Siderophore inspired tetra- and octadentate antenna ligands for luminescent Eu(III) and Tb(III) complexes

Article abstract of DOI:10.1016/j.jinorgbio.2016.01.006

Following the success of the siderophore-inspired 1,2-hydroxypyridonate (HOPO) and 2-hydroxisophthalamide (IAM) chromophores in Eu(III) and Tb(III) luminescence, we designed three new ligands bearing both chromophores. Syntheses of the octadentate ligands

Full text of DOI:10.1016/j.jinorgbio.2016.01.006

Siderophore inspired tetra- and octadentate antenna ligands for luminescent
Eu(III) and Tb(III) complexes
Lena J. Daumann, Philipp Werther, Michael J. Ziegler, Kenneth N. Ray-
mond
PII:
DOI:
Reference:
S0162-0134(16)30008-3
doi: 10.1016/j.jinorgbio.2016.01.006
JIB 9895
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
1 October 2015
5 January 2016
7 January 2016
Please cite this article as: Lena J. Daumann, Philipp Werther, Michael J. Ziegler, Ken-
neth N. Raymond, Siderophore inspired tetra- and octadentate antenna ligands for lu-
minescent Eu(III) and Tb(III) complexes, Journal of Inorganic Biochemistry (2016), doi:
10.1016/j.jinorgbio.2016.01.006
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ACCEPTED MANUSCRIPT
Graphical Abstract
Three antenna ligands bearing 1,2-hydroxypyridonate (HOPO) and 2-hydroxisophthalamide (IAM)
chromophores are reported. Photophysical characterization of the Gd(III), Eu(III) and Tb(III) complexes
revealed localized triplet states for the chromophores and that they need to reside on a primary amine of
the spermine backbone to be efficient in lanthanide excitation.
Siderophore inspired tetra- and octadentate
antenna ligands for luminescent Eu(III) and
Tb(III) complexes
Leave this area blank for abstract info.
Lena J. Daumann, Philipp Werther, Michael J. Ziegler and Kenneth N. Raymond
Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and
Department of Chemistry, University of California, Berkeley, California 94720

ACCEPTED MANUSCRIPT
Siderophore inspired tetra- and octadentate antenna ligands for luminescent Eu(III)
and Tb(III) complexes
Lena J. Daumann, Philipp Werther, Michael J. Ziegler and Kenneth N. Raymond
Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and Department of Chemistry, University of California,
Berkeley, California 94720
In memory of Professor Graeme Hanson and his contributions to Bioinorganic Chemistry and Electron Paramagnetic Resonance
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Following the success of the siderophore-inspired 1,2-hydroxypyridonate (HOPO) and 2-
hydroxisophthalamide (IAM) chromophores in Eu(III) and Tb(III) luminescence, we designed
three new ligands bearing both chromophores. Syntheses of the octadentate ligands 3,4,3-LI-
IAM-1,2-HOPO and 3,4,3-LI-1,2-HOPO-IAM, where the chromophores are attached to
different positions in the (LI = linear) spermine backbone, are reported in addition to a
tetradentate ligand based on 1,5-diaminopentane. The Gd(III) complexes were prepared and
revealed localized triplet states typical for the IAM and HOPO chromophores. Photophysical
characterization of the Eu(III) and Tb(III) complexes revealed that the chromophores need to
reside at a primary amine of the spermine backbone to be efficient in lanthanide excitation.
These systems help us to understand the antenna effect in siderophore inspired chromophores
and could be potential targets for sensing and biological imaging applications.
Available online
Keywords:
luminescent lanthanides
siderophore inspired ligands
antenna
Europium
Terbium
1. Introduction
photoluminescence quantum yield (Фtot) as high as 20% for the
tetradentate 5LI-1,2-HOPO ligand, while the salicylate-type IAM
Lanthanides (Ln) (or rare earth elements (REE) when
including Sc and Y)[1] are featured in a multitude of applications
in technology,[2-6] research[7] and medicine[8-10] and have
chromophore triplet state is better matched to sensitize Tb(III)
with excellent quantum yields of 36% with the tetradentate 5LI-
IAM ligand or as high as 60% in octadentate IAM cages in
recently been found to have
a
role in nature as
metalloenzymes.[11-13] It is especially due to their favorable
luminescent properties (long lifetimes, narrow emission bands,
large effective Stokes’ shifts) that they have found their way in
many applications, like dissociation enhanced lanthanide
fluorescence immunoassay (DELFIA), homogeneous time
resolved fluoroimmunoassays (HTRF) and luminescence
resonance energy transfer (LRET) assays.[14, 15] In consequence
of their forbidden f-f-transitions, indirect excitation via an
organic chromophore (antenna effect) is employed. Siderophore
inspired ligands have been used to efficiently chelate Ln and
actinides (Ac). For example, the octadentate ligand 3,4,3-LI-1,2-
HOPO (Figure 1, LI stands for a linear, unbranched ligand
backbone) based on the spermine backbone, has been reported to
be an excellent actinide sequestering agent in vivo.[16-20]
However, due to the high acidity of the 1,2-hydroxypyridonate
(1,2-HOPO) moieties in this ligand, mixed 1,2-HOPO and
catechol amide (CAM) or 3,2-HOPO spermine ligands have been
developed.[21-23] By altering the chelating moieties, different
affinities and stabilities in various pH can be achieved. 3,4,3-LI-
1,2-HOPO has also been reported as being an excellent antenna
for sensitizing both Ln and Ac.[16, 20, 24] The hydroxamate
inspired 1,2-HOPO chromophore has been shown to be an
especially efficient sensitizer for Eu(III) luminescence, with
aqueous solution.[14, 25]
Figure
1 New siderophore inspired ligands (left) and
previously reported highly efficient antenna and f-element
sequestering ligands (right)

ACCEPTED MANUSCRIPT
Here we report the synthesis of three new ligands (Scheme 1-
CAUTION The coupling reagent O-(Benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) used in
some of the syntheses is potentially explosive and should be
handled with care.
3), two octadentate and one tetradentate, based on the IAM and
1,2-HOPO chelating chromophores (Figure 1) and discuss the
effects of different antennas on photophysical properties such as
Фtot and triplet state to aid design and deeper understanding of
new luminescent probes and sequestering agents. The Eu(III) and
Tb(III) complexes of the reported ligands have been
characterized by high resolution Electrospray Injection Mass
Spectrometry (ESI-MS) and photophysical measurements. In
addition, a new and more efficient synthesis to the previously
reported benzyl protected IAM precursor is reported here.
Density Functional Theory (DFT) calculations were conducted to
elucidate the structure around the metal ions and to assess the
ligand abilities to fully coordinate the metal ions.
2-Hydroxyisophthalic acid (2). This compound was
synthesized after a slight modification of a previously published
procedure.[29] Potassium hydroxide (115 g, 2.05 mol) was
combined with water (24 mL) in a stainless steel beaker and 3-
methylsalicylic acid 1 (19.2 g, 0.126 mol) and lead(IV) oxide
(115 g, 0.481 mol) were added. The mixture was stirred with a
glass rod and heated to 250°C for 45 min. Subsequently the
orange mixture was cooled to ~ 100°C and poured into water (1
L). Orange lead(II) oxide and red lead were filtered off on a glass
frit. The filtrate was partially neutralized (pH~10) with
concentrated hydrochloric acid (150 mL) and saturated sodium
sulfide was added until no further precipitation of black lead(II)
sulfide occurred. The black solution was heated briefly to
coagulate lead(II) sulfide (40°C, 10 min) and the black
suspension filtered through a glass frit. Subsequently the filtrate
was acidified to pH 1 with concentrated hydrochloric acid (120
mL) while cooling on ice to precipitate 2-hydroxyisophtalic acid
(2) as white solid. The product was collected on a glass frit and
first dried in air and then in high vacuum overnight. Yield
2. Experimental
2.1 Materials and Methods
Ground state density functional theory (DFT) calculations
were performed at the Molecular Graphics and Computational
Facility, College of Chemistry, University of California,
Berkeley using Gaussian 09.[26] The ground state geometries of
the complexes were optimized using the B3LYP functional,
treating the light atoms with the 6-31G(d,p) basis set and the
Eu(III) with the effective core potential MWB52.[27, 28]
Nuclear Magnetic Resonance (NMR) spectra were obtained
using (unless otherwise noted) a Bruker AV600 spectrometer
1
17.91 g (78 %). H NMR (DMSO, 400 MHz): δ 6.89 (t, 1H, J =
7.7 Hz), 7.94 (d, 2H, J = 7.7 Hz). 13C NMR (d6-DMSO,
150 MHz) δ 117.5; 118.1; 135.9; 162.0; 169.4. FTIR (ν, cm-1)
2968 (b, O-H str); 1687 (s, C=O str); 1154 (s, C-O str); 755 (m,
arH). n-ESI HRMS (MeOH): 181.0142 (calc. 181.0142,
[C8H5O5]-, [M-H]-).
1
1
operating at 600 (151) MHz for H (or 13C, respectively). H (or
13C) chemical shifts are reported in ppm relative to the solvent
resonances, taken as δ 7.26 (77.0), 2.50 (39.5) and 4.78 (49.2),
respectively for CDCl3, (CD3)2SO and CD3OD while coupling
constants (J) are given in Hz. Fourier-Transform Infra-Red (FT-
Dibenzyl-2-(benzyloxy)isophthalate (3). This compound was
synthesized after a modification of a previously published
procedure.[30] 2-Hydroxyisophthalic acid (2) (5.0 g, 0.027 mol),
dry potassium carbonate (15.18 g, 0.109 mol) and benzyl
bromide (18.78 g, 0.109 mol, 13.06 mL) were added to
dimethylformamide (DMF, 35 mL) in a flame dried flask under a
nitrogen atmosphere. The reaction mixture was stirred for 16 h at
75°C. Subsequently the solution was cooled down to room
temperature, filtered and the solid washed with DMF (5 x 1 mL).
The filtrate was evaporated to dryness in high vacuum (60°C
water bath) to yield a yellow, viscous oil. The crude product was
purified by column chromatography (SiO2, gradient from 0 % to
10 % MeOH in dichloromethane). Purified 3 crystalized after
drying in high vacuum as yellow needles. Yield 6.32 g (51 %).
1H NMR (CDCl3, 400 MHz) δ 5.04 (s, 2H), 5.29 (s, 4H), 7.21 (t,
IR) spectroscopy was conducted using
a
Nicolet 380
spectrometer from Thermo Electron (ZnSe crystal). High-
Resolution Electrospray Ionization (HR-ESI)-Mass spectrometry
was performed at the QB3/Chemistry Mass Spectrometry
Facility, University of California, Berkeley.
2.2 Ligand and Complex Syntheses
1H, J = 7.7 Hz), 7.29-7.35 (m, 15H), 7.96 (d, 2H, J = 7.7 Hz). 13
C
NMR (CDCl3, 150 MHz) δ 67.2; 77.9; 123.7; 127.3; 127.8;
128.2; 128.3; 128.4; 128.5; 135.0; 135.6; 136.8; 158.0; 165.5. IR
(ν, cm-1) 3033, 2946 (m, CH2 str); 1720 (s, C=O str); 1248 (s, C-
O-C str); 734 (m, arH). p-ESI HRMS (MeOH) 453.1695 (calc.
453.1697,
[C29H25O5]+);
475.1514
(calc.
475.1516,
[C29H25NaO5]+).
2-(benzyloxy)isophthalic acid (4). This compound was
synthesized after a modification of a previously published
procedure.[30] Dibenzyl 2-(benzyloxy)isophthalate (3) (2.91 g,
6.43 mmol) was dissolved in methanol (50 mL) and sodium
hydroxide (0.771 g, 19.3 mmol, in 8 mL water) were added. In
order to dissolve the starting material completely, more methanol
(13 mL) was added and the solution was sonicated (final solvent
composition: MeOH/H2O 8/1). Subsequently the mixture was
stirred at room temperature for 17 h. The solvent was removed in
vacuo, then the residue was co-evaporated first with water
(15 ml) then diethyl ether (15 mL). The yellow residue was taken
up with half saturated sodium chloride solution (50 mL) and
extracted with dichloromethane (2 x 25 mL). Starting material
Scheme 1 Synthesis of 3,4,3-LI-IAM-1,2-HOPO (10).
4

ACCEPTED MANUSCRIPT
was recovered from the combined and dried organic phases. The
79.4, 125.4, 129.0, 129.0, 129.1, 129.2, 129.5, 133.9, 134.0,
135.6, 154.5, 165.9, 166.3 p-ESI HRMS (MeOH) 737.4023 (calc.
aqueous phase was acidified to pH 1 with concentrated
hydrochloric acid and the white precipitate of (4) was filtered and
dried in vacuo overnight. Yield: 1.46 g (83 %). 1H NMR (DMSO,
600 MHz) δ 5.03 (s, 2H); 7.29-7.39 (m, 4H); 7.47 (d, 2H, J = 7.0
Hz); 7.84 (d, 2H, J = 7.7 Hz), 13.25 (bs, 2H). 13C NMR (DMSO,
150 MHz) δ 76.7; 123.9; 128.1; 128.4; 128.6; 133.7; 137.4;
156.4; 167.5; 176.0. IR (ν, cm-1) 2870 (b, O-H str); 2685 (m, CH2
str); 1720 (s, C=O str); 1244 (s, C-O-C str); 766 (m, arH). n-ESI
737.4021,
[C42H53N6O6]+);
369.2050
(calc.
369.2047,
[C42H54N6O6]2+).
3,4,3-LI-IAMBn-1,2-HOPOBn (8). 3,4,3-LI-IAMBn (7)
(362 mg, 491 µmol), 1,2-HOPOBn (9)[16] (289 mg, 1.18 mmol),
DMAP (5.4 mg, 44 µmol), TBTU (379 mg, 1.18 mmol) were
suspended in 20 ml DCM. DIPEA (254 mg, 1.96 mmol, 343 µL,
ρ 0.74 g/cm3) was added to start the reaction and it was stirred at
room temperature for 30 h until TLC showed no further
conversion. After removal of the solvent under reduced pressure,
the crude product was subjected to column chromatography
(SiO2, gradient from 0 % to 10 % MeOH in DCM) to afford a
white solid. Yield: 394 mg (67 %). ESI MS, LC-ESI MS data as
well as TLC experiments indicated partial cleavage of benzyl
protecting groups during the purification step. Thus the crude (8)
was used directly in the next step. p-ESI HRMS (MeOH):
1191.5160 (calc. 1191.5186, [C68H71N8NaO12]+); 1213.4967
(calc. 1213.5005, [C68H70N8NaO12]+); 1229.4748 (calc.
1229.4745, [C68H70N8KO12]+).
-
HRMS (MeOH): 271.0609 (calc. 271.0612, C15H11O5 , [M-H]-).
2-(Benzyloxy)-1,3-phenylene)bis((2-thioxothiazolidin-3-
yl)methanone (5). 2-(Benzyloxy)isophthalic acid (4) (2.00 g,
7.35 mmol), 2-mercaptothiazoline (1.84 g, 15.4 mmol), 4-
dimethylaminopyridine (DMAP, 80.8 mg, 661 µmol), TBTU
(5.66 g, 17.6 mmol) were dissolved in dichloromethane (60 mL).
After addition of N,N-diisopropylethylamine (DIPEA, 3.80 g,
29.4 mmol, 5.13 mL), the mixture turned yellow immediately,
and was stirred at room temperature for 3.5 h. After removal of
the solvent in vacuo, 5 was purified with column chromatography
(SiO2, 100 % dichloromethane (DCM)) to afford a yellow
powder. Yield 2.00 g (57 %). 1H NMR (CDCl3, 400 MHz) δ 3.02
(t, 4H, J = 7.3 Hz), 4.41 (t, 4H, J = 7.3 Hz), 5.02 (s, 2H), 7.21 (t,
1H, J = 7.7 Hz), 7.31-7.40 (m, 5H), 7.48 (d, 2H, J = 7.6 H). 13C
NMR (CDCl3, 100 MHz) δ 29.2, 56.1, 77.6, 124.2, 128.1, 128.9,
129.2, 132.6, 136.7, 153.8, 167.6, 201.2. IR (ν, cm-1) 2939 (m,
CH2 str); 1673 (s, C=O str); 1221 (s, C-O str); 1148 (s, C=S str);
752 (m, arH). p-ESI HRMS (MeOH) 497.0081 (calc. 497.0092,
[C21H18N2NaO3S4]+).
3,4,3-LI-IAM-1,2-HOPO (10). 3,4,3-LI-IAMBn-1,2-HOPOBn
(8) (206 mg, 173 µmol) was dissolved in dry dichloromethane
(5 mL) and cooled on an ice bath. Boron tribromide (1.73 g,
6.92 mmol, 656 µL, ρ 2.64 g/cm3) was added and the mixture
was stirred overnight and allowed to warm to room temperature.
Subsequently the solvent and excess boron tribromide were
removed under high vacuum. The orange residue was cooled
with liquid nitrogen and methanol was added until no further gas
evolution was observed (ca. 50 mL). The solution was allowed to
warm to room temperature and refluxed for 2.5 h. Then the
solution was concentrated (to ca 1 mL) and filtered. Diethyl ether
was added to the filtrate to precipitate a beige solid. The solvent
was removed in vacuo and orange oily residue of 10 was dried in
2-(Benzyloxy)-N-methyl-3-(2-thioxothiazolidine-3-carbonyl)
benzamide (6). A solution of methylamine (1.7 mL, 3.39 mmol,
2M in tetrahydrofuran (THF)) in DCM/MeOH (95:5, 200 mL)
was added slowly over 48 h to a solution of 5 (1.61 g,
3.39 mmol) in 5 mL dichloromethane using a syringe pump.
After removal of the solvent, the yellow residue was dissolved in
dichloromethane (50 mL) and was washed with 1M KOH
solution (2 x 30 mL). The combined organic phases were dried
and the solvent was removed under reduced pressure.
Subsequently the crude (6) was purified by column
chromatography (SiO2, gradient from 0 % to 10 % EtOAc in
1
high vacuum. Yield 128 mg (89 %). H NMR (CD3OD, 600
MHz) δ 1.49-2.01 (m, 8H), 2.93 (bs, 6H), 2.95 (s, 3H), 3.46-3.64
(m, 8H), 3.30-3.31 (m, 4H), 6.53-6.85 (m, 4H), 6.96 (s, 2H),
7.46-7.71 (m, 2H), 7.91-8.00 (m, 4H). n-ESI HRMS (MeOH)
829.3135 (calc. 829.3162, [C40H45N8O12]-). IR (ν, cm-1) 2868 (m,
CH2 str); 1631, 1527 (s, C=O str); 1431 (m, CH2 def); 795, 756
(m, arH).
1
DCM) to yield 0.74 g (56 %) of 6 as a yellow powder. H NMR
(CDCl3, 400 MHz) δ 2.85 (d, 3H, J = 4.9 Hz), 3.14 (t, 2H, J =
7.3 Hz), 4.51 (t, 2H, J = 7.3 Hz), 4.98 (s, 2H,), 7.27 (t, 1H, J =
7.7 Hz), 7.34-7.41 (m, 5H), 7.48 (dd, 1H, J = 7.6, 1.8 Hz), 8.14
(dd, 1H, J = 7.8, 1.8 Hz). 13C NMR (CDCl3, 100 MHz) δ 26.8;
28.9; 55.9; 78.5; 125.1; 127.9; 128.3; 129.1; 129.1; 129.9; 132.6;
134.6; 136.1; 154.5; 165.6; 167.7; 201.8. p-ESI HRMS (MeOH)
387.0825 (calc. 387.0832, [C19H19N2O3S2]+); 409.0644 (calc.
409.0651, [C19H18N2NaO3S2]+); 425.0385 (calc. 425.0390,
[C19H18KN2O3S2]+).
2-Methoxy-3-(methoxycarbonyl)benzoic acid (13) 2-Methoxy-
isophtalic acid (11) (2.75 g, 14.0 mmol) and K2CO3 (5.81 g, 42.1
mmol) were suspended in DMF (50 mL). Methyl iodide (2.6 mL,
5.97 g, 42.1 mmol) was added slowly at room temperature. After
the mixture was stirred at 80°C overnight, the solvent was
evaporated in vacuo. The solid residue was dissolved in DCM
(150 mL) and washed with water and brine (50 mL,
respectively). The organic layer was dried over Na2SO4 and the
solvent was removed to deliver dimethyl 2-methoxyisophthalate
(12) as a yellow oil (2.24 g, 71 %), which was used directly in
the next step. The yellow oil (1.28 g, 5.7 mmol) was dissolved in
methanol (15 mL) and KOH (320mg, 5.7 mmol) was added. The
mixture was refluxed for 4 h, and then concentrated in vacuo.
The solid residue was dissolved in water (5 mL) and the aqueous
layer was acidified with 6M HCl solution until an off-white oil
separated. The aqueous layer was extracted with DCM
(3x20 mL). The organic layers were combined, washed with
water and brine and dried over Na2SO4. The solvent was
evaporated and 2-methoxy-3-(methoxycarbonyl)benzoic acid 13
3,4,3-LI-IAMBn (7). A solution of 6 (988 mg, 2.56 mmol) and
DIPEA (0.424 mL, 2.43 mmol) in dichloromethane (300 mL)
was added dropwise at room temperature over 24h to a solution
of spermine (254 mg, 1.22 mmol) in dichloromethane (35 mL).
After the complete addition, the reaction mixture was stirred for
another 24h. Subsequently the solvent was removed from the
colorless solution under reduced pressure and the crude product
was subjected to column chromatography (Al2O3 basic, gradient
0% to 10% MeOH in dichloromethane) to yield 3,4,3-LI-IAMBn
(7) as a white solid. Yield: 308 mg (34%). 1H NMR (CDCl3, 600
MHz) δ 1.36 (m, 4H), 1.62 (p, 4H, J = 6.4 Hz), 2.44 (m, 4H,),
2.57 (t, 4H, J = 6.4 Hz), 2.88 (d, 6H, J = 4.8 Hz), 3.45 (q, 4H, J =
6.1 Hz), 4.94 (s, 4H), 7.26 (t, 2H, J = 7.7 Hz), 7.34-7.41 (m,
10H,), 7.49-7.52 (m, 2H), 7.94-7.95 (m, 4H), 7.98-8.00 (m, 2H).
13C NMR (CDCl3, 150 MHz) δ 26.9, 28.0, 29.2, 39.1, 48.1, 49.8,
1
was obtained as a white solid. Yield: 673 mg (56 %) H-NMR
(CDCl3, 400 MHz) δ 3.96 (s, 3H), 4.05 (s, 3H), 7.33 (t, 1H, J =
8.5 Hz), 8.08 (d, 1H, J = 8.5 Hz), 8.30 (d, 1H, J = 8.5 Hz), 11.1
(bs, 1H).
5

ACCEPTED MANUSCRIPT
spermine (519 mg, 2.6 mmol) in DCM (50 mL). The mixture was
stirred for 18h at rt until TLC showed full conversion. The
reaction mixture was washed with 1M KOH (2 x 150 mL)
solution, subsequently concentrated in vacuo. The crude product
was purified with column chromatography (Al2O3 basic, 0.5%
MeOH in dichloromethane) yielding the pure product (17) as
1
yellowish residue. Yield: 968 mg (57 %). H NMR (CDCl3, 600
MHz) δ 1.27 (m, 4H), 1.66 (m, 4H), 2.15 (s, 2H), 2.31 (m, 4H),
2.55 (m, 4H), 3.41 (s, 4H), 5.92 (s, 4H), 6.35 (d, J = 6.2 Hz, 2H),
6.64 (d, J = 10.2 Hz, 2H), 7.26 (dd, J = 6.2, 7.2 Hz, 2H), 7.22-
7.55 (m, 10H) 8.55 (bs, 2H) 13C NMR (CDCl3, 150 MHz) δ 27.0,
27.1, 39.2, 47.7, 48.2, 79.4, 105.9, 123.6, 138.2, 128.5, 129.5,
130.4, 138.3, 143.3, 158.6, 160.3. p-ESI HRMS m/z 657.3387
+
(calc. 657.3395 for C36H45O6N6 ).
3,4,3-Li-1,2-HOPOBn-IAMMe (18). 3,4,3-Li-1,2-HOPOBn
(17) (220 mg, 335 μmol) was dissolved in DCM (60 mL). TBTU
(231 mg, 720 μmol), DMAP (4 mg, 33 μmol) and 2-methoxy- 3-
(methylcarbamoyl)benzoic acid (15) (141 mg, 720 μmol) were
added. The reaction was started by adding DIPEA (234 μl, 173
mg, 1.34 mmol) and stirred for 3 h at room temperature. The
solvent was evaporated and column chromatography (SiO2,
DCM:MeOH 0-10%) yielded the protected ligand 3,4,3-Li-1,2-
HOPOBn-IAMMe as a white solid. Yield 92 mg (26 %). ESI
MS, LC-ESI MS data as well as thin layer chromatography
(TLC) experiments indicated partial cleavage of benzyl
protecting groups during the purification step. Thus the crude
(18) was used directly in the next step. p-ESI HRMS m/z
+
Scheme 2 Synthesis of 3,4,3-LI-1,2-HOPO-IAM (19).
1039.4555 (calc. 1039.4560 for C56H63O12N8 ).
Methyl 2-methoxy-3-(methylcarbamoyl)benzoate (14) 2-
methoxy-3-(methoxycarbonyl)benzoic acid (13) (516 mg,
2.45 mmol) was dissolved in DCM (5 mL). TBTU (1.97 g,
6.14 mmol), DMAP (0.75 mg, 6.14 mmol) and methylamine (2M
in THF, 3.68 mL, 7.36 mmol) were added. The reaction was
started by adding DIPEA (214 μL, 159 mg, 1.23 mmol) and
stirred for 3 h at room temperature. The solvent was evaporated
and column chromatography (SiO2, DCM:MeOH, 0-10 %
3,4,3-Li-1,2-HOPO-IAM (19). The protected 3,4,3-Li-1,2-
HOPOBn-IAMMe ligand (403 mg, 388 μmol) was dissolved in
dry DCM (10 mL) under inert conditions and cooled on ice. Neat
BBr3 (1.47 ml, 3.89 g, 15.5 mmol) was slowly added and the
mixture was warmed up to room temperature and stirred
overnight. The solvent was evaporated in vacuo. The solid
residue was quenched with methanol while cooling with liquid
nitrogen. After no further reaction could be observed, the reaction
was heated to reflux for 3 h in an open flask, while adding
constantly methanol to maintain a steady level. The mixture was
concentrated to ~1 ml and filtrated. Addition of diethylether
precipitated the deprotected ligand 3,4,3-Li-1,2-HOPO-IAM
(19) as a brown residue, which was dried in vacuo. Yield: 303
gradient)
delivered
methyl
2-methoxy-3-(methyl
carbamoyl)benzoate as an off-white liquid. Yield: 770 mg (140
%, contained TBTU impurity). 14 was used without further
1
purification in the next step. H NMR (CDCl3, 600 MHz) δ 3.03
(d, 3H, J = 5.0 Hz), 3.89 (s, 3H), 3.94 (s, 3H), 7.25-7.28 (m, 1H),
7.67-7.72 (bs, 1H), 7.91 (d, 1H, J = 8.2 Hz), 8.24 (d, 1H, J = 8.2
Hz). p-ESI HRMS (MeOH) 224.0914 (calc. 224.0917,
[C11H14O4N]+)
1
mg (64 %). H NMR (CD3OD, 600 MHz) δ 1.22-1.91 (m, 8H);
2.79 (d, 6H); 2.93-3.56 (m, 12H); 6.37-7.62 (m, 12H). n-ESI
–
HRMS m/z 829.3237 (calc. 829.3162 for C40H45O12N8 , (L-H)-).
IR (ν, cm-1) 2830 (m, CH2 str); 1631,1526 (s, C=O str); 1432 (m,
2-Methoxy-3-(methylcarbamoyl)benzoic acid (15) Methyl 2-
CH2 def); 794, 747 (m, arH).
methoxy-3-(methylcarbamoyl)
benzoate
(14)
(770 mg,
Syntheses of the EuIII and TbIII complexes The complexes
were synthesized following a general procedure: A ~1M solution
of LnCl3·6H2O (0.9 eq, 29.2 µmol) in methanol was prepared and
added to a solution of 10 or 19 (1 eq, 32.5 µmol) in methanol
(~3 mL) by what a white precipitate was formed instantaneously.
After stirring for 5 min, pyridine (10 eq, 325 µmol) was added
and the mixture was heated at 60°C for two hours. Subsequently,
the mixture was cooled down to room temperature and
centrifuged. The residue was extracted with methanol (2 x 3 mL)
in order to remove uncoordinated ligand and the white solid was
dried in high vacuum.
3.45 mmol) was dissolved in methanol (5 mL). An aqueous 1 M
KOH solution (5.17 mL, 5.17 mmol) was added and the mixture
was stirred at room temperature for 16 h. The solvent was
evaporated and the solid residue redissolved in water (5 mL). The
aqueous layer was extracted with DCM (3 x 5 mL), and then
acidified with 6 M HCl solution. A yellowish oil separated,
which was extracted with DCM (3 x 15 mL). The organic layers
were combined, dried over Na2SO4 and the solvent was
evaporated to yield 2-methoxy-3-(methylcarbamoyl)benzoic acid
1
(15) as a white solid. Yield: 296 mg (41 %) H NMR (CDCl3,
600 MHz) δ 3.05 (d, J = 5.0 Hz, 3H), 3.94 (s, 3H), 7.31 (t, 1H, J
= 7.2 Hz), 7.60-7.65 (bs, 1H), 8.10 (d, J = 7.2 Hz,1H), 8.25 (d, J
= 7.2 Hz, 1H).13C NMR (CDCl3, 150 MHz) δ 27.1, 64.0, 123.8,
124.8, 128.2, 135.5, 136.6, 136.7, 158.4, 165.6. p-ESI HRMS
(MeOH) 210.0757 (calc. 210.0761 for C10H12N+).
[Eu(3,4,3-LI-IAM-1,2-HOPO)]C5H5NH Yield: 39 %. n-ESI
HRMS (MeOH) 979.2124 (calc. 979.2140, [EuC40H42N8O12]-). IR
(ν, cm-1) 1598, 1540 (s, C=O str); 1433 (m, CH2 def); 1180 (m,
C-OH, str); 801, 762, 696 (m, arH, pyH).
3,4,3-Li-1,2-HOPOBn (17) 1,2-HOPOBn thiazolide (16)
(1.78 g, 5.4 mmol) and DIPEA (0.87 ml, 0.66 g, 5.1 mmol) were
dissolved in DCM (100 mL) and added over 1 h to a solution of
[Tb(3,4,3-LI-IAM-1,2-HOPO)]C5H5NH Yield: quantitative.
n-ESI
HRMS
(MeOH)
985.2167
(calc.
985.2181,
6

ACCEPTED MANUSCRIPT
[TbC40H42N8O12]-). IR (ν, cm-1) 2969 (m, CH2 str); 1588, 1530 (s,
C=O str); 1433 (m, CH2 def); 1170 (m, C-OH, str); 801, 762, 696
(m, arH, pyH).
NMR (MeOD, 600 MHz) δ 1.40 (m, 2H); 1.59 (m, 2H); 2.28 (s,
3H); 3.28-3.40 (m, 4H); 6.48 (d, 1H); 6.64 (d, 1H); 6.88 (t, 1H);
7.38 (dt, 1H); 7.88 (m, 2H). p-ESI HRMS (MeOH) 415.1619
(calc. 415.1623, [C20H23N4O6]-). IR (ν, cm-1) 1637,1529 (s, C=O
str); 1429 (m, CH2 def); 1154 (m, C-O-H str); 804, 754 (m, arH).
[Eu(3,4,3-LI-1,2-HOPO-IAM)]PyH Yield: 78 %. n-ESI
HRMS (MeOH) 977.2106 (calc. 977.2126, [EuC40H42O12N8]–).
IR (ν, cm-1) 2969 (m, CH2 str); 1588, 1530 (s, C=O str); 1433 (m,
CH2 def); 1170 (m, C-OH, str); 801, 762, 696 (m, arH, pyH).
Eu(III) and Tb(III) complexes of 5LI-IAM-1,2-HOPO (22)
Due to the low quantity of ligand obtained, the complexes
were made in situ for photophysical measurements. Two equiv.
of ligand were dissolved in DMSO (0.01 M) and one equivalent
of LnCl3 solution in DMSO (0.05 M) was added. Subsequently,
ten equivalents of pyridine were added and the solutions left to
equilibrate at room temperature prior to mass spectrometric
analysis and photophysical measurements.
[Tb(3,4,3-LI-1,2-HOPO-IAM)]PyH Yield: 28 %. n-ESI
HRMS (MeOH) 985.2183 (calc. 985.2181, [TbC40H42O12N8]–).
IR (ν, cm-1) 2959 (m, CH2 str); 1598, 1520 (s, C=O str); 1433 (m,
CH2 def); 1151 (m, C-OH, str); 801, 753, 645 (m, arH, pyH).
[Eu(5LI-IAM-1,2-HOPO)2]- p-ESI HRMS (MeOH) 979.2262
(calc. 979.2283, [C40H44EuN8O12]-).
[Tb(5LI-IAM-1,2-HOPO)2]- p-ESI HRMS (MeOH) 987.2319
(calc. 987.2338, [C40H44TbN8O12]-).
2.3 Photophysical Measurements
Sample preparation for absorption, Фtot,
ε
and
τ
measurements: The complexes of the octadentate complexes
were dissolved in DMSO (1 mg/ml) or in situ generated in the
same solvent in case of the tetradentate complexes and then
diluted into tris(2-aminoethyl)amine (TRIS) buffered saline
(TBS) (20 mM buffer, pH 7.4, 100 mM NaCl) to a final
concentration of ~0.5 x 10-5 M. UV-visible absorption spectra
were recorded with an HP 8453 diode array UV-vis absorption
spectrometer. Relative quantum yields were determined by the
optically dilute method (eq (1)) using the same excitation
wavelength for both the reference (Quinine Sulfate, purchased
from Sigma-Aldrich, meets USP testing specifications) and
samples (λex = 330 nm) following a previously described protocol
in aqueous medium.[16, 31] Please note, that we used a
previously described protocol from our group here for
determination of Фtot for consistency. However, it should be
mentioned that optimized protocols are now available.[32-34]
Quinine sulfate (QS) in 0.5 M H2SO4 (Φ = 0.546) was used as the
aqueous fluorescence quantum yield standard.[31]
Scheme 3 Synthesis of 5LI-IAM-1,2-HOPO (22).
5LI-1,2-HOPOBn (20). 1,2-HOPOBn thiazolide (16) (1 g,
2.9 mmol) was dissolved in chloroform (150 mL) and added over
20 hours to a solution of 1,5-diaminopentane (1.3 g, 12.7 mmol)
in dichloromethane (5 mL) at room temperature using a syringe
pump. The solution was stirred overnight and subsequently
washed with 1M NaOH in 20% brine (3x 100 mL), brine (2x
100 mL) and then dried over sodium sulfate. The solvent was
removed in high vacuum and the pale brown oil was dried in high
1
vacuum for two days. Yield 650 mg, 68 %. H NMR (CDCl3) δ
1.26 (m, 2H); 1.36 (m, 2H); 1.45 (m, 2H); 2.57 (m, 2H); 3.29 (m,
2H); 5.25 (s, 2H); 6.39 (d, 1H); 6.63 (dd, 1H); 7.19-7.45 (m, 8H);
7.69 (s, 1H). 13C-NMR (150 MHz, CDCl3) δ (ppm) 24.0, 28.7,
30.2, 40.0, 41.4, 79.3, 106.6, 123.8, 127.9, 128.6, 129.4, 130.1,
138.8, 142.8, 158.1, 160.1. p-ESI HRMS (MeOH) 330.1811
(calc. 330.1812, [C18H24N3O3]+).
eq (1)
A is the absorbance at the excitation wavelength (λ) and E is
the integrated photoluminescence intensity. The subscripts ‘x’
and ‘QS’ refer to the sample and reference respectively. The plot
of integrated emission intensity (i.e. EQS) vs. absorbance at 330
nm (i.e. AQS) yields a linear plot with a slope which can be
equated to the quinine sulfate quantum yield ΦQS.[31]
Instrumentation details (HORIBA Jobin Yvon IBH FluoroLog-3
spectrofluorimeter) and protocols were the same as recently
published. Time-gated phosphorescence spectra of the in situ
generated gadolinium complexes were measured in
methanol:ethanol (1:4 v/v) at 77 K (λex = 330 nm, delay: 0.1 ms)
using the same HORIBA Jobin Yvon IBH FluoroLog-3
spectrofluorimeter. The Gd-triplet phosphorescence spectra were
fitted using Gaussian functions with OriginPro 6.1 software.[35,
36] The in situ preparation protocol was as follows: One
equivalent of 10 or 19 or two equivalents of 22 were dissolved in
MeOH (0.01 M), respectively, and one equivalent of GdCl3
hexahydrate solution in MeOH (0.05 M) was added.
Subsequently, ten equivalents of pyridine were added and the
solutions left to equilibrate at room temperature prior to mass
spectrometric analysis and photophysical measurements. To
confirm successful formation of the Gd(III) complexes in situ, n-
5LI-IAMBn-1,2-HOPOBn (21). 5LI-1,2-HOPOBn (20)
(125 mg, 0.38 mmol) and IAMMeNHBn thiazolide (6) (123 mg,
0.32 mmol) were dissolved in dichloromethane (5 mL) and
stirred at room temperature for 24 hours. The pale yellow
solution was then evaporated and the crude oil purified with
column chromatography (100% DCM, then 5% MeOH in DCM).
1
21 was obtained as a beige oil. Yield 67 mg, 35%. H NMR
(CDCl3) δ 1.29 (m, 2H); 1.31 (m, 2H); 1.50 (m, 2H); 2.91 (d,
3H); 3.29 (m, 4H); 4.82 (s, 2H); 5.22 (s, 2H); 6.30 (dd, 1H); 6.54
(d, 1H); 7.13-7.39 (m, 14H); 7.48 (d, 1H), 7.83 (dd, 2H). 13C-
NMR (150 MHz, CDCl3) δ (ppm) 23.2, 26.6, 27.6, 28.6, 39.7,
78.5, 79.4, 105.9, 123.7, 124.9, 128.4, 128.5, 128.7, 128.9, 129.2,
129.4, 130.2, 133.1, 133.3, 133.4, 135.2, 138.1, 142.9, 153.9,
158.5, 160.3, 165.6, 166.5. p-ESI HRMS (MeOH) 619.2511
(calc. 619.2527, [C34H36N4O6Na]+).
5LI-IAM-1,2-HOPO (22). 5LI-IAMBn-1,2-HOPOBn (21)
(67 mg, 0.11 mmol) was dissolved in a 1:1 mixture of
concentrated hydrochloric acid and glacial acetic acid (2 mL) and
stirred at room temperature for four days. The solvent was
removed in high vacuum and then the beige oil co-evaporated
1
with MeOH to yield 22 as a beige foam. Yield 47 mg, 97% H
7

ACCEPTED MANUSCRIPT
Figure 2 DFT optimized structures of the Europium complexes of the three mixed ligands.
ESI HRMS were recorded of the samples in methanol. Spectra
are included in the supporting information.
mode and for the complexes with octadentate ligands adducts
with HBr were found in the addition to the [ML]- species. The
free octadentate ligands also showed formation of HBr adducts.
The high affinity for anions has been previously observed for
IAM ligands and complexes like the BH(2,2)IAM cage.[25]
[Gd(5LI-IAM-1,2-HOPO)2]- n-ESI HRMS (MeOH) 986.2330
(calc. 986.2325, [C40H44GdN8O12]-).
[Gd(3,4,3-LI-IAM-1,2-HOPO)]- n-ESI HRMS (MeOH) 984.2172
(calc. 984.2169, [GdC40H42N8O12]-).
3.2. DFT calculations
[Gd(3,4,3-LI-1,2-HOPO-IAM)]- n-ESI HRMS (MeOH) 984.2172
Despite considerable efforts, no X-ray suitable crystals were
obtained for the complexes. We thus conducted geometry
optimizations to assess the ability of the ligands to fully
coordinate Ln(III) ions. Input structures were based on recently
published crystal structures of Eu(III) complexes of related 3,4,3-
LI-1,2-HOPO and 5LIO-1,2-HOPO ligands.[16, 37] The
structures are shown in Figure 2 and demonstrate the ability of
the ligands to shield the metal ions efficiently from solvent
molecules.
(calc. 984.2169, [GdC40H42N8O12]–).
3. Results and Discussion
3.1. Ligand and Complex Syntheses
The octadentate ligand syntheses, characterization and
purification proved to be challenging (Schemes 1 and 2), due to
1
1) rotamer formation (visible in the H-NMR spectra) of the 3,4-
3-LI-IAMBn and 3,4,3-LI-1,2-HOPOBn precursors, 2) the
decreased nucleophilicity of the secondary amines in 3,4-3-LI-
IAMBn and 3,4,3-LI-1,2-HOPOBn due to formation of
intramolecular hydrogen bonds and thus decreased reactivity in
the final coupling step. This has been previously observed in
spermine based, mixed 1,2-HOPO/catechol ligands, leading to
yields as low as 5%.[22] Thus a different activating mechanism
was considered and the coupling reagent TBTU yielded the best
results with up to 70% yield. 3) We observed partial cleavage of
the benzylic protecting groups which occurred during purification
of 7 and 17 and thus full NMR characterization was conducted
after deprotection with BBr3 (which was found to be superior to
HCl/AcOH deprotection for the octadentate ligands). The
tetradentate 5LI-IAM-1,2-HOPO ligand was obtained under high
dilution/slow addition conditions in moderate yield (Scheme 3).
The ligands and complexes were, among other methods,
characterized by HR-ESI-mass spectrometry and the
corresponding spectra can be found in the supporting
information. The complexes were observed in the negative ion
3.3. Photophysical characterization
Matching the triplet state of an antenna to the emitting state of
a lanthanide is one of the key factors in designing highly
luminescent reporter molecules.[14, 38] Although in theory the
singlet state can also transfer energy to the lanthanide ion, the
short lifetime makes this pathway less efficient and is thus
considered of little importance.[39, 40] In the IAM chromophore
for example the lowest singlet and triplet states are close in
energy (ΔS1-T0-0 = 850 cm-1) favoring a high rate of intersystem
crossing and an excitation via the triplet state.[14] For 1,2-HOPO
excitation via the triplet state is also considered to be the
dominating pathway.[41] In order to determine the triplet states
of the ligands, phosphorescence spectra were collected of the
gadolinium complexes of the mixed IAM-1,2-HOPO ligands at
77K (Figure 3). This ion lacks ligand accessible states for energy
transfer and thus only ligand centered phosphorescence is
observed form which one can deduct the ligand’s triplet state.
8

ACCEPTED MANUSCRIPT
this work, we observed for the tetradentate mixed
chromophore ligand 5LI-IAM-1,2-HOPO 22 two distinctly
separated peaks at 24300 and 21500 cm-1 in the
phosphorescence spectrum, that correspond well to the triplet
states in single chromophore (IAM or 1,2-HOPO) ligands. In
the mixed 3,4,3-LI-IAM-1,2-HOPO 10 ligand a similar
scenario is observed with both the IAM and 1,2-HOPO triplet
states (25300 and 21500 cm-1) being observed. In the 3,4,3-LI-
1,2-HOPO-IAM ligand 19, however, the IAM chromophore
phosphorescence is of low intensity (at 25200 cm-1). It is
suggested this is due to the importance for this chromophore to
reside on a primary amine (yielding a secondary amide bond a
with the spermine backbone) rather than on a secondary amine
of the backbone. Furthermore, due to the closely spaced triplet
states of 1,2-HOPO and IAM triplet-triplet ET might also be
possible (Figure 4). Interestingly, the absorbance spectrum of
[Eu(3,4,3-LI-IAM-1,2-HOPO)]- (dashed black line, Figure 5)
is blue shifted compared to the complexes with ligands 19 and
22, indicating a different singlet state energy for ligand 10. We
have also recorded the excitation spectra (Figure S10) and
while the complexes follow the same trend (10 blue shifted in
respect to complexes with 19 and 22), the maxima of the
excitation spectra of the complexes are all slightly red-sifted in
comparison to the absorbance spectra, which is in accordance
with previous data for the 3,4,3-LI-1,2-HOPO complex. The
1
0.8
0.6
0.4
0.2
0
400
450
500
550
600
Wavelength [nm]
Figure 3 Phosphorescence spectra at 77K in MeOH/EtOH (1:4).
[Gd(3,4,3-LI-1,2-HOPO)]- (purple, dashed line), [Gd(3,4,3-LI-
IAM-1,2-HOPO)]- (black, solid line), [Gd(3,4,3-LI-1,2-HOPO-
IAM)]- (red, solid line), [Gd(5LI-IAM-1,2-HOPO)]- (blue, solid
line), [Gd(5LI-IAM)]- (green, dashed line), [Gd(5LI-1,2-HOPO)]-
(orange, dashed line).
For completeness, the spectra of the 5LI-IAM, 5LI-1,2-HOPO
and 3,4,3-LI-1,2-HOPO complexes were also recorded under the
same conditions and are included in Figure 3. The herein
experimentally determined triplet state for the 5LI-IAM ligand
(24400 cm-1) is in good agreement with the values previously
reported and demonstrates why this chromophore is such an
excellent sensitizer for terbium (5D4 = 20500 cm-1, ΔT1-5D4 =
2750-3900 cm-1). In order to facilitate efficient energy transfer
(ET), the gap between the lanthanide emitting state and the
antenna’s triplet state should be greater than 2000 cm-1 otherwise
significant energy back transfer (EBT) can occur.
1.2
1
0.8
0.6
0.4
0.2
0
280
300
320
340
360
380
400
Wavelength [nm]
Figure 5 Absorbance spectra of Eu(III) (solid lines) and Tb(III)
(dashed lines) of the complexes with 5LI-IAM-1,2-HOPO (22)
(blue), 3,4,3-LI-1,2-HOPO-IAM (19) (red) and 3,4,3-LI-IAM-
1,2-HOPO (10) (black) in TBS, pH 7.4
1.2
Normalized Emission Intensity
0.1
Figure 4 Energy level diagram showing energy transfer pathways
from the different chromophores to the emissive states of Eu(III)
and Tb(III). The red arrows (→) indicate that ET can occur both
from the IAM and 1,2-HOPO triplet states for Eu(III) while for
Tb(III) excitation from IAM is proposed to be the main pathway
(→). In addition, Tb(III) can transfer energy back onto the 1,2-
HOPO triplet state after excitation by IAM. Energy back transfer
pathways not shown for clarity.
1
0.08
0.06
0.8
0.04
0.02
0.6
0
580
590
600
Wavelength [nm]
0.4
0.2
0
The triplet state of the previously reported octadentate 3,4,3-
LI-1,2-HOPO ligand is slightly higher (22400 cm-1) than the one
of 5LI-1,2-HOPO (21500 cm-1) but both ligands are well suitable
to act as antenna for europium (5D0 = 19030 cm-1, ΔT1-5D0 =
3370-2470 cm-1). While the IAM chromophore has been reported
600
640
680
720
as moderately efficient antenna for europium[42] (ΔT1-5D0
~
4800 cm-1), the 1,2-HOPO chromophore is not suited as terbium
Wavelength [nm]
sensitizer, due to the small energy gap (ΔT1-5D4 ~ 1000 cm-1). In
Figure 6 Europium emission spectra of the complexes in TBS,
pH 7.4, λex = 330 nm. Inset: close-up view of the MD-transition,
with the ligands 5LI-IAM-1,2-HOPO (22) (blue), 3,4,3-LI-1,2-
HOPO-IAM (19) (red) and 3,4,3-LI-IAM-1,2-HOPO (10)
(black).
9

ACCEPTED MANUSCRIPT
Table 1 Photophysical data of the complexes
ε
λmax
[nm]
bright-
ness
krad
τrad
Фtot
[%]
q
τH2O
[ms]
τD2O
[ms]
knonrad
[s-1]
T0-0
[M-1cm-1]
complex
ηEu
ηsens
[s-1]
[µs]
[cm-1]
[M−1cm−1
]
[Eu(5LI-IAM-1,2-HOPO)2]-
[Tb(5LI-IAM-1,2-HOPO)2]-
502
-
1992
-
0.357
-
0.119
-
4.3
0.5
0.2
-
0.71
-
1.08
-
904
-
24004
21024
335
334
103
24300,
21500
11
9
18814
334
[Eu(3,4,3-LI-1,2-HOPO-IAM)]-
[Tb(3,4,3-LI-1,2-HOPO-IAM)]-
[Eu(3,4,3-LI-IAM-1,2-HOPO)]-
472
-
2119
-
0.308
-
0.016
-
0.5
0
0.0
-
0.65
-
0.79
-
1061
-
25200,
21321
16987
16171
333
326
-
3
478
2093
0.242
0.009
0.2
0.3
0.51
0.71
1495
25300,
21500
[Tb(3,4,3-LI-IAM-1,2-HOPO)]-
[Eu(3,4,3-LI-1,2-HOPO)]- [16]
[Eu(5LI-1,2-HOPO)2]- [41]
[Tb(5LI-IAM)2]- [42]
-
-
-
-
2.3
-
-
-
-
16998
17700
18800
25100
325
315
331
337
39
276
389
903
586
620
-
1705
1476
-
0.469
0.490
-
0.397
0.420
-
15.6
20.7
36.0
0.04
0.05
-0.1
0.81
0.74
2.52
1.14
0.99
2.81
623
740
-
22400
21500
24400
emission spectra recorded are shown in Figure 6 for Eu(III) and
in the supporting information for Tb(III) (Figure S11). The
former complexes show pure red luminescence due to the band
~612 nm. Interestingly the complex with 3,4,3-LI-IAM-1,2-
HOPO (10) shows a shift and significant broadening of the
The lifetime measurements for the europium complexes
revealed that no water molecules are bound to the complexes in
aqueous solution,[46] and that all three ligands are effective in
shielding the complexes from quenching water molecules. This is
in line with the DFT geometry optimizations described above. It
should be noted, however, that the decay of the octadentate
Eu(III) complexes could be better fit to a bi-exponential decay,
and suggested the presence of a small portion of a short lived
species. The presence of multiple species is not unusual has been
previously reported for octadentate 1,2-HOPO ligands.[48]
Further analysis of the europium emission data was conducted
after previously reported methods by Beeby et al and Werts et al,
[49, 50] where Фtot is the product of the europium centered
5
hypersensitive D0-7F2 transition, indicating a different geometry
around the Eu ion with this ligand.[43] Photoluminescence
quantum yield (Фtot) measurements of the europium and terbium
complexes of the mixed ligands revealed that the ligands were
poor antennas when 1,2-HOPO and IAM chromophores were
combined. The tetradentate 5LI-IAM-1,2-HOPO ligand was
better in sensitizing europium with a Фtot of 4.3% than terbium
(0.5%) but inferior to 5LI-1,2-HOPO (Eu Фtot = 21%) and 5LI-
IAM (Tb Фtot = 36%).[44, 45] The 3,4,3-LI-1,2-HOPO-IAM
ligand was better than the 3,4,3-LI-IAM-1,2-HOPO ligand in
sensitizing europium (Eu Фtot = 0.5 and 0.2%, respectively). A
vice versa trend was observed for terbium (Tb Фtot = 0 and 2.3%,
respectively). This is attributed to the fact that the chromophore
bound to the secondary amines in the spermine backbone plays a
less important role as sensitizer which was shown previously in
the 3,4,3-LI-1,2-HOPO ligand, where the R2N-1,2-HOPO
chromophore is less efficient in sensitizing Eu than the RNH-1,2-
HOPO chromophore.[16] We propose that the poor ability of the
ligands to sensitize terbium is due to two main pathways. Firstly,
the energy of the IAM chromophore could be partially transferred
to the 1,2-HOPO chromophore which lies too close to the
emitting state of terbium. Secondly, when IAM transfers the
energy onto terbium, EBT to the 1,2-HOPO causes non-radiative
quenching, which is proposed to be the main pathway. Moreover,
the triplet state population of the IAM chromophore is negligible
(Figure 3, red spectrum) and explains further why the 3,4,3-LI-
1,2-HOPO-IAM ligand is not suitable as terbium antenna. To
rule out that the low Фtot of the mixed ligand complexes are due
to non-radiative quenching from water molecules, we conducted
lifetime measurements in TBS and D2O. Quenching of lanthanide
luminescence by vibrational modes of coordinating solvent
molecules is an important factor when designing luminescent
probes. Since O-D oscillators are less likely to contribute to non-
radiative quenching, the number of water molecules (q) can be
deducted from the lifetimes in water (τH2O) and deuterated water
(τD2O), respectively, and using the empirically derived equation
(2) from Horrocks et al.[46, 47]
efficiency (ηEu) and the efficiency of the ligand (ηsens
)
eq (4)
eq (5)
The europium centred efficiency, or sometimes also called
intrinsic quantum yield, is significantly (25-50%) lower in the
mixed complexes than in complexes with 5LI-1,2-HOPO.
Simultaneously, the ligand centred sensitization efficiency (ηsens
)
is lower and follows a certain trend: 5LI-IAM-1,2-HOPO (22) >
3,4,3-LI-1,2-HOPO-IAM (19) > 3,4,3-LI-IAM-1,2-HOPO (10).
The almost two-fold higher ηsens in 19 compared to 10 confirms
the previous observation that the R2N-1,2-HOPO chromophore is
less efficient in sensitizing Eu than the RNH-1,2-HOPO
chromophore.[16] The radiative lifetime (τrad) is the
approximated lifetime in the absence of non-radiative processes
and can be obtained from the estimated radiative lifetime
constant krad
in s-1
eq (6)
AMD,0 is the spontaneous emission probability of the magnetic
dipole transition (5D0→7F1) and is equal to 14.65 s-1, refers to
the refractive index of the medium (here, water = 1.334) and the
last term is the ratio of integrated emission intensity of the total
5
emission (5D0→7FJ, J = 0-6) divided by the integrated D0→7F1
emission (580-600 nm).[50-53] Please note, due to experimental
limitations the 5D0→7F5 and 5D0→7F6 transitions were not
recorded and we stress that the total emission intensity Itot is thus
only a low estimate. Shortening τrad has been discussed as being
one of the most important steps in generating highly emissive
europium complexes.[43, 54] The data shown in Table 1
eq (2)
eq (3)
in ms-1
10

ACCEPTED MANUSCRIPT
confirms this claim. Finally, the rate of non-radiative decay
(knonrad) can be deducted from the lifetime in water (τH2O) and krad.
Note Some of this technology is licensed to Lumiphore, Inc. in
which KNR has a financial interest.
in s-1 eq (7)
Supplementary Material
Compared to the 3,4,3-LI-1,2-HOPO ligand, non-radiative
decay rates of the two mixed octadentate ligands are twofold
enhanced.[16] Keeping in mind that quenching through water
molecules is not the main pathway; the data in Table 1 suggests
that the antenna itself contributes to the quenching of the
luminescence. ET between the closely spaced triplet states (and
possibly also the singlet states) of IAM and 1,2-HOPO and EBT
between europium and the two chromophores leads to the poor
photophysical performance of the complexes. This result is
surprising as previously groups have reported the use of
synergistic two-antenna systems, where either a protein serves as
first antenna which then transferred the energy onto an organic
chromophore or where two chromophores (e.g. hfac and N,N-
dimethyl-N,N-bis(2-hydroxy-3,5-
ESI-Mass spectra of the Eu(III), Tb(III) and Gd(III)
complexes, additional emission spectra and excitation spectra are
available in the supporting information.
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We report the first synthesis of three ligands incorporating
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supported by the NSF CHE-0233882 and CHE-0840505 grants.
The authors thank Dr. Nicola Alzakhem for helpful discussions.
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Highlights
•
•
•
2-Hydroxisophthalamide (IAM) and 1,2-hydroxypyridonate (HOPO) mixed ligands are prepared
Photophysical properties of Tb and Eu complexes are reported
When bound to a primary amine the above chromophores are better in sensitizing Tb and Eu
12