EuIII complexes of octadentate 1-hydroxy-2-pyridinones: Stability and improved photophysical performance

Article abstract of DOI:10.1071/CH09314

The luminescence properties of lanthanoid ions can be dramatically enhanced by coupling them to antenna ligands that absorb light in the UV-visible and then efficiently transfer the energy to the lanthanoid centre. The synthesis and the complexation of Ln

Full text of DOI:10.1071/CH09314

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2009, 62, 1300–1307
www.publish.csiro.au/journals/ajc
III
Eu Complexes of Octadentate 1-Hydroxy-2-pyridinones:
∗
Stability and Improved Photophysical Performance
Evan G. Moore,A,B Anthony D’Aléo,
and Kenneth N. Raymond
A,B
Jide Xu,A,B
A,B,C
AChemical Sciences Division, Lawrence Berkeley National Laboratories, 1 Cyclotron Road,
Mail Stop 70A1150, Berkeley, CA 94720, USA.
B
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Corresponding author. Email: raymond@socrates.berkeley.edu
C
The luminescence properties of lanthanoid ions can be dramatically enhanced by coupling them to antenna ligands that
absorb light in the UV-visible and then efficiently transfer the energy to the lanthanoid centre. The synthesis and the
complexation of LnIII cations (Ln = Eu, Gd) for a ligand based on four 1-hydroxy-2-pyridinone (1,2-HOPO) chelators
III
appended to a ligand backbone derived by linking two l-lysine units (3LI-bis-LYS) is described. This octadentate Eu
−
complex ([Eu(3LI-bis-LYS-1,2-HOPO)] ) has been evaluated in terms of its thermodynamic stability, UV-visible absorp-
tion and luminescence properties. For this complex, the conditional stability constant (pM) is 19.9, which is an order of
III
magnitude higher than diethylenetriaminepentacetic acid at pH = 7.4. This Eu complex also shows an almost two-fold
increase in its luminescence quantum yield in aqueous solution (pH = 7.4) when compared with other octadentate ligands.
Hence, despite a slight decrease of the molar absorption coefficient, a much higher brightness is obtained for [Eu(3LI-
−
III
bis-LYS-1,2-HOPO)] . This overall improvement was achieved by saturating the coordination sphere of the Eu cation,
yielding an increased metal-centred efficiency by excluding solvent water molecules from the metal’s inner sphere.
Manuscript received: 2 June 2009.
Manuscript accepted: 12 September 2009.
Introduction
and N–H vibrations of the medium) and allowing better lumi-
nescence properties (higher luminescence quantum yields and
longer luminescence lifetimes).
We report herein the synthesis of a new octadentate ligand
composed of two l-lysine groups covalently linked via their car-
boxylic acid functional groups with a propyl diamine chain to
form the diamide backbone, 3LI-bis-LYS.The resulting four pri-
maryaminefunctionalgroupsofthe3LI-bis-LYSbackbonewere
Chelated lanthanoid ions represent a unique class of lumines-
cent compounds that feature both narrow-band emission in the
visible and near infrared (NIR) regions as well as long lumi-
nescent excited-state lifetimes, due to the Laporte forbidden
[
1,2]
character of the f–f transitions involved.
Their relative resis-
tance toward oxygen quenching allows work in aerated solution,
making these chelate complexes ideal candidates for biological
[
3–6]
used to attach four 1-hydroxy-2-pyridinone (1,2-HOPO) groups
applications.
In particular, terbium and europium are of pri-
III
(
Scheme 1) via amidation and this ligand was complexed to Eu
mary interest because their higher luminescence quantum yields,
together with long luminescence lifetimes, enable time-gating of
their emission,
short-lived autofluorescence from the sample. To optimize their
luminescence properties, the LnIII cation also requires satura-
tion of its coordination sphere, using a light-absorbing ligand
that can also act as antenna.[ The energy difference between
the lanthanoid emitting level and the molecular triplet excited
state of the ligand is of paramount importance and sensitization
efficiency can strongly depend on the energy gap between these
−
to give [Eu(3LI-bis-LYS-1,2-HOPO)] , which has been evalu-
ated in terms of its thermodynamic stability, and photophysical
properties.
[
5–10]
dramatically lowering background due to
Results and Discussion
11]
Synthesis and Design of Ligands
The ligands (Scheme 1) feature the 1,2-HOPO group, which acts
III
as a bidentate ligand to complex Ln ions efficiently, and the
[
12,13]
excited states.
resulting octadentate ligand topology has been shown to form
stable ML complexes (where 3LI-bis-LYS-1,2-HOPO ligand
is a tetra-bidentate ligand composed of four such 1,2-HOPO
moieties). The 1,2-HOPO units can also be linked to form a
For optimal lanthanoid luminescence, octadentate ligands
are of particular interest because they generally fill the coor-
dination sphere of the metal, protecting it from its environment
III
[14,15]
[16]
(
deleterious non-radiative quenching of the Ln ion from O–H
tetradentate ligand using aliphatic
or aromatic spacers
∗
This paper is dedicated to a giant of Australian chemistry, Alan Sargeson. The senior author had the great good fortune to spend his first sabbatical leave
in Australia, much of it at the Australian National University. It left a lasting influence regarding coordination chemistry and its interface to biology and
medicine.
©
CSIRO 2009
10.1071/CH09314
0004-9425/09/101300

RESEARCH FRONT
EuIII Complexes of Octadentate 1-Hydroxy-2-pyridinones
1301
O
O
H
H
N
N
O
N
N
N
H
N
O
O
H
OH
O
O
N
NH
HN
O
N
O
OH
N
O
OH
HO
O
OH
O
HN
O
OH
N
H
3LI-bis-LYS-1,2-HOPO
N
N
O
O
N
N
N
H
OH
O
OH
O
O
OH
O
N
NH
O
N
N
O
N
H
N
H
HO
O
H(2,2)-1,2-HOPO
5
LI-1,2-HOPO
Scheme 1. Chemical structure of the discussed ligands.
O
O
O
NHBoc
(
(
1)
2)
BocHN
N
H
N
H
O
Cl
O
NH
HN
O
BocHN
OH
NHCbz
H N
NH2
O
O
2
1
2
TFA
DCM
O
N
Cl
O
O
4
BnO
O
NH2
H N
N
H
N
2
H
NH2
NH2
3
OBn O
N
O
O
H
N
O
O
N
N
N
N
H
H
H
O
N
NH
HN
O
OBn
O
BnO
OBn
N
HCl/AcOH
O
O
5
OH
N
O
O
O
H
O
N
O
N
N
H
N
H
H
O
N
NH
HN
O
OH
N
HO
OH
N
O
O
O
3LI-bis-LYS-1,2-HOPO
Scheme 2. Synthetic pathway for 3LI-bis-LYS-1,2-HOPO.
via the amide functional groups. For the octadentate topology,
we recently reported on the N,N,N ,N -tetrakis-(2-aminoethyl)-
in the backbone bearing 1,2-HOPO units, which causes lower
luminescence quantum yields.
ꢀ
ꢀ
[17]
ethane-1,2-diamine-(H(2,2)-) backbone bearing four 1,2-HOPO
The protected backbone 2 was prepared by coupling the
activated l-lysine, protected at its two N-termini, to propylene
diamine by activating the carboxylic acid with isobutyl chloro-
formate. This intermediate was deprotected in acidic medium to
give 3.
[
17]
units (Scheme 1).
In the present case, the backbone of the
3LI-bis-LYS-1,2-HOPO ligand differs in terms of the linking
point, by the identity of the linking atom and by the length of
the linking central group (i.e. propyl instead of ethyl). Hence,
for H(2,2)-1,2-HOPO, the central ethyl chain that connects the
two tetradentate moieties is substituted via a nitrogen atom in
position 3, whereas 3LI-bis-LYS-1,2-HOPO is substituted via a
carbon atom in position 1 (Scheme 2). It is not anticipated that
this difference will increase the degrees of freedom of the sys-
tem, but it is expected to change the arrangement of the ligand
The benzyl-protected 1,2-HOPO chromophore was prepared
as reported elsewhere[ and its acid chloride version (4) was
prepared in situ using thionyl chloride.[ This acid chloride (4,
4.4 equiv.) was combined with one equivalent of 3 to furnish
the benzyl-protected 1,2-HOPO octadentate ligand 5. Subse-
quent reflux in 1:1 (v/v) conc. HCl and glacial acetic acid
gave the desired ligand (3LI-bis-LYS-1,2-HOPO) in good yields.
18]
19]
III
around the Ln cation. The substitution of the nitrogen by a
III
carbon was done in order to avoid having a protonated nitrogen
The Ln complexes (Ln = Eu, Gd) were prepared by refluxing

RESEARCH FRONT
1
302
E. G. Moore et al.
(
a)
(b)
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
1.5
1
0
0
.0
.5
.0
Ϫ0.5
Ϫ1.0
Ϫ1.5
0
.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
550
600
650
700
log[(DTPA)/(L)]
λ [nm]
Fig. 1. (a) Luminescence spectrum showing the typical decrease of luminescence intensity on addition of diethylenetriaminepentacetic acid (DTPA);
−
__
−
(
b) DTPA competition batch titration of [Eu(3LI-bis-LYS-1,2-HOPO)]
(
) and [Eu(H(2,2)-1,2-HOPO)] (red line) versus DTPA. The x intercept indicates
the difference in pEu between EuDTPA and the two complexes.
−
−
Table 1. pEus of [Eu(5LI-1,2-HOPO)2] , [Eu(H(2,2)-1,2-HOPO)] ,
Complex Stability
−
and [Eu(3LI-bis-LYS-1,2-HOPO)] , determined by competitions batch
The stability of the EuIII complex in aqueous medium was
titration using diethylenetriaminepentacetic acid (DTPA) (pEu = 19.04)
determined by competition batch titration (Fig. 1) versus
diethylenetriaminepentacetic acid (DTPA) as a known com-
petitor (pEu = 19.04 at pH = 7.4). By analogy to pH, pEu is
defined as the negative log of the concentration of free metal in
in 0.1 M aqueous TRIS buffer, pH = 7.4, and 0.1 M KCl
pEu
3+
−
A
solution (pEu = −log[Eu ]free) at a specified set of standard
[
[
[
Eu(5LI-1,2-HOPO)2]
18.35
−
B
Eu(H(2,2)-1,2-HOPO)]
Eu(3LI-bis-LYS-1,2-HOPO)]
21.2
19.9
conditions (chosen usually as [Eu]T = 1 µM, [L]T = 10 µM,
−
◦
pH = 7.4, 25 C, and0.1 M KCl).ThepEuvaluesthereforeoffera
convenient and meaningful way to compare relative chelate sta-
bilities between various ligands, regardless of differing ligand
ASee ref. [16]; Bsee ref. [18].
−
protonation behaviour. In the cases of [Eu(5LI-1,2-HOPO)2]
−
and [Eu(H(2,2)-1,2-HOPO)] , the pEus were determined to
2
1
1
0000
5000
0000
[15]
[17]
be 18.35
and 21.2,
respectively, an increase of almost
three orders of magnitude in stability due to the enhanced
chelate effect arising from an octadentate versus bis-tetradentate
complex.
For [Eu(3LI-bis-LYS-1,2-HOPO)] , the pEu was evaluated
to be 19.9, which represents a value almost one order of magni-
tude higher than the DTPA competitor.A loss in thermodynamic
−
−
stability when compared with [Eu(H(2,2)-1,2-HOPO)] (one
orderofmagnitudehigherthan[Eu(3LI-bis-LYS-1,2-HOPO)] )
−
5
000
0
can be readily observed. This difference in stability can be
attributed to a more ideal geometry adopted by the ligand around
III
the Eu centre using the flexible H(2,2) backbone, and sug-
gests that the 3LI-bis-LYS-1,2-HOPO scaffold presents some
constraint, yielding a loss of stability (Table 1).
3
00
350
400
450
500
λ [nm]
Fig. 2. UV-visible spectra of [Eu(3LI-bis-LYS-1,2-HOPO)] (__) and
[
at pH = 7.4.
−
UV-visible Absorption Spectroscopy
−
Eu(H(2,2)-1,2-HOPO)] (red line) in 0.1 M aqueous Tris buffer solution
The UV-visible absorption spectra of the EuIII complexes in
0
.1 M aqueous Tris buffer at pH = 7.4 are shown in Fig. 2
and the data are summarized in Table 2. The absorption
one equivalent of ligand with one equivalent of the appropriate
LnCl3·6H2O using pyridine as a base to ensure deprotonation of
the N-hydroxyl groups. The complex was then precipitated and
washed with diethyl ether to yield an analytically pure hydrated
complex. Full characterization of the ligand and complexes and
synthetic details are reported in the Experimental section.
maxima are at ∼331, 333, and 341 nm for the [Eu(5LI-1,2-
−
−
HOPO)2] , [Eu(3LI-bis-LYS-1,2-HOPO)] , and [Eu(H(2,2)-
−
1,2-HOPO)] complexes respectively. The lowest-energy
transition for these spectra can be assigned to a π–π* tran-
[
15]
sition with some n−π* character, as described elsewhere.
−
The maximum absorption of [Eu(3LI-bis-LYS-1,2-HOPO)] is

RESEARCH FRONT
EuIII Complexes of Octadentate 1-Hydroxy-2-pyridinones
1303
Table 2. UV-visible absorption data of the studied EuIII complexes in 0.1 M aqueous TRIS buffer (pH = 7.4) and triplet excited state energies
III
determined for the corresponding Gd complexes at 77 K
0
.1 M aqueous TRIS buffer, pH =−71.4
77 KA
−
max
abs
−1
]
T0–0 [nm cm 1]
λ
[nm]
ε [M cm
−
[
[
[
Eu(5LI-1,2-HOPO)2]
331
341
333
18800
18200
15760
21260
21980
22120
−
Eu(H(2,2)-1,2-HOPO)]
Eu(3LI-bis-LYS-1,2-HOPO)]
−
B
B
ADetermined in a solid matrix at 77 K (1:4 (v/v) MeOH:EtOH) using the GdIII complexes. BContaining 1% (v/v) DMSO for solubility.
in accordance with our previously reported results with other
measured in 0.1 M aqueous TRIS buffer solution at pH = 7.4.
III
Eu bis-tetradentate complexes, as illustrated with [Eu(5LI-1,2-
The luminescence lifetimes were also measured in the corre-
−
abs
HOPO)2] (λ = 331 nm) while the slight bathochromic shift
sponding deuterated solvents, in order to estimate the number of
−
[21]
in going from [Eu(3LI-bis-LYS-1,2-HOPO)] to [Eu(H(2,2)-
,2-HOPO)] reveals a stabilization of the first excited state
bound solvent molecules in the inner sphere (i.e. q in water
)
−
1
using the empirical Horrock’s equations. The relevant radiative
andnon-radiativeparameterswerealsodeterminedfollowingthe
using the H(2,2) backbone.
work of Verhoeven[ and Beeby,
22]
[23]
and a complete summary
of these data are reported in Tables 3 and 4.
The molar absorption coefficients are within experimen-
−
1
−1
for the
tal error, with typical values of ∼18000 M cm
−
−
−
[
Eu(5LI-1,2-HOPO)2] and [Eu(H(2,2)-1,2-HOPO)] com-
The emission spectrum of [Eu(3LI-bis-LYS-1,2-HOPO)] is
III
plexes, whereas a slightly decreased value is apparent for
typical of our previously reported Eu 1,2-HOPO derivatives,
−
−1
−1
7
5
[
Eu(3LI-bis-LYS-1,2-HOPO)] (15760 M cm ). This 16%
with a very intense FJ ← D0 (J = 2) transition at ∼612 nm
decrease of the molar absorption coefficient for [Eu(3LI-bis-
and a weaker J = 4 transition at ∼680–700 nm. For [Eu(H(2,2)-
−
−
−
LYS-1,2-HOPO)] compared with [Eu(H(2,2)-1,2-HOPO)]
1,2-HOPO)] , the J = 4 peak is slightly more intense (Fig. 4).
affects the brightness (here defined as the product of the molar
absorption coefficient with the luminescence quantum yield),
These changes have been shown to originate from differing
metal-centred symmetries, and in the present case, it is due
to the presence of one water molecule in the inner sphere
−
giving an advantage to [Eu(H(2,2)-1,2-HOPO)] .
−
III
for [Eu(H(2,2)-1,2-HOPO)] , yielding a nine-coordinate Eu
Luminescence of GdIII Complexes
complexversusaneight-coordinatestructureforcomplexessuch
−
as [Eu(5LI-1,2-HOPO)2] , which lack a water molecule in the
To estimate the energies of the ligand-based triplet excited state,
7
5
inner sphere. Notably, the crystal field splitting of the FJ ← D0
III
the Gd complexes were prepared and studied. Gadolinium is
(
J = 1) transition at ∼580 nm can be used to infer the site sym-
7
a 4f lanthanoid ion with the same oxidation state, a similar
III
metry of the Eu cation. Although the transition in this case
is quite broad, which precludes a definitive identification of the
exact point group, the observation of only two peaks suggests
that from the three most common coordination polyhedra, the
best match to the observed luminescence spectra is obtained for
the trigonal dodecahedral (D2d) geometry as noted elsewhere for
4
f electronic configuration, and similar size as the europium
6
cation (4f ), but with an inaccessible metal-centred electronic
excited state (the 6P7/2 is at 32224 cm ). For these com-
plexes at 77 K, in a frozen solution, an emission band with
a maximum at ∼500 nm can be seen. This emission is red-
shifted compared with the S0 → S1 transitions, observed at
−1
[15]
similar derivatives.
∼
330–340 nm by UV-visible absorption, and can be assigned
As can also be seen from Table 3, the luminescence quantum
to emission from the triplet excited state, which lies at lower
energy than the corresponding singlet excited state. From these
spectra, it appears the triplet excited states of the complexes are
almost coincident when considering the approximate onset of
the emission, varying only slightly from 452 nm for [Gd(3LI-
yields are different for each complex. As reported elsewhere,
−
[
Eu(5LI-1,2-HOPO)2] presents an optimum luminescence
[15]
quantum yield for the bis-tetradentate complexes of ∼20.7%,
−
while [Eu(H(2,2)-1,2-HOPO)] has a limited value of 3.6%
owing to the quenching by a water molecule in the inner
−
−
bis-LYS-1,2-HOPO)] and [Gd(5LI-1,2-HOPO)2] to 455 nm
for [Gd(H(2,2)-1,2-HOPO)] , respectively. The almost identi-
III [17]
sphere of the Eu .
The measured luminescence quantum
−
−
yield of [Eu(3LI-bis-LYS-1,2-HOPO)] reveals a value of 7.0%,
cal triplet excited-state energy should therefore give the same
energy transfer efficiency in the sensitization process.
which is almost a two-fold increase when compared with
−
[
Eu(H(2,2)-1,2-HOPO)] .
−
1
The slight decrease of 145 cm
in the energy for the
This improved value of ΦEu, the overall europium quantum
yield, will also affect the brightness of the complexes (see above)
triplet excited state follows the trends observed for the first
singlet excited-state transitions (Fig. 3). This should give an
intersystem crossing rate slightly in favour of [Eu(3LI-bis-
(
Table3), andthistimetheimprovementfavoursthe[Eu(3LI-bis-
−
LYS-1,2-HOPO)] complex, yielding a maximum brightness of
1
that the red-shifted absorption spectrum of the [Eu(H(2,2)-1,2-
HOPO)] complex yields a brightness that reverts to slightly
−
LYS-1,2-HOPO)] , because the gap between the singlet and
−1
−1
100 M cm at 333 nm. Above ∼358 nm, however, we note
−
1
the triplet excited states ꢀE, is closer to the 5000 cm that
[
20]
1
is the ideal gap predicted by Verhoeven et al.
(ꢀE( S*–
−
3
−1
−1
T*) = 7350 cm
and 7900 cm
for [Gd(3LI-bis-LYS-1,2-
favour the latter (Fig. 5).
−
−
HOPO)] and [Eu(H(2,2)-1,2-HOPO)] , respectively).
Indeed, for both eight-coordinate complexes, the decrease
in luminescence quantum yield (and also of the molar absorp-
Luminescence of EuIII Complexes
−
tion coefficient for [Eu(3LI-bis-LYS-1,2-HOPO)] ) when com-
−
The steady-state emission spectra, the luminescence quantum
yields and luminescence lifetimes of the EuIII complexes were
pared with the bis-tetradentate [Eu(5LI-1,2-HOPO)2] induces
a three-fold decrease in brightness for the former structures,

RESEARCH FRONT
1
304
E. G. Moore et al.
Table 3. Photophysical data of the investigated EuIII complexes
B
0
.1 M aqueous TRIS buffer, pH = 7.4
77 KA
τ [µs]
Brightness
−
1
−1
H2O
D2O
[M cm ]
φtot
τ
[µs]
τ
[µs]
q
−
[
[
[
Eu(5LI-1,2-HOPO)2]
0.207
0.036
0.070
737
480
712
996
1222
917
0.0
1.1
0.1
778
914
754
3900
655
1100
−
Eu(H(2,2)-1,2-HOPO)]
Eu(3LI-bis-LYS-1,2-HOPO)]
−
AMeasured in a solid matrix at 77 K (1:4 (v/v) MeOH:EtOH). BDefined here as the product of the molar extinction coefficient and the overall luminescence
quantum yield.
Table 4. Photophysical data of the investigated complexes in 0.1 M aqueous TRIS buffer at pH = 7.4
kr [s 1]
−
knr [s
−1
]
ηEu
ηsens
φtot
τ [µs]
τrad [µs]
−
A
[
[
[
Eu(5LI-1,2-HOPO)2]
0.207
0.036
0.070
737
480
712
1728
3000
2254
579
333
444
778
1750
951
0.426
0.160
0.316
0.485
0.225
0.222
−
Eu(H(2,2)-1,2-HOPO)]
Eu(3LI-bis-LYS-1,2-HOPO)]
−
ASee ref. [18].
1
0
0
.0
.8
.6
1.0
0.8
0.6
0.4
0.2
0.0
0.4
0
.2
0
.0
400
500
600
700
550
600
650
700
λ [nm]
λ [nm]
Fig. 3. Triplet excited-state emission spectra for [Gd(3LI-bis-LYS-1,2-
−
__
−
Fig. 4. Normalized luminescence spectra of [Eu(3LI-bis-LYS-1,2-
HOPO)]
:4 (v/v) MeOH:EtOH) (λex = 340 nm).
( )and[Gd(H(2,2)-1,2-HOPO)] (redline)insolidmatrix(77 K,
−
__
−
HOPO)]
TRIS buffer solution at pH = 7.4 (λex = 340 nm).
( ) and [Eu(H(2,2)-1,2-HOPO)] (red line) in 0.1 M aqueous
1
although the gain in stability still remains more important for
aqueous measurements, and more specifically for biological
measurements.
The luminescence lifetimes were determined in 0.1 M aque-
ous TRIS buffer and reveal values of 712 µs for [Eu(3LI-
1
1
200
000
800
600
400
200
0
−
bis-LYS-1,2-HOPO)] that are of the same order as those
−
for [Eu(5LI-1,2-HOPO)2] (737 µs), and much longer than
that of [Eu(H(2,2)-1,2-HOPO)] , which has a decay time of
480 µs. The difference between [Eu(5LI-1,2-HOPO)2] and
Eu(H(2,2)-1,2-HOPO)] has been shown to result from the
−
−
∼
−
[
presence of a water molecule in the inner sphere of the latter,
and the similarity in lifetimes suggests that [Eu(3LI-bis-LYS-
−
1
,2-HOPO)] does not have an aqua ligand coordinated to the
III
Eu cation (see below).
In order to determine whether [Eu(3LI-bis-LYS-1,2-
−
HOPO)] also possesses an aqua ligand, the luminescence
3
00
350
400
450
500
lifetime was also measured in deuterated water, yielding a
λ [nm]
value of 917 µs, which is comparable with that of [Eu(5LI-1,2-
−
Fig. 5. Brightness of [Eu(3LI-bis-LYS-1,2-HOPO)] (__) and [Eu(H(2,2)-
1,2-HOPO)] (red line) in 0.1 M aqueous TRIS buffer at pH = 7.4.
−
HOPO)2] (996 µs). The q number can therefore be obtained
[21]
−
(
Table 3) using the improved Horrocks equation,
revealing

RESEARCH FRONT
EuIII Complexes of Octadentate 1-Hydroxy-2-pyridinones
1305
a value of 0.1, and hence confirming the lack of aqueous sol-
chain. The resulting tetramine was functionalized by 1,2-HOPO
chelates, again via amide bond formation, resulting in a novel
octadentate ligand. On addition of Eu , this ligand was deter-
vent access toward the metal. This difference compared with
−
III
[
Eu(H(2,2)-1,2-HOPO)] mainly explains the large increase of
luminescence quantum yield, by minimizing solvent-induced
non-radiative deactivation pathways.
mined to form a complex more thermodynamically stable than
DTPA by ∼one order of magnitude, albeit lower than our pre-
−
Luminescence lifetimes were also determined at 77 K,
in a solid matrix (Table 3), to determine whether back
energy transfer between the excited-state donor triplet and the
excited-state acceptor manifold of the lanthanoid is present,
or alternatively whether quenching via a low-lying Ligand
to Metal Charge Transfer (LMCT) state can occur. As can
be seen from Table 3, no such quenching occurs as there
is only a small difference between the luminescence life-
times at room temperature in solution and in a solid matrix
viously reported [Eu(H(2,2)-1,2-HOPO)] . The photophysical
III
III
study of both Eu and Gd complexes has revealed the triplet
excitedstateisideallylocatedtosensitizetheEu cation, andthe
III
luminescence quantum yield presents a two-fold increase com-
−
pared with [Eu(H(2,2)-1,2-HOPO)] . This improvement has
been attributed to a more complete saturation of the metal’s
coordination sphere, and also results in a longer luminescence
−
lifetime for [Eu(3LI-bis-LYS-1,2-HOPO)] , similarly to other
bis-tetradentate ligand topologies comprising the 1,2-HOPO
moiety. Despite a slight decrease in the molar absorption coef-
ficient, this increase of luminescence quantum yield also yields
a much brighter complex. Finally, as the sensitization efficiency
is the same for both octadentate complexes, the main limitation
−
at 77 K. Hence, [Eu(3LI-bis-LYS-1,2-HOPO)] possesses a
3
5
−1
triplet state ideally located (the T* to D1 gap is 2090 cm
see above) for efficient energy transfer to the europium D1
;
5
[
12,13]
level.
Following the work ofVerhoeven et al.[22] and Beeby et al.,[23]
the efficiency of the sensitization can be estimated using a
method that defines the overall europium quantum yield of lumi-
nescence (φEu) as the product of the efficiency of the intersystem
crossing (ηISC), the efficiency of the energy transfer (ηET) and
the efficiency of metal-centred luminescence (ηEu):
of [Eu(H(2,2)-1,2-HOPO)] when compared with [Eu(3LI-bis-
−
−
LYS-1,2-HOPO)] is attributed to the metal-centred efficiency
while, when comparing the octadentate chelators with the bis-
tetradentate, a current limitation we encounter is a decrease in
the sensitization efficiency, which is almost two times less for
octadentate versus bis-tetradentate ligands. We attribute this in
part to changes in the coordination geometry that affect the
efficiency of ligand-to-metal energy transfer.
φEu = ηISCηETηEu = ηsensηEu
In this equation, ηEu can be calculated knowing the ratio
Experimental
of the total integrated emission intensity to the intensity of the
5
7
General
D0 → F1 transition (yielding the radiative decay rate constant,
TLC was performed using precoated Kieselgel 60 F254 plates.
Flash chromatography was performed using EM Science sil-
ica gel 60 (230–400 mesh). NMR spectra were obtained using
either Bruker AM-300 or DRX-500 spectrometers operating
kr, (and hence τrad)) and the non-radiative decay rate constant,
knr, can then be deduced knowing kr and τobs (the luminescence
lifetime of the considered complex).
As can be seen from Table 4, the radiative and non-radiative
1
13
−
at 300 (75) MHz and 500 (125) MHz for H (or C) respec-
decay rates are rather close for [Eu(3LI-bis-LYS-1,2-HOPO)]
1
13
−
tively. H (or C) chemical shifts are reported in ppm relative
and [Eu(5LI-1,2-HOPO)2] and are comparable with other 1,2-
HOPO-based Eu complexes that lack a water molecule in their
III
to the solvent resonances, taken as δ 7.26 (δ 77.0) and δ 2.49
[
14,15]
(δ 39.5) respectively for CDCl3 and (CD3)2SO while cou-
inner sphere.
The observed differences between these two
−
pling constants (J) are reported in Hz. The following standard
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. Fast-atom bom-
bardment mass spectra (FABMS) were performed using
complexes (higher kr and lower knr for [Eu(5LI-1,2-HOPO)2] )
reveal the better metal-centred efficiency for the bis-tetradentate
systems (ηEu are 42.6 and 31.6% respectively for [Eu(5LI-1,2-
1
−
−
HOPO)2] and [Eu(3LI-bis-LYS-1,2-HOPO)] ).
−
For [Eu(H(2,2)-1,2-HOPO)] , the low value for ηEu obtained
3-nitrobenzyl alcohol (NBA) or thioglycerol/glycerol (TG/G)
(
16.0%) can be attributed to the water molecule in the inner
as the matrix. Elemental analyses were performed by
the Microanalytical Laboratory, University of California,
Berkeley, CA.
sphere, which quenches the emission. More importantly, it
should be highlighted that the sensitization efficiencies are effec-
tively the same for both octadentate complexes (ηsens of 22.2
−
and 22.5% for [Eu(3LI-bis-LYS-1,2-HOPO)] and [Eu(H(2,2)-
−
Synthesis
1
,2-HOPO)] respectively) illustrating that while one of the
−
ε
main limitations for [Eu(H(2,2)-1,2-HOPO)] is attributed to
the metal centred efficiency, more generally the octadentate
ligand topologies containing 1,2-HOPO chelates result in sensi-
tization efficiencies that are almost two times lower than those of
the bis-tetradentate ligands. Unfortunately, the reason for these
differences are not yet clear, and remain a topic under current
investigation, as further optimization of this crucial parameter
will yield highly stable octadentate complexes with superior
3LI-bis(Boc-N -Z-lysine) 2
ε
Under N2, a solution of Boc-N -Z-lysine (1.14 g, 3 mmol) in
◦
dry THF (25 mL) was cooled to −15 C and neutralized with
N-methylmorpholine (0.33 mL, 3 mmol). Isobutyl chlorofor-
mate (0.40 mL, 3 mmol) was added under stirring. After 2 min,
1,3-diaminopropane (0.13 mL, 0.15 mmol) was added.The reac-
tion mixture warmed to room temperature and was evaporated to
dryness; the residue was dissolved in dichloromethane (DCM)
extracted with 5% citric acid solution and brine successively.
The organic phase was separated and loaded on a flash silica
column. Elution with 2–7% methanol in DCM allowed the sepa-
III
Eu -based luminescence performance.
Conclusion
ε
We have described the synthesis of an octadentate ligand
comprising two l-lysine groups attached via amidation of
their carboxylic acid functional groups with a propyl diamine
ration of 3LI-bis(Boc-N -Z-lysine) (2) (1.0 g, 84% based on the
free amine) as a thick pale yellow oil, which was solidified on
standing overnight.

RESEARCH FRONT
1
306
E. G. Moore et al.
δH (300 MHz, CDCl3) 1.36 (s,br, 4H), 1.41 (s, 18H), 1.48
m, 4H), 1.62 (s,br, 4H), 3.16 (s,br, 6H), 3.40 (s,br, 2H), 4.04
m, 2H), 5.07 (s,br, 6H), 5.30 (s,br, 2H), 7.09 (s,br, 2H), 7.29–
pure hydrated complex as a white solid in ∼60% yield (Found: C
43.87, H 4.58, N 13.28. Anal. calc. for C44H48N11O14Eu·5H2O:
C 44.15, H 4.88, N 12.87%).
(
(
7
3
1
.40 (m, 10H). δC (75 MHz, [D6]DMSO) 22.3, 27.9, 29.0, 31.7,
6.4, 40.1, 54.2, 65.9, 79.2, 127.2, 127.5, 128.0, 136.3, 155.6,
Optical Spectroscopy
+
56.3, 172.7. m/z (HR-MS FAB+) 799.4575 (MH ).
UV-visible absorption spectra were recorded on a Varian Cary
300 double-beam absorption spectrometer. Emission spectra
3
LI-bis-LYS-1,2-HOPOBn 5
were acquired on a HORIBA Jobin Yvon IBH FluoroLog-3
spectrofluorimeter, equipped with three-slit double-grating exci-
ε
3
LI-bis(Boc-N -Z-lysine) (0.80 g, 1 mmol) was dissolved in
1
0 mL of a 1:1 (v/v) mixture of trifluoroacetic acid (TFA) and
−1
tation and emission monochromators (2.1 nm mm dispersion,
dichloromethane (DCM) and stirred at room temperature for
h. The volatiles were removed under vacuum; the residue
−1
1
200 grooves mm ). Spectra were reference corrected for both
3
the excitation light-source variation (lamp and grating) and the
emission spectral response (detector and grating). Lumines-
cence 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 flash-
lamp (Jobin Yvon, 5000XeF) was used as the light source,
with an input pulse energy (100 nF discharge capacitance)
of ∼50 mJ, yielding an optical pulse duration of less than
was mixed with Pd/C (10%, 100 mg) in methanol (20 mL) and
hydrogenated at 2760 kPa H2 overnight in a Parr bomb. The
catalyst was filtered off with a fine glass frit and the filtrate
was evaporated to dryness, yielding 3LI-bis-LYS amine (3)
(
2,6-diamino-hexanoic acid (3-(2,6-diamino-hexanoylamino)-
3
propyl)amide). δH (300 MHz, D2O) 1.31 (quin, J 7.5, 4H), 1.57
3
3
(
m, 6H), 1.75 (m, 4H), 2.86 (t, J 7.5, 4H), 3.14 (t, J 7.5, 4H),
3
3
3
.81 (t, J 6.6, 2H). δC (75 MHz, [D6]DMSO) 21.5, 26.5, 27.9,
0.6, 37.0, 39.2, 53.3, 169.7.
300 ns at full width at half maximum. Spectral selection was
Compound 3 was dissolved in DCM (20 mL) and a biphasic
achieved by passage through the same double-grating excita-
tion monochromator. Emission was monitored perpendicular to
the excitation pulse, again with spectral selection achieved by
passage through the double-grating emission monochromator
solution of potassium carbonate (4 g of K2CO3 in 10 mL water)
was added. This biphasic solution was cooled in an ice-water
bath.A solution of 1,2-HOPOBn chloride (4) (1.25 g, 5 mmol) in
DCM (40 mL) was added drop wise to the above stirred mixture
over 1 h and the mixture was allowed to warm up overnight.
The organic phase was separated and the evaporated residue was
loaded onto a flash silica column. Elution with 2–7% methanol
−1 −1
2.1 nm mm dispersion, 1200 grooves mm ). A thermoelec-
(
trically cooled single-photon detection module (HORIBA Jobin
Yvon IBH, TBX-04-D) incorporating fast rise time photomul-
tiplier, wide bandwidth preamplifier and picosecond constant-
fraction discriminator was used as the detector. Signals were
acquired using an IBH DataStation Hub photon-counting mod-
ule and data analysis was performed using the commercially
available DAS 6 decay analysis software package from HORIBA
Jobin Yvon IBH. Goodness of fit was assessed by minimizing
in DCM allowed the separation of 3LI-bis-LYS-1,2-HOPOBn
ε
(
5) (0.89 g, 72%) (based on 3LI-bis(Boc-N -Z-lysine)), giving
a thick pale yellow oil that solidified overnight. δH (300 MHz,
3
3
[
D6] DMSO) 6.56 (d, 2H, J 7.2, HOPO-H), 6.67 (dd, 2H, J
3
9
3
1
.0, 1.5, HOPO-H), 7.44 (d+d, 2H, J 9.0, HOPO-H), 7.90 (s,br,
H, Py-H), 11.39 (s, 2H, amide H). δC (75 MHz, CDCl3) 105.2,
10.7, 119.6, 136.9, 140.9, 141.2, 149.8, 157.3, 159.3.
2
the reduced Chi squared function, χ , and a visual inspection
of the weighted residuals. Each trace contained at least 10000
pointsandthereportedlifetimevaluesresultedfromatleastthree
3LI-bis-LYS-1,2-HOPO
independent measurements. Typical sample concentrations for
3
LI-bis-LYS-1,2-HOPOBn (5) (0.5 g, 0.4 mmol) was dis-
−5
both absorption and fluorescence measurements were ∼10
–
solved in concentrated HCl (12 M)/glacial acetic acid (1:1,
0 mL), and stirred at room temperature for 2 days. Removal of
the solvent gave a beige foam as the deprotected product. Yield
.27 g, 75%. δH (300 MHz, [D6]DMSO) 1.20–1.80 (m, 14H,
−6
10
M and 1.0-cm cells in quartz Suprasil or equivalent were
2
used for all measurements. Quantum yields were determined by
the optically dilute method (with optical density <0.1) using the
following equation:
0
3
CH2), 3.08 (m, 4H, CH2), 3.16 (m, 4H, CH2), 4.31 (q, 2H, J
3
4
2
2
r
4
.5, CH2), 6.25 (dd, 2H, J 6.9, J 1.5, HOPO-H), 6.41 (dd, 2H,
Φ /Φ = [A (λ )/A (λ )][I(λ )/I(λ )][n /n ][D /D ],
x
r
r
r
x
x
r
x
x
x
r
3
4
3
4
J 6.9, J 1.5, HOPO-H), 6.55 (dd, 2H, J 6.0, J 1.5, HOPO-
3
4
H), 6.60 (dd, 2H, J 6.3, J 1.5, HOPO-H), 7.32–7.42 (m, 4H,
HOPO-H), 7.95 (s,br, 2H, amide H), 8.74 (s,br, 2H, amide H),
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 inten-
sity. The subscripts ‘x’ and ‘r’ refer to the sample and reference
respectively. For quantum yield calculations, an excitation wave-
length of 340 nm was used for both the reference and sample;
hence the I(λr)/I(λx) term is removed. Similarly, the refractive
3
9.04 (d, 2H, J 5.1, amide H). δC (75 MHz, CDCl3) 22.8, 28.4,
29.2, 31.6, 36.4, 48.7, 53.4, 103.9, 105.0, 119.4, 137.2, 137.5,
141.7, 142.5, 157.6, 160.2, 160.3, 171.0(Found: C 48.86, H 5.73,
N 14.36. Anal. calc. for C39H46N10O14·HCl·2.5H2O: C 48.77,
H 5.87, N 14.59%).
2
x
2
r
indices term, n /n , was taken to be identical for the aqueous
−
[Eu(3LI-bis-LYS-1,2-HOPO)]
reference and sample solutions. Hence, a plot of integrated emis-
sion intensity (i.e. Dr) versus absorbance at 340 nm (i.e. Ar(λr))
yields a linear plot with a slope that can be equated to the refer-
ence quantum yield Φr. Quinine sulfate in 0.5 M (1.0 N) sulfuric
In a 25-mL round-bottom flask, 3LI-bis-LYS-1,2-HOPO
(
1 equiv.) was suspended in methanol (10 mL). Europium(iii)
chloride hexahydrate (1.02 equiv.) in methanol (10 mL) and two
drops of pyridine were added.The solutions were heated to reflux
for 4 h, then cooled to room temperature. Slow evaporation of
the methanol at room temperature overnight afforded the desired
complexes as their pyridinium salts, which were collected by fil-
tration and washed thoroughly with diethyl ether, resulting in the
[
24]
acid was used as the reference (Φr = 0.546).
By analogy, for
the sample, a plot of integrated emission intensity (i.e. Dx) ver-
sus absorbance at 340 nm (i.e. Ax(λx)) yields a linear plot and
Φx can then be evaluated. The values reported in the manuscript
are the average of four independent measurements.

RESEARCH FRONT
EuIII Complexes of Octadentate 1-Hydroxy-2-pyridinones
1307
Competition Batch Titrations for pEu Determination
[7] G. Marriott, R. M. Clegg, D. J. Arndt-Jovin, T. M. Jovin, Biophys. J.
1991, 60, 1374. doi:10.1016/S0006-3495(91)82175-0
The general procedure used to determine the pEu values of the
three ligands was adapted from a previously described study
[
8] A. Beeby, S. W. Botchway, I. M. Clarkson, S. Faulkner, A. W. Parker,
D. Parker, J. A. G. Williams, J. Photochem. Photobiol. B 2000, 57, 83.
doi:10.1016/S1011-1344(00)00070-1
[
25,26]
of gadolinium
complexes.
and is similar to those reported for other
Different volumes of a standardized DTPA stock
[
17]
[
9] S. Faulkner, S. J. A. Pope, B. P. Burton-Pye, Appl. Spec. Rev. 2005, 40,
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 0.1 M aqueousTris buffer
instead of adjusting the pH to 6.0 as was done in past studies,[
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 sam-
ples. 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 complexed ligand in each solution were deter-
mined from the luminescence spectra, using the emission spectra
of the fully complexed ligand as a standard. These concentra-
tions were used for the log versus log plots (Fig. 1b) to yield the
difference in pEu between the competing DTPA and ligand of
interest.
1
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[
[
[
[
Acknowledgement
This work was partially supported by the National Institutes of Health (NIH)
(
grant HL69832) and supported by the Director, Office of Science, Office of
Basic Energy Sciences, and the Division of Chemical Sciences, Geosciences,
and Biosciences of the US Department of Energy at Lawrence Berkeley
National Laboratory (LBNL) under Contract no. DE-AC02–05CH11231.
This technology is licensed to Lumiphore, Inc. in which some of the authors
have a financial interest. Professor Gilles Muller is thanked for access to the
low-temperature time-resolved luminescence facilities.
[
[
[
[
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