C57H93N14O17S).
◦
in CH2Cl2 (200 mL) at -78 C. The mixture was stirred for 12 h
at room temperature and then washed with 1 M HCl (2 ¥ 75 mL),
saturated aqueous NaHCO3 (75 mL), and brine (100 mL). The
organic extract was dried, the solvent was evaporated and the
residue was triturated with hexanes–Et2O (9 : 1, 100 mL) and
stored at 0 ◦C for 1 h. The solid was isolated by filtration to
afford 14.4 g (66%) of the title compound. The spectral properties
corresponded to those reported in the literature.34
Alkylation of DO3A tert-butyl ester (2) with N-bromoacetyl
Gly-OBn (3) and reductive removal of the benzyl group
Labeling of conjugates 6a and 6b with Oregon Green and
To a solution of DO3A-tert-butyl ester (2, 200 mg, 0.39 mmol)
in MeCN (10 mL), were added K2CO3 (140 mg, 1.01 mmol) and
N-bromoacetyl Gly-OBn (3, 111 mg, 0.39 mmol). The mixture
was stirred for 18 h at 60 ◦C and subsequently concentrated to
metalation with TmCl3 · 6H2O
Separate solutions of conjugates 6a (24.3 mg, 7.50 ¥ 10-6 mol)
or 6b (5.4 mg, 4.20 ¥ 10-6 mol) in water (6a, 2 mL; 6b, 600 mL)
2564 | Org. Biomol. Chem., 2010, 8, 2560–2566
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
were added to separate solutions of Oregon Green maleimide (3.5
mg, 7.50 ¥ 10-6 mol, to conjugate with 6a; 1.9 mg, 4.20 ¥ 10-6
mol, to conjugate with 6b) in MeCN (600 mL). The mixtures were
stirred for 48 h at rt in the dark, after which time they were frozen
and lyophilized to afford an orange solid residues. These were
dissolved in a mixtures of water (1.5 mL) and MeCN (500 mL),
to conjugate with 6a or water (600 mL) and MeCN (600 mL), to
conjugate with 6b and separate solutions of TmCl3·6H2O (0.1 M,
225 mL, 2.25 ¥ 10-5 mol to conjugate with 6a; 0.1 M, 126 mL,
1.26 ¥ 10-5 mol to conjugate with 6b) were added. The mixtures
were stirred for 48 h at rt in the dark and were purified as follows.
The mixture containing the dual probe 1 was subjected to size
exclusion chromatography as described in General experimental
procedures. Fractions containing the product were combined and
were lyophilized to afford 22.5 mg (78%) of the dual probe 1
as an orange solid; HRMS (ESI) m/z: found 3863.7924 [M -
2H]+ (3863.8110 calcd for C166H249F2N53O40STm). The mixture
containing the Cat D cleavage product 1cleaved was subjected to
semi-preparative HPLC as described in the General experimental
procedures. Conjugate 6b (Method C, 3.3 mg, 41%); orange
solid; HPLC: tR 14.7 min; HRMS (ESI) m/z: found 1906.6208
[M+H]+ (1906.6175 calcd for C81H101F2N15O24STm). The absence
of unchelated Tm3+ in the resulting conjugates 1 and 1cleaved was
confirmed as described previously.36
a laser-scanning confocal microscope using a 63 ¥ 1.4 numerical
aperture oil immersion lens. The contrast agent was visualized
using a 488 nm excitation laser and a 500–530 nm emission filter
set. Red fluorescence from the Cat D–mCherry fusion protein as
imaged using a 543 nm excitation laser and LP 560 filter set.
Cathepsin D enzymatic cleavage of 1
Cleavage of 1 (40 pmol) was performed with Cat D (0.01 units)
from bovine spleen in 100 ml of 150 mM NaCl with 20 mM
citrate-phosphate buffer at a pH of 4.5. Cleavage proceeded at
37 ◦C for 5 min and the reaction was stopped by the addition of
the inhibitor pepstatin A (2.5 mg). The resulting mixtures were
analyzed by UPLC-MS(ESI-TOF) operating under conditions
described earlier, vide supra. The identity of the initial cleavage
product, 1cleaved, was confirmed by independent synthesis. The
synthetic 1cleaved possessed the same retention time as the product
of initial cleavage of 1 by Cat D.
MRI studies with 1cleaved
Detection of 1cleaved by MRI was verified using a 9.4 Tesla, 31 cm
diameter bore Varian (Palo Alto, CA) small animal MRI scanner.
Three samples were prepared for imaging. Sample 1 contained
3 mM 1cleaved dissolved in 80% water–20% acetonitrile. Sample 2
contained 3 mM of an unmetallated version of 1cleaved without the
Oregon Green label (conjugate 6b) dissolved in 80% water–20%
acetonitrile. Sample 3 contained only 80% water–20% acetonitrile.
All three samples were placed in the MRI scanner (21 ◦C) and
two single slice images were acquired using a fast low angle shot
(FLASH) pulse sequence (TE/TR = 2.5/5.3 ms, 25.6 mm ¥ 25.6
mm field of view, 128 ¥ 128 matrix, 3 mm thick, 5 s pre-delay).
The first image was considered the control image. Preceding the
second image, a WALTZ-16 (480 ms, 6 mT) preparation pulse was
centered on the bulk water frequency to generate OPARACHEE
contrast. The percent change in signal intensity within each sample
(% change) was calculated on a pixel-by-pixel basis by taking the
difference between the signal intensity in the first FLASH control
image (I0) and the FLASH image acquired following the WALTZ-
16 preparation pulse (Is) and normalizing to the average signal
intensity within each sample in the control FLASH image.
Cell culture
SN56 cells were a gift from Dr Jane Rylett (The University of
Western Ontario). SN56 cells were derived from septum neurons
(Septal neurons X neuroblastoma N18TG2)38 and present a num-
ber of neuronal features including expression of synaptic vesicle
proteins39 and neuronal type calcium channels. These features
are increased by differentiation.40,41 Cells were maintained in
Dulbecco’s modified Eagle’s medium supplemented with 5% fetal
bovine serum and 1% penicillin/streptomycin. Cell differentiation
was performed with the addition of 1 mM dibutyryl-cyclic AMP
in the same medium without fetal bovine serum for 24 h. Transient
transfections were performed using Lipofectamine 2000 according
to the manufacturer’s instructions.
Constructs
An mCherry (a red monomeric fluorescent protein) cDNA was a
gift from Dr Roger Tsien (University of California, San Diego).
mCherry was amplified using PCR using primers that appended
5¢ BamH1 and 3¢ Not1 restriction sites and used to replace EGFP
in the pEGFP-N1 expression vector plasmid. A cDNA encoding
cat D was amplified using primers that delete the terminal stop
codon and append 5¢Xho1 and 3¢HindIII restriction sites and
cloned into the mCherry expression vector. This plasmid drives
high level expression of the inserted cat D-mCherry fusion protein
immediate early promoter of CMV.
Acknowledgements
The authors gratefully acknowledge funding received from the
Ontario Institute for Cancer Research (OICR), Canada to support
this work. Gifts of materials are gratefully acknowledged from
Professors Roger Tsein (University of California, San Diego) and
Jane Rylett (The University of Western Ontario).
Notes and references
1 H. Rochefort, Breast Cancer Res. Treat., 1990, 16, 3.
2 A. Losch, M. Schindl, P. Kohlberger, J. Lahodny, G. Breitenecker, R.
Horvat and P. Birner, Gynecol. Oncol., 2004, 92, 545.
3 I. Dvalishvili, L. Charkviani, T. Charkviani, G. Turashvili and G.
Burkadze, Georgian Med. News, 2005, 27.
4 S. Konno, J. P. Cherry, J. A. Mordente, J. R. Chapman, M. S.
Choudhury, C. Mallouh and H. Tazaki, World J. Urol., 2001, 19, 234.
5 H. Rochefort, F. Capony and M. Garcia, Cancer Metastasis Rev., 1990,
9, 321.
Cellular uptake and confocal laser scanning microscopy
Cells were grown in 35 mm glass-bottomed culture dishes for
confocal studies. Cells were incubated at 37 ◦C for 30 min with
compound 1 added to media (50 mM). After incubation, the
media and contrast agent were removed and replaced with Hanks
buffered saline solution. Confocal microscopy was performed on
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2560–2566 | 2565
©

View Article Online
6 G. Leto, F. M. Tumminello, M. Crescimanno, C. Flandina and N.
Gebbia, Clin. Exp. Metastasis, 2004, 21, 91.
7 E. Liaudet-Coopman, M. Beaujouin, D. Derocq, M. Garcia, M.
Glondu-Lassis, V. Laurent-Matha, C. Prebois, H. Rochefort and F.
Vignon, Cancer Lett., 2006, 237, 167.
25 T. Jiang, E. S. Olson, Q. T. Nguyen, M. Roy, P. A. Jennings and R. Y.
Tsien, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 17867.
26 For the recent review on the use of cell penetrating peptides in drug
delivery see: K. M. Stewart, K. L. Horton and S. O. Kelly, Org. Biomol.
Chem., 2008, 6, 2242.
8 P. Benes, V. Vetvicka and M. Fusek, Crit. Rev. Oncol. Hematol., 2008,
68, 12.
9 A. M. Cataldo, J. D. Barnett, S. A. Berman, J. Li, S. Quarless, S.
Bursztajn, C. Lippa and R. A. Nixon, Neuron, 1995, 14, 671.
10 A. M. Cataldo and R. A. Nixon, Proc. Natl. Acad. Sci. U. S. A., 1990,
87, 3861.
11 A. L. Schwagerl, P. S. Mohan, A. M. Cataldo, J. P. Vonsattel, N. W.
Kowall and R. A. Nixon, J. Neurochem., 1995, 64, 443.
12 R. E. Kohnken, U. S. Ladror, G. T. Wang, T. F. Holzman, B. E. Miller
and G. A. Krafft, Exp. Neurol., 1995, 133, 105.
27 MEROPS database http://merops.sanger.ac.uk. The core cleavage
sequence (ILFFRL) registered three hits: Cathepsin E, Pepsin F and
Cad3 peptidase. The biodistribution of Cat D is much different that Cat
E (ref. 31), especially in the brain which is one of the primary targets
of interest for this work since Cat D is associated with Alzheimer’s
disease. Pepsin F is a digestive protease and is of little consequence
for contrast agents that are administered intravenously and Cad3 is a
digestive protease from Musca domestica (housefly).
28 E. Vinogradov, H. He, A. Lubag, J. A. Balschi, A. D. Sherry and R. E.
Lenkinski, Magn. Reson. Med., 2007, 58, 650.
13 N. Chevallier, J. Vizzavona, P. Marambaud, C. P. Baur, M. Spillantini,
P. Fulcrand, J. Martinez, M. Goedert, J. P. Vincent and F. Checler,
Brain Res., 1997, 750, 11.
29 E. Vinogradov, S. Zhang, A. Lubag, J. A. Balschi, A. D. Sherry and R.
E. Lenkinski, J. Magn. Reson., 2005, 176, 54.
30 Oregon Green absorbs at 491 nm and emits at 515 nm.
31 Y. Yasuda, T. Kageyama, A. Akamine, M. Shibata, E. Kominami, Y.
Uchiyama and K. Yamamoto, J. Biochem., 1999, 125, 1137.
32 J. Higaki, R. Catalano, A. W. Guzzetta, D. Quon, J. F. Nave´, C. Tarnus,
H. D’Orchymont and B. Cordell, J. Biol. Chem., 1996, 271, 31885.
33 For the recent review on the functionalization of cyclen and its simple
derivatives see: M. Suchy´ and R. H. E. Hudson, Eur. J. Org. Chem.,
2008, 4847.
34 Synthesis of compound 3 has been recently described, see: R. Scheffe-
laar, R. A. K. Nijenhuis, M. Paravidino, M. Lutz, A. L. Spek, A. W.
Ehlers, F. J. J. de Kanter, M. B. Groen, R. V. A. Orru and E. Ruijter, J.
Org. Chem., 2009, 74, 660.
35 For a recent review on DOTA peptide conjugates see: L. M. De Leo´n-
Rodr´ıguez and Z. Kova´cs, Bioconjugate Chem., 2008, 19, 391.
36 A. Barge, G. Cravotto, E. Gianolio and F. Fedeli, Contrast Media Mol.
Imaging, 2006, 1, 184.
37 J. Marciniszyn, J. A. Hartsuck and J. Tang, J., J. Biol. Chem., 1976, 251,
7088.
14 For the recent review on the diagnostics of Alzheimer’s disease see: P.
Lewczuk and J. Wiltfang, Proteomics, 2008, 8, 1292.
15 For the seminal paper introducing this concept see: K. M. Ward, A. H.
Aletras and R. S. Balaban, J. Magn. Reson., 2000, 143, 79.
16 For the recent reviews on PARACEST MRI CAs see: (a) M. Woods,
D. E. Woessner and A. D. Sherry, Chem. Soc. Rev., 2006, 35, 500;
(b) A. D. Sherry and M. Woods, Annu. Rev. Biomed. Eng., 2008, 10,
391.
17 R. S. Zhang, M. Merrit, D. E. Woessner, R. Lenkinski and A. D. Sherry,
Acc. Chem. Res., 2003, 36, 783.
18 T. Chauvin, P. Durand, M. Bernier, H. Meudal, B. T. Doan, F. Noury,
´
B. Badet, J. C. Beloeil and E. To´th, Angew. Chem., Int. Ed., 2008, 47,
4370.
19 (a) B. Yoo and M. D. Pagel, J. Am. Chem. Soc., 2006, 128, 14032; (b) B.
Yoo, M. S. Raam, R. M. Rosenblum and M. D. Pagel, Contrast Media
Mol. Imaging, 2007, 2, 189.
20 B. Yoo, V. R. Sheth and M. D. Pagel, Tetrahedron Lett., 2009, 50,
4459.
21 F. Wojciechowski, M. Suchy´, A. X. Li, H. A. Azab, R. Bartha and R.
H. E. Hudson, Bioconjugate Chem., 2007, 18, 1625.
22 A. X. Li, F. Wojciechowski, M. Suchy´, C. K. Jones, R. H. E. Hudson,
R. Menon and R. Bartha, Magn. Reson. Med., 2008, 59, 374.
23 M. Suchy´, A. X. Li, R. Bartha and R. H. E. Hudson, Bioorg. Med.
Chem., 2008, 16, 6156.
38 D. N. Hammond, H. J. Lee, J. H. Tonsgard and B. H. Wainer, Brain
Res., 1990, 512, 190.
39 J. Barbosa, A. R. Massensini, M. S. Santos, S. I. Meireles, R. S. Gomez,
M. V. Gomez, M. A. Romano-Silva, V. F. Prado and M. A. M. Prado,
J. Neurochem., 1999, 73, 1881.
40 C. Kushmerick, M. A. Romano-Silva, M. V. Gomez and M. A. M.
Prado, Brain Res., 2001, 916, 199.
41 J. K. Blusztajn, A. Venturini, D. A. Jackson, H. J. Lee and B. H. Wainer,
J. Neurosci., 1992, 12, 793.
24 M. Suchy´, A. X. Li, R. Bartha and R. H. E. Hudson, Org. Biomol.
Chem., 2008, 6, 3588.
2566 | Org. Biomol. Chem., 2010, 8, 2560–2566
This journal is
The Royal Society of Chemistry 2010
©