Lanthanide-binding peptides with two pendant aminodiacetate arms: Impact of the sequence on chelation

Article abstract of DOI:10.1039/c2dt11686c

Lanthanide complexes with a series of hexapeptides - incorporating two unnatural chelating amino acids with aminodiacetate groups, Ada₁ and Ada₂ - have been examined in terms of their speciation, structure, stability and luminescence properties. Whereas Ada₂ acts as a tridentate donor in all cases, Ada₁ may act as a tetradentate donor thanks to the coordination of the amide carbonyl function assisted by the formation of a six-membered chelate ring. The position of the Ada₁ residue in the sequence is demonstrated to be critical for the lanthanide complex speciation and structure. Ada₁ promotes the coordination of the backbone amide function to afford a highly dehydrated Ln complex and an S-shape structure of the peptide backbone, only when found in position 2. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2dt11686c

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PAPER
Lanthanide-binding peptides with two pendant aminodiacetate arms: Impact
of the sequence on chelation†
Agnieszka Niedźwiecka,a Federico Cisnetti,a,b Colette Lebrun,a Christelle Gateaua and Pascale Delangle*a
Received 6th September 2011, Accepted 21st December 2011
DOI: 10.1039/c2dt11686c
Lanthanide complexes with a series of hexapeptides—incorporating two unnatural chelating amino acids
with aminodiacetate groups, Ada1 and Ada2—have been examined in terms of their speciation, structure,
stability and luminescence properties. Whereas Ada2 acts as a tridentate donor in all cases, Ada1 may act
as a tetradentate donor thanks to the coordination of the amide carbonyl function assisted by the
formation of a six-membered chelate ring. The position of the Ada1 residue in the sequence is
demonstrated to be critical for the lanthanide complex speciation and structure. Ada1 promotes the
coordination of the backbone amide function to afford a highly dehydrated Ln complex and an S-shape
structure of the peptide backbone, only when found in position 2.
carboxylates (Asp, Glu), phenolates (Tyr), or amides (Asn, Gln,
Introduction
backbone peptide linkage) which have low affinity for Ln3+ in
Metal-binding peptides or pseudopeptides are interesting candi-
dates as models of biological metal transporters,1 efficient toxic
metal chelators2 or to design metal-based probes.3,4 In particular,
the application of the unique magnetic and spectroscopic proper-
ties of trivalent lanthanide ions (Ln3+)5 to magnetic resonance
imaging6 and optical imaging of cells7 is of increasing interest.
Therefore, Ln-peptide complexes have been investigated by
several research groups to benefit both from Ln-based spectro-
scopic properties and the biological peptide scaffold. Ln-com-
plexing peptides or proteins have been elaborated de novo4 or
starting from naturally occurring Ca2+ binding loops as Ca2+ and
Ln3+ have similar ionic radii.8,9 Lanthanide Binding Tags (LBT)
developed by B. Imperiali’s group are short peptides optimized
for tight Ln3+ binding and water exclusion from the coordination
sphere in order to obtain efficient sensitized terbium lumines-
cence.10,11 These LBTs have been coupled to proteins to investi-
gate their structure, function and dynamics by X-ray
crystallography, paramagnetic induced NMR data, or Ln-lumi-
nescence-based techniques.3 However, the above-mentioned
examples show that there is an intrinsic limitation of the stability
of lanthanide-peptide complexes if only natural amino acids are
used. Indeed, the latter bear only simple binding groups like
comparison to synthetic multidentate ligands of the poly(amino-
carboxylate) family.
Peptides containing unnatural chelating amino acids were
recently designed in our group to obtain Ln-binding peptides of
enhanced stability with a ligating site embedded in the peptide
framework.12,13 Two synthetic amino acids, which bear tridentate
aminodiacetate chelating groups (Adan) were introduced in short
peptide sequences to obtain P11, P22 and P33 (Scheme 1). These
compounds may be viewed as models of polyaminocarboxylate
ligands, namely EDTA or TMDTA, with a peptide spacer instead
of an alkyl chain. In such systems, the peptide backbone is used
as a non-innocent spacer between the coordinating groups and a
central Pro-Gly unit was chosen to favour the formation of a
β-turn containing structure.14
We have shown that the length of the alkyl chain between the
main peptide backbone and the aminodiacetate moiety (1, 2 or
3 methylene units) has a marked effect on both the stability and
structure of the complexes. The most flexible peptide P33 forms
low stability complexes in which the two aminodiacetate moi-
eties tend to act as independent chelating groups. Interestingly,
the peptide P22 provides exclusively a stable mononuclear Ln
complex (β110 = 1012.1) with a triply hydrated Tb3+ ion.12 The
use of shorter alkyl chains in P11 seemed very promising to us as
they favour the coordination of a backbone carbonyl due to the
formation of a six-membered chelate ring and afford higher
aINAC, Service de Chimie Inorganique et Biologique (UMR_E 3 CEA
UJF), Commissariat à l’Energie Atomique, 17 Rue des Martyrs, 38054
Grenoble Cedex, France. E-mail: pascale.delangle@cea.fr; Fax: +33 4
3878 5090; Tel: +33 4 3878 9822
bPresent address: Clermont Université, Université Blaise Pascal,
Laboratoire SEESIB, UMR CNRS 6504, 24 Avenue des Landais, 63177
Aubière Cedex, France
†Electronic supplementary information (ESI) available: pH-metric titra-
tions, isotope patterns of polymetallic complexes, fluorescence of Trp,
fluorescence titrations with EuCl3, determination of luminescence life-
times, competitive titration with TbCl3, NMR tables. See DOI: 10.1039/
c2dt11686c
Scheme 1 Design of lanthanide-binding peptides incorporating Adan
residues.
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denticity. This induces a lower hydration state of the Tb3+ ion in
the complex.13 These differences in the Ln3+ ion coordination by
P11 and P22 modify profoundly the structures of the peptides as
revealed by the solution NMR structures of the two complexes
LaP22 and LuP11. In LaP22, the Ada2PGAda2 motif forms a type
II β-turn and the peptide backbone adopts a U-shape structure
with the La3+ ion located above the peptide backbone plane. On
the contrary, LuP11 revealed an extended S-shape structure due
to the extra coordination of the amide carbonyl of Ada1(2),
which prevents the formation of the β-turn and drives the Ln3+
ion in the proline plane and closer to the Trp indole sensitizer
(Fig. 1).
Scheme 2 Synthesis of Fmoc-Ada1(tBu)2-OH.
In this article, we report the synthesis and Ln complexation
properties of two novel hexapeptides P12 and P21 which contain
one Ada1 and one Ada2 non natural chelating amino acids.
These two peptides were expected to exhibit intermediate proper-
ties between P22 and P11. We demonstrate that the position of the
Ada1 residue in the sequence is critical for the Ln complex spe-
ciation and structure. The spectroscopic properties of the Ln
complexes with the series of Pnn′ peptides (n,n′ = 1,2) demon-
strate that P12 forms a mononuclear complex similar to LnP11
without the drawback of polymetallic complexes formation. On
the contrary, P21 behaves like P22 with Ada1 inducing a more
complicated speciation. The position of the chelating amino acid
Ada1 in the peptide sequence is therefore critical to promote the
coordination of the backbone carbonyl which leads to dehy-
drated Ln complexes.
protection was removed in a strong acid medium and the result-
ing amine was alkylated with BrCH2CO2tBu at 0 °C to afford
compound 3 in 43% yield over two steps. Conducting this reac-
tion at higher temperature resulted in lower yields due to Fmoc
deprotection. The final Fmoc-Ada1(tBu)2-OH was recovered
after hydrogenolytic debenzylation. The coupling of Fmoc-
Ada1(tBu)2-OH with a chiral amine (α-methylbenzylamine)
either in an enantiopure or a racemic form allowed us to prove
the enantiomeric purity of the latter compound. Indeed, the
1
coupling products were examined by H NMR and splitting of
some signals (especially NH doublets) occurred only in the
coupling product with the racemic amine, indicating that the
Fmoc-Ada1(tBu)2-OH synthon was enantiopure.
Peptides P11, P12 and P21 (Scheme 1) were then manually
assembled by solid phase peptide synthesis (SSPS) starting from
Fmoc-Adan(tBu)2-OH and natural commercial amino acid
derivatives, on Rink amide MBHA resin using standard con-
ditions as performed previously for P22.12 No significant by-pro-
ducts were detected, showing that the unnatural amino acid
Fmoc-Ada1(tBu)2-OH displayed normal reactivity in the on-resin
synthetic steps as the two longer chains analogues Ada2 and
Ada3. Before resin cleavage, N-terminal acetylation was per-
formed. As the choice of the resin ensured the isolation of pep-
tides as C-terminal amides, peptides were recovered after
simultaneous resin cleavage and side-chain deprotection with
both extremities protected. The pure peptides were obtained in a
50 mg scale after preparative HPLC purification. The identity
and purity of the peptides were confirmed by electrospray ioniz-
ation mass spectrometry and standard combination of 1D and 2D
1H-NMR techniques.17 ROESY spectra for both peptides
(500 MHz, 298 K) showed no though-space correlations other
that sequential ones. This, along with little dispersion of the NH-
amide and glycine Hα resonances is indicative of flexible confor-
mations in solution as expected for linear oligopeptides (Tables
S1 to S3†).
Results and discussion
1. Synthesis of Ada1 containing peptides P11, P12 and P21
The synthesis of small peptides incorporating unnatural amino
acids (Adan) bearing aminodiacetate groups was performed by
solid phase peptide synthesis (SPPS) using standard Fmoc/tBu
strategy.
The synthon Fmoc-Ada2(tBu)2-OH, which is directly used in
the SSPS, was previously prepared in good yield starting from
commercially available Nα-Fmoc protected amino acid deriva-
tives.12 The synthesis of the protected Nα-Fmoc-Ada1(tBu)2-OH
amino acid is described in Scheme 2. The latter compound was
prepared following a method adapted from Kazmierski15 as per-
formed previously for the synthesis of Fmoc-Ada3(tBu)2-OH.12
Commercial Nα-Fmoc and Nβ-Boc protected L-diaminopropionic
acid, Fmoc-Dpr(Boc)-OH, 1, was benzylated using a published
procedure16 to obtain compound 2 in 82% yield. Then, the Boc
2. Peptide protonation
The protonation states of Adan amino acids in the sequences
affect the structure, the solubility and the metal-binding proper-
ties of the peptide scaffolds. Therefore, the acid/base properties
of P11 and P21 were investigated with pH-metric titrations.
Measured pKas are reported in Table 1 (see Fig. S1 for titration
curves†). Four pKas were determined for each peptide: two pKas
(> 6) correspond to the ammonium functions and two pKas (< 4)
Fig. 1 NMR solution structures (lowest energy) of LuP11 and LaP22
,
evidencing the S-shape and U-shape of the backbones, respectively.12,13
3240 | Dalton Trans., 2012, 41, 3239–3247
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Table 1 Equilibrium constants of Pnn′ peptides and LnPnn′
complexesa
Equilibrium constants in
water solution of L
P11
P12
P21
P22b
pKa1
pKa2
pKa3
7.2(1)
6.4(1)
3.0(1)
∼2
8.4(1)
6.2(1)
2.8(1)
∼2
9.0(5)
10.5(5)
8.8(1)
8.2(1)
2.8(1)
∼2
9.1(5)
12.1(5)
pKa4
logβp11H0= 7 (TbL)
logβ110 (TbL)
10.3(5)c
10.8(5)
9.5(5)
11.0(5)d
a The experiments were performed at ionic strength I = 0.1 M (KCl) at
pH = 7
25 °C. logβ
are the conditional stability constants at pH = 7.0 and
110
logβ110 are the global stability constants; b From reference 12. c From
reference 13. d Calculated, assuming that the pKa values for P12 and P21
are the same.
are assigned to carboxylic functions. Not all carboxylic acids
(pKa5 and pKa6 are < 2) could be detected due to sample
dilution. The values of pKa1 and pKa2 are essential for the calcu-
lation of the stability constants from the measured conditional
values at pH = 7.0, as described later in this paper.
Fig. 2 Titrations of P12 (left) and P21 (right) (20 μM) with TbCl3 in
HEPES buffer (10 mM, pH 7). Evolution of tryptophan fluorescence
and Tb-centred luminescence upon excitation at 280 nm (inserts: evol-
ution of the luminescence intensity at λmax).
The first pKa of P11 corresponds to the deprotonation of Ada1
ammonium function. Therefore its value is lower than the value
obtained with P22,12 due to the larger withdrawing effect of the
peptide backbone amide functions through the short side-chains
of Ada1 in P11. As expected, the first pKa of P21 is in the same
range as for P22 because both correspond to the deprotonation of
the ammonium group of an Ada2 residue. In summary, the three
peptide basicities follow the order: P22 > P21 > P11. Moreover, it
is expected that P12 and P21 display a similar behaviour towards
protonation as these two peptides do not show specific secondary
structures that could affect the pKa values.
monometallic europium complex (EuP12). On the contrary, P21
forms significant amounts of the polymetallic species Eu2P21
and Eu3P212 in excess of metal ion. The isotope patterns of these
complexes match the theoretical isotope patterns as seen in
Fig. S2.† Moreover, signals of the free peptide with 1 equiv. or
excess of EuCl3 are detected with P21 and not with P12. The
ES-MS experiments point to a rather complicated speciation
with the peptide P21 whereas P12 forms only the mononuclear
europium complex. These behaviours were corroborated by cir-
cular dichroism measurements.
As described before, CD titrations of the peptide ligands with
lanthanide ions are very sensitive to metal complexation. In par-
ticular, polymetallic complexes formed in excess of metal ion
3. Formation and stability of LnPnn′ complexes
A tryptophan residue was inserted in the peptide sequence to
enable the energy transfer from the indole moiety (λexc,max
Trp
=
280 nm) to the Tb3+/Eu3+ ion coordinated in a close proximity.
Thanks to the tryptophan sensitizer, lanthanide luminescence can
be easily detected. The LnPnn′ complexes exhibit interesting
spectroscopic characteristics, such as long-lived luminescence
lifetimes, large Stokes shifts and sharp emission bands in the
visible range. The titration of peptide solutions in HEPES buffer
(10 mM, 0.1 M KCl, pH = 7.0) with Tb3+ show linear evolutions
Trp
of Trp fluorescence (λ
= 350 nm) and gradual growths
em,max
of D4→7FJ bands (J = 3–6) of Tb luminescence, from 0 to 1
equiv. of metal added. These behaviours are consistent with the
formation of a unique TbPnn′ complex, in excess of peptide
(Fig. 2). A plateau is obtained after one Tb3+ equiv. with P22 and
P12. However the signals still slightly evolve in the presence of
excess Tb3+with P11 and P21, due to the formation of other metal
complexes.
5
Electrospray mass spectrometry (ES-MS) and circular dichro-
ism (CD) confirmed these observations. Samples analyzed by
ES-MS contained one Pnn′ peptide and europium chloride in
different proportions, in AcONH4 buffer (20 mM, pH = 7.0).
The spectra recorded in positive ionization mode are presented
in Fig. 3. P12 complexation results exclusively in the
Fig. 3 ES+-MS spectra of solutions containing P12 or P21 peptide
(60 μM) and EuCl3 (1 and 2 equiv.) in AcONH4 buffer (20 mM, pH =
7.0). The less intense peaks of sodium and potassium adducts were
removed for simplification. The signals at m/z = 587.2 and 733.3 corre-
spond to the polymetallic complexes Eu2P21 and Eu3P212, respectively.
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have significantly different CD signatures in comparison to
monometallic complexes. Indeed the formation of the former
leads to more elongated peptide structures in comparison with
the mononuclear complexes with the two Adan coordinated to
the same metal ion. Using such CD titrations, we previously
demonstrated that P11 forms higher nuclearity complexes after
one Ln3+ equiv.,13 while P22 gave only a mononuclear complex,
which does not transform in excess of metal ion.12 The titrations
of the peptides with Tb3+ show isodichroic points from 0 to 1
equiv. of added metal (P11: 220 nm,13 P22: 208 nm,12 P12:
227 nm and P21: 220 nm), which proves the formation of a
unique complex (Fig. 4). After 1 Tb3+ equiv., the two com-
pounds show different behaviours. CD spectra recorded with P12
do not evolve, which confirms the absence of polymetallic com-
plexes. On the contrary, higher nuclearity complexes with a
different CD signature (in red Fig. 4 bottom) are evidenced with
P21.
constants logβ110 were calculated from the conditional constants
at pH = 7.0 and the pKas of the peptides. It appears that P22
forms the most stable Tb complex with a logβ110 = 12.1. The
three other complexes have logβ110 values ranging from 10.5 to
11.0. This shows that the additional coordination of Ada1(2) car-
bonyl does not enhance the stabilities of TbP11 and TbP12.
Nevertheless, the latter complexes demonstrate higher stabilities
at neutral pH, due to their lower overall basicity.
4. Photophysical properties
Tryptophan fluorescence. The evolution of the tryptophan flu-
orescence shows interesting features that point to different beha-
viours of P11 and P12 in comparison to P22 and P21. Several
effects determine Trp fluorescence evolution upon Ln3+ binding:
indole environment modification, energy transfer to the Ln3+ ion
and in some cases photoinduced electron transfer. The compari-
son of Trp fluorescence of P12 with different Ln ions is shown as
an example in Fig. S5.†
Overall, ES-MS, CD and luminescence titrations highlight the
formation of polymetallic complexes with P11 and P21, whereas
P22 and P12 induce a simple speciation with the formation of a
single mononuclear LnP complex.
With Eu3+, quenching of the Trp fluorescence is observed for
the four peptides as a result of photoinduced electron transfer
(PET).18 Indeed this quenching mechanism has been demon-
strated to highly contribute with Eu3+ and Yb3+, the two most
readily reduced Ln3+ ions.19 For that reason, the four peptides
show a 34% (P22) to 81% (P12) decrease of Trp luminescence
upon Eu3+ addition, in comparison with the free peptide (Fig. S5
and S6†).
The indole environment modification upon complexation can
be estimated by recording the fluorescence spectra of the free
peptide and its La complex, since La3+ has no accessible energy
levels for energy transfer and is not susceptible to PET. The
spectra recorded with Tb3+ give exactly the same results as with
La3+, which demonstrates that the energy transfer from Trp to
Tb3+ is undetectable, even though the latter ion is significantly
sensitized upon binding to the peptides as seen in the time-
resolved Tb3+ luminescence spectra shown in Fig. 2. Therefore,
the evolution of Trp fluorescence during the titration with Tb3+
reflects only changes in the environment of the indole moiety
upon Tb3+ complexation rather than a Trp → Tb3+ energy trans-
fer.12,20 The complexation of Tb3+ or La3+ by P11 and P12
induces a decrease of the Trp fluorescence of 40% and 52%,
respectively. On the contrary, for P22 and P21 the Trp fluor-
escence increases of 40% and 26%, respectively. These data indi-
cate that the indole environment, which is closely related to the
peptide conformation, is similar for P11 and P12 Tb complexes
on the one hand and for P22 and P21 on the other hand.
The conditional stability constants of TbPnn′ complexes at pH
= 7.0, βp11H7, were determined by competition experiments
between two peptides, which Tb complexes have significantly
different time-resolved luminescence intensities. Such a compe-
tition, performed with peptide P22 as a reference of known
affinity for Tb allowed us previously to obtain the affinity con-
stant of TbP11.13 Similar experiments were run to obtain TbP12
and TbP21 stabilities—values are reported in Table 1. Typical
experiments are presented in Fig. S3 and S4.† The values of the
conditional stability constants determined at a specific pH are
dependent on the basicity of the ligands. Therefore, to compare
the stabilities of the four TbPnn′ complexes, the stability
Luminescence lifetimes and hydration states. The lumines-
cence lifetimes of Tb and Eu in the LnPnn′ complexes in H2O
and D2O solutions were measured in order to obtain the
hydration number q of these complexed ions with the empirical
equations by Parker et al.21 To avoid underestimation of lumines-
cence lifetimes in D2O because peptides are accompanied by
H2O hydration molecules, τD2O was determined as the extrapo-
lated limit of the luminescent decay rates in solutions of increas-
ing D2O molar fractions tending to an H2O-free solution
(Fig. S7†). The experimental values of τH2O and τD2O with the
calculated hydration numbers are reported in Table 2. For LnP21
and LnP22, hydration numbers close to 3 are in agreement with
Fig. 4 Circular dichroism titration of 20 μM water solutions of P12
(top) and P21 (bottom) with Tb(OTf)3 at pH = 7.0. Titration with 0.33
Tb equiv. until 1 equiv. (black curves) and 0.5 equiv. until 2 equiv (red
curves).
3242 | Dalton Trans., 2012, 41, 3239–3247
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Table 2 Luminescence lifetimes and calculated hydration numbers for
TbPnn′ and EuPnn′ complexes
for water). The quantum yield of the lanthanide luminescence
step can then be calculated ΦEu = 0.09. This figure is lower than
values obtained with totally dehydrated complexes (q = 0)26 due
to the O–H vibrations of the coordinated water molecule in
EuP12, which efficiently quench the Eu luminescence. However,
monohydrated Eu complexes with macrocyclic DOTA deriva-
tives bearing an acetophenone sensitizing group, give similar
values of ΦEu ≈ 0.08–0.09,24 in agreement with a rather efficient
metal-centred emission in EuP12. Finally, the sensitization
Complex
τH2O/ms
τD2O/ms
q
TbP11a
TbP12
TbP21
TbP22b
2.00(2)
2.36(2)
1.09(1)
1.11(1)
3.44(3)
3.13(3)
3.51(3)
3.71(3)
0.7(1)
0.2(1)
2.9(1)
2.9(1)
EuP11
EuP12
EuP21
EuP22
0.51(1)
0.60(1)
0.31(1)
0.30(1)
1.34(1)
1.54(1)
1.98(2)
2.13(2)
1.2(2)c
0.9(2)c
3.0(5)
3.1(5)
efficiency is evaluated according to eqn (1): ηsens = 0.017. The
Eu
tot
low quantum yield determined experimentally (Φ = 1.5 ×
10−3) is therefore mainly assigned to an inefficient sensitization
process, which is expected with Trp as a Eu-sensitizer due to
competitive quenching of the S1 Trp excited state by photoin-
duced electron transfer as explained above.
a Values from reference 13. b Values from reference 12. c Calculated
without taking into account the quenching effect of individual amide
N–H oscillators. Considering the quenching effect of one amide N–H
oscillator (0.075 ms−1 contribution) gives q = 1.1 (EuP11) and 0.8
(EuP12).
The energy transfer efficiency is highly dependent on the
donor–acceptor distance with a 1/r6 relationship. The distance
for 50% efficiency of intramolecular energy transfer by a dipole–
dipole mechanism – also called the Förster distance – lies close
to 3.5 Å for Tb3+ sensitization by Trp.20,27 Thus, the energy
transfer dramatically decreases when the donor–acceptor distance
becomes greater than 3.5 Å. This steep dependence of the
energy transfer on the donor–acceptor distance was indeed
demonstrated experimentally with a series of octadentate ligands
bearing a phenanthridinium chromophore at various distances
from the emitting Ln3+ ion.28 The distances between the Trp
indole sensitizer and the accepting Ln3+ ion are 5.7 Å in LnP11
and 8.7 Å in LaP22 according to the NMR structures of these
complexes.12,13 They may be compared to the 7 Å distance
measured in the X-ray structure of the Tb3+-LBT developed by
B. Imperiali.11 Since the aromatic indole group is not directly
coordinated to the metal, these distances are significantly longer
than the Förster distance and therefore the energy transfer is
clearly the limiting parameter in the quantum yields of Tb3+
complexes. Even though the energy transfer from Trp to Tb3+ is
not very efficient, the latter ion is significantly sensitized upon
binding to the peptides as previously described in other Ln-
binding peptides or proteins.10,11,20,29
both peptides behaving as hexadentate ligands like [Ln(EDTA)
(H2O)q]− complexes.21,22 On the contrary, longer luminescence
lifetimes in H2O are obtained for LnP12 and LnP11, which
exhibit much lower hydration numbers, inferior or close to
1. This can be related to the coordination of an extra donor
group, as previously evidenced in the NMR solution structure of
LuP11, which showed that the backbone carbonyl of Ada1(2)
was coordinated to the Lu3+ ion. These data demonstrate that the
carbonyl group of the Ada1 residue is able to coordinate the
Ln3+ ion only if placed in position 2 in the hexapeptide
sequence.
Quantum yields. The quantum yields of LnPnn′ (Ln = Eu, Tb
and n,n′ = 1,2) were measured following the procedure of Bünzli
et al.23 In our case, the convenience of this protocol is the use of
Eu and Tb tris(dipicolinates) as secondary standards, which exci-
tation band (λexc,max (Eu(dpa)3/Tb(dpa)3) = 279 nm) perfectly
overlap with the excitation band of tryptophan (λexc,max (Trp) =
Tb
Eu
280 nm). The values Φ = 2–6 × 10−3 and Φ = 1–2 × 10−3
tot
tot
are rather small as demonstrated in the literature for Eu3+ or Tb3+
sensitization by tryptophan.9,20
With Eu, the two important contributions to the overall
Eu
tot
Europium lifetimes and CH deactivation. It has been shown
that CH oscillators close to the metal ion can quench europium
luminescence due to high-energy vibrational CH modes of the
ligand itself.30 A contribution of +45 s−1 has been evaluated for
the luminescence quenching due to a proton located 3.75 Å
away from the Eu centre.21 This effect could explain the signifi-
cant differences observed in the europium lifetimes measured in
D2O in the four complexes. Indeed EuP21 and EuP22 have quite
large τD2O values around 2 ms, whereas EuP12 and EuP11
display significantly lower τD2O values 1.34–1.54 ms. This indi-
cates that a quenching mechanism is present in the two latter
complexes due to non exchangeable CH protons of the ligand.
The examination of the solution structures previously determined
for LaP22 and LuP11 shows that the S-shape of the backbone in
LuP11 due to the coordination of one amide carbonyl (Ada1(2))
locates the Hα proton of Ada1(5) very close to the Ln3+ ion (ca
2.8 Å). On the contrary, the U-shape structure of LaP22 moves
the two Hα of the Ada2 residues far away from the cation (ca
6 Å). The environments of the Ln3+ ion in the two complexes
are very similar except the Hα proton of Ada1(5), therefore the
quantum yield Φ , i.e. the metal-centred visible light emission,
ΦEu, and the sensitization efficiency, ηsens, can be evaluated (eqn
(1)).24–26
Eu
tot
Φ
¼ ηsens ꢀ ΦEu
ð1Þ
Indeed, the quantum yield ΦEu of the lanthanide luminescence
step can be estimated from the observed metal ion lifetime in the
complex and the radiative lifetime τR of the Eu(5D0) level, given
by eqn (2).24–26
ꢀ
ꢁ
1
Itot
¼ AMD;0n3
ð2Þ
τR
IMD
Where n is the refractive index of the medium, AMD,0 (= 14.65
s−1) is the spontaneous emission probability for the 5D0→7F1
transition and Itot/IMD is the ratio of the total area of the corrected
Eu3+ emission spectrum to the area of the 5D0→7F1 band.
This method was applied to EuP12 which shows the most
intense metal-centred emission spectrum. We experimentally
measured Itot/IMD = 4.18 and calculated τR = 6.9 ms (n = 1.334
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difference in the CH contribution to Eu luminescence quenching
between the two complexes may be tentatively assigned to the
close proximity of Hα(Ada1(5)) and the metal centre in EuP11.
Considering that the energy transfer is inversely proportional to
the distance between the two centres in a 1/r6 relationship like in
the general energy transfer formula developed by Förster,31 this
difference is expected to be 45×(3.75/2.8)6 ≈ 260 s−1. This
value is in very good accordance with the experimental differ-
ence calculated between the two complexes, i.e. τD2O−1(EuP11)
− τD2O−1(EuP22) = 277 s−1. Therefore, the shortening of the
τD2O value observed for EuP11 and EuP12 with respect to EuP22
and EuP21 can reasonably be assigned to the presence of the
proton Hα Adan(5) at a short distance to the metal centre, which
deactivates Eu luminescence. The smaller CH deactivation evi-
the amide carbonyl function assisted by the formation of a six-
membered chelate ring. We demonstrated previously that P11
behaves as a Ln heptadentate ligand, with the carbonyl function
of Ada1(2) coordinated to the metal ion. This induces a dehy-
dration of the Ln ion, which has better luminescence properties
as well as an S-shape conformation of the peptide backbone pre-
venting the formation of a β-turn. Moreover, as Ada1 may act as
a tetradentate donor, P11 gives binuclear complexes in excess of
metal, in which the two Ada1 moieties act independently.
In this paper, we investigated two novel hexapeptides P12 and
P21, which incorporate one Ada1 and one Ada2 in positions 2 and
5—i.e. on either side of the PG spacer—to rationalize the effect
of Ada1 position in the peptide sequence on the complexation
properties. We demonstrated that Ada1 acts as a tetradentate donor
only in P12—i.e. if placed in position 2 in the sequence. Indeed,
P21 does not promote the coordination of the Ada1 carbonyl
group and forms a mononuclear complex with a triply hydrated
Ln3+ ion, which transforms into polymetallic adducts in excess of
metal. The examination of the luminescence properties of the
series of Tb and Eu complexes indicates that LnP21 and LnP22
have similar structures, assigned to a U-shape complex as deter-
mined previously by solution NMR for LaP22. On the contrary,
P12 gives a well-defined speciation with the formation of a unique
highly dehydrated mononuclear complex (qTb = 0.2), which does
not evolve into polymetallic species in excess of metal. So, when
Ada1 is introduced in position 2 in the peptide sequence (P12 and
P11) its carbonyl amide function is involved in the coordination
of the Ln3+ ion instead of forming a H-bond in a β-turn like for
Ada2, which induces an S-shape structure like in LuP11. These
results highlight the determination of the global shape of the
complex through a single-site backbone complexation.
denced with EuP12 (τD2O = 649 s−1) with respect to EuP11
−1
−1
(τD2O = 746 s−1) may be attributed to the higher compacity of
the latter complex, which incorporates two unnatural amino acids
Ada1 with very short chelating side chains. This effect together
with the Ln hydration numbers is in accordance with similar U-
shape structures for LnP22 and LnP21 complexes with triply
hydrated Ln3+ and similar S-shape structures for LnP11 and
LnP12 with dehydrated Ln3+ (q = 0–1) due to coordination of the
backbone carbonyl in position 2.
Structural interpretation. The photophysical properties of
LnP12 and LnP21 compared with LnP11 and LnP22 allow us to
predict the solution structures of these complexes, although we
could not obtain satisfactory data for NMR structure compu-
1
tation. Indeed, the H NMR spectra of LuP12 and LaP21 (Tables
S4–5†) are indicative of only one complex in solution, but 2D
NOESY or ROESY spectra did not give enough through space
correlations to calculate the structures of the latter species.
However, the photophysical properties of LnP21 and LnP22 com-
plexes point to similar structures with a triply hydrated Ln3+ ion
and a U-shape conformation of the peptide backbone like in the
NMR structure of LaP22.12 Indeed, both Ln complexes show a
metal hydration state of 3 and an increase of Trp fluorescence
upon Tb binding, which indicate a similar Trp environment in
the structures. On the contrary, LnP12 and LnP11 complexes
exhibit dehydrated Ln3+ ions, a decrease of Trp fluorescence
upon Tb binding and short Eu luminescence lifetimes in D2O,
indicative of deactivation by CH groups of the ligand. All these
characteristics allow us to conclude to the coordination of the
carbonyl group of Ada1(2) inducing a S-shape conformation of
the peptide backbone, as evidenced previously in the NMR
structure of LuP11.13
The stability constants logβ110 ∼ 11 (TbP11 and TbP12) have
smaller values than logβ110 measured for P22, which indicates
that the coordination of an extra donor—i.e. the carbonyl group
of Ada1(2)—does not stabilize the mononuclear Tb3+ complex.
This may be explained by the metal dehydration contributions
associated to the coordination of a neutral amide donor group32
and also by the absence of the H-bond which is present in the
β-turn of TbP22. Interestingly at physiological pH, TbP11 and
TbP12 complexes display higher stabilities than TbP22 due to
their lower overall basicity thanks to the electron-withdrawing
effect of the peptide chain.
In summary, P12 is very promising as it shows an intermediate
behaviour between the two hexapeptides P22 and P11: a simple
speciation with the formation of a well-defined mononuclear
complex having the luminescence and structural properties of
LnP11. Its insertion in a more sophisticated framework for the
molecular recognition of biomolecules is now considered.
Conclusions
A series of peptides incorporating two unnatural aminoacids
with aminodiacetate chelating groups Adan (n = 1,2) was studied
to rationalize the impact of the sequence on the properties of Ln
complexes, i.e. metal hydration state, coordination numbers and
peptide structure in the complex. The analysis of the Ln com-
plexes reveals that the side-chain lengths of the Adan moieties
and their positions in the peptide sequence have dramatic effects
on the properties and structures of the complexes. Indeed
whereas Ada2 acts as a tridentate donor, Ada1 may act either as a
tri- or tetradentate donor thanks to the facultative coordination of
Experimental
General remarks
Reagents and solvents were purchased from Aldrich and Nova-
biochem and used without further purification unless specified.
Organic products were characterized by NMR using a Varian
Mercury 400 MHz spectrometer at 298 K. Analytical and pre-
parative peptide RP-HPLC were performed with LaChrom and
LaPrep systems (see below for details).
3244 | Dalton Trans., 2012, 41, 3239–3247
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Abbreviations. Ac2O (acetic anhydride), AcOEt (ethyl
acetate), AcONH4 (ammonium acetate), Ar (aromatic), Bn
(benzyl), Bu (butyl), Boc (tert-butyloxycarbonyl), CbzCl
(benzyl chloroformate), DMAP (4-dimethylaminopyridine),
DMF (N,N-dimethylformamide), DIEA (N,N-diisopropylethyla-
mine), Dpr (L-diaminopropionic acid), DTT (dithiothreitol);
Et2O (diethyl ether), EtOH (ethanol), Fmoc (9-fluorenyl-methy-
loxycarbonyl), PyBOP [(benzotriazole-1-yloxy)tris(pyrrolidino)
phosphonium hexafluorophosphate], tBu (tert-butyl), TFA
(trifluoroacetic acid), TIS (triisopropylsilane), TNBS (2,4,6-trini-
trobenzenesulfonic acid).
solvents in vacuo, the resulting product was purified by silica-gel
column chromatography (elution gradient: toluene to toluene/
ether, v/v = 9/1) to yield 3 (720 mg, 43% yield) as a colorless
oil. 1H NMR (500 MHz, CDCl3, 298 K): δ (ppm) = 7.80 (2H, d,
3
3J(H,H) = 7.3 Hz, Ar); 7.75 (2H, t, J(H,H) = 8.3 Hz, Ar); 7.43
3
(2H, t, J(H,H) = 7.3 Hz, Ar); 7.45–7.28 (6H, m, Ar); 7.21 (1H,
3
d, J(H,H) = 7.0 Hz, NH(Fmoc)); 5.25, 5.21 (2H, AB system,
JAB = 12.4 Hz, CH2(Bn)); 4.47 (1H, dd, 3J1(H,H) = 9.2 Hz,
3J2(H,H) = 6.4 Hz, CH2(Fmoc)a); 4.37 (1H, m, Hα); 4.31 (2H,
m, CH2 (Fmoc)b and CH(Fmoc); 3.48, 3.39 (4H, AB system,
JAB = 17.8 Hz, CH2COOtBu); 3.21 (2H, m, Hβ); 1.51 (9H, s,
tBu). 13C NMR (125 MHz, CDCl3, 298 K): δ (ppm) = 171.8
(COOBn)); 171.4 (COOtBu); 157.1 (CO(Fmoc)); 144.7, 144.4,
141.7 (2C) (Cq (Fmoc)); 135.8 (Cq(Bn)); 129.5, 129.2, 129.0
(CAr-H(Bn)); 128.0, 127.5, 125.9, 120.3 (CAr-H(Fmoc)); 82.2
(Cq(tBu)); 67.5 (2C) (CH2(Bn), (CH2(Fmoc)); 57.1 (CH2-
COOtBu); 55.5 (Cβ); 54.1 (Cα)); 47.6 (CH(Fmoc)); 28.6 (tBu).
ES+-MS (AcONH4, pH = 7.0): m/z = 667.3 [M+Na]+.
Synthesis of Fmoc-Ada1-(tBu)2-OH
Fmoc-Dpr(Boc)-OBn (2). A solution of compound 1 (1.500 g,
3.52 mmol, 1 eq) in CH2Cl2 (40 mL) was treated with DIEA
(920 μL, 682.4 mg, 5.28 mmol, 1.5 eq) and DMAP (43.0 mg,
0.352 mmol, 0.1 eq). The resulting mixture was cooled at 0 °C
and a solution of CbzCl (545 μL, 660.5 mg, 3.88 mmol, 1.1 eq)
in CH2Cl2 (10 mL) was added dropwise over 15 min. After
3.5 h stirring at 0 °C, TLC analysis (CH2Cl2/AcOEt v/v = 9/1)
indicated complete conversion. The mixture was washed twice
with 20 mL of 5% KHSO4 and dried over Na2SO4. After fil-
tration and removal of the solvents in vacuo, purification by
silica-gel column chromatography (CH2Cl2/AcOEt v/v = 9/1)
Fmoc-Ada1(tBu)2-OH. Compound 3 (700 mg, 1.11 mmol)
was dissolved in 50 mL of absolute ethanol and hydrogenated (3
bar, overnight) over 100 mg 10% Pd/C. Fmoc-Ada1(tBu)2-OH
was obtained as a hygroscopic glassy solid (568 mg, 94% yield)
after filtration of the reaction mixture through a Celite pad and
solvent evaporation. 1H NMR (500 MHz, CDCl3, 298 K): δ
3
3
(ppm) = 7.79 (2H, d, J(H,H) = 7.4 Hz, Ar); 7.63 (2H, d, J(H,
1
afforded 2 in 82% yield (1.494 g). H NMR (500 MHz, CDCl3,
3
H) = 7.4 Hz, Ar); 7.43 (2H, t, J(H,H) = 7.4 Hz, Ar); 7.34 (2H,
3
298 K): δ (ppm) = 7.79 (2H, d, J(H,H) = 7.5 Hz, Ar); 7.63
t, 3J(H,H) = 7.4 Hz, Ar); 6.06 (1H, d, 3J(H,H) = 4 Hz,
NH(Fmoc)); 4.40 (2H, m, CH2 (Fmoc)); 4.26–4.20 (2H, m, CH
(Fmoc) and Hα); 3.59, 3.51 (4H, AB system, JAB = 18.0 Hz,
CH2COOtBu); 3.48 (1H, m, Hβa); 2.79 (1H, m, Hβb); 1.51
(18H, s, tBu). 13C NMR (125 MHz, CDCl3, 298 K): δ (ppm) =
172.4 (COOH); 171.1(COOtBu); 156.5 (CO (Fmoc)); 144.2,
144.1, 141.7 (2C) (Cq (Fmoc)); 128.2, 127.5, 125.6, 120.4
(CAr-H (Fmoc)); 83.1(Cq (tBu)); 67.7 (CH2 (Fmoc)); 57.7
(CH2COOtBu); 57.4 (Cβ); 51.9 (Cα); 47.5 (CH (Fmoc)); 28.5
(tBu). ES+-MS (AcONH4, pH = 7.0): m/z = 577.3 [M+Na]+;
elemental analysis calcd (%) for C30H8N2O8.1.5 H2O: C 61.95,
H 7.10, N 4.82; found: C 61.64, H 6.87, N 4.87.
(2H, d, 3J(H,H) = 7.2 Hz, Ar); 7.45–7.32 (9H, m, Ar); 6.01 (1H,
3
d, J(H,H) = 6.3 Hz, NH(Fmoc)); 5.24, 5.21 (2H, AB system,
JAB = 12.4 Hz, CH2(Bn)); 4.81 (1H, m, NH(Boc)); 4.48 (1H, m,
Hα); 4.45–4.36 (2H, m, CH2(Fmoc)); 4.25 (1H, m, CH(Fmoc));
3.60 (2H, m, Hβ); 1.47 (9H, s, tBu). 13C NMR (125 MHz,
CDCl3, 298 K): δ (ppm) = 170.8 (COOBn); 156.5 (CO(Fmoc));
144.3, 144.2, 141.7 (2C) (Cq(Fmoc)); 135.6 (Cq(Bn)); 129.1,
129.0, 128.8, (CAr-H (Bn)); 128.1, 127.5, 125.6, 120.4 (CAr-H-
(Fmoc)); 80.5 (Cq (tBu)); 68.0 (CH2(Bn)); 67.6 (CH2(Fmoc));
55.6 (Cα); 47.6 (CH(Fmoc)); 42.6 (Cβ); 28.7 (tBu). ES+-MS
(AcONH4, pH = 7.0): m/z = 516.8 [M+H]+.
Fmoc-Ada1(tBu)2-OBn (3). A solution of compound
2
Control of Fmoc-Ada1(tBu)2-OH enantiopurity by 1H
NMR. A solution of Fmoc-Ada1(tBu)2-OH (30 mg, 54 μmol)
and α-methylbenzylamine (14 μL, 13.3 mg, 110 μmol, 2 eq,
either in enantiopure (S) or in racemic form) in 0.5 mL of
CH2Cl2 was treated with PyBOP (54 mg, 104 μmol, 2 eq),
DIEA (57 μL, 42 mg, 327 μmol, 6 eq) and stirred for 3 h. The
resulting mixture of products was separated by silica-gel column
chromatography (CH2Cl2/AcOEt v/v = 15/85) and each fraction
(1.383 g, 2.61 mmol) in TFA/CH2Cl2 (70 mL, v/v = 1/2) was
stirred for 2 h at room temperature. The progress of Boc depro-
tection was monitored by TLC analysis (CH2Cl2/AcOEt v/v = 9/
1). When the reaction was finished, solvents were removed in
vacuo and excess TFA was discarded by azeotropic evaporation
with H2O (3 × 20 mL), EtOH (2 × 20 mL) and CHCl3 (2 ×
20 mL). The ES-MS analysis of the resulted solid shows the
molecular peak belonging to Fmoc-Dpr-OBn (m/z = 417.2
[M+H]+). The solid was dissolved in DMF (15 mL) and tert-
butyl bromoacetate (848 μL, 1.120 g, 5.74 mmol, 2.2 eq),
KHCO3 (2.613 g, 26.1 mmol, 10 eq) and NBu4I (964 mg,
2.61 mmol, 1 eq) were added. The reaction mixture was stirred
for 4 h at 0 °C, and then an extra equivalent of tert-butyl
bromoacetate was added. After an additional 2 h at 0 °C, water
(60 mL) and diethyl ether (60 mL) were added to the solution.
The phases were separated and the product was further extracted
with diethyl ether (3 × 50 mL). The combined organic phases
were washed with saturated Na2S2O3 (50 mL), water (50 mL)
and dried over Na2SO4. After filtration and removal of the
1
was identified by 1D H NMR. In the spectra of the adducts, a
splitting of the NH amide resonances was detected only for the
coupling products with the racemic amine. This indicates that no
racemization of Fmoc-Ada1(tBu)2-OH occurred during the syn-
thesis (assuming that 2% diastereomeric product would be
detected by NMR, ee > 96%).
Peptide synthesis
The hexapeptides were assembled manually by solid-phase
peptide synthesis on Rink Amide MBHA resin (substitution
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Dalton Trans., 2012, 41, 3239–3247 | 3245

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0.59 mmol g−1, 150 mg) using Fmoc chemistry. The synthesis
was started by an initial deprotection of the commercially resin-
bound Fmoc with DMF/piperidine (v/v = 4/1). Couplings were
performed with Nα-Fmoc-protected amino acids (2 equiv.),
PyBOP (2 equiv.), and DIEA (6 equiv.) in DMF for 30 min. In
the case of Fmoc-Adan(tBu)2-OH the coupling reaction was
monitored by a TNBS test.33 For incomplete reactions, a second
coupling with Fmoc-Adan(tBu)2-OH (0.5 equiv.) PyBOP
(1 equiv.), and DIEA (4 equiv.) was performed. After each coup-
ling, the resin was treated with DMF/pyridine/Ac2O (v/v/v =
7/2/1) to acetylate unreacted amino groups (2 × 2 min). Fmoc
deprotection was achieved with DMF/piperidine (v/v = 4/1)
(3 × 3 min). The yield of each peptide coupling was monitored
by UV-Vis spectroscopy (ε300 = 7800 L mol−1 cm−1 for the
piperidine adduct of dibenzofulvene). After the final Fmoc
deprotection, the peptide was acetylated as described above. The
peptide was freed from the resin and the side-chain protections
were removed by treatment with a cleavage cocktail consisting of
200 mg DTT dissolved in 20 mL TFA/TIS/H2O (v/v/v = 92/4/4).
After 2.5 h of stirring, the solution was evaporated to yield a
yellow oil, which was triturated in Et2O until a white powder
was obtained. This solid was analysed C18 Reverse Phase
HPLC [Merck Purospher® STAR endcapped, 4.6 × 250 mm,
5 μm particles, solvent A = H2O/TFA (v/v = 99.925/0.075),
solvent B = CH3CN/H2O/TFA (v/v/v = 90/10/0.1), elution gradi-
ent: from vA/vB = 1/9 to vA/vB = 4/6 in 15 min, flow rate 1 mL
min−1, UV monitoring at 280 nm]. HPLC analysis indicated that
the solid consists essentially of one product. The solid residue
was dissolved in water/acetonitrile (v/v = 9/1) and easily purified
by C18 reversed-phase high-performance liquid chromatography
[RP-HPLC, Merck Purospher®, 250 × 40 mm, 10 μm C18 par-
ticles, solvent A = H2O/TFA (v/v = 99.925/0.075), solvent B =
CH3CN/H2O/TFA (v/v/v = 90/10/0.1), elution gradient: from
between the measurements. Experimental data were refined
using the computer program Hyperquad 2000.34 All equilibrium
constants were expressed as concentration rather than activity
ratios. The ionic product of water at 25 °C and 0.1 M ionic
strength was pKw = 13.78.35 The initial concentration of the
ligand was determined by UV-vis spectroscopy (ε280 = 5690 L
mol−1 cm−1 for tryptophan residues), and proton content was
adjusted to fit the observed values (since the peptides are present
in the lyophilised solid as a mixture of protonation states, the
proton content was reproducible for a given peptide). All values
and errors (one standard deviation) reported represent the
average of three independent experiments.
Preparation of aqueous solutions
The 3 mM and 30 mM aqueous solutions of lanthanides were
prepared by dissolving corresponding salts (TbCl3·6H2O,
Tb(OTf)3, EuCl3·6H2O, LuCl3·6H2O and LaCl3·7H2O) in deio-
nized water. The precise concentrations were determined by col-
orimetric titration with 5mM EDTA (Fisher Chemicals) in the
presence of xylenol orange indicator. Peptide solutions in
HEPES buffer (10 mM, 0.1 M KCl, pH = 7.0) were prepared
directly before the use. The precise concentrations were estab-
lished by UV absorption at 280 nm using the known extinction
coefficient of tryptophan (ε280 = 5690 L mol−1 cm−1). HEPES
buffer (10 mM, 0.1 M KCl, pH = 7.0) was prepared from solid
4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (Fluka) and
potassium chloride in deionized H2O (or D2O). The pH (or pD)
was adjusted to 7.0 with KOH (or NaOD).
Electrospray mass spectrometry of EuPnn′ complexes
The mass spectrometry measurements were performed with a
LXQ-type Thermo Scientific spectrometer equipped with an
electrospray ionization source and a linear trap detector. The
samples of EuPnn′ complexes were prepared by adding EuCl3
(0, 0.5, 1.0, 2.0 equiv.) to a peptide solution (60 μM) in
AcONH4 buffer (20 mM, pH = 7.0). Resulting mixtures where
injected into the spectrometer at a flow rate of 5 μL min−1. 2kV
voltage and 250 °C capillary temperature were applied.
10 vA/vB = 1/9 to vA/vB = 4/6 in 15 min, flow rate 75 mL min−1
]
to yield the desired peptide as a white powder.
P11: Ac-WAda1PGAda1G-NH2, Yield of the on-resin synthesis
(UV): 76%, Isolated mass: 44.5 mg (isolated yield assuming that
the solid is P11·2TFA: 45%). ES+ MS (AcONH4, pH 7): m/z =
861.2 [M+H]+, ES−-MS (AcONH4, pH = 7.0): m/z = 859.2 [M
− H]−; RP-HPLC: tR = 10.8 min, >95% purity (NMR).
P12: Ac-WAda1PGAda2G-NH2, Yield of the on-resin synthesis
(UV): 60%, Isolated mass: 40.5 mg (isolated yield assuming that
the solid is P12·2TFA: 36%). ES+-MS (AcONH4, pH 7): m/z =
875.3 [M+H]+, ES−-MS (AcONH4, pH = 7.0): m/z = 873.3 [M
− H]−; RP-HPLC: tR = 11.0 min, >95% purity (NMR).
Circular dichroism measurements
The CD spectra were collected on an Applied Photophysics Chir-
ascan spectrometer in a 1 cm path length quartz cell at 25 °C.
The 20 μM solutions of free peptides and Tb-peptide complexes
(0.5, 1, 1.5, 2 equiv. of Tb(OTf)3) were prepared in deionized
water and the pH was adjusted to 7.0 with KOH. Subsequently,
the CD measurements were performed in 280–200 nm range
with a 1 nm interval, 2 s time constant, 1 nm bandwidth and
three scans. The CD signal was reported in molar ellipticity per
P21: Ac-Wada2PGAda1G-NH2, Yield of the on-resin synthesis
(UV): 82%, Isolated mass: 54 mg (isolated yield assuming that
the solid is P21·2TFA: 48%). ES+-MS (AcONH4, pH 7.0): m/z =
875.3 [M+H]+, ES−-MS (AcONH4, pH = 7.0): m/z = 873.3 [M
− H]−; RP-HPLC: tR = 10.9 min, >95% purity (NMR).
α-amino acid residue ([θ] in units of deg cm2 mol−1; [θ] = θobs
/
(10lcn), with θobs the observed ellipticity in m°, l the optical path
pH-metric titration
length of the cell in cm, c the peptide concentration in mol L−1
and n the number of residues in the peptide (n = 6 in this case)).
,
Continuous potentiometric titrations with 0.1 M KOH were con-
ducted in 5 mL aqueous solutions of peptide (0.5 mM) with KCl
(0.1 M) as background electrolyte. The reverse titrations with 0.1
M HCl were performed after each experiment to check whether
equilibration had been achieved. In a typical experiment, 90
points (2 μL increment) were measured with a 2 min delay
Luminescence measurements
The luminescence measurements were performed on a LS50B
spectrofluorimeter connected to a computer equipped with
3246 | Dalton Trans., 2012, 41, 3239–3247
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J. Am. Chem. Soc., 2011, 133, 286; A. M. Pujol, C. Gateau, C. Lebrun
and P. Delangle, Chem.–Eur. J., 2011, 17, 4418.
FLWINLAB 2.0. The spectra were recorded in a 1 cm path
length quartz luminescence cell at 25 °C, by exciting the sample
at 280 nm and recording the emission with maximum at 350 nm
(tryptophan fluorescence), 545 nm (Tb-centred luminescence) or
618 nm (Eu-centred luminescence). For fluorescence measure-
ments, excitation slit: 3.0 nm and emission slit: 8.0 nm were
applied. The terbium and europium emission spectra were
recorded after 0.05 ms delay, with gate time/excitation slit/emis-
sion slit: 1 ms/10 nm/5 nm and 0.5 ms/10 nm/15 nm for Tb3+
and Eu3+, respectively. The 430 nm cut-off filter was used
during all tryptophan-sensitized luminescence measurements.
The competition titrations were performed with delays chosen
to maximize the differences in Tb emission intensities of the two
complexes (Fig. S3–S4†). The experimental data were fitted with
fixed values of the conditional stability constant of the reference
peptide and the ratio of the luminescence intensities measured in
the same conditions.
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The quantum yield measurements were performed according
to Chauvin et al.23 in a 1 cm path length luminescence cell at
25 °C on aqueous solutions of TbPnn′, EuPnn′(50 μM, A280
=
0.3), [Tb(dpa)3]3− (0.107 mM, A280 = 0.295) and [Eu(dpa)3]3−
(0.110 mM, A280 = 0.295) in HEPES buffer (10 mM, 0.1 M
KCl, pH = 7.0). The standard solutions of [Tb(dpa)3]3− and
[Eu(dpa)3]3− were prepared by mixing Tb3+/Eu3+ with dipicoli-
nic acid (dpa) in 1/3 equivalent ratio in HEPES buffer (10 mM,
0.1 M KCl, pH = 7.0) and stirring the resulting mixture during
5 min. The quantum yields Φ have been calculated using the
equation Φx/Φr = (Ex/Er) × (Ar(λr)/Ax(λx)), where x refers to the
sample and r to the reference; E is the integrated luminescence
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intensity and A is the absorbance of the solution at the excitation
Tb
dpa
wavelength λ. The values of quantum yields Φ = 18.4 2.5%
18 D. Parker, Coord. Chem. Rev., 2000, 205, 109.
Eu
dpa
(0.107 mM, A280 = 0.295) and Φ = 20.4 2.5% (0.110 mM,
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in the error range.23
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This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 3239–3247 | 3247