Optimization of the Sensitization Process and Stability of Octadentate Eu(III) 1,2-HOPO Complexes

Article abstract of DOI:10.1021/acs.inorgchem.5b00748

The synthesis of a series of octadentate ligands containing the 1-hydroxypyridin-2-one (1,2-HOPO) group in complex with europium(III) is reported. Within this series, the central bridge connecting two diethylenetriamine units linked to two 1,2-HOPO chromophores at the extremities (5-LIN-1,2-HOPO) is varied from a short ethylene chain (H(2,2)-1,2-HOPO) to a long pentaethylene oxide chain (H(17O5,2)-1,2-HOPO). The thermodynamic stability of the europium complexes has been studied and reveals these complexes may be effective for biological measurements. Extension of the central bridge results in exclusion of the inner-sphere water molecule observed for [Eu(H(2,2)-1,2-HOPO)]^- going from a nonacoordinated to an octacoordinated Eu(III) ion. With the longer chain length ligands, the complexes display increased luminescence properties in aqueous medium with an optimum of 20% luminescence quantum yield for the [Eu(H(17O5,2)-1,2-HOPO)]^- complex. The luminescence properties for [Eu(H(14O4,2)-1,2-HOPO)]^- and [Eu(H(17O5,2)-1,2-HOPO)]^- are better than that of the model bis-tetradentate [Eu(5LIN^Me-1,2-HOPO)₂]^- complex, suggesting a different geometry around the metal center despite the geometric freedom allowed by the longer central chain in the H(mOn,2) scaffold. These differences are also evidenced by examining the luminescence spectra at room temperature and at 77 K and by calculating the luminescence kinetic parameters of the europium complexes. (Graph Presented).

Full text of DOI:10.1021/acs.inorgchem.5b00748

Article
pubs.acs.org/IC
Optimization of the Sensitization Process and Stability of
Octadentate Eu(III) 1,2-HOPO Complexes
Anthony D’Aleo, Evan G. Moore, Jide Xu, Lena J. Daumann, and Kenneth N. Raymond*
́
Department of Chemistry, University of California, Berkeley, California 94720-1460, United States
Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: The synthesis of a series of octadentate ligands
containing the 1-hydroxypyridin-2-one (1,2-HOPO) group in
complex with europium(III) is reported. Within this series, the
central bridge connecting two diethylenetriamine units linked
to two 1,2-HOPO chromophores at the extremities (5-LIN-1,2-
HOPO) is varied from a short ethylene chain (H(2,2)-1,2-
HOPO) to a long pentaethylene oxide chain (H(17O5,2)-1,2-
HOPO). The thermodynamic stability of the europium
complexes has been studied and reveals these complexes may
be effective for biological measurements. Extension of the
central bridge results in exclusion of the inner-sphere water
molecule observed for [Eu(H(2,2)-1,2-HOPO)]− going from a
nonacoordinated to an octacoordinated Eu(III) ion. With the longer chain length ligands, the complexes display increased
luminescence properties in aqueous medium with an optimum of 20% luminescence quantum yield for the [Eu(H(17O5,2)-1,2-
HOPO)]− complex. The luminescence properties for [Eu(H(14O4,2)-1,2-HOPO)]− and [Eu(H(17O5,2)-1,2-HOPO)]− are
better than that of the model bis-tetradentate [Eu(5LINMe-1,2-HOPO)2]− complex, suggesting a different geometry around the
metal center despite the geometric freedom allowed by the longer central chain in the H(mOn,2) scaffold. These differences are
also evidenced by examining the luminescence spectra at room temperature and at 77 K and by calculating the luminescence
kinetic parameters of the europium complexes.
detection,15,16 and for use in high-throughput screening.17,18
INTRODUCTION
■
For instance, several Ln(III) chelates (with Ln = Tb or Eu) are
commercially available (e.g., Lance, PerkinElmer; Lan-
thaScreen, Invitrogen, CisBio), and fluorescent assay platforms
such as Dissociation-Enhanced Lanthanide Fluorescent Im-
muno-Assay (DELFIA)19 are well developed, offering increased
sensitivity compared to colorimetric assay formats such as the
Enzyme-Linked Immuno-Sorbent Assay (ELISA).20 Since these
complexes are also expected to be useful in FRET-type
experiments,21 high brightness together with good aqueous
solubility and stability are an added requirement.10,22,23
It has been shown that octadentate 1-hydroxypyridin-2-one
(1,2-HOPO) ligands hold high promise for biological
applications and in radionuclide decorporation.24−28 Here, we
report on the synthesis, thermodynamic stability, and photo-
physical properties of several octadentate 1,2-HOPO deriva-
tives with the aim of increasing the luminescent properties of
the respective Eu(III) complexes in aqueous solution. The
ligands are composed of two diethylamine units which bridge
two 1,2-HOPO moieties connected by a tertiary nitrogen atom
(e.g., 5LINMe-1,2-HOPO). For the octadentate ligands, two of
such units are connected by either aliphatic or oligo-ethylene
The dangers and drawbacks inherent with radioactivity-based
biological assay methods together with the low sensitivity of
MRI agents have yielded a major shift toward luminescence
measurements and visualization techniques due to their
demonstrated and dramatically increased sensitivity.1−3 In
such techniques, the low background signal and the wide
variety of detection wavelengths make these measurements
highly appealing.3 For coordination compounds to be utilized
for biological luminescence purposes, several parameters have
to be optimized.4−7 First, the observed emission should display
insensitivity toward the environment. This is especially
important for labeling applications with biomolecules.
Furthermore, in addition to high thermodynamic and kinetic
stability, to prevent unwanted release of metal, the complex
should have high overall luminescence quantum yield and
brightness. The latter parameter can be determined from the
product of the luminescence quantum yield and the molar
absorption coefficient. In this respect, lanthanide ions are
appealing since they possess intrinsic long-lived excited state
lifetimes and can be tuned to have high luminescence quantum
yields.8−10 For these reasons, their use has now spread to
traditional clinical environments,11 and applications have grown
from their more traditional use as chemical shift reagents12−14
toward clinical assays for DNA sequencing,10 for antioxidant
Received: April 2, 2015
Published: July 7, 2015
© 2015 American Chemical Society
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DOI: 10.1021/acs.inorgchem.5b00748
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Article
a
Scheme 1. Synthesis of H(m,2)-1,2-HOPO
a
Reagents and conditions: (a) 1,2-HOPOBn-thiaz, Et3N, DCM, RT, 16 h; (b) conc HCl/glacial HOAc (1:1), 3 days, RT.
a
Scheme 2. Synthesis of H(mOn,2)-1,2-HOPO
a
Reagents and conditions: (a) MsCl, Et3N, DCM, 0 °C to RT, 4 h; (b) NaN3, EtOH, 80 °C, 16 h; (c) H2 (500 psi), 5−10% Pd/C, 8 h; (d) Z-
aziridine, tert-butyl alcohol, 80 °C, 16 h; (e) H2 (500 psi), 5−10% Pd/C, 8 h; (f) 1,2-HOPOBn acid chloride, K2CO3, H2O/DCM biphasic, 8 h; (g)
conc HCl/glacial HOAc (1:1), 3 days, RT.
F254 plates. Flash chromatography was performed using EM Science
Silica Gel 60 (230−400 mesh). NMR spectra were obtained using
either a Bruker AM-300 or a DRX-500 spectrometer operating at 300
glycol chains (resulting in four 1,2-HOPO units which compose
the octadentate ligand topology). The stability of such
complexes has been determined and shows that these
complexes are stable in aqueous medium, allowing measure-
ments at submicromolar concentration. The luminescence
properties in terms of their molar absorption coefficients,
luminescence quantum yields, luminescence lifetimes, and
brightness are investigated, together with the pattern of the
Eu(III) emission spectra both at room temperature and at 77 K.
1
1
(75) and 500 (125) 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 (δ 77.0), δ 2.49 (δ 39.5), and δ 3.31 δ (49.0),
respectively, for CDCl3, (CD3)2SO, and CD3OD, while coupling
constants (J) are reported in Hertz. The following standard
1
abbreviations are used for characterization of H NMR signals: s =
singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m =
multiplet, dd = doublet of doublets. Both low-resolution mass (FAB)
and high-resolution mass (HRESI) spectra were obtained from the
Micromass/Analytical Facility operated by the College of Chemistry,
University of California, Berkeley, CA. Elemental analyses were
performed by the Microanalytical Laboratory, University of California,
Berkeley, CA.
EXPERIMENTAL SECTION
■
General. All of the reagents and solvents used were of analytical
grade and purchased commercially; H(2,2)-1,2-HOPO and 5LINMe
-
1,2-HOPO were prepared as reported elsewhere.29 Thin-layer
chromatography (TLC) was performed using precoated Kieselgel 60
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CAUTION! Some of the syntheses reported herein involve the use of
organic azides. These are potentially explosive, and appropriate care should
be taken when handling the respective compounds.
filtrate was evaporated to dryness to leave a pale yellow oil as product.
Yield: 0.23 g (84%). 1H NMR (500 MHz, CDCl3): δ 0.84 (t, J = 5 Hz,
4H), 0.90 (t, J = 5 Hz, 8H), 1.10 (t, J = 5 Hz, 8H), 1.66 (t, J = 5 Hz,
4H). 13C NMR (125 MHz, CDCl3): δ 38.6, 53.4, 53.9, 70.1. MS (FAB
+) (m/z): calcd for [C12H33N6O]+ 277.3, found 277.3.
Synthesis. The syntheses of H(m,2)-1,2-HOPO and H(mOn,2)-
1,2-HOPO ligands are shown in Schemes 1 and 2, respectively.
H(3,2)-1,2-HOPOBn (6b). To a solution of 1,2-HOPOBn-
thiazolide26 (1.38 g, 4 mmol) and Et3N (0.4 g, 4 mmol) in dry
dichloromethane (30 mL) was added neat H(3,2)-amine (5b) (220
mg, 0.9 mmol). The mixture was stirred overnight, the solvent was
then removed, and the residue was loaded onto a flash silica column.
Elution with 2−6% methanol in dichloromethane allows the
separation of the benzyl-protected precursor as pale yellow oil (0.73
g, 71% based on amine). 1H NMR (500 MHz, CDCl3): δ 1.03 (s, 2H),
1.87 (s, 4H), 2.13 (s, 8H), 3.08 (s, br, 8H), 5.22 (s, 8H), 6.15 (d, J = 6
Hz, 4H), 6.52 (d, J = 9 Hz, 4H), 7.15 (t, J = 9 Hz, 4H), 7.30−7.34 (m,
12H), 7.44 (m, 8H), 7.49 (s, 4H). 13C NMR (125 MHz, CDCl3): δ
25.3, 37.6, 51.2, 52.8, 79.4, 105.2, 123.5, 128.5, 129.4, 133.2, 138.1,
142.9, 158.4, 160.7, 162.2. MS (FAB+) (m/z): calcd for
[C63H67N10O12]+ 1155.6, found 1155.6.
H(3,2)-1,2-HOPO (7b). Compound 6b (0.87 g, 0.75 mmol) was
dissolved in concentrated HCl (12 M)/glacial acetic acid (1:1, 20 mL)
and stirred at room temperature for 3 days. Filtration followed by
removal of the solvent gives a beige residue, which was washed with
ether to give the product (0.52 g, 87%) as a beige solid. 1H NMR (500
MHz, DMSO-d6): δ 1.67 (s, br, 2H), 2.70−2.85 (m, 12H), 3.07 (s, br,
4H), 5.97 (dd, J = 6 Hz, 4H), 6.02 (dd, J = 9 Hz, 4H), 6.67 (dd, J = 9
Hz, 4H). 13C NMR (500 MHz, CD3OD): δ 20.5, 36.3, 51.7, 54.7,
109.9, 121.5, 138.8, 140.7, 160.3, 163.8. Anal. Calcd for C35H42N10O12·
2HCl·1.5H2O: C, 46.98; H, 5.29; N, 15.66. Found: C, 47.19; H, 5.22;
N, 15.55. MS (ESI−) (m/z): calcd for [C35H41N10O12]− 793.3, found
793.1.
H(5O,2)-1,2-HOPOBn (6d). To a mixture of compound 5d (0.14 g,
0.5 mmol) and 30% potassium carbonate solution (5 mL) in
dichloromethane (20 mL) with cooling by an ice bath, a solution of
1,2-HOPOBn acid chloride from 0.75 g (3 mmol) of 1,2-HOPOBn
acid in dry dichloromethane (35 mL) was added dropwise in 2 h with
vigorous stirring. The mixture was warmed to room temperature with
stirring, until TLC indicated the reaction was complete. The organic
phase was separated and loaded on a flash silica column. Elution with
2−7% methanol in dichloromethane allows the separation of the
benzyl-protected precursor H(5O,2)-1,2-HOPOBn (0.42g, 71% based
1
on the free amine) as a thick pale yellow oil. H NMR (300 MHz,
CDCl3): δ 2.14 (s, 4H), 2.32 (s, 8H), 2.83 (s, 4H), 3.06 (s, 8H), 5.15
(s, 8H), 6.05 (s, 4H), 6.34 (s, 4H), 7.04 (s, 4H), 7.20 (s, 12H), 7.32 (s,
8H). 7.63 (s, 4H). 13C NMR (75 MHz, CDCl3): δ 37.3, 52.0, 52.7,
78.8, 104.8, 122.9, 128.1, 128.9, 129.7, 133.1, 138.0, 143.0, 158.1,
160.3. MS (FAB+) (m/z): calcd for [C64H69N10O13]+ 1185.5, found
1185.6.
H(5O,2)-1,2-HOPO (7d). H(5O,2)-1,2-HOPOBn was deprotected
with concentrated HCl (12 M)/glacial acetic acid (1:1) as mentioned
above for deprotecting 6b. The ligand was obtained as a beige solid.
Yield: 81%. 1H NMR (500 MHz, DMSO-d6): δ 3.40 (s, 8H), 3.52 (s,
4H), 3.70 (s, 8H), 3.86 (s, 4H), 6.41 (d, J = 6 Hz, 4H), 6.60 (d, J = 9
Hz, 4H), 7.40 (d, J = 9 Hz, 4H), 9.11 (t, 2H, J = 6 Hz), 10.48 (s, 4H).
13C NMR (75 MHz, CD3OD): δ 36.7, 55.0, 55.4, 66.3, 110.1, 121.6,
138.9, 141.4, 160.6, 163.9. Anal. Calcd for C36H44N10O13·2HCl·H2O:
C, 47.22; H, 5.28; N, 15.30. Found: C, 47.54; H, 5.35; N, 14.95. MS
(FAB+) (m/z): calcd for [C36H45N10O13]+ 825.3, found 825.3.
H(8O2,2)-CBZ (4e). This compound was prepared by the similar
procedure as described for compound 4d except 2-[2-(2-amino-
ethoxy)-ethoxy]-ethylamine (0.15 g, 1 mmol) was used instead of
5LIO-amine. Separation and purification were performed as described
above. H(8O2,2)-CBZ was obtained as a pale beige thick oil. Yield:
H(4,2)-1,2-HOPOBn (6c). This compound was prepared by the
similar procedure as compound 6b, except H(4,2) amine (5c 234 mg,
0.9 mmol) was used instead of H(3,2) amine (5b). Separation and
purification were performed as described above. The benzyl-protected
precursor was obtained as a pale yellow oil (0.72 g, 68% based on
1
amine). H NMR (500 MHz, CDCl3): δ 0.85 (s, 4H), 1.95 (s, 4H),
1
2.29 (s, 8H), 3.15 (s, br, 8H), 5.24 (s, 8H), 6.18 (s, 4H), 6.50 (d, J = 9
Hz, 4H), 7.14 (m,4H), 7.30−7.34 (m, 12H), 7.43−7.50 (m, 12H). 13C
NMR (125 MHz, CDCl3): δ 23.9, 37.4, 52.1, 53.0, 78.8, 105.0, 122.9,
128.1, 128.9, 129.7, 133.1, 137.8, 142.9, 158.1, 160.3. MS (FAB+) (m/
z): calcd for [C64H69N10O12]+ 1169.5, found 1169.5.
0.64 g, 74%. H NMR (300 MHz, CDCl3): δ 2.53 (s, 12H), 3.16 (s,
8H), 3.23 (s, 4H), 3.35 (s, 4H), 5.03 (s, 8H), 7.28 (s, 20H). 13C NMR
(75 MHz, CDCl3): δ 38.8, 52.6, 53.6, 66.3, 69.2, 69.9, 127.8, 128.0,
128.3, 136.6, 156.5. MS (FAB+) (m/z): calcd for [C46H61N6O10]+
857.4, found 857.5.
H(4,2)-1,2-HOPO (7c). Compound 6c (0.49 g, 0.42 mmol) was
deprotected with concentrated HCl (12 M)/glacial acetic acid (1:1) as
described for deprotection of 6b. Compound 7c was obtained as a
beige solid (0.36 g, 90%). 1H NMR (500 MHz, DMSO-d6): δ 1.81 (s,
br, 4H), 3.15 (s, 4H), 3.25 (s, 8H), 3.55 (s, 8H), 6.43 (d, J = 6 Hz,
4H), 6.59 (d, J = 9 Hz, 4H), 7.40 (d, J = 9 Hz, 4H), 9.13 (t, J = 5.6 Hz,
4H), 10.96 (s, br, 4H). 13C NMR (100 MHz, CD3OD): δ 22.1, 36.3,
49.8, 54.3, 109.7, 121.7, 139.1, 140.9, 160.3, 163.7. Anal. Calcd for
C36H44N10O12·2HCl·2.5H2O: C, 46.78; H, 5.56; N, 15.15. Found: C,
46.82; H, 5.23; N, 14.89. MS (ESI−) (m/z): calcd for
[C36H43N10O12]− 807.3, found 807.3.
H(8O2,2)-Amine (5e). This compound was prepared by the similar
procedure for preparing compound 5d except compound 4e (0.86 g, 1
mmol) was used instead of compound 4d. A pale yellow oil was
1
obtained as the product, yield 0.27 g (85%). H NMR (300 MHz,
D2O): δ 2.49 (t, J = 5 Hz, 4H), 2.54 (t, J = 5 Hz, 8H), 2.78 (t, J = 5
Hz, 8H), 3.34 (t, J = 5 Hz, 4H), 3.40 (t, J = 5 Hz, 4H); 13C NMR (125
MHz, D2O): δ 36.9, 51.3, 51.7, 68.1, 69.3; MS (FAB+) (m/z): Calcd
for [C14H37N6O2]+ 321.3, Found: 321.3
H(8O2,2)-1,2-HOPOBn (6e). This compound was prepared by a
similar procedure for preparing compound 6d, except compound 5e
(0.5 mmol) was used instead of compound 5d. Separation and
purification were performed as described above. The benzyl-protected
precursor 6e (0.41g, 68% based on the free amine) was obtained as a
H(5O,2)-CBZ (4d). 2,2′-Oxybis(ethan-1-amine) (5LIO-amine) (0.21
g, 2 mmol) and benzyl aziridine-1-carboxylate (1.77 g, 10 mmol) were
mixed in tert-butanol (30 mL) at room temperature under N2. The
mixture was stirred under an N2 atmosphere at 80 °C for 16 h, when
TLC showed the completeness of the reaction. The volatiles were
removed under vacuum, and the residue was dissolved in dichloro-
methane. The appropriate fractions of a gradient flash silica gel column
(1−7% methanol in dichloromethane) were collected and evaporated
1
thick pale yellow oil. H NMR (300 MHz, CDCl3): δ 2.31 (s, 4H),
2.42 (s, 8H), 2.63 (s, 4H), 2.85 (s, 4H), 3.14 (s, 8H), 5.32 (s, 8H),
6.20 (d, J = 6 Hz, 4H), 6.48 (d, J = 9 Hz, 4H), 7.08 (s, 4H), 7.34 (s,
16H), 7.50 (s, 8H). 13C NMR (75 MHz, CDCl3): δ 37.4, 52.2, 53.0,
68.8, 69.0, 79.0, 104.9, 123.0, 129.1, 130.1, 133.3, 138.2, 143.2, 158.2,
160.4. MS (FAB+) (m/z): calcd for [C66H73N10O14]+ 1229.5, found
1229.7.
1
to dryness to give a pale beige thick oil. Yield: 1.28 g, 79%. H NMR
(300 MHz, CDCl3): δ 2.53 (s, br, 12H), 3.17 (s, br, 4H), 3.83 (s, br,
8H), 5.04 (s, br, 8H), 7.29 (s, br, 20H). 13C NMR (75 MHz, CDCl3):
δ 38.8, 53.0, 53.6, 69.3, 128.0, 128.1, 128.4, 136.6, 156.4. MS (FAB+)
(m/z): calcd for [C44H57N6O9]+ 813.4, found 813.5.
H(5O,2)-Amine (5d). H(5O,2)CBZ (4d 0.83 g, 1 mmol) and 0.1 g
of 5% Pd/C catalyst were combined in methanol (25 mL). The
mixture was hydrogenated (500 psi pressure, room temperature)
overnight in a Parr bomb. After removing the catalyst by filtration, the
H(8O2,2)-1,2-HOPO (7e). Compound 6e was deprotected with
concentrated HCl (12 M)/glacial acetic acid (1:1) as mentioned above
for preparing compound 7b. An off-white solid was obtained as
product. Yield: 80%. 1H NMR (500 MHz, DMSO-d6): δ 3.36 (s, 8H),
3.47 (s, 4H), 3.62 (s, 4H), 3.67 (q, J = 5 Hz, 8H), 3.85 (s, 4H), 6.43
(dd, J = 6 Hz, 4H), 6.60 (d, J = 9 Hz, 4H), 7.41 (d, J = 9 Hz, 4H), 9.11
(t, J = 5.6 Hz, 2H), 10.56 (s, 4H). 13C NMR (125 MHz, CD3OD): δ
36.4, 49.3, 55.0, 66.0, 71.5, 110.2, 121.0, 139.0, 141.1, 159.9, 163.2.
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Anal. Calcd for C38H48N10O14·2HCl·2H2O: C, 46.68; H, 5.57; N,
14.32. Found: C, 46.69; H, 5.71; N, 13.98. MS (FAB+) (m/z): calcd
for [C38H49N10O14]+ 869.3, found 869.3.
3.56 (s, 8H), 3.65 (s, 8H), 3.82 (s, 4H), 6.41 (d, J = 7.0 Hz, 4H), 6.61
(d, J = 9.0 Hz, 4H), 7.41 (d, J = 8.0 Hz, 4H), 9.09 (s, 4H). 13C NMR
(75 MHz, CD3OD): δ 36.5, 54.9, 66.1, 71.3, 71.5, 111.7, 120.7, 140.4,
141.8, 160.0, 162.9. Anal. Calcd for C40H52N10O15·2HCl·5H2O: C,
44.65; H, 5.99; N, 13.02. Found: C, 44.63; H, 5.98; N, 12.74. MS
(FAB+) (m/z): calcd for [C40H53N10O15]+ 913.4, found 913.3.
Pentaethylene Glycol Dimesylate (1g). This compound was
prepared by the similar procedure as described for compound 1f,
except pentaethylene glycol (0.5 mmol) was used instead of
tetraethylene glycol. Separation and purification were performed as
described above. A pale yellow thick oil was obtained as product. Yield:
Tetraethylene Glycol Dimesylate (1f). To a solution of 3.9 g (20
mmol) of tetraethylene glycol and 4 g of Et3N (2.0 equiv) in dry
dichloromethane (30 mL) at 0 °C under N2 atmosphere was added 3
mL of methanesulfonyl chloride in dry dichloromethane (10 mL) via a
Teflon cannula with a glass capillary tip over 30 min. The reaction
mixture was stirred at this temperature for 4 h and then treated with
30 mL of a cold saturated aqueous NaHCO3 solution. The mixture
was extracted with 3 × 30 mL of dichloromethane. The combined
organic layers were dried (MgSO4) and evaporated to give a crude
product that was purified by column chromatography (2−7%
methanol/dichloromethane). Yield: 6.3 g (90%) colorless thick oil.
1H NMR (300 MHz, CDCl3): δ 3.06 (s, 6H), 3.63 (m, 8H), 4.18 (m,
4H). 13C NMR (75 MHz, CDCl3): δ 36.8, 68.2, 69.1, 69.7, 69.8. MS
(FAB+) (m/z): calcd for [C10H23O9S2]+ 351.1, found 351.1.
1
90%. H NMR (300 MHz, CDCl3): δ δ 3.09 (s, 6H), 3.64 (m, 12H),
3.77 (m, 4H), 4.38 (m, 4H). 13C NMR (75 MHz, CDCl3): δ 37.4,
67.8, 69.0, 69.8, 69.9. MS (FAB+) (m/z): calcd for [C12H27O10S2]+
395.1, found 395.1.
Pentaethylene Glycol Diazide (2g). This compound was prepared
by the similar procedure as described for compound 2f, except
pentaethylene glycol dimesylate was used instead of tetraethylene
glycol dimesylate. Separation and purification were performed as
described above. Colorless thick oil was obtained as product. Yield:
Tetraethylene Glycol Diazide (2f). A solution of 1f (3.5 g, 10
mmol) and sodium azide (2.2 equiv) in 50 mL of ethanol was heated
at reflux for 8 h. After cooling to room temperature, the ethanol was
removed in vacuo, and the remaining mixture was diluted with 100 mL
of dichloromethane. The solution was washed twice with water (50
mL), dried over anhydrous sulfate, and concentrated in vacuo to give
the crude product, which was purified by silica gel chromatography
eluting with a gradient of 2−5% methanol in dichloromethane to
afford the product as colorless oil (1.9 g). Yield: 77% based on
1
77% based dimesylate. H NMR (300 MHz, CDCl3): δ 3.34 (t, J = 5
Hz, 4H), 3.62 (m, 16H). 13C NMR (75 MHz, CDCl3): δ 50.5, 69.9,
70.5. MS (FAB+) (m/z): calcd for [C10H21N6O4]+ 289.2, found 289.2.
Pentaethylene Glycol Diamine (3g). Compound 3g was prepared
by catalytical hydrogenation as described for compound 5f, except
compound 2g was used instead of 2f. Yield: 80%. 1H NMR (300 MHz,
CDCl3): δ 1.11 (s, 4H), 2.52 (t, J = 5 Hz, 4H), 3.17 (t, J = 5 Hz, 4H),
3.33 (m, 12H). 13C NMR (75 MHz, CDCl3): δ 41.3, 69.1, 69.8, 72.9.
MS (FAB+) (m/z): Calcd for [C10H25N2O4]+ 237.2, Found: 237.2.
H(14O4,2)-Cbz (4g). This compound was prepared by the similar
procedure as described for preparing compound 4d, except penta-
ethylene glycol diamine (0.47 g, 2 mmol) was used instead of 5LIO-
amine. Separation and purification were performed as described abve.
Compound 4g was obtained as a beige thick oil. Yield: 1.5 g (79%
based on diamine). 1H NMR (300 MHz, CDCl3): δ 2.60 (t, J = 5 Hz,
12H), 3.18 (t, J = 5 Hz, 8H), 3.40 (m, 6H), 3.44 (s, 8H), 5.04 (s, 8H),
5.80 (s, 4H), 7.27 (s, 20H). 13C NMR (300 MHz, CDCl3): δ 38.9,
52.8, 53.3, 66.0, 66.2, 69.9, 70.1, 70.2, 127.7, 127.9, 128.2, 136.6, 156.6.
MS (FAB+) (m/z): calcd for [C50H69N6O12]+ 945.5, found 945.5.
H(14O4,2)-Amine (5g). H(14O4,2)CBZ (0.95 g, 1 mmol) was
deprotected by catalytic hydrogenation as described for preparing
compound 5e. A 0.37 g (90%) amount of H(14O4,2)-amine was
obtained as a colorless thick oil. 1H NMR (400 MHz, D2O): δ 2.70 (s,
4H), 2.82 (t, J = 5 Hz, 8H), 3.12 (t, J = 5 Hz, 8H), 3.56 (s, 4H), 3.70
(s, 8H). 13C NMR (100 MHz, D2O): δ 39.2, 55.1, 58.5, 69.0, 71.0,
72.0. MS (FAB+) (m/z): calcd for [C18H45N6O4]+ 409.4, found 409.3.
H(14O4,2)-1,2-HOPOBn (6g). This compound was prepared by the
similar procedure as described for preparing compound 6d, except
compound 5g (0.21 g, 0.5 mmol) was used instead of compound 5d.
Separation and purification were performed as described above.
Compound 6g was obtained as a pale yellow thick oil. Yield: 0.49 g
1
dimesylate. H NMR (300 MHz, CDCl3): δ 3.39 (t, J = 4.8 Hz, 4H),
3.65 (m, 12H). 13C NMR (75 MHz, CDCl3): δ 50.2, 69.6, 70.2. MS
(FAB+) (m/z): calcd for [C8H16N6O3]+ 245.1, found 245.1.
Tetraethylene Glycol Diamine (3f). The tetraethylene glycol
diazide (1.9 g) was dissolved in 40 mL of ethanol and hydrogenated
at 0−5 °C (cooling with a water bath) and 500 psi in the presence of
10% Pd/C (0.3 g). Filtration of the catalyst and evaporation of the
solvent gave 1.3 g (90%) of compound 3f. 1H NMR (300 MHz,
CDCl3): δ 1.60 (s, 4H), 2.81 (m, 4H), 3.45 (m, 12H), 3.57 (m, 12H).
13C NMR (75 MHz, CDCl3): δ 41.3, 69.1, 69.8, 72.9. MS (FAB+) (m/
z): calcd for [C8H21N2O3]+ 193.2, found 193.1.
H(11O3,2)-Cbz (4f). This compound was prepared by the similar
procedure as described for compound 4d, except tetraethylene glycol
diamine (0.39 g, 2 mmol) was used instead of 5LIO-amine. Separation
and purification were performed as described above. H(11O3,2)-Cbz
1
was obtained as a beige thick oil. Yield: 1.4 g, 79%. H NMR (300
MHz, CDCl3): δ 2.53 (s, 12H), 3.17 (s, 4H), 3.83 (s, 8H), 5.04 (s,
8H), 7.29 (s, 20H). 13C NMR (75 MHz, CDCl3): δ 38.8, 50.0, 53.8,
66.0, 69.3, 69.9, 127.6, 127.7, 128.1, 136.5, 156.5. MS (FAB+) (m/z):
calcd for [C48H65N6O11]+ 901.5, found 901.6.
H(11O3,2)-Tetraamine (5f). H(11O3,2)-Cbz (0.9 g, 1 mmol) was
dissolved in 30 mL of methanol and hydrogenated at 25 °C and 500
psi in the presence of 10% Pd/C (0.2 g). Filtration of the catalyst and
evaporation of the solvent gave 0.30 g (90%) of H(11O3,2)-
1
tetraamine. H NMR (400 MHz, D2O): δ 2.68 (s, 4H), 2.80 (t, J =
1
5 Hz, 8H), 3.09 (t, J = 5 Hz, 8H), 3.50 (s, 4H), 3.65 (s, 8H). 13C
NMR (100 MHz, D2O): δ 39.7, 54.1, 54.5, 71.0, 72.0. MS (FAB+)
(m/z): calcd for [C16H41N6O3]+ 365.3, found 365.3.
(75% based on the free amine). H NMR (500 MHz, CDCl3): δ 2.21
(s, 4H), 2.48 (s, 8H), 3.11 (m, 12H), 3.19 (s, 12H), 5.28 (s, 8H), 6.16
(d, J = 7 Hz, 4H), 6.54 (d, J = 9 Hz, 4H), 7.17 (dd, J = 9 Hz, 4H), 7.32
(m, 12H), 7.49 (m, 12H). 13C NMR (100 MHz, CDCl3): δ 37.6, 52.1,
53.1, 53.3, 69.2, 69.7, 77.2, 78.8, 104.6, 122.9, 128.1, 128.8, 129.8,
133.3, 137.9, 143.1, 158.1, 160.5. MS (FAB+) (m/z): calcd for
[C70H81N10O16]+ 1317.6, found 1317.4.
H(14O4,2)-1,2-HOPO (7g). H(14O4,2)-1,2-HOPOBn was depro-
tected with concentrated HCl (12 M)/glacial acetic acid (1:1) as
mentioned above for compound 7b. An off-white foam was obtained
as product. Yield: 90%. 1H NMR (300 MHz, CD3OD): δ 3.26 (s, 4H),
3.28 (s, 4H), 3.56 (s, 8H), 3.38 (m, 16H), 3.66 (s, 8H), 3.72 (s, 4H),
6.57 (d, J = 8 Hz, 8H), 7.28 (d, J = 8 Hz, 4H). 13C NMR (75 MHz,
CD3OD): δ 36.5, 54.9, 55.1, 66.0, 67.0, 71.3, 71.4, 109.8, 121.4, 138.9,
141.2, 160.1, 163.4. Anal. Calcd for C42H56N10O16·2HCl·4H2O: C,
45.78; H, 6.04; N, 12.71. Found: C, 45.52; H, 5.95; N, 12.47. MS
(FAB+) (m/z): calcd for [C42H57N10O16]+ 956.4, found 956.3.
Hexaethylene Glycol Dimesylate (1h). This compound was
prepared by the similar procedure as described for compound 1f,
H(11O3,2)-1,2-HOPOBn (6f). This compound was prepared by the
similar procedure as described for compound 6d, except compound 5f
(0.5 mmol) was used instead of compound 5d. Separation and
purification were performed as described above. Compound 6f was
obtained as a thick pale yellow oil. Yield: 0.48 g, 75% based on the free
1
amine. H NMR (500 MHz, CDCl3): δ 2.32 (s, 4H), 2.46 (m, 8H),
3.00 (m, 8H), 3.03 (s, 4H), 3.06 (s, 4H), 3.18 (m, 8H), 5.30 (s, 8H),
6.15 (d, J = 7 Hz, 4H), 6.52 (d, J = 9 Hz, 4H), 7.17 (d, J = 9.0 Hz,
4H), 7.32 (m, 12H), 7.49 (m, 8H). 13C NMR (125 MHz, CDCl3): δ
37.4, 52.3, 53.1, 53.3, 69.1, 69.3, 69.6, 78.8, 104.6, 122.9, 128.1, 128.9,
129.6, 137.9, 143.1, 158.1, 160.5. MS (FAB+) (m/z): calcd for
[C69H77N10O15]+ 1273.6, found 1273.2.
H(11O3,2)-1,2-HOPO (7f). Compound 6f was deprotected with
concentrated HCl (12 M)/glacial acetic acid (1:1) as mentioned above
for compound 7b. An off-white solid was obtained as product. Yield:
1
85%. H NMR (300 MHz, DMSO-d6): δ 3.38 (s, 8H), 3.45 (s, 4H),
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except hexaaethylene glycol (0.5 mmol) was used instead of
tetraethylene glycol. Separation and purification were performed as
described above. A pale yellow thick oil was obtained as product. Yield:
90%. 1H NMR (300 MHz, CDCl3): δ 3.03 (s, 6H), 3.56 (s, 8H), 3.60
(m, 6H), 3.70 (m, 4H), 4.32 (m, 4H). 13C NMR (75 MHz, CDCl3): δ
37.4, 67.8, 69.0, 69.8, 69.9. MS (FAB+) (m/z): calcd for
[C14H31O11S2]+ 439.1, found 439.1.
HOPO, and H(8O2,2)-1,2-HOPO) or isopropanol (complexes with
ligands H(11O3,2)-1,2-HOPO, H(14O4,2)-1,2-HOPO, and H-
(17O5,2)-1,2-HOPO), and air dried, yielding the desired complexes
as beige powders (60−89%).
Eu(H(3,2)-1,2-HOPO). Yield: 60%. Anal. Calcd for EuC35H39N10O12·
2H2O: C, 42.91; H, 4.42; N, 14.30. Found: C, 42.73; H, 4.51. N, 14.01.
HRMS-ESI (m/z): [M − H]− calcd for 151EuC35H38N10O12 941.1875,
found 941.1855. The observed isotopic distribution pattern matched
the calculated one (Figure S1, Supporting Information).
Eu(H(4,2)-1,2-HOPO). Yield: 78%. Anal. Calcd for EuC36H41N10O12·
H2O: C, 44.31; H, 4.44; N, 14.05. Found: C, 44.14; H, 4.56; N, 14.05.
HRMS-ESI (m/z): [M − H]− calcd for 151EuC36H40N10O12 955.2031,
found 955.2021. The observed isotopic distribution pattern matched
the calculated one (Figure S2, Supporting Information).
Eu(H(5O,2)-1,2-HOPO). Yield: 89%. Anal. Calcd for Eu-
C36H41N10O13·2H2O: C, 42.82; H, 4.49; N, 13.87. Found: C, 43.01;
H, 4.67; N, 13.60. HRMS-ESI (m/z): [M − H]− calcd for
151EuC36H40N10O13 971.1980, found 971.1987. The observed isotopic
distribution pattern matched the calculated one (Figure S3,
Supporting Information).
Eu(H(8O2,2)-1,2-HOPO). Yield: 70%. Anal. Calcd for Eu-
C38H45N10O14·5H2O: C, 41.20; H, 5.00; N, 12.64. Found: C, 41.13;
H, 5.05; N, 12.50. HRMS-ESI (m/z): [M − H]− calcd for
151EuC38H44N10O14 1015.2243, found 1015.2231. The observed
isotopic distribution pattern matched the calculated one (Figure S4,
Supporting Information).
Eu(H(11O3,2)-1,2-HOPO). Yield: 73%. Anal. Calcd for Eu-
C40H49N10O15·6H2O: C, 41.07; H, 5.26; N, 11.97. Found: C, 41.34;
H, 5.01; N, 11.71. HRMS-ESI (m/z): [M − H]− calcd for
151EuC40H48N10O15 1059.2505, found 1059.2481. The observed
isotopic distribution pattern matched the calculated one (Figure S5,
Supporting Information).
Eu(H(14O4,2)-1,2-HOPO). Yield: 63%. Anal. Calcd for Eu-
C42H53N10O16·5H2O: C, 42.18; H, 5.31; N, 11.71. Found: C, 42.03;
H, 5.06; N, 11.55. HRMS-ESI (m/z): [M − H]− calcd for
151EuC42H52N10O16 1103.2767, found 1103.2740. The observed
isotopic distribution pattern matched the calculated one (Figure S6,
Supporting Information).
Eu(H(17O5,2)-1,2-HOPO). Yield: 58%. Anal. Calcd for Eu-
C44H57N10O17·6H2O: C, 42.01; H, 5.53; N, 11.13. Found: C, 41.84;
H, 4.94; N, 10.87. HRMS-ESI (m/z): [M − H]− calcd for
151EuC44H56N10O17 1147.3023, found 1147.3003. The observed
isotopic distribution pattern matched the calculated one (Figure S7,
Supporting Information).
Hexaethylene Glycol Diazide (2h). This compound was prepared
by the similar procedure as described for compound 2f, except
hexaethylene glycol dimesylate was used instead of tetraethylene glycol
dimesylate. Separation and purification were performed as described
above. A colorless thick oil was obtained as product (yield 75% based
1
on dimesylate). H NMR (300 MHz, CDCl3): δ 3.36 (m, 4H), 3.65
(m, 20H). 13C NMR (75 MHz, CDCl3): δ 50.0, 69.4, 69.9, 70.0. MS
(FAB+) (m/z): calcd for [C12H25N6O5]+ 333.2, found: 333.2.
Hexaethylene Glycol Diamine (3h). Compound 3h was prepared
by catalytic hydrogenation as for preparing compound 5f, except
compound 2h was used instead of 2f. Yield: 82% 1H NMR (300 MHz,
CDCl3): δ 1.11 (s, 4H), 2.80 (t, J = 5 Hz, 4H), 3.45 (t, J = 5 Hz, 4H),
3.58 (m, 16H). 13C NMR (75 MHz, CDCl3): δ 40.8, 69.2, 69.5, 69.6,
72.4, 77.2. MS (FAB+) (m/z): calcd for [C12H29N2O5]+ 281.2, found
281.2.
H(17O5,2)-Cbz (4h). This compound was prepared by the similar
procedure as described for preparing compound 4d, except compound
3h (0.56 g, 2 mmol) was used instead of 5LIO-amine. Separation and
purification were performed as described above. Compound 4h was
obtained as a beige thick oil. Yield: 1.5 g (75% based on diamine). 1H
NMR (300 MHz, CDCl3): δ 2.61 (s, 12H), 3.19 (m, 8H), 3.44 (m,
8H), 3.48 (s, 12H), 5.05 (s, 8H), 5.79 (s, 4H), 7.29 (s, 20H). 13C
NMR (300 MHz, CDCl3): δ 39.0, 53.0, 54.1, 66.3, 70.0, 70.2, 70.3,
70.4, 77.2, 127.9, 128.0, 128.3, 136.7, 156.7. MS (FAB+) (m/z): calcd
for [C52H72N6O13]+ 989.5, found 989.5.
H(17O5,2)-Amine (5h). H(17O3,2)-Cbz (0.9 g, 1 mmol) was
dissolved in 20 mL of methanol and hydrogenated at 25 °C (cooling
with a water bath) and 500 psi in the presence of 10% Pd/C (0.2 g).
Filtration of the catalyst and evaporation of the solvent gave 0.39 g
1
(90%) of H(17O5,2)-amine. H NMR (400 MHz, D2O): δ 2.75 (s,
4H), 2.87 (m, 8H), 3.22 (m, 8H), 3.54 (s, 4H), 3.72 (s, 16H). 13C
NMR (100 MHz, D2O): δ 39.0, 55.3, 58.7, 69.3, 71.3, 72.2. MS (FAB
+) (m/z): calcd for [C20H49N6O5]+ 453.3, found 453.4.
H(17O5,2)-1,2-HOPOBn (6h). This compound was prepared by the
similar procedure as described for preparing compound 6d, except
compound 5h (230 mg, 0.5 mmol) was used instead of compound 5d.
Separation and purification were performed as described above.
Compound 6h was obtained as a pale yellow thick oil. Yield: 0.48 g
1
Optical Spectroscopy. UV−vis absorption spectra were recorded
on a Varian Cary 300 double-beam absorption spectrometer. Emission
spectra were acquired with a HORIBA Jobin Yvon IBH FluoroLog-3
spectrofluorimeter, equipped with 3-slit double-grating excitation and
emission monochromators (2.1 nm/mm dispersion, 1200 grooves/
mm). Spectra were reference corrected for both the excitation light
source variation (lamp and grating) and the emission spectral response
(detector and grating). Luminescence lifetimes were determined on
the same HORIBA Jobin Yvon IBH FluoroLog-3 spectrofluorimeter,
adapted for time-correlated single-photon counting (TCSPC) and
multichannel scaling (MCS) measurements. A submicrosecond Xenon
flashlamp (Jobin Yvon, 5000XeF) was used as the light source, with an
input pulse energy (100 nF discharge capacitance) of ca. 50 mJ,
yielding an optical pulse duration of less than 300 ns at fwhm. Spectral
selection was achieved by passage through the same double-grating
excitation monochromator. Emission was monitored perpendicular to
the excitation pulse, again with spectral selection achieved by passage
through the double-grating emission monochromator (2.1 nm/mm
dispersion, 1200 grooves/mm). A thermoelectrically cooled single-
photon detection module (HORIBA Jobin Yvon IBH, TBX-04-D)
incorporating fast rise time PMT, wide bandwidth preamplifier and
picosecond constant fraction discriminator was used as the detector.
Signals were acquired using an IBH DataStation Hub photon counting
module, and data analysis was performed using the commercially
available DAS 6 decay analysis software package from HORIBA Jobin
(72% based on the free amine). H NMR (500 MHz, CDCl3) δ 2.40
(s, 4H), 2.52 (s, 8H), 3.11 (m, 4H), 3.23 (s, 20H), 3.31(m, H), 5.31
(s, 8H), 6.19 (d, J = 6 Hz, 4H), 6.58 (d, J = 9 Hz, 4H), 7.22 (dd, J =
9.0 Hz, 4H), 7.34 (m, 12H), 7.52 (m, 12H). 13C NMR (100 MHz,
CDCl3): δ 37.6, 52.2, 53.2, 69.6, 69.7, 77.0, 77.4, 78.77, 104.5, 122.8,
128.0, 128.8, 129.7, 133.3, 137.9, 143.1, 158.1, 160.5. MS (FAB+) (m/
z): calcd for [C72H85N10O17]+ 1361.6, found 1361.7.
H(17O5,2)-1,2-HOPO (7h). H(17O5,2)-1,2-HOPOBn was depro-
tected with concentrated HCl (12 M)/glacial acetic acid (1:1) as
mentioned above for preparing 7b. An off-white foam was obtained as
1
product. Yield: 90%. H NMR (300 MHz, CD3OD): δ 3.34 (s, 8H),
3.39 (s, 4H), 3.46 (s, 16H), 3.66 (s, 8H), 3.72 (s, 4H), 6.57 (d, J = 8
Hz, 8H), 7.28 (d, J = 8 Hz, 4H). 13C NMR (75 MHz, CD3OD): δ
36.5, 54.9, 55.1, 66.0, 71.2, 71.4, 71.5, 109.6, 121.4, 138.9, 141.1, 160.1,
163.4. Anal. Calcd for C44H60N10O17·2HCl·5H2O: C, 45.40; H, 6.24;
N, 12.03. Found: C, 45.09; H, 6.35; N, 11.86. MS (FAB+) (m/z):
calcd for [C44H60N10O17]+ 1001.4, found 1001.4.
Preparation of Eu Complexes. To a solution of ligand (0.01 mmol)
in MeOH (5 mL) in a 10 mL round-bottom flask was added a solution
of 1.0 equiv of EuCl3·6H2O in MeOH (1 mL), and the mixture was
stirred for 15 min; pyridine (15 μL) was then added. The mixture was
heated to reflux temperature for 4 h with stirring. Upon cooling, a
white solid formed which was collected by centrifuge, washed with a
small amount (∼3 mL) of methanol (complexes with ligands H(2,2)-
1,2-HOPO, H(3,2)-1,2-HOPO, H(4,2)-1,2-HOPO, H(5O,2)-1,2-
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Figure 1. (a) Luminescence spectrum showing the typical decrease of luminescence intensity upon addition of increasing amounts of DTPA; (b)
DTPA competition batch titration of [Eu(H(3,2)-1,2-HOPO)]− (black squares, line), [Eu(H(5O,2)-1,2-HOPO)]− (red circles, line), and
[Eu(H(11O3,2)-1,2-HOPO)]− (blue triangles, line) versus DTPA. The x intercept indicates the difference in pEu between EuDTPA (pEu = 19.04)
and the two complexes.
Chart 1. Chemical Structures of the Tetradentate Model Compound 5LINMe-1,2-HOPO (left) and the Octadentate 1,2-HOPO
Ligands Investigated (center, right)
Yvon IBH. Goodness of fit was assessed by minimizing the reduced chi
squared function, χ2, and a visual inspection of the weighted residuals.
Each trace contained at least 10 000 points, and the reported lifetime
values resulted from at least three independent measurements. Typical
sample concentrations for both absorption and fluorescence measure-
ments were ca. 10−5−10−6 M, and 1.0 cm cells in quartz Suprasil or
equivalent were used for all measurements. Quantum yields were
determined by the optically dilute method (with optical density < 0.1)
using the following equation
and 10−8 M. An excitation wavelength of 340 nm was used, and
emission lifetimes were monitored at 612 nm. Samples were analyzed
immediately after dilution into buffer and then again after several
different time intervals.
Competition Batch Titrations for pEu and pZn Determi-
nation. The general procedure used to determine the pEu values of
the ligands was adapted from an already described study using Gd31,32
and are similar to those already reported for other complexes.29
Different volumes of a standardized DTPA stock solution were added
to solutions of constant ligand, metal, and electrolyte concentrations.
In the current work, the pH of all solutions was kept constant at 7.4
with TRIS buffer instead of adjusting the pH to 6.0 as was done in past
studies,31 and the solutions were diluted to identical volumes. After
stirring the solutions for 24 h to ensure thermodynamic equilibrium
was reached, the pH was again checked just before analyzing the
samples spectrophotometrically. The concentrations of each ligand
relative to DTPA used in the final data analysis ranged from 1:1 to
1:1000 (L:DTPA). Concentrations of free and complexed ligand in
each solution were determined from the luminescence spectra at
identical pH and concentrations. These concentrations were used for
the log/log plots (Figure 1) to give the difference in pEu between the
competing DTPA and ligand of interest. In a similar way, pZn was
determined by using a solution of ZnCl2 in water as a competitor
instead of DTPA.
2
Φ /Φ = [Ar(λr)/Ax(λx)][I(λr)/I(λx)][nx /nr2][D /D]
x
r
x
r
where A is the absorbance at the excitation wavelength (λ), I is the
intensity of the excitation light at the same wavelength, n is the
refractive index, and D is the integrated luminescence intensity. The
subscripts x and r refer to the sample and reference, respectively. For
quantum yield calculations, an excitation wavelength of 340 nm was
utilized for both the reference and the sample; hence, the I(λr)/I(λx)
term is removed. Similarly, the refractive indices term, nx2/nr2, was
taken to be identical for the aqueous reference and sample solutions.
Hence, a plot of integrated emission intensity (i.e., IDr) vs absorbance
at 340 nm (i.e., Ar) yields a linear plot with a slope which can be
equated to the reference quantum yield Φr. Quinine sulfate in 0.5 M
(1.0 N) sulfuric acid was used as the reference (Φr = 0.546).30 By
analogy, for the sample, a plot of integrated emission intensity (i.e.,
Dx) versus absorbance at 340 nm (i.e., Ax) yields a linear plot, and Φx
can then be evaluated. The values reported in the manuscript are the
average of four independent measurements.
RESULTS AND DISCUSSION
■
Design of the Ligands and Synthesis. The tetradentate
ligand 5LINMe-1,2-HOPO was prepared as described else-
where,29 and all octadentate ligands were prepared in a similar
way to the previously reported H(2,2)-1,2-HOPO ligand. Since
For the lifetime dilution and time-dependent measurements, the
samples were initially dissolved in DMSO to give a concentration of 1
mM and then diluted into TRIS buffered saline (20 mM TRIS, 100
mM NaCl, pH 7.4) to the desired final concentrations of 10−5, 10−6,
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the complex ([Eu(H(2,2)-1,2-HOPO)]−) has been shown to
have one water molecule in its inner sphere,29 which limits its
overall quantum yield, the central ethyl chain was substituted
by longer aliphatic chains (propyl and butyl) or polyethylene
glycol (PEG) chains yielding a more flexible ligand backbone
for the two essentially “5LINMe-1,2-HOPO-like” motifs to bind
in a similar way to the model bis-tetradentate [Eu(5LINMe-1,2-
HOPO)2]− complex, in order to achieve enhanced optical
properties, vide infra. Noticeably, the addition of the PEG
groups is also expected to increase the solubility of the ligand,
and this alteration has proven successful in increasing the
solubility of luminescent lanthanide complexes.23,33,34 For these
ligands, we adopted the H(XOn,Y) notation, where the X index
refers to the total number of atoms in the central chain between
the two bridgehead tertiary nitrogen atoms which connect each
pair of terminal 1,2-HOPO units, and the Y index refers to the
number of atoms between the bridgehead tertiary nitrogen
atom and the N atom of the 1,2-HOPO amide linkage.
Additional nomenclature after the X refers to the number (n) of
ether oxygen atoms (O) in the central chain. For example, for
H(11O3,2), there are two carbons atoms between the 1,2-
HOPO amide and the bridgehead tertiary nitrogen atoms,
while the central chain contains a total of 11 atoms, of which
three are oxygen atoms (see Chart1).
Thermodynamic Stability. One practical concern related
to the use of europium chelators for biological applications is
the thermodynamic stability of the complexes. It should also be
noted that the kinetic inertness of the corresponding complexes
is similarly a very important factor to consider for biological
applications.3,38,39 In biological media, many factors can
influence the stability of the metal complex, such as the
competition of proton, endogenous cations (e.g., Ca2+, Zn2+,
Mg2+) and anions (hydroxide, phosphate), and also natural
chelators such as transferrin or albumin. In order to determine
their thermodynamic stabilities, spectrophotometric titration
studies were performed in terms of the pEu value for all 1,2-
HOPO derivatives. Analogous to pH, pEu is defined as the
negative log of the concentration of free metal in solution
(pEu= −log [Eu3+]free) at a specified set of standard conditions
(typically [Eu]T = 1 μM, [L]T = 10 μM, pH = 7.4, 25 °C, and
0.1 M KCl). The evaluated pEu values therefore offer a
convenient way to compare relative chelate thermodynamic
stabilities between various ligands, regardless of their differing
protonation behavior.
The method chosen to determine this conditional
thermodynamic stability parameter was competition batch
titration using the potent octadentate chelator, diethylenetri-
amine pentaacetic acid (DTPA) as the competing ligand which
is widely used in industry. The FDA has approved CaNa3-
DTPA injection and ZnNa3DTPA injection for treatment of
individuals with known or suspected internal contamination
with plutonium, americium, or curium to increase the rates of
elimination (www.fda.gov/drugs/EmergencyPreparedness/).
In this experiment, the concentrations of ligands and Eu(III)
as well as the pH were kept constant while the concentration of
DTPA was progressively increased. Figure 1a shows the
luminescence spectra obtained for one of the complexes and
the evolution upon addition of varying amounts of DTPA.
From the luminescence data, the resulting concentrations of
free and complexed ligand were determined, and a plot of
log([EuL]−/[EuDTPA]2−) vs log([DTPA]/[L]) was con-
structed (Figure 1b), which directly yields the difference in
pEu between the studied ligands and DTPA (i.e., from the x
axis intercept, ΔpEu = log([DTPA]/[L] when log([EuL]−/
[EuDTPA]2− = 0, or, alternately, this is the concentration of
DTPA which generates equal partition of Eu between the
described ligands and DTPA). Using the known pEu of 19.04
for DTPA,40 the pEu of all Eu(III) complexes were calculated
using luminescence spectroscopy.
The syntheses of the H(m,2)-1,2-HOPO ligands were
straightforward (Scheme 1). The backbone amines of H(3,2)-
1,2-HOPO and H(4,2)-1,2-HOPO, N,N,N′,N′-tetrakis(2-ami-
noethyl)-propane-1,2-diamine, and N,N,N′,N′-tetrakis(2-
amino-ethyl)-butane-1,4-diamine were prepared as reported
elsewhere.35
The H(mOn,2)-1,2-HOPO ligands were synthesized from
the corresponding α,ω-glycol-diamine (Scheme 2). While 2-(2-
amino-ethoxy)-ethylamine (5LIO-diamine) (3d) and 2-[2-(2-
amino-ethoxy)-ethoxy]-ethylamine (3e) are commercially
available, other oligoethylene glycol diamines (3f−h) were
prepared from the corresponding oligoethylene glycols as
shown in Scheme 2. The oligoethylene glycols were converted
to corresponding mesylates (1f−h), which were transformed
into the diazides (2f−h) by reaction with sodium azide. The
diamines (3f−h) were prepared from the diazides (2f−h) by
catalytic hydrogenation over Pd/C. The protected H(mOn,2)-
tetraamines (4d−h) were synthesized by the reaction of the
appropriate oligoethylene glycol diamines (3d−h) with benzyl
aziridine-1-carboxylate, with subsequent deprotection of the
Cbz group by hydrogenation giving H(mOn,2)-tetraamines in
good yields. Amide coupling of the H(mOn,2)-tetraamines with
1,2-HOPOBn-thiazolide or 1,2-HOPOBn acid chloride36,37
yields the benzyl-protected ligands (6d−h), which were
deprotected under acidic conditions with 1:1 (v/v) AcOH/
HCl (12 M) to yield the target 1,2-HOPO ligands (7d−h).
The Eu(III) complexes were prepared by refluxing equal
equivalents of the appropriate ligand with EuCl3·6H2O in
methanol using pyridine as a base to ensure complete
complexation. The desired complexes were then precipitated,
isolated by centrifuging, and washed with either methanol or
isopropanol to yield analytically pure hydrated complexes. Since
the same results were obtained by mixing equal equivalents of
the ligand with EuCl3·6H2O (and allowing to equilibrate
overnight), the Gd(III) complexes were prepared in situ using
the latter method. The full characterization of the ligands and
isolated complexes and synthetic details are reported in the
Experimental Section.
The stability values in terms of pEu (see Table 1) vary
slightly from one ligand to the other spanning from 19.9 to 21.2
Table 1. Thermodynamic Parameters Related with the
Stability of The Eu(III) and Zn(II) Complexes with the
Discussed Ligands in TRIS Buffered Solution (pH = 7.4)
pEu
pZn
H(2,2)-1,2-HOPO29
H(3,2)-1,2-HOPO
21.2(1)
17.5(1)
18.4(2)
19.2(1)
20.4(1)
20.4(1)
20.3(1)
20.0(1)
17.3(1)
17.2(4)
13.7(5)
14.7(4)
15.6(4)
16.9(4)
17.4(3)
16.9(4)
16.8(3)
14.8(4)
H(4,2)-1,2-HOPO
H(5O,2)-1,2-HOPO
H(8O2,2)-1,2-HOPO
H(11O3,2)-1,2-HOPO
H(14O4,2)-1,2-HOPO
H(17O5,2)-1,2-HOPO
5LINMe-1,2-HOPO29
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for most of the octadentate ligands except for [Eu(H(3,2)-1,2-
HOPO)]− and [Eu(H(4,2)-1,2-HOPO)]− (pEu = 17.5 and
18.4, respectively). These values establish that highly stable
europium complexes are formed with thermodynamic stabilities
slightly to moderately higher than that of DTPA. Most of the
octadentate complexes possess pEu’s higher than the bis-
tetradentate model ([Eu(5LINMe-1,2-HOPO)2]− (pEu = 17.3),
which can be attributed to the enhanced chelate effect arising
from an octadentate versus bis-tetradentate topology, in
addition to increased ligand preorganization. Interestingly,
[Eu(H(2,2)-1,2-HOPO)]− is the most thermodynamically
stable complex within this series (pEu = 21.2). This result is
somewhat surprising since the backbone of all the complexes is
similar but can be rationalized presumably by an essentially
ideal nonacoordinated geometry of the ensuing complex
(where the ninth site is occupied by one water molecule as
shown elsewhere29) and the existence of intramolecular H-
bonding interactions between the tertiary amines previously
noted elsewhere34 which stabilizes the Eu(III) complex. For
[Eu(H(3,2)-1,2-HOPO)]−, [Eu(H(4,2)-1,2-HOPO)]−, and
[Eu(H(5O,2)-1,2-HOPO)]−, the smaller pEu values suggest
that the complexation geometry is slightly different with
complexes slightly more constrained compared to [Eu(H-
(8O2,2)-1,2-HOPO)]−, [Eu(H(11O3,2)-1,2-HOPO)]−, [Eu-
(H(14O4,2)-1,2-HOPO)]−, and [Eu(H(17O4,2)-1,2-
HOPO)]−. All of the latter complexes, with long central
chains, possess pEu values around ca. 20, which are one order
of magnitude lower than [Eu(H(2,2)-1,2-HOPO)]− but one
order higher than the benchmark DTPA. Such impressive
aqueous stability allows measurements to be performed at
submicromolar concentration without observable decomposi-
tion of the complexes.
Notably, for the longer chain backbones, we also considered
the possibility of bimetallic dimeric [Eu2L2]2− complex
formation. Although the pEu’s determined in this manner
cannot differentiate between [ML]− and [M2L2]2− complexes,
the single-exponential decay behavior we observe suggests only
a single emitting species in solution, which based on
thermodynamic grounds should be the monomeric EuL
complex. Furthermore, we conducted additional luminescence
lifetime experiments upon serial dilution of the [Eu(H(8O2,2)-
1,2-HOPO)]−, [Eu(H(11O3,2)-1,2-HOPO)]−, [Eu(H-
(14O4,2)-1,2-HOPO)]−, and [Eu(H(17O5,2)-1,2-HOPO)]−
complexes at three different concentrations representing the
range of concentrations used for quantum yield measurements
(10−5 and 10−6 M) and also at nanomolar concentration (10
nM). At very low concentrations, any potentially dimeric
[Eu2L2]2− complexes (or polymeric [EunLn]n− species) would
be thermodynamically disfavored due to their second-order (or
higher) concentration dependence. However, the observed
luminescence lifetimes obtained immediately after dilution and
again after several different time intervals (10 min, 1 h, 4 days)
remained unchanged within experimental error, with all
complexes exhibiting monoexponential decays, providing
additional evidence that the complex observed in solution is
indeed the monomeric [ML]− species.
612 nm using the intense J = 2 transition. This high stability is
due to the rather low pKa (∼5) of the 1,2-HOPO moieties.41
Similarly, no decomposition of the complexes was observed
when measurements were performed in 20 mM CaCl2 solution,
revealing the absence of affinity of 1,2-HOPO ligands for this
metal cation (Figure S9, Supporting Information).
However, in the presence of Zn(II), at 20 mM in TRIS buffer
(pH = 7.4), a total loss of the Eu(III) luminescence was
observed. As a consequence, the stability of the Eu(III)
complex versus the Zn(II) ion was measured in terms of the
pZn. These pZn were measured in an analogous way to the
described DTPA batch titration, substituting DTPA by a
solution with a known concentration of ZnCl2.
As seen in Table 1, the pZn values are all much lower (by 3
to 4 orders of magnitude) than the pEu, demonstrating the
weaker interaction of the 1,2-HOPO ligands with Zn(II) and
therefore the specificity of such ligands to preferentially bind
the Eu(III) cation in biological media where the free Zn(II)
concentration is comparably low. It is interesting to note that
the same trend of the Eu(III) ions is followed with the Zn(II)
ion (Figure S10, Supporting Information).
UV−Vis Absorption Spectroscopy. The UV−vis absorp-
tion data for each of the Eu(III) complexes in TRIS buffer
solution (pH = 7.4) are summarized in Table 2. Each of the
Table 2. UV−Vis Absorption Data of the Studied Eu(III)
Complexes in TRIS Buffer (pH = 7.4); Brightness at
Maximum Absorption and Triplet Excited State Energies
TRIS buffer
pH = 7.4
a
λ
abs
ε (M−1
cm−1
·
brightness
77 K, T0−0/
max
(nm)
)
(M−1·cm−1
)
nm (cm−1
21 980
)
[Eu(H(2,2)-1,2-
29
341
18 200
17 700
17 900
15 900
15 350
15 070
15 200
15 000
18 050
655
HOPO)]−
[Eu(H(3,2)-1,2-
HOPO)]−
339
337
337
336
334
336
336
332
655
21 900
22 390
22 000
22 320
22 120
22 020
21 690
22 010
[Eu(H(4,2)-1,2-
HOPO)]−
555
[Eu(H(5O,2)-1,2-
HOPO)]−
1065
1720
2485
2920
2940
3120
[Eu(H(8O2,2)-1,2-
HOPO)]−
[Eu(H(11O3,2)-1,2-
HOPO)]−
[Eu(H(14O4,2)-1,2-
HOPO)]−
[Eu(H(17O5,2)-1,2-
HOPO)]−
[Eu(5LINMe-1,2-
29
HOPO)2]−
a
Determined in a solid matrix at 77 K (methanol:ethanol, 1:4 v/v)
using the Gd complexes. Estimated error in ε and brightness (ε ×
ϕTot) are 15% and 20%, respectively
spectra have absorption maximal around 335−340 nm (Figure
2). Those bands are composed of two electronic transitions; at
higher energy, a purely π−π* transition and at slightly lower
energy (ca. 320 nm) a π−π* transition with some n−π*
character, as evidenced previously from TD-DFT calcula-
tions.9,42 Absorption maxima are slightly shifted toward higher
energy upon increasing the bridge length, and this has
previously been proposed to be due to a small interaction
between the terminal 5LINMe-1,2-HOPO units.29 This
interaction gives maxima blue shifting from 341 nm for
[Eu(H(2,2)-1,2-HOPO)]− to 334 nm for [Eu(H(17O5,2)-1,2-
To ensure that the thermodynamic stability of the complexes
is high enough for biological measurements, their stability vs
slightly basic or acidic conditions or vs Ca(II) or Zn(II) was
also estimated. As can be seen from Figure S8, Supporting
Information, the 1,2-HOPO moiety is highly stable to basic or
acidic conditions (TRIS solution at pH = 6.1, 7.4, 8.5), yielding
no change in emission intensity over days when measured at
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Figure 3. Time-gated phosphorescence spectra of [Gd(H(2,2)-1,2-
HOPO)]− (black), [Gd(H(3,2)-1,2-HOPO)]− (red), [Gd(H(4,2)-
1,2-HOPO)]− (blue), [Gd(H(5O,2)-1,2-HOPO)]− (magenta), and
[Gd(H(11O3,2)-1,2-HOPO)]− (green) in methanol:ethanol (1:4 v/
v) at 77 K (λex = 330 nm, delay 0.1 ms).
Figure 2. UV−vis absorption spectra of [Eu(H(2,2)-1,2-HOPO)]−
(black), [Eu(H(3,2)-1,2-HOPO)]− (red), Eu(H(4,2)-1,2-HOPO)]−
(blue), [Eu(H(5O,2)-1,2-HOPO)]− (aqua), [Eu(H(8O2,2)-1,2-
HOPO)]− (magenta), [Eu(H(11O3,2)-1,2-HOPO)]− (olive), Eu-
(H(14O4,2)-1,2-HOPO)]− (orange), and [Eu(H(17O5,2)-1,2-
HOPO)]− (dark red) in 0.1 M TRIS buffer (pH = 7.4).
triplet excited state energies should not provide any large
difference in the sensitization efficiency between complexes,
since all triplet excited states possess almost the same energy
gap with respect to the 5D2 (E = 21 519 cm−1) and the 5D1 (E
= 19 028 cm−1) accepting levels of europium.
HOPO)]− (as low as 332 nm for the previously reported
[Eu(5LINMe-1,2-HOPO)2]− complex). At the same time, the
molar absorption coefficients decrease considerably as the
length of the central bridge is increased, by as much as 15% for
[Eu(H(17O5,2)-1,2-HOPO)]− compared to other 1,2-HOPO
complexes previously reported.9,29,43
It should be noticed that no differences were observed when
comparing the UV−vis absorption spectra of the gadolinium
and europium complexes. Furthermore, inspection of the UV−
vis properties of the free ligand under the same conditions
reveals the same blue shift of the absorption maximum upon
increasing the length of the central bridge. This result reveals
that the effect observed with europium (and gadolinium) arises
from an interaction between the terminal 5LINMe-1,2-HOPO
motifs within one octadentate ligand.
Luminescence of Eu Complexes. As expected from the
difference in crystal field, the nonacoordinated and octacoordi-
nated complexes present some significant differences in their
luminescence pattern, with different relative intensities and
splitting for all transitions (see Figure 4) giving an unusual type
of spectrum for [Eu(H(2,2)-1,2-HOPO)]− compared to all 1,2-
HOPO octacoordinated derivatives. For all octacoordinated
complexes, as can be seen in Figure 4, the emission spectra are
7
typical with very intense J = 2 transitions (5D0 → F2). The
intensity of the J = 1 band (5D0 → 7F1) changes as compared to
the overall intensity (Figure 4), yielding different luminescence
radiative parameters (vide infra).44,45 Also, the splitting pattern
of the J = 1 transition changes, which clearly indicates a change
in the geometry around the metal center. Of interest is also the
similarities in pattern and spectra of [Eu(H(2,2)-1,2-HOPO)]−
and [Eu(H(3,2)-1,2-HOPO)]−, with intense J = 1 and 4 bands
(when compared to the J = 2), suggesting that the emission
observed for [Eu(H(3,2)-1,2-HOPO)]− may also arise from a
nonacoordinated species (as previously observed for [Eu(H-
(2,2)-1,2-HOPO)]−).29 For [Eu(H(4,2)-1,2-HOPO)]−, as
shown in Figure 5, we note that the J = 4 transition is
intermediate between [Eu(H(2,2)-1,2-HOPO)]− and [Eu(H-
(5O,2)-1,2-HOPO)]− (as an example of all other complexes
with longer bridges), suggesting the presence of two different
emitting species in solution (one nona- and the other
octacoordinated). This change in pattern is also observed at
77 K, in solid matrix, supporting the change of geometry
around the Eu(III) ion (see Figure 5b). Importantly, the
position of the J = 0 transitions is unique for all differing
emitting complexes in solution, but the broadness of this
transition in this case (even at 77 K) precludes any definitive
Luminescence of Gd Complexes. Estimation of the
energies of the ligand-based triplet excited state were
determined using the Gd(III) complexes. Gadolinium was
7
chosen since it is a 4f lanthanide cation having a similar
6
electronic configuration and size as the europium cation (4f )
but lacking any accessible metal-based low-energy electronic
excited states. At room temperature, only a broad weak
emission centered between 380 and 400 nm can be seen for the
Gd(III) complexes, which can be attributed to the poorly
emissive singlet excited state of the 1,2-HOPO chromophore in
complex with the gadolinium cation.9,42 At 77 K, in a 1:4 (v/v)
methanol:ethanol solid matrix, a broad emission band at ca. 500
nm is observed (Figure 3). This emission, red shifted compared
to the singlet excited state, can be assigned to phosphorescence
from the triplet excited state, which is lower in energy than the
singlet excited state observed at room temperature. Selective
time-gated phosphorescence spectra (delay 0.1 ms) of the
gadolinium complexes at 77 K are depicted in Figure 3, and the
energies associated with these triplet excited states are reported
in Table 2. From these values, it appears the lowest energy
triplet excited state of the complexes all have approximately the
5
conclusion. As shown in Figure 5b, the D0 → 7F1 transition is
composed of three peaks for all europium(III) complexes at
room temperature and at 77 K in solid matrix. While the
broadness of the transition again precludes a definitive
determination of the exact point group of the complex, such
multiplicity suggests that from the three most common
same energy (22 050
210 cm−1) with a very small (<1%)
standard deviation. This result suggests that the small
interaction observed for the singlet excited state is absent (or
weak enough to not be observed). Such small differences in the
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Figure 4. (a) Luminescence spectra of [Eu(H(2,2)-1,2-HOPO)]− (black), [Eu(H(3,2)-1,2-HOPO)]− (red), [Eu(H(4,2)-1,2-HOPO)]− (green),
and [Eu(H(5O,2)-1,2-HOPO)]− (blue) and (b) [Eu(H(8O2,2)-1,2-HOPO)]− (black), [Eu(H(11O3,2)-1,2-HOPO)]− (red), [Eu(H(14O4,2)-1,2-
HOPO)]− (green), and [Eu(H(17O5,2)-1,2-HOPO)]− (blue) at room temperature in 0.1 M TRIS buffer at pH = 7.4 (λex = 340 nm).
Figure 5. Luminescence spectra and highlight of the J = 0 and 1 transitions of [Eu(H(2,2)-1,2-HOPO)]− (black), [Eu(H(3,2)-1,2-HOPO)]− (red),
[Eu(H(4,2)-1,2-HOPO)]− (green), and [Eu(H(5O,2)-1,2-HOPO)]− (blue) and [Eu(H(8O2,2)-1,2-HOPO)]− (magenta), [Eu(H(11O3,2)-1,2-
HOPO)]− (olive), [Eu(H(14O4,2)-1,2-HOPO)]− (purple), and [Eu(H(17O5,2)-1,2-HOPO)]− (dark red) at 77 K in solid matrix
(ethanol:methanol 4:1) (λex= 340 nm).
coordination polyhedra, the best match to the observed
luminescence spectra is obtained for the bicapped trigonal
prism (C2v) geometry as noted elsewhere for similar
derivatives.46
In addition to the steady state emission spectra, the
luminescence quantum yields and luminescence lifetimes of
the Eu(III) complexes were also measured in aqueous solution
with 0.1 M TRIS buffer pH = 7.4 and in deuterated solution to
estimate the number of inner-sphere water molecules (i.e., q)
using the improved Horrock’s equation.47 All photophysical
characterizations are summarized in Table 3.
As can be readily seen, the central bridge influences all the
luminescence properties by inducing constraint on the
complexation geometry for shorter bridges. Increasing the
chain length results in a subsequent increase of the
luminescence efficiency, going from 0.031 to 0.196 for
[Eu(H(4,2)-1,2-HOPO)]− and [Eu(H(17O5,2)-1,2-
HOPO)]−, respectively (see also Figure 8a). In more detail,
the luminescence quantum yields are in the same order from
[Eu(H(2,2)-1,2-HOPO)]− to [Eu(H(4,2)-1,2-HOPO)]−; then
a constant increase is observed until reaching a plateau for
[Eu(H(14O4,2)-1,2-HOPO)]− and [Eu(H(17O5,2)-1,2-
HOPO)]− (Figure 8a). Noticeably, the maximum quantum
Table 3. Photophysical Data of the Investigated Eu
Complexes
a
0.1 M TRIS buffer pH = 7.4
77 K
τ (μs)
914
ϕTot
τ (μs) τD (μs)
q
[Eu(H(2,2)-1,2-
29
0.036 480
1222
1.1
HOPO)]−
[Eu(H(3,2)-1,2-
HOPO)]−
0.037 552;
811;
369
0.3;
1.0
1040;
781
253
0.031 649;
236
0.067 651;
304
[Eu(H(4,2)-1,2-
HOPO)]−
803;
338
0; 1.1
0; 1.1
0
902; 645
823; 608
748
[Eu(H(5O,2)-1,2-
HOPO)]−
825;
462
[Eu(H(8O2,2)-1,2-
HOPO)]−
0.112 697
0.165 668
0.192 700
0.196 704
0.173 728
913
888
961
962
1000
[Eu(H(11O3,2)-1,2-
HOPO)]−
0.1
765
[Eu(H(14O4,2)-1,2-
HOPO)]−
0.1
819
[Eu(H(17O5,2)-1,2-
0.1
826
HOPO)]−
[Eu(5LINMe-1,2-
29
0.1
860
HOPO)2]−
a
Measured in a solid matrix at 77 K (methanol:ethanol 1:4 v/v).
Estimated error in ϕTot and τ are 15% and 10%, respectively
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yield obtained is higher than that of the model bis-tetradentate
complex ([Eu(5LINMe-1,2-HOPO)2]−), suggesting that the
geometry of the complexed ligand is different in octadentate
structures versus bis-tetradentate structures.
LMCT state occurs. In the present case, as can be seen from
Table 3, no such quenching can be evidenced since there is
only a small difference between the luminescence lifetimes in
solution and at low temperature (77 K) in frozen solid
solutions.
As demonstrated elsewhere, the luminescence lifetime of
[Eu(H(2,2)-1,2-HOPO)]− is short because of a single water
molecule in its inner sphere (τ = 480 μs).29 For the shorter
central bridges, from [Eu(H(3,2)-1,2-HOPO)]− to [Eu(H-
(5O,2)-1,2-HOPO)]−, the luminescence decay traces (Figure
S11, Supporting Information) only gave satisfactory fits when
modeled as biexponential decays, composed of both a short
component (τ = 253, 236, and 304 μs for [Eu(H(3,2)-1,2-
HOPO)]−, [Eu(H(4,2)-1,2-HOPO)]−, and [Eu(H(5O,2)-1,2-
HOPO)]−, respectively) and a longer component (τ = 552,
649, and 651 μs for [Eu(H(3,2)-1,2-HOPO)]−, [Eu(H(4,2)-
1,2-HOPO)]−, and [Eu(H(5O,2)-1,2-HOPO)]−, respectively).
This biexponential luminescence decay behavior emphasizes
the presence of two different species in solution with these
shorter bridges. From [Eu(H(8O2,2)-1,2-HOPO)]− to [Eu-
(H(17O5,2)-1,2-HOPO)]−, the measured luminescence life-
times are all monoexponential and in the same range (between
650 and 720 μs) in 0.1 M TRIS buffer solution (pH = 7.4),
while in deuterated water, the luminescence lifetimes vary from
825 to 915 μs (Table 3, Figure 8b).
In terms of their overall luminescence, since the
luminescence quantum yield does not take into account the
absorptivity of the molecule, a more accurate way to rank the
overall efficiency of these compounds is to examine their
brightness, typically defined as the product of the luminescence
quantum yield with the molar absorption coefficient. For these
complexes, as highlighted by the UV−vis absorption study, the
molar absorption coefficient decreases by 15% in going from
the shorter [Eu(H(2,2)-1,2-HOPO)]− to the longer derivatives
(from [Eu(H(8O2,2)-1,2-HOPO)]− to [Eu(H(17O5,2)-1,2-
HOPO)]−, vide supra). This advantage to the shorter bridge
complexes is counterbalanced by the large difference in
quantum yield going from 3% to almost 20% for the complexes
with longer bridges. This results in an increased brightness by
extending the central bridge of these types of chelators going
from 655 M−1·cm−1 for [Eu(H(2,2)-1,2-HOPO)]− to 2940
M−1·cm−1 for [Eu(H(17O5,2)-1,2-HOPO)]−, respectively
(Figure 6). This latter brightness value is as large as the one
obtained for the best bis-tetradentate ligands, typified by
[Eu(5LINMe-1,2-HOPO)2]− (3400 M−1·cm−1).
The lifetime differences (between 0.1 M aqueous TRIS
buffer and deuterated water) can be related to the hydration
states of the complexes.47 Estimates of q reveal no water
molecule in the inner sphere for all complexes with bridges
longer than that of [Eu(H(8O2,2)-1,2-HOPO)]−. Importantly,
the obvious luminescence quantum yield differences between
[Eu(H(8O2,2)-1,2-HOPO)]− and [Eu(H(17O5,2)-1,2-
HOPO)]− are not accompanied by any relevant changes in
their luminescence lifetimes. This suggests that while the triplet
excited state energies undoubtedly play an important role in the
sensitization process differences, the efficiency of the
intersystem crossing and the “quantity of energy” accessing
the triplet excited state is also a crucial factor that affects the
luminescence quantum yield.48 As explained above, from
[Eu(H(3,2)-1,2-HOPO)]− to [Eu(H(5O,2)-1,2-HOPO)]−,
biexponential decays were obtained (in 0.1 M aqueous TRIS
buffer at pH = 7.4 and in deuterated water at room temperature
or at 77 K in solid matrix), revealing the presence of two
emitting species with ligands having 3−5 atoms in the central
bridge. The subsequent measured luminescence lifetimes in
deuterated water reveal the presence of two types of complexes,
one hydrated and one not. This can be explained by geometric
constraints due to the central bridge; the H(2,2) bridge allows
only the formation of hydrated complex, while extension of the
chain length of the bridge allows better protection of the metal
center after complexation by increasing the degrees of freedom
between the two terminal 5LINMe-1,2-HOPO motifs. This
conclusion is supported by the obtained q = 0.3 value for
[Eu(H(3,2)-1,2-HOPO)]−, which suggests that the propyl
chain favors the formation of both an eight- and a nine-
coordinate species, since the chain is presumably not long
enough to form a single eight-coordinate complex species but is
too long to form a single nine-coordinate complex as obtained
for [Eu(H(2,2)-1,2-HOPO)]−.
Figure 6. Brightness of [Eu(H(2,2)-1,2-HOPO)]− (black), [Eu(H-
(3,2)-1,2-HOPO)]− (red), [Eu(H(4,2)-1,2-HOPO)]− (blue), [Eu(H-
(5O,2)-1,2-HOPO)]− (aqua), [Eu(H(8O2,2)-1,2-HOPO)]− (magen-
ta), [Eu(H(11O3,2)-1,2-HOPO)]− (olive), [Eu(H(14O4,2)-1,2-
HOPO)]− (blue), and [Eu(H(17O5,2)-1,2-HOPO)]− (dark red) in
0.1 M aqueous TRIS buffer at pH = 7.4.
Calculated Eu Parameters. As demonstrated else-
where,44,45 the efficiency of the sensitization can be estimated
using a method that defines the overall luminescence quantum
yield (ϕEu) as the product of the efficiency of the intersystem
crossing (ηISC), the efficiency of the energy transfer (ηET), and
the efficiency of metal-centered luminescence (ηEu): ϕEu
=
η
ISCηETηEu = ηsensηEu. In this equation, the ηISCηET term is
termed the sensitization efficiency, ηsens (ηsens= ηISCηET). All
luminescence parameters τR (the pure radiative luminescence
lifetime) and kR and knR (the radiative and nonradiative rate
constants) can be deduced from the corrected steady state
emission spectrum using a value of AMD,0 = 14.65 s−1 for the
spontaneous emission probability of the 5D0−7F1 purely
magnetic dipole-allowed transition.49−54 It should be noted
Luminescence lifetimes were also determined at 77 K, in a
solid matrix (Table 3), which have allowed us to determine
whether back energy transfer between the donor triplet excited
state and the acceptor manifold excited state of the lanthanide
is present or alternately whether quenching via a low-lying
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Table 4. Photophysical Data of the Investigated Complexes Containing Only One Species in Aqueous TRIS pH = 7.4 (see
Supporting Information for details)
ϕTot
τ (μs)
τrad (μs)
kR (s−1
)
knR (s−1
)
ηEu
ηsens
29
[Eu(H(2,2)-1,2-HOPO)]−
0.036
0.112
0.165
0.192
0.196
0.173
480
697
668
700
704
728
3000
1770
1630
1326
1348
1770
333
566
615
754
742
566
1750
869
882
674
679
807
0.160
0.395
0.411
0.528
0.522
0.412
0.225
0.284
0.402
0.364
0.375
0.420
[Eu(H(8O2,2)-1,2-HOPO)]−
[Eu(H(11O3,2)-1,2-HOPO)]−
[Eu(H(14O4,2)-1,2-HOPO)]−
[Eu(H(17O5,2)-1,2-HOPO)]−
29
[Eu(5LINMe-1,2-HOPO)2]−
that this approach has its limitations and can lead to large
errors, especially when multiple species are present in solution.
Hence, these parameters were calculated for five of the
octadentate complexes and the model complex which have
only one species in solution at pH = 7.4, and the resulting
values are reported in Table 4.
As detailed earlier (vide supra), geometric changes around
the Eu(III) cation can be seen by integrating the J = 1 transition
over the entire spectrum, resulting in a decrease of the intensity
of I(0,1)/ITOT (Figure 7) for all complexes as a function of the
number of atoms in the central bridge.
As can be readily seen from Table 4, there are some striking
similarities among the kR and knR values that were also found
for the previously reported [Eu(5LINMe-1,2-HOPO)2]−.46 In
detail, the radiative decay rate is smaller than the nonradiative
decay for all complexes until [Eu(H(14O4,2)-1,2-HOPO)]−,
yielding a metal-centered efficiency inferior to 50%, while for
[Eu(H(14O4,2)-1,2-HOPO)]− and [Eu(H(17O5,2)-1,2-
HOPO)]−, the radiative and nonradiative decay are equal,
allowing an optimized metal-centered efficiency around 50% to
be obtained. This limitation is in line with the results already
published for tetradentate 1,2-HOPO derivatives where 50%
efficiency seems to be a limit in 0.1 M aqueous TRIS buffer for
the 1,2-HOPO derivatives.9,37,42 Indeed, we note that this
increase in the sensitization efficiency by increasing the chain
length can partially explain the change of the luminescence
quantum yield (Figure 8a), but the observed change can not
only be attributed to this phenomenon. The other limitation
results from the sensitization process efficiency as illustrated by
the value of 28.4% for [Eu(H(8O2,2)-1,2-HOPO)]− vs 40.2%
for [Eu(H(11O3,2)-1,2-HOPO)]−). This result demonstrates
that the change in geometry between [Eu(H(8O2,2)-1,2-
HOPO)]− and [Eu(H(11O3,2)-1,2-HOPO)]− (both com-
plexes being octacoordinated) strongly affects the metal-
centered efficiency (as expected) and also influences the
sensitization efficiency. This metal-centered efficiency can be
further evidenced by looking at the evolution of the radiative
lifetimes as a function of the bridge length (Figure 8b).
The values obtained for [Eu(H(11O3,2)-1,2-HOPO)]− are
very close to those obtained for [Eu(5LINMe-1,2-HOPO)2]−
(such as ηsens, ηEu, knR, quantum yield) and could suggest a
similar geometry for these two complexes. While [Eu(H-
(14O4,2)-1,2-HOPO)]− and [Eu(H(17O5,2)-1,2-HOPO)]−
Figure 7. Variation of the ratio I(0,1)/ITOT as a function of the number
of atoms in the central bridge. Vertical bars represent the error on each
point.
■
●
▲
Figure 8. (a) Variation of the luminescence quantum yield ( ), metal-centered efficiency ( ), and sensitization efficiency ( ) as a function of the
■
●
number of atoms in the central bridge. (b) Variation of the luminescence lifetimes ( ) [in the square, ( ) second component of the luminescence
lifetimes] and radiative luminescence lifetimes ( ) as a function of the number of atoms in the central bridge.
▲
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have a lower ligand-centered sentitization (small ηsens compared
to Eu(H(11O3,2)-1,2-HOPO)]− and [Eu(5LINMe-1,2-
HOPO)2]−), the former two complexes exhibit minimal
quenching (large ηEu, q close to zero, small knR). It is proposed
that the longer backbones in these two ligands allow the four
1,2-HOPO to provide optimal shielding of the europium ion
from solvent water molecules.
Prof. Gilles Muller (San Jose State University) for the use of a
low-temperature time-resolved luminescence spectrometer.
L.J.D. is grateful for a postdoctoral fellowship of the Alexander
von Humboldt Foundation.
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