Picolinate-appended tacn complexes for bimodal imaging: Radiolabeling, relaxivity, photophysical and electrochemical studies

Article abstract of DOI:10.1016/j.jinorgbio.2019.110978

Based on our previous works involving two 1,4,7-triazacyclononane (tacn)-based ligands Hno2py1pa (1-Picolinic acid-4,7-bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane) and Hno1pa (1-Picolinic acid-1,4,7-triazacyclononane), we report here the synthesis of analogues bearing picolinate-based π-conjugated ILCT (Intra-Ligand Charge Transfer) transition antenna (HL1, HL2), using regiospecific N-functionalization of the tacn skeleton and their related transition metal complexes (e.g. Cu²⁺, Zn²⁺ and Mn²⁺). Coordination properties as well as their photophysical and electrochemical properties were investigated in order to quantify the impact of such antenna on the luminescent or relaxometric properties of the complexes. The spectroscopic properties of the targeted ligands and metal complexes have been studied using UV–Vis absorption and fluorescence spectrocopies. While the zinc complex formed with HL1 possesses a moderate quantum yield of 5%, complexation of Cu²⁺ led to an extinction of the luminescence putatively attributed to a photo-induced electron transfer, as supported by spectroscopic and electrochemical evidences. The [Mn(L2)]⁺ complex is characterized by a fluorescence quantum yield close to 8% in CH₂Cl₂. The potential interest of such systems as bimodal probes has been assessed from radiolabeling experiments conducted on HL1 and ⁶⁴Cu²⁺ as well as confocal microscopy analyses and from relaxometric studies carried out on the cationic [Mn(L2)]⁺ complex. These results showed that HL1 can be used for radiolabeling, with a radiochemical conversion of 40% in 15 min at 100 °C. Finally, the relaxivity values obtained for [Mn(L2)]⁺, r_1p = 4.80 mM^{? 1}·s^{? 1} and r_2p = 8.72 mM^{? 1}·s^{? 1}, make the Mn(II) complex an ideal candidate as a probe for Magnetic Resonance Imaging.

Full text of DOI:10.1016/j.jinorgbio.2019.110978

Journal Pre-proof
Picolinate-appended tacn complexes for bimodal imaging:
Radiolabeling, relaxivity, photophysical and electrochemical
studies
Amaury Guillou, Margaux Galland, Amandine Roux, Balázs
Váradi, Réka Anna Gogolák, Patricia Le Saëc, Alain Faivre-
Chauvet, Maryline Beyler, Christophe Bucher, Gyula Tircsó,
Véronique Patinec, Olivier Maury, Raphaël Tripier
PII:
S0162-0134(19)30597-5
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110978
JIB 110978
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
16 September 2019
11 December 2019
24 December 2019
Please cite this article as: A. Guillou, M. Galland, A. Roux, et al., Picolinate-appended
tacn complexes for bimodal imaging: Radiolabeling, relaxivity, photophysical and
electrochemical studies, Journal of Inorganic Biochemistry (2018), https://doi.org/
10.1016/j.jinorgbio.2019.110978
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Picolinate-Appended Tacn Complexes for Bimodal Imaging:
radiolabeling, relaxivity, photophysical and electrochemical studies
Amaury Guillou,a Margaux Galland,b Amandine Roux,b Balázs Váradi,c Réka Anna Gogolák,c
Patricia Le Saëc,d Alain Faivre-Chauvet,d Maryline Beyler,a Christophe Bucher,b Gyula
Tircsó,c Véronique Patinec,*,a Olivier Maury*,b, and Raphaël Tripier*,a
a
Univ Brest, UMR-CNRS 6521 CEMCA,
6 avenue Victor le Gorgeu, 29200 Brest, France.
raphael.tripier@univ-brest.fr
b
Univ Lyon, ENS Lyon, CNRS, Université Lyon 1, Laboratoire de Chimie, UMR 5182, 46 allée d’Italie, 69364
Lyon, France. Olivier.Maury@ens-lyon.fr
c
Department of Physical Chemistry, Faculty of Science and Technology, University of Debrecen, Egyetem tér 1, H-
4032 Debrecen, Hungary.
d
Université de Nantes, Centre de Recherche en Cancérologie et Immunologie Nantes Angers (CRCINA), Unité
INSERM 1232 – CNRS 6299, 8 quai Moncousu, BP 70721, 44007 Nantes Cedex, France.
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ABSTRACT
Based on our previous works involving two 1,4,7-triazacyclononane (tacn)-based ligands
Hno2py1pa (1-Picolinic acid-4,7-bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane) and
Hno1pa (1-Picolinic acid-1,4,7-triazacyclononane), we report here the synthesis of analogues
bearing picolinate-based π-conjugated ILCT (Intra-Ligand Charge Transfer) transition
antenna (HL1, HL2), using regiospecific N-functionalization of the tacn skeleton and their
related transition metal complexes (e.g. Cu2+, Zn2+and Mn2+). Coordination properties as well
as their photophysical and electrochemical properties were investigated in order to quantify
the impact of such antenna on the luminescent or relaxometric properties of the complexes.
The spectroscopic properties of the targeted ligands and metal complexes have been studied
using UV-Vis absorption and fluorescence spectrocopies. While the zinc complex formed
with HL1 possesses a moderate quantum yield of 5%, complexation of Cu2+ led to an
extinction of the luminescence putatively attributed to a photo-induced electron transfer, as
supported by spectroscopic and electrochemical evidences. The [Mn(L2)]+ complex is
characterized by a fluorescence quantum yield close to 8% in CH2Cl2. The potential interest
of such systems as bimodal probes has been assessed from radiolabeling experiments
conducted on HL1 and 64Cu2+ as well as confocal microscopy analyses and from relaxometric
studies carried out on the cationic [Mn(L2)]+ complex. These results showed that HL1 can be
used for radiolabeling, with a radiochemical conversion of 40% in 15 minutes at 100°C.
Finally, the relaxivity values obtained for [Mn(L2)]+ r1p = 4.80 mM-1.s-1 and r2p = 8.72 mM-
,
1.s-1, make the Mn(II) complex an ideal candidate as a probe for Magnetic Resonance
Imaging.
1. Introduction
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Current molecular imaging and therapy techniques used in medicine, such as Positron
Emission Tomography (PET), Optical Imaging (OI), Magnetic Resonance Imaging (MRI) or
RadioImmunoTherapy (RIT) exploit the intrinsic radioactive, optical or magnetic properties
of transition metals (i.e. 52Mn2+, 64Cu2+, 68Ga3+, 89Zr4+, etc…), lanthanides (i.e. Gd3+, Eu3+,
Yb3+) or organic fluorophores to produce visualization techniques for diagnosis or direct
therapy of disease, especially for cancer 1,2. Each technique presents its own advantages
and drawbacks. For example, the main limitations of fluorescence imaging is the lack of depth
penetration of the incident or emitted light while PET presents a low resolution and MRI lacks
sensitivity. PET, MRI and OI thus stand as fully complementary techniques that could be
advantageously combined to achieve more accurate diagnosis 3. Such combinations can be
realized through the development of multimodal probes involving coordination complexes as
key functional /building elements [4,5]. Beyond multimodality, the selected metal-containing
probes to be used in biological media must comply with essential stability and solubility
standards.
Given the growing importance of bimodal imaging, the design of functional
polyazamacrocyclic ligands has recently become a very active field of research [6-9]. Despite
being the smallest saturated polyazamacrocycle, the tacn (1,4,7-triazacyclononane)
macrocycle has proved to be capable, when appropriately N-functionalized, to stabilize a wide
range of metal ions starting from the smallest transition metal ions to the largest lanthanides
10-12. In line with this strategy, some of us have recently developed a series of N-
functionalized tacn chelators bearing picolinate pendants (Hno2py1pa and Hno1pa, Figure 1)
used, after complexation with Cu2+ or Mn2+, as PET 13 or MR 14 imaging agents. Indeed,
Cu2+ is widely used in nuclear medicine due to the availability of 64Cu isotope (+ emitter, t1/2
= 12.7 h) suitable for PET imaging. On the other hand, high spin (S= 5/2) Mn2+ complexes
have been considered as viable alternatives to the gadolinium (III) complexes currently used
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in MRI 15. In order to further implement an optical imaging modality to these scaffolds, we
envisaged to functionalize the picolinate pendant arm by suitable electron rich -conjugated
chromophore selected for their ability to act as effective antennas for lanthanide sensitization
under one or two-photon excitation (Figure 1) [16,17].
[Figure 1 here]
In this paper, we present the synthesis and detailed characterizations of two original ligands
HL1, HL2 and of their corresponding metal complexes with Cu2+, Zn2+ and Mn2+ (including
radiolabeling with 64Cu2+). Their photophysical, electrochemical and relaxivity properties
have been thoroughly investigated to evaluate their potential as bimodal probes.
2. Experimental
Reagents were purchased from ACROS Organics and from Aldrich Chemical Co. Reagents
used in radiolabeling process are « trace select » grade. 1,4,7-triazacyclononane (tacn) was
purchased from CheMatech (Dijon, France). Acetonitrile, THF and water solvents were
distilled before use. HRMAs analyses were performed at ICOA, Orléans, France. NMR
spectra were recorded at the “Services communs” of the University of Brest. 1H and 13C NMR
spectra were recorded with Bruker Avance 500 (500 MHz), Bruker Avance 400 (400 MHz),
or Bruker AMX-3 300 (300 MHz) spectrometers. Dialysis purification were performed using
Float-A-Lyzer® dialysis device (MWCO = 100-500 D). Briefly, the membrane was activated
using a 20% iPrOH in H2O (>18.2 MΩ·cm at 25 ºC) and then washed 3 times with water. The
sample was loaded and the device was put in 500 mL of H2O and stirred for 4 h, the process
was repeated twice. The synthesis of the functionalized picolinate pendant a (Scheme 1),
featuring dialkylamino phenylethynyl conjugated skeleton, was achieved through a
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modification of a previously published procedure (see in Supporting Informations, Text S1
and Figures S1-S6) 18,19. This key fragment was then further “decorated” by oligo-ethylene
glycol substituents to improve the solubility of the resulting complexes in biological media.
All the spectra corresponding to HL1 and HL2 characterization are in Supporting
Informations, Figures S7-S15.
2.1. Synthesis of the ligands
Ligand HL1. A solution of the mesylated antenna a (Methyl 4-[4-ethynyl-N,N-bis(3,6,9-
trioxodecyl)aniline]-6-[(methylsulfonyloxy)methyl]-2-picolinate) (600 mg, 0.92 mmol)
(Scheme 1) in acetonitrile (30 mL) was added dropwise to a solution of no2py (4,7-
bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane) (288 mg, 0.92 mmol). The solution was
stirred at room temperature for 4 days, filtered and the solvent evaporated under reduced
pressure. The crude product was purified by chromatography on neutral Alumina (CHCl3 to
CHCl3/MeOH 85/15). The purified compound was then dialyzed to yield 1-{4-[4-ethynyl-
N,N-bis(3,6,9-trioxodecyl)aniline]-2-methoxycarbonyl}pyridyn-2-ylmethyl-4,7-bis(pyridin-2-
ylmethyl)-1,4,7-triazacyclononane (compound 1) as a yellow oil (439 mg, 55 %).
1H NMR (CDCl3, 300 MHz, 298 K) δ (ppm) 2.85-2.90 (m, 11H, CH2 tacn) 3.34 (s, 6H, CH3)
3.51-3.61 (m, 24H, CH2) 3.83 (s, 4H, CH2) 3.90 (s, 2H, CH2) 3.95 (s, 3H, CH3) 6.63 (d, 2H,
3J= 9 Hz) 7.07 (t, 2H, 3J= 5 Hz) 7.34 (d, 2H, 3J= 9 Hz) 7.49 (d, 2H, 3J= 8 Hz) 7.59 (t, 2H, 3J=
3
3 Hz) 7.78 (s, 1H) 8.00 (s, 1H) 8.46 (d, 2H, J= 4 Hz). 13C NMR (CDCl3, 75 MHz, 298 K) δ
(ppm) 50.6 (CH2 N) 52.7 (CH3) 55.3, 55.6 (CH2 tacn) 58.8 (CH3) 63.9 (CH2) 64.4 (CH2)
68.1, 70.3, 70.4, 70.5, 71.7 (CH2) 85.0 (Cqt) 96.7 (Cqt) 107.8 (Cqt) 111.2 (CH) 121.7 (CH)
123.1 (CH) 125.0 (CH) 127.1 (CH) 133.2 (CH) 133.6 (Cqt) 136.1 (CH) 147.0 (Cqt) 148.4
(Cqt) 148.6 (Cqt) 165.4 (C=O). ESI-HRMS m/z calcd. for [C48H66N7O8+H]+ 868.4967,
found 868.4962.
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The intermediate 1 (100 mg, 0.11 mmol) was then dissolved in THF (4 mL) and 1M KOH(aq)
(2 mL) was added. The mixture was stirred at 55°C for 3 h and concentrated under reduced
pressure. The crude was dissolved in H2O (3 mL) and the pH was adjusted to 3 with HCl 1M.
The
product
was
dialyzed
yielding
1-{2-carboxy-4-[4-ethynyl-N,N-bis(3,6,9-
trioxodecyl)aniline]}pyridyn-2-ylmethyl-4,7-bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane
HL1 as a yellow oil (97 mg, 99%).
1H NMR (D2O, 500 MHz, 298 K) δ (ppm) 2.73 – 2.95 (m, 8H, CH2 tacn) 3.26 – 3.30 (m, 9H,
CH3 PEG) 3.45 – 3.51 (m, 28H, CH2 tacn + CH2 PEG) 3.79 (s, 2H) 3.88 (s, 2H) 6.61 (m, 2H)
7.15 – 7.17 (m, 3H) 7.19 – 7.25 (m, 2H) 7.35 (m, 2H) 7.73 (m, 1H) 7.80 (m, 1H) 8.41 (s, 1H).
13C NMR (D2O, 126 MHz, 298 K) δ (ppm) 51.5, 51.9 (CH2 tacn) 52.8 (CH2 N) 53.8 (CH2
tacn) 60.9 (CH3) 62.9 (CH2) 64.5 (CH2) 65.3 (CH2) 70.6, 71.2, 72.3, 72.5, 72.8, 73.9, 74.9
(CH2) 88.8 (Cqt) 99.9 (Cqt) 110.1 (Cqt) 114.8 (CH) 125.7 (CH) 126.6 (CH) 127.3 (CH) 128.9
(CH) 136.1 (Cqt) 136.4 (CH) 140.8 (CH) 151.7 (CH) 156.5 (Cqt) 156.8 (Cqt) 160.7 (Cqt)
173.8 (C=O). ESI-HRMS m/z calcd. for [C47H65N7O8+H]2+ 427.7441, found 427.7446.
Ligand HL2. To a solution of 1,4,7-triazacyclononane (450 mg, 3.48 mmol) in distilled
ethanol (70 mL) containing molecular sieves was added benzaldehyde (369 mg, 3.48 mmol).
The reaction mixture was stirred for 4 h, then filtered through a pad of Celite® and the solvent
was removed under reduced pressure, yielding 10-phenyl-1,4,7-triazabicyclo[5.2.1]decane 2
as a white solid (750 mg, 95%).
1H NMR (CDCl3, 300 MHz, 298 K) δ (ppm) 2.89-2.93 (4H, m, CH2 tacn); 2.99-3.03 (2H, m,
CH2 tacn); 3.07-3.17 (4H, m, CH2 tacn); 3.32-3.39 (2H, m, CH2 tacn); 5.66 (1H, s, CH
3
3
3
aminal); 7.18 (1H, t, J= 7.3 Hz, CHAr); 7.29 (2H, t, J= 7.3 Hz, CHAr); 7.50 (2H, d, J= 7.3
Hz, CHAr). 13C NMR (CDCl3, 75 MHz, 298 K) δ (ppm) 49.3, 49.6, 58.8 (CH2 tacn) 88.3 (CH)
126.6, 126.7, 128.2 (CHAr) 145.8 (Cqt).
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Compound 2 (400 mg, 1.84 mmol) was dissolved in acetonitrile (100 mL) and K2CO3 (508
mg, 3.68 mmol) was added before addition of a solution containing a (1.2 g,
1.84 mmol) (Scheme 1) in acetonitrile (50 mL) over a period of 30 min. The reaction was
stirred at room temperature for 2 days. Filtration through Celite® and evaporation of the
solvent gave a crude yellow oil that was purified by column chromatography (Al2O3,
CH2Cl2 100% to CH2Cl2 / MeOH / isopropylamine 90: 7: 3) to yield deprotected compound
1-{4-[4-ethynyl-N,N-bis(3,6,9-trioxodecyl)aniline]-2-methoxycarbonyl}pyridyn-2-ylmethyl-
1,4,7-triazacyclononane 3 as a yellow oil (781 mg, 62 % over two steps).
1H NMR (CDCl3, 300 MHz, 298 K) δ (ppm) 2.74 (s, 8H, CH2 tacn) 2.85 (s, 4H, CH2 tacn)
3.00 (s, 2H, NH) 3.35 (s, 6H, CH3) 3.51 – 3.62 (m, 24H, CH2) 3.96 (s, 2H, CH2) 3.98 (s, 3H,
3
3
CH3) 6.94 - 6.98 (d, J = 8.9 Hz, 2H, CH) 7.35 - 7.38 (s, J = 8.8 Hz, 2H, CH) 7.61 (s, 1H,
CH) 8.01 (s, 1H, CH). 13C NMR (CDCl3, 75 MHz, 298 K) δ (ppm) 46.7, 47.1, 50.9 (CH2 tacn)
53.1 (CH3) 53.2 (CH2) 59.1 (CH3) 61.7 (CH2) 68.4, 70.7, 70.7, 70.8, 72.0 (CH2 PEG) 85.1
(Cqt) 97.4 (Cqt) 108.1 (Cqt) 111.5 (CH) 125.5 (CH) 127.1 (CH) 133.6 (CH) 134.2 (Cqt)
147.5 (Cqt) 148.7 (Cqt) 161.0 (Cqt) 165.6 (C=O). ESI-HRMS m/z calcd. for [C36H56N5O8]+
686.4123, found 686.4124.
Finally, compound 3 (200 mg, 0.29 mmol) was dissolved in THF and 1M KOH
(2 mL) was added. The mixture was stirred at 50°C for 3 h and then concentrated under
reduced pressure. The crude product was purified by semi-preparative HPLC yielding the
final
ligand
1-{2-carboxy-4-[4-ethynyl-N,N-bis(3,6,9-trioxodecyl)aniline]}pyridyn-2-
ylmethyl-1,4,7-triazacyclononane HL2 as a yellow oil (188 mg, 97%).
1H NMR (D2O, 300 MHz, 298 K) δ (ppm) 3.12 (m, 4H), 3.25-3.34 (m, 12H) 3.47-3.58 (m,
19H) 3.68-3.78 (m, 13H) 4.18 (s, 2H, CH2 αN) 7.07-7.10 (d, 3J = 9 Hz, 2H) 7.48 (s, 1H) 7.97-
8.00 (d, 3J = 9 Hz, 3H). 13C NMR (D2O, 75 MHz, 298 K) δ (ppm) 46.1, 47.5, 51.6, 54.7, 60.0
(CH2) 60.8 (CH) 69.7, 72.3, 72.7, 73.8 (CH2 PEG) 117.3 (CHAr) 121.1 (Cqt) 129.0 (CHAr)
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129.3 (Cqt) 131.0 (CHAr) 134.1 (CHAr) 149.4, 151.5, 152.8, 162.2 (Cqt) 198.0 (C=O). ESI-
HRMS m/z calcd. for [C35H54N5O8]+ 672.3966, found 672.3966.
2.2. Synthesis and characterisation of the complexes
General procedure for the synthesis of the [MIIL1] complexes (MII = Cu2+/Zn2+)
Ligand HL1 (10 mg, 0.01 mmol) was dissolved in ultra-pure deionized H2O (5 mL), and the
pH of the solution was adjusted to pH = 5. Then M(ClO4)2·6H2O (0.01 mmol) was added and
the pH was adjusted to 6.6. The mixture was stirred for 24 h at 60°C. The resulting crudes
were purified by semi-preparative HPLC (see in Supporting Informations, Figures S16-S19)
and lyophilized.
[Cu(L1)](ClO4): yellow oil (10 mg, 94%). ESI-HRMS m/z calcd. for [C47H63N7O8Cu]2+
458.2011, found 458.2015.
[Zn(L1)](ClO4): yellow oil (9.8 mg, 90%). ESI-HRMS m/z calcd. for [C47H63N7O8Zn]2+
459.7014, found 459.7009.
Synthesis of [MnL2]+ complex
A procedure similar to the one used for HL1 complexes has been followed starting from HL2
(0.01 mmol) and MnCl2·4H2O (0.01 mmol) to lead to [Mn(L2)](Cl) complex as a yellow oil
(6.8 mg, 60%). Our attempts to characterize the Mn2+ complex via HR-MS unfortunately
failed although the HPLC obtained confirmed its formation /existence and purity (see in
Supporting Informations, Figure S20). Additionally, as explained below, the pH-
potentiometic and relaxation experiments also confirmed its formation.
2.3. Radiolabeling with 64Cu
64CuCl2 in 0.1 M HCl was added in a molar ratio 1:1 equivalent into 0.1 M acetate pH = 5.5
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buffered ligand solution. The reaction mixture was incubated at 100°C for 15 min. Then the
radiolabeling was quenched through addition of a calculated volume of 1 mM EDTA pH = 5
to obtain a ratio EDTA-to-ligand of 10 and the mixture was stirred at room temperature for 10
min. 2 µL of this solution was taken out and deposited on a silica gel plate (Silica Gel
60F254, Merck). The thin layer chromatography was developed for 7 cm using a mixture of
ammonium chloride 20% in water:methanol (1:1/v:v). The plate was then removed from the
solvent and exposed for 5 min on a storage phosphor plate, and this was revealed using a
radiometric phosphor imager Cyclone® Plus (PerkinElmer) and analyzed with the
Optiquant® software to obtain a radiochromatogram.
2.4. r1p and r2p relaxivities of the Mn2+ complex
Relaxation times of water protons were measured at 60 MHz with a Bruker Minispec MQ-60
NMR Analyzer. The temperature of the sample holder was set (25.00.2°C) and controlled
with the use of a circulating water bath. The longitudinal relaxation times (T1) were measured
by using the inversion recovery method (180°–t–90°) by averaging 5-6 identical data points
obtained at 14 different t values. The transverse relaxation times (T2) were measured by using
the Carr-Purcell-Meiboom-Gill sequence (CPMG) sequence again by averaging 5-6 identical
data points.
The r1p and r2p relaxivities of the complex were determined by using batch samples which
were prepared under nitrogen atmosphere having the ligand present at a 1.5 mM concentration
(the pH in these samples was kept constant at pH = 8.12 with the use of HEPES buffer
o
(I = 0.15 M NaCl, 25 C)). Various amounts of MnCl2 were added to these solutions and the
relaxation times of the solutions were measured. By relying on the species distribution curves
under these conditions, only one Mn2+ ion containing species is present in solution which is
the [Mn(L2)] complex. The curve obtained by plotting 1/T1,2p as a function of Mn2+
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concentration for the samples with [L]>[Mn2+] gives a straight line, with a slope that is equal
to r1p and r2p relaxivities respectively. To confirm the complex formation and its pH range, a
sample containing 1 mM of the ligand and Mn2+ ion (Vtot= 10 mL) was titrated with NaOH
under N2 protected atmosphere and the 1/T1,2p data was collected in the pH range of 3.0-11.0.
The given data co-plotted with the species distribution curve calculated based on the stability
constants determined by pH-potentiometric method were in excellent agreement.
2.5.Potentiometric measurements of the Mn2+ complex
The protonation constants of the ligands were determined by pH-potentiometric titrations
carried out with a Metrohm 888 Titrando titration workstation using a Metrohm 6.0233.100
combined electrode. The titrated solutions (6.00 mL) were thermostated at 25C and the ionic
strength in the samples was set to 0.15 M NaCl. The samples were stirred and kept under inert
gas atmosphere (N2) to avoid the effect of CO2. For the pH-calibration of the electrode
standard buffers (KH-phthalate (pH=4.005) and borax (pH=9.177)) were used. The
calculation of [H+] from the measured pH values was performed with the use of the method
proposed by Irving et al. 20 by titrating a 0.01 M HCl solution (I=0.15 M NaCl) with a
standardized NaOH solution (from the data collected in the pH range of 1.75-2.2). The KMLOH
was calculating with equation (1).
[ML]
[MLOH][H ]
KMLOH

(1)
The differences between the measured and calculated pH values were used to obtain the [H+]
concentrations from the pH-data obtained in the titrations. The ion product of water was
determined from the same experiment in the pH range 11.2-11.9.
The concentration of the ligand stock solution was determined by pH-potentiometric titration
from the titration data obtained in the presence and absence of large Mn2+ excess. In the pH-
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potentiometric titrations of the ligand 186 data pairs were recorded in the pH range of 2.77-
11.85, while the sample containing the Mn2+ ion (one equivalent) was titrated in the pH range
of 3.46-11.86 when 126 data pairs were collected and fitted. The protonation and stability
constants were calculated from the titration data with the PSEQUAD program 21.
2.6. Spectroscopic experiments
Absorption spectra were recorded in diluted solution ca. 10-2 or 10-3 mM in CH2Cl2
(spectrophotometric grade) on a JASCO V-650 spectrophotometer. Emission and excitation
spectra were recorded using a Horiba-Jobin-Yvon Fluorolog-3 fluorimeter. The steady state
luminescence was excited by unpolarized light from a 450 W xenon continuous wave lamp
and detected at an angle of 90° for measurements of dilute solutions (10 mm quartz cuvette)
either by a Hamamatsu R928 or Peltier cooled R2658 photomultiplier tube. Luminescence
quantum yield Φ were measured in diluted solutions with an absorbance lower than 0.1 and
were determined using coumarin or quinine as references (Φr = 0.45 and 0.54 respectively).
2.7. Electrochemistry
The electrochemical studies were performed in a glovebox (Jacomex) (O < 1 ppm, H O < 1
2
2
ppm)
with
a
home-designed
3-electrode
cell
(WE:
glassy
carbon,
RE: Ag/AgCl/NaCl (3 M), CE: graphite rod). The potential of the cell was controlled by using
an AUTOLAB PGSTAT 302 (Ecochemie) potentiostat monitored by using a computer. The
glassy carbon electrode was carefully polished before each voltammetry experiment with a 1
μm alumina aqueous suspension and ultrasonically rinsed in water and then with acetone.
Exhaustive electrolysis was performed with a graphite rod working electrode. The ultrapure
(18 MΩ) deoxygenated water was used as received and kept under argon in the glovebox after
degassing. Sodium perchlorate (Sigma-Aldrich, 99.99%) was used as a background
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electrolyte in 0.1 M concentration without purification.
1. Results and Discussion
1.1. Synthesis of the ligands and complexes
The synthesis of 1-{2-carboxy-4-[4-ethynyl-N,N-bis(3,6,9-trioxodecyl)aniline]}pyridyn-2-
ylmethyl-4,7-bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane HL1 (Scheme 1) started from
the previously described 4,7-bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane no2py
compound 13, which reacted in dry acetonitrile with 1 equivalent of the mesylated antenna a
(Methyl
4-[4-ethynyl-N,N-bis(3,6,9-trioxodecyl)aniline]-6-[(methylsulfonyloxy)methyl]-2-
picolinate) to give intermediate 1, isolated with a 51% yield after purification by column
chromatography. Deprotection of the methyl ester group was then readily achieved in a
KOH/THF medium, the targeted product HL1 being eventually purified by dialysis to
eliminate the remaining salts. The preparation of 1-{2-carboxy-4-[4-ethynyl-N,N-bis(3,6,9-
trioxodecyl)aniline]}pyridyn-2-ylmethyl-1,4,7-triazacyclononane HL2 involved an alternative
approach to the one used for the synthesis of the parent Hno1pa ligand 14, mostly to avoid
the use of strongly acidic conditions hardly incompatible with the structure of fragment a. The
simultaneous
N-protection of two secondary amines available on the tacn skeleton was achieved with 1
equivalent of benzaldehyde at room temperature in anhydrous EtOH. The resulting aminal
product 2 was obtained without purification as a white solid with 97% yield. This protected
macrocycle then reacted with 1 equivalent of the mesylated antenna a. It should be mentioned
that the purification of the crude product on activated alumina led to the cleavage of the
aminal protection to produce the desired mono-N-functionalized tacn 1-{4-[4-ethynyl-N,N-
bis(3,6,9-trioxodecyl)aniline]-2-methoxycarbonyl}pyridyn-2-ylmethyl-1,4,7-
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triazacyclononane 3 with 62% yield (calculated for two steps; 1- N-alkylation; 2- deprotection
of the aminal-bridged phenyl group). Finally, hydrolysis of the methyl ester function was
carried out in 1 M KOH/THF at 55°C for 3 h. The targeted ligand HL2 was obtained as a
yellow oil with a 95% yield after purification, performed via semi-preparative HPLC. All
final ligands and reaction intermediates have been fully characterized by standard techniques
(see Supporting Information for details, Figure S7 to S15).
Finally, the targeted [Cu(L1)]+ and [Zn(L1)]+ complexes have been obtained after heating at
60°C for 24 h in a neutral aqueous mixture of HL1 with 1 molar equivalents of copper and
zinc perchlorate, respectively. After evaporation of the solvent followed by dialysis, both
complexes have been isolated as yellow oils with 94 and 90% yield, respectively. The HPLC
and HR-MS data collected of these complexes are provided in the ESI section (Supporting
Information for details, Figures S16 to S19). The complexation of HL2 with manganese (II)
was also attempted following a similar procedure and the desired complex was isolated in
60% yield. All our attempts to obtain consistent MS data failed, but experimental evidences
supporting the formation of manganese complex in solution have nevertheless been provided
by potentiometric titration and relaxation experiments, allowing to isolate the monometallic
complex [Mn(L2)]+. The analytical HPLC data are provided in Supporting Information
(Figure S20).
[Scheme 1 here]
1.2. Copper-64 radiolabeling
Radiolabeling studies have been conducted on freshly prepared ammonium acetate buffer (pH
64
5.5) solutions containing HL1 and CuCl2 (58 µM). After stirring the mixture for 15 min at
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100°C, the radiochemical conversion was monitored by radio-TLC. The resulting thin layer
radiochromatograms showing the measured radioactive emission on a storage phosphor
screen after 15 min, is depicted in Figure 2. In contrast to the quantitative radiolabeling
observed with the parent Hno2py1pa ligand, we found that the labeled complex [64Cu(L1)]+
is formed with a rather modest yield of about 40%. This result supports the conclusion that the
picolinate-based ligand is not best suited to chelate 64Cu and that further modifications of the
ligand will be needed to enhance the complexation.
[Figure 2 here]
1.3. Potentiometric and relaxometric measurements
Potentiometric experiments were performed in order to gain insights about the complexation
properties of HL2 (see titration curves in Supporting Information, Figure S21). We initially
assumed that the presence of the antenna should not affect the complexation ability of the
ligand and that similar binding constants should be obtained with HL2 and Hno1pa. The
acid-base and the complex formation constants were firstly determined in aqueous media and
the data obtained with HL2 were systematically compared to the values collected with the
parent Hno1pa ligand [14]. All acid-base and metal complexation studies were performed by
pH-potentiometric titrations in aqueous solution at 25.0 ± 0.1 °C and 0.15 ± 0.01 M NaCl.
The stepwise formation constants KiH calculated for HL2 are collected in Table 1 along with
those determined for Hno1pa, while overall (log βiH) species distribution diagrams calculated
are shown in Supporting Information (respectively Table S1 and Figure S22). With an overall
protonation constant (log β3) of 22.12, HL2 possesses nearly the same total basicity as
Hno1pa (log β3 = 21.12) [14]. Both ligands display three protonation constants and the first
one (K1 ≈ 11.4) can be assigned to the protonation of one of the secondary amines of the
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macrocycle. The second protonation (log K2 ≈ 7.46) occurs also at the secondary amine
nitrogen of the tacn skeleton. Finally, the third protonation constant (log K3 ≈ 3.25), attributed
to the protonation of the carboxylic function of the picolinate arm, was found to be
significantly larger than the one determined for Hno1pa.
[Table 1 here ]
The complexation of Mn(II) was evaluated from pH-potentiometric titration experiments
carried out in a sample containing a 1:1 metal-to-ligand ratio. Since complexation was not
quantitative at acidic pH, the stability constants associated to the formation of [MnL2]+ could
be assessed in the absence of competitor. The calculated equilibrium constants are reported in
Table 2 while the speciation distribution curves calculated with the use of these data are
shown in Figure 2.
[Table 2 here]
The formation constant determined for [MnL2]+ complex is slightly larger (Δlog KMnL = 0.28)
than that obtained in the same conditions with Hno1pa. A second formation constant of 11.62
was measured for the hydroxo complex [MnL2(OH)]. A detailed analysis of the species
distribution diagram indicates that the complexation of HL2 starts at pH > 4 and becomes
quantitative near to the physiological pH (pH=7.4) up to pH=10.
For a better comparison of the thermodynamic stability constant of the Mn(II) complex
formed with HL2 and values obtained in the literature for different Mn(II)-based contrasts
agents, the pM values (pM = −log [Mn+]) that take into account the different basicity of the
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ligands were also calculated (Table 2) using the conditions described by Tóth É et al. for
Mn(II) complexes ([L2] = [Mn(II)] = 10 µM at pH 7.4) [26]. The pMn value of 7.21 obtained
for HL2 is nearly identical to the one obtained for the complex of Hno1pa (ΔpMn = 0.13) but
it is still slightly lower than the value calculated for [Mn(PyC3a)] described by Caravan et
al. [15] and for the complex formed with the tri-N-acetate tacn ligand H3nota [24] . To
conclude, while our first attempts to confirm the formation of the [Mn(L2)]+ complex were
not successful, we were able to prove the formation of this complex by pH-potentiometry,
relaxometry and HPLC techniques. The introduction of an extended π-conjugated system at
the para position of the picolinate arm does not impact the coordination properties of the
ligand and it also leads to a thermodynamically stable Mn(II) complex possessing comparable
stability to other tacn based chelates cited before.
The relaxation properties of [Mn(L2)]+ have then been measured by low field NMR
relaxometry and then compared to data measured in the same conditions with the free ligand
HL2, with particular focus on the proton relaxivity, r1p, which refers to the relaxation
enhancement of water protons promoted by a 1 mM concentration of the Mn2+ cation. The r1p
values obtained for the Mn2+ complex [Mn(L2)]+ was recorded simultaneously with
potentiometric titration data collected during a titration experiment. The relative relaxivity
data and the molar fraction obtained by potentiometric measurements are plotted together as a
function of pH on the graph depicted in Figure 3.
[Figure 3 here]
The r1p value obtained for the [Mn(L2)]+ complex at 60 MHz, 298 K and pH = 8.15 is
4.80 mM-1.s-1. This value is in agreement with the presence of one water molecule in the inner
sphere of the Mn2+ center, which was already described for the parent complex formed with
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Hno1pa 14. The relaxivity of [Mn(L2)]+ is found to be much larger than those reported in
literature for other Mn2+ contrast agents, including several ones concerning the FDA-
approved Gd3+-based contrast agents (for example Dotarem® in water r1p = 3.4 mM-1.s-1 at 20
MHz
(313 K) and 2.9 mM-1.s-1 at 60 MHz (310 K)) 27. Interestingly, the complex [Mn(L2)]+ also
possesses a r2p value of 8.72 mM-1.s-1 which was unfortunately not determined for the parent
Mn2+ complex ([Mn(no1pa)]+).
It can be reasonnably assumed that the significant increase in the r1p and r2p values is related
to the large increase in mass resulting from the introduction of both the π-conjugated antenna
and of the hydrosolubilizing PEG groups attached to the tacn platform. This interesting result
opens the way for the use of such complex as a T1 or T2 shortening MRI relaxation agent.
1.4. Photophysical and electrochemical properties.
The photophysical properties (absorption, emission, quantum yield) of the complexes were
investigated in organic solvent (CH2Cl2). Most of these data are collected in Table 3, while
the absorption/emission spectra are given in Supporting Information section (Figures S23-
S26). The absorption and emission spectra of all the investigated compounds show a broad
band in the visible range which can be assigned to an Intra-Ligand Charge Transfer transition
(ILCT) from the N-donor atom to the picolinic acceptor unit. As expected, the [M(L1)]+ (M =
Cu, Zn) complexes present very similar spectra due to their comparable Lewis acidity.
Nevertheless, the comparison of their luminescent quantum yield shows a modest 5% yield in
the case of zinc, similar to what is observed for the free ligand (Figure S22 in Supporting
Information) that is strongly reduced to less than 1% in the case of copper. This quenching is
not observed in the case of [Mn(L2)]+, featuring a slightly blue shifted emission with a
quantum yield efficiency of about 8%.
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[Table 3 here]
The quenching monitored upon copper (II) complexation is a common phenomenon which
has been widely described in the literature 28-30. It is generally explained by a photo-
induced electron transfer mechanism whose thermodynamic feasibility can be readily
assessed from the following Rehm-Weller equation (2) 31.
( )
( )
(2)
In this equation Eox(D) corresponds to the oxidation potential of the ILCT transition antenna
unit and Ered(A) to the reduction potential of the Cu(II) complex while E* refers to the
excited-state energy and C represents a Coulombic term (neglected in this experiment).
The Ered(A) and Eox(D) values have been readily obtained upon carrying electrochemical
measurements. The cyclic voltammogram (CV) recorded for [Cu(L1)]+ in aqueous medium
(0.1 M LiClO4) by using a carbon working electrode is shown in Figure 4. The curve displays
an irreversible oxidation process centered at Epeak = +0.91 V attributed to the oxidation of the
donor part of the fluorescent antenna (Eox(D)), together with a quasi-reversible
copper-centered reduction wave at E1/2 = -0.54 V (Ep = 120mV at 100mV/s) which was
approximated to the formal potential E°‟([CuII(L1)]+/[CuI(L1)]) = Ered(A). Finally, the
excited-state energy for this complex was estimated by the zero-phonon energy, E00
corresponding to the intersection of the absorption and emission curves and a value of
2.72 eV was found in CH2Cl2 (Figure S23 in Supporting Information). According to the
Rehm-Weller equation, the resulting ΔG value was found to be roughly -1.27 eV. This
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negative value allows us to predict that the electron transfer process is favorable in this
complex and is a reasonable explanation for the observed luminescence quenching.
[Figure 4 here]
The apparent reversibility of the reduction wave observed at E1/2 = -0.54 V suggests that the
electrogenerated copper(I) complex [CuI(L1)] is stable at the CV time scale and that there is
no chemical step (isomerization, degradation etc.) coupled to the electron transfer under these
experimental conditions. A similar behavior was observed in DMF electrolyte (Figure 5B).
In-situ spectro-electrochemical (SEC) analyses have thus been carried out in the latter
conditions to obtain further insights into the nature and stability of the [CuI(L1)] species.
SEC measurements carried out in the UV-Vis range using an optically transparent thin-layer
electrochemical cell showed that the one electron reduction of the [CuII(L1)]+ leads to a
gradual disappearance of the signal centered at λmax = 397 nm at the expense of a new
absorption band growing at λmax = 377 nm (Figure 5A). These changes were found to proceed
through a well-defined isobestic point at 385 nm only during the first stage of the reduction.
Keeping the potential of the working electrode at Eapp = -1.1V for a prolonged time indeed led
to a progressive increase of the intensity at 377 nm (dashed line in Figure 5A). The whole
process was however found to be reversible as the intensity of the initial signal attributed to
[CuII(L1)]+ could be recovered upon scanning the electrode potential back to Eapp = 0 V.
Overall these experiments reveal that the [CuI(L1)] seems stable at the electrolysis time scale
and that its absorption band is hypsochemically shifted by about 20 nm compared to that of
the copper(II) complex in agreement with the reduced Lewis acidity of Cu(I) compared to
Cu(II).
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[Figure 5 here]
1.5. Confocal Microscopy
Finally, preliminary cell imaging experiments have been undertaken using the most
luminescent [Zn(L1)]+ on MDA-MB 468 cell-line. In our hand this complex was not able to
stain spontaneously living cells by passive internalisation processes as already observed for
other cationic complexes [16,17,32]. Consequently, PFA-fixed cells have been used to
illustrate the proof-of-concept of fluorescence bioimaging of this complex. (Figure 5) 10 µM
of [Zn(L1)]+ were added to the culture medium and incubated for 1h with fixed cells. The
images were acquired under 385 nm excitation and a broadband detection between 500 to 600
nm. The slight green luminescence in the cells (Figure 6) indicates that the cationic complex
is able to accumulate in fixed cells, more precisely in the perinuclear cytoplasmic area and in
the nucleoli as observed for lanthanide species featuring similar π-conjugated antennas 16-
17.
[Figure 6 here]
2. Conclusion
In this article, we describe the results of our first attempt to implement a luminescence
modality on existing scaffold suitable for 64Cu-PET or Mn(II)-based MRI applications by
functionalizing the picolinate pendant arm with a donor π-conjugated chromophore antenna in
order to combine two imaging modalities in a single candidate. The cationic complexes
[M(L1)]+ (M = Cu, Zn) and [M(L2)]+ (M = Mn) have been prepared and fully characterized.
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The results we obtained are contrasted: in the case of copper, the picolinate functionalization
induced a modification of the chelation properties leading to an uncompleted radiolabeling
and the emission properties of the antenna have been quenched by a photo-induced electron
transfer process; however, results obtained with Zn2+ clearly show the viability of the concept
since cells images have been obtained. In the case of manganese, the results are more
promising than it was found for the parent complex formerly since the [Mn(L2)]+ complex
presents improved relaxation properties better than that of the [Gd(DOTA)] (the active
ingredient of Dotarem® which is the most trusted contrast agent on the market) accompanied
with acceptable luminescence properties. This complex presents a double-imaging modality
potential (MRI and optical imaging) and is the first step towards the design of a real bimodal
probe.
3. Conflict of interest
The authors declare no conflict of interest.
4. Acknowledgements
The authors acknowledge support of the Ministère de l‟Enseignement Supérieur et de la
Recherche, the Centre National de la Recherche Scientifique, the Institut National de la Santé
et de la Recherche Médicale and the Agence Nationale de la Recherche (SADAM ANR-16-
CE07-0015-02). R.T. thanks the „„Service Commun de RMN‟‟ of the University of Brest.
Gy.T. acknowledges financial support arriving form the Hungarian National Research,
Development and Innovation Office (NKFIH K-120224 project), János Bolyai Research
Scholarship of the Hungarian Academy of Sciences and European Regional Development
Fund under the project GINOP-2.3.2-15-2016-00008.
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References
1 E.W. Price, C. Orvig, Chem. Soc. Rev. 43 (2013) 260–290.
[2] E. Boros, A.B. Packard, Chem. Rev. 119, 2 (2019) 870-901
[3] L.E. Jennings, N.J. Long, Chem. Commun. (2009) 3511–3524.
4 A. Louie, Chem. Rev. 110 (2010) 3146–3195.
5 E. Ranyuk, R. Lebel, Y. Bérubé-Lauzière, K. Klarskov, R. Lecomte, J.E. van Lier, B.
Guérin, Bioconjugate Chem. 24 (2013) 1624–1633.
[6] T. Joshi, M. Kubeil, A. Nsubuga, G. Singh, G. Gasser, H. Stephan, ChemPlusChem 83
(2018) 554–564.
[7] T.J. Wadas, E.H. Wong, G.R. Weisman, C.J. Anderson, Chem. Rev. 110 (2010) 2858–
2902.
[8] J. Wahsner, E.M. Gale, A. Rodríguez-Rodríguez, P. Caravan, Chem. Rev. 119 (2019),
957-1057.
[9] A.J. Amoroso, S.J.A. Pope, Chem. Soc. Rev. 44, 14 (2015) 4723–4742.
[10] R.M. Izatt, K. Pawlak, J.S. Bradshaw, R.L. Bruening, Chem. Rev. 91, 8 (1991) 1721–
2085.
11 M. Le Fur, M. Beyler, N. Le Poul, L.M.P. Lima, Y. Le Mest, R. Delgado, C. Platas-
Iglesias, V. Patinec, R. Tripier, Dalton Trans. 45, 17 (2016) 7406–7420.
12 A. Nonat, D. Imbert, J. Pécaut, M. Giraud, M. Mazzanti, Inorg. Chem. 48, 9 (2009)
4207–4218.
[13] M. Roger, L.M.P. Lima, M. Frindel, C. Platas-Iglesias, J.-F. Gestin, R. Delgado, V.
Patinec, R. Tripier, Inorg. Chem. 52 (2013) 5246–5259.
[14] E. Molnár, N. Camus, V. Patinec, G.A. Rolla, M. Botta, G. Tircsó, F.K. Kálmán, T.
Fodor, R. Tripier, C. Platas-Iglesias, Inorg. Chem. 53 (2014) 5136–5149.
22

Journal Pre-proof
[15] E.M. Gale, I.P. Atanasova, F. Blasi, I. Ay, P. Caravan, J. Am. Chem. Soc. 137 (2015)
15548–15557.
[16] A.T. Bui, M. Beyler, Y.-Y. Liao, A. Grichine, A. Duperray, J.-C. Mulatier, B. Le
Guennic, C. Andraud, O. Maury, R. Tripier, Inorg. Chem. 55 (2016) 7020–7025.
[17] A.T. Bui, M. Beyler, A. Grichine, A. Duperray, J.-C. Mulatier, Y. Guyot, C. Andraud,
R. Tripier, S. Brasselet, O. Maury, Chem. Commun. 53 (2017) 6005–6008.
[18] A. D‟Aléo, A. Picot, A. Beeby, J.A. Gareth Williams, B. Le Guennic, C. Andraud, O.
Maury, Inorg. Chem. 47 (2008) 10258–10268.
[19] A. D‟Aléo, A. Picot, P.L. Baldeck, C. Andraud, O. Maury, Inorg. Chem. 47 (2008)
10269–10279.
[20] H.M. Irving, M.G. Miles, L.D. Pettit, Anal. Chim. Acta, 38 (1967) 475–488.
[21] L. Zekany, I. Nagypal, in Comput. Methods Determ. Form. Constants (Ed.: D.J.
Leggett), Springer US, Boston, MA (1985) pp. 291–353.
[22] A.A. Belal, L.H. Abdel-Rahman, A.H. Amrallah, J. Chem. Eng. Data 42 (1997) 1075–
1077.
[23] B. Drahoš, V. Kubíček, C.S. Bonnet, P. Hermann, I. Lukeš, E. Tóth, Dalton Trans. 40
(2011) 40, 1945-1951.
[24] S. Cortes, E. Brucher, C.F.G.C. Geraldes, A.D. Sherry, Inorg. Chem., 29 (1990) 5-9.
[25] A. Sá, C.S. Bonnet, C.F. Geraldes, É. Tóth, P.M. Ferreira, J.P. André, Dalton Trans.
2013, 42(13), 4522-4532.
[26] C. Paul-Roth, K.N. Raymond, Inorg. Chem. 34 (1995) 1408–1412.
[27] M. Rohrer, H. Bauer, J. Mintorovitch, M. Requardt, H.-J. Weinmann, Invest. Radiol. 40
(2005) 715-724.
[28] Z. Liu, Z. Yang, T. Li, B. Wang, Y. Li, D. Qin, M. Wang, M. Yan, Dalton Trans. 40
(2011) 9370–9373.
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Journal Pre-proof
[29] L. Sun, D. Hao, W. Shen, Z. Qian, C. Zhu, Microchim. Acta 177 (2012) 357–364.
[30] N. Chopin, M. Médebielle, O. Maury, G. Novitchi, G. Pilet, Eur. J. Inorg. Chem. 36
(2014) 6185–6195.
[31] D. Rehm, A. Weller, Isr. J. Chem. 8 (1970) 259–271.
[32] J.W. Walton, A. Bourdolle, S.J. Butler, M. Soulie, M. Delbianco, B.K. McMahon, R.
Pal, H. Puschmann, J.M. Zwier, L. Lamarque, O. Maury, C. Andraud, D. Parker, Chem.
Commun. 49 (2013) 1600–1602.
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Tables
Table 1. Stepwise (log KiH) protonation constants of HL2 in aqueous solution at 25.0 ± 0.1 °C
and 0.15 ± 0.01 M NaCl.
Equilibrium reaction
HL2
Hno1pa [14]
log K
L + H+ ⇄ HL
HL + H+ ⇄ H2L
H2L+ H+ ⇄ H3L
11.41(1)
7.46(3)
3.25(3)
11.33
7.30
2.49
Table 2. Stability constants (log K) for the metal complexes of the ligands in aqueous solution
at 25.0 ± 0.1 °C and 0.15 ± 0.01 M NaCl.
log
KMnL
14.14
8.33
log
KMnL
pMn
Reference
OH
H3PyC3a
Tacn
-
-
8.17
6.37
15
22
H3nota
14.9
-
7.76 ; 7.94
5.73
23; 24
25
25
25
14
NODAHep
NODAHAa
NODABA
Hno1pa
HL2
10.98
10.15
9.9
10.28
10.56(4)
-
9.65
-
11.94
11.62(5)
5.76
5.60
7.08
7.21
This work
a
the formation of a protonated complex was also observed for the given system (log
KMnLH=4.95)
Table 3. Photophysical data measured in CH2Cl2 for studied ligands and metal complexes
Compound
[Cu(L1)]+
[Zn(L1)]+
[Mn(L2)]+
Solvent
CH2Cl2
CH2Cl2
CH2Cl2
λabs (nm)
410
λem (nm)
507
Φa (%)
< 1
5
407
315
570
464
8
a Coumarin 153 in MeOH (Φ = 45%) for L1 species and Quinine sulfate in
H2SO4 1N (Φ = 54%) for L2 species as standards
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Figures and Schemes Captions
Figure 1. Tacn-based ligands investigated as part of the present article.
Figure 2. Thin layer radiochromatograms of 64Cu(CO2Me)2 (A) and [64Cu(L1)]+ (B) and % of
64Cu emission; 58 µM ligand and 15 min incubation time in ammonium acetate buffer at
100°C.
Figure 3. Species distribution diagram of the complex [Mn(L2)]+ calculated in the pH range
of 3 to 11 (calculated by using 1 mM of Mn2+ and ligand; plain lines) and the plots of the 1/T1
() and 1/T2 (s-1) () values evaluated at 60 MHz and 0.15 M NaCl at 298 K (dashed lines).
Figure 4. Cyclic voltammogram (0.1 v.s-1, GC carbon WE  =3mm, H2O, pH 7, 0.1 M
LiClO4) and absorption/emission spectra of [Cu(L1)]+ (CH2Cl2).
Figure 5. A) UV−Vis abs. spectra recorded during the one-electron reduction of [CuII(L1)]+
in DMF (0.1 M TBAP). The reduction was carried out in an optically transparent thin-layer
electrochemical cell (l = 1mm) using a platinum gauze working electrode whose potential was
linearly scanned (10 mV/s) between 0 and -1.1 V. The spectrum recorded at the end of the
reduction is shown as a dashed line; B) Voltammetric curves of a DMF (TBAP 0.1 M)
solution of [Cu(L1)]+ recorded at a stationary carbon working electrode (Ø = 3 mm, E vs
Ag+/Ag (10−2 M), ν = 0.1 V·s−1).
Figure 6. Confocal microscopy images of MDA-MB cells before (A) and after 1h of
incubation with the [Zn(L1)]+ at 10 µM (B)
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Scheme 1. Synthesis of the ligands and complexes studied in this work. i) adapted from ref
13; ii) benzaldehyde, molecular sieves, EtOH, rt 4h; iii) antenna a, K2CO3, CH3CN, rt, 72h;
iv) 1M KOH, THF, 55°C, 12h. v) MII(ClO4)2·6H2O (MII = Cu2+ / Zn2+), H2O, pH 6.6, 60°C,
24h; vi) MnCl2·4H2O, H2O, neutral pH, 60°C, 24h.
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Figures and Schemes
Figure 1.
Figure 2.
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Figure 3.
Figure 4.
Eox(D)
10 mA
x
E’
Ered(A)
-1
0
1
E(V) vs E[AgCl/Ag]
29