Induced europium CPL for the selective signalling of phosphorylated amino-acids and: O -phosphorylated hexapeptides

Article abstract of DOI:10.1039/c6dt01212d

Two bright, europium(iii) complexes based on an achiral heptadentate triazacyclononane ligand bearing two strongly absorbing chromophores have been evaluated for the selective emission and CPL signalling of various chiral O-phosphono-anions. Binding of O-

Full text of DOI:10.1039/c6dt01212d

Dalton
Transactions
View Article Online
View Journal
PAPER
Induced europium CPL for the selective signalling
of phosphorylated amino-acids and
Cite this: DOI: 10.1039/c6dt01212d
O-phosphorylated hexapeptides†
Emily R. Neil, Mark A. Fox, Robert Pal and David Parker*
Two bright, europium(III) complexes based on an achiral heptadentate triazacyclononane ligand bearing
two strongly absorbing chromophores have been evaluated for the selective emission and CPL signalling
of various chiral O-phosphono-anions. Binding of O-phosphono-Ser and Thr gives rise to a strong
induced CPL signature and a favoured Δ complex configuration is adopted. A similarly large induced CPL
signal arises when [Eu·L1]2+ binds to lysophosphatidic acid (LPA), where the strong binding (log K 5.25
(295 K)) in methanol allowed its detection over the range 5 to 40 μM. Strong and chemoselective binding
to the phosphorylated amino-acid residues was also observed with a set of four structurally related hexa-
peptides: in one case, the sign of the gem value in the ΔJ = 1 transition allowed differentiation between
the binding to O-P-Ser and O-P-Tyr residues.
Received 29th March 2016,
Accepted 19th April 2016
DOI: 10.1039/c6dt01212d
www.rsc.org/dalton
lanthanide(III) centre to be observed selectively over the milli-
second timescale, long after ligand fluorescence has
Introduction
We introduce a dynamically racemic probe, [Eu·L1]Cl2, that can decayed.8–10 The emissive lanthanide excited state may be per-
bind to a range of structurally different chiral O-phosphono turbed in a number of different ways.11 In particular, changes
anions, and signal the binding of the analyte via induction of in the ligand field that take place following reversible binding
circularly polarised luminescence.
to the metal or the ligand, cause significant variations in emis-
Rather erratic progress has been made in the design of sion spectral form, lifetime and polarisation.1,12–15 The latter
lanthanide-based probes for O-phosphono oxy-anions, and type of modulation is of particular use in signalling selectively
many recent examples simply offer detection via emission the presence of chiral species in solution.
quenching or via displacement assays of limited utility1 The
Emission spectra of LnIII complexes are well known to be
parent phosphate di-anion is usually considered to bind to a sensitive to changes in the coordination environment deter-
lanthanide centre through a single negatively charged oxygen mining the ligand field.16–18 With europium(III) emission
atom. The hypothesis of monodentate binding is based on the spectra, the oscillator strength of the magnetic-dipole (MD)
analysis of kinetic emission and NMR spectroscopic data for a allowed ΔJ = 1 transition (ca. 590 nm) is normally independent
wide range of complexes, based on 1,4,7,10-tetraazacyclodode- of the ligand environment. The ΔJ = 2 and ΔJ = 4 transitions
cane (12-N4) ligand systems.2,3 For these examples, which are (ca. 615 and 700 nm respectively) are electric-dipole (ED)-
less sterically demanding than the 1,4,7-triazacyclononane allowed. In each case, they are hypersensitive to ligand pertur-
(9-N3) analogues described more recently, the available experi- bation. To a good first approximation, their intensities are
mental data is in line with the preference of phosphate to act directly proportional to the square of the ligand polarisabil-
5
7
as a monodentate ligand to a single metal centre, as reflected ities. Electric-quadrupole allowed transitions, e.g. D0 to F2/4
,
in X-ray structural data (e.g. for Na+, Ca2+, Zn2+).4,5 gain ED strength via a quadrupole/induced dipole (Ln3+ion/
The development of CPL probes has been dominated by ligand donor) mechanism of coupling. The induced dipoles on
strongly emissive lanthanide(III) complexes, as they serve as the ligands, in turn, are caused by direct coupling to the elec-
pure spherical emitters with large emission dissymmetry tric dipole components of the radiation field. In this way, the
factors.6,7 Time-gating techniques allow the signal from the electric dipole strength of 4f–4f transitions is related to ligand
dipolar polarisabilities and to the directional dependence (i.e.
the anisotropy) of these polarisabilities.19,20 The perturbation
of the ligand field has a major impact on the emission profile
Department of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK.
E-mail: david.parker@dur.ac.uk
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
of Eu(III) complexes. Variation of the axial donor in mono-
capped square-antiprismatic, 9-coordinate complexes, for
c6dt01212d
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
7
example, has a major effect on the relative intensity of the F2
The ligand L2 has been reported earlier, and forms a mono-
← 5D0 transition,21 and has been exploited in signalling revers- cationic complex with Eu(III). The introduction of a methyl-
ible anion binding to the metal centre. Examples of anion sig- ammonium group in the meta-position of the N-benzyl moiety,
nalling and sensing have evolved, including practicable assays gives rise to a dicationic complex. It was hypothesised that the
for lactate, bicarbonate and citrate, in complex bio-fluids.22–25
primary electrostatic binding interaction to any coordinated
In an achiral environment, a racemic mixture of Eu(III) com- anion would be stronger with [Eu·L1]2+ vs. [Eu·L2]+, aided in
plexes does not exhibit CPL. Following addition of a chiral certain cases by directed hydrogen bonding between the
agent, a net CPL signal can be observed in principle. When a ammonium group, serving as a hydrogen bond donor, and, for
chiral anion binds reversibly to the metal centre of a racemic example, a metal-coordinated phosphate group acting as a
complex, diastereoisomeric complexes of differing relative stabi- hydrogen bond acceptor.
lity are formed and may lead to an induced CPL signal, whose
relative intensity is a function of the binding selectivity and con-
formational rigidity of the complex, on the emission timescale.
Recently, a series of very bright Eu(III) complexes has been
reported, in which 1,4,7-triazacyclononane (9-N3) acts as the
core ligand structure. Up to three pyridyl-alkynylaryl groups can
Results and discussion
Ligand and complex synthesis and characterisation
be introduced to serve as the chromophore, allowing sensitised
emission to take place.26–32 In particular, coordinatively unsatu-
rated complexes based on heptadentate ligands have been pre-
pared33 that bind reversibly to anions in aqueous media.
In this work, we compare and contrast the behaviour of the
Eu(III) complexes of the ligands L1 and L2, (Scheme 1) and
examine the issues related to chemoselective signalling of
binding for a range of chiral O-phosphono anions, allowing an
evaluation of their scope and utility as chiral probes for CPL.
At the outset, it was hypothesised that the greater steric
demand at the europium centre would disfavour chelation of
anions with a small bite angle, such as carbonate or a simple
carboxylate. Such thinking was based on the experimental
observation that [Eu·L2]+ does not bind a solvent molecule in
water or methanol solution, consistent with the notion that
the N-benzyl creates a significant steric demand as a result of
free rotation about the N–C and C–phenyl bonds, thereby inhi-
biting hydrogen bonding to the second sphere of solvation
that would otherwise stabilise the metal-coordinated solvent,
with respect to dissociation.
It was necessary first to synthesise an aromatic precursor con-
taining a charged amino group that also contains an electro-
philic benzylic substituent at the meta position. An
N-methylbenzylamine group was selected, as it has an appro-
priate pKa value (∼9.7), ensuring that it is positively charged at
ambient pH and is not too sterically bulky. It also offers scope
for stabilising hydrogen-bonding interactions.
The synthetic pathway began with formation of the mono-
amide, 2: reaction of 1 with oxalyl chloride furnished the acid
chloride, followed by addition of methylamine to generate the
amide, 2. Simultaneous reduction of the amide and ester
groups using LiAlH4 in THF gave the amino-alcohol, 3. A
reasonable yield was achieved by using the classical ‘Fieser
work-up’ procedure: the granular aluminium salts that formed
were easily removed by filtration. Protection of the amine with
a formamide group was achieved using 2,2,2-trifluoroethyl-
formate (TFEF). The formamide group was selected as it is
removed under basic conditions. Therefore, deprotection
could occur at the same time as hydrolysis of the methyl ester
groups of the ligand. The final step in the synthesis of the pre-
Scheme 1
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
Scheme 2
cursor was conversion of the alcohol, 4, to the mesylate, 5, in H2O and the emission intensity was significantly quenched,
under standard conditions, (Scheme 2). probably due to self-aggregation. Therefore, in order to get an
The synthesis of the ligand was completed by alkylation of understanding of the number of metal bound solvent mole-
the secondary amine, 6, with the mesylate, 5, in MeCN in the cules, the rate of radiative decay of europium(III) emission was
presence of potassium carbonate. Treatment with base fol- determined in methanol and d4-methanol. The solvation state,
lowed by complexation afforded the cationic complex, q, was estimated using literature methods, modified to allow
[Eu·L1]2+, which was purified by reverse phase HPLC. The high for the presence of only one quenching OH oscillator. The
absorbance of each complex in the range 332 to 352 nm arises number of metal-bound solvent molecules was estimated to be
from the strong ICT band.23,28 Several photophysical measure- zero in each case, consistent with the data obtained for
ments were made to characterise the chloride salt of the [Eu·L2]+ in water, reported earlier (Scheme 3).34
complex [Eu·L1]2+ and data compared to the control N-benzyl
The two Eu(III) complexes do not possess a bound solvent
complex, [Eu·L2]+, (Table 1). The complex was not fully soluble molecule. In each case, the steric demand imposed by the
benzylic substituent suppresses solvent coordination. The lack
of variation of the emission spectrum in water compared to
methanol is consistent with the rate data analysis.
Table 1 Photophysical properties of Eu(III) complexes of L1 and L2
(295 K, MeOH)
[Eu·L2]+
[Eu·L1]2+
Anion binding behaviour
λ/nm
348
352
The anion binding behaviour towards hydrogenphosphate,
bicarbonate and lactate was examined first, prior to a study of
the more complex phosphorylated amino acid and hexapeptide
anions (Scheme 4).
ε/mM−1 cm−1
Φ
36.0
0.18
0.83
0.93
35.0
0.18
0.77
1.01
k (MeOH)/ms−1
k (CD3OD)/ms−1
Scheme 3
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
Scheme 4 Structures of the three common O-phosphono amino acids, the non-phosphorylated hexapeptide Gly-Ala-Pro-Tyr-Lys-Phe (GAPYKF)
and the phosphorylated hexapeptides Gly-Ala-Pro-O-P-Tyr-Lys-Phe (GAPY*KF), Gly-O-P-Ser-Pro-Phe-Lys-Phe (GS*PFKF) and Gly-O-P-Ser-Pro-O-
P-Tyr-Lys-Phe (GS*PY*KF).
Incremental addition of aqueous solutions containing a 4.37 and 3.15( 0.07) for [Eu·L1]2+ and [Eu·L2]+ respectively. The
given anion, to each Eu complex in turn, was monitored by higher affinity with the dicationic complex may simply be
emission spectroscopy examining changes in the relative inten- attributed to the greater attractive electrostatic term. With
sity and form of the Eu(III) emission spectrum. The relative added hydrogenphosphate, whilst [Eu·L2]+ showed no change
intensity of the ΔJ = 2 and ΔJ = 1 emission bands was plotted in spectral form with added anion, [Eu·L1]2+ exhibited a sig-
as a function of anion concentration and the variation was nificant modulation, and a binding constant of 4.2 ( 0.1) was
fitted to a 1 : 1 binding model by non-linear least-squares estimated (ESI). Given that the analogue of [Eu·L2]+, where the
fitting, to give an estimate of the association constant.
N-benzyl group is replaced by NH, underwent decomplexation
Each Eu(III) complex (5 μM, 295 K, 50/50 MeOH/water, of the Eu(III) ion with added hydrogenphosphate, the origins
apparent pH 7.4, 10 mM HEPES) exhibited no change in spec- of the enhanced stability of the phosphate adduct of [Eu·L1]2+
tral form in aqueous methanol, following addition of hydro- were assessed using DFT computational methods.
gencarbonate in one thousand fold excess, consistent with the
Stereochemistry of a model hydrogenphosphate adduct: a DFT
study
absence of anion binding at the metal centre. The binding of
lactate had earlier been examined with [Eu·L2]+ and a set of
three analogues with differing sensitising groups and overall As the paramagnetic europium(III) complexes are rather
charge. Lactate bound reversibly to give a ternary adduct in difficult to model computationally, optimised geometries of
each case, for which the estimated binding constants were the analogous diamagnetic yttrium(III) complex was investi-
Fig. 1 Yttrium complexes [Y·L1]2+ and [Y·L2]+ explored here as model complexes in computations for [Eu·L1]2+ and [Eu·L2]+ respectively.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
gated. It has been shown earlier that they serve as suitable minimum located without such an N-hydrogen interaction is
models for the Eu analogues;26,35–38 consistent with the fact 26.6 kJ mol−1 higher.
that the Y(III) ion differs in ionic radius from the Eu(III) ion by
The binding energy of [Y·L2]+ with hydrogenphosphate is
only 0.05 Å. The functional/basis set of B3LYP/3-21G* gives 342.6 kJ mol−1 which is lower than the binding energy of
model geometries of yttrium complexes with reasonable confi- [Y·L1]2+ with the phosphate by 40.4 kJ mol−1. This suggests
dence34
and is arguably superior
to alternative that the stronger binding involving [Y·L1]2+ is a combination of
reported26,35–37,39,40 functionals and basis sets. The binding of the higher electrostatic binding interaction and the directed
one hydrogenphosphate molecule to the model geometry hydrogen bonding between the ammonium group and the
[Y·L1]2+ (Fig. 1) was examined here at B3LYP/3-21G* and com- phosphate group.
pared to the behaviour of the aqua, acetate, lactate, hydrogen-
The binding energies listed (Table 2) follow the experi-
carbonate and carbonate adducts. The corresponding adducts mental observations of an absence of binding between
of the model geometry [Y·L2]+ (Fig. 1) were also explored for [Eu·L1]2+ and [Eu·L2]+ with water molecules and are consistent
comparison and Table 2 summarises the relative binding ener- with the spectral evidence for binding between [Eu·L1]2+ and
gies of [Y·L1]2+ and [Y·L2]+.
[Eu·L2]+ and lactate anions. The higher affinity of [Eu·L1]2+
Of the many minima located for the hydrogenphosphate compared to [Eu·L2]+ with the lactate is in agreement with
complex [Y·L1(HOPO3)], the lowest energy minima are dis- computed binding energies (Table 2). However, the spectral
cussed in which hydrogenphosphate is a monodentate ligand observations that [Eu·L1]2+ and [Eu·L2]+ bind neither with
(Fig. 2), resulting in an eight coordinate Y centre. There are no hydrogencarbonate nor with acetate anions, and, in the case of
examples in the CCDC of hydrogenphosphate chelating to a [Eu·L2]+, with hydrogenphosphate are not in agreement with
Eu centre in a monomeric complex. Directed hydrogen bonds DFT-computed binding energies. The absence of binding may
occur between the negatively charged phosphate oxygen and be explained by two effects: first the fact the N-benzyl group is
the ammonium NH proton that contribute to stronger hydro- rotating quickly on the emission timescale creates a greater
genphosphate binding in [Y·L1]2+ compared to [Y·L2]+ observed effective steric demand at the metal centre; secondly, the
experimentally for the europium analogues. The lowest energy ligand hydrophobicity means there is a smaller second sphere
of solvation which would otherwise stabilise the metal-
coordinated solvent with respect to dissociation. Such sol-
vation stabilisation models would require considerable compu-
tational efforts, and are beyond the scope of this study.
Table 2 Binding energies (kJ mol−1) for [Y·L1]2+ and [Y·L2]+ as models
of the Eu(III) complexes, [Eu·L1]2+ and [Eu·L2]+
Binding behaviour of the chiral anions and CPL studies
[Y·L1]2+
[Y·L1]2+
Emission spectral changes for [Eu·L1]2+ were investigated fol-
lowing the addition of O-P-Ser, O-P-Thr and O-P-Tyr, in a
MeOH–H2O solvent system (1 : 1, v/v), maintaining the appar-
ent pH at 7.4 using a HEPES buffer (10 mM). The form of the
total emission spectrum of [Eu·L1]2+ was similar to that follow-
ing addition of inorganic phosphate, and the changes that did
occur were relatively subtle, except in the hypersensitive ΔJ = 4
NH⋯O interaction No NH⋯O interaction [Y·L2]+
H2O
HCO3
27.2
117.6
124.7
133.8
356.4
46.4
129.2
129.9
161.3
342.6
327.9
−
159.7
−
CH3CH(OH)CO2 163.5
−
CH3CO2
192.6
383.0
406.1
2−
HPO4
2−
CO3
Fig. 2 Top and side elevations showing the two lowest energy minima for [Y·L1(HOPO3)], in which the substituted phenyl groups are omitted for
clarity; there is a directed hydrogen bond (green) between the negatively charged phosphate oxygen and the ammonium NH proton.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
transitions, where isoemissive points could be distinguished induced CPL spectrum of equal and opposite form, with iden-
(Fig. 2). Moreover, a broadening from one into three tran- tical gem values at 593 nm. Comparative analysis with the CPL
sitions in the ΔJ = 1 manifold was observed. In each case, it spectrum of the control N-benzyl complex, [Eu·L2]+, following
was possible to measure an estimated binding constant addition of R and S-lactate, allowed a tentative assignment of
assuming a 1 : 1 stoichiometry for the ternary adduct. The the absolute configuration of the complex adduct, as
stability constant for binding of each O-P-amino acid was cal- Δ-[Eu·L1·O-P-S-Ser] and Λ-[Eu·L1·O-P-R-Ser].
culated to be log K = 4.80 0.05, revealing that there is no pre-
Three phosphorylated peptides were selected for this study,
ference for a particular phosphorylated amino acid.
varying in the phosphorylated residue (Ser or Tyr) and the
Such behaviour contrasts to previous work based on 12-N4 number of phosphorylated sites (mono or di). The corres-
europium(III) complexes, where a preference for O-P-Tyr was ponding non-phosphorylated peptide, GAPYKF, was also
observed in pure water, most likely as a result of the lower studied as a control. These systems had been used earlier in
hydration energy of the tyrosine moiety.41 However, the study related emission studies using 12-N4 based cationic
involving [Eu·L1]2+ was carried out in a 1 : 1 MeOH–H2O complexes.41
solvent system, to aid solubility. Selective solvation of the
In order to try and gain insight into the solution state struc-
complex by a cluster of methanol molecules is likely to be tures of the three phosphorylated peptides, electronic circular
occurring,42 and so the effect of the differential anion dichroism (ECD) and 2D 1H NMR methods were used to assess
hydration energy is diminished. It is worth noting that no peptide conformation. The hexapeptides each contain
a
change in the emission spectrum was observed following proline residue, which can often induces a turn in peptide,
addition of a 1000-fold excess of the non-phosphorylated particularly when followed by an aromatic residue.43 Weak
amino acids. Binding must occur through the anionic phos- ECD activity (around 200–230 nm) was observed for each of
phate group.
the three phosphorylated peptides. The hexapeptides
The change in emission spectral form was accompanied by GAPY*KF and GS*PY*KF exhibited ECD spectra that were very
the induction of a strong CPL signal, upon adding O-P-Ser or similar in form, consistent with the assignment of an open
O-P-Thr (Fig. 3). The emission dissymmetry values, gem (where conformation peptide.44 In the case of GS*PFKF, the ECD spec-
gem = 2(IL − IR)/(IL + IR) for the ΔJ = 1 transition) were bigger trum was slightly more well-defined with a positive and nega-
for O-P-Thr (gem (592.5 nm) = +0.08 vs. +0.04 for O-P-Ser), tive component at 220 and 200 nm respectively, indicating that
perhaps as a result of the additional methyl group providing a the mono-phosphorylated hexapeptide adopts a more struc-
slightly higher degree of conformational rigidity at the metal tured preferred conformation in solution.
centre. No induced CPL signal could be recorded for the
The induced CPL response of [Eu·L1]2+ was investigated, fol-
[Eu·L1·O-P-Tyr] adduct. From the total emission studies, it is lowing addition of a 20-fold excess of each peptide. The
clear that the phosphorylated amino acid binds to the lantha- addition of the non-phosphorylated control peptide, GAPYKF,
nide centre in a similar manner to O-P-Ser and O-P-Thr. There- resulted in no induced CPL, providing further evidence that
fore, an alternative explanation must explain the lack of the peptide binds to the europium(III) centre via the phosphate
induced chiroptical activity: the chiral centre is more remote group, (Fig. 4). In the case of the O-P-Tyr peptide, GAPY*KF,
in the O-P-Tyr molecule, and simply explains the lack of CPL the CPL spectral form resembles that of the O-P-amino acids,
activity observed. Addition of R- and S-O-P-Ser resulted in an O-P-Ser and O-P-Thr). However, it is interesting to recall that
Fig. 3 Variation of the Eu(III) total emission (left) as a function of added O-phosphono-Ser and the limiting CPL spectral profile for [Eu·L1] (right).
The insets show the fit (line) to the experimental data points and an expansion of the ΔJ = 1 CPL transition for 50 µM added R-O-phosphono-Ser
(red) and the S-enantiomer (blue) (λexc 352 nm, apparent pH 7.4; 50% H2O/MeOH, 10 mM HEPES, 5 µM complex). The CPL spectrum was nearly
identical for addition of O-phosphono-Thr, but no CPL signal was observed with added O-phosphono-Tyr.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
Fig. 4 Limiting CPL spectra for [Eu·L1]2+, induced following addition of a 20-fold excess of peptide: (top left)-GAPY*KF; (top right)-GS*PFKF;
(bottom left)-GS*PY*KF; (bottom right)-GAPYKF (10 µM complex, λexc 352 nm, 295 K, MeOH).
the simple O-P-Tyr amino acid did not induce a CPL response.
The di-phosphorylated peptide, contains both a phosphory-
In contrast, the O-P-Tyr peptide induced significant CPL, with lated serine and tyrosine residue. The induced CPL is much
a measured emission dissymmetry value of gem (592.5 nm) = weaker than for the other two peptides, and appears to be an
+0.10, which is amongst the highest induced gem values additive sum of the two separate spectra. One possible expla-
observed with
complex.12,13,45
a
dynamically racemic europium(III) nation is that the complex has no preferential binding site, as
the estimated affinity constants for the O-P-amino acids are
Another interesting observation, was the induced CPL spec- the same (vide supra). Therefore, the emission spectrum
tral response of the O-P-Ser peptide, GS*PFKF; this was oppo- observed is a combination of the lanthanide complex bound to
site in sign and form to that of GAPY*KF. The gem value at the the O-P-Ser of the peptide and the O-P-Tyr residue. The gem
same transition, λ = 592.5 nm, was −0.04 and therefore much values for the induced CPL were bigger for the O-P-Tyr residue,
smaller than the O-P-Tyr peptide. Such behaviour, suggests which may explain why the major chiral emissive species for
that it may be the chiral structure of the entire peptide, when the GS*PY*KF adduct of [Eu·L1]2+, generated an induced CPL
bound to the lanthanide centre that is associated with the spectrum that more closely resembles that created by the
induced CPL response.
O-P-Tyr hexapeptide.
Fig. 5 (left): variation in the Eu(III) emission spectral profile for [Eu·L1]2+ as a function of added GAPY*KF showing the fit (line) to the experimental
data points, assuming a 1 : 1 binding model; (right)- changes in the ΔJ = 1 manifold of the CPL spectrum for [Eu·L1]2+, following peptide addition,
showing the fit to the data points (10 µM complex, λexc 352 nm, 295 K, MeOH).
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
Investigation of the binding affinities of the three hexapep- in the fluorescence intensity of the ligand.47 In a study using a
tides with [Eu·L1]2+ was carried out by monitoring the emis- coordinatively unsaturated europium(III) salophene complex of
sion and induced emission dissymmetry values, gem, as a ill-defined speciation, an apparent selectivity of binding was
function of peptide concentration. Emission spectral changes demonstrated in methanol for a number of competitive ana-
with [Eu·L1]2+ were very subtle, following addition of the phos- lytes, such as serum albumin, phospholipids (phosphatidyl-
phorylated peptide, GAPY*KF. It was much more informative serine, phosphatidylinositol, phosphatidylethanolamine) and
to monitor the switching on of CPL with the system, particu- selected carboxylates (phosphate and citrate). However, selecti-
larly because the gem values were as large as +0.10 (Fig. 5). The vity over bicarbonate and lactate (anions present at the highest
change in the intensity of the ΔJ = 2 and ΔJ = 1 transitions of physiological concentration) was not discussed. Furthermore,
the emission spectrum was plotted as a function of added con- the chemical origin for the particularly high affinity of the
centration of peptide, and the data was fitted to a 1 : 1 binding europium(III) complex for LPA was neither apparent nor investi-
isotherm, following iterative least-squares fitting. The esti- gated. The study monitored the fluorescence response of the
mated binding affinity in methanol, log K = 5.9 ( 0.1), was very europium(III) complex in methanolic extracts of human serum,
large and consistent with the value obtained by plotting the spiked with LPA. However, no information regarding the com-
induced gem values against concentration of peptide, log K = position of the extract was given, making it difficult to deter-
6.1( 0.1). Such behaviour implies that the charged amino mine the nature of the background medium in which LPA was
complex, [Eu·L1]2+, has
a strong binding affinity with ‘selectively’ detected. This particular lanthanide probe relied
GAPY*KF, and that the major chiral species in solution is the on short-lived ligand-centred emission to detect the
most emissive species. A slightly higher binding affinity was biomolecule.
calculated from the CPL data suggesting that it is associated
The anion LPA has a lower pKa2 than phosphatidic acid
with formation of the favoured isomer (Δ).
(PA), the related phospholipid with a simple phosphate head
A similar experiment was carried out for the peptide group, illustrated by the calculated values in a phosphatidyl-
GS*PFKF, however the data did not fit well to a 1 : 1 binding choline bilayer (PA: pKa1 3.2 0.3, pKa2 7.92 0.03; LPA: pKa1
model, and a stability constant could not be reliably estimated. 2.9
0.3, pKa2 7.47
0.03).48 Intramolecular hydrogen
In the case of the di-phosphorylated peptide, GS*PY*KF, bonding is observed between the hydroxyl group on the gly-
unusual behaviour was observed following the sequential cerol backbone of the molecule and the phospho-monoester
addition of the peptide to the europium(III) complex. The head group in the crystal structure, and is believed to persist at
induced CPL signal at 592.5 nm (ΔJ = 1) initially gave rise to physiological pH (Fig. 6).48,49 Therefore, ionisation of the phos-
increasingly negative values, until a critical concentration was phate hydroxyl group of LPA occurs more easily than for PA
reached. At this point, the sign of the CPL reversed and (and other phosphate anions), generating a higher negative
increasing positive gem values were recorded, until the limiting charge on the analyte, facilitating proposed binding to a posi-
spectral response was achieved. Such behaviour provides tively charged probe, for example [Eu·L1]2+.
further evidence that there is only a very small energy differ-
ence between the binding affinities of the two phosphorylated
sites (O-P-Ser and O-P-Tyr). Nonetheless, at higher concen-
tration of added peptide, the most emissive observed chiral
species has a positive gem value at 592.5 nm, corresponding to
binding at the O-P-Tyr residue.
[Eu·L1]2+ as a chiral probe for oleoyl-lysophosphatidic acid
Oleoyl-L-α-lysophosphatidic acid (LPA) is
a phospholipid
present at a physiological concentration of <0.1–6.3 µM and at
significantly increased levels (up to about 40 µM) in ovarian
cancer cells, (Scheme 5).46
Some attempts have been made to develop lanthanide
probes able to detect LPA in MeOH, by monitoring an increase
Fig. 6 Intramolecular hydrogen bonding of lysophosphatidic acid (LPA)
and phosphatidic acid (PA), R = oleoyl acid chain.
Scheme 5 Structures of lysophosphatidic acid (LPA) and a putative Eu(III)–salophene complex.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
LPA must bind to the lanthanide centre via the phosphate analytes, allowed a tentative assignment of the configuration
head group, in the same mode discussed for the phosphory- of the complex adduct as Δ-[Eu·L2·L-LPA]. It is useful to recall
lated amino acids and peptides in the preceding sections.
that the O-P-S-amino acids also induced a similar CPL spectral
response, resulting in the same Δ-assignment of helicity.
Following the promising results of the emission and CPL
studies, further work set out to investigate the binding selecti-
vity of the complex and LPA in a competitive environment. The
nature of the ‘signature’ induced CPL response would provide
further evidence for determination of the species bound to the
europium centre.
No spectral evidence for an interaction was found for
addition of other phospholipid species including phosphati-
dyl-serine, -inositol and -ethanolamine. The addition of HSA
(human serum albumin) resulted in no change in the emis-
sion spectral form, although significant quenching of the
emission intensity was observed at a protein concentration
>0.2 mM, with no induced CPL. The binding affinity for LPA
may be compared to other physiologically relevant anions,
including lactate, bicarbonate and phosphate, (Table 3).
It is important to note that the affinity constant for LPA
complexation was estimated in 100% MeOH and so is expected
to be higher than the value measured in MeOH–H2O (1 : 1,
v/v), due to the lower solvation energy of the anion. However,
with that in mind, analysis of the affinity constants revealed
that selective binding of LPA may be possible in a competitive
Emission and induced CPL studies with LPA
The emission spectral form changes following addition of LPA
to [Eu·L1]2+ were particularly apparent in the ΔJ = 1 transition,
which broadened from a single manifold, into three distinct
transitions, (Fig. 7). Such a change resembled the emission
spectral response of [Eu·L1]2+ following addition of the other
phosphate anions investigated (i.e. HPO42−, O-P-amino acids
and O-P-peptides). Upon increasing the concentration of LPA,
characteristic changes in the total emission spectrum were
observed until a limiting spectrum was achieved, beyond
which the total intensity continued to increase consistently
across all transitions, with no further change to the emission
spectral form. Such behaviour may be attributed to the long
lipophilic chains in the molecule, which may aggregate to
form a micelle, reducing the degree of quenching from second
sphere solvent molecules. The changes in the local solvent per-
mittivity may slightly change the ICT excited state energy level
and favour the intramolecular energy transfer process, thereby
significantly enhance the europium emission intensity.
The limit of detection for LPA was 5 µM; the limiting spec-
trum was reached at a concentration of 40 µM, which is in the
desired range for LPA detection in ovarian cancer cells. The
estimated stability constant in methanol, log K = 5.25, was
high, demonstrating that there is indeed a strong binding
affinity in the ternary adduct, [Eu·L1·LPA].
The induced CPL response (ESI) resembled that observed
with O-P-Ser, O-P-Thr and the phosphorylated hexapeptides,
with little activity observed in the ΔJ = 3 and ΔJ = 4 region. An
emission dissymmetry value, gem, of +0.04 at 593 nm was
measured. This value is lower than that measured for the
O-P-Tyr phosphorylated peptide, suggesting that there is less
conformational rigidity, consistent with the structure of LPA,
which contains a long, aliphatic carbon chain.
Table 3 Affinity constants, log K, for analyte complexation with
[Eu·L1]2+ and physiological concentrations of relevant biomolecules.
(MeOH–H2O 1 : 1,10 mM HEPES, 295 K)
Concentration in serum/mM
log K
−
HCO3
24–27
*
Lactate
HPO4
HSA
LPA
0.6–2.3
1.2–1.3
0.5–0.75
0–0.05
4.37
4.20
*
2−
5.25a
a Affinity constant estimated in 100% MeOH due to solubility of the
biomolecule; (*) no evidence for a significant binding interaction was
observed.
Comparison of the CPL spectrum (ESI) with the parent
N-benzyl complex, [Eu·L2]+, on addition of a number of chiral
Fig. 7 (left): variation of the Eu(III) spectral emission profile for [Eu·L1]2+ as a function of added LPA, showing the fit to the experimental data points
for a 1 : 1 binding model (5 µM complex, MeOH, λexc 352 nm, 295 K). (right): increased emission intensity, without change of spectral form, in the
range 100–600 µM LPA, consistent with micelle formation with an aggregation constant of 0.8 ( 0.04) mM.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
environment. In aqueous methanolic solution (1 : 1, v/v; appar- Dimethyl 6,6′-((7-(3-((N-methylformamido)methyl)benzyl)-
ent ‘pH’ 7.4), simulating an extracellular fluid containing HSA 1,4,7-triazonane-1,4-diyl)bis(methylene))bis(4-((4-methoxy-2,6-
(0.4 mM), sodium lactate (2.3 mM), hydrogen phosphate dimethylphenyl)ethynyl)picolinate), 7
(0.9 mM), citrate (0.13 mM) and sodium bicarbonate (30 mM),
the observed emission spectrum of [Eu·L1]2+ (5 µM) was the
same as that observed for the complex alone. LPA was titrated
into the solution in 5 µM aliquots until 100 µM was added.
However, neither a change in the emission spectrum nor an
induction of CPL was observed, under these conditions.
Summary and conclusions
This study has demonstrated that [Eu·L1]2+ binds selectively
via the phosphate group to the O-phosphono amino acids and
The bis-alkylated ligand, 6, (23 mg, 0.031 mmol) and K2CO3
(5 mg, 0.037 mmol) were dissolved in anhydrous CH3CN
(2.5 mL) and bubbled with argon (20 minutes). The mesylate 5
(8 mg, 0.031 mmol) was added and the mixture was stirred
under argon at 65 °C and monitored by LC-MS. After 24 h the
reaction was cooled and filtered to remove excess potassium
salts. The solvent was removed under reduced pressure and the
crude material was purified by flash column chromatography
(silica, gradient elution starting from 100% CH2Cl2 to 10%
CH3OH in CH2Cl2 in 1% increments) to give 7 as a glassy solid
(13 mg, 46%). TLC analysis Rf 0.32 (silica, 10% CH3OH in
model phosphorylated peptides and
a CPL response is
induced. Accurate determination of stability constants can be
achieved by monitoring the CPL spectral changes, as an
alternative to europium(III) emission spectral modulation.
Such an approach is particularly useful in cases where the
changes in the total emission response are small or indistinct.
The sign of the gem value in the ΔJ = 1 transition allows
differentiation between O-P-Ser and O-P-Tyr residues. The mag-
nitude of the induced CPL emission dissymmetry value for the
O-P-Tyr hexapeptide, GAPY*KF was +0.10; such a value is
amongst the highest that have been recorded for a dynamically
racemic europium(III) system.12,13,45 The large gem value allows
quick and easy detection of this peptide in solution when
using CPL as the detection technique.
1
CH2Cl2); H NMR (295 K, 600 MHz, CDCl3) δH 8.25, 8.11 (1H, 2
× s, NCOH,
7.49–7.14 (4H, m, Ph-H), 6.62 (4H, s, Ar-H2/2′
NCH2Ph), 4.39 (1H, s, NCH′2Ph), 4.12 (4H, s, py-CH2
s, CO2CH3), 3.80 (6H, s, OCH3), 3.47–2.99 (12H, br m, rings Hs),
2.87, 2.74 (3H, 2 × s, NCH3, NCH′3), 2.47 (12H, s, Ar-CH3
); 13C
NMR (295 K, 150 MHz, CDCl3) δC 165.3 (CO2CH3), 162.9
(NCO ),
H), 160.4 (Ar-C1), 157.8 (py-C6), 148.0 (Ar-C4), 143.1 (Ar-C3/3′
137.4 (Ph-C), 134.6 (Ph-C), 130.3 (Ph-C), 129.6 (Ph-C), 129.5 (Ph-
C), 128.8 (Ph-C), 128.0 (py-C5), 125.9 (py-C3), 113.8 (py-C4
), 112.9
(Ar-C2/2′), 94.8 (alkyne C5), 93.0 (alkyne C6
), 61.9 (ring Cs), 59.7
(ring Cs), 55.4 (OCH3), 53.6 (CO2CH3), 53.1 (py-CH2), 51.7 (ring
); m/z
̲
NCOH′
̲
), 8.02 (2H, s, py-H3
̲
), 7.62 (2H, s, py-H5
̲
),
), 4.49 (1H, s,
), 3.93 (6H,
̲
The detection of LPA using [Eu·L1]2+ as a lanthanide probe
has also been demonstrated, within the range 5–40 µM. This
result is the first example of induced CPL from an LPA adduct.
The total emission and circularly polarised luminescence of
the probe was monitored as a function of added lysophosphati-
dic acid. Due to the bright, strongly emissive nature of the
lanthanide complex, detection of LPA was readily achieved.
Analysis of the europium emission and CPL spectra revealed
information about the structure of the ternary adduct, improv-
ing upon previous examples of lanthanide LPA probes. The
absolute configuration of the [Eu·L1·L-LPA] adduct was tenta-
tively assigned as Δ, consistent with the configuration
assigned for the adducts with the O-P-L-amino acids.
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
̲
Cs), 47.7 (NCH̲ 2Ph), 34.6, 29.7 (NCH̲ 3), 21.5 (Ar-CH̲ 3
(HRMS+) 905.4599 [M + H]+ (C54H61N6O7 requires 905.4602).
[Eu·L1]Cl2
Further work may seek to enhance the scope of the use of
this, or analogous water-soluble complexes, in the detection
and recognition of more complex and structurally diverse
series of peptides, for example, those incorporating phos-
phorylated tyrosine residues.
Experimental
Details of the synthesis of the stated precursors 1–5, general
experimental aspects and spectroscopic, analytical and compu-
tational methods are given in the ESI.† The synthesis of
[Eu·L2]+ and compound 6 have been reported earlier.34
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
An aqueous solution of sodium hydroxide 0.3 M (0.5 mL) was 12 K. Okutani, K. Nozaki and M. Iwamura, Inorg. Chem., 2014,
added to a solution of ligand 7 (13 mg, 14 µmol) in methanol 53, 5527–5537.
(0.5 mL). The mixture was stirred at 70 °C for 48 h. The reac- 13 M. Iwamura, Y. Kimura, R. Miyamoto and K. Nozaki, Inorg.
tion was cooled to r.t. and aqueous hydrochloric acid (0.1 M) Chem., 2012, 51, 4094–4098.
was added until pH 7 was achieved. The crude solid was puri- 14 C. M. G. dos Santos, A. J. Harte, S. J. Quinn and
fied by preparative HPLC, tR = 16.5 min. The solid was passed T. Gunnlaugsson, Coord. Chem. Rev., 2008, 252, 2512–2527.
down an ion exchange column to remove the presence of 15 G.-L. Law, D. Parker, S. L. Richardson and K.-L. Wong,
formate. Europium chloride hexahydrate (3 mg, 8 µmol) was Dalton Trans., 2009, 8481–8484.
added and the pH was readjusted to 6.5 by addition of 16 L. M. P. Lima and R. Tripier, Curr. Inorg. Chem., 2011, 1,
aqueous sodium hydroxide (0.1 M). The reaction was stirred at 36.
65 °C for 24 h. The solvent was removed under reduced 17 A. M. Funk, K.-L. N. A. Finney, P. Harvey, A. M. Kenwright,
pressure to give the complex [Eu·L1]Cl2 as a white solid (5 mg,
68%). m/z ESI (MeOH no column) 1044 [M + H]+; λexc (MeOH)
E. R. Neil, N. J. Rogers, P. Kanthi Senanayake and
D. Parker, Chem. Sci., 2015, 6, 1655–1662.
18 A. M. Funk, P. Harvey, K.-L. N. A. Finney, M. A. Fox,
A. M. Kenwright, N. J. Rogers, P. K. Senanayake and
D. Parker, Phys. Chem. Chem. Phys., 2015, 17, 16507–
16511.
= 352 nm; Φem (MeOH) = 0.18; ε (MeOH) 36 000 M−1 cm−1
τ (MeOH) = 0.77 ms.
;
19 S. F. Mason, Struct. Bonding, 1980, 39, 43.
20 M. F. Reid and F. S. Richardson, Chem. Phys. Lett., 1983, 95,
501–506.
21 R. S. Dickins, D. Parker, J. I. Bruce and D. J. Tozer, Dalton
Trans., 2003, 1264–1271.
Acknowledgements
We thank the ERC (FCC 266804) and The Royal Society for
support.
22 R. Pal, D. Parker and L. C. Costello, Org. Biomol. Chem.,
2009, 7, 1525–1528.
23 D. G. Smith, G. Law, B. S. Murray, R. Pal, D. Parker and
K.-L. Wong, Chem. Commun., 2011, 47, 7347–7349.
Notes and references
1 (a) S. J. Butler and D. Parker, Chem. Soc. Rev., 2013, 42, 24 D. G. Smith, R. Pal and D. Parker, Chem. – Eur. J., 2012, 18,
1652–1666; (b) W. T. Xu, Y. F. Zhou, D. C. Huang, M. Y. Su, 11604–11613.
K. Wang, M. Xiang and M. C. Hong, J. Mater. Chem. C, 25 R. Pal, A. Beeby and D. Parker, J. Pharm. Biomed. Anal.,
2015, 3, 2003–2015; (c) S. Nadella, J. Sahoo, 2011, 56, 352–358.
P. S. Subramanian, A. Sahu, S. Mishra and M. Albrecht, 26 M. Soulié, F. Latzko, E. Bourrier, V. Placide, S. J. Butler,
Chem. – Eur. J., 2014, 20, 6047–6053; (d) D. F. Caffrey and
T. Gunnlaugsson, Dalton Trans., 2014, 43, 17964–17970.
2 R. S. Dickins, S. Aime, A. S. Batsanov, A. Beeby, M. Botta,
R. Pal, J. W. Walton, P. L. Baldeck, B. Le Guennic,
C. Andraud, J. M. Zwier, L. Lamarque, D. Parker and
O. Maury, Chem. – Eur. J., 2014, 20, 8636–8646.
J. I. Bruce, J. A. K. Howard, C. S. Love, D. Parker, 27 J. W. Walton, A. Bourdolle, S. J. Butler, M. Soulie,
R. D. Peacock and H. Puschmann, J. Am. Chem. Soc., 2002,
124, 12697–12705.
3 J. I. Bruce, R. S. Dickins, L. J. Govenlock, T. Gunnlaugsson,
M. Delbianco, B. K. McMahon, R. Pal, H. Puschmann,
J. M. Zwier, L. Lamarque, O. Maury, C. Andraud and
D. Parker, Chem. Commun., 2013, 49, 1600–1602.
S. Lopinski, M. P. Lowe, D. Parker, R. D. Peacock, 28 N. H. Evans, R. Carr, M. Delbianco, R. Pal, D. S. Yufit and
J. J. B. Perry, S. Aime and M. Botta, J. Am. Chem. Soc., 2000,
122, 9674–9684.
4 R. S. Alexander, Z. F. Kanyo, L. E. Chirlian and
D. W. Christianson, J. Am. Chem. Soc., 1990, 112, 933–937.
D. Parker, Dalton Trans., 2013, 42, 15610–15616.
29 M. Delbianco, V. Sadovnikova, E. Bourrier, G. Mathis,
L. Lamarque, J. M. Zwier and D. Parker, Angew. Chem., Int.
Ed., 2014, 53, 10718–10722.
5 B. Schneider and M. Kabeláč, J. Am. Chem. Soc., 1998, 120, 30 M. Delbianco, L. Lamarque and D. Parker, Org. Biomol.
161–165.
Chem., 2014, 12, 8061–8071.
6 G. Muller, Dalton Trans., 2009, 9692–9707.
7 R. Carr, N. H. Evans and D. Parker, Chem. Soc. Rev., 2012,
41, 7673–7686.
31 S. J. Butler, M. Delbianco, N. H. Evans, A. T. Frawley, R. Pal,
D. Parker, R. S. Puckrin and D. S. Yufit, Dalton Trans., 2014,
43, 5721–5730.
8 F. Zinna and L. Di Bari, Chirality, 2015, 27, 1–13.
9 J.-C. G. Bunzli and S. V. Eliseeva, Chem. Sci., 2013, 4, 1939–
1949.
10 J. M. Zwier, H. Bazin, L. Lamarque and G. Mathis, Inorg.
Chem., 2014, 53, 1854–1866.
32 S. J. Butler, L. Lamarque, R. Pal and D. Parker, Chem. Sci.,
2014, 5, 1750–1756.
33 S. J. Butler, B. K. McMahon, R. Pal, D. Parker and
J. W. Walton, Chem. – Eur. J., 2013, 19, 9511–9517.
34 E. R. Neil, M. A. Fox, R. Pal, L.-O. Palsson, B. A. O’Sullivan
and D. Parker, Dalton Trans., 2015, 44, 14937–14951.
11 F. Kielar, C. P. Montgomery, E. J. New, D. Parker,
R. A. Poole, S. L. Richardson and P. A. Stenson, Org. 35 T. T. da Cunha, J. Jung, M.-E. Boulon, G. Campo,
Biomol. Chem., 2007, 5, 2975–2982.
F. Pointillart, C. L. M. Pereira, B. L. Guennic, O. Cador,
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
K. Bernot, F. Pineider, S. Golhen and L. Ouahab, J. Am. 42 S. Dixit, J. Crain, W. C. K. Poon, J. L. Finney and
Chem. Soc., 2013, 135, 16332–16335. A. K. Soper, Nature, 2002, 416, 829–832.
36 K. Sénéchal-David, A. Hemeryck, N. Tancrez, L. Toupet, 43 J. Yao, H. J. Dyson and P. E. Wright, J. Mol. Biol., 1994, 243,
J. A. G. Williams, I. Ledoux, J. Zyss, A. Boucekkine, 754–766.
J.-P. Guégan, H. Le Bozec and O. Maury, J. Am. Chem. Soc., 44 A. Perczel and G. D. Fasman, Protein Sci., 1992, 1, 378–395.
2006, 128, 12243–12255.
45 R. Carr, R. Puckrin, B. K. McMahon, R. Pal, D. Parker and
L.-O. Pålsson, Methods Appl. Fluoresc., 2014, 2, 024007.
46 Y. Xu, Z. Shen, D. W. Wiper, M. Wu, R. E. Morton, P. Elson,
A. W. Kennedy, J. Belinson, M. Markman and G. Casey,
JAMA, J. Am. Med. Assoc., 1998, 280, 719–723.
47 O. Alptürk, O. Rusin, S. O. Fakayode, W. Wang,
J. O. Escobedo, I. M. Warner, W. E. Crowe, V. Král,
J. M. Pruet and R. M. Strongin, Proc. Natl. Acad.
Sci. U. S. A., 2006, 103, 9756–9760.
37 F. Pointillart, B. L. Guennic, O. Maury, S. Golhen, O. Cador
and L. Ouahab, Inorg. Chem., 2013, 52, 1398–1408.
38 R. S. Dickins, A. S. Batsanov, J. A. K. Howard, D. Parker,
H. Puschmann and S. Salamano, Dalton Trans., 2004, 70–
80.
39 A. Bourdolle, M. Allali, J.-C. Mulatier, B. L. Guennic,
J. M. Zwier, P. L. Baldeck, J.-C. G. Bünzli, C. Andraud,
L. Lamarque and O. Maury, Inorg. Chem., 2011, 50, 4987–
4999.
48 E. E. Kooijman, K. M. Carter, E. G. van Laar, V. Chupin,
K. N. J. Burger and B. de Kruijff, Biochemistry, 2005, 44,
17007–17015.
40 E. R. Neil, A. M. Funk, D. S. Yufit and D. Parker, Dalton
Trans., 2014, 43, 5490–5504.
41 P. Atkinson, Y. Bretonnière, D. Parker and G. Muller, Helv. 49 I. Pascher and S. Sundell, Chem. Phys. Lipids, 1985, 37, 241–
Chim. Acta, 2005, 88, 391–405. 250.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016