Luminescent lanthanide-binding peptides: Sensitising the excited states of Eu(iii) and Tb(iii) with a 1,8-naphthalimide-based antenna

Article abstract of DOI:10.1039/c1ob06567j

The investigation into the luminescence properties of a lanthanide-binding peptide, derived from the Ca-binding loop of the parvalbumin, and modified by incorporating a 1,8-naphthalimide (Naph) chromophore at the N-terminus is described. Here, the Naph is used as a sensitising antenna, which can be excited at lower energy than classical aromatic amino acids, such as tryptophan (the dodecapeptide of which was also synthesised and studied herein). The syntheses of the Naph antenna, its solid phase incorporation into the dodecapeptide, and the NMR investigation into the formation of the corresponding lanthanide complexes in solution is presented. We also show that this Naph antenna can be successfully employed to sensitize the excited states of both europium and terbium ions, the results of which was used to determined the stability constants of their formation complexes, and we demonstrated that our peptide 'loop' can selectively bind these lanthanide ions over Ca(ii).

Full text of DOI:10.1039/c1ob06567j

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PAPER
Luminescent lanthanide-binding peptides: sensitising the excited states of
Eu(III) and Tb(III) with a 1,8-naphthalimide-based antenna†
Ce´lia S. Bonnet,*a,b Marc Devocellec and Thorfinnur Gunnlaugsson*a
Received 2nd August 2011, Accepted 13th September 2011
DOI: 10.1039/c1ob06567j
The investigation into the luminescence properties of a lanthanide-binding peptide, derived from the
Ca-binding loop of the parvalbumin, and modified by incorporating a 1,8-naphthalimide (Naph)
chromophore at the N-terminus is described. Here, the Naph is used as a sensitising antenna, which can
be excited at lower energy than classical aromatic amino acids, such as tryptophan (the dodecapeptide
of which was also synthesised and studied herein). The syntheses of the Naph antenna, its solid phase
incorporation into the dodecapeptide, and the NMR investigation into the formation of the
corresponding lanthanide complexes in solution is presented. We also show that this Naph antenna can
be successfully employed to sensitize the excited states of both europium and terbium ions, the results
of which was used to determined the stability constants of their formation complexes, and we
demonstrated that our peptide ‘loop’ can selectively bind these lanthanide ions over Ca(II).
populated by using sensitizing antennae, often embedded, or
Introduction
conjugated, via short spacers into macrocyclic structures.18 In
The use of lanthanide(III) complexes in structural analysis,1–3 for
particular, the use of polyaminocarboxylate based ligands, such
probing supramolecular interactions,4,5 or in various biomedical
applications5–9 is of great current interest in chemistry. This is
in particularly due to the unique magnetic and photophysical
properties that these ions possess.10 Gd(III) complexes and con-
jugates have been employed as MRI relaxation contrast agents,
while, with the exception of La(III) and Lu(III), most of the
remaining lanthanides have unique luminescent properties, which
have been explored in the development of various luminescent
supramolecular devices. This includes the development of lu-
minescent switches, sensors, logic gate mimics, imaging agents,
etc. using ions such as Eu(III) and Tb(III), which emit in the
visible ranges,11–13 whereas ions such as Nd(III), Yb(III) emit in
the near infra-red; an area of particular interest for biological,
and telecommunication applications.14–16 The main advantages of
using these ions in the development of luminescent probes and
devices is attributed to their long-lived excited states and line-like
emission bands which occurs at long wavelengths. These features
overcome interferences from short-lived background fluorescence
and light scattering from biological matter.17 As these are Lapporte
forbidden transitions, these excited states are most effectively
as cyclen, has been widely investigated for such applications.19–27
In contrast, the use of peptides that can be synthetically modified
with such sensitizing antennae, and possess donor moieties that
can enable the binding of lanthanide ions, has been much less
explored. Such systems could be of significant value, particularly
for use in biological media, for instance, with the view of generating
novel luminescent protein mimics, employed to investigate protein-
protein or protein-substrate interactions.
Due to the similar ionic radii of Ca(II), the lanthanides have been
used as substitutes for Ca(II) ions in various Ca-binding peptides
and proteins,28–31 or with the aim of developing luminescent
peptides.32,33 For the latter, such structures usually possess amino
acids with aromatic residues, such as tryptophan, tyrosine and
phenylalanine, which can be used to sensitize the excited state of
Tb(III).34 Recently, this area of research has attracted significant
attention, particularly through the work of Imperiali et al.,
who has developed lanthanide-binding tags appended to various
proteins (LBTs).34,35 These have been used to elucidate protein
structure,3,36 detect protein-protein interactions and to phase
X-ray crystallographic data.37 Some other examples within this
area of research includes that of Va´zquez et al.,38 who developed
novel sensors for investigating protein-substrate interactions,
Franklin et al.39–40 who investigated the DNA binding affinity and
hydrolysis of such lanthanide based peptides. Recently, Ito et al.41
have also developed lanthanide complexes containing the cyclic
peptide c(RGDfK) to visualize the a(v)b(3)-integrin-expressing
tumor cells, while Delangle et al.42 have developed Gd(III)-based
complexes of cyclic decapeptide with two independent faces as
“regioselectively addressable functionalised templates” (RAFTs).
aSchool of Chemistry, Centre for Synthesis and Chemical Biology, Trinity
College Dublin, Dublin, 2, Ireland. E-mail: gunnlaut@tcd.ie; Fax: +353 1
671 2826; Tel: +353 1 896 3459
bCentre de Biophysique Mole´culaire, CNRS, Rue Charles Sadron, 45071,
Orle´ans Cedex 2, France. E-mail: celia.bonnet@cnrs-orleans.fr
cCentre for Synthesis and Chemical Biology, Department of Pharmaceutical
and Medicinal Chemistry, Royal College of Surgeons in Ireland, 123 St.
Stephen’s Green, Dublin, 2, Ireland
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06567j
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All the above examples have been formed by using natural
occurring amino acids, and while these complexes are clearly useful
for in vitro studies, there are intrinsic limitations for their use
in vivo. Firstly, the stability of the lanthanide-peptide complexes
formed are limited, as such peptides bear only simple binding
groups such as carboxylates, phenolates, or amides. Secondly,
only few of these amino acids can be employed as antennae.
Consequently, modified amino acids have to be used to introduce
strong donor groups for reinforcing metal complexation43,44 or to
improve the luminescence properties.45,46
We have recently reported preliminary results for the use
of 1,8-naphthalimide (Naph) structures to sensitize the excited
states of both Eu(III) and Tb(III).47,48 This sensitizing antenna
is particularly attractive as it can be excited at lower energy
(345 nm), and by simple synthetic modifications, the photophysical
properties can be easily tuned. Hence, we have employed this
structure in our laboratory in various supramolecular systems and
devices.49,50 Herein, we give full account of the use of this antenna
within peptide structures designed to form stable complexes with
lanthanide ions for use in biological applications.
using a mixture of TFA/TIS/H2O 95/2.5/2.5, and purified by
reverse-phase HPLC, and characterised by Maldi-Tof MS (See
ESI).
The synthesis of the Naph derivative is shown in Fig. 1, and was
formed in one step by reacting glycine (1.2 eq.) with naphthalic
anhydride (1 eq.) in DMF at 150 ◦C over 24 h. The resulting
brown oil was purified by precipitation from methanol to give a
white solid in 56% yield (see Experimental section).
The peptide sequence DADGDGAIEPEE was synthesised
using standard automated solid phase peptide synthesis ac-
cording to the Fmoc/tBu strategy and HBTU-HOBt-DIEA
coupling chemistry in NMP. The Naph antenna was then
coupled manually at the N-terminus of the peptide sequence
using
a double coupling procedure. The resulting modi-
fied polymer-bound peptide Naph-Asp(tBu)-Ala-Asp(tBu)-Gly-
Asp(tBu)-Gly-Ala-Ile-Glu(tBu)-Pro-Glu(tBu)-Glu(tBu) was then
cleaved from the resin and the side chains were depro-
tected using TFA/thioanisole/TIS/EDT/H2O (v/v/v/v/v =
77/5.75/5.75/5.75/5.75), and P1 was finally purified by reverse-
phase HPLC, and characterised by their ESMS (see ESI†).
The peptide sequence chosen for this investigation is shown in
Fig. 1 as the dodecapeptide P1; designed on the Ca(II) binding
loop of the parvalbumin protein, an amino acid sequence which
includes three Glu and three Asp amino acids; each one able to
provide a chelating carboxylate residues for binding to Ln(III). In
P1, the Naph antenna, the synthesis of which is shown in Fig. 1,
was incorporated at the N-terminus of the peptide. Furthermore,
we also developed P2 for use in comparison studies. This system
lacks the Naph antenna, but has instead, a Trp moiety that can be
employed as such an antenna.
Photophysical evaluation of P1 and P2
The photophysical properties of P1 and P2 were investigated in
10 mM HEPES buffer solution in the presence of 0.1 M NaCl, to
maintain a constant ionic strength. The UV-Vis absorption, the
fluorescence emission and the excitation spectra, were recorded
both in the absence, as well as in the presence of one equivalent of
either Eu(III) or Tb(III).
The UV-vis absorption spectrum of P1 was dominated by a
broad band centred at 344 nm (log e = 4.04) which was assigned
to the n–p* transition of the Naph antenna (see Fig. S3, ESI†).
Excitation into this band gave rise to a fluorescence emission
typical of such Naph moieties, with lmax = 393 nm. In analogous
manner, the Trp-antenna-based peptide P2 was analysed in
buffered solution, and the UV-vis absorption spectrum showed
a broad band with lmax at 280 nm, which had the typical Trp
structure. Excitation of this band gave rise to the characteristic
Trp emission centred at 334 nm.
Upon addition of one equivalent of TbCl3 or EuCl3 to P1, no
significant changes were seen in the UV-vis absorption spectra of
P1, but the characteristic emission spectra of Eu(III) or Tb(III)
ions were observed as shown in Fig. 2a and 2c respectively. This
emission is most likely due to an energy transfer mechanism
occurring from the Naph, to the Eu(III) and Tb(III) excited states.
This was confirmed by recording the fluorescence excitation
spectra of both systems in the presence of Eu(III) and Tb(III), by
fixing the emission at 616 nm (5D4 → F2 transition) and 544 nm
7
7
(5D0 → F5 transition), respectively (see ESI, Fig. S4–S5†). The
Fig. 1 The two dodecapeptides P1 and P2 developed in this study and
the synthesis of the Naph antenna that was incorporated into P1.
excitation spectra were found to be structurally similar to the
absorption spectra of these systems, confirming the successful
energy transfer from these antennae to the Eu(III) and Tb(III)
excited states. Additionally, in the case of Tb(III), a new band
centred at 270 nm was observed and attributed to the formation
of a 4f–5d transition (which could be MLCT in nature).52,53 In a
similar manner, P2 was analysed and similar results were observed
to those described above (see ESI, Fig. S7–S9†). As expected, P2
was also able to sensitise the Tb(III) excited state, and the relative
intensity of the emission at 544 nm was significantly greater than
Results and discussion
Syntheses of the dodecapeptides P1 and P2
The dodecapeptide sequence DADGDGWIEPEE of the model
peptide P2 was synthesised using standard manual solid-phase
synthesis based on Fmoc/tBu protection strategy.51 The peptide
was cleaved off the resin and the side chains were deprotected
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Fig. 3 The overall changes in the Eu(III) emission upon titrating P1
(21 mM) with EuCl3 at pH 7.0 (10 mM HEPES) and in 0.1 M NaCl.
Inset: corresponding titration profile at 617 nm and the best fit obtained
using the non-linear regression analysis programme SPECFIT.
Fig. 2 (a) Emission spectra of P1 (30 mM) in the presence of Eu(III)
(17 mM) with excitation of Naph at 345 nm (slit width 5 nm for excitation
and emission). (b) Emission spectra of P2 (30 mM) in the presence of Eu(III)
(17 mM) with excitation of Trp at 280 nm (slit width 20 nm for excitation
and 10 nm for emission). (c) Emission spectra of P1 (30 mM) in the presence
of Tb(III) (17 mM) with excitation of Naph at 345 nm (slit width 20 nm
for excitation and emission). (d) Emission spectra of P2 (30 mM) in the
presence of Tb(III) (17 mM) with excitation of Trp at 280 nm (slit width
5 nm for excitation and 2.5 nm for emission).
same behaviour than P2 towards Ln(III) complexation, where
the metal-centred luminescence increased upon formation of the
lanthanide peptide complex, within the use of one equivalent of
the lanthanide ions. However, beyond the use of one equivalent,
the Eu(III) emission reduced in intensity, see inset in Fig. 3. This
was attributed to the formation of mononuclear and binuclear
species, the latter resulting in a quenching of the luminescence
intensity as previously observed.56–58 Indeed, upon further analysis
of these changes using the non-linear regression analysis program
SPECFIT, this hypothesis was confirmed, as these changes were
best fitted to two step binding equilibria.
that seen for P1. It is possible that this is partially due to closer
proximity of this antenna to the lanthanide in comparison to P1,
where the Naph antenna is located at the N-terminus. But there is
also certainly a difference in the mechanism of the energy transfer
to the Tb(III) excited state (5D4 = 20 500 cm-1) which is thought
to occur via the triplet state of the Trp (24 830 cm-1),55 and which
might occur through the singlet state of the Naph (27 980 cm-1),
because the triplet state is too low in energy (18 540 cm-1).54 On the
contrary, the relative intensity of Eu(III) luminescence at 616 nm
was found to be significantly greater in the case of P1 than that
seen for P2, Fig. 2a and 2b, despite the difference in the location of
these two antennae within the peptide structures. Even though we
did not quantify the efficiency of the energy transfer for these two
systems, it is clear from these results that the Naph antenna can
be successfully employed for populating the Eu(III) excited state,
and that it does so more efficiently than Trp. This can be explained
because the T1 energy of Naph is better suited for populating Eu(III)
(5D0 = 17 200 cm-1)9 than the T1 energy of Trp, and additionally
in the case of Trp a non-radiatively deactivated LMCT could also
be formed.
The stability constants determined from the fitting of the
changes in the Eu(III) emission of both P1 and P2 are summarized
in Table 1. They show that both P1 and P2 give rise to similar
binding constants for these ions. Unfortunately, due to the low
solubility of the Tb2P1 species, we were unable to perform full
titrations on this system, and, hence, unable to determine the
Tb(III) affinity for P1. From Table 1, we can, however, conclude
that the formation of the binuclear complexes is more difficult in
terms of binding affinity than the formation of the mononuclear
complexes. The stepwise stability constant is ca. 5 for all the
systems; nearly two orders of magnitude lower than the formation
of the mononuclear species. This difference can be explained by
unfavourable electrostatic repulsion between the two lanthanide
ions. This has previously been observed, even for systems with a
large cavity, such as the one of a modified a-cyclodextrin (where the
˚
distance between the two Ln(III) ions is 5.74 A in the solid state),
and for which the stepwise formation constant of the binuclear
species is nearly four orders of magnitude lower than the one of the
mononuclear species.59 Concerning the mononuclear complex, the
stability constants are of the same order of magnitude than the 17-
amino acid peptide developed by Imperiali et al. (log bTb = 7.2) and
optimised by screening methods, where the coordination sphere of
Photophysical titrations of P1 and P2 using Eu(III) and Tb(III)
We next investigated the formation of these lanthanide-based
peptides by carrying out careful spectroscopic titration in solu-
tions, where the fluorescence emission and the lanthanide emission
spectra were monitored. A typical evolution of the time-resolved
Eu(III) emission for the titration of P1, upon excitation of the
antenna at 345 nm, is shown in Fig. 3. Here the characteristic line-
like and narrow emission bands of the Eu(III) emission became
apparent upon binding of the ion to the peptide; the binding
of which was monitored by observing the changes in the Eu(III)
DJ = 2 transition at 617 nm. The results are shown as an inset
in Fig. 3. First, it should be pointed out that P1 displays the
Table 1 Stability constants of Ln-P complexes in HEPES buffer 10 mM,
pH = 7.0, 0.1 M NaCl at 298 K. bmn = [LnmPn]/[Ln]m[P]n
Tb(III)
Eu(III)
P1
P2
log b11
log b21
log b11
log b21
—
—
6.8 (0.1)
11.9 (0.1)
6.8 (0.1)
11.8 (0.1)
6.75 (0.08)
11.9 (0.1)
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Table 2 Stability constants of M-P2 complexes in HEPES buffer 10 mM,
pH = 7.0, 0.1 M NaCl at 298 K. bpmn = [MpLnmP2n]/[M]p[Ln]m[P]n
Tb(III) is fully saturated, without any water molecule
coordinated.35 It is also higher than the stability constants reported
for natural Ca(II) binding loops in calmodulin, where the affinity
was found to be 5.5 for Tb(III) and 6.2 for Eu(III).60 Finally, and
possibly most importantly, the presence of the new sensitizer,
the Naph antenna, does not significantly alter the affinity of the
peptide sequence for Eu(III); being log b = 6.8 for P1, compared to
log b = 6.75 for the unmodified version P2.
In addition to explore the binding of the lanthanide ions by
P1 and P2, we were also interested in assessing the selectivity of
our system for these lanthanide ions with respect to endogenous
cations such as Ca(II), Cu(II), and Zn(II). In order to do that, we
chose the model compound P2 and we performed competition
experiments starting with the Tb(III) complex Tb2P2 (typically,
[Tb(III)] = 5 ¥ [P2] at the start of the titration), and we monitored
the decrease of the Tb(III) luminescence intensity upon addition
of the desired cation. A complete quenching of the luminescence
intensity was observed upon addition of 5 eq. of Cu2+, as is
demonstrated in Fig. 4, whereas the addition of 1000 eq. of Ca(II)
resulted in 60% of quenching (Fig. S10, ESI†), and the addition of
2000 eq. of Zn(II) resulted in less than 40% of quenching, making
it impossible to determine a stability constant from these changes.
For Cu(II), the best model to fit the data using SPECFIT involved
the formation of mixed species between M(II)/Ln(III) and P2,
whereas for Ca(II) only the formation of CaP2 was observed. The
results are summarized in Table 2. The Ca(II) complex formed
with P2 is clearly less stable than the one formed with native
Ca(II)
Cu(II)
log b101
log b111
4.61 (0.05)
7.53 (0.05)
11.7 (0.1)
parvalbumin (log bCaP ~ 8).61 This could be attributed to the
modification made from the native Parvalbumin binding loop,
and also from the fact that other residues, not strictly from the
binding loop could contribute to the stability of the complex in
the naturally occurring protein. We achieved a good selectivity
for Ln(III) over Ca(II) as the stability of Ln(III) complexes is
two order of magnitude higher than seen for the corresponding
Ca(II) complex. Previous work in this field has shown that the
Ln(III) complexes formed with the first calmodulin domain are
one order of magnitude more stable than the corresponding Ca(II)
complexes.60
Elucidation of the structure of the metallo-peptides in solution
In order to gain a better insight into the structure of the metallo-
peptides formed, we used circular dichroism experiments to
monitor their formation. The CD spectra of P1, P1 with one
and two eq. of Eu(III), P2, P2 with one and two eq. of Tb(III)
are shown Fig. S11–S12 in ESI.† The same trend was observed
for both systems upon binding to the lanthanides: a stronger
negative band at 200 nm was observed for the free peptides,
than for the metallo-peptides. This is characteristic of a random
coil configuration in the absence of lanthanide ions.62 Moreover,
additional changes were observed upon addition of the lanthanide
ions such as the transition centred at 220 nm became more negative
which is indicative of some folding interactions. However, we were
unable to identify any typical b-sheet or a-helix signatures from
these spectral changes, and no significant changes were observed
that allowed us to distinguish between the mononuclear and the
binuclear species in these spectra. Because of this, we thus decided
to carry out NMR experiments on these systems to get more
information on the structure of the resulting metallo-peptides.
The NMR titration of both P1 and P2 were undertaken using
the diamagnetic ion, La(III), and Eu(III) which is paramagnetic,
in 90 : 10 H2O:D2O solutions. Selected spectra from these investi-
gations are shown as stack-plots in Fig. 5. On all occasions, we
only observed changes in the 1H NMR spectra up to the addition
Fig. 4 Evolution of the luminescence spectra of P2 (18.8 mM)-Tb(III)
(75.2 mM) upon titration by CuCl2 and after excitation of Trp at 280 nm
at pH 7.0 (10 mM HEPES) and in 0.1 M NaCl. Inset: The corresponding
titration profile at 550 nm and the best fit obtained using SPECFIT.
Fig. 5 Partial 1H NMR spectra (600 MHz, 298 K) of the titration of P2 with La3+ in a 9/1 H2O/D2O solution.
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Table 3 1H NMR (600 MHz) chemical shifts (d ppm) for P2, basified P2
(P2 + 6 eq. NaOH) and its La(III) complexes in H2O/D2O (v/v = 9/1) at
298 K
of 2 equivalents of metal ion added, with no significant changes
occurring at higher equivalents. This confirms the formation
of 1 : 1 and 2 : 1 complexes (see also Fig. S13, ESI†). In the
case of P1, we could not determine the structure because in
H2O/D2O solutions, the NH proton resonances were too broad,
and becoming almost invisible when the pH was adjusted to 7.
Although in the presence of La(III) the NH signals appear again
(see Fig. S14, ESI†), they remained too broad for any further
analysis. However, the NH-resonances were better resolved for
P2 and its complexes, which allowed us to analyse those changes
in more details. This difference between P1 and P2 is possibly
due to a difference in dynamics between the two peptides in
solution. For the titration of P2 with La(III) the addition of less
than one equivalent of La(III) resulted in a broadening in the
NMR spectra, suggesting the formation of a labile 1 : 1 complex,
with dynamic equilibrium between the free and the bound form
of P2. Upon addition of one equivalent of La(III), the spectrum
sharpened significantly, making it possible to obtain a complete
assignment of the resonances. To achieve this, we carried out t-
ROESY experiments. A partial spectrum is shown in Fig. 6a,
and demonstrates that while we did not observe enough long-
range correlations to allow for a full structure determination,
a correlation between the NH of Asp1 and the Hg of Ile8
was observed. This suggests a folding of the P2 peptide upon
complexation with the La(III) ion, which further confirms the
conformation changes observed in the CD spectra above. Further
analysis of the chemical shifts of the different protons identified
in these experiments, showed that the Hb protons of the three Asp
and the Ha protons of Trp and Ile were all shifted upon interaction
of P2 with La(III). These results are summarised in Table 3, and
the changes in the NMR spectra suggest a direct coordination
P2
P2 + 6 eq. NaOH
P2La
P2La2
Asp1 Hb
Asp1 Hb
Asp1 Hb
Glu 9 Hb
Hg
2.72–2.80
2.77–2.83
2.78
1.93–2.05
2.43
2.51–2.62
2.64
2.63
2.39–2.69
2.63–2.99
2.60–3.03
1.89–2.18
2.42–2.63
2.66–3.05
2.63–3.07
1.77–2.20
1.88–2.25
Glu 11 Hb 1.84–2.00
Hg 2.42
1.95–2.22
1.92–2.24
2.00–2.25
2.20–2.41
2.03–2.24
4.72
2.20–2.41
Glu 12 Hb 1.93–2.09
Hg
Trp Ha
Ile Ha
2.41
4.60
3.99
4.58
9.96
4.90
4.46
4.95
4.37
of the three Asp in P2 to the La(III) ion, as well as the amide
backbone of the Trp. Importantly, the coordination of the amide
of the Trp in the position 7 of the Ca-binding loop is consistent
with what has been observed for typical coordination of metal ions
in EF hand motif for other peptide-Ln complexes both by X-ray
in the solid state35 and by NMR in solution.40 We had previously
determined the number of water molecules directly coordinated
to the Ln(III) ion in the mononuclear complex by luminescence
lifetime measurements. We found a q value of ~3 for both P1 and
P2 complexes,47 showing the coordinatively unsaturated nature of
the lanthanide ions within P1 and P2. These results, together with
the stability constant values confirm that the introduction of Naph
does not affect the coordination sphere of the Ln(III). Moreover,
these results are consistent with the direct coordination of three
Asp, and suggest that possibly one or two of the Asp residues are
coordinating to the ion in a bidentate manner in P2. Upon addition
of a second equivalent of La(III) to P2 further shifts were observed
for the side chains of two of the three Glu residues, suggesting
that the second metal ion is coordinated within the Glu pocket
of P2. This was further confirmed by the t-ROESY correlations,
Fig. 6b, observed between the Hg of Ile8 and the NH of Glu12,
and between the Hg of Ile8 and the C-terminal NH2. This is also
an indication of the folding of the peptide at the C-terminus. It
should be noted that a correlation is also observed between the
NH of Glu9 and the CH3 at the N-terminus, confirming that we
still have a folding of the N-terminus part of the peptide.
Conclusions
In summary, we have developed a peptide, based on the Ca-binding
loop of the parvalbumin, bearing a sensitising naphthalimide
chromophore that can used to populate both the excited states
Eu(III) and Tb(III). The Naph has been coupled to a glycine
and then easily introduced at the N-terminus of the peptide
using standard peptide solid-phase synthesis. The binding loop
used and studied through the use of a similar peptide bearing
a Trp chromophore does show a good selectivity for Ln(III)
over Cu(II), Zn(II), and more importantly Ca(II). The presence
of two coordination pockets composed of Asp and Glu, and
corresponding to the formation of 1/1 and 2/1 Ln/P complexes
respectively was evidenced. The introduction of the Naph antenna
does not affect the stability of the lanthanide complexes formed
with the peptide, and does not change the number of water
molecules directly coordinated to the Ln(III) ions. This is a first
1
Fig. 6 Partial H t-ROESY spectra at 600 MHz, 298 K with (a) [P2] =
[La] = 0.63 mM and (b) [P2] = [La]/2 = 0.63 mM.
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step towards the optimisation of peptide- lanthanide complexes as
the use of lower excitation energies than the natural amino acids
can provide is compulsory for further possible in vivo applications.
77/5.75/5.75/5.75/5.75) for 2 h. After concentration the residue
was precipitated in cold diethylether to yield a white powder. The
solid residue was dissolved in MeOH/H2O (v/v = 60/40) and
purified by RP-HPLC to yield Naph-DADGDGAIEPEE as a
white powder. Maldi-Tof -MS: m/z = 1475.4 [M+K+].
Experimental section
Peptide P2: DADGDGWIEPEE. The peptide with pro-
tected side chains Ac-Asp(tBu)-Ala-Asp(tBu)-Gly-Asp(tBu)-Gly-
Trp(Boc)-Ile-Glu(tBu)-Pro-Glu(tBu)-Glu(tBu)-NH2 was assem-
bled manually by solid-phase peptide synthesis on Rink-amide
resin (substitution 0.6 mmol g-1) using Fmoc chemistry.51 Cou-
plings were performed with N-a-Fmoc-protected amino acids (2
eq.), PyBOP (2 eq.) and DIEA (pH ª 8–9) in DMF. Coupling
reaction was monitored by TNBS test. For non complete reactions,
a second coupling was achieved followed by DMF/pyridine/Ac2O
(v/v/v = 7/2/1) treatment. Fmoc deprotection was achieved
with DMF/piperidine (v/v = 4/1). After the last coupling, the
acetylation was performed with a DMF/pyridine/Ac2O 7/2/1
mixture. The peptide was cleaved from the resin and deprotected by
treatment with TFA/TIS/H2O (v/v/v = 95/2.5/2.5) for 2 h. After
concentration the residue was precipitated in cold diethylether to
yield a white powder. The solid residue was dissolved in water and
purified by RP-HPLC to yield Ac-DADGDGWIEPEE-NH2 as a
white powder. Maldi-Tof -MS: m/z = 1394.8 [M+Na+].
General
All chemicals were purchased at the purest grade commercially
available and were used without further purification unless other-
wise mentioned. Solvents used were HPLC grade unless otherwise
mentioned. Dichloromethane for peptide synthesis was distilled
prior to use. Water was purified with a Waters Milli-Q system to
give a specific resistance <15 MX cm.
Chromatographic analysis and purification were performed
on a BioCAD SPRINT Perfusion Chromatography Workstation
(PerSeptive Biosystems) using Phenomenex Gemini columns (5
˚
A, C18, 4.6mmd/250mmL (analytic) 100mmd:250mmL (semi-
preparative), solvent A = H2O/TFA (v/v = 99.9/0.1), solvent B =
CH3CN/TFA (v/v = 99.9/0.1), flow rate: 1 mL min-1 (analytical)
4.5 mL min-1 (semi-preparative). The gradient employed was 2
to 60% B in 18 column volumes with UV monitoring at 214 and
350 nm (for P1) or only 214 nm for P2.
The peptides were characterized by Matrix Assisted Laser
Desorption Ionisation- Time Of Flight- Mass Spectroscopy (a-
cyano-4-hydroxy-cinnamic acid matrix).
Preparation of liquid samples
Mass spectrum of Naph was determined using electrospray on a
Micromass LCT spectrometer. High-resolution mass spectra were
determined relative to a standard of leucine enkephaline.
The peptide concentration was systematically determined by
measuring the UV-absorption of the tryptophan residue for P2
(lmax = 280 nm, emax = 5690 cm-1 M-1), and the Naph residue
for P1 (lmax = 345 nm, emax = 11 500 cm-1 M-1). Metal stock
solutions were prepared by dissolving LnCl3·6H2O or LnOTf3 in
H2O or D2O. Their exact Ln3+ concentrations were systematically
determined by colorimetric titration in acetate buffer (pH = 4.5)
using standardized H2Na2edta solution and xylenol orange as an
indicator,63 except for Ca(II) solutions, which were titrated at pH
12 using Calcon as an indicator.
Synthesis
1,3-Dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid (Naph).
Glycine (3.7 mmol) and 1,8-naphthalic anhydride (3 mmol) were
mixed together with 20 mL of DMSO in a pressure tube. The
◦
mixture was heated at 150 C to allow the dissolution, and was
kept under continuous stirring at this temperature during 24h. It
was then evaporated to give a brown solid. The crude product was
washed with methanol and filtrated to give a white solid in 56%
yield.1H NMR (DMSO-d6, 40 MHz), d (ppm): 8.54 (m, 4H), 7.92
(t, J = 7.76 Hz, 2H), 7.76 (s, 2H).
Luminescence titrations
Solutions were all made at pH = 7.0 in 10 mM HEPES buffer,
0.1 M NaCl. UV-Vis spectra were recorded on a Varian UV-
Vis spectrophotometer from 250 to 500 nm using excitation
and emission slit width of 1 nm with a medium scan speed.
Luminescence and lifetimes were measured on a Varian Cary
Eclipse Fluorescence spectrophotometer following an excitation
at 345 nm for P1, and at 280 nm for P2. The following settings
were as follow: for fluorescence mode: PMT detector: 600 mV, scan
speed: 600 nm min-1, averaging time: 0.1 s, data interval: 1 nm. For
phosphorescence mode: delay time: 0.1 ms, flash count: 1, PMT
detector: 1000 mV, data interval: 1 nm, total decay time: 0.02 s,
gate time: 10 ms.
Spectra were analysed by using the program SPECFIT, which
employs a singular value decomposition algorithm and refines the
data according to a global least-squares analysis procedure.64,65
The values obtained are the average of at least three titrations.
Terbium and europium luminescence lifetimes were measured
by recording the decay of the emission intensity at 545 nm and
616 nm, respectively, after excitation at 345 nm for P1 and 280 nm
for P2. It was also checked that direct excitation at 368 nm and
13C NMR (DMSO-d6, 150 MHz), d (ppm): 169.2 (COOH),
163.0 (CO), 134.9, 131.4, 131.1, 127.36, 127.33, 121.5 (Car), 41.1
(CH2). ESI-MS m/z: [L + Na]+ calculated for C14H9NO4Na:
278.04; Found, 278.05.
Peptide P1: Naph-DADGDGAIEPEE. The polymer-bound
precursor peptide Asp(tBu)-Ala-Asp(tBu)-Gly-Asp(tBu)-Gly-
Ala-Ile-Glu(tBu)-Pro-Glu(tBu)-Glu(tBu) was prepared by stan-
dard solid phase peptide synthesis according to the Fmoc-tBu
strategy with HBTU/HOBt/DIEA coupling chemistry, in NMP
solvent. Single coupling cycles using a 10-fold excess of Fmoc-
amino acid derivatives to resin-bound peptide were employed.
Assembly of the amino acid sequence, starting from a Rink
Amide MBHA resin was carried out on an automated peptide
synthesizer (Applied Biosystems 433A). The Naphthalimide (2
eq.) was then coupled manually using PyBOP (2 eq.). Cou-
pling reaction was monitored using the qualitative Kaiser Test.
The peptide was cleaved from the resin and deprotected by
treatment with TFA/thioanisole/TIS/EDT/H2O (v/v/v/v/v =
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396 nm give the same results. The lifetimes were measured in
such conditions that only the 1/1 complex is present in solution.
The signals were analyzed as single-exponential decays. As the
peptide is highly hydrated, the lifetimes were measured for different
H2O/D2O ratio ranging from 0.2/0.8 to 1/0, and the lifetime in
D2O was extrapolated from the measured values.
10 (a) J. P. Leonard, C. B. Nolan, F. Stomeo and T. Gunnlaugsson, Top.
Curr. Chem., 2007, 281, 1; (b) T. Gunnlaugsson and J. P. Leonard,
Chem. Commun., 2005, 3114; (c) D. Parker, Chem. Soc. Rev., 2004, 33,
156.
11 (a) J. Massue, S. J. Quinn and T. Gunnlaugsson, J. Am. Chem. Soc.,
2008, 130, 6900; (b) C. S. Bonnet, J. Massue, S. J. Quinn and T.
Gunnlaugsson, Org. Biomol. Chem., 2009, 7, 3074; (c) N. S. Murray, S.
P. Jarvis and T. Gunnlaugsson, Chem. Commun., 2009, 4959.
12 (a) C. S. Bonnet and T. Gunnlaugsson, New J. Chem., 2009, 33, 1025;
(b) S. J. A. Pope and R. H. Laye, Dalton Trans., 2006, 3108; (c) S. J.
A. Pope, B. P. Burton-Pye, R. Berridge, T. Khan, P. J. Skabara and S.
Faulkner, Dalton Trans., 2006, 2907.
13 C. P. Montgomery, B. S. Murray, E. J. New, R. Pal and D. Parker, Acc.
Chem. Res., 2009, 42, 925.
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S. Petoud and E. Toth, Chem. Commun., 2008, 6591.
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Commun., 2011, 47, 206.
NMR experiments
All the NMR experiments were performed on a Bruker Avance
II 600 MHz spectrometer equipped with a 5 mm TCI cryoprobe.
The spectra were recorded at 298 K in H2O/D2O (v/v, 95/5)
using watergate W5 when necessary66 with a bandwidth of 7200
Hz and a power of 4.05 dB for the Watergate pulse. 2D NMR
spectra were acquired in phase-sensitive mode with TPPI for
quadrature detection in the indirect dimension using 2048¥128
matrices. TOCSY experiments were performed by using a MLEV-
17 spin-lock sequence with a mixing time of 70 ms. Off-resonance
ROESY experiments were recorded with a mixing time of 300 ms
(5000 Hz spin-lock). The spectra of P2 were recorded at 0.63 mM.
A titrated solution of NaOH was added and spectra were recorded
up to 6 equivalents of NaOH added with respect to P2. A titrated
solution of Ln(III) (La or Eu) was then added and spectra were
recorded up to 4 equivalents of metal ion with respect to P2.
The same procedure was followed with P1 and the corresponding
spectra were recorded with a concentration of P1 of 0.20 mM.
16 J. C. G. Bunzli and S. V. Eliseeva, J. Rare Earths, 2010, 28, 824.
17 C. M. G. dos Santos, A. J. Harte, S. J. Quinn and T. Gunnlaugsson,
Coord. Chem. Rev., 2008, 252, 2512.
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19 (a) S. E. Plush and T. Gunnlaugsson, Dalton Trans., 2008, 3801; (b) S. E.
Plush and T. Gunnlaugsson, Org. Lett., 2007, 9, 1919; (c) M. Cantuel,
C. Lincheneau, T. Buffeteau, L. Jonusauskaite, T. Gunnlaugsson, G.
Jonusauskas and N. D. McClenaghan, Chem. Commun., 2010, 46,
2486; (d) J. P. Leonard, C. M. G. Dos Santos, S. E. Plush, T. McCabe
and T. Gunnlaugsson, Chem. Commun., 2007, 129; (e) J. P. Leonard,
P Jensen, T. McCabe, J. E. O’Brien, R. D. Peacock, P. E. Kruger
and T. Gunnlaugsson, J. Am. Chem. Soc., 2007, 129, 10986; (f) T.
Gunnlaugsson, J. P. Leonard, K. Senechal and A. J. Harte, J. Am.
Chem. Soc., 2003, 125, 12062; (g) T. Gunnlaugsson and J. P. Leonard,
Dalton Trans., 2005, 3204.
20 K. Se´ne´chal-David, S. J. A. Pope, S. Quinn, S. Faulkner and T.
Gunnlaugsson, Inorg. Chem., 2006, 45, 10040.
Circular dichroism
21 B. McMahon, P. Mauer, C. P. McCoy, T. C. Lee and T. Gunnlaugsson,
J. Am. Chem. Soc., 2009, 131, 17542.
◦
CD spectra were recorded at 25 C on a JASCO’s J-810 dichro-
22 R. Pal and D. Parker, Org. Biomol. Chem., 2008, 6, 1020.
23 F. Kielar, C. P. Montgomery, E. J. New, D. Parker, R. A. Poole, S. L.
Richardson and P. A. Stenson, Org. Biomol. Chem., 2007, 5, 2975.
24 J. Yu, D. Parker, R. Pal, R. A. Poole and M. J. Cann, J. Am. Chem.
Soc., 2006, 128, 2294.
25 R. J. Aarons, J. K. Notta, M. M. Meloni, J. Feng, R. Vidyasagar, J.
Narvainen, S. Allan, N. Spencer, R. A. Kauppinen, J. S. Snaith and S.
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graph in a cell with a 0.1 cm path length. The peptide concentration
was 100 mM in 10 mM HEPES, NaCl 0.1 M at pH 7.4. All
spectra were obtained from 260 to 180 nm at intervals of 1 nm,
a band width of 2 nm. The contribution of the buffer was always
subtracted and three spectra were averaged for each sample. CD
spectra are reported in molar ellipticity per a-amino acid residue.
26 S. J. A. Pope, B. J. Coe, S. Faulkner, E. V. Bichenkova, X. Yu and K. T.
Douglas, J. Am. Chem. Soc., 2004, 126, 9490.
27 W. S. Perry, S. J. A. Pope, C. Allain, B. J. Coe, A. M. Kenwright and S.
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Acknowledgements
We particularly like to thank Science Foundation Ireland
(SFI) for RFP 2008, RFP 2009 funding and SFI (grant
06/RFP/CHO024/EC07) for the acquisition of the peptide
synthetizer, and PRTLI for Cycle 4 funding. We would like to
thank particularly Dr John O’Brien (TCD), Dr Dilip Rai (CSCB,
UCD), and Ce´line Petit and Ste´phane Desgranges (CSCB, RCSI)
for their help.
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