Dual-functional gadolinium-based copper(II) probe for selective magnetic resonance imaging and fluorescence sensing

Article abstract of DOI:10.1021/ic202322f

A unique gadolinium complex, Nap-DO3A-Gd, comprising a naphthylamine luminescent moiety, a di-2-picolylamine (DPA) binding chelator, and a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) moiety has been designed and synthesized as a dual-functional probe for selective magnetic resonance imaging and fluorescent sensing of copper(II) in living cells. Nap-DO3A-Gd exhibited a turn-on manner of relaxivity changes and a fluorescent quenching toward Cu²⁺. Through the introduction of naphthalamide into the Gd³⁺ contrast agent platform to restrict the coordination ability of the DPA chelator and with Gd³⁺ coordinating to the DPA moiety to turn away the interferences of other metal cations from Cu ²⁺ detection, the probe featured selective relaxivity changes toward Cu²⁺ over other metal ions and brought unique Cu²⁺- specific luminescent responses. The probe was water-soluble with the luminescent detection limit established at 6 ppb and was successfully used for luminescence imaging detection of copper(II) in living cells. The results demonstrated the efficiency and advantage of our approach in the development of a dual-modality image.

Full text of DOI:10.1021/ic202322f

Article
pubs.acs.org/IC
Dual-Functional Gadolinium-Based Copper(II) Probe for Selective
Magnetic Resonance Imaging and Fluorescence Sensing
Xiaolin Zhang,† Xu Jing,† Tao Liu,† Gang Han,‡ Huaqiang Li,† and Chunying Duan*,†
†State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China
‡Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts
01605, United States
ABSTRACT: A unique gadolinium complex, Nap-DO3A-Gd,
comprising a naphthylamine luminescent moiety, a di-2-picolyl-
amine (DPA) binding chelator, and a 1,4,7,10-tetraazacyclodode-
cane-1,4,7-triacetic acid (DO3A) moiety has been designed and
synthesized as a dual-functional probe for selective magnetic
resonance imaging and fluorescent sensing of copper(II) in living
cells. Nap-DO3A-Gd exhibited a turn-on manner of relaxivity
changes and a fluorescent quenching toward Cu2+. Through the
introduction of naphthalamide into the Gd3+ contrast agent platform
to restrict the coordination ability of the DPA chelator and with
Gd3+ coordinating to the DPA moiety to turn away the interferences
of other metal cations from Cu2+ detection, the probe featured
selective relaxivity changes toward Cu2+ over other metal ions and
brought unique Cu2+-specific luminescent responses. The probe was water-soluble with the luminescent detection limit
established at 6 ppb and was successfully used for luminescence imaging detection of copper(II) in living cells. The results
demonstrated the efficiency and advantage of our approach in the development of a dual-modality image.
between copper regulation and its physiological or pathological
consequences is fascinating and led to extensive interest in the
development of new ways to study the aspects of copper-ion
accumulation, trafficking, and export in living systems by live-
cell optical imaging and MRI.29−31 Because copper ions are
involved in numerous molecular processes globally within an
intact and complex whole body, rather than within an isolated
in vitro system, the complementary nature of a dual-functional
MRI/fluorescent emission probe that takes advantage of the
exquisite sensitivity of optical techniques and the high spatial
resolution of MRI methods will benefit the biological imaging
copper ions.32 Inspired by the elegant smart contrast agents for
the β-galactosidase activity described by Meade et al.1,4 and the
synthetic approach of the naphthylamine-based probes for
copper(II) ions,33−35 herein, we reported a new approach to
design and synthesize a dual-functional MRI/fluorescent
emission probe through the incorporation of a Gd3+ contrast
agent platform to a luminescent-active naphthylamine moiety
having di-2-picolylamine (DPA) as a tridentate chelator. We
envisioned that the DPA tridentate chelator coordinated to
copper(II) strongly to relieve steric congestion around the Gd3+
center,36−40 benefiting the increase of proton relaxivity. Also,
the incorporation of a larger naphthylamine moiety will modify
the coordination ability of DPA, from which the improvement
INTRODUCTION
■
Magnetic resonance imaging (MRI) has become increasingly
popular in experimental molecular imaging and clinical because
it is noninvasive and capable of producing three-dimensional
representations of opaque organisms with high spatial and
temporal resolution.1−6 While the combination of metal-based
MRI contrast agents with selective molecular recognition
elements provides a promising new class of chemosensors for
molecular imaging of essential s-block and d-block metal ions in
biological systems, the quantitative measurements using MRI as
a single modality are still challenging because the local
concentration of the contrast agent is unknown, and the
sensitivity of the MRI technique is relatively low compared to
other modalities such as optical imaging or positron emission
tomography.7−18 In this regard, the attachment of a
luminescent probe to an MRI contrast agent offers a potential
powerful approach for the quantitative integration of molecular
and cellular information about complex biological signaling
networks at a system level because the fluorescent metal
sensors exhibit excellent sensitivity and multiplexed detection
capabilities at the cellular level and have been widely exploited
in biotechnology and biomedical research fields.19−25
Of the transition-metal ions in the human body, copper is the
third most abundant essential trace element. The alternations in
its cellular homeostasis are connected to serious neuro-
degenerative diseases including Alzheimer’s disease, familial
amyotrophic lateral sclerosis, Menkes and Wilson’s diseases,
and prion diseases.26−28 Understanding the relationships
Received: October 27, 2011
Published: February 8, 2012
© 2012 American Chemical Society
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of the selectivity toward copper ions over other metal ions,
especially for that of zinc(II) ions, is expected.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Nap-DO3A-Gd
Probe. Nap-DO3A-Gd was synthesized according to the
synthetic route outlined in Scheme 1. 3 was gained through the
a
Scheme 1. Synthetic Procedure for Nap-DO3A-Gd
Figure 1. ESI-MS spectra of Nap-DO3A-Gd in the solution of
methanol and water (1:2, v/v; m/z 500−600) in the absence of copper
ions (a) and after the addition of copper ions (b). Insets: theoretical
and experimental isotopic patterns at m/z 511.04, 522.02, 541.50,
564.33, and 575.48 in parts a and b.
three new peaks at m/z 541.50, 564.33, and 575.48. These
peaks were assigned to [Cu(Nap-DO3A-Gd)]2+, [Cu(Nap-
DO3A-Gd)(C2H5OH)]2+, and [Cu(Nap-DO3A-Gd)-
(CH3OH)(H2O)2]2+, respectively, according to the exact
comparison of the intense peaks with the simulation on the
basis of natural isotopic abundances. This result provides
evidence of the formation of a 1:1 stoichiometric complexation
species in solution. Upon the addition of zinc ions, instead of
copper ions, no complexation species were detected in the ESI-
MS spectra under the same experimental conditions, which
suggests the better selectivity of Nap-DO3A-Gd to Cu2+ than
Zn2+ in solution.
MRI Responses toward Cu2+. The ability of Cu2+ to
modulate the longitudinal relaxivity of Nap-DO3A-Gd was
determined using T1 measurements at a proton frequency of 20
MHz (pH 7.0; Figures 2 and 3). In the absence of Cu2+, the
a
Conditions: (a) 2-pyridinecarboxaldehyde, C2H5OH, reflux (83%);
(b) sodium triacetoxyborohydride, CH3OH, reflux (80%); (c) 2,6-
bis(chloromethyl)pyridine, potassium carbonate, CH3CN, reflux
(60%); (d) tristerbutyl ester of DO3A, NaHCO3, CH3CN, reflux
(48%); (e) trifluoroacetic acid, CH2Cl2, rt (90%); (f) Gd-
(NO3)3·6H2O, MeOH/H2O, rt (51%).
Schiff base reaction and the reduction reaction of 1. The
nucleophilic substitution of bis(chloromethyl)pyridine with 3
gave 4 in a 60% yield. 4 was reacted with tri-tert-butyl ester of
1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) to
produce 5 via alkylation. 5 was then treated with trifluoroacetic
acid to remove the tert-butyl protecting groups and reacted
overnight with Gd(NO3)3·6H2O. Nap-DO3A-Gd was purified
via semipreparatory high-performance liquid chromatography
(HPLC) on a reversed-phase column. Analytical HPLC−mass
spectrometry (MS) was used to confirm the purity and identity
of the collected fractions, and its Gd3+ content was analyzed
using inductively coupled plasma optical emission spectroscopy
(ICP-OES).
Figure 2. Relaxivity responses of Nap-DO3A-Gd (0.2 mM) to Cu2+
with various concentrations (T1 measurements at a proton frequency
of 20 MHz) and T1-weighted phantom MRI images: (a) image of only
Nap-DO3A-Gd (0.2 mM) in water; (b) image of Nap-DO3A-Gd (0.2
mM) and copper ions (0.6 mM) in water.
Electrospray MS (ESI-MS) of Nap-DO3A-Gd showed three
peaks at m/z 511.04, 522.02, and 1021.32, assignable to
[H2(Nap-DO3A-Gd)]2+, [NaH (Nap-DO3A-Gd)]2+, and [H-
(Nap-DO3A-Gd)]+, respectively (Figure 1). The addition of
Cu2+ caused the disappearance of the peaks and brought out
relaxivity of Nap-DO3A-Gd (0.2 mM) was 5.53 mM−1 s−1,
which gradually increased to 7.87 mM−1 s−1 with the addition
of Cu2+ until 3 equiv of copper ions had been added. Adding
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Table 1. Luminescence Lifetimes (ms) and Calculated
Number of Inner-Sphere Water Molecules (q) for Nap-
DO3A-Tb (0.2 mM) in the Absence and Presence of Cu2+
a
(0.6 mM)
τH O/ms
τD O/ms
q
2
2
Nap-DO3A-Tb
Nap-DO3A-Tb + Cu2+
0.73
0.23
0.77
0.27
0.28
2.58
a
Luminescence measurements of the terbium(III) analogues were
performed in the absence or presence of excess Cu(NO3)2. Samples
were excited at 280 nm, and the emission maxium at 544 nm was used
to determine luminescence lifetimes. The number of bound water
molecules was estimated using Horrocks’ equation:39 q = 4.2(1/τH O
/
2
ms − 1/τD O/ms), where τ is the luminescence lifetime in H2O or
D2O.
2
Figure 3. MRI responses (T1 measurements at a proton frequency of
20 MHz) of Nap-DO3A-Gd (0.2 mM) upon the addition of metal
ions of interest (0.6 mM).
of Cu2+ (Figure 4). Fitting of the fluorescence titration curve of
Cu2+ triggered about 42% enhancement in the relaxivity. The
observed relaxivity increases for Nap-DO3A-Gd are stable over
time, indicating that Gd3+/DO3A has excellent stability and
remains intact in the presence of Cu2+.14 The apparent KA for
the 1:1 Cu2+/Nap-DO3A-Gd complex is 8.5 × 104 M−1. Also,
the acquired T1-weighted images of Nap-DO3A-Gd (0.2 mM)
with a commercial 0.5 T magnet in the absence and presence of
copper ions (0.6 mM) readily visualized differences in the
copper levels in a biologically relevant micromolar range.
The relaxivity changes of Nap-DO3A-Gd were Cu2+
specificity over other physiologically important metal ions
including Ca2+, Mg2+, Na+, K+, Fe2+, Fe3+, Cu+, and Mn2+. Even
upon the addition of 2 mM (10 equiv) of metal ions such as
Ca2+, Mg2+, Na+, and K+, the relaxivity of Nap-DO3A-Gd
hardly changed in an obvious manner. Interestingly, Nap-
DO3A-Gd exhibited high excellent selectivity toward Cu2+ over
Zn2+; the addition of Zn2+ (0.6 mM) did not affect the relaxivity
of Nap-DO3A-Gd. The better selectivity compared to the
reported copper(II)-responsive MRI probes37,38 is reasonably
attributed to the introduction of naphthalimide into the Gd3+
contrast agent platform that restricts the coordination ability of
the DPA chelator to that of zinc ions; only the ions exhibiting
stronger binding ability could replace the Gd3+ ions to
coordinate with the DPA donors. We also observed the MRI
responses toward Cu2+ with a pH range from 5.5 to 8.0, and no
significant variation was observed.
Nap-DO3A-Gd (5 μM) revealed a 1:1 stoichiometry of the
Figure 4. Family of fluorescence spectra of Nap-DO3A-Gd (5 μM, 1:2
CH3OH/H2O, pH 7.0) upon the addition of a Cu(NO3)2 aqueous
solution. The intensity was recorded at 550 nm. Excitation at 400 nm.
Inset: Titration profiles of Nap-DO3A-Gd upon the addition of Cu2+
showing fluorescence intensities at 550 nm.
Nap-DO3A-Gd/Cu2+ species with an association constant of
6.31 × 106 M−1.41 The related higher association constant
calculated from the fluorescent titration than that from the MRI
experiment might be due to the fact that the free copper ions
also can induce the quench of fluorescence. The fluorescence
quantum yield42 decreased to 8% of the original one (from
0.054 to 0.0042) with EC50 (the concentration of Cu2+ in the
case of the luminescence quench: 50%) being ca. 0.25 μM.
Under optimized conditions, the fluorescence intensity of Nap-
DO3A-Gd (0.1 μM) was nearly proportional to the copper
concentration, with the detection limit about 6 ppb. Because
the average concentration of blood copper in the normal group
is 100−150 μg/L (15.7−23.6 μM),43 Nap-DO3A-Gd thus is
able to detect copper(II) in the normal physiological condition.
Furthermore, the fluorescence quench of Nap-DO3A-Gd
induced by Cu2+ could be recovered when a solution of
glutathione was added. This indicated that the chelation of
Cu2+ was reversible.
From a view of the mechanism, in the solution of a free Nap-
DO3A-Gd probe, water molecules are limited in their inner-
sphere access to the Gd3+ center by steric hindrance from the
two bulky pyridine groups, which cap the DO3A moiety,
therefore minimizing proton relaxivity. Upon binding Cu2+, the
Gd3+ ion is exposed to bulk water, thereby changing the
relaxivity from weak to strong with DPA coordinated to the
better acceptors of the copper ions. The fluorescence decay
rates of the terbium(III) analogue in water and D2O with and
without copper(II) present were measured to calculate the
number of inner-sphere water molecules (q; Table 1). The
increase of q from 0.28 in the absence of Cu2+ to 2.58 after the
addition of Cu2+ confirmed that Cu2+ binding to Nap-DO3A-
Gd increases the number of the inner-sphere water
molecules.38−41
The fluorescence responses of Nap-DO3A-Gd (10 μM)
exhibited excellent selectivity toward Cu2+ over other metal
ions. No significant spectral changes were observed for Nap-
DO3A-Gd (10 μM) in the presence of alkali- or alkaline-earth
metals, such as Na+, K+, Mg2+, and Ca2+ (Figure 5).
Competition experiments revealed that the Cu2+-induced
fluorescence was unaffected in the presence of the metal
Fluorescence Detection toward Cu2+. Nap-DO3A-Gd
displays a fluorescence band centered at 550 nm. Upon the
addition of Cu(NO3)2 to the solution of Nap-DO3A-Gd, the
fluorescence emission at 550 nm gradually decreased and then
remained constant after the addition of approximately 1 equiv
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Figure 5. Fluorescence responses of Nap-DO3A-Gd (10 μM, 1:2
CH3OH/H2O, pH 7.0) to various metal ions with excitation
wavelength at 400 nm. Blue bars represent the emission intensities
of Nap-DO3A-Gd (10 μM) after the addition of the appropriate metal
ions (10 μM Ag+, Al3+, Ba2+, Cd2+, Cr3+, Fe2+, Zn2+, Ni2+, Cu+, and
Cu2+ and 100 μM Mg2+, Na+, Ca2+). Red bars represent the emission
intensities that occur upon the subsequent addition of 10 μM Cu2+ to
the above-mentioned solutions, respectively. Emission intensities were
recorded at 550 nm, and the excitation wavelength was at 400 nm.
Figure 6. Fluorescence images of Cu2+ in HeLa cells: (a) Fluorescence
image of HeLa cells incubated with Nap-DO3A-Gd (10 μM); (b)
bright-field transmission image of HeLa cells incubated with Nap-
DO3A-Gd (10 μM); (c) fluorescence image of HeLa cells incubated
with Nap-DO3A-Gd for 15 min, washed three times, and further
incubated with 10 μM Cu(NO3)2 for 15 min; (d) bright-field
transmission image of HeLa cells incubated with Nap-DO3A-Gd (10
μM) and Cu(NO3)2 (10 μM).
cations mentioned above. Interestingly, while DPA-functional-
ized naphthalimide-based probes exhibited zinc-specific fluo-
rescence enhancement in aqueous media,33−35 the fluorescence
of Nap-DO3A-Gd was hardly affected by the presence of zinc
ions, and the Cu2+-induced fluorescent responses were also
unaffected in the presence of zinc ions in solution. The
excellent selectivity toward Cu2+ over zinc ions is attributed to
the fact that the coordination of the DPA chelator to Gd3+
restricts the probe to complexate zinc ions; only the Cu2+ ions
exhibiting stronger binding ability is able to replace the Gd3+
coordinating to the DPA donors. Nap-DO3A-Gd responded to
Cu2+ in the pH range 5.5−8.0, with the fluorescence intensity
varying by less than 10%, facilitating the detection of Cu2+ in
aqueous media at physiological pH values.
Fluorescence imaging of intracellular Cu2+ was observed
under a Nikon eclipse TE2000-5 inverted fluorescence
microscope with a 20× objective lens. As shown by our
fluorescence imaging experiments (Figure 6), staining of HeLa
cells with a 10 μM solution of Nap-DO3A-Gd at room
temperature for 30 min led to obvious intracellular green
fluorescence. When cells stained with Nap-DO3A-Gd were
incubated with 10 μM Cu2+ in phosphate-buffered saline (PBS;
pH 7.4) for 30 min and washed, the obvious quenching of the
fluorescence intensity was observed. The results indicate that
cells were permeable to Nap-DO3A-Gd and copper ions could
be detected in living cells.
In vivo experiments were also carried out to examine if Nap-
DO3A-Gd could be used to detect Cu2+ in living organisms
(Figure 7).44 Five-day-old zebrafish were incubated with Nap-
DO3A-Gd (10 μM) in PBS for 30 min and washed three times
with PBS (pH 7.4). The obvious green fluorescence was
observed and indicated that Nap-DO3A-Gd was successfully
taken up into various organs of the zebrafish. However, the
zebrafish incubated with Nap-DO3A-Gd (10 μM) and then
treated with Cu2+ displayed a very weak fluorescence signal,
indicating that copper ions in zebrafish were fluorescently
detected by Nap-DO3A-Gd. These experiments show that
Nap-DO3A-Gd can detect copper ions both in cultured cells
and in whole organisms. For all images, the microscope
settings, such as brightness, contrast, and exposure time, were
held constant to compare the relative intensity of intracellular
Cu2+ fluorescence.
Figure 7. Fluorescence and bright-field images of a 5-day-old zebrafish
incubated with Nap-DO3A-Gd: (a−c) fluorescence images of a
zebrafish incubated with Nap-DO3A-Gd (10 μM); (d−f) bright-field
images of a zebrafish from panels a−c; (g−i) fluorescence images of a
zebrafish incubated with Nap-DO3A-Gd (10 μM) and further
incubated with Cu2+ (10 μM); (j−l) bright-field images of a zebrafish
from panels g−i. Excited with blue light (400−460 nm).
CONCLUSIONS
■
In conclusion, we designed and synthesized a dual-modality
MRI and fluorescent copper-specific probe in aqueous solution.
Nap-DO3A-Gd exhibited a turn-on manner of relaxivity
changes and a fluorescent quenching toward Cu2+. It provides
the possibility of its application as the dual-modality probe in
biological systems. Through the introduction of bulky
naphthalimide into the Gd3+ contrast agent platform to restrict
the coordination ability of the DPA chelator, the relaxivity
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Zebrafish incubation and imaging zebrafish were maintained at 28
°C. For mating, male and female zebrafish were maintained in one
tank on a 12 h light/12 h dark cycle. Egg spawning was triggered by
light stimulation in the morning. Almost all eggs were fertilized
immediately. Five-day-old zebrafish were maintained in an E3 embryo
medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2,
0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, and 10−5%
methylene blue; pH 7.5). The 5-day-old zebrafish were incubated in an
E3 medium containing 10 μM Nap-DO3A-Gd for 30 min at room
temperature. After washing three times with PBS to remove the
remaining Nap-DO3A-Gd in solution, the zebrafish were further
incubated in solution containing 10 μM Cu2+ for 30 min at room
temperature. These zebrafish were imaged by fluorescence microscopy.
Synthesis of Compound 1. To a solution of 4-bromoanhydride
naphthalene (5 g, 18.2 mmol) in ethylene glycol monomethyl ether
was added morpholine (4.74 g, 54.3 mmol). After 4 h under reflux
conditions, the reaction was cooled to room temperature and yellow
needle crystals were filtered. Crude product (4-morpholinoanhydride
naphthalene) was used directly without further purification. To a
solution of ethylenediamine (4.2 g, 70 mmol) in ethanol was added 4-
morpholinoanhydride naphthalene (2 g, 7 mmol), and the resulting
mixture was refluxed for 2 h. The reaction was cooled to room
temperature and concentrated via rotary evaporation. The crude
product was purified on a silica gel column, eluting with 10% methanol
in dichloromethane. Compound 1 was collected in 60% yield as an
orange solid. Rf = 0.5 [SiO2; 9:1 (v/v) dichloride/methanol]. 1H NMR
(400 MHz, DMSO-d6): δ 8.32−8.49 (m, 3H, ArH), 7.80 (t, 1H, J =
8.0 Hz, ArH), 7.34 (d, 1H, J = 8.0 Hz, ArH), 4.04 (t, 2H, J = 8.0 Hz,
−CH2CH2−), 3.91 (t, 4H, J = 4.0 Hz, −CH2CH2−), 2.50−3.21 (m,
8H, 4H for −CH2CH2, −2H for −CH2CH2−, 2H for −NH2). 13C
NMR (100 MHz, DMSO-d6): δ 164.2, 163.7, 155.8, 132.5, 131.0,
130.9, 129.6, 126.5, 125.7, 123.2, 116.6, 115.5, 79.6, 66.7, 53.5, 43.1.
TOF-ESI-MS. Calcd for [C18H19N3O3 + H]+: m/z 326.1505. Found:
m/z 326.1508.
changes of Nap-DO3A-Gd showed excellent selectivity toward
Cu2+ over other metal ions, especially for that of Zn2+. Also,
with the Gd3+ coordinated to the DPA moiety to turn away the
interferences of other metal cations from Cu2+ detection, the
luminescence responses were Cu2+-specific.
EXPERIMENTAL SECTION
■
Instruments and Reagents. All reagents and solvents were of
analytical reagent grade and were used without further purification. 2-
Pyridinecarboxaldehyde, 1,4,7,10-tetraazacyclododecane(cyclen), and
sodium triacetoxyborohydride were purchased from Sigma-Aldrich.
Metal salts were provided by Shanghai Fourth Chemical Reagent
Company (China). Thin-layer chromatography was performed on
silica gel 60F-254 glass plates. Reaction products were purified by flash
chromatography using silica gel. Reversed-phase chromatography was
performed by Dalian Zhonghuida Scientific Instrument Company
1
using C18 columns. H and 13C NMR spectra were measured on a
Varian INOVA 400 M spectrometer. ESI-MS analyses were carried out
on a HPLC-Q-Tof MS spectrometer using methanol as the mobile
phase. Fluorescence spectra of solutions were measured on an
Edinburgh FS920 spectrometer. ICP-OES was performed on a Perkin-
Elmer Optima 2000 DV ICP-OES. NMR imaging was performed on
an NMR NMI20-Analyst Analyzing & Imaging system (Shanghai
Niumag Corp.) using a 0.5 T magnet, point resolution = 256 × 128
mm, section thickness = 2 mm, TE = 23 ms, TR = 500 ms, and
number of acquisitions = 4.
General Procedure for Detecting Spectra. Stock solutions
(10−3 M) of Nap-DO3A-Gd were prepared in water (pH 7.0). Stock
solutions (10−2 M) of nitrate salts of various metal ions of Na+, Ca2+,
Mg2+, Cu2+, Zn2+, Cd2+, Al3+, Fe3+, Cr3+, Ba2+, Ag+, and Ni2+ and sulfate
salts of Fe2+ were prepared in water for spectrometric analysis. Cu2+
fluorescence was titrated at an excitation wavelength of 400 nm. Before
spectroscopic measurements, solutions were freshly prepared by
diluting the corresponding high-concentration stock solution. For
each spectrum, 2 mL of a probe solution was added to a 1-cm quartz
cell, to which different stock solutions of cations were gradually added
using a microsyringe. The added volume of a cationic stock solution
was always less than 100 μL to minimize changes in the total volume
of the testing solution. Fluorescence quantum yield was determined
using rhodamine B (Φf = 0.69 in ethanol) as the standard.42 All
spectroscopic measurements were performed at least in triplicate and
averaged. Also, the quantum yield was calculated using equation
Synthesis of Compound 2. Compound 1 (1 g, 3 mmol) was
dissolved in ethanol with 2-pyridinecarboxaldehyde (0.39 g, 3.6
mmol). The reaction was stirred and refluxed for 6 h. The yellow solid
was precipitated, and the reaction was cooled to room temperature
and filtered. Compound 3 was obtained as a yellow powder. Yield:
1
1.26 g, 83%. Rf = 0.3 [SiO2; 40:1 (v/v) dichloride/methanol]. H
NMR (400 MHz, CDCl3): δ 8.60 (m, 2H, 1H for −NCH−, 1H for
ArH), 8.54 (d, 1H, J = 8.0 Hz, ArH), 8.42 (m, 2H, ArH), 7.99 (d, 1H, J
= 8.0 Hz, ArH), 7.21 (m, 2H, ArH), 7.26−7.30 (m, 2H, ArH), 4.58 (t,
2H, J = 8.0 Hz, −CH2CH2−), 4.01−4.05 (m, 6H), 3.27 (t, 4H, J = 4.0
Hz, −CH2CH2−). 13C NMR (100 MHz, CDCl3): δ 164.4, 163.9,
163.3, 155.7, 154.5, 149.3, 136.6, 132.6, 130.2, 130.1, 129.9, 126.1,
125.8, 124.7, 123.2, 121.4, 117.1, 114.9, 67.0, 58.6, 53.4, 40.4. TOF-
ESI-MS. Calcd for [C24H22N4O3 + H]+: m/z 415.1770. Found: m/z
415.1785.
2
⎛
⎝
⎞
⎠
η
η
(I /A
unk unk
)
unk
⎜
⎜
⎟
⎟
Φ
= Φ
std
unk
(I /A
)
std std
std
where Φunk and Φstd are the radiative quantum yields of the sample
and standard, Iunk and Istd are the integrated emission intensities of the
corrected spectra for the sample and standard, Aunk and Astd are the
absorbances of the sample and standard at the excitation wavelength,
and ηunk and ηstd are the indices of refraction of the sample and
standard solutions, respectively. Excitation and emission slit widths
were modified to adjust the luminescent intensity in a suitable range.
Fluorescence Microscopy Imaging in Cultured Cells and
Zebrafish. Fluorescence images were observed under a Nikon eclipse
TE2000-5 inverted fluorescence microscope with a 20× objective lens
excited with blue light (400−460 nm). For all images, the microscope
settings, such as brightness, contrast, and exposure time, were held
constant to compare the relative intensities of intracellular Cu2+
fluorescence.
HeLa cell incubation and imaging HeLa cells were cultured at 37 °C
in a 1640 medium supplemented with 10% FCS (Invitrogen). Cells
were seeded in 24-well flat-bottomed plates. After 12 h, HeLa cells
were incubated with 10 μM Nap-DO3A-Gd (in a culture medium) for
30 min at room temperature, washed three times with PBS, incubated
with 10 μM Cu2+ for another 30 min, and rinsed three times with PBS.
These HeLa cells were subjected to fluorescence imaging of
intracellular Cu2+.
Synthesis of Compound 3. Sodium triacetoxyborohydride (1.5 g,
7.2 mmol) was added to a solution of compound 2 (1 g, 2.4 mmol) in
methanol and refluxed for 2 h. Then the reaction mixture was cooled
to room temperature. After concentration under reduced pressure, the
mixture was poured into a KOH solution and extracted with
dichloromethane, dried over MgSO4, and evaporated under reduced
pressure. The crude product 3 was purified on flash silica gel using
dichloromethane/methanol (40:1) as the eluent. Yield: 0.8 g, 80%. Rf
1
= 0.5 [SiO2; 40:1 (v/v) dichloride/methanol]. H NMR (400 MHz,
CDCl3): δ 8.59 (d, 1H, J = 8.0 Hz, ArH), 8.53 (d, 1H, J = 8.0 Hz,
ArH), 8.49 (d, 1H, J = 8.0 Hz, ArH), 8.42 (d, 1H, J = 8.0 Hz, ArH),
7.71 (d, 1H, J = 8.0 Hz, ArH), 7.58 (d, 1H, J = 8.0 Hz, ArH), 7.30 (d,
1H, J = 8.0 Hz, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH), 7.11 (t, 1H, J =
8.0 Hz, ArH), 4.39 (t, 2H, J = 6.0 Hz, −CH2CH2−), 4.00−4.03 (m,
6H, 4H for −CH2CH2−, 2H for −CH2−), 3.27 (t, 4H, J = 4.0 Hz,
−CH2CH2−), 3.06 (t, 2H, J = 6.0 Hz, −CH2CH2−). 13C NMR (100
MHz, CDCl3): δ 164.6, 164.2, 159.6, 155.7, 149.3, 136.4, 132.6, 131.3,
130.1, 130.0, 126.2, 125.8, 123.3, 122.3, 121.9, 117.2, 115.0, 67.0, 54.8,
53.5, 47.4, 39.8. TOF-ESI-MS. Calcd for [C24H24N4O3 + H]+: m/z
417.1927. Found: m/z 417.1941.
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Inorganic Chemistry
Article
Synthesis of Compound 4. 2,6-Bis(chloromethyl)pyridine was
synthesized as described.45 Benzoyl peroxide (1.25 g, 4.65 mmol) was
added in batches to a solution of 2,6-dimethylpyridine (10 g, 0.093
mol) and N-chlorosuccinimide (24.8 g, 0.186 mol) in carbon
tetrachloride. The reaction was refluxed for 24 h until the raw
material was consumed completely. The mixture was cooled to room
temperature and filtered. The solvent was evaporated under reduced
pressure. The crude product was purified on flash silica gel using
CH2Cl2/petroleum ether (1:1, v/v) as the eluent to yield a white solid
[2,6- bis(chloromethyl)pyridine]. 1H NMR (400 MHz, CDCl3): δ
7.77 (t, 1H, J = 7.6 Hz, ArH), 7.44 (d, 2H, J = 7.6 Hz, ArH), 4.67 (s,
4H, −CH2−). 13C NMR (100 MHz, CDCl3): δ 156.4, 138.2, 122.1,
46.4. The solution of compound 3 (600 mg, 1.44 mmol) in acetonitrile
was slowly added to a solution of 6-bis(chloromethyl)pyridine (757
mg, 4.32 mmol) and potassium carbonate (796 mg, 5.76 mmol) in
acetonitrile. The reaction mixture was stirred and refluxed for 8 h.
After the solvent was evaporated under reduced pressure, the crude
product was purified by silica gel column chromatography using
CH2Cl2/MeOH (50:1, v/v) as the eluent to afford a yellow solid.
Yield: 480 mg, 60%. Rf = 0.50 [SiO2; 50/1 (v/v) dichloride/
(m, 26H). TOF-ESI-MS. Calcd for [C45H55N9O9 + H]+: m/z
866.4201. Found: m/z 866.4203.
Synthesis of Nap-DO3A-Gd. Gd(NO3)3·6H2O (32 mg, 0.086
mmol) and compound 6 (50 mg, 0.057 mmol) were combined in a
solution of methanol and water (1:1, v/v). The pH was adjusted to 6−
7 with added portions of dilute aqueous NaOH, and the reaction was
stirred at room temperature overnight, maintaining the solution at pH
6−7 with added portions of NaOH. The solvent was removed by
rotary evaporation, and the crude product was purified by reversed-
phase C18 chromatography, eluting with MeOH/H2O to yield a yellow
solid (30 mg, 51%). The purity and identity of the collected fractions
were confirmed by analytical HPLC−MS. The Gd content was
measured using ICP-OES. TOF-ESI-MS. Calcd for [M + H+]: m/z
1021.3. Found: m/z 1021.3. The appropriate isotope distribution was
observed.
Synthesis of Nap-DO3A-Tb. Nap-DO3A-Tb was synthesized in a
manner analogous to that of Nap-DO3A-Gd using Tb(NO3)3·6H2O.
The purity and identity of the collected fractions were confirmed by
analytical HPLC−MS. TOF-ESI-MS. Calcd for [M + H+]: m/z
1022.3. Found: m/z 1022.2. Calcd for [M + Na+]: m/z 1044.3. Found:
m/z 1044.2. The appropriate isotope distribution was observed.
1
methanol]. H NMR (400 MHz, CDCl3): δ 8.52 (d, 1H, J = 8.0 Hz,
ArH), 8.41−8.51 (m, 3H, ArH), 7.72 (d, 1H, J = 8.0 Hz, ArH), 7.38
(d, 1H, J = 8.0 Hz, ArH), 7.29−7.31 (m, 3H, ArH), 7.24 (d, 1H, J =
8.0 Hz, ArH), 7.18 (d, 1H, J = 4.0 Hz, ArH), 7.01 (d, 1H, J = 4.0 Hz,
ArH), 4.55 (s, 2H, −CH2−), 4.40 (t, 2H, J = 6.0 Hz, −CH2CH2−),
4.04 (t, 4H, J = 6.0 Hz, −CH2CH2−), 3.93 (s, 2H, −CH2−), 3.90 (s,
2H, −CH2−), 3.30 (t, 4H, J = 6.0 Hz, −CH2CH2−), 2.94 (t, 2H, J =
6.0 Hz, −CH2CH2−). 13C NMR (100 MHz, CDCl3): δ 164.2, 163.7,
159.7, 155.6, 148.8, 137.0, 136.1, 132.5, 131.1, 130.0, 129.9, 126.1,
125.9, 123.4, 122.8, 122.0, 121.7, 120.6, 117.3, 115.0, 67.0, 60.4, 60.1,
53.5, 51.8, 46.7, 37.9. TOF-ESI-MS. Calcd for [C31H30ClN5O3 + H]+:
m/z 556.2115. Found: m/z 556.2123.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: cyduan@dlut.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the NSFC (Grants 91122031 and
21102014). We also thank Shanghai Niumag Corp. for help
with measurement of the relaxivity and MRI experiments.
Synthesis of Compound 5. Tri-tert-butyl ester of 1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid (DO3A) was synthesized as
described.46 To the solution of cyclen (1 g, 5.8 mmol) in acetonitrile
was slowly added dry sodium bicarbonate (1.95 g, 4.0 equiv) and
bromoacetic acid/tert-butyl ester (3.74 g, 19.1 mmol, 3.3 equiv) in an
ice bath. Then the mixture was stirred for 18 h and filtered. The filtrate
was evaporated to dryness. The crude product was recrystallized from
toluene. Compound 4 (400 mg, 0.72 mmol) and tri-tert-butyl ester of
DO3A (370 mg, 0.72 mmol) were dissolved in acetonitrile (50 mL)
under argon. An excess of NaHCO3 (242 mg, 2.88 mmol) was added
to the solution. The mixture was refluxed for 8 h and filtered, and the
solvent was evaporated. The residue was purified using silica gel
column chromatography, eluting with dichloromethane/methanol
(40:1) to give 5 as a pale-yellow solid. Yield: 360 mg, 48%. Rf =
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