Responsive Mn(II) complexes for potential applications in diagnostic Magnetic Resonance Imaging

Article abstract of DOI:10.1016/j.bmc.2010.07.064

The investigation of new Mn(II)-based MRI/Molecular Imaging probes responsive to the enzyme tyrosinase for potential diagnostic applications is herein described. The expression of the enzyme tyrosinase, an oxidoreductase, is up-regulated in melanoma cance

Full text of DOI:10.1016/j.bmc.2010.07.064

Bioorganic & Medicinal Chemistry 19 (2011) 1115–1122
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Responsive Mn(II) complexes for potential applications in diagnostic
Magnetic Resonance Imaging
Gabriele A. Rolla a, Lorenzo Tei a, Marianna Fekete a, Francesca Arena b, Eliana Gianolio b, Mauro Botta a,
⇑
a Dipartimento di Scienze dell’Ambiente e della Vita, Università degli Studi del Piemonte Orientale ‘Amedeo Avogadro’, Viale T. Michel 11, 15121 Alessandria, Italy
b Centre for Molecular Imaging, Dipartimento di Chimica IFM, Università degli Studi di Torino, Via Nizza 52, 10126 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The investigation of new Mn(II)-based MRI/Molecular Imaging probes responsive to the enzyme tyrosi-
nase for potential diagnostic applications is herein described. The expression of the enzyme tyrosinase,
an oxidoreductase, is up-regulated in melanoma cancer cells. Three novel ligands (L1, L2 and L3) were
Received 5 March 2010
Revised 15 July 2010
Accepted 27 July 2010
Available online 2 August 2010
designed as modified acyclic polyaminocarboxylate chelates by introducing an L-tyrosine residue in place
of an aminoacetate unit. The corresponding Mn(II) complexes were fully characterised by 1H NMR relaxo-
metric techniques in aqueous media. The responsive activity towards the expression of tyrosinase was
then assessed by monitoring the 1H 1/T1 relaxivity changes during incubation experiments in buffered
solutions containing tyrosinase at different concentrations and in B16F10 melanoma cell homogenate.
New insight on the mechanism of action of these systems was gained by measuring the magnetic field
dependence of the relaxivity and ESR spectra of the incubated solutions. The systems developed showed
responsive activity to tyrosinase with a relaxation enhancement spanning from 50% (MnL1) to 350%
(MnL3) which augurs well for the development of diagnostic probes to detect melanoma cancer.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Manganese
Responsive Contrast Agents
Tyrosinase
Molecular Imaging
MRI
Relaxometry
1. Introduction
inates a high MRI signal before being excreted via hepato-biliary
route. Although only Mn-DPDP is clinically approved, the search
Over the last three decades Magnetic Resonance Imaging (MRI)
has become one of the techniques of choice for diagnostic Imaging.
In parallel, the development of efficient contrast enhancing agents
(CAs), able to evidence further anatomical details and physiologic
information, has yielded very effective tools to access supplemen-
tary insight. Currently Gd(III) complexes are by far the MRI CAs
most used in clinical practice.1–4
for stable Mn(II)-chelates for possible use as MRI CAs has
brought to the synthesis and characterisation of numerous Mn(II)
complexes endowed with interesting properties.8 Another news-
worthy application of Mn(II)-based CAs, although only restricted
to animal study, is the widely exploited application of MnCl2
MRI, often referred to as MEMRI (Manganese Enhanced Magnetic
Resonance Imaging).9 The scope of this technique comprises the
detection of neuronal cell layers in brain structures;10,11 the
study of local brain or cardiac function activity (exploiting Mn(II)
as a biological calcium analogue); tracing neuronal projections
(exploiting the fact that once inside cells in a specific brain re-
gion, Mn(II) will move along appropriate neuronal pathways in
an anterograde direction).12
A further challenge in the field of MRI CAs is represented by the
development of effective protocols for Molecular Imaging, recently
defined as ‘the visualisation, characterisation and measurement of
biological processes at the molecular and cellular levels in humans
and other living systems’.13 A possible approach is the develop-
ment of probes responsive to specific biochemical events which
promote the accumulation of CAs as a consequence of a ‘catalytic’
action of the target of interest (enzymatic activity,14–19 pH
change,20 pO2 change,21 temperature change22). Reporters based
on this mode of action can in principle accumulate in large num-
bers reaching high local concentration and allowing the detection
of targets of interest.
However, as Mn2+ is a paramagnetic ion with five unpaired
electrons (high magnetic moment) and long electronic relaxation
time (S state electronic structure), high-spin Mn(II) complexes
have also attracted interest as potential MRI CAs. In fact, manga-
nese is an essential heavy metal playing a key role as cofactor in
a number of important enzymes such as manganese superoxide
dismutase and glutamine synthetase.5,6
A
rich biology has
evolved to absorb and transport manganese, thus numerous bio-
logical structures can complex Mn2+ ions with high affinity. Not-
withstanding this, only one Mn(II)-based CA, the liver specific
Teslascan™ or Mangafodipir™ (Mn-DPDP, DPDP = dipyridoxal
diphosphate), is currently approved for clinical practice.7 Its
mode of action is radically different from that of Gd(III)-based
CAs: once the complex reaches the liver it slowly releases the
metal ion which, after coordination by bio-macromolecules, orig-
⇑
Corresponding author. Tel.: +39 0131360253; fax: +39 0131360250.
E-mail address: mauro.botta@mfn.unipmn.it (M. Botta).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.064

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G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
Figure 1. Ligands discussed in this paper.
Prompted by the need of novel Mn(II)-based CAs and willing to
address the demand for new Molecular Imaging tools able to probe
relevant biochemical events associated with specific pathologies,
we designed new Mn(II) complexes, endowed with sufficiently
high thermodynamic and kinetic stability, responsive to tyrosinase
activity. Tyrosinase is an oxidoreductase over-expressed in mela-
noma cells which has already been object of considerable interest
as a target for developing both imaging probes18,23 and pro-drug
based cancer chemotherapy.24 The aminoacid
L-tyrosine is a natu-
ral substrate of tyrosinase which converts it into melanins via a
cascade of reactions sketched in Scheme 1.
To probe this approach, we devised simple ligands based on the
core structure of H4EDTA (ethylenediamine-N,N0-tetraacetic acid),
H5DTPA (diethylenetriamine-N,N,N0,N00,N00-pentaacetic acid) and
H4DTTA (diethylenetriamine-N,N,N0,N00-tetraacetic acid), namely
L1, L2 and L3 (Fig. 1), where an aminocarboxylate unit is substituted
Scheme 2. Reagents and conditions: (i) Na2CO3, BrCH2CO2t-Bu, MeCN, rt, 18 h, 71%;
(ii) 3, Na2CO3, MeCN heated under reflux, 48 h, 55%; (iii) (a) KOH in MeOH–H2O, rt,
4 h; (b) TFA, i-Pr3SiH, CHCl3, rt, 18 h.
with an L-tyrosine residue. We postulated that the coordination
sphere of the corresponding Mn(II) complexes could be altered as
a consequence of the action of tyrosinase with a concomitant
change in relaxivity that can be monitored. In fact, the complex
destabilisation would result in a controlled release of metal ion,
eventually taken up by biological macromolecules, with an ex-
pected, remarkable increase of relaxivity. In fact it is well known
that the interaction of Mn2+ ions with proteins can give rise to high
values of relaxivity.1 Under this hypothesis, the resulting relaxivity
enhancement would arise as a specific consequence of tyrosinase
expression for the detection of melanocytes in a possible in vivo
application.
In a similar manner, protected L-tyrosine (5, Scheme 3) was re-
acted with three equivalents of 3 (Na2CO3, MeCN, under reflux) to
give the protected DTPA-like ligand 6. L2 was then obtained by
deprotection of 6 under acidic conditions (TFA, CHCl3, rt).
The DTTA-like ligand L3 was synthesised starting from N-benzyl
protected L-tyrosine (7, Scheme 4), obtained from 1 by reductive
amination with benzaldehyde (PhCHO, NaBH(OAc)3, C2H4Cl2, rt).
Alkylation with 2-bromoethyltrifluoromethansulfonate26 (2,6-luti-
dine, toluene, 0 °C to rt) gave the intermediate 8 which was cou-
pled with tris-t-butyl-ethylendiaminotriacetate (9)27 (Na2CO3,
MeCN heated under reflux) to give the protected ligand 10. Depro-
tection of esters and ethers (KOH in MeOH–H2O, rt; TFA, CHCl3, rt)
followed by removal of benzyl protecting group (Pd/C, H2, MeOH,
rt) yielded L3 as white solid.
2. Results and discussion
2.1. Ligand synthesis
2.2. Complex preparation and relaxometric characterisation
The synthesis of the proposed ligands started with protected
-tyrosine (1, Scheme 2), which was monoalkylated with t-butyl
The Mn(II) complexes were prepared by adding to a solution of
the ligand (pH 6.5, 298 K) small volumes of a stock solution of
L
bromoacetate (Na2CO3, MeCN, rt) to give 2. Following a strategy
developed by Rapoport and Williams,25 2 was coupled with di-
t-butyl-2-bromoethyl iminodiacetate (3) (Na2CO3, MeCN heated
under reflux) obtaining the protected ligand 4 in 55% yield after
chromatographic purification. Compound 4 was then deprotected
(KOH in MeOH–H2O, rt, followed by TFA, CHCl3, rt) to yield L1 as
white powder.
Scheme 3. Reagents and conditions: (i) Na2CO3, MeCN, heated under reflux, 48 h,
51%; (ii) TFA, i-Pr3SiH, CHCl3, rt, 18 h.
Scheme 1. Biosynthesis of melanines starting from L-tyrosine.

G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
1117
Scheme 4. Reagents and conditions: (i) 2-bromoethyl trifluoromethanesulfonate,
2,6-lutidine, toluene, 0 °C to rt, 48 h, 38%; (ii) Na2CO3, MeCN, heated under reflux,
36 h, 32%; (iii) (a) KOH in MeOH–H2O, rt, 8 h; (b) TFA, CHCl3, rt, 12 h; (c) Pd/C, H2,
MeOH, rt, 18 h.
Mn(NO3)2 (pH 1.7, 25 °C) and maintaining the pH at 6.5 with di-
luted NaOH. The complexation process was monitored by measur-
ing the change in the longitudinal water proton relaxation rate (R1)
at 20 MHz as a function of the concentration of Mn(II) added. The
straight line obtained has a slope that corresponds to the relaxivity,
r1p, of the complex, according to the well known equation:
R1 ¼ r1p½MnLꢀ þ R1w
ð1Þ
where R1w is the diamagnetic contribution and corresponds to
0.38 sꢁ1 at 20 MHz and 298 K. The r1p values of the complexes were
independently assessed by measuring R1 and determining the con-
centration of the solution by mineralisation.28 These r1p values
found are 3.7, 1.7 and 1.6 mMꢁ1 sꢁ1 for MnL1, MnL2 and MnL3,
respectively. The relaxivity of MnL1 is quite similar to that of the
parent complex MnEDTA, to indicate that the replacement of an
aminocarboxylate with a L-tyrosine did not alter the complexation
properties of the ligand. Thus in MnL1 the metal ion is heptacoordi-
nated with one water molecule in its inner coordination sphere
(q = 1). The low relaxivity measured for MnL2 and MnL3 may be
attributed to the absence of a bound (i.e., inner sphere) water mol-
ecule (q = 0) and then to the occurrence of the only outer sphere
mechanism of relaxation.1–4 Also in this case the results reproduce
quite closely those found for MnDTPA.1 The relaxivity of the three
complexes was measured also as a function of applied field in the
range 0.01–70 MHz at 283, 298 and 310 K, (Fig. 2). These Nuclear
Magnetic Relaxation Dispersion (NMRD) profiles were then fitted
to the established theory for paramagnetic relaxation and the val-
ues of the physico-chemical parameters describing the solute–sol-
vent interaction obtained (Table 1).1–4 Whereas, the value of most
of the parameters is similar for the three complexes and in line with
published data for related complexes,29 the electronic relaxation
Figure 2. 1/T1 1H NMRD profiles of MnL1 (top), MnL2 (middle) and MnL3 (bottom)
at pH 6.5 and at 283 K (open triangles), 298 K (filled circles) and 310 K (open
squares).
times for MnL2 appears sensibly longer (shorter
D
2) than for
MnL3, consistent with a higher local symmetry around Mn ion. Fi-
nally, the constant value of r1p with pH in the interval 4–12
(20 MHz, 298 K) confirmed the integrity and stability of the com-
plexes with respect to coordination equilibria and/or hydrolytic
processes.
Table 1
Best-fit parameters obtained from the analysis of the 1/T1 NMRD profiles of the Mn-
complexes
Parameter
MnL1
MnL2
MnL3
D2a (sꢁ2 1019
)
6.7 0.3
29.1 1.3
75.4 0.6
2.92
3.8 0.2
25.5 1.1
/
/
6.5 0.2
22.3 0.7
/
/
298sV (ps)
a
298sR (ps)
rb (Å)
qc
2.3. Incubation experiments
1
0
0
Incubation experiments of MnL1, MnL2 and MnL3 with tyrosi-
nase, at 37 °C in buffered conditions (50 mM phosphate, pH 6.5;
5 mM hepes, pH 7.4), were carried out monitoring the variation
of R1 at 20 MHz for dilute solutions of the complex (0.4–1.5 mM)
as a function of incubation time (0–20 h). In particular, for each
complex three experiments were run by varying the loading of en-
zyme (50, 250 and 500 U). For all the experiments an increase of
the longitudinal water proton relaxation rate was measured and
the degree of this effect was expressed with an enhancement coef-
ad (Å)
3.2 0.1
2.24
3.2 0.1
2.24
3.4 0.1
2.24
De (cm2 sꢁ1 10ꢁ5
)
a
Parameters of the electronic relaxation times: the trace of the square of the
zero-field splitting tensor,
D
2, and the correlation time describing the modulation of
the zero-field splitting, sV
.
b
Mean Mn–H (water) distance of the coordinated water molecule.
Fixed during the fitting.
Distance of closest approach of the bulk water molecules to the metal ion.
Relative diffusion coefficient of solute and solvent, fixed during the fitting.
c
d
e

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G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
t
*
ficient,
e
, defined as the ratio of R1 after a time t of incubation (R1)
centration of MnL1 was converted into the metal aquaion,
over the corresponding initial value of R1 (R0):
whereas the remaining part is likely to correspond either to
unmodified MnL1 complex or to low-relaxing oligomers that con-
tribute only marginally to the observed NMRD profile. Further evi-
dence of the release of free Mn(II) ions was obtained by measuring
ESR spectra on the same mixture. In fact, while Mn(II) aquaion
originates a typical ESR multiplet (Fig. 5B-i), the Mn(II) chelates
generally do not give rise to an ESR signal in aqueous media at
room temperature. Thus, while MnL1 is ESR silent (Fig. 5B-ii), after
its incubation with tyrosinase the ESR signal is partially restored
(Fig. 5B-iii), suggesting some release of Mn2+ ions.
1
ꢀꢂ ¼ ðRt ꢁ R1wÞ=ðR0 ꢁ R1w
The parameter eꢂF can be defined as the limiting value of
Þ
ð2Þ
. The sta-
1
1
*
e
bility of the Mn(II) complexes in the absence of enzyme was proved
by incubation under identical conditions of concentration, pH, buf-
fer and temperature to those used in the experiment with the en-
zyme. No changes in R1 with time could be measured in the
absence of tyrosinase. The incubation experiment of MnL1 was car-
ried out on a 0.4 mM solution in 50 mM phosphate buffer (pH 6.5;
37 °C) and the relaxation rate was measured at 20 MHz at intervals
of ca. 10–20 min. A relaxation enhancement is observed that
reaches a plateau in about 120/150 min. The largest enhancement
corresponds to a relaxivity increase of 51% (ꢀꢂF ¼ 1:51) and is ob-
served using 500 U of enzyme after ca. 3 h of incubation (Fig. 3A).
In order to investigate the mechanism responsible for the ob-
served relaxation enhancement, the 1/T1 NMRD profile of the solu-
tion containing MnL1 and the enzyme after incubation was
measured at 37 °C. As compared with the corresponding profile
of the intact complex, the relaxivity after the incubation period is
increased over the entire magnetic field range (Fig. 4A). In particu-
lar, the relaxivity gain is more pronounced at low frequencies (<ca.
1 MHz) where it is possible to detect a dispersion at ca. 0.3 MHz
attributable to the contact term. This result suggests the presence
in the incubated solution of Mn(II) aquaion. To check this hypoth-
esis we measured the NMRD profile under identical experimental
condition (50 mM phosphate buffer; pH 6.5; 37 °C) of a equimolar
solution of Mn(NO3)2. The shape of this profile is quite similar to
that reported in Figure 4A but its amplitude is about 3.6 times lar-
ger. From these data we can estimate that part of the initial con-
In order to check a possible active role of the strongly coordinat-
ing environment associated with 50 mM phosphate buffer in the
Mn(II) decomplexation and to better reproduce the physiologic
pH conditions, the incubation experiment was repeated in 5 mM
hepes buffer at pH 7.4. Surprisingly, the enhancement observed
is almost negligible. Probably tyrosinase catalyses the formation
of low stability structures from which manganese can be released
by strongly coordinating anions such as phosphate. On the other
hand, in the presence of the non coordinating hepes buffer the
decomplexation of Mn2+ ions is markedly reduced.
Analogous incubation experiments were carried out on MnL2
and MnL3 and a good enhancement was observed both in 50 mM
phosphate buffer (pH 6.5) and in 5 mM hepes buffer (pH 7.4)
(Fig. 3B and C). For both complexes the relaxation enhancements
observed are significantly larger than those found for MnL1. The
eꢂF values corresponding to the incubation in the presence of
500 U of tyrosinase are 2.91 and 3.56 for MnL2 and MnL3, respec-
tively. These high values reflect the fact that whereas MnL1 has
q = 1 and a relaxivity of ca. 2.9 mMꢁ1 sꢁ1 at 37 °C, MnL2 and
MnL3 have q = 0 and then relaxivity values of only 1.5 and
*
Figure 3. Enhancement factor
e
, at 20 MHz and 310 K, as a function of incubation time for buffered solutions of MnL1 (A), MnL2 (B) and MnL3 (C) in the presence of different
tyrosinase concentration (black circles: 50 U; red squares: 250 U; blue triangles: 500 U).
Figure 4. (A) 1/T1 1H NMRD profiles at 310 K and pH 6.5 of a 0.6 mM solution of MnL1 in phosphate buffer after incubation with 500 U of tyrosinase (open squares) and of a
0.6 mM solution of MnL1 in water (filled circles); (B) X-band ESR spectra at 25 °C of: (i) 0.6 mM aqueous solution of Mn(NO3)2; (ii) 0.6 mM aqueous solution of MnL1 (pH 6.5);
(iii) 0.6 mM solution of MnL1 in phosphate buffer (pH 6.5) after incubation at 310 K with 500 U of tyrosinase.

G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
1119
Figure 5. (A) 1/T1 1H NMRD profiles at 310 K and pH 7.4 of a 1.5 mM solution of MnL2 in hepes buffer after incubation with 500 U of tyrosinase (open squares) and of a
1.5 mM solution of MnL2 in water (filled circles); (B) X-band ESR spectra at 25 °C of: (i) 1.5 mM aqueous solution of Mn(NO3)2; (ii) 1.5 mM aqueous solution of MnL2 (pH 7.4);
(iii) 1.5 mM solution of MnL2 in hepes buffer (pH 7.4) after incubation at 310 K with 500 U of tyrosinase.
1.3 mMꢁ1 sꢁ1 at the same temperature. Then, these two complexes
combine a higher stability (higher denticity of the ligand) to a low-
er intrinsic relaxivity that allows for larger enhancement upon
enzymatic activity. 1H NMRD profiles and ESR spectra revealed
that, whereas for MnL2 the enhancement effect seems to be asso-
ciated with a complex destabilization and release of free Mn2+
ion (Fig. 5), for MnL3 a different mechanism of enzymatic activity
appears to be involved. In fact, the ESR spectrum of the mixture
after incubation does not show any presence of free Mn2+ ions
and the NMRD profile shows a relaxivity peak around 40 MHz
which is characteristic of the presence of slowly tumbling oligo-
meric species ( Fig. 6). Polymerisation is an expected reactivity
pathway for tyrosinase, as previously exploited for Gd-based tyros-
inase and myeloperoxidase responsive agents.17,18 Remarkably,
this pathway was observed only in the case of MnL3 and not for
MnL1 and MnL2. We surmised a correlation of the different reactiv-
ity with the presence in MnL3 of a secondary amine in the tyrosine
residue which might promote a reaction pathway similar to that of
melanin biosynthesis (i.e., formation of polyphenolic biopolymers).
It is worth noting that, while in the case of MnL1 and MnL2 the
enzymatic activity causes destabilization of the complex, the for-
mation of oligomers observed for MnL3 seems not to be associated
with destabilization of the monomeric units.
vivo, MnL2 and MnL3 were incubated with B16F10 murine mela-
noma cell homogenate. B16F10 melanoma cells have been used
for the enzymatic assays, as this cell line has been reported to pos-
sess high tyrosinase activity related to its natural melanin pigmen-
tation.30–32
Cellular tyrosinase activity was assayed according to the meth-
od of Tomita et al.33 using
L-DOPA as substrate and following the
conversion of DOPA to DOPAchrome via DOPA quinone. B16F10
cells (ca. 1 ꢃ 106) were washed with PBS, added to 0.5 mL of a
1 mM L-DOPA solution, sonicated in order to induce cell lysis,
and the absorbance at 475 nm was measured over time by main-
taining the temperature of the reaction mixture at 310 K
(Fig. 7A). The increase of absorbance with time reports about the
DOPAchrome formation and so about the cells tyrosinase activity.
In these proliferation conditions, the enzymatic activity of one mil-
lion of cells has been estimated to be ca. 30 U (the extinction coef-
ficient of DOPAchrome at 475 nm is 3700 Mꢁ1 cmꢁ1).
Then, MnL2 and MnL3 were tested on B16F10 lysates. B16 cells
(ca. 5 ꢃ 106) were washed with PBS, added to 100
lL of a 1 mM
solution of MnL2 or MnL3, sonicated to induce cell lysis and the
proton longitudinal relaxation rate at 20 MHz was measured over
time by maintaining the temperature of the reaction mixture at
310 K (Fig. 7B). An increase of the observed relaxation rate as a
function of time was observed, as previously found for the incuba-
tion experiments with the isolated tyrosinase (Fig. 3B and C). The
relatively low relaxation enhancements observed in Figure 7B are
comparable to those obtained with the lower (50 U) enzyme load-
ing used in the experiments reported in Figure 3B and C. This is
2.4. Incubation experiments on B16 melanoma cell homogenate
In order to evaluate more closely the potential application of
the designed Mn(II) complexes to the detection of tyrosinase in
Figure 6. (A) 1/T1 1H NMRD profiles at 310 K and pH 7.4 of a 1.5 mM solution of MnL3 in hepes buffer after incubation with 500 U of tyrosinase (open squares) and of a
1.5 mM solution of MnL3 in water (filled circles); (B) X-band ESR spectra at 25 °C of: (i) 1.5 mM aqueous solution of Mn(NO3)2; (ii) 1.5 mM aqueous solution of MnL3 (pH 7.4);
(iii) 1.5 mM solution of MnL3 in hepes buffer (pH 7.4) after incubation at 310 K with 500 U of tyrosinase.

1120
G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
Figure 7. (A) Absorbance at 475 nm as a function of time for B16F10 cells homogenates added to a 0.5 mL of a 1 mM
L-DOPA solution; (B) relaxivity enhancement factors as a
function of time for solutions of MnL2 (0.74 mM; black circles) and MnL3 (0.56 mM; red squares) incubated in B16F10 homogenates.
consistent with the rather low tyrosinase activity measured on the
cells lysates when compared to the experiments carried on in the
presence of 250 U and 500 U of tyrosinase.
the relaxation rates, the standard inversion-recovery method was
employed (16 experiments, 2 scans) with a typical 90° pulse width
of 3.5 ms, and the reproducibility of the T1 data was 0.5%.
The exact concentrations of manganese were determined by
mineralisation of samples in HNO3 and by measuring their water
proton relaxation rates.28 The temperature was controlled with a
Stelar VTC-91 airflow heater equipped with a calibrated copper–
constantan thermocouple (uncertainty of 0.1 °C). The proton
1/T1 NMRD profiles were measured on a fast field-cycling Stelar
SmarTracer relaxometer over a continuum of magnetic field
strengths from 0.00024 to 0.25 T (corresponding to 0.01–10 MHz
proton Larmor frequencies). The relaxometer operates under com-
puter control with an absolute uncertainty in 1/T1 of 1%. Addi-
tional data points in the range 15–70 MHz were obtained on the
Stelar Spinmaster spectrometer.
3. Conclusions
In this study, new Mn(II) complexes responsive to the enzyme
tyrosinase were synthesised and their responsive activity was eval-
uated in vitro both by incubation with isolated tyrosinase and by
incubation in B16 cell homogenate. A remarkable relaxation
enhancement effect was detected, especially in the experiments
with the outer sphere complexes MnL2 and MnL3, confirming the
hypothesis initially postulated. In principle it was expected that
the different loading of enzyme would effect the rate of change
of R1 but not its limiting value (eꢂF). The observed dependence of
the enhancement factor after 20 h of incubation from the loading
of tyrosinase used could be due to a relatively low turnover of
the enzyme; therefore, higher loading would allow a larger amount
of substrate to be converted by the enzyme, thus giving rise to lar-
ger relaxation enhancement effects.
The mechanism of action proposed is based on the destabilisa-
tion of Mn(II) complexes followed by release of free Mn2+ ion in a
similar manner as with MnDPDP, approved for clinical use. NMRD
profiles and ESR spectra of the mixtures of Mn(II) complexes and
tyrosinase/B16 cell homogenate confirmed release of some free
Mn2+ ion in the case of MnL1 and MnL2, whereas for MnL3 the forma-
tion of oligomeric species seems responsible for the increased relax-
ivity. As the expression of tyrosinase is associated with melanoma
cancer, a method able to detect it in vivo could potentially find
application as a diagnostic tool for early diagnosis of melanoma.
4.2. Synthesis of ligands
4.2.1. Methyl (2S)-2-(tert-butoxycarbonylmethyl-amino)-3-(4-
tert-butoxy-phenyl)-propionate (2)
Neat tert-butyl bromoacetate (487
drop-wise over 2 min into suspension of Na2CO3 (1.10 g;
10.4 mmol) and -Tyr(OtBu)-OMe (1) (1.00 g; 3,47 mmol) in MeCN
lL; 3.30 mmol) was added
a
L
(10 mL). After being vigorously stirred 18 h at rt the mixture was
centrifuged; the solid was washed with EtOAc (3 ꢃ 15 mL) and
the solution was concentrated and purified by column chromatog-
raphy (petroleum ether/EtOAc 5:1–3:1) to give a colourless oil
(900 mg; 71%). 1H NMR (CDCl3) 400 MHz: d = 1.28 (s, 9H, t-Bu),
1.39 (s, 9H, t-Bu), 2.91 (d, 2H, J = 7.0 Hz, ArCH2), 3.18 (d, 1H,
J = 17.0 Hz , CHCO2t-Bu), 3.26 (d, 1H, J = 17.0 Hz , CHCO2t-Bu),
3.50 (t, 1H, J = 7.0 Hz, CHCO2Me), 3.57 (s, 3H, CO2Me), 6.86 (d,
2H, J = 8.4 Hz, Ar), 7.04 (d, 2H, J = 8.4 Hz, Ar) ppm. 13C NMR (CDCl3)
100 MHz: d = 28.0 (CH3, t-Bu), 28.8 (CH3, t-Bu), 38.9 (CH2, ArCH2),
49.9 (CH2), 51.6 (CH3, CO2Me), 62.3 (CH, CHCO2Me), 78.2 (C, t-
Bu), 81.2 (C, t-Bu), 124.1 (CH, Ar), 129.6 (CH, Ar), 131.7 (C, Ar),
154.1 (C, Ar), 170.6 (C, CO), 174.1 (C, CO) ppm. ESI-MS (m/z):
366.3 (M+H+); 388.3 (M+Na+).
4. Experimental
4.1. Materials and instrumentation
All chemicals were purchased from Sigma–Aldrich Co., Alfa Ae-
sar Co. and Bachem Co. and were used without purification unless
otherwise stated. Compounds tert-butyl 2-bromoethylethylenea-
minodiacetate (3),25 2-bromoethyl trifluoromethanesulfonate26
and tris-t-butyl ethylenediaminotriacetate (9)27 were prepared fol-
lowing previously reported procedures. NMR spectra were re-
corded on a on a JEOL Eclipse Plus 400 spectrometer operating at
9.4 Tesla. ESI mass spectra were recorded on a Waters SQD 3100.
ESR spectra were recorded using a JEOL FA-200 ESR X-band spec-
trometer with JEOL ES-LC11 flat cell for aqueous samples analysis.
ESR spectroscopic analyses were carried out under the following
conditions: temperature 298 K; magnetic field 328 40 mT; field
modulation width 0.3 mT; field modulation frequency 100 KHz;
time constant 0.03 s; sweep time 2 min; microwave frequency
9.226 GHz, microwave power 10 mW. For the measurement of
4.2.2. Methyl (2S)-2-((2-(bis-tert-butoxycarbonylmethyl-
amino)-ethyl)-tert-butoxycarbonylmethyl-amino)-3-(4-tert-
butoxy-phenyl)-propionate (4)
A suspension of 2 (900 mg; 2.46 mmol), tert-butyl 2-bromoeth-
yl iminodiacetate (3) (1.73 g; 4.93 mmol) and Na2CO3 (783 mg,
3.78 mmol) in MeCN (9 mL) was heated under reflux. After 24 h,
more 3 (200 mg) was added and the reaction was continued for
further 24 h. Then the reaction mixture was centrifuged and the
solid residue was washed with EtOAc (3 ꢃ 15 mL). The resulting
solution was concentrated and purified by column chromatogra-
phy (petroleum ether/EtOAc 5:1–3:1) to give a colourless oil
(864 mg; 55%). 1H NMR (CDCl3) 400 MHz: d = 1.29 (s, 9H, t-Bu),

G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
1121
1.44 (s, 27 H, CO2t-Bu), 2.70–2.99 (m, 6H, CH2), 3.36 (d, 2H,
J = 17.7 Hz, 2CH), 3.43 (d, 2H, J = 17.7 Hz, CH2CO2t-Bu), 3.50 (s,
2H, CH2CO2t-Bu), 3.55 (s, 3H, CO2Me), 3.75 (t, 1H, J = 8.2 Hz,
CHCO2Me), 6.85 (d, 2H, J = 8.2 Hz, Ar), 7.09 (d, 2H, J = 8.2 Hz, Ar)
ppm. 13C NMR (CDCl3) 100 MHz: d = 27.8 (CH3, t-Bu), 28.6 (CH3,
t-Bu), 35.7 (CH2, ArCH2), 50.5 (CH2, NCH2), 50.8 (CH3, CO2Me),
52.4, 52.9, 55.7 (CH2, NCH2), 65.8 (CH, CHCO2Me), 77.7 (C, t-Bu),
80.2 (C, t-Bu), 80.4 (C, t-Bu), 123.6 (CH, Ar), 129.4 (CH, Ar), 132.6
(C, Ar), 153.4 (C, Ar), 170.3 (C, CO), 170.7 (C, CO), 172.4 (C, CO)
ppm. ESI-MS (m/z): 637.5 (M+H+); 659.5 (M+Na+).
3.55 mmol) was added in one portion. After 24 h NaBH4 (70 mg,
1.85 mmol) was added in one portion and the resulting mixture
was stirred for additional 20 min before being quenched with a sat-
urated solution of NaHCO3 (5 mL) and extracted in CH2Cl2
(3 ꢃ 15 mL). The organic layer was dried over Na2SO4, concentrated
in vacuo and purified by column chromatography (petroleum
ether/EtOAc 5:1–3:1) to give a light yellow oil (813 mg, 67%). 1H
NMR (CDCl3) 400 MHz: d = 1.41 (s, 9H, t-Bu), 3.00 (d, 2H,
J = 7.0 Hz, ArCH2), 3.59 (t, 1H, J = 7.0 Hz, CHCO2Me), 3.69 (s, 3H,
CO2Me), 3.71 (d, 1H, J = 13.3 Hz, CHPh), 3.90 (d, 1H, J = 13.3 Hz,
CHPh), 6.99 (d, 2H, J = 8.4 Hz, Ar), 7.14 (d, 2H, J = 8.4 Hz, Ar), 7.27–
7.36 (m, 5H, Bn) ppm. 13C NMR (CDCl3) 100 MHz: d = 28.6 (CH3, t-
Bu), 38.9 (CH2, ArCH2), 51.3 (CH3, CO2Me), 51.7 (CH2, NCH2), 61.9
(CH, CHCO2Me), 77.9 (C, t-Bu), 123.9 (CH, Ar), 126.8 (CH, Ar),
127.9 (CH, Ar), 128.1 (CH, Ar), 129.4 (CH, Ar), 132.0 (C, Ar), 139.3
(C, Ar), 153.9 (C, ArO), 174.8 (C, CO) ppm. ESI-MS (m/z): 342.1
(M+H+); 364.2 (M+Na+); 380.3 (M+K+).
4.2.3. (2S)-2-((2-(Bis-carboxymethyl-amino)-ethyl)-carboxy
methyl-amino)-3-(4-hydroxy-phenyl)-propionic acid (L1)
A
solution of 4 (864 mg, 1.36 mmol) and KOH (91 mg,
1.63 mmol), in 1:1 MeOH–H2O (3 mL) was stirred at rt for 4 h;
more KOH (180 mg, 3.21 mmol) was added and the reaction mix-
ture was kept stirring for further 4 h. Then MeOH was removed
in vacuo and the mixture was lyophilised after being acidified to
pH 2. The resulting solid was triturated under acidic water (pH
4.2.7. Methyl (2S)-2-(benzyl-(2-bromo-ethyl)-amino)-3-(4-tert-
butoxy-phenyl)-propionate (8)
2) then suspended in CHCl3 (7 mL) containing i-Pr3SiH (5 lL) and
treated with TFA (2 mL). The reaction mixture was stirred for
18 h and then concentrated in vacuo to give a white solid. 1H
NMR (D2O + NaOD, pD = 8.5) 400 MHz: d = 2.86–3.25 (m, 4H,
CH2CH2), 3.39–3.42 (m, 2H, CH2), 3.61 (d, 1H, J = 7.0 Hz, CH2CO2H),
3.64 (d, 1H, J = 7.0 Hz, CH2CO2H), 3.84 (d, 1H, J = 3.3 Hz, NCHCO2H),
3.96 (d, 2H, J = 17.6 Hz, CH2CO2H), 4.05 (d, 2H, J = 17.6 Hz,
CH2CO2H), 6.84 (d, 2H, J = 8.8 Hz, Ar), 7.16 (d, 2H, J = 8.8 Hz, Ar)
ppm. 13C NMR (D2O + NaOD) 100 MHz: d = 34.5 (CH2, ArCH2),
49.6, 50.7, 53.3, 54.1 (CH2, NCH2), 66.2 (CH, NCHCO2H), 119.5
(CH, Ar), 128.3 (CH, Ar), 130.1 (C, Ar), 157.9 (C, Ar), 171.3 (C, CO),
173.5 (C, CO) ppm. ESI-MS (m/z): 397.3 (MꢁH+).
Neat 2-bromoethyl trifluoroethanesulfonate (892 mg, 3.47
mmol) was added into a solution of 7 (741 mg, 2.17 mmol) and
2,6-lutidine (784 lL, 6.73 mmol) stirring in anhydrous toluene
(10 mL) at 0 °C. The resulting reaction mixture was stirred at rt for
24 h. More 2-bromo trifluoroethanesulfonate (300 mg, 1.17 mmol)
was added and the reaction mixture was kept stirring for further
24 h, then diluted with EtOAc (20 mL) and washed with water
(2 ꢃ 10 mL) and brine (2 ꢃ 10 mL). The organic layer was dried over
Na2SO4, concentrated in vacuo and purified by column chromatog-
raphy (petroleum ether/EtOAc 5:1–3:1) to give a colourless oil
(372 mg, 38%). 1H NMR (CDCl3) 400 MHz: d = 1.41 (s, 9H, t-Bu),
3.13–3.22 (m, 3H), 3.67 (t, 2H, J = 6.2 Hz, CH2), 3.75 (s, 3H, CH3),
3.74 (m, 1H, CHPh), 4.00 (d, 1H, J = 13.9 Hz , CHPh), 4.81 (t, 2H,
J = 6.2 Hz, CH2), 6.97 (d, 2H, J = 8.4 Hz, Ar), 7.09 (d, 2H, J = 8.4 Hz,
Ar), 7.20–7.36 (m, 5H, Bn) ppm. 13C NMR (CDCl3) 100 MHz:
d = 26.2 (CH2, CH2Br), 28.8 (CH3, t-Bu), 35.7 (CH2, ArCH2), 51.4
(CH3, CO2Me), 53.0 (CH2, Bn), 64.9 (CH, CHCO2Me), 74.4 (CH2,
CH2N), 78.3 (C, t-Bu), 124.2 (CH, Ar), 127.3 (CH, Ar), 128.3 (CH, Ar),
128.6 (CH, Ar), 129.6 (CH, Ar), 132.8 (C, Ar), 139.0 (C, Ar), 153.9 (C,
ArO), 172.9 (C, CO) ppm. ESI-MS (m/z): 448.1, 450.2 (M+H+); 470.4,
472.0 (M+H+).
4.2.4. tert-Butyl (2S)-2-(bis-(2-(bis-tert-butoxycarbonylmethyl-
amino)-ethyl)-amino)-3-(4-tert-butoxy-phenyl)-propionate (6)
A suspension of 3 (449 mg, 1.28 mmol), L-Tyr(OtBu)-OtBu (5)
(150 mg, 0.425 mmol) and Na2CO3 (180 mg, 1.7 mmol) in MeCN
(3 mL) was heated under reflux for 48 h. The reaction mixture
was centrifuged and the solid residue was washed with EtOAc
(3 ꢃ 10 mL). The resulting solution was concentrated and purified
by column chromatography (petroleum ether/EtOAc 5:1–3:1) to
give a light-yellow oil (182 mg; 51%). 1H NMR (CDCl3) 400 MHz:
d = 1.29 (s, 9H, t-Bu), 1.38 (s, 45 H, CO2t-Bu), 2.63–2.99 (m, 10H),
3.51 (s, 8H, CH2CO2t-Bu), 3.57–3.61 (m, 1H, NCHCO2t-Bu), 6.84
(d, 2H, J = 8.2 Hz, Ar), 7.08 (d, 2H, J = 8.2 Hz, Ar) ppm. 13C NMR
(CDCl3) 100 MHz: d = 28.1 (CH3, t-Bu), 28.8 (CH3, t-Bu), 35.4 (CH2,
ArCH2), 50.2, 53.3, 53.4, 56.0 (CH2, NCH2), 65.7 (CH, CHCO2t-Bu),
78.0 (C, t-Bu), 80.2 (C, t-Bu), 80.7 (C, t-Bu), 123.8 (CH, Ar), 129.7
(CH, Ar), 133.4 (C, Ar), 153.5 (C, Ar), 170.5 (C, CO) ppm. ESI-MS
(m/z): 836.6 (M+H+); 858.6 (M+Na+).
4.2.8. Methyl (2S)-2-(benzyl-(2-((2-(bis-tert-butoxycarbon-
ylmethyl-amino)-ethyl)-tert-butoxycarbonylmethyl-amino)-
ethyl)-amino)-3-(4-tert-butoxy-phenyl)-propionate (10)
A suspension of 8 (372 mg, 0.830 mmol), tris-tert-butyl ethylen-
ediaminotriacetate (9) (368 mg, 0.913 mmol) and Na2CO3 (264 mg,
2.49 mmol) in MeCN (3 mL) was heated under reflux for 36 h. The
reaction mixture was diluted with EtOAc (15 mL), washed with
water (2 ꢃ 5 mL) and brine (5 mL). The organic layer was dried over
Na2SO4, concentrated in vacuo and purified by column chromatog-
raphy (petroleum ether/EtOAc 5:1–3:1) to give a light-yellow oil
(219 mg, 32%). 1H NMR (CDCl3) 400 MHz: d = 1.31 (s, 9H, t-Bu),
1.38 (s, 27H, CO2t-Bu), 2.71–2.91 (m, 10H, CH2), 2.89 (dd, 1H,
J = 14.0, 7.8 Hz, ArCH), 3.03 (dd, 1H, J = 14.0, 7.8 Hz, ArCH), 3.40 (s,
4H, CH2CO2t-Bu), 3.59 (d, 1H, J = 14.1 Hz, BnCH), 3.63 (s, 3H,
CO2Me), 3.65 (t, 1H, J = 7.8 Hz, NCH CO2Me), 3.91 (d, 1H,
J = 14.1 Hz, BnCH), 6.90 (d, 2H, J = 7.9 Hz, Ar), 7.01 (d, 2H,
J = 7.9 Hz, Ar), 7.06–7.22 (m, 5H, Bn) ppm. 13C NMR (CDCl3)
100 MHz: d = 28.1 (CH3, t-Bu), 28.9 (CH3, t-Bu), 35.3 (CH2, ArCH2),
51.2 (CH3, CO2Me), 51.6, 52.4, 52.6, 53.2, 55.9, 56.0, 56.2 (CH2,
NCH2), 64.3 (CH, CHCO2Me), 78.2 (C, t-Bu), 80.9 (C, t-Bu), 81.1 (C,
t-Bu), 124.0 (CH, Ar), 127.0 (CH, Ar), 128.1 (CH, Ar), 128.7 (CH, Ar),
129.7 (CH, Ar), 133.1 (C, Ar), 139.3 (C, Ar), 153.7 (C, Ar), 170.5 (C,
CO), 172.9 (C, CO) ppm. ESI-MS (m/z): 770.5 (M+H+); 792.5 (M+Na+).
4.2.5. (2S)-2-(Bis-(2-(bis-carboxymethyl-amino)-ethyl)-amino)-
3-(4-hydroxy-phenyl)-propionic acid (L2)
A solution of 6 (182 mg, 0.218 mmol) in CHCl3 (2 mL) contain-
ing i-Pr3SiH (5 lL) and treated with TFA (400 lL). The reaction
mixture was stirred for 18 h and then concentrated in vacuo to give
a white solid. 1H NMR (D2O + NaOD) 400 MHz: d = 2.45–2.90 (m,
10H), 3.08 (s, 8H), 3.26 (m, 1H), 6.47 (d, 2H, J = 8.4 Hz, Ar), 6.91
(d, 2H, J = 8.4 Hz, Ar) ppm. 13C NMR (D2O + NaOD, pD = 8.5)
100 MHz: d = 35.3 (CH2, ArCH2), 48.9, 53.0, 59.3 (CH2, NCH2), 70.3
(CH, CHCO2H), 118.8 (CH, Ar), 124.8 (C, Ar), 130.6 (CH, Ar), 164.6
(C, Ar), 179.8 (C, CO) ppm. ESI-MS (m/z): 498.7 (MꢁH+).
4.2.6. Methyl (2S)-2-benzylamino-3-(4-tert-butoxy-phenyl)-
propionate (7)
A solution of
aldehyde (360
(10 mL) was stirred for 30 min and then NaBH(OAc)3 (736 mg,
L-Tyr(OtBu)-OMe (1) (1.0 g, 3.47 mmol) and benz-
lL, 3.54 mmol) in anhydrous 1,2-dichloroethane

1122
G. A. Rolla et al. / Bioorg. Med. Chem. 19 (2011) 1115–1122
4.2.9. (2S)-2-(2-((2-(Bis-carboxymethyl-amino)-ethyl)-
carboxymethyl-amino)-ethylamino)-3-(4-hydroxy-phenyl)-
propionic acid (L3)
5 mM hepes buffer at pH 7.4 (100 lL) and sonicated to induce cell
lysis. The variation of the longitudinal water proton relaxation rate
at 20 MHz was followed over time.
A solution of 10 (160 mg, 0.208 mmol) and KOH (35.0 mg,
0.623 mmol), in 1:1 MeOH–H2O (2 mL) was stirred at rt for 8 h. Then
MeOH was removed in vacuo and the mixture was lyophilised after
being acidified to pH 2. The resulting solid was triturated under
acidic water (pH 2) and then suspended in CHCl3 (2 mL) containing
Acknowledgements
This work was supported by MIUR (PRIN 2007) and carried out
under the frame of ESF COST Action D38. We thank Dr. Claudio Cas-
sino for help with the measurement of the ESR spectra.
i-Pr3SiH (5 lL) and treated with TFA (400 lL). The reaction mixture
was stirred for 12 h and then concentrated in vacuo to give a white
solid. The resulting solid was suspended in MeOH (5 mL) and treated
with 10% Pd/C (25 mg); the reaction mixture was stirred under an
atmosphere of H2 for 18 h, then filtered over Celite and concentrated
in vacuo to give a white solid. 1H NMR (D2O + NaOD, pD = 8.5)
400 MHz: d = 2.44–3.01 (m, 10H), 3.35 (s, 6H), 3.80–4.03 (m, 1H),
6.68 (d, 2H, J = 8.3 Hz, Ar), 6.95 (d, 2H, J = 8.3 Hz, Ar) ppm. 13C NMR
(D2O + NaOD) 100 MHz: d = 37.2 (CH2, ArCH2), 46.5, 51.8, 52.5,
55.5, 58.3, 59.7 (CH2, NCH2), 65.3 (CH, CHCO2H), 115.7 (CH, Ar),
129.3 (CH, Ar), 135.4 (C, Ar), 156.1 (C, ArOH), 176.3 (C, CO), 176.9
(C, CO) ppm. ESI-MS (m/z): 440.1 (MꢁH+).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.064.
References and notes
1. Lauffer, R. B. Chem. Rev. 1987, 87, 901.
2. Caravan, P.; Ellison, J.; McMurry, T.; Lauffer, R. Chem. Rev. 1999, 99, 2293.
3. Tòth, E.; Helm, L.; Merbach, A. E. Top. Curr. Chem. 2002, 221, 61.
4. Aime, S.; Botta, M.; Terreno, E. Adv. Inorg. Chem. 2005, 57, 173.
5. Li, Y.; Huang, T. T.; Carlson, E. J.; Melov, S.; Ursell, P. C.; Olson, J. L.; Noble, L. J.;
Yoshimura, M. P.; Berger, C.; Chan, P. H. Nat. Genet. 1995, 11, 376.
6. Wedler, F. C.; Denman, R. B. Curr. Top. Cell Regul. 1984, 24, 153.
7. Lim, K. O.; Stark, D. D.; Leese, P. T.; Pfefferbaum, A.; Rocklage, S. M.; Quay, S. C.
Radiology 1991, 178, 79.
8. Kubicek, V.; Toth, E. Adv. Inorg. Chem. 2009, 61, 63.
9. Koretsky, A. P.; Silva, A. C. NMR Biomed. 2004, 17, 527.
10. Watanabe, T.; Natt, O.; Boretius, S.; Frahm, J.; Michaelis, T. Magn. Reson. Med.
2002, 48, 852.
4.3. Incubation experiments
4.3.1. General incubation with tyrosinase
A solution of Mn(II) complex (MnL1, MnL2 or MnL3) in 50 mM
phosphate buffer at pH 6.5 (100
buffer at pH 7.4 (100 L; 297 nmol) was treated with a solution
of tyrosinase (10 L; 50 U, 50 L; 250 U or 100 L; 500 U) in
lL; 333 nmol) or in 5 mM hepes
l
11. Aoki, I.; Wu, Y. J.; Silva, A. C.; Lynch, R. M.; Koretsky, A. P. Neuroimage 2004, 22,
1046.
12. Pautler, R. G.; Silva, A. C.; Koretsky, A. P. Magn. Reson. Med. 1998, 40, 740.
13. Mankoff, D. A. J. Nucl. Med. 2007, 48, 18N.
l
l
l
50 mM phosphate buffer at pH 6.5 or in 5 mM hepes buffer at pH
7.4 and further diluted with the correspondent buffer, when neces-
14. Lowe, M. P. Curr. Pharm. Biotechnol. 2004, 5, 519.
15. Moats, R. A.; Fraser, S. E.; Meade, T. J. Angew. Chem., Int. Ed. 1997, 36, 726.
16. Giardiello, M.; Lowe, M. P.; Botta, M. Chem. Commun. 2007, 4044.
17. Aime, S.; Geninatti Crich, S.; Gianolio, E.; Giovenzana, G. B.; Tei, L.; Terreno, E.
Coord. Chem. Rev. 2006, 250, 1562.
18. Querol, M.; Bennett, D. G.; Sotak, C.; Kang, H. W.; Bogdanov, A., Jr.
ChemBioChem 2007, 8, 1637.
19. Chen, J. W.; Pham, W.; Weissleder, R.; Bogdanov, A., Jr. Magn. Reson. Med. 2004,
52, 1021.
20. Aime, S.; Botta, M.; Milone, L.; Terreno, E. Chem. Commun. 1996, 1265.
21. Aime, S.; Botta, M.; Gianolio, E.; Terreno, E. Angew. Chem., Int. Ed. 2000, 39, 747.
22. Aime, S.; Botta, M.; Fasano, M.; Terreno, E.; Kinchesh, P.; Calabi, L.; Paleari, L.
Magn. Reson. Med. 1996, 35, 648.
23. Chen, J. W.; Pham, W.; Weissleder, R.; Bogdanov, A., Jr. Mag. Reson. Med. 2004,
52, 1021.
sary, to reach the final volume of 200 lL. The variation of the lon-
gitudinal water proton relaxation rate was followed over 20 h.
4.3.2. Cell culture
B16F10 melanoma cells (mouse) were obtained from American
Type Culture Collection (ATCC, Manassas, USA), grown in Dul-
becco’s modified Eagle’s medium (DMEM) containing 10% foetal
bovine serum from Lonza (Lonza Sales AG, Verviers. Belgium),
100 U/mL penicillin and 100 mg/mL streptomycin and maintained
at 310 K under 5% CO2 conditions. After 3 days culture, the cells
were harvested with trypsine/EDTA, counted with the Trypan-blue
exclusion test, and assayed for tyrosinase activity.
24. Jawaid, S.; Khan, T. H.; Osborn, H. M. I.; Williams, N. A. O. Anticancer Agents
Med. Chem. 2009, 9, 717.
25. Williams, M. A.; Rapoport, H. J. Org. Chem. 1993, 58, 1151.
26. Chi, D. Y.; Kilbourn, M. R.; Katzenellenbogen, J. A.; Welch, M. J. J. Org. Chem.
1987, 52, 658.
27. Achilefu, S.; Wilhelm, R. R.; Jimenez, H. N.; Schmidt, M. A.; Srinivasan, A. J. Org.
Chem. 2000, 65, 1562.
28. Crich, S. G.; Biancone, L.; Cantaluppi, V.; Esposito, D. D. G.; Russo, S.; Camussi,
G.; Aime, S. Magn. Reson. Med. 2004, 51, 938.
29. Aime, S.; Anelli, P. L.; Botta, M.; Brocchetta, M.; Canton, S.; Fedeli, F.; Gianolio,
E.; Terreno, E. J. Biol. Inorg. Chem. 2002, 7, 58.
4.3.3. Cellular tyrosinase activity assay
Ca. 1 ꢃ 106 B16F10 cells were washed with PBS, added to 0.5 mL
of a 1 mM L-DOPA solution, sonicated to induce cell lysis and the
absorbance at 475 nm was measured over time by maintaining
the temperature of the reaction mixture at 37 °C. An extinction
coefficient of 3700 Mꢁ1 cmꢁ1 was used for the quantification of
the DOPAchrome formation.
30. Pawelek, J. M. J. Invest. Dermatol. 1976, 66, 201.
31. Petrescu, S. M.; Petrescu, A. J.; Titu, H. N.; Dwek, R. A.; Platt, F. M. J. Biol. Chem.
1997, 272, 15796.
32. Liu, S. H.; Pan, I. H.; Chu, I. M. Biol. Pharm. Bull. 2007, 30, 1135.
33. Tomita, Y.; Maeda, K.; Tagami, H. Pigment Cell Res. 1992, 5, 357.
4.3.4. MnL2 and MnL3 incubation with B16-F10 homogenates
Ca. 5 ꢃ 106 cells were washed with PBS, harvested with tryp-
sine/EDTA and added to a 1 mM solution of MnL2 or MnL3 in