Synthesis and characterization of a cell-permeable bimodal contrast agent targeting β-galactosidase

Article abstract of DOI:10.1016/j.bmc.2011.03.023

Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular β-galactosidase (β-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell-penetrating peptide D-Tat_49-57 and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl ₃. Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8 ± 0.6 mM^-1 s^-1, 123 MHz, ～21 °C) relative to the commercial Gd-DOTA (4.0 ± 0.12 mM^-1 s^-1, 123 MHz, ～21 °C). The activation of Gd-DOTA-k(FR)-Gal- CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the β-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in β-gal expressing C6/LacZ and enzyme deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed non-specific background signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities.

Full text of DOI:10.1016/j.bmc.2011.03.023

Bioorganic & Medicinal Chemistry 19 (2011) 2529–2540
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of a cell-permeable bimodal contrast
agent targeting b-galactosidase
Aneta Keliris a, , Thomas Ziegler b, Ritu Mishra a, , Rolf Pohmann a, Martin G. Sauer c, Kamil Ugurbil a,à
,
⇑
Jörn Engelmann a,
⇑
a Max Planck Institute for Biological Cybernetics, Spemannstrasse 41, Tüebingen, Germany
b Institute of Organic Chemistry, University of Tuebingen, Auf der Morgenstelle 18, Tüebingen, Germany
c Pediatric Hematology & Oncology, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of mag-
netic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prere-
quisite for their visualization is the development of cell-permeable imaging probes, which can
specifically interact with the target that characterizes the cellular or molecular process of interest. Here,
we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellu-
lar b-galactosidase (b-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as
cleavable spacer, incorporated between the cell-penetrating peptide D-Tat49–57 and reporter moieties
(Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe,
Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subse-
quent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand
Received 22 September 2010
Revised 5 February 2011
Accepted 9 March 2011
Available online 13 March 2011
Keywords:
b-D-Galactopyranose
Solid phase synthesis
Gadolinium complex
Cellular imaging
14 with GdCl3. Gd-DOTA-k(FR)-Gal-CPP showed
a considerably higher relaxivity enhancement
Intracellular MRI contrast agent
(16.8 0.6 mMꢀ1 sꢀ1, 123 MHz, ꢁ21 °C) relative to the commercial Gd-DOTA (4.0 0.12 mMꢀ1 sꢀ1
,
123 MHz, ꢁ21 °C). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy
that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internal-
ization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the b-gal
expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in b-gal expressing C6/LacZ and enzyme defi-
cient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES
measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells com-
pared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-
k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around
cell nuclei. This cellular distribution was most likely responsible for the observed non-specific back-
ground signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal
probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
cell. These biocatalysts can serve as specific indicators of disease
processes2,3 and remain popular as reporter molecules for reveal-
Molecular imaging is one of the most dynamically evolving re-
search fields aimed at providing tools for noninvasive, quantitative
visualization of fundamental molecular and cellular processes
in vivo.1 Of many clinically essential targets, enzymes are enticing
tags for the monitoring of biological events taking place inside the
ing gene expression in biological systems.4,5 To detect enzyme
activity, and hence indirectly gene expression, specific imaging
probes are required, which upon conversion produce a characteris-
tic signal measurable by the respective imaging modality.
Although mainly optical and nuclear-based approaches have thus
far been used for visualization of marker enzyme activity, the con-
tribution of magnetic resonance imaging (MRI) has been increasing
over the years. The vast potential of MRI for molecular imaging
applications derives from its ability to produce images of opaque
organisms noninvasively, at high spatial and temporal resolution
and without the use of ionizing radiation.
⇑
Corresponding authors. Tel.: +49 7071 601 704; fax: +49 7071 601 702.
E-mail addresses: aneta.brud@tuebingen.mpg.de (A. Keliris), joern.engelmann@
tuebingen.mpg.de (J. Engelmann).
Present address: Dept. for Biological & Biomedical Sciences, Durham University,
Durham, UK.
à
Basically, MR images are generated by using the NMR signal of
water molecules, the intensity of which depends on the relaxation
Present address: Center for Magnetic Resonance Research, Dept. of Radiology,
University of Minnesota, Minneapolis, USA.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.023

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A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
times (T1-longitudinal, T2-transverse) and density of water within a
given volume. To enhance MR image contrast in the region of inter-
est being examined, contrast agents (CAs) are commonly applied as
imaging probes for MRI. These imaging agents function by acceler-
ating relaxation times of nearby water molecule protons,6 where
their efficacy for inducing such changes is defined by the term
‘relaxivity’ (ri = 1,2).7 The majority of CAs nowadays employed in
clinical practice are based on the paramagnetic gadolinium metal
ion (Gd3+) caged within the cavity of a poly(amino carboxylate) li-
gand. The chelating of Gd3+ prevents the toxicity of this metal ion
while still permitting the efficient transmission of its paramagnetic
effect by the rapid exchange of coordinated water with the bulk
solvent. Clinically employed CAs are mostly non-specific and re-
stricted to the extracellular space. Although useful for providing
anatomical information, such CAs are incapable of reporting on ac-
tual biochemical events themselves (i.e., gene expression and en-
zyme activity). Therefore, the development of a new generation
of highly specific CAs is a vital prerequisite for research in areas
aiming at in vivo monitoring of enzyme location and activity by
means of MRI. Over the last decade, many efforts were made to de-
vise enzyme-sensing MRI CAs. In general, the mechanism of their
activation, leading to a change in their relaxivity via modulation
Figure 1. Schematic structure of Gd-DOTA-k(FR)-Gal-CPP. The dashed lines
separate the building blocks.
b-galactosidase. In this CA, a galactose moiety (Gal) serving as
the enzymatically cleavable linker was inserted between the
cell-penetrating peptide (CPP) D-Tat49–57 and the Gd3+-based MRI
reporter moiety, which is a derivative of Gd-DOTA chelate. The
enzymatic activation of this CA following cellular uptake would
require enzymatic cleavage of the transporting peptide and subse-
quent cellular entrapment of the MRI reporter component only in
LacZ-expressing cells, thereby leading to signal amplification. Thus,
the unconverted CA should effectively efflux from cells in the ab-
sence of the target enzyme. To exploit the enzymatic cleavage
reaction occurring at the anomeric center of the sugar unit,
D-Tat49–57 was incorporated at the C-1 position of galactose. The
MRI imaging reporter was introduced at the C-6 position to main-
tain enzymatic activity.14,15 Hydroxyls at C-2, C-3, and C-4 re-
mained unsubstituted because they are essential for binding of
galactose moiety to the active site of enzyme and catalytic hydro-
lysis at anomeric center.16 To enable the detection of specific cellu-
lar retention also by optical imaging, we integrated a fluorescein
moiety (FR) into the structure of Gd-DOTA-k(FR)-Gal-CPP. We have
of water hydration number (q) or rotational correlation time (sr),
was driven by the enzyme-catalyzed formation or cleavage of
chemical bonds. Accordingly, alterations such as increasing the q
number (r1 is linearly proportional to q) or slowing down the
molecular tumbling rate by increasing the size (longer sr) would
translate to elevated relaxivity of the Gd3+-complexes. Recent
developments contributing towards the creation of enzyme-
responsive CAs were initiated with the synthesis of b-EgadMe.8
This probe was designed to report on b-galactosidase activity, an
enzyme encoded by the bacterial LacZ reporter gene commonly
used for tracking gene expression. The authors used a modulation
of q in the inner coordination sphere of the Gd3+-chelate, occurring
upon cleavage of the galactose moiety by b-gal, to achieve r1
changes and thereby MR image contrast enhancement. However,
due to its lack of transmembrane permeability, b-EgadMe had to
be introduced into the cells by microinjection. A different approach
for obtaining enzyme-hydrolysable MRI CAs explored the changes
chosen D-amino acids for synthesis of the CPP, D-Tat49–57, (making
it stable to proteases) to ensure that the observed intracellular
accumulation of MRI and FR imaging moieties resulted from enzy-
matic reaction on the sugar and not from peptide degradation. This
activation–retention strategy via enzyme-mediated activation had
proven successful for radionuclide imaging of reporter gene
expression.17 Here, we present efficient synthesis and physico-
chemical evaluation of this bimodal, cell membrane-permeable,
b-gal-targeting probe. In addition, in vitro assays were performed
using b-gal-positive and enzyme deficient cells for the assessment
of translocation and specific retention of Gd-DOTA-k(FR)-Gal-CPP
inside target-containing cells.
of rotational correlation time (sr) through enzyme-controlled mod-
ulation of the affinity of the imaging probe for proteins.9–11 In this
case, the enzymatically cleavable moiety is introduced into the
protein binding unit to prevent its strong interactions in the ab-
sence of enzyme. Thus, upon enzymatic reaction, cleavage of the
masking group results in the subsequent binding of the fast tum-
bling Gd3+-complex to proteins to yield slowly tumbling molecular
adducts with higher relaxivity. Together with developments in
generating enzyme-hydrolysable MRI CAs, other groups utilized
an opposite activation mechanism involving chemical bond forma-
tion specifically mediated by target enzymes (i.e., polymerases or
oxidoreductases).10–12 In this instance, the Gd3+-complex bears a
moiety which can be specifically oxidized by a certain enzyme to
generate reactive product species that subsequently oligomerize
in situ to produce slowly rotating larger molecules with higher
relaxivity. Although the examples mentioned above demonstrate
the feasibility of monitoring enzymatic activity, and thereby visu-
alization of gene expression by MRI, the field is still in its infancy.13
A decisive constraint on the existing enzyme-responsive MRI CAs
regarding their applicability for in vivo monitoring of intracellular
enzyme activity, is clearly their lack of efficient cell membrane
permeability. Thus, further advances are required to facilitate the
optimal translation of such enzyme-based approaches to in vivo
models.
2. Results and discussion
2.1. Synthesis of Gd-DOTA-k(FR)-Gal-CPP
The bimodal probe Gd-DOTA-k(FR)-Gal-CPP (Fig. 1) was ob-
tained using a facile building block strategy, which involved the
preparation of basic segments and then linking them together by
Fmoc-mediated solid phase synthesis (SPS). We started with the
synthesis of the Fmoc-protected galactose derivative 11 (Scheme 1)
serving as the key building block for our CA. This monosaccharide
was designed in such a way that would enable the efficient incor-
poration of D-Tat49–57 and the DOTA ligand/FR moiety at its respec-
tive 1-O and 6-O positions during sequential synthesis on a
polymeric support. We based the synthesis of 11 mainly on trans-
formations, which involved thiogalactosides, because these versa-
tile and robust glycosyl donors allow a wide scope of synthetic
manipulations. Accordingly, the thiophenyl galactose donor 1
With the aim of devising an enzyme-activated MRI CA capable
of crossing cell membranes, we synthesized and evaluated the
model contrast agent Gd-DOTA-k(FR)-Gal-CPP (Fig. 1) targeting

A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
2531
mixture even after using the longer reaction time of 48 h. Attempts
to increase the conversion by elevating the temperature (55 °C),
using a different solvent (THF), or an excess of alkylating agent
added in several aliquots after certain time periods, failed to im-
prove the overall reaction yield. Thus, of the reaction conditions
tested, the etherification method initially applied proved to be
the best to provide 6. At this point the PMB ethers had to be ex-
changed with acetate esters in order to assist high glycosylation
selectivity towards the b-galactoside 9. Although an efficient acidic
cleavage of PMB ethers using 5% or 10% TFA in DCM (v/v) has been
reported elsewhere,24 both conditions failed to provide 7. In con-
trast, oxidative cleavage of PMB groups by DDQ yielded 7 at 60%.
Next, the free hydroxyl groups were reacted with acetic anhydride
in pyridine to give acetate protected 8 (85%). In the following step,
tert-butyl 3-hydroxypropionate was subjected to glycosylation
with the thiogalactose donor 8 using NBS/Me3SiOTf as an activa-
tor.25 The resulting b-glycosylated product 9, formation of which
was facilitated by the participation of the neighboring 2-O-acetyl
group, was obtained at a yield of 50%. The cleavage of the tert-butyl
ester in 9 with TFA/DCM (1:1) occurred concomitantly with the
partial hydrolysis of the Cbz-protecting group. The removal of
Cbz was completed by catalytic hydrogenation over Pd/C at 2 bar
of hydrogen to yield the intermediate 10. Finally, the amino group
was protected in the reaction of 10 with Fmoc succinimide (Fmoc-
OSu) to give the desired building block 11 at a yield of 64%.
We proceeded further with the manual SPS of conjugate 13 as
presented in Scheme 2. First, the side chain protected peptide D-
Tat49–57 (r(Pbf)k(Boc)k(Boc)r(Pbf)r(Pbf)q(Trt)r(Pbf)r(Pbf)r(Pbf)) was synthe-
sized on pre-loaded Wang resin. Thereafter, the monosaccharide
11 (3 equiv), activated with HATU (3 equiv)/DIPEA (6 equiv) in
DMF, was coupled to the N-amino terminus of the peptide. This
step was followed by Fmoc deprotection and conjugation of a
Fmoc/Dde protected
D-lysine (Fmoc-D-k(Dde)-OH) to the amino
functionality on the sugar moiety. The attached lysine residue
served as a linker for the incorporation of the macrocyclic chelate
1226 (DOTA (t-Bu)3) and fluorescein at its respective primary amino
groups. After 12 was coupled to the a-NH2 group of the side chain
protected lysine, the Dde group was removed by treatment with 2%
hydrazine hydrate in DMF. Subsequently, the fluorophore was
introduced at the e-NH2 group of lysine in a reaction with fluores-
Scheme 1. Synthesis of building block 11. Reagents and conditions: (i) PhSH,
BF3ꢂEt2O, DCM; (ii) NaOHcat., MeOH; (iii) pyridine, Et3N, TBDMSCl, 0 °C to rt; (iv)
NaH, PMBBr, DMF, 0 °C to rt; (v) 1 M TBAF/THF, THF; (vi) Br(CH2)3NHCbz, NaH,
DMF, 0 °C to rt; (vii) DDQ, H2O/DCM (1:30), rt; (viii) Ac2O, pyridine; (ix)
Br(CH2)2COOtBu, NBS, TMSOTf, DCM, ꢀ50 °C; (x) (a) DCM/TFA 1:1; (b) H2/Pd,
EtOH; (xi) Fmoc-OSu, Na2CO3, DCM, water.
cein isothiocyanate (FITC) (4 equiv) and DIPEA (8 equiv) in DMF for
12 h. Finally, the entire molecule was cleaved off the polymeric
support using TFA/TIPS/m-cresol/water with the simultaneous
deprotection of all acid-labile protecting groups of the amino acid
side chains as well as tert-butyl esters on the macrocyclic chelator.
Subsequent O-deacetylation of released conjugate 13 proved to be
challenging. Our initial attempts to remove the acetates by hydra-
zinolysis, prior to release of the molecule from resin,27 led to
decomposition of the O-glycosidic linkage under these acidic con-
ditions. Therefore, deprotection of the acetate esters, which are
known to indirectly stabilize the acid-labile bonds,28 was per-
formed in solution following the detachment of conjugate 13 from
the polymeric support. Commonly used procedures such as
Zemplén deprotection or treatment with methanolic ammonia
were tested. However, under the conditions employed, only an
incomplete O-deacetylation of 13 was observed leading to a main
product containing a single acetyl group as revealed by LC–ESI-
MS analysis. The observed resistance to deprotection might be
associated with the previously reported difficulties in cleaving
the acetate ester at 4-OH of galactose moieties in polyfunctional
systems due to steric hindrance.29 Finally, treatment of 13 with
hydrazine hydrate in MeOH (1:6 v/v)30 for 12 h was found to be
optimal to generate 14. Metalation of this ligand with gadolinium
was accomplished by adding GdCl3 (0.9 equiv) to a solution of 14
(1 equiv) in ultrapure water. Chelation was completed after stirring
at room temperature for 72 h, as monitored by LC–ESI-MS. The
was prepared with a 70% yield using a commercially available
penta-O-acetyl-b-D
-galactopyranoside as the starting material.18
Following quantitative O-deacetylation, the primary hydroxyl
group was silylated with tert-butyldimethylsilyl chloride in pyri-
dine-triethylamine to furnish 3 (80%). Hydroxyl groups were pro-
tected as p-methoxybenzyl (PMB) ethers using p-methoxybenzyl
bromide and NaH in dry DMF. The resulting product 4 was treated
with TBAF in THF to give 5 (69%). The PMB ethers introduced as
protecting groups facilitated the subsequent incorporation of a
propylene spacer linked Cbz-protected amino functionality into
the sugar moiety via an ether bond created at the primary hydroxyl
group of 5. So far, examples of mono-alkylation at the 6-O position
of galactopyranosides with higher n-alkanes (n P 3) are rare in the
literature19–21 and proved to be difficult in several cases.22 Hence,
to obtain our propylene-tethered derivative 6, we executed the
Williamson reaction as the method of ether preparation most com-
monly used in organic chemistry. Benzyl 3-bromopropylcarba-
mate23 and in situ generated monosaccharide alkoxide of 5 were
reacted in DMF at room temperature to give 6 at a moderate yield
of 45% after purification. As indicated by TLC and LC–ESI-MS, a
small amount of substrate 5 was still present in the reaction

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A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
Scheme 2. Synthesis of Gd-DOTA-k(FR)-Gal-CPP. Reagents and conditions: (i) 11, HATU, DIPEA, DMF, 3 h; (ii) Fmoc-Lys(Dde)-OH, HATU, DIPEA, DMF, 3 h; (iii) DOTA-(t-Bu)3,
HATU, DMF, 24 h; (iv) (a) 2% hydrazine hydrate, DMF, 2 ꢃ 4 min; (b) FITC, DIPEA, DMF, 12 h; (v) TFA/m-cresol/TIPS/H2O (90:5:2.5:2.5), 4 h; (vi) hydrazine hydrate/MeOH (1:6
v/v), 12 h; (viii) GdCl3ꢂ6H2O, 40 °C/12 h, rt 3 days.
absence of free Gd3+ metal ions was confirmed by a competitive as-
say with DTPA (see Section 4). The crude product was purified by
RP-HPLC, dialyzed and lyophilized to yield the desired Gd-DOTA-
k(FR)-Gal-CPP, the structure of which was verified by ESI-MS.
Hence, the building block approach presented here, which involved
different branches of chemistry, proved to be a viable synthetic
strategy for devising a bimodal Gd-DOTA-k(FR)-Gal-CPP conjugate.
constructs. Fast and independent local motions of Gd3+ containing
segments attached to macromolecules can significantly diminish
an expected increase of relaxivity for slow rotating high molecular
weight conjugates.35 Thus an increased relaxivity is more likely in
case that the fast internal motions of attached Gd3+ chelates are re-
stricted.36 We presume that the observed high longitudinal relax-
ivity of Gd-DOTA-k(FR)-Gal-CPP was obtained as result of
effective increase in global sr as a consequence of the higher
molecular weight and restricted local motion of the Gd3+ chelate
moiety. The latter is believed to be an effect of two mainly contrib-
uting factors. First, the influence on the local motions might origi-
nate from the steric hindrance created by the neighbored
fluorophore in the close spatial vicinity of the Gd3+ chelate moiety.
We assume that a second contribution to slow rotational dynamics
of Gd-DOTA-k(FR)-Gal-CCP might be given by the slowed down
rotation of the Gd3+ chelate moiety due to its coupling at the
2.2. Longitudinal relaxivity
The efficacy of Gd-DOTA-k(FR)-Gal-CPP to shorten the water
proton relaxation time T1 was studied at
a proton Larmor
frequency of 123 MHz at room temperature (ꢁ 21 °C). The r1 of
Gd-DOTA-k(FR)-Gal-CPP in water (16.8 0.6 mMꢀ1 sꢀ1) was sub-
stantially higher than that of Gd-DOTA (4.0 0.12 mMꢀ1 sꢀ1) mea-
sured under the same conditions. It is well known that conjugation
of bulky molecules (i.e., peptides,31 proteins32 or dendrimers33) to
small Gd-DOTA like complexes reduces their molecular tumbling
a-amino group attached to the sterically crowded secondary
-carbon of lysine linker. In order to explore the impact of the
a
rate, increases the rotational correlation time (
s
r), and conse-
structural arrangement in close vicinity of Gd3+ chelate on the
measured relaxivity, we synthesized a second conjugate, Gd-
DOTA-Gal-k(FR)-CPP, shown in Figure 2 (for the synthetic details,
see Section 4). The rational for the design of this molecule was to
quently yields MRI probes with elevated relaxivity.34 However,
the grafting of small Gd3+ complexes to large macromolecules is
not always associated with an effective increase of sr for such

A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
2533
conditions in complete serum containing culture medium. Cells
were treated afterwards with trypan blue to quench external and
surface-bound fluorescence as well as fluorescence from cells with
disrupted cell membrane integrity.39 As demonstrated by fluores-
cence spectroscopy, Gd-DOTA-k(FR)-Gal-CPP was very efficiently
transported into the cells in a concentration dependent manner
(Fig. 3a) and without inducing cytotoxicity up to 20 lM (data not
shown). We selected a long treatment time (18 h) in these experi-
ments, because the efficiency of cellular labeling with CA was nota-
bly increased (on average twofold) after prolonged incubation
times (data not shown). The high cellular uptake of Gd-DOTA-
k(FR)-Gal-CPP was of great importance for the subsequent MRI
imaging of cells because of the relatively low sensitivity of MRI,
which requires large amounts of CA to be delivered for visualiza-
tion of intracellular targets. Quantitative results of fluorescence
spectroscopy revealed a reproducibly greater fluorescence signal
from C6/LacZ cells compared to enzyme deficient C6 cells over
the whole range of tested concentrations (Fig. 3a). The largest sta-
tistically significant difference in the measured fluorescence be-
Figure 2. Schematic structure of Gd-DOTA-Gal-k(FR)-CPP.
reduce steric hindrance nearby the Gd3+ chelate and thus allow fas-
ter local motions by moving the lysine linker with fluorescein to
the peptide segment of the conjugate. Accordingly, the Gd-DOTA
chelate was directly attached to the aminopropyl spacer of the gal-
actose core whereas the lysine-FR spacer was inserted between the
galactose moiety and D-Tat49–57 peptide. With this rearrangement
we expected decrease in relaxivity due to the increased internal
motions of Gd-DOTA moiety. In fact, the measured relaxivity of
Gd-DOTA-Gal-k(FR)-CPP (10.0 0.8 mMꢀ1 sꢀ1) was significantly
lower compared to r1 values obtained for Gd-DOTA-k(FR)-Gal-
CPP, even though the molecular weight of both CAs was identical.
It appears evident, that position of the fluorophore with lysine lin-
ker relative to the Gd-DOTA chelate had a considerable influence
on the r1 values of the CAs investigated here. On the other hand,
the relaxivity of Gd-DOTA-Gal-k(FR)-CPP was still considerably
higher than that of Gd-DOTA itself (4.0 0.12 mMꢀ1 sꢀ1) showing
the influence of a slowed down tumbling rate with increasing
molecular size of conjugates. In agreement with our assumption,
increase of molecular weight as well as steric hindrance of the local
motions of the Gd3+ chelate additively contributed to the slower
rotational dynamics of Gd-DOTA-k(FR)-Gal-CPP and thus its mark-
edly elevated relaxivity. However, this phenomenon requires more
thorough investigations. Only Gd-DOTA-k(FR)-Gal-CPP, as the
more efficient CA, was further examined in cell studies.
2.3. Internalization studies by fluorescence spectroscopy
A vital prerequisite for visualization of intracellular targets is
clearly the efficient delivery of imaging probes into the cell. Of
the well-known CPPs, Tat basic domain peptides have been
exploited extensively for translocation of different molecules
across cell membranes.37 Their efficacy proved to be highly depen-
dent on the size and nature of the transported cargo.38 In the
present study, we used fluorescence spectroscopy to assess the
efficiency of internalization as well as specific retention of Gd-
DOTA-k(FR)-Gal-CPP in rat glioma cells (C6/LacZ) expressing
b-galactosidase. The parental cell line (C6), deficient in the target
enzyme, was used in all experiments as a control. Due to the pro-
posed mode of activation for Gd-DOTA-k(FR)-Gal-CPP, after incu-
bation with CA, a stronger fluorescent signal would be expected
from C6/LacZ cells compared to the enzyme deficient C6 cell line
as a result of enzyme-induced cellular retention of imaging moie-
ties via cleavage of the D-Tat49–57 permeation peptide. To test our
enzyme targeting probe, both cell types were incubated with differ-
ent concentrations of Gd-DOTA-k(FR)-Gal-CPP under physiological
Figure 3. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP and localization after labeling
of C6/LacZ and C6 rat glioma cells in serum containing complete medium for 18 h at
37 °C. External fluorescence was quenched with trypan blue and subsequent
washes with HBSS. (a) Fluorescence spectroscopy. Values are means SEM (n = 2–4
with six replicates each), ⁄p <0.05, ⁄⁄⁄p <0.001, statistically significantly different
compared to C6 cells (Student’s t-test). (b) Fluorescence microscopy. Nuclei: blue
(Hoe), green (FR fluorescence of contrast agent). The bar represents 20 lM.

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A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
tween both cell lines was found at the 10
lM labeling concentra-
higher increase of R1,cell values was observed in C6/LacZ compared
tion of Gd-DOTA-k(FR)-Gal-CPP. Hence, these data imply that the
bimodal probe investigated here showed the expected higher cel-
lular retention in transgenic b-galactosidase expressing cells, de-
spite the presence of strong non-specific fluorescence in enzyme
deficient C6 cells.
to enzyme deficient C6 cells. However, this difference in R1,cell val-
ues was not statistically significant. The gadolinium content in
these cells was further quantified by ICP-AES of corresponding cell
lysates. The results revealed an about four times higher concentra-
tion of gadolinium in b-gal containing C6/LacZ cells (0.85
0.05 Gd fmol/cell) compared to enzyme deficient C6 cells (0.21
0.01 Gd fmol/cell). Hence, our results demonstrated a significantly
higher loading of C6/LacZ cells by the MRI reporter compared to C6
cells and remain in agreement with the fluorescence spectroscopy
data. However, it is also obvious that the cellular relaxation rate
R1,cell of intact cells does not linearly correlate with the internalized
gadolinium concentration. The non-significant difference in cellu-
lar relaxation rates R1,cell between both cell lines might be ascribed
to the lower relaxivity of the smaller residual MRI probe accumu-
lating in C6/LacZ cells upon the cleavage of D-Tat49–57 peptide. The
converted MRI probe is expected to have faster rotational dynam-
ics compared to parent Gd-DOTA-k(FR)-Gal-CPP conjugate, thereby
lower r1. Therefore, even though the Gd-loading of C6/LacZ cells
was much higher compared to C6 cells, the cellular relaxation rate
R1,cell would be influenced to a lesser extent by the cleaved probe,
and probably in our case not enough to create a substantial differ-
ence in R1,cell between both cell lines. Moreover, the predominantly
vesicular localization of the dual-labeled probe, observed by fluo-
rescent microscopy, would certainly reduce the dynamic efflux of
the uncleaved Gd-DOTA-k(FR)-Gal-CPP, thereby leading to a signif-
icant non-specific background signal contributing to the overall
signal detected in either cell line. In addition, the largely vesicular
distribution of the probe in cells can cause so called relaxation
quenching by restricting the number of water molecules which
have access to the contrast agent.48,49 Endosomal entrapment pre-
sumably reduced the extent of interactions between cytosolic en-
zyme and our conjugate. The detected higher accumulation of
imaging reporters in C6/LacZ cells suggested, however, that a cer-
tain amount of conjugate could escape the vesicles and retained its
intended functionality. Examples of maintained biological func-
tionality of endocytically internalized cargo molecules were dis-
cussed in the literature.50,51 El-Andaloussi et al. reported, that
entrapped fluorescent cargo molecules could to some extent es-
cape the endosomes and exhibit its biological activity, even though
only an exclusively punctuate vesicular distribution was detected
in microscopic images. It was stated in several reports that small
amounts of cargo released from endosomes could not be detected
by microscopy in the presence of the much stronger fluorescence
from the vesicles but also because of concentrations below
2.4. Fluorescence microscopy studies
Following fluorescence spectroscopy, we performed fluores-
cence microscopy with the same cells. The microscopic images of
labeled cells showed that green fluorescent conjugate was pre-
dominantly localized in vesicles displayed as bright punctuate dots
around nuclei (Fig. 3b). The observed cellular distribution pattern
might indicate endocytosis as the main route of internalization of
Gd-DOTA-k(FR)-Gal-CPP. Endocytosis was recently proposed by
other groups as a common translocation mechanism of CPPs.40
Nonetheless, there are still several uncertainties regarding this
mechanism because many factors can radically influence the effi-
ciency of cellular uptake as well as subcellular distribution of per-
meation peptides with integrated cargos.38,41 Furthermore,
preserved functionality of the CPP-associated cargo also proved
to be ambiguous. Reports demonstrating restricted contact of
CPP-transported cargo with cytosolic targets due to endosomal
encapsulation42 as well as examples of the opposite, documenting
efficient interactions43,44 can be found in the literature. For exam-
ple, efficient activation by proteases and cellular retention of cargo
transported by Tat only in target-containing cells has been re-
ported45 proving the successful utilization of this peptide for en-
zyme targeting approaches.
2.5. MR imaging in cells
To assess the ability of our CA to increase the cellular relaxation
rates (R1,cell (1/T1)), we performed spin-lattice relaxation time (T1)
measurements of cells incubated with Gd-DOTA-k(FR)-Gal-CPP
using a 3T human MR scanner. A labeling concentration of 10 lM
was chosen for the MRI cell studies because of the results of fluo-
rescence spectroscopy showing the largest difference in the cellu-
lar accumulation of the imaging probe between b-gal expressing
C6/LacZ and deficient of enzyme C6 cells at this concentration of
externally applied CA. Accordingly, confluent cultures of C6/LacZ
and parental C6 cells were incubated with 10 lM of Gd-DOTA-
k(FR)-Gal-CPP for 18 h. Control cells were treated in the same
way but without CA in the culture medium. The relaxation rates
of the samples were determined in an axial slice through the pellet.
The MR results showed a substantial positive contrast enhance-
ment in C6/LacZ as well as C6 cells after loading with 10 lM of CA
(Fig. 4). The used labeling concentration, 10–30 times lower than
those typically reported for delivery of CPP coupled Gd-DOTA che-
lates into cells,46,47 was sufficient to observe an increase of cell-
associated T1 values. This might be due to the high longitudinal
relaxivity of Gd-DOTA-k(FR)-Gal-CPP conjugate (see Section 2.2).
The increase of cellular relaxation rates R1,cell (1/T1) of labeled
C6/LacZ as well as C6 cells was statistically significant compared
to control cells without CA as indicated in Figure 5. A slightly
Figure 5. Cellular relaxation rates R1,cell in C6/LacZ and C6 cells after labeling with
10 lM of Gd-DOTA-k(FR)-Gal-CPP for 18 h at 37 °C. Cells were trypsinized,
centrifuged and re-suspended in 1.5 ml Eppendorf tubes at 2 ꢃ 107 cells/500
ll in
complete serum containing medium for MRI studies. Control: cells incubated
identically with culture medium without CA. Values are means SEM (n = 2), ⁄⁄p
<0.01, ⁄⁄⁄p <0.001 statistically significantly different compared to respective
controls (Student’s t-test).
Figure 4. T1-weighted images of C6/LacZ cells incubated with 10 lM of Gd-DOTA-
k(FR)-Gal-CPP for 18 h. (a) Control without contrast agents and b) cells with Gd-
DOTA-k(FR)-Gal-CPP.

A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
2535
0.2
l
M will be masked by autofluorescence.50,52,53 We assume that
Polarimeter 341 (Perkin–Elmer, Germany) using a 1 dm quartz cell
and the concentrations are reported in g/100 ml. Analytical and
semi-preparative Reversed-Phase High Performance Liquid Chro-
matography (RP-HPLC) were performed at room temperature on
a Varian PrepStar Instrument (Australia) equipped with PrepStar
SD-1 pump heads. UV absorbance was measured using a ProStar
335 photodiode array detector at 214 and 245 nm. Analytical RP-
the cytosolic localization of our conjugate was not detectable by
fluorescence microcopy for the same reasons. Furthermore, the dis-
crepancies in the quantitative measurements from ICP-AES, MR,
and optical imaging can be also related to the sensitivity difference
between these methods.54 Nonetheless, all methods showed a ten-
dency for higher loading with imaging reporters in enzyme con-
taining C6/LacZ cells, indicating
a
specific accumulation of
HPLC was performed on
250 mm, particle size 5 m, particle pore diameter 100 Å; Varian,
Germany) and preparative RP-HPLC was performed on a Polaris 5
18-Ether column (21.2 ꢃ 250 mm, 5 m, 100 Å; Varian, Germany)
or Polaris C8-Ether column (100 ꢃ 250 mm, 5 m, 100 Å). The com-
a
Polaris
C
18-Ether column (4.6 ꢃ
bimodal probe in the presence of the enzymatic target. To improve
the interaction of the contrast agent with the cytosolic target, a
more efficient release from the vesicles or its direct uptake into
the cytoplasm are required. We are currently working on such
strategies.55
l
C
l
l
pounds were purified using one of the following methods. Method
1: a linear gradient was used with the mobile phase starting from
90% of solvent A (0.1% TFA/water), 10% of solvent B (0.1% TFA/ace-
tonitrile) isocratic for 5 min and increased to 60% B over 20 min,
moving to 90% B over 3 min, and 90% B isocratic up to 30 min.
Method 2: a linear gradient was used with the mobile phase start-
ing from 80% of solvent C (0.05% TFA/water), 10% of solvent D
(0.05% TFA/acetonitrile) isocratic for 5 min and increased to 60%
D over 20 min, moving to 90% D over 3 min, and 90% D isocratic
up to 30 min. The flow rate used for analytical HPLC was
1 ml/min and for a preparative RP-HPLC 4 ml/min or 10 ml/min.
3. Conclusions
In summary, we report the synthesis of a novel dual-labeled
imaging probe Gd-DOTA-k(FR)-Gal-CPP, based on a galactose moi-
ety with incorporated Gd-DOTA, FR and transduction (D-Tat49–57
)
moieties. This contrast agent was produced using a multistep
building block strategy. Gd-DOTA-k(FR)-Gal-CPP showed a sub-
stantial increase of r1 values compared to the commercially avail-
able Gd-DOTA chelate. The insertion position of fluorescein-lysine
spacer versus the attachment position of Gd-DOTA chelate was
found to have a significant effect on the resultant longitudinal
relaxivity values of the synthesized Gd3+ complexes. In vitro cell
studies revealed that Gd-DOTA-k(FR)-Gal-CPP was very efficiently
internalized into cells in a concentration dependent fashion. The
higher accumulation of imaging reporters in enzyme containing
C6/LacZ cells was observed by fluorescent spectroscopy, MRI and
ICP-AES measurements. Nonetheless, structural modifications are
still required to circumvent the predominantly vesicular entrap-
ment of the CA investigated here, and these are currently under-
way. Despite restricted interactions with the target enzyme, we
nevertheless developed an efficient cell-permeable bimodal CA,
which can be potentially used for cell imaging by MRI as well as
optical modalities.
All the injected solutions were filtered through
a nylon-66
Millipore filter (0.45 mm) prior to purification. Electrospray Ioniza-
tion-Mass Spectrometry (ESI-MS) spectra were measured on an SL
1100 system (Agilent, Germany) with ion-trap detection in positive
and negative mode. HR-FT-ICR mass spectra were generated on an
APEX 2 spectrometer (Bruker Daltonic, Germany) with ESI. Liquid
Chromatography–Electrospray Ionization-Mass Spectrometry
(LC–ESI-MS) analysis of samples was performed using an in-house
system consisting of an analytical RP-HPLC Beckman System Gold
LC 126 (Germany) and the ESI-MS SL 1100 system (Agilent, Ger-
many). The LC system was equipped with 508 Autosampler. UV
absorbance was measured using UV 168 detector at 214 and
245 nm. RP-HPLC was performed using a Polaris C18-Ether column
(4.6 ꢃ 250 mm, particle size 5
lm, particle pore diameter 100 Å,
Varian, Germany). NMR spectra were recorded on a Bruker Avance
II 300 MHz ‘Microbay’ spectrometer (1H: CDCl3 internal reference
at 7.27 ppm, DMSO at 2.5 ppm, TMS at 0 ppm); (13C, 75 MHz: inter-
nal reference CDCl3 at 77.0 ppm, DMSO at 39.51 or TMS at 0 ppm).
Abbreviations: ps-pseudo-singlet, os-overlapping singlet, pd-
pseudo-doublet. All measurements were performed at room tem-
perature. Solvent residual signals in 1H and 13C NMR spectra were
assigned based on published data.56 Proton and carbon signal
assignments were confirmed by 1H–1H-COSY and 1H–13C-COSY
(HSQC) experiments. Gadolinium concentrations of Gd-DOTA-
k(FR)-Gal-CPP, Gd-DOTA-Gal-k(FR)-CPP, and cell lysates were
determined at Mikroanalytisches Labor Pascher (Remagen,
Germany) by ICP-AES on an Icap 6500 by Thermo Instruments.
4. Experimental
4.1. General methods
All reagents and solvents were purchased from Acros Organics,
Fluka, Sigma–Aldrich or Merck (Germany) and used without fur-
ther purification unless otherwise stated. Tetraazacyclododecane
(cyclen) was obtained from Strem Chemicals (USA). The used
Fmoc-protected
D
-amino acid derivatives (Fmoc-
D-Arg(Pbf)-OH,
Fmoc- -Lys(Boc)-OH, Fmoc-
D
D
-Gln(Trt)-OH, Fmoc- -Lys(Dde)-OH),
D
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU), and Wang resin pre-loaded with Fmoc/Pbf pro-
tected
D
-arginine (0.4 mmol/g, 100–200 mesh) were obtained from
4.2. Experimental procedures and spectral data
Novabiochem, United Kingdom. 2-(1-H-7-Azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) was
purchased from McTony (Canada). Dry, anhydrous solvents: DMF,
dichloromethane (DCM), acetonitrile (ACN), methanol (MeOH) as
Sure-Seal bottles with molecular sieves, N,N-Diisopropylethyl-
amine (DIPEA), and hydroxybenzotriazole (HOBt) were purchased
from Acros Organics (Belgium). Water was purified using a Milli-
Q Synthesis purifier (Millipore, Germany). Column chromatogra-
phy was carried out using silica gel, mesh size 70–230 Å (Merck,
Germany). Aluminum sheet silica gel plates with 0.2-mm-thick Sil-
ica Gel 60 F254 (Merck, Germany) were used to run thin-layer chro-
matography (TLC). The compounds were visualized by UV254 light
or charring with 5% H2SO4/EtOH (for carbohydrates) or I2 vapor.
4.2.1. Phenyl-1-thio-b-D-galactopyranoside (2)
O-Acetyl deprotection of 1 (150 mmol, 66 g) was carried out in
dry MeOH (500 ml) in the presence of sodium methoxide. Progress
was monitored by TLC (MeOH/ethyl acetate 1:9, Rf = 0.25). After
completion, the reaction mixture was neutralized with Dow-
exÒ50Wx8-100 ion-exchange resin (pH ꢁ6), filtered, and concen-
trated to give 2 (40 g, 98%) in the form of an oil. The spectral
data were consistent with that reported in the literature.18
4.2.2. Phenyl 6-(O-tert-butyldimethylsilyl)-1-thio-b-D-
galactopyranoside (3)
To a solution of 2 (78.96 mmol, 21.5 g) in dry pyridine (310 ml)
tert-butyldimethylsilyl chloride (TBDMSCl) (118.4 mmol, 17.84 g)
Optical rotations [a]D were measured at 20 °C with a Perkin–Elmer

2536
A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
was added portionwise, followed by triethylamine addition at
ꢀ10 °C over 20 min under nitrogen. The reaction mixture was stir-
red at ꢀ10 °C for 1 h, slowly warmed up to room temperature and
stirred for the next 20 h until the starting material was consumed,
as monitored by TLC (MeOH/ethyl acetate 9:1, Rf = 0.9). The mix-
ture was diluted with DCM (400 ml), poured into ice-cold water
and extracted with DCM (4ꢃ). The combined organic layers were
washed with saturated aqueous NaHCO3, brine, dried over Na2SO4,
filtered, and concentrated. The organic residue was purified by col-
umn chromatography using hexane/ethyl acetate to yield 3 as a
hexane/ethyl acetate as eluent (hexane/ethyl acetate 1:1,
Rf = 0.49) to yield 5 at 69% as a white foam. ½a D20
= ꢀ2.4 (c 0.8,
ꢄ
CHCl3).1H NMR (300 MHz, CDCl3): d = 3.35–3.52 (m, 2H, H-5, 1H,
H-6), 3.55 (dd, 1H, H-3, J3–2 = 9.3 Hz, J3–4 = 2.83 Hz), 3.75–3.85
(m, 11H, H-6, CH3, H-4), 3.90 (t, 1H, H-2, J = 9.44 Hz), 4.56 (d, 1H,
OCH2, J = 11.52 Hz), 4.62 (d, 1H, H-1, J1–2 = 9.63 Hz), 4.65–4.79
(m, 4H, OCH2), 4.88 (d, 1H, OCH2, J = 11.33 Hz), 6.82–6.94 (m, 6H,
ArH), 7.14–7.37 (m, 9H, ArH), 7.48–7.57 (m, 2H, ArH). 13C NMR
(75 MHz, CDCl3): d = 55.23 (CH3), 62.23 (C-6), 72.68 (OCH2), 72.74
(C-4), 73.64 (OCH2), 75.26 (OCH2), 77.23 (C-2), 78.72 (C-5), 83.91
(C-3), 87.75 (C-1), 113.71, 113.73, 113.84, 127.06, 128.79, 129.21,
129.90, 130.27, 130.37, 130.45, 131.37, 134.08, 159.26 (Ar). HRMS
yellow viscous oil (80%, 24.42 g). ½a D20
ꢄ
= ꢀ45.6 (c 0.8, MeOH). 1H
NMR (300 MHz, DMSO-d6): d = 0.05, 0.07 (2s, 2 ꢃ 3H, SiCH3), 0.9
(s, 9H, C(CH3)3, 3.37–3.50 (m, 2H, H-2, H-3), 3.52–3.64 (m, 1H,
H-5), 3.68–3.77 (m, 3H, H-6, H-4), 4.54 (pd, 1H, OH), 4.64 (d, 1H,
J1–2 = 9.1 Hz, H-1), 4.97 (pd, 1H, OH), 5.18 (pd, 1H, OH), 7.20–7.36
(m, 3H, Ar-H), 7.45–7.51 (m, 2H, Ar-H). 13C NMR (75 MHz,
DMSO-d6): d = ꢀ5.28, ꢀ5.44 (SiCH3), 17.98 (C(CH3)3), 25.81
(C(CH3)3), 62.89 (C-6), 68.50, 69.16, 74.56 (C-4, C-2, C-3), 79.04
(C-5), 87.80 (C-1), 126.03 128.71, 129.07, 135.72 (ArH).
(EI) (+) for C36H40O8S: [M+Na]+(found) = 655.23398, [M+Na]+
655.23361.
=
(calcd)
4.2.5. Phenyl 2,3,4-tri-O-(4-methoxybenzyl)-6-O-(3-
benzyloxycarbonylaminopropyl)-1-thio-b-D-galactopyranoside
(6)
Sodium hydride (47.6 mmol, 1.9 g, 60% dispersion in mineral
oil) was added portionwise at ꢀ10 °C to a solution of 5 (14.5 mmol,
9.16 g) in DMF (95 ml) under nitrogen. The reaction mixture was
stirred for 30 min at ꢀ10 °C and N-benzyloxycarbonyl-3-bromo-
propylamine (58 mmol, 15.8 g) dissolved in DMF (13 ml) was
added. Stirring was continued at ꢀ10 °C for 30 min and then at
room temperature. The progress of the reaction was monitored
by TLC. After 24 h the reaction mixture was cooled to 0 °C, MeOH
(7 ml) was slowly added to destroy the excess of sodium hydride.
The mixture was stirred for 10 min, further diluted with ethyl ace-
tate (250 ml) and poured into ice-cold water. The organic layer was
separated and the water layer was extracted with ethyl acetate
(3ꢃ), DCM (1ꢃ). The organic layers were combined, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography with hexane/ethyl ace-
tate as eluent (hexane: ethyl acetate 1:1, Rf = 0.6), to give 5.37 g
HRMS (EI) (+) for
C
18H30O5SSi: [[M+Na]+(found) = 409.14752,
M+Na]+(calcd) = 409.14754.
4.2.3. Phenyl 2,3,4-tri-O-(4-methoxybenzyl)-6-O-(tert-
butyldimethylsiloxypropyl)-1-thio-b- -galactopyranoside (4)
D
To a solution of 3 (32.8 mmol, 12.64 g) and 4-methoxybenzyl
bromide (115 mmol, 23 g, 16.6 ml) in anhydrous DMF (400 ml), so-
dium hydride (115 mmol, 4.6 g, 60% dispersion in mineral oil) was
added at ꢀ10 °C. The reaction mixture was stirred at ꢀ10 °C for
30 min and the next 4 h at room temperature. The progress of
the reaction was monitored by TLC (hexane/ethyl acetate 1:1,
Rf = 0.7). The reaction mixture was cooled to 0 °C, diluted with cold
water and extracted with diethyl ether (3ꢃ), followed by ethyl ace-
tate extraction (2ꢃ). The combined organic layers were washed
with water and brine, dried over Na2SO4, filtered, and concentrated
under vacuum. The residue was purified by column chromatogra-
phy on silica gel using hexane/ethyl acetate to yield 4 as a yellow-
(45%) of 6 as a white viscous oil. ½a D20
ꢄ
= +6.8 (c 0.9, CHCl3). 1H
NMR (300 MHz, CDCl3): d = 1.63–1.75 (m, 2H, CH2NH), 3.1–3.29
(m, 2H, CH2NH), 3.3–3.65 (m, 5H, H-5, H-6, OCH2CH2, H-3), 3.7–
3.95 (m, 12H, H-6, CH3, H-2, H-4), 4.54 (d, 1H, OCH2,
J = 11.33 Hz), 4.60 (d, 1H, H-1, J1–2 = 9.82 Hz), 4.64–4.75 (m, 4H,
OCH2), 4.88 (d, 1H, OCH2, J = 11.33 Hz), 4.95–5.2 (2 os, 3H, PhCH2,
NH)), 6.8–6.91 (m, 6H, ArH), 7.1–7.4 (m, 14H, ArH), 7.5–7.6 (m,
2H, ArH). 13C NMR (75 MHz, CDCl3): d = 22.60 (CH2), 38.86
(CH2NH), 55.22 (CH3), 66.47 (C-6), 69.47, 69.64 (OCH2CH2, OCH2Ph)
72.43 (OCH2),, 73.11 (C-4), 73.87, 75.20 (OCH2), 77.20 (C-2), 77.30
(C-5), 83.92 (C-3), 87.77 (C-1), 113.55, 113.68, 113.78, 126.93,
128.0, 128.43, 128.69, 129.16, 129.48, 129.89, 130.38, 130.53,
130.81, 131.34, 134.36, 136.61, 156.29, 159.07, 159.17, 159.23
(Ar). HRMS (EI) (+) for C47H53NO10S: [M+K]+(found) = 862.30217,
[M+K]+(calcd) = 862.30218.
ish, viscous oil (70%, 17.1 g). ½a D20
ꢄ
= +1.6 (c 1.0, CHCl3). 1H NMR
(300 MHz, CDCl3): d = 0.02, 0.03 (2s, 6H, SiCH3), 0.87 (s, 9H,
C(CH3)3)), 3.40 (pt, 1H, H-5, J = 6.52), 3.54 (dd, 1H, H-3,
J3–2 = 11.9 Hz, J3–4 = 2.64 Hz), 3.58–3.76 (m, 2H, H-6), 3.80, 3.82
(os, 9H, CH3), 3.85–3.95 (m, 2H, H-4, H-2), 4.55 (d, 1H,
J = 11.4 Hz), 4.60 (d, 1H, H-1, J1–2 = 9.63 Hz), 4.62–4.76 (os, 4H,
OCH2), 4.90 (d, 1H, J = 10.95 Hz), 6.8–6.92 (m, 6H, ArH), 7.10–7.25
(m, 3H, ArH), 7.26–7.35 (m,6H, ArH), 7.49–7.51 (m, 2H, ArH). 13C
NMR (75 MHz, CDCl3): d = ꢀ5.51, ꢀ5.27 (SiCH3), 18.16 (C(CH3)3),
25.86 (C(CH3)3), 55.23 (CH3), 61.72 (C-6), 72.44 (OCH2), 73.14
(C-4), 74.00 (OCH2), 75.18 (OCH2), 77.03 (C-2), 78.96 (C-5), 83.92
(C-3), 87.79 (C-1), 113.51, 113.69, 113.77, 126.76, 128.69, 129.19,
129.30, 129.36, 129.92, 130.42, 130.49, 130.63, 131.13, 134.51,
158.99, 159.17, 159.22 (Ar). HRMS (EI) (+) for
C42H54O8SSi:
[M+Na]+(found) = 769.32018, [M+Na]+(calcd) = 769.32009.
4.2.6. Phenyl 6-O-(3-benzyloxycarbonylamidopropyl)-1-thio-b-
D-galactopyranoside (7)
4.2.4. Phenyl 2,3,4-tri-O-(4-methoxybenzyl)-6-hydroxy-1-thio-
Compound 6 (6.20 mmol, 5.2 g) was dissolved in DCM (90 ml)
b-
D
-galactopyranoside (5)
A 1 M solution of TBAF in THF (15.6 ml) was added dropwise at
and water (3 ml) was added followed by DDQ addition
(27.83 mmol, 6.32 g). The mixture was stirred for 24 h at room
temperature, diluted with DCM, filtered and washed with satu-
rated aqueous NaHCO3, Na2S2O3, and brine. The organic layer
was dried over Na2SO4, filtered, and concentrated. The residue
was purified on silica gel by flash column chromatography using
MeOH/DCM as eluent (MeOH/ethyl acetate 1:9, Rf = 0.6) to yield
0 °C under nitrogen to a suspension of compound 4 (22.35 mmol,
16.67 g) and pulverized molecular sieves (4 Å) in freshly distilled
THF (117 ml). The reaction mixture was allowed to warm up to
room temperature (ꢁ1 h) and was stirred for the next 6 h. After
completion (monitored by TLC), the reaction mixture was filtered
through Celite 545, which was washed thoroughly with ethyl ace-
tate. The collected filtrate was concentrated under vacuum and the
residue was re-dissolved in ethyl acetate, washed with saturated
aqueous NaHCO3 and water. The organic layer was dried over
Na2SO4, filtered, and concentrated in vacuo. The crude product
was purified on silica gel by column chromatography using
7 as a white solid (1.7 g, 60%). ½a D20
ꢄ
= ꢀ30.2 (c 0.9, MeOH). 1H
NMR (300 MHz, CDCl3): d = 1.47–1.85 (m, 2H, CH2), 3.05–3.52 (m,
4H, CH2NH, OCH2CH2), 3.53–3.75 (m, 4H, H-3, H-5, OH), 3.76–
4.41 (m, 5H, H-6, H-2, H-4, OH), 4.56 (d, 1H, H-1, J1–2 = 9.63 Hz),
5.04 (s, 2H, OCH2Ph), 5.26 (br s, NH), 7.14–7.38 (m, 8H, ArH),
7.45–7.57 (m, 2H, ArH). 13C NMR (75 MHz, CDCl3): d = 29.72

A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
2537
(CH2), 38.16 (CH2NH), 66.64 (OCH2Ph)), 68.78 (OCH2CH2), 68.95 (C-
4), 69.55 (C-6), 69.97 (C-2), 74.79, 77.20 (C-5, C-3), 88.66 (C-1),
113.64, 127.53, 128.04, 128.46, 128.86, 131.74, 136.50, 156.64
(Ar). HRMS (EI) (+) for C23H29NO7S: [M+Na]+(found) = 486.15571,
[M+Na]+(calcd) = 486.15569.
4.2.9. 3-((2,3,4-Tri-O-acetyl-6-O-((N-9-fluoroenylmethoxycarbo-
nyl)amino)propyl))-b- -galactopyranos-1-yl)-propanoic acid (11)
D
To a solution of 9 (1.27 mmol, 0.56 g) in DCM (24 ml) neat TFA
(24 ml) was added at 0 °C and the mixture was stirred at room
temperature for 15 h, then co-evaporated with toluene to give a
mixture of crude 10 and a partially deprotected intermediate with
the remaining Cbz group. The organic residue was dissolved in eth-
anol (50 ml), 10% Pd/C (0.2 g) and a few drops of formic acid were
added (pH ꢁ4). The reaction mixture was flushed with nitrogen,
then with hydrogen and stirred for 8 h under a hydrogen atmo-
sphere (2 bar). After flushing with nitrogen, the solution was fil-
tered by Celite 545, sorely washed with ethanol and
concentrated to give crude 10. This material was dissolved in a
mixture of dioxane/water (90 ml, 1:1 v/v) and solid Na2CO3
(3.0 mmol, 0.32 g) was added. The reaction mixture was ultrasoni-
cated for 15 min, cooled to 0 °C, Fmoc-OSu (1.9 mmol, 0.64 g)
added and stirring continued for the next 6 h. The reaction mixture
was acidified with aqueous HCl (pH ꢁ5), diluted with water and
the water layer was extracted with DCM (3ꢃ). The organic layer
was dried over Na2SO4, filtered, and concentrated in vacuo. The or-
ganic residue was purified on silica by column chromatography
starting with hexane/ethyl acetate (1:1) followed by MeOH/DCM
eluent (MeOH/DCM 1:9, Rf = 0.4) to yield 0.5 g (64%) of 11 as a
4.2.7. 2,3,4-Tri-O-acetyl-6-O-(3-benzyloxycarbonylamidopro-
pyl)-1-thio-b-D-galactopyranoside (8)
Pyridine (13 ml) was placed in a flask (under nitrogen), cooled
to ꢀ10 °C and acetic anhydride (10.7 ml) was added. Compound
7 (1.57 g, 3.4 mmol) was dissolved in 3 ml of pyridine and added
dropwise to the pyridine/acetic anhydride mixture at ꢀ10 °C. The
reaction mixture was allowed to warm up to room temperature
and stirred overnight. The progress of the reaction was checked
by TLC. After 24 h, the reaction mixture was diluted with ethyl ace-
tate (100 ml) and poured into ice-cold water. The organic layer was
separated and the water layer was extracted with ethyl acetate
(3ꢃ). The organic layers were combined, dried over Na2SO4, fil-
tered, and concentrated. The organic residue was purified by col-
umn chromatography on silica gel using hexane/ethyl acetate to
give 8 (1.7 g, 85%) as a colorless syrup (hexane: ethyl acetate 1:1,
Rf = 0.7). ½a 2D0
ꢄ
= +2.0 (c 0.9, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 1.63–1.76 (m, 2H, CH2), 1.97, 2.09, 2.11 (3s, 3 ꢃ 3H, CH3CO),
3.14–3.30 (m, 2H, CH2NH), 3.32–3.61 (m, 2H, H-6, 2H, OCH2CH2),
3.76–3.87 (m, 1H, H-5), 4.72 (d, 1H, H-1, J1–2 = 10 Hz), 4.99–5.18
(m, 4H, H-3, PhCH2, NH), 5.24 (dd, 1H, H-2, J2–1 = 10 Hz,
J2–3 = 10 Hz), 5.44 (pd, 1H, H-4, J3–4 = 3 Hz), 7.24–7.39 (m, 8H,
ArH), 7.45–7.53 (m, 2H, ArH). 13C NMR (75 MHz, CDCl3):
d = 20.55, 20.62, 20.81 (COCH3), 29.44 (CH2CH2NH), 38.30
(CH2NH), 66.41 (PhCH2), 67.49, 67.73 (C-2, C-4), 68.60 (OCH2),
69.22 (C-6), 72.05 (C-3), 77.75 (C-5), 86.59 (C-1), 127.97, 128.86,
132.74, 136.74 (Ar), 156.45 (NHCOO), 169.45, 169.94, 170.34
(CH3CO). HRMS (EI) (+) for C29H35NO10S: [M+K]+(found) = 628.16124,
[M+K]+(calcd) = 628.16133.
greenish oil. ½a D20
ꢄ
= +2.8 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3):
d = 1.65–1.8 (m, 2H, CH2CH2NH), 1.97, 2.04, 2.14 (3s, 3 ꢃ 3H,
COCH3, 2.53–2.69 (m, 2H, CH2COOH), 3.1–3.6 (m, 6H, CH2NH,
CH2OCH2, Ha-6, Hb-6), 3.75–3.94 (m, 2H, H-5, OCH2CH2COOH),
4.0–4.15 (m, 1H, OCH2CH2COOH), 4.17–4.29 (pt, 1H, CHFmoc),
4.32–4.57 (m, 3H, OCH2Fmoc, H-1), 4.95–5.09 (dd, 1H, J3–4
=
3.2 Hz, J3–2 = 10.4 Hz, H-3), 5.12–5.22 (dd, 1H, H-2, J2–1 = 8.0 Hz,
J2–3 = 10.4 Hz), 5.3 (s, 1H, NH), 5.42 (pd, 1H, H-4), 7.27–7.35 (t,
2H, Fmoc), 7.36–7.45 (t, 2H, Fmoc), 7.60 (d, 1H, Fmoc), 7.76 (d,
1H, Fmoc). 13C NMR (75 MHz, CDCl3): d = 20.53, 20.64 (COCH3),
29.36 (CH2CH2NH), 34.68 (CH2COOH), 38.19 (CH2NH), 47.25
(CHFmoc), 65.37 (OCH2CH2COOH), 66.36 (NHCOOCH2), 67.64 (C-
4), 68.37 (C-6), 68.92 (C-2), 69.09 (CH2OCH2), 70.90 (C-3), 71.90
(C-5), 101.57 (C-1), 119.91, 124.99, 126.98, 127.62, 143.91 (Fmoc),
156.62 (NHCO), 169.61, 170.08, 170.52 (COCH3), 175.19 (COOH).
4.2.8. 3-(2,3,4-Tri-O-acetyl-6-O-(3-benzyloxycarbonylamido-
propyl))-b-D-galactopyranos-1-yl)-tert-butyl propionate (9)
To a solution of 8 (2.6 mmol 1.53 g), in dry DCM (90 ml) under
nitrogen tert-butyl 3-hydroxypropionate (5.2 mmol, 0.76 g), in
DCM (1 ml) and pulverized molecular sieves (4 Å) were added.
The reaction mixture was stirred for 1 h at room temperature
and then cooled to ꢀ50 °C. NBS (6.72 mmol, 1.2 g) was added fol-
lowed by addition of Me3SiOTf (0.5 mmol, 0.11 g) and stirring con-
tinued for the next 45 min at which time the substrate 8 was fully
consumed as detected by TLC. The reaction mixture was diluted
with DCM (150 ml), filtered through Celite 545 and washed thor-
oughly with DCM. The collected filtrate was washed with saturated
aqueous NaHCO3, aqueous Na2S2O3, brine, and water. The organic
layer was dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified using hexane/ethyl acetate as eluent by
column chromatography on silica gel (hexane/ethyl acetate 3:1,
HRMS (EI) (+) for
C
33H39NO13
:
[M+K]+(found) = 696.20540,
[M+K]+(calcd) = 696.20530.
4.2.10. Solid phase synthesis of 13
The peptide was synthesized manually using a Heidolph Syn-
thesis 1 synthesizer (Heidolph, Germany) by the Fmoc SPPS
method on a pre-loaded polysterene-based Wang resin containing
Fmoc/Pbf protected
D-arginine (loading 0.4 mmol/g). The resin
(0.08 mmol, 200 mg) was initially washed with DCM (2ꢃ) and
swelled for 30 min in DCM, followed by further washing with
DMF (6ꢃ). The Fmoc group was removed by treatment with 20%
piperidine/DMF (2 ꢃ 10 min). The resin was drained and washed
with DMF (4ꢃ) after each deprotection and coupling step. The
D-
Rf = 0.45) to give 9 (0.81 g, 50%) as a colorless syrup. ½a D20
ꢄ
= ꢀ6.9
amino acids were coupled by adding a pre-activated mixture of
an appropriate Fmoc-protected -amino acid (4 equiv) [Fmoc/
Boc- -lysine, Fmoc/Pbf- -arginine, Fmoc/Trt- -glutamine], HBTU
(c 0.9, CHCl3). 1H NMR (300 MHz, CDCl3): d = 1.44 (s, 9H,
C(CH3)3), 1.61–1.84 (m, 2H, CH2CH2), 1.97, 2.04, 2.13 (3s, 3 ꢃ 3H,
CH3CO), 2.28–2.62 (m, 2H, CH2COO), 3.08–3.63 (m, 6H, CH2O,
CH2NH), 3.67–3.87 (m, 2H, H-5, H-6), 3.93–4.10 (m, 1H, H-6),
4.48 (d, 1H, J1,2 = 7.7 Hz, H-1), 5.0 (dd, 1H, H-3, J3–2 = 10.4 Hz,
J3–4 = 3.4 Hz), 5.09 (os, 2H, PhCH2O), 5.11–5.27 (m, 2H, H-2, NH),
5.41 (d, 1H, H-4, J3–4 = 3.1 Hz), 7.24–7.43 (m, 5H, ArH). 13C NMR
(75 MHz, CDCl3): d = 20.56, 20.65, 20.74 (COCH3), 28.04 (C(CH3)3),
29.43 (CH2), 35. 83 (CH2) 38.41 (CHNH), 65.45 (CH2OC-6), 66.40
(CH2OC-1), 67.63 (C-4), 68.47 (C-6), 68.96 (C-2), 69.35 (PhCH2O),
71.01 (C-3), 71.89 (C-5), 80.69 (C(CH3)3), 101.30 (C-1), 127.98,
128.01, 128.44 (Ph), 136.77 (Ph), 156.46 (NHCO), 169.45, 170.05,
D
D
D
D
(3.6 equiv)/HOBt (3.6 equiv) in DMF (2 ml) and DIPEA (8 equiv)
and stirring the mixture for 1 h. The Fmoc-protected peptide
D-Tat49–57 (Fmoc-r(Pbf)k(Boc)k(Boc)r(Pbf)r(Pbf)q(Trt)r(Pbf)r(Pbf)r(Pbf)
) was
treated with 20% piperidine/DMF and washed thoroughly with
DMF (6ꢃ). Coupling of the sugar building block 11 was performed
by adding a pre-activated solution of Fmoc-protected compound
11 (3 equiv), HATU (3 equiv) in DMF (3 ml) and DIPEA (6 equiv)
to the peptide linked resin. After 3 h of coupling under nitrogen
atmosphere, the resin was washed with DMF (4ꢃ). After Fmoc
group deprotection on the sugar moiety, Fmoc-
was coupled to the free amino group of the sugar. The Fmoc-
Lys(Dde)-OH (4 equiv) and HATU (4 equiv) were dissolved in
D-Lys(Dde)-OH
170.27 (CH3CO), 170.44 (CH2COO). HRMS (EI) (+) for C30H43NO13
:
D-
[M+NH4]+(found) = 643.30736 [M+NH4]+(calcd) = 643.30727.

2538
A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
DMF (3 ml), DIPEA (8 equiv) was added and the resulting solution
was allowed to react for 3 h under nitrogen. The Fmoc group was
removed by treatment with piperidine and the resin was washed
The absence of free Gd3+ ions was verified in exemplary samples
by a competitive assay with DTPA. The solution of 14 and GdCl3
was stirred for 3 days and divided to two parts. One half was con-
centrated and purified by HPLC as described above. The second half
was treated with excess of DTPA and stirred for additional 12 h and
was subsequently purified by HPLC, dialyzed and lyophilized. No
significant differences were observed in the relaxation rates R1
measured at 123 MHz between both samples (equally concen-
trated) indicating the absence of free gadolinium in our CA after
purification.
with DMF (4ꢃ). Coupling of the DOTA-(t-Bu)3 ester 12 to the
a-
NH2 group of lysine was carried out by adding a pre-activated solu-
tion of 12 (4 equiv) with HATU (4 equiv) in DMF (3 ml) and DIPEA
(8 equiv) to the resin and the reaction was allowed to continue for
24 h under nitrogen. The Dde group of the lysine linker was re-
moved by treatment with 2% hydrazine hydrate in DMF
(2 ꢃ 4 min) and the fluorophore was coupled to
e-NH2 group of ly-
sine within 12 h (FITC (4 equiv): DIPEA (8 equiv), DMF (3 ml)). All
coupling steps were followed by Kaiser test on the resin to indicate
presence (deprotection) or absence (coupling) of free amino
groups. The resin was washed with DMF (4ꢃ), DCM (4ꢃ), and
MeOH (6ꢃ) and dried under vacuum. The resin attached conjugate
was cleaved off the resin with TFA/water/TIPS/m-cresol
(90:5:2.5:2.5 v/v/v/v) for 4 h and washed with TFA (2 ml). The
crude product was precipitated with cold (ꢀ20 °C) tert-butyl
methyl ether (MTBE). The precipitate was washed with an addi-
tional amount of MTBE (2ꢃ), centrifuged and re-suspended in neat
TFA. After precipitation with cold MTBE and centrifugation, the
crude product was dissolved in H2O/t-BuOH (2:1 v/v) and lyophi-
lized to afford crude 13 as orange powder. This conjugate was puri-
fied by semi-preparative RP-HPLC using method 1 (Polaris 5 ether
column, flow rate 10 ml/min). The product was characterized by
ESI-MS. The detected molecular ions were consistent with the cal-
culated mass of 13 (2659.34). ESI-MS (+) m/z: found 1331.2
[(M+2H)2+], 887.8 [(M+3H)3+], 666.1 [(M+4H)4+], 390.1 (fragment).
ESI-MS (ꢀ) m/z: found 2686.8 [(MꢀH)1ꢀ], 1343.3 [(Mꢀ2H)2ꢀ];
calcd for C108H173GdN38O31S 2688.21. ESI-MS (+) m/z: found
1345.1 [(M+2H)2+], 897.0 [(M+3H)3+], 673.2 [(M+4H)4+], 390 (FR).
4.2.13. Synthesis of Gd-DOTA-Gal-k(FR)-CPP
A side chain protected D-Tat49–57 (Fmoc-r(Pbf)k(Boc)k(Boc)r(Pbf)
r(Pbf)q(Trt)r(Pbf)r(Pbf)r(Pbf)) was synthesized on Fmoc/Pbf D-Arg pre-
loaded Wang resin (0.08 mmol, 200 mg) using the same protocol
as for conjugate 13. The Fmoc group was then removed by treat-
ment with 20% piperidine/DMF (2ꢃ) and resin was washed with
DMF (4ꢃ). Next Fmoc-D-Lys-(Dde)-OH (4 equiv) pre-activated with
HBTU (3.6 equiv)/HOBt (3.6 equiv) in DMF (2 ml) and DIPEA
(8 equiv) were added and the reaction was stirred for 1 h. After
Fmoc group deprotection, the monosaccharide 11 (3 equiv) was
coupled to the lysine linker using HATU (3 equiv) and DIPEA
(6 equiv) in DMF (3 ml). The reaction was completed after stirring
for 3 h, as indicated by Kaiser test. The Fmoc group on the sugar
residue was then deprotected and a solution of 12 (4 equiv) pre-
activated with HATU (4 equiv) and DIPEA (8 equiv) in DMF (3 ml)
was added to the resin. After stirring for 24 h, the resin was thor-
oughly washed with DMF and treated with 2% hydrazine hydrate
4.2.11. O-Acetyl deprotection of 13 (14)
The acetyl protected compound 13 was dissolved in dry meth-
anol, cooled to 0 °C and hydrazine hydrate (1:6 v/v with methanol)
was added slowly. The resulting reaction mixture was stirred at
0 °C for 30 min and then stirring was carried out at room temper-
ature. The progress of the reaction was monitored by LC–ESI-MS.
After 12 h, the reaction mixture was cooled to 0 °C and acetone
was added slowly to achieve pH ꢁ5. The organic solvents were re-
moved in vacuo. The crude product was re-dissolved in water,
lyophilized and purified by semi-preparative RP-HPLC using
method 1 (Polaris 5 C18-ether column, flow rate 10 ml/min). The
obtained fractions were collected and solvents removed under
vacuum, followed by dissolving the sample in ultra pure water
and lyophilization. Ligand 14 was characterized by ESI-MS. The de-
tected molecular ions were consistent with the calculated mass of
14 (2533.31). ESI-MS (+) m/z: found 1268.2 [(M+2H)2+], 845.8
[(M+3H)3+], 634.6 [(M+4H)4+].
in DMF (2 ꢃ 4 min). The
e-NH2 of the lysine linker was reacted
with FITC (4 equiv) and DIPEA (8 equiv) in DMF (3 ml) for 12 h.
All coupling and deprotection steps were followed by a Kaiser test
on the resin. The resin-bound conjugate was cleaved off by the pro-
cedure described for 13. The acetate ester groups were deprotected
according to the procedure described for 14. The obtained ligand
was purified by semi-preparative RP-HPLC and characterized by
ESI-MS. The following preparation of Gd-DOTA-Gal-k(FR)-CPP
was obtained using a protocol described for Gd-DOTA-k(FR)-Gal-
CPP. The crude product was purified by RP-HPLC (Polaris C8-Ether
column, flow rate 4 ml/min) using method 2, dialyzed (48 h) and
lyophilized to provide Gd-DOTA-Gal-k(FR)-CPP. Its structure was
verified by ESI-MS. ESI-MS (+) m/z: found 1346.2 [(M+2H)2+],
897.6 [(M+3H)3+], 767.4 [(M-FR)+3H)2+], 672.7 [(M+4H)4+], 390
(FR fragment), calcd for C108H173GdN38O31S 2688.21.
4.2.12. Preparation of the Gd3+ complex of ligand 14 (Gd-DOTA-
k(FR)-Gal-CPP)
4.3. Concentration estimation
The concentration of ligand 14 was determined based on the
absorbance of fluorophore. Ligand 14 (1 equiv) was dissolved in
ultrapure water and a titrated solution of GdCl3 (0.9 equiv) in
water was added dropwise (pH was kept above 5.5 for the duration
of the gadolinium addition). The pH was adjusted to 6–6.5 with
0.1 M NaOH. The solution was stirred at 40 °C for 12 h and then
stirring was continued at room temperature for 3 days. The pH
was checked periodically and adjusted to 6.5. The formation of
Gd3+ complex was confirmed by analyzing aliquots of the reaction
mixture by LC–ESI-MS. The crude product was purified by RP-
HPLC, to remove ligand 14 and small impurities, using method 2
(Polaris C8-Ether column, flow rate 4 ml/min) and afterwards dia-
lyzed (Float-ALyser, cellulose ester membranes, MWCO 1000;
Spectrum Laboratories Inc., Germany) and lyophilized to provide
Gd-DOTA-k(FR)-Gal-CPP as yellow to orange solid. The detected
molecular ions were consistent with the calculated mass of prod-
uct (2688.21 g/mol).
Fluorescein-labeled conjugates were dissolved in Milli-Q water
to obtain a 10 mM solutions by weight and the pH was adjusted to
ꢁ7. To determine the real concentration of these stock solutions,
the absorbance of a 1:100 dilution in Dulbecco’s Modified Eagle’s
Medium (DMEM; Biochrom AG, Germany) was measured in a
Fluostar Optima multiplate reader (BMG Labtech, Germany) at
485 nm with ratiometric correction of turbidity at 690 nm. The
concentration of stock solutions was calculated assuming a molar
extinction coefficient (efluorescein) of 81,000 l/(mol cm). All further
dilutions were done in proportion to the calculated concentration
of the stock solutions.
4.4. Cell culture conditions
C6/LacZ cells (C6/lacZ7, ATCCÒ No.: CRL-2303™, USA) express-
ing b-galactosidase and C6 rat glioma cells (University of Tübingen,
Germany) were cultured as monolayers at 37 °C in a 5% CO2

A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
2539
humidified atmosphere in DMEM supplemented with 10% fetal bo-
vine serum (FBS), 4 mM -glutamine, 100 g/ml streptomycin and
(MAGNETOM Tim Trio, Siemens Healthcare, Germany), using a
12-channel RF Head coil and slice selective measurements from a
slice with a thickness of 1 mm positioned through the cell pellet.
Relaxation times (T1) were measured using an inversion-recovery
sequence, with an adiabatic inversion pulse followed by a turbo-
spin-echo readout. Numbers of MR images acquired were in the
range 10–15, with the time between inversion and readout varying
from 23 to 3000 ms. With a repetition time of 10 s, 15 echoes were
acquired per scan and averaged six times. All experiments scanned
2562 voxels in a field-of-view of 110 mm in both directions result-
ing in a voxel volume of 0.43 ꢃ 0.43 ꢃ 1 mm3.
L
l
100 U/ml penicillin (all purchased from Biochrom AG, Germany).
The culture medium of C6/LacZ cells was additionally supple-
mented with 0.1 mM non-essential amino acids (NEM, Biochrom
AG, Germany). Both cell lines were passaged by trypsinization with
trypsin/EDTA 0.05:0.02% (v/v) in phosphate-buffered saline (PBS;
Biochrom AG, Germany) every second to third day.
4.5. Cellular uptake by fluorescence spectroscopy
Data analysis was performed by fitting of relaxation curves with
self-written routines under MATLAB 7.1 R14 (The Mathworks Inc.,
United States). The series of T1 relaxation data were fitted to the
following equation:
C6 and C6/LacZ cells were grown in 96-well microplates. After
reaching 70–80% confluence (24 h), cells were treated with differ-
ent concentrations of Gd-DOTA-k(FR)-Gal-CPP in complete culture
medium for 18 h under routine culture conditions. Subsequently,
the supernatant was removed and the nuclear stain Hoechst
T1 series with varying t ¼ TI :
S ¼ S0 ð1 ꢀ expðꢀt=TIÞ þ SðTI ¼ 0Þ expðꢀt=T1Þ:
33342 (Hoe) in complete culture medium (100 ll/well) was added
to cells in order to estimate their number per well (correlates to
the DNA content).57 After 30 min of incubation, cells were washed
with Hank’s buffered salt solution (HBSS; Biochrom AG, Germany),
Nonlinear least-squares fitting of three parameters S0, S
,
ðTI¼0Þ
and T1 was done for manually selected regions of interest with
the Trust-Region Reflective Newton algorithm implemented in
MATLAB. The quality of the fit was controlled by visual inspection
and by calculating the mean errors and residuals. The obtained T1
values of the cell pellet were converted to R1,cell (= 1/T1). These
were expressed as % of control R1,cell of cells incubated under the
same conditions in the absence of CA.
treated with cold trypan blue (TB) (0.05% (w/v) in PBS, 100 ll/well)
for 3 min to quench extracellular fluorescence and repeatedly
washed with HBSS. Cell-associated FR fluorescence (Ex 485 nm/
Em 530 nm) and cell number based on Hoe fluorescence (Hoe, Ex
346 nm/Em 460 nm) were measured in the multiplate reader
(Fluostar Optima, BMG Labtech, Germany). Cells incubated in the
absence of Gd-DOTA-k(FR)-Gal-CPP were used as controls and
wells without cells but treated with Hoechst, Trypan Blue and
washed as described above were used as blanks. Hoe fluorescence
was also used for the evaluation of cytotoxicity of CA. Experiments
were run at least two times for each given concentration of
Gd-DOTA-k(FR)-Gal-CPP with six replicates each.
4.8. Longitudinal relaxivity (r1) of Gd3+ complexes
Relaxation times T1 of aqueous solutions of Gd3+ complexes
were determined at 123 MHz (3 T) and room temperature
(ꢁ21 °C) as described above. Three to five samples with concentra-
tions of Gd-DOTA-k(FR)-Gal-CPP (800 ll) in the range 5–40 lM
4.6. Microscopy
were prepared in Milli-Q water. Pure water was used as a blank.
Two times 200 l of each sample were transferred to a 96-well
plate and the absorbance was measured at 485 nm to evaluate
the exact concentration of CA. Afterwards, samples for each con-
centration were combined again and 380 ll aliquots were trans-
ferred to Eppendorf cups for MR measurement (two replicates
per concentration). Relaxation rate values were plotted on the ex-
act concentration (in mM) and linear regression was done. The
slope of the obtained curve showed the corresponding relaxivity.
l
Following fluorescence spectroscopy, microscopy on the same
cells was performed without fixation using a Zeiss Axiovert
200 M microscope (Germany) with an LD Plan NeoFluor 40X objec-
tive. The conditions used for all imaging experiments were kept
constant. Cellular localization of fluorescein linked conjugates
was monitored by sample irradiation with blue light at 470 nm
and observing emission at 525 nm. Hoechst-related fluorescence
was viewed by excitation at 365 nm and emission at 460 nm. Try-
pan blue fluorescence was observed by irradiation at 535 and
emission at 645 nm. Additionally, cell morphology was observed
by capturing phase contrast images with differential interference
contrast (DIC) microscopy of the same areas used for fluorescence
detection. VolocityÒ Acquisition and Visualization software
(Improvision/Perkin–Elmer, UK) was used for the image acquisi-
tion and processing.
4.9. Statistical analysis
Data are expressed as means standard error of the mean
(SEM). Two mean values of interest were tested for statistically sig-
nificant differences by unpaired student’s t-test using GraphPad
Prism version 5.02 for Windows (GraphPad Software, USA). When
comparing more than two groups, a one-way analysis of variance
(ANOVA) with Tukey’s post-test for multiple comparisons was per-
formed. p-values of <0.05 were considered statistically significant.
4.7. MR imaging studies in cells
Exponentially growing C6/LacZ and C6 cells were incubated
with 10 l
M of Gd-DOTA-k(FR)-Gal-CPP in 175 cm2 tissue culture
Acknowledgments
flasks for 18 h at 37 °C. After washing twice with HBSS and once
with PBS, cells were trypsinized and centrifuged. Cell viability
was assessed by trypan blue staining. Subsequently, 2 ꢃ 107 cells,
The authors thank Professor Hamprecht, University of
Tübingen, for the kind gift of the C6 rat glioma cells. The authors
are particularly grateful to Ms. Hildegard Schulz, High Field
Magnetic Resonance Center, Max Planck Institute for Biological
Cybernetics, for the help in performing the biological experiments.
This work was funded by the Max Planck Society, supported by the
German Ministry for Education and Research, BMBF (FKZ
01EZ0813), and was performed in the frame of COST action D38.
re-suspended in 500 ll of fresh complete culture medium without
CA, were transferred to 1.5 ml Eppendorf tubes. Tubes with culture
medium only and cells without CA in culture medium were used as
controls. Before performing MR measurements, cells were allowed
to settle in the tubes. Cell pellets were imaged at room tempera-
ture (ꢁ21 °C) with a clinical 3 T (123 MHz) human MR scanner

2540
A. Keliris et al. / Bioorg. Med. Chem. 19 (2011) 2529–2540
28. Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1988, 27, 1697.
Supplementary data
29. Brocke, C.; Kunz, H. Synthesis-Stuttgart 2004, 525.
30. Sjolin, P.; Elofsson, M.; Kihlberg, J. J. Org. Chem. 1996, 61, 560.
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