The Ligand Cap Affects the Coordination Number but Not Necessarily the Affinity for Anions of Tris-Bidentate Europium Complexes

Article abstract of DOI:10.1021/acs.inorgchem.0c00137

To evaluate the effect of ligand geometry on the coordination number, number of inner-sphere water molecules, and affinity for anions of the corresponding lanthanide complex, six tris-bidentate 1,2-hydroxypyridonate (HOPO) europium(III) complexes with different cap sizes were synthesized and characterized. Wider or more flexible ligand caps, such as in Eu^III-TREN-Gly-HOPO and Eu^III-3,3-Gly-HOPO, enable the formation of nine-coordinate europium(III) complexes bearing three inner-sphere water molecules. In contrast, smaller or more rigid caps, such as in Eu^III-TREN-HOPO, Eu^III-2,2-Li-HOPO, Eu^III-3,3-Li-HOPO, and Eu^III-2,2-Gly-HOPO, favor eight-coordinate europium(III) complexes that have only two inner-sphere water molecules. Notably, there is no correlation between the number of inner-sphere water molecules and the affinity of the Eu(III) complexes for phosphate. Some q = 2 (Eu^III-TREN-HOPO, Eu^III-3,3-Li-HOPO, and Eu^III-2,2-Gly-HOPO) and some q = 3 (Eu^III-TREN-Gly-HOPO) complexes have no affinity for anions, whereas one q = 2 complex (Eu^III-2,2-Li-HOPO) and one q = 3 complex (Eu^III-3,3-Gly-HOPO) have a high affinity for phosphate. For the latter two systems, each inner-sphere water molecule is replaced with a phosphate anion, resulting in the formation of EuLPi₂ and EuLPi₃ adducts, respectively.

Full text of DOI:10.1021/acs.inorgchem.0c00137

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The Ligand Cap Affects the Coordination Number but Not
Necessarily the Affinity for Anions of Tris-Bidentate Europium
Complexes
́
Sheng-Yin Huang, Michelle Qian, and Valerie C. Pierre*
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ABSTRACT: To evaluate the effect of ligand geometry on the coordination number,
number of inner-sphere water molecules, and affinity for anions of the corresponding
lanthanide complex, six tris-bidentate 1,2-hydroxypyridonate (HOPO) europium(III)
complexes with different cap sizes were synthesized and characterized. Wider or more
flexible ligand caps, such as in EuIII-TREN-Gly-HOPO and EuIII-3,3-Gly-HOPO, enable
the formation of nine-coordinate europium(III) complexes bearing three inner-sphere
water molecules. In contrast, smaller or more rigid caps, such as in EuIII-TREN-HOPO,
EuIII-2,2-Li-HOPO, EuIII-3,3-Li-HOPO, and EuIII-2,2-Gly-HOPO, favor eight-coor-
dinate europium(III) complexes that have only two inner-sphere water molecules.
Notably, there is no correlation between the number of inner-sphere water molecules
and the affinity of the Eu(III) complexes for phosphate. Some q = 2 (EuIII-TREN-
HOPO, EuIII-3,3-Li-HOPO, and EuIII-2,2-Gly-HOPO) and some q = 3 (EuIII-TREN-
Gly-HOPO) complexes have no affinity for anions, whereas one q = 2 complex (EuIII-
2,2-Li-HOPO) and one q = 3 complex (EuIII-3,3-Gly-HOPO) have a high affinity for
phosphate. For the latter two systems, each inner-sphere water molecule is replaced with a phosphate anion, resulting in the
formation of EuLPi2 and EuLPi3 adducts, respectively.
plexes, the eight and nine coordination states are very close
in energy. Members of this class that are eight-coordinate thus
have two inner-sphere water molecules in the ground state,
whereas nine-coordinate ones have three (Figure 1). The small
difference in energy between the eight- and nine-coordinate
states leads to the characteristic fast water exchange rate
observed in GdIII members of this class that are in turn key to
the high relaxivity of their macromolecular derivatives.23−25
Previous work by our group12,26−28 and others3,22,29−31 has
demonstrated that stabilization of the nine-coordinate state
over the eight-coordinate one can be achieved either by
addition of hydrogen-bonding moieties close to the open
coordination site or by minor modification of the ligand cap.
Of those two parameters, the effect of the ligand cap on the
structure and coordination number of the lanthanide complex
remains difficult to predict. For instance, even though the
parent EuIII-TREN-HOPO [1 (Figure 2)] complex is eight-
coordinate with two coordinated water molecules,32 the
derivative EuIII-3,3-Gly-HOPO (6) is nine-coordinate with
INTRODUCTION
■
Due to their unique photophysical and magnetic properties,
coordination complexes of lanthanide ions are increasingly
being used for biomedical applications. Three of those
applications [luminescent probes,1 contrast agents for
magnetic resonance imaging (MRI),2,3 and sequestration of
anions4,5] are dependent on the geometry that the ligand
imparts on the lanthanide complex, which in turn governs the
number of inner-sphere or coordinated water molecules, q,
and, possibly, the affinity of the complex for anions. The design
of numerous responsive luminescent lanthanide probes6,7 and
contrast agents2,8,9 depends on modulating the number of
inner-sphere water molecules, often via displacement through
reversible coordination of an anion. Those labile inner-sphere
water molecules decrease the intensity and shorten the lifetime
of luminescent lanthanide probes, most commonly terbium or
europium complexes. Inner-sphere water molecules are also
key to the relaxivity of gadolinium complexes used as MRI
contrast agents. Displacing those water molecules results in a
significant decrease in the longitudinal relaxivity of GdIII-based
contrast agents. We10−12 and others13−19 have employed this
strategy for the development of responsive lanthanide-based
luminescent probes and MRI contrast agents.
Received: January 14, 2020
Literature precedence,20 including crystal structures21 and
variable-pressure NMR spectroscopy,22 suggests that for tris-
bidentate hydroxypyridinonate-based lanthanide(III) com-
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Figure 1. Tripodal, tris-bidentate lanthanide complexes whose CN of
8 is more stable than the CN of 9 have two inner-sphere water
molecules, whereas those whose CN of 9 is more stable than the CN
of 8 have three inner-sphere water molecules.
Figure 3. Chemical structures of GdIII complexes comprising different
ligand caps: (a) GdIII-TREN-1-Me-3,2-HOPO (1), (b) GdIII-Ser3-
TREN-1-Me-3,2-HOPO (2), (c) GdIII-TREN-Gly-1-Me-3,2-HOPO
(3), (d) GdIII-TRPN-1-Me-3,2-HOPO (4), (e) GdIII-TACN-3,2-
HOPO (5), and (f) GdIII-TACN-1,2-HOPO (6).
which they can be displaced by anions. Because we have
previously demonstrated that the nature of the podand affects
the affinity of the corresponding lanthanide complex for
anions,26 all complexes in this study comprise the same binding
moiety, 1,2-hydroxypyridinonate. 1,2-HOPO was chosen due
to its optimal basicity that leads to the formation of stable
lanthanide complexes and due to its strong ability to sensitize
EuIII, leading to highly emissive complexes that facilitate
investigation of hydration number and anion coordina-
tion.12,32,35,36 Moreover, because the complex charge and the
presence of hydrogen-bonding moieties are known to influence
both the number of inner-sphere water molecules27,28 and the
affinity of the complex for anions,26 all complexes in this series
bear no charge at neutral pH and do not contain pendant
alcohol or amine groups.
Our hypothesis was that expanding the ligand cap would
facilitate coordination of a third water molecule on the
lanthanide center by stabilizing the nine-coordinate state over
the eight-CN one. We further postulated that increasing the
number of inner-sphere water molecules would not necessarily
increase the affinity of the EuIII complex for anions, because
this property is also affected by ligand basicity37 and steric
hindrance at the coordination site. As shown in Figure 1,
adding a third inner-sphere water molecule does not
necessarily entail that the coordinated waters are adjacent to
one another, as would be necessary for the coordination of
bidentate ligands. With this in mind, six hydroxypyridinonate-
based EuIII complexes were synthesized and investigated
(Figure 2). They range from small ligand caps such as EuIII-
TREN-HOPO (1) and EuIII-2,2-Li-HOPO (3) to ones of
moderate sizes, such as EuIII-2,2-Gly-HOPO (5) and EuIII-3,3-
Li-HOPO (4), to the larger EuIII-TREN-Gly-HOPO (2) and
EuIII-3,3-Gly-HOPO (6).
Figure 2. Chemical structures of EuIII complexes comprising different
ligand caps: (a) EuIII-TREN-HOPO (1), (b) EuIII-TREN-Gly-HOPO
(2), (c) EuIII-2,2-Li-HOPO (3), (d) EuIII-3,3-Li-HOPO (4), (e)
EuIII-2,2-Gly-HOPO (5), and (f) EuIII-3,3-Gly-HOPO (6).
three inner-sphere water molecules.12,26 The former has no
affinity for common anions,32 whereas the latter has strong
affinity for phosphate26 at neutral pH and cyanide above pH
10.12 Similarly, most GdIII complexes with hydroxypyridino-
nate ligands (Figure 3) are eight-coordinate with q = 2,
including the parent GdIII-TREN-1-Me-3,2-HOPO (Figure
3a)33 and its derivatives GdIII-Ser3-TREN-1-Me-3,2-HOPO
(Figure 3b),30 GdIII-TREN-Gly-1-Me-3,2-HOPO (Figure
3c),29 and GdIII-TRPN-1-Me-3,2-HOPO (Figure 3d).34 On
the other hand, the ligand cap TACN favors the nine-
coordinate, q = 3 geometry, as was observed in Gd-TACN-3,2-
HOPO (Figure 3e) and Gd-TACN-1,2-HOPO (Figure 3f).34
It is not known whether this increase in q favors coordination
of endogeneous anions.
Ligand Design. The reports discussed above indicate that
how the ligand cap affects the coordination number of tripodal
lanthanide complexes remains poorly understood. Herein, we
report a systematic study of the effect of the ligand cap on the
solution structure of lanthanide complexes, specifically on their
number of inner-sphere water molecules and on the ease with
B
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RESULTS AND DISCUSSION
Scheme 2. Synthesis of EuIII-2,2-Li-HOPO (3) and EuIII-3,3-
Li-HOPO (4)
■
a
Synthesis. The EuIII-TREN-HOPO (1) and EuIII-3,3-Gly-
HOPO (6) complexes were synthesized as previously reported
by Jocher et al.32 and Huang et al.,26 respectively. EuIII-TREN-
Gly-HOPO (2) was synthesized as shown in Scheme 1. In the
a
Scheme 1. Synthesis of EuIII-TREN-Gly-HOPO (2)
a
Experimental conditions: (a) (COCl)2, DMF (catalyst), CH2Cl2, rt,
1 h; (b) NEt3, CH2Cl2, 6 h; (c) HBr/AcOH, rt, 6 h; (d) EuCl3·6H2O,
py, CH3OH, 50 °C, 6 h.
a
Experimental conditions: (a) NEt3, CH2Cl2, 6 h; (b) TFA, CH2Cl2,
rt, 2 h; (c) 2, NEt3, CH3CN, rt, 6 h; (d) HBr/AcOH, rt, 6 h; (e)
EuCl3·6H2O, py, CH3OH, 50 °C, 6 h.
a
Scheme 3. Synthesis of EuIII-2,2-Li-Gly-HOPO (5)
first step, tris(2-aminoethyl)amine (TREN) was acylated with
BOC-protected and N-hydroxysuccinimide-activated glycine
(Boc-Gly-OSu) to yield the BOC-protected TREN-Gly
backbone (7). Deprotection of the amines with trifluoroacetic
acid exposed the three amino groups onto which were
conjugated the benzyl-protected and N-hydroxysuccinimide-
activated 1,2-hydroxypyridinone podand, HOPO(Bn)-OSu
(8). The advanced intermediate HOPO(Bn)-OSu (8) was
previously synthesized in one step from commercial materials
following literature procedures.38 Deprotection of 9 under
acidic conditions yielded the free ligand TREN-Gly-HOPO
(10) that was subsequently complexed with EuCl3·6H2O in
the presence of pyridine to give the final EuIII-TREN-Gly-
HOPO (2).
The complexes EuIII-2,2-Li-HOPO (3) and EuIII-3,3-Li-
HOPO (4) were synthesized as shown in Scheme 2. This
synthetic scheme makes use of the acid chloride of the benzyl-
protected 1,2-hydroxypyridinone podand (11) because the N-
hydroxysuccinimide-activated analogue, HOPO(Bn)-OSu (8),
used in the previous scheme is not sufficiently reactive to
enable coupling to the secondary amine of the linear
backbones. HOPO(Bn) (11) was synthesized according to
the literature procedure.39 The activated podand was
synthesized from the corresponding carboxylic acid (11)
with oxalyl chloride in the presence of a catalytic amount of
DMF. The acid chloride intermediate reacted immediately
with either diethylenetriamine or dipropylenetriamine to yield
the protected ligand 2,2-Li-HOPO(Bn) (12) or 3,3-Li-
HOPO(Bn) (13), respectively. Deprotection under strong
acidic conditions yielded the free ligands that were
subsequently complexed with EuCl3·6H2O in the presence of
pyridine to give the final EuIII-2,2-Li-HOPO (3) and EuIII-3,3-
Li-HOPO (4) complexes.
a
Experimental conditions: (a) TBTU, NEt3, CH2Cl2, 6 h; (b) TFA,
CH2Cl2, rt, 2 h; (c) 2, NEt3, CH3CN, rt, 6 h; (d) HBr/AcOH, rt, 6 h;
(e) EuCl3·6H2O, py, CH3OH, 50 °C, 6 h.
first step, BOC-protected glycine was conjugated to di-BOC-
protected diethylenetriamine under standard coupling con-
ditions to form the protected backbone 16. Deprotection
under acid conditions exposed the three primary amines that
were then coupled with the activated podand HOPO(Bn)-OSu
(8) to yield the protected ligand 2,2-Gly-HOPO(Bn) (17).
Deprotection under strong acidic conditions followed by
complexation with EuCl3·6H2O in the presence of a mild base
yielded the final complex EuIII-2,2-Gly-HOPO (5).
The Ligand Cap Affects the Coordination Number
and Number of Inner-Sphere Water Molecules of the
EuIII Complex. Mass spectrometry analysis of each EuIII
complex confirms that every ligand favors the formation of
The last complex, EuIII-2,2-Gly-HOPO (5), was synthesized
according to Scheme 3 following a procedure similar to that
reported for the synthesis of EuIII-3,3-Gly-HOPO (6).26 In the
C
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mononuclear EuL complexes (Figures S14, S20, S33, S49, S54,
and S67), in agreement with the many reports of similar
lanthanide complexes.4,12,20,32,37 The structure of each complex
in solution was further evaluated by luminescence lifetime
spectroscopy, which enables determination of the number of
inner-sphere water molecules and thus, by extension, the
coordination number of each EuIII complex. The number of
inner-sphere water molecules, q, can be determined as
follows:40−43
different q values if steric hindrance caused by the methyl
groups of 1-Me-3,2-HOPO prevents coordination of a third
water molecule.
It should be noted that seemingly minor modifications to the
cap of the ligand can have a significant impact on the difference
in energy levels of eight- and nine-coordinate lanthanide
complexes even for other ligand classes, including the more
common polyaminocarboxylate ligands. Elongation of one of
the arms of GdIII-DOTA (Figure 4a) by adding one extra
q = 1.2[(1/τH O − 1/τD O) − 0.25 − 0.075x]
(1)
2
2
where q is the number of water molecules directly coordinated
on the EuIII center, τH O and τD O are the luminescence
2
2
lifetimes of the complex in protonated and deuterated water,
respectively, and x is the number of exchangeable N−H
oscillators close to the EuIII center. The strong ability of the
1,2-hydroxypyridinonate podand to sensitize EuIII lumines-
cence facilitates this study.
The measured luminescence lifetimes and calculated number
of inner-sphere water molecules, q, of each complex are
reported in Table 1. As one can see from these data, wider or
Figure 4. Chemical structures of GdIII complexes comprising different
ligands: (a) GdIII-DOTA, (b) GdIII-D03A-Nprop, and (c) GdIII-
TRITA.
methylene carbon [Gd-DO3A-Nprop (Figure 4b)] results in a
15-fold increase in the water exchange rate. Similarly,
elongation of the cyclen cap by adding one extra methylene
[Gd-TRITA (Figure 4c)] increases the water exchange rate
nearly 600-fold.46 These observations are in agreement with a
dissociative mechanism of ligand exchange in which the nine-
coordination number (CN) q = 1 ground state is closer in
energy to the eight-CN q = 0 intermediate for the elongated
derivatives compared to the parent complex.
Table 1. Luminescence Lifetimes (τ) in H2O and D2O,
Numbers of Inner-Sphere Coordinated Water Molecules
(q), Total Quantum Yields (Φt) of EuIII Complexes in the
−
Absence and Presence of HPO42−/H2PO4 , and Changes in
the Number of Inner-Sphere Water Molecules (Δq) upon
a
Phosphate Coordination
τ
τ
D2O
(ms)
H2O
Φt
The total quantum yields of each EuIII complex, Φt, are listed
in Table 1. Interestingly, although coordinated water molecules
quench much of the EuIII-centered emission, the total quantum
yields of the six tripodal HOPO europium complexes do not
correlate with their number of inner-sphere water molecules.
For instance, EuIII-TREN-Gly-HOPO has the same quantum
yield as EuIII-TREN-HOPO and EuIII-2,2-Gly-HOPO,
although the former is q = 3 while the latter two are q = 2.
Likewise, the total quantum yields of the q = 2 complexes vary
over an order of magnitude from 0.27% (EuIII-2,2-Li-HOPO)
to 3.8% (EuIII-3,3-Li-HOPO). It is noteworthy that similar
observations were also reported by the Raymond group48,49
and were attributed to the dependence of lanthanide
sensitization on the geometry of the ligand around the
complex. In particular, lanthanide sensitization is highly
dependent on the distance separating the sensitizer (HOPO)
from the europium center, a distance that is, in part, a function
of the ligand cap.48−50
(ms)
q
Δq (%)
b
EuIII-TREN-HOPO
0.22
0.22
0.18
0.45
0.37
0.53
0.30
0.54
1.9
2.9
2.3
0.1
1.3
1.3
0.27
0.36
EuIII-TREN-Gly-HOPO
EuIII-2,2-Li-HOPO
EuIII-2,2-Li-HOPO and 10 equiv
of Pi
2
3
EuIII-3,3-Li-HOPO
EuIII-2,2-Gly-HOPO
0.19
0.17
0.20
0.60
0.31
0.27
0.47
0.82
2.1
2.2
2.8
0.2
3.8
1.3
0.82
1.0
c
EuIII-3,3-Gly-HOPO
EuIII-3,3-Gly-HOPO and 10 equiv
c
of Pi
a
Experimental conditions: [EuIII complexes] = 7.6 μM, pH 7.4, λex of
b
c
335 nm, delay time of 0.1 ms. From ref 32. From ref 26.
Unsubstituted coumarin was used as the reference with a reported
quantum yield of 0.32% in water for quantum yield measurements.45
more flexible ligand caps, such as TREN-Gly and 3,3-Gly,
enable the formation of nine-coordinate europium(III)
complexes bearing three inner-sphere water molecules. In
contrast, smaller or more rigid caps, such as TREN, 2,2-Li, 3,3-
Li, and 2,2-Gly, favor eight-coordinate europium(III) com-
plexes that have only two inner-sphere water molecules. The
difference in behavior of EuIII-TREN-Gly-HOPO (2), which
has three inner-sphere water molecules, and GdIII-TREN-Gly-
1-Me-3,2-HOPO (Figure 3c), which has two,29 is surprising
given as they have the same ligand cap. The difference between
those two complexes is minor. EuIII, which is adjacent to GdIII
in the lanthanide series, is slightly larger (ionic radii of 1.120 Å
for EuIII and 1.105 Å for GdIII),44 which could be sufficient to
favor coordination of a third water molecule on 2 given how
close the eight-CN and nine-CN are in energy. The smaller
size of the podand 1,2-HOPO of 2 (Figure 2b) compared to
the 1-Me-3,2-HOPO (Figure 3c) could also contribute to the
The Ligand Cap Affects the Affinity of the EuIII
Complex for Anions. An assumption often made about
lanthanide complexes is that the higher the number of “open”
coordination sites filled with water molecules, the higher the
affinity of the complex for anions. This is not necessarily the
case, because the affinity of a lanthanide complex LnL for
anions is also a function of the steric hindrance around the
open site, the basicity or strength of ligand L, and the charge of
complex LnL among other parameters.1,6,47 All six complexes
of this study have the same neutral charge, and all consist of
the same binding moiety, 1,2-dihydroxypyridinonate. Their
different ligand caps, which give the complexes different
geometries in solution, enable evaluation of a possible direct
correlation between the number of inner-sphere water
molecules of the complex and its affinity for anions.
D
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We have previously demonstrated that for tripodal
lanthanide complexes of this class, the anion with the highest
affinity for the lanthanide is inorganic phosphate.26 Coordina-
tion of phosphate to the lanthanide center occurs with
displacement of the inner-sphere water molecules. Because the
fourth overtone of the O−H vibration of water quenches most
of the EuIII-centered luminescence, replacement of water
molecules with phosphate anions results in a significant
increase in EuIII-centered luminescence intensity and lifetime
(Figure 5). This property, which is the basis of many EuIII- and
TbIII-based luminescent probes, enables rapid evaluation of the
affinity of anions for lanthanide complexes.
complexes [EuIII-TREN-HOPO (1), EuIII-3,3-Li-HOPO (4),
and EuIII-2,2-Gly-HOPO (5)] do not show noticeable affinity
for phosphate. No increase in EuIII-centered luminescence
intensity is observed even after addition of 10 equiv of
phosphate at pH 7.4. One of the two q = 3 EuIII complexes,
EuIII-TREN-Gly-HOPO (2), also does not have noticeable
affinity for inorganic phosphate at neutral pH. It is clear from
these observations that for neutral tripodal EuIII complexes,
there is no correlation between the number of inner-sphere
water molecules and the affinity of the complex for phosphate.
Some q = 2 and q = 3 complexes have high affinity for
phosphate, and other q = 2 and q = 3 complexes have no
noticeable one.
Interestingly, the increase in the luminescence intensity of
EuIII-2,2-Li-HOPO and EuIII-3,3-Gly-HOPO upon phosphate
coordination, 10- and 20-fold, respectively (Figure 6), does not
match the increase in Φt (Table 1). This discrepancy is to be
expected given that the total quantum yield of the phosphate
adduct is a function of both the number of inner-sphere water
molecules and the geometry of the ligand around the EuIII
center. Given the bulkier size of phosphate compared to water,
the displacement of two (for EuIII-2,2-Li-HOPO) or three (for
EuIII-3,3-Gly-HOPO) water molecules by phosphate anions is
expected to alter the geometry of the lanthanide complex,
resulting in decreased sensitization efficiency and hence a
smaller increase in total quantum yields.
Figure 5. Mechanism of the luminescence response of EuIII complexes
to phosphate. In the absence of phosphate, the water molecules
coordinated to the EuIII center quench most of the lanthanide-
centered emission. Displacement of the inner-sphere water molecules
by coordinating phosphate eliminates this quenching mechanism,
resulting in a significant increase in the EuIII-centered phosphor-
escence intensity and lifetime.
The response of the six HOPO-based EuIII complexes to
other common endogenous anions was also tested by time-
gated luminescence spectroscopy. As shown in Figure 7 (white
bars), none of the six complexes have any affinity for F−, Cl−,
Of the six HOPO-based EuIII complexes evaluated, only two
have affinity for phosphate in water at neutral pH: EuIII-2,2-Li-
HOPO (3), which is q = 2, and EuIII-3,3-Gly-HOPO (6),
which is q = 3 (Figure 6). Three of the four q = 2 EuIII
Br−, I−, HCO3 /CO32−, SO4 , CH3CO2 , and NO3 in water
at pH 7.4. Sequential addition of 10 equiv of phosphate to
solutions containing the europium complex and the competing
anion indicated that the presence of these anions did not
interfere substantially with the luminescence response, or a
lack of response, of the EuIII complexes toward phosphate
(Figure 7, gray bars). Compared to their polyamino-
carboxylate counterparts, HOPO-based EuIII complexes
demonstrate unique resistance to the coordination of fluoride,
bicarbonate, and acetate.1,7,10,11,51−53
−
−
−
−
The composition of the complex formed in solution between
phosphate and either EuIII-2,2-Li-HOPO (3) or EuIII-3,3-Gly-
HOPO (6) was evaluated by three complementary techniques.
Luminescence lifetime studies confirmed the lack of any
residual water molecule in the phosphate adducts formed with
EuIII-2,2-Li-HOPO or EuIII-3,3-Gly-HOPO (Table 1 and
Figure S3). Jobs’ plots confirmed the 1:2 and 1:3 EuL:Pi
ratios of 3 and 6, respectively (Figure S8).26 Finally, elemental
analysis by energy dispersive X-ray microscopy in conjunction
with scanning electron microscopy (SEM-EDS) (Table S1)
also confirmed the binding ratios,26 indicating that in both
cases, all inner-sphere water molecules on the EuIII center were
displaced, and that each phosphate anion replaced a single
water molecule. All three analyses indicate that EuIII-2,2-Li-
HOPO (3), which is q = 2, forms a EuLPi2 complex whereas
EuIII-3,3-Gly-HOPO (6), which is q = 3, forms a EuLPi3
complex, where Pi is inorganic phosphate.
Figure 6. Integrated time-gated luminescence intensity of EuIII
complexes in the absence (white bars) and presence (gray bars) of
10 equiv of phosphate. Experimental conditions: [EuIII complex] = 7.6
μM in H2O, pH 7.4, λexcitation = 335 nm, excitation slit width of 10 nm,
emission slit width of 5 nm, time delay of 0.1 ms, I is the integrated
time-gated luminescence intensity from 550 to 750 nm, and Io is the
integrated time-gated luminescence intensity in the absence of
phosphate. Luminescence spectra were recorded 15 min after mixing
to ensure that thermodynamic equilibrium was reached (see Figure
S1). The results are means the standard deviation (n = 3).
Determination of the composition of the final EuL:Pi
composition enables us to determine the formation constants
from titrations monitored by time-gated luminescence spec-
troscopy (Figure 8a). The stepwise association constants of the
EuIII complexes for phosphate, Ka, are defined as follows
E
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Figure 7. Time-delayed luminescence response of EuIII complexes to common anions in water: (a) EuIII-TREN-HOPO (1), (b) EuIII-TREN-Gly-
HOPO (2), (c) EuIII-2,2-Li-HOPO (3), (d) EuIII-3,3-Li-HOPO (4), (e) EuIII-2,2-Gly-HOPO (5), and (f) EuIII-3,3-Gly-HOPO (6). White bars
represent the time-delayed relative luminescence intensity after the addition of 10 equiv of the appropriate anions (NaF, NaCl, NaBr, NaI,
NaHCO3, Na2SO4, NaOAc, and NaNO3). Gray bars represent the time-delayed relative luminescence intensity after the subsequent addition of 10
equiv of phosphate. Experimental conditions: [EuIII complex] = 7.6 μM in H2O, pH 7.4, λexcitation = 335 nm, excitation slit width of 10 nm, emission
slit width of 5 nm, time delay of 0.1 ms, I is the integrated time-gated luminescence intensity from 550 to 750 nm, and Io is the integrated time-
gated luminescence intensity in the absence of phosphate. Luminescence spectra were recorded 15 min after mixing to ensure that thermodynamic
equilibrium was reached.
Ka1 EuL + P F EuL·P
(2)
(3)
(4)
obtained by fitting the titration data with the appropriate
models as described by Thordarson.54
i
i
As shown in Figure 8b, both EuIII-2,2-Li-HOPO and EuIII-
3,3-Gly-HOPO display a slower increase in luminescence
intensity at a lower concentration of phosphate systems, which
is characteristic of a cooperativity effect. As a result, in both
systems, Ka1 ≤ Ka2 (Table 2). Although unusual, such a
cooperativity is not unprecedented for lanthanide complexes.
In the case of lanthanide-based dipicolinic acid receptors,55−57
Jones and Silber attributed such a cooperativity to a decrease in
the electron density of the lanthanide center upon coordina-
tion of the first anion. This was postulated to increase the
Ka2 EuL·P + P F EuL·P
i
i
i2
Ka3 EuL·P + P F EuL·P
i2
i
i3
where Pi denotes HPO42−/H2PO4 . The cumulative constant,
β, refers to the overall association of all phosphates on the EuIII
complex as follows:
−
β
EuL + nP F EuL·P
(5)
i
in
where n = 2 for EuIII-2,2-Li-HOPO and n = 3 for EuIII-3,3-Gly-
HOPO. Stepwise and cumulative binding constants were
F
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parameters such as ligand basicity, number and size of chelate
rings, bite angles, and complex charge also affect formation
constants. A nine-coordinate q = 3 LnL complex with a
hexadentate ligand is thus not necessarily less stable than a
nine-coordinate q = 1 LnL complex with a heptadentate ligand.
The stability of metal complexes can be determined by
determining the pKa’s of the ligand and that of the metal
complexes, often by either potentiometry or spectrophotom-
etry. Tripodal ligands and complexes of this class often have
four or more and two or more pKa’s, respectively. Combined
with their low solubility, the lack of distinct features in their
titration profiles can make their interpretation difficult. Instead,
we have determined the stability of each complex at a given pH
(pH 7.4) by direct competition with diethylenetriaminepenta-
acetic acid (DTPA), a ligand whose europium complex has
been well characterized thermodynamically.58,59 As previously
reported, the equilibrium, depicted in eq 6, enables direct
determination of the stability of the complex at that pH in
terms of pM.
EuIIIL(aq) + DTPA(aq) F EuIII‐DTPA(aq) + L(aq)
(6)
The pM is defined by the concentration of free (uncomplexed)
metal in solution as follows:
Figure 8. (a) Time-delayed luminescence intensity of EuIII-2,2-Li-
pM = −log[M]free when [L]total = 10−5 M and [M]total = 10−6 M
2−
HOPO upon addition of increasing concentrations of HPO4
/
(7)
−
H2PO4 . (b) Increase in the time-delayed luminescence intensity of
EuIII-2,2-Li-HOPO (empty green circles) and EuIII-3,3-Gly-HOPO
Because the pM of Eu-DTPA is known, the concentration of
the competing DTPA ligand needed to partition equally the
metal ion between the hydroxypyridinone ligand and DTPA
[log([DTPA]/[L]) when log([M-DTPA]/[M-L]) = 0]
directly gives the pM of the ligand L at that pH. The more
stable the complex, the higher its pM value. Advantageously,
EuIII-DTPA has a substantially lower quantum yield compared
to those of the HOPO-based EuIII complexes, such that
formation of the EuIII-DTPA complex at the detriment of the
EuIIIL complex results in a substantial decrease in time-gated
luminescence intensity, which can be easily monitored by
spectroscopy.
The time-gated luminescence spectra of the HOPO-based
complexes (1−6) upon addition of DTPA are shown in
Figures S9 and S10. Interestingly, only three HOPO-based
complexes transmetallate upon addition of DTPA: EuIII-
TREN-HOPO (1), EuIII-TREN-Gly-HOPO (2), and EuIII-
2,2-Gly-HOPO (5). For those complexes, further competition
titration with DTPA (Figure S9a−d) enabled determination of
the pM values listed in Table 3. As expected on the basis of the
(filled blue triangles) upon titration with HPO42−/H2PO4
.
−
Experimental conditions: [EuIII complex] = 7.6 μM in H2O, pH
7.4, λexcitation = 335 nm, excitation slit width of 10 nm, emission slit
width of 5 nm, time delay of 0.1 ms, I is the integrated time-gated
luminescence intensity from 550 to 750 nm, and Io is the integrated
time-gated luminescence intensity in the absence of phosphate.
Luminescence spectra were recorded 15 min after mixing to ensure
that thermodynamic equilibrium was reached (see Figure S1). The
results are means the standard deviation (n = 3).
Table 2. Cumulative (β) and Stepwise (Ka) Association
Constants of EuIII-2,2-Li-HOPO and EuIII-3,3-Gly-HOPO in
Complexes with Phosphate
a
β
Ka1 (M−1
)
Ka2 (M−1
)
Ka3 (M−1
)
EuIII-2,2-Li-HOPO
EuIII-3,3-Gly-
HOPO
2.5 × 1010
3.2 × 1014
8.5 × 104
4.7 × 103
2.9 × 105
1.9 × 105
−
3.6 × 105
a
M−2 for EuIII-2,2-Li-HOPO and M−3 for EuIII-3,3-Gly-HOPO.
electropositivity of the remaining open coordination sites,
which enhanced binding of the second anion.
Table 3. pM Values of Tripodal EuIII Complexes
Interestingly, the two europium(III) complexes bind
phosphate strongly in water, but with different affinities. The
first and second stepwise binding constants of EuIII-2,2-Li-
HOPO (3), for which q = 2, are both higher than those of
EuIII-3,3-Gly-HOPO (6), for which q = 3. Although the source
of this difference is unknown, it could potentially be due to the
increased steric hindrance at the open coordination site for the
nine-CN EuIII-3,3-Gly-HOPO compared to the eight-CN EuIII-
2,2-Li-HOPO.
pM
EuIII-TREN-HOPO
EuIII-TREN-Gly-HOPO
EuIII-2,2-Gly-HOPO
19.1
18.1
17.5
literature, EuIII-TREN-HOPO (1) is the most stable complex
with a pM value close to that of EuIII-DTPA.58,59 EuIII-TREN-
Gly-HOPO (2) and EuIII-2,2-Gly-HOPO (5) are both less
stable, with pM values that are 18.1 and 17.5 lower than that of
the parent tripodal complex EuIII-TREN-HOPO, respectively.
These observations are in agreement with previous work by the
Raymond group that demonstrated that modification of the
TREN backbone via insertion of a glycine spacer into the cap
The Ligand Cap Affects the Stability of the EuIII
Complex. Another assumption sometimes made about
lanthanide complexes is that the lower the denticity of the
ligand L, the lower the stability of the lanthanide complex LnL.
However, the stability of a lanthanide coordination complex is
not related only to the denticity of the ligand. Other
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can significantly decrease the stability of the corresponding
lanthanide complexes.3,29
EXPERIMENTAL SECTION
■
General Considerations. Unless stated otherwise, all chemicals
were purchased from commercial suppliers and used without further
purification. Deuterated solvents were obtained from Cambridge
Isotope Laboratories (Tewskbury, MA). Distilled water was further
purified by a Millipore Simplicity UV system (resistivity of 18 × 106
Ω). All organic extracts were dried over anhydrous MgSO4(s). Flash
chromatography was performed on Merck Silica Gel. Modified silica
gel was prepared by heating the silica gel in 37% HCl(aq) at 50 °C for
6 h, further washing it with deionized water until the pH of the filtrate
was neutral, and drying it under reduced pressure at 100 °C. The
collected silica gel was subsequently suspended in toluene with 1%
(v/v) hexadecyltrimethoxysilane. The mixture was stirred at 100 °C
for 6 h, after which the mixture was filtered, rinsed with toluene and
ethyl acetate, and dried under reduced pressure at 100 °C.
Surprisingly, three of the HOPO-based complexes do not
transmetallate upon addition of excess DTPA. Instead, EuIII-
2,2-Li-HOPO (3), EuIII-3,3-Li-HOPO (4), and EuIII-3,3-Gly-
HOPO (6) form ternary Eu(L)(DTPA) complexes as shown
in equilibrium 8. In these cases, a single DTPA ligand replaces
the inner-sphere water molecules of the HOPO-based EuL,
resulting in a significant increase in time-gated luminescence
intensity. While the formation of the ternary complex does not
enable us to determine the pM of the HOPO-based EuL
complex, the formation of a stable ternary complex suggests
that those HOPO ligands remain strong chelators of
europium(III).
1H NMR and 13C NMR spectra were recorded at room
temperature on a Bruker Advance III 400 instrument at 400 and
100 MHz, respectively, or a Bruker Advance III AV 500 instrument at
500 and 125 MHz, respectively, at the LeClaire-Dow instrumentation
facility of the Department of Chemistry of the University of
Minnesota. The residual solvent peaks were used as internal
references. Data for 1H NMR are recorded as follows: chemical
shift (δ, parts per million), multiplicity (s, singlet; d, doublet; t, triplet;
q, quartet; br, broad; m, multiplet), coupling constant (hertz),
integration. Data for 13C NMR are recorded as follows: chemical shift
(δ, parts per million). Low-resolution (LR) and high-resolution (HR)
electrospray ionization time-of-flight mass spectrometry (ESI/TOF-
MS) data were recorded on a Bruker BioTOF I instrument at the
LeClaire-Dow instrumentation facility of the Department of
Chemistry of the University of Minnesota. Ultraviolet−visible spectra
were recorded on a Varian Cary 100 Bio Spectrophotometer. Data
were collected over the range of 200−800 nm. Luminescence data
were acquired on a Varian Cary Eclipse fluorescence spectropho-
tometer using a quartz cell with a path length of 1 cm and a chamber
volume of 400 μL. Time-gated luminescent spectra were recorded
with a time delay of 0.1 ms and a gate time of 5 ms. Sample solutions
were allowed to equilibrate for 15 min before measurement of their
luminescence spectra, as initial studies demonstrated that this time
was sufficient to achieve thermodynamic equilibrium (Figure S1).
Every data point was measured in triplicate from three independently
prepared samples. Luminescence data were processed with Scilab
6.0.2 and QtiPlot 0.9.8.9 software. All pH measurements were
performed using a Thermo Scientific Ag/AgCl refillable probe and a
Thermo Orion 3 Benchtop pH meter. Characterization by scanning
electron microscopy−energy dispersive X-ray spectroscopy (SEM-
EDS) was performed on a JEOL 6500 instrument at the Character-
ization Facility at the University of Minnesota. High-performance
liquid chromatography (HPLC) data were collected on a Varian
Prostar model 210 instrument, coupled with an Agilent ZORBAX
Eclipse XDB-C18 column, and a Varian ProStar 335 diode array
detector. Unless specified otherwise, HPLC measurements were
performed at a flow rate of 1.0 mL min−1 with the following elution
condition: 15% CH3CN/85% water from 0 to 2 min, followed with a
linear gradient to 85% CH3CN/15% water from 2 to 23 min, 85%
CH3CN/15% water from 23 to 26 min, with a linear gradient to 15%
CH3CN/85% water from 26 to 30 min, and 15% CH3CN/85% water
from 26 to 32 min. Luminescence lifetimes were measured as reported
in the literature.32
EuIIIL(aq) + DTPA(aq) F EuIII(L)(DTPA)(aq)
(8)
Interestingly, there is no correlation between the number of
inner-sphere water molecules, q, and the preference for
transmetalation over the formation of ternary complexes.
Some q = 2 (1 and 5) and some q = 3 (2) complexes
transmetallate upon addition of excess competing DTPA;
instead, other q = 2 (3 and 4) and q = 3 (6) complexes favor
the formation of ternary complexes. It is noteworthy, though,
that the two complexes that do have high affinity for phosphate
[EuIII-3,3-Gly-HOPO (6) and EuIII-2,2-Li-HOPO (3)] both
favor formation of ternary complexes with DTPA, although
they do not have strong affinity for either carbonate or acetate.
It is possible that the inner-sphere water molecules of these
two complexes suffer from less steric hindrance, which
facilitates coordination of phosphate and polydentate anions.
CONCLUSION
■
The structure and behavior of lanthanide(III) complexes of
tripodal tris-bidentate ligands in solution are highly dependent
on the geometry that the cap imparts on the ligand. For 1,2-
hydroxypyridinonate-based europium(III) complexes, the eight
and nine coordination states are very close in energy and either
state can be preferentially stabilized. Smaller ligand caps such
as TREN, 2,2-Li, 3,3-Li, and 2,2-Gly favor solution structures
in which the europium(III) is eight-coordinate. Larger caps
such as TREN-Gly and 3,3-Gly, on the other hand, favor nine-
coordinate structures. A larger ligand cap and a higher number
of inner-sphere water molecules do not correlate with a higher
affinity for anions. Both eight-coordinate, q = 2 (EuIII-2,2-Li-
HOPO) and nine-coordinate, q = 3 (EuIII-3,3-Gly-HOPO)
complexes can have a high affinity for phosphate. Likewise,
some eight-coordinate, q = 2 complexes (EuIII-TREN-HOPO,
EuIII-3,3-Li-HOPO, and EuIII-2,2-Gly-HOPO) and some nine-
coordinate, q = 3 complexes (EuIII-TREN-Gly-HOPO) have
no noticeable affinity for small anions. Regardless of the
number of inner-sphere water molecules, formation of the
phosphate EuL complex occurs via displacement of one water
molecule per phosphate. EuIII-2,2-Li-HOPO, which is a q = 2
complex, thus leads to a EuLPi2 complex, whereas EuIII-3,3-
Gly-HOPO, which is a q = 3 complex, leads to EuLPi3.
Phosphate binding does not appear to alter the coordination
number of the complex. As a whole, regardless of the geometry
of the ligand, 1,2-hydroxypyridinonate lanthanide(III) com-
plexes retain excellent resistance to the coordination of
bicarbonates, acetates, and fluoride.
EuIII-TREN-HOPO (1). The free ligand TREN-HOPO was
prepared according to the reported procedure with successful
synthesis of a pure compound established by 1H NMR, ESI-MS,
and HPLC.32 TREN-HOPO (20 mg, 0.036 mmol) and EuCl3·6H2O
(13 mg, 0.036 mmol) were dissolved in a 1:1 (v/v) mixture of
methanol and Milli-Q water (5 mL). Pyridine (26 μL, 0.36 mmol)
was subsequently added to the reaction mixture, which was stirred at
60 °C for 15 h. The reaction mixture was allowed to cool to room
temperature. The suspension was then filtered and rinsed with
methanol. The residue was further dried under reduced pressure to
H
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yield the final EuIII complex 1 as a beige powder (20 mg, 80%). ESI-
HRMS: m/z 708.0922 ([M + H]+) (calcd 708.0887).
EuIII-TREN-Gly-HOPO (2). The deprotected ligand 10 (20 mg,
0.025 mmol) and EuCl3·6H2O (9 mg, 0.025 mmol) were dissolved in
a 1:1 (v/v) mixture of methanol and Milli-Q water (5 mL). Pyridine
(20 μL, 0.25 mmol) was subsequently added to the reaction mixture,
which was stirred at 60 °C for 15 h. The reaction mixture was allowed
to cool to room temperature. The suspension was then filtered and
rinsed with methanol. The residue was further dried under reduced
pressure to yield the final EuIII complex 2 as a beige powder (20 mg,
90%). ESI-HRMS: m/z 879.1536 ([2M + 2H]2+) (calcd 879.1567).
1-(Benzyloxy)-N,N-bis{2-[1-(benzyloxy)-6-oxo-1,6-dihydro-
pyridine-2-carboxamido]ethyl}-6-oxo-1,6-dihydropyridine-2-
carboxamide (12). Oxalyl chloride (400 μL, 4.48 mmol) and a drop
of DMF were added to a suspension of the benzyl-protected 1,2-
hydroxypyridinone carboxylic acid 11 (1.00 g, 4.08 mmol) in
anhydrous CH2Cl2 (10 mL). The reaction mixture was stirred at
room temperature under a steady N2(g) flow connected to a bubbler
containing 25% NaOH(aq) to scrub evolved HCl(g) for 1 h. The
volatiles were removed under reduced pressure to yield a viscous
amber liquid, which was used immediately without further
purification. The intermediate was dissolved in anhydrous CH2Cl2
(20 mL) and cooled with an ice bath under a flow of N2(g). A
solution of diethylenetriamine (146 μL, 1.36 mmol) and triethylamine
(568 μL, 4.08 mmol) in anhydrous CH2Cl2 (5 mL) was added to the
solution of the intermediate acid chloride at 0 °C. The reaction
mixture was allowed to warm to room temperature and further stirred
at room temperature for 6 h. The reaction mixture was then washed
with 1 N HCl(aq) (20 mL) and 10% NaHCO3(aq) (20 mL) and
dried over anhydrous MgSO4(s). The solvent was removed under
reduced pressure to give the crude product as a viscous oil that was
further purified by flash chromatography over silica eluting with a 4%
CH3OH/96% CH2Cl2 mixture to yield the protected ligand 12 a
white foaming liquid (864 mg, 81%). 1H NMR (500 MHz, CDCl3): δ
7.45−7.42 (m, 4H), 7.35−7.27 (m, 13H), 7.18 (dd, J1 = 7 Hz, J2 = 2
Hz, 1H), 7.11 (dd, J1 = 7 Hz, J2 = 2 Hz, 2H), 6.59−6.51 (m, 3H),
6.22 (dd, J1 = 7 Hz, J2 = 1 Hz, 1H), 6.12 (dd, J1 = 7 Hz, J2 = 1 Hz,
1H), 6.05 (dd, J1 = 7 Hz, J2 = 1 Hz, 1H), 5.58 (d, J = 8 Hz, 1H), 5.26
(q, J1 = 9 Hz, J2 = 6 Hz, 2H), 5.17 (dd, J1 = 16 Hz, J2 = 9 Hz, 2H),
4.98 (d, J = 8 Hz, 1H), 3.94−3.85 (m, 1H), 3.56−3.46 (m, 1H),
3.35−3.10 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 162.7, 160.8,
160.5, 158.3, 158.2, 157.9, 142.4, 142.1, 141.9, 138.2, 138.0, 137.9,
133.3, 133.12, 133.06, 129.8, 129.3, 129.2, 128.42, 128.37, 123.6,
122.8, 105.7, 105.4, 103.0, 79.1 78.93, 78.88, 48.1, 45.2, 37.9. ESI-
HRMS: m/z 807.2570 ([M + Na]+) (calcd 807.2749).
1-(Benzyloxy)-N,N-bis{3-[1-(benzyloxy)-6-oxo-1,6-dihydro-
pyridine-2-carboxamido]propyl}-6-oxo-1,6-dihydropyridine-
2-carboxamide (13). Oxalyl chloride (390 μL, 4.49 mmol) and a
drop of DMF were added to a suspension of the benzyl-protected 1,2-
hydroxypyridinone carboxylic acid 11 (1.00 g, 4.08 mmol) in
anhydrous CH2Cl2 (10 mL). The reaction mixture was stirred at
room temperature under a steady N2(g) flow connected to a bubbler
containing 25% NaOH(aq) to scrub evolved HCl(g) for 1 h. The
volatiles were removed under reduced pressure to yield a viscous
amber liquid, which was used immediately without further
purification. The intermediate was dissolved in anhydrous CH2Cl2
(20 mL) and cooled with an ice bath under a flow of N2(g). A
solution of diethylenetriamine (190 μL, 1.36 mmol) and triethylamine
(650 μL, 4.66 mmol) in anhydrous CH2Cl2 (5 mL) was added to the
solution of the intermediate acid chloride at 0 °C. The reaction
mixture was allowed to warm to room temperature and further stirred
at room temperature for 6 h. The reaction mixture was then washed
with 1 N HCl(aq) (20 mL) and 10% NaHCO3(aq) (20 mL) and
dried over anhydrous MgSO4(s). The solvent was removed under
reduced pressure to give the crude product as a viscous oil that was
further purified by flash chromatography over silica eluting with a 5%
CH3OH/95% CH2Cl2 mixture to yield the protected ligand 13 as a
white foaming liquid (936 mg, 85%). 1H NMR (500 MHz, CDCl3): δ
7.55−7.49 (m, 2H), 7.49−7.40 (m, 4H), 7.38−7.34 (m, 3H), 7.31−
7.28 (m, 1H), 7.26−7.10 (m, 10H), 6.60 (dd, J1 = 9 Hz, J2 = 1 Hz,
1H), 6.47 (dd, J1 = 9 Hz, J2 = 1 Hz, 2H), 6.24 (dd, J1 = 7 Hz, J2 = 1
Hz, 1H), 6.12 (dd, J1 = 7 Hz, J2 = 1 Hz, 1H), 5.85 (dd, J1 = 7 Hz, J2 =
EuIII-3,3-Gly-HOPO (6). The free ligand 3,3-Gly-HOPO was
prepared according to the reported procedure with successful
synthesis of a pure compound established by 1H NMR, ESI-MS,
and HPLC.26 The ligand 3,3-Gly-HOPO (20 mg, 0.033 mmol) and
EuCl3·6H2O (12 mg, 0.033 mmol) were dissolved in a 1:1 (v/v)
mixture of methanol and Milli-Q water (5 mL). Pyridine (27 μL, 0.33
mmol) was subsequently added to the reaction mixture, which was
stirred at 60 °C for 15 h. The reaction mixture was allowed to cool to
room temperature. The suspension was then filtered and rinsed with
methanol. The residue was further dried under reduced pressure to
yield the final EuIII complex 6 as a beige powder (22 mg, 89%). ESI-
HRMS: m/z 750.0968 ([M + H]+) (calcd 750.1028), m/z 771.0859
([2M + 2Na]2+) (calcd 771.0841).
Tri-tert-butyl ({[Nitrilotris(ethane-2,1-diyl)]tris(azanediyl)}-
tris(2-oxoethane-2,1-diyl))tricarbamate (7). Tris(2-aminoethyl)-
amine (TREN, 183 μL, 1.22 mmol) and triethylamine (682 μL, 4.90
mmol) were added to a solution of Boc-glycine-N-hydroxysuccini-
mide (Boc-Gly-OSu, 1000 mg, 3.673 mmol) in CH2Cl2 (10 mL). The
reaction mixture was stirred at room temperature for 6 h, after which
it was washed with 10% NaHCO3(aq) (10 mL) and dried over
anhydrous MgSO4(s). The solvents were then removed under
reduced pressure, and the residual viscous product was purified by
flash chromatography over silica eluting with an 8% CH3OH/92%
CH2Cl2 mixture to yield 7 as a white foaming liquid (604 mg, 80%).
1H NMR (400 MHz, CDCl3): δ 5.92 (br, 2H), 3.79 (d, J = 6 Hz, 6H),
3.23 (br, 6H), 2.52 (m, 6H), 1.40 (s, 27H). 13C NMR (100 MHz,
CDCl3): δ 170.46, 156.49, 79.91, 54.45, 44.10, 37.91, 28.28. ESI-
HRMS: m/z 640.3712 ([M + Na]+) (calcd 640.3640).
N,N′,N″-({[Nitrilotris(ethane-2,1-diyl)]tris(azanediyl)}tris(2-
oxoethane-2,1-diyl))tris[1-(benzyloxy)-6-oxo-1,6-dihydropyri-
dine-2-carboxamide] (9). Trifluoroacetic acid (10 mL) was added
to a solution of the BOC-protected ligand cap 7 (500 mg, 0.810
mmol) in CH2Cl2 (10 mL). The reaction mixture was stirred at room
temperature for 1 h, after which the volatiles were removed under
reduced pressure to yield the deprotected ligand cap that was used
immediately without further purification. Benzyl-protected N-
hydroxysuccinimide-activated 1,2-hydroxypyridinone podand HOPO-
(Bn)-OSu 8 (831 mg, 2.43 mmol)38 was added to a solution of the
deprotected ligand cap in CH3CN (10 mL). Triethylamine (564 μL,
4.05 mmol) was then slowly added to the reaction mixture, which was
further stirred at room temperature for 6 h. The reaction mixture was
then washed with 10% NaHCO3(aq) (10 mL) and dried over
anhydrous MgSO4(s) to yield a viscous liquid. This crude product was
purified by flash chromatography over silica eluting with an 8%
CH3OH/92% CH2Cl2 mixture to yield a white foaming liquid that
was further recrystallized with CH3OH/CH2Cl2 to yield the protected
1
ligand 9 as a white foaming liquid (687 mg, 85%). H NMR (500
MHz, CDCl3): δ 8.23 (br, 3H), 7.40 (d, J = 7 Hz, 6H), 7.28−7.25 (m,
9H), 7.17 (t, J = 7 Hz, 3H), 7.01 (br, 3H), 6.57 (d, J = 7 Hz, 3H),
6.31 (d, J = 7 Hz, 3H), 5.24 (s, 6H), 3.89 (m, 6H), 2.85 (br, 6H),
2.10 (br, 6H). 13C NMR (125 MHz, CDCl3): δ 168.5, 160.6, 158.2,
142.0, 138.1, 132.9, 130.1, 129.3, 128.4, 123.7, 105.8, 79.1, 54.0, 43.0,
37.6. ESI-HRMS: m/z 1021.3897 ([M + Na]+) (calcd 1021.3815).
N,N′,N″-({[Nitrilotris(ethane-2,1-diyl)]tris(azanediyl)}tris(2-
oxoethane-2,1-diyl))tris(1-hydroxy-6-oxo-1,6-dihydropyri-
dine-2-carboxamide) (TREN-Gly-HOPO, 10). The protected
ligand 9 (500 mg, 0.501 mmol) was dissolved in a 1:1 (v/v) mixture
of glacial acetic acid (5 mL) and HBr(aq) (37%, 5 mL). The reaction
mixture was stirred at room temperature for 6 h, after which the
volatiles were removed under reduced pressure. The residue was then
purified by flash chromatography over modified silica eluting with a
gradient of 100% H2O to 5% CH3OH/95% H2O to yield the final
1
deprotected ligand 10 as a beige foaming liquid (303 mg, 83%). H
NMR (400 MHz, D2O): δ 7.34 (t, J = 9 Hz, 3H), 6.79 (d, J = 7 Hz,
3H), 6.64 (d, J = 9 Hz, 3H), 4.09 (s, 6H), 3.62 (br, 6H), 3.47 (br,
6H). 13C NMR (100 MHz, D2O): δ 172.0, 162.6, 160.7, 139.0, 136.5,
120.3, 109.8, 54.0, 42.9, 34.7. ESI-HRMS: m/z 729.2591 ([M − Br]+)
(calcd 729.2587).
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1 Hz, 1H), 5.55 (d, J = 8 Hz, 1H), 5.32−5.14 (m, 4H), 5.01 (d, J = 8
Hz, 1H), 3.28−3.17 (m, 1H), 3.17−3.01 (m, 3H), 2.94−2.83 (m,
2H), 2.83−2.71 (m, 2H), 1.37−1.25 (m, 2H), 1.25−1.10 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 161.3, 160.4, 160.3, 157.8, 157.7,
142.7, 142.4, 142.3, 138.0, 133.1, 133.0, 132.9, 130.1, 129.94, 129.85,
129.3, 129.2, 129.0, 128.30, 128.27, 128.2, 123.3, 123.2, 122.4, 104.6,
102.2, 79.2, 79.1, 78.9. ESI-HRMS: m/z 835.3053 ([M + Na]+)
(calcd 835.3062).
washed with 10% citric acid(aq) (20 mL) and 10% NaHCO3(aq) (20
mL). The organic layer was dried over anhydrous MgSO4(s) and
concentrated under reduced pressure. The crude product was then
purified by flash chromatography over silica eluting with a 40% ethyl
acetate/60% hexanes mixture to yield the protected cap 16 as a white
1
foaming liquid (1.89 g, 90%). H NMR (400 MHz, CDCl3): δ 5.53
(br, 1H), 5.42 (br, 1H), 5.21 (br, 1H), 3.90 (br, 2H), 3.41 (br, 2H),
3.32 (br, 2H), 3.24 (br, 2H), 1.42−1.31 (m, 27H). 13C NMR (100
MHz, CDCl3): δ 169.8, 156.2, 155.9, 155.7, 79.6, 79.4, 79.2, 47.4,
46.3, 42.0, 38.8, 28.23, 28.22, 28.20. ESI-HRMS: m/z 483.2794 ([M
+ Na]+) (calcd 483.2789).
1-Hydroxy-N,N-bis[2-(1-hydroxy-6-oxo-1,6-dihydropyri-
dine-2-carboxamido)ethyl]-6-oxo-1,6-dihydropyridine-2-car-
boxamide (2,2-Li-HOPO, 14). The protected ligand 12 (500 mg,
0.638 mmol) was dissolved in a 1:1 (v/v) mixture of glacial acetic acid
and HBr(aq) (37%, 10 mL). The reaction mixture was stirred at room
temperature for 6 h. The volatiles were then removed under reduced
pressure, and the residue was purified by flash chromatography over
modified silica eluting with a gradient of 100% H2O to 5% CH3OH/
95% H2O to yield the deprotected ligand 14 as a beige foaming liquid
1-(Benzyloxy)-N-(2-{2-[1-(benzyloxy)-6-oxo-1,6-dihydropyr-
idine-2-carboxamido]-N-{2-[1-(benzyloxy)-6-oxo-1,6-dihydro-
pyridine-2-carboxamido]ethyl}acetamido}ethyl)-6-oxo-1,6-di-
hydropyridine-2-carboxamide (17). Trifluoroacetic acid (10 mL)
was added to a solution of the protected cap 16 (500 mg, 1.09 mmol)
in CH2Cl2 (10 mL). The reaction mixture was stirred at room
temperature for 1 h. The volatiles were removed under reduced
pressure to yield the deprotected ligand cap that was used
immediately without further purification. The benzyl-protected N-
hydroxysuccinimide-activated 1,2-hydroxypyridinone podand [8,
HOPO(Bn)-OSu, 1.11 g, 3.26 mmol] was added to a solution of
the deprotected ligand cap in CH3CN (10 mL). Triethylamine (697
μL, 4.36 mmol) was then slowly added to the reaction mixture, which
was stirred at room temperature for 6 h. The reaction mixture was
then washed with 10% NaHCO3(aq) (10 mL), dried over anhydrous
MgSO4(s), and concentrated under reduced pressure to yield a
viscous liquid. The crude product was further purified by flash
chromatography over silica eluting with a 4% CH3OH/96% CH2Cl2
mixture to yield the protected ligand 17 as a foam (780 mg, 85%). 1H
NMR (500 MHz, CDCl3): δ 7.80 (br, 1H), 7.51−7.45 (m, 2H),
7.44−7.36 (m, 7H), 7.34−7.28 (m, 6H), 7.25−7.18 (m, 4H), 7.17−
7.10 (m, 2H), 6.62 (d, J = 9 Hz, 1H), 6.54 (dd, J1 = 8 Hz, J2 = 1 Hz,
2H), 6.29 (d, J = 7 Hz, 1H), 6.25 (d, J = 7 Hz, 1H), 6.19 (d, J = 7 Hz,
1H), 5.24 (s, 4H), 5.22 (s, 2H), 3.86 (d, J = 5 Hz, 2H), 3.35 (br, 4H),
3.31−3.25 (m, 2H), 3.23−3.18 (m, 2H). 13C NMR (125 MHz,
CDCl3): δ 168.6, 161.0, 160.7, 159.8, 158.4, 158.3, 142.7, 142.4,
141.7, 138.10, 138.06, 137.9, 133.3, 133.2, 133.0, 130.2, 130.1, 130.0,
129.34, 129.26, 128.51, 128.50, 128.4, 124.1, 123.6, 106.6, 105.7,
105.4, 79.2, 79.0, 77.3, 77.2, 77.0, 76.7, 46.4, 45.8, 41.3, 38.4, 38.2.
ESI-HRMS: m/z 864.3072 ([M + Na]+) (calcd 864.2964).
1
(295 mg, 90%). H NMR (400 MHz, CD3OD): δ 7.54−7.42 (m,
3H), 6.75 (dd, J1 = 9 Hz, J2 = 1 Hz, 2H), 6.73−6.63 (m, 3H), 6.47
(dd, J1 = 7 Hz, J2 = 1 Hz, 1H), 3.93−3.83 (m, 2H), 3.81−3.67 (m,
2H), 3.67−3.55 (br, 4H). 13C NMR (100 MHz, CD3OD): δ 164.4,
162.9, 162.7, 160.5, 160.3, 160.1, 142.4, 142.0, 141.5, 139.9, 139.0,
138.7, 121.0, 120.9, 120.8, 109.3, 108.8, 106.3, 49.2, 45.9, 39.1, 38.5.
ESI-HRMS: m/z 537.1346 ([M + Na]+) (calcd 537.1340).
1-Hydroxy-N,N-bis[3-(1-hydroxy-6-oxo-1,6-dihydropyri-
dine-2-carboxamido)propyl]-6-oxo-1,6-dihydropyridine-2-
carboxamide (3,3-Li-HOPO, 15). The protected ligand 13 (500
mg, 0.616 mmol) was dissolved in a 1:1 (v/v) mixture of glacial acetic
acid and HBr(aq) (37%, 10 mL). The reaction mixture was stirred at
room temperature for 6 h. The volatiles were then removed under
reduced pressure, and the residue was purified by flash chromatog-
raphy over modified silica eluting with a gradient of 100% H2O to 5%
CH3OH/95% H2O to yield the deprotected ligand 15 as a beige
1
foaming liquid (287 mg, 86%). H NMR (500 MHz, CD3OD): δ
7.51−7.40 (m, 3H), 6.71 (dd, J1 = 9 Hz, J2 = 1 Hz, 2H), 6.64 (dd, J1 =
7 Hz, J2 = 1 Hz, 1H), 6.57 (dd, J1 = 9 Hz, J2 = 1 Hz, 1H), 6.54 (dd, J1
= 7 Hz, J2 = 1 Hz, 1H), 6.38 (dd, J1 = 7 Hz, J2 = 1 Hz, 1H), 3.61 (br,
2H), 3.49−3.39 (m, 2H), 3.33 (br, 1H), 3.30−3.25 (m, 3H), 2.00−
1.93 (m, 2H), 1.93−1.78 (m, 2H). 13C NMR (125 MHz, CD3OD): δ
163.7, 162.6, 162.5, 160.3, 160.2, 160.0, 142.8, 142.2, 141.7, 139.9,
139.1, 138.7, 120.9, 120.7, 120.6, 108.9, 108.5, 105.7, 47.7, 43.6, 38.4,
38.2, 29.2, 27.9. ESI-HRMS: m/z 565.1699 ([M + Na]+) (calcd
565.1653).
EuIII-2,2-Li-HOPO (3). The deprotected ligand 14 (20 mg, 0.039
mmol) and EuCl3·6H2O (14 mg, 0.039 mmol) were dissolved in a 1:1
(v/v) mixture of CH3OH and Milli-Q water (5 mL). Pyridine (31 μL,
0.39 mmol) was subsequently added to the reaction mixture, which
was stirred at 60 °C for 15 h. The reaction mixture was allowed to
cool to room temperature. The suspension was filtered and rinsed
with methanol. The residue was further dried under reduced pressure
to yield the final EuIII complex 3 as a beige powder (22 mg, 95%).
ESI-HRMS: m/z 695.0479 ([M + CH3OH − H]−) (calcd 695.0606),
m/z 709.0388 ([M + COOH2 − H]−) (calcd 709.0399).
N-(2-{Bis[2-(1-hydroxy-6-oxo-1,6-dihydropyridine-2-
carboxamido)ethyl]amino}-2-oxoethyl)-1-hydroxy-6-oxo-1,6-
dihydropyridine-2-carboxamide (2,2-Gly-HOPO, 18). The
protected ligand 17 (500 mg, 0.594 mmol) was dissolved in a 1:1
(v/v) mixture of glacial acetic acid and HBr(aq) (37%, 10 mL). The
reaction mixture was stirred at room temperature for 6 h, after which
the volatiles were removed under reduced pressure. The residue was
then purified by flash chromatography over modified silica eluting
with a gradient of 100% H2O to 5% CH3OH/95% H2O to yield the
1
EuIII-3,3-Li-HOPO (4). The deprotected ligand 15 (20 mg, 0.037
mmol) and EuCl3·6H2O (14 mg, 0.037 mmol) were dissolved in a 1:1
(v/v) mixture of CH3OH and Milli-Q water (5 mL). Pyridine (30 μL,
0.37 mmol) was subsequently added to the reaction mixture, which
was stirred at 60 °C for 15 h. The reaction mixture was allowed to
cool to room temperature. The suspension was filtered and rinsed
with methanol. The residue was further dried under reduced pressure
to yield the final EuIII complex 4 as a beige powder (23 mg, 88%).
ESI-HRMS: m/z 715.0605 ([M + Na]+) (calcd 715.0633).
tert-Butyl [2-(Bis{2-[(tert-butoxycarbonyl)amino]ethyl}-
amino)-2-oxoethyl]carbamate (16). Triethylamine (1.27 mL,
9,14 mmol) was added to a solution of (tert-butoxycarbonyl)glycine
(Boc-Gly-OH, 800 mg, 4.57 mmol), di-tert-butyl [azanediylbis-
(ethane-2,1-diyl)]dicarbamate (1.39 g, 4.57 mmol), and 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate
(TBTU, 1.47 mg, 4.57 mmol) in anhydrous CH2Cl2 (25 mL). The
reaction mixture was stirred at room temperature for 12 h and then
deprotected ligand, a beige foaming liquid 18 (305 mg, 90%). H
NMR (400 MHz, D2O): δ 7.51−7.39 (m, 3H), 6.77−6.52 (m, 6H),
4.30 (br, 2H), 3.64 (br, 6H), 3.56 (br, 2H). 13C NMR (100 MHz,
D2O): δ 169.9, 162.3, 162.1, 161.7, 159.69, 159.66, 159.62, 140.1,
139.9, 139.2, 138.9, 138.6, 120.9, 120.7, 109.5, 108.33, 108.30, 45.9,
44.7, 41.5, 37.5, 37.3. ESI-HRMS: m/z 572.1798 ([M + H]+) (calcd
572.1736).
EuIII-2,2-Gly-HOPO (5). The deprotected ligand 18 (20 mg, 0.035
mmol) and EuCl3·6H2O (13 mg, 0.035 mmol) were dissolved in a 1:1
mixture of CH3OH (2.5 mL) and Milli-Q water (2.5 mL). Pyridine
(28 μL, 0.35 mmol) was subsequently added to the reaction mixture,
which was stirred at 60 °C for 15 h. The reaction mixture was allowed
to cool to room temperature. The suspension was filtered and rinsed
with methanol. The residue was further dried under reduced pressure
to yield the final EuIII complex 5 as a beige powder (21 mg, 84%).
ESI-HRMS: m/z 744.0604 ([M + Na]+) (calcd 744.0535).
J
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c00137.
■
sı
Characterization data, including H and 13C NMR data
1
of intermediates and final ligands, ESI-MS data of the
intermediates, ligands, and EuIII complexes, HPLC
chromatograms of the ligands, ultraviolet−visible spectra
of the EuIII complexes, and luminescence decays of the
EuIII complexes in the absence and presence of
phosphate, determination of quantum yields of all EuIII
complexes and phosphate adducts, Job plots of the EuIII
complexes with phosphate, SEM-EDS analysis of the
ternary complex, and data fitting (PDF)
(10) Page, S. E.; Wilke, K. T.; Pierre, V. C. Sensitive and Selective
Time-Gated Luminescence Detection of Hydroxyl Radical in Water.
Chem. Commun. 2010, 46 (14), 2423−2425.
(11) Peterson, K. L.; Margherio, M. J.; Doan, P.; Wilke, K. T.; Pierre,
V. C. Basis for Sensitive and Selective Time-Delayed Luminescence
Detection of Hydroxyl Radical by Lanthanide Complexes. Inorg.
Chem. 2013, 52 (16), 9390−9398.
AUTHOR INFORMATION
Corresponding Author
■
́
Valerie C. Pierre − Department of Chemistry, University of
(12) Huang, S.-Y.; Pierre, V. C. A Turn-on Luminescent Europium
Probe for Cyanide Detection in Water. Chem. Commun. 2018, 54
(66), 9210−9213.
Minnesota, Minneapolis, Minnesota 55455, United States;
orcid.org/0000-0002-0907-8395; Phone: (+1) 612 625
0921; Email: pierre@umn.edu
(13) Bruce, J. I.; Dickins, R. S.; Govenlock, L. J.; Gunnlaugsson, T.;
Lopinski, S.; Lowe, M. P.; Parker, D.; Peacock, R. D.; Perry, J. J. B.;
Aime, S.; Botta, M. The Selectivity of Reversible Oxy-Anion Binding
in Aqueous Solution at a Chiral Europium and Terbium Center:
Signaling of Carbonate Chelation by Changes in the Form and
Circular Polarization of Luminescence Emission. J. Am. Chem. Soc.
2000, 122 (40), 9674−9684.
Authors
Sheng-Yin Huang − Department of Chemistry, University of
Minnesota, Minneapolis, Minnesota 55455, United States;
orcid.org/0000-0002-3278-3826
Michelle Qian − Department of Chemistry, University of
Minnesota, Minneapolis, Minnesota 55455, United States;
orcid.org/0000-0002-4815-1014
(14) Shuvaev, S.; Pal, R.; Parker, D. Selectively Switching on
Europium Emission in Drug Site One of Human Serum Albumin.
Chem. Commun. 2017, 53 (50), 6724−6727.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c00137
(15) Pal, R.; Parker, D.; Costello, L. C. A Europium Luminescence
Assay of Lactate and Citrate in Biological Fluids. Org. Biomol. Chem.
2009, 7 (8), 1525−1528.
Author Contributions
(16) Butler, S. J. Quantitative Determination of Fluoride in Pure
Water Using Luminescent Europium Complexes. Chem. Commun.
2015, 51 (54), 10879−10882.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(17) Tripier, R.; Platas-Iglesias, C.; Boos, A.; Morfin, J.-F.;
̀
Charbonniere, L. Towards Fluoride Sensing with Positively Charged
Funding
Lanthanide Complexes. Eur. J. Inorg. Chem. 2010, 2010 (18), 2735−
The authors acknowledge the support of the National Science
Foundation provided by INFEWS N/P/H2O:SusChEM CHE-
1610832. S.-Y.H. was supported in part by a John Wertz
Fellowship from the Department of Chemistry of the
University of Minnesota. M.Q. was supported in part by a
UROP fellowship from the University of Minnesota.
2745.
(18) Lima, L. M. P.; Lecointre, A.; Morfin, J.-F.; de Blas, A.; Visvikis,
̀
D.; Charbonniere, L. J.; Platas-Iglesias, C.; Tripier, R. Positively
Charged Lanthanide Complexes with Cyclen-Based Ligands: Syn-
thesis, Solid-State and Solution Structure, and Fluoride Interaction.
Inorg. Chem. 2011, 50 (24), 12508−12521.
(19) Verma, K. D.; Massing, J. O.; Kamper, S. G.; Carney, C. E.;
MacRenaris, K. W.; Basilion, J. P.; Meade, T. J. Synthesis and
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(20) Xu, J.; Churchill, D. G.; Botta, M.; Raymond, K. N.
Gadolinium(III) 1,2-Hydroxypyridonate-Based Complexes: Toward
MRI Contrast Agents of High Relaxivity1. Inorg. Chem. 2004, 43 (18),
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Notes
The authors declare no competing financial interest.
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