Lanthanide(III) complexes with two hexapeptides incorporating unnatural chelating amino acids: Secondary structure and stability

Article abstract of DOI:10.1002/chem.200900747

Unnatural metal-chelating amino acids bearing aminodiacetate side-chains have been introduced into two hexapeptides to obtain efficient lanthanide-binding peptides. The synthesis of the enantiopure Fmoc-Ada.,(tBu)₂-OH synthons is described with overall yields of 32 and 50% for n = 2 and n = 3 side-chain carbon atoms, respectively. The two peptides AcWAda_nPGAda_nGNH₂ (Pⁿ) were synthesized from the protected synthons by standard solid-phase peptide synthesis. Studies of the lanthanide complexes of the two peptides P" by luminescence titrations, mass spectrometry, circular dichroism, and solution NMR spectroscopy demonstrate that the Ada., chain length has a dramatic effect on the complexation properties. Indeed, the flexible compound P³ forms a mononuclear complex of moderate stability (β₁₁ = 10 ^9.9), which tends to transform into a binuclear species in the presence of excess of the metal ion. Interestingly, the more compact peptide P² provides stable Ln³⁺ complexes with the exclusive formation of the mononuclear LnP² adduct. The stability constant of TbP² is two orders of magnitude higher (β₁₁ = 10 ^12.1) than that measured for P³. The 800 MHz NMR spectrum of the La³⁺ complex of P² evidences a well-defined type II ss-turn as well as a hydrophobic Trp(indole)-Pro interaction. These interactions exemplify the non-innocent character of the peptide spacer in the complex LaP² as well as the role of a peptide secondary structure in the stabilization of metal complexes.

Full text of DOI:10.1002/chem.200900747

DOI: 10.1002/chem.200900747
LanthanideACHTUNGTRENNUNG(III) Complexes with Two Hexapeptides Incorporating Unnatural
Chelating Amino Acids: Secondary Structure and Stability
Federico Cisnetti, Christelle Gateau, Colette Lebrun, and Pascale Delangle*[a]
Abstract: Unnatural metal-chelating
amino acids bearing aminodiacetate
side-chains have been introduced into
two hexapeptides to obtain efficient
lanthanide-binding peptides. The syn-
thesis of the enantiopure Fmoc-Adan-
chroism, and solution NMR spectrosco-
py demonstrate that the Adan chain
length has a dramatic effect on the
complexation properties. Indeed, the
flexible compound P3 forms a mononu-
clear complex of moderate stability
(b11 =109.9), which tends to transform
into a binuclear species in the presence
of excess of the metal ion. Interesting-
ly, the more compact peptide P2 pro-
vides stable Ln3+ complexes with the
exclusive formation of the mononu-
clear LnP2 adduct. The stability con-
stant of TbP2 is two orders of magni-
tude higher (b11 =1012.1) than that mea-
sured for P3. The 800 MHz NMR spec-
trum of the La3+ complex of P2 eviden-
ces a well-defined type II b-turn as well
ACHTUNGTRENNUNG(tBu)2-OH synthons is described with
overall yields of 32 and 50% for n=2
and n=3 side-chain carbon atoms, re-
spectively. The two peptides AcWA-
danPGAdanGNH2 (Pn) were synthe-
sized from the protected synthons by
standard solid-phase peptide synthesis.
Studies of the lanthanide complexes of
the two peptides Pn by luminescence ti-
trations, mass spectrometry, circular di-
as a hydrophobic TrpACTHNGUTERNNU(G indole)–Pro in-
teraction. These interactions exemplify
the non-innocent character of the pep-
tide spacer in the complex LaP2 as well
as the role of a peptide secondary
structure in the stabilization of metal
complexes.
Keywords: amino acids · bioinor-
ganic chemistry
· lanthanides ·
peptides · structure elucidation
Introduction
thanide cations as magnetic probes for structural NMR
study in chemistry[4] and biology,[5] and emissive Tb3+ and
Eu3+ complexes as optical probes with various applications
in biology.[6] The introduction of Ln3+ ions into living organ-
isms is a challenge for the coordination chemist because of
their intrinsic toxicity. Ln3+ ions need to be encapsulated in
ligands to form kinetically inert and/or thermodynamically
stable complexes in vivo. Poly(aminocarboxylate) ligands
have been extensively investigated because they carry a
Lanthanide trivalent ions (Ln3+) are normally absent in
living organisms. However, due to an ionic radius similar to
that of the ubiquitous calcium ion (Ca2+), Ln3+ cations can
bind to proteins in Ca2+-binding sites.[1] Common Ca2+
-
binding loops in proteins such as calmodulin have 12 amino
acids bearing side-chains with oxygen donors, such as acids
or amides. In contrast to the absence of endogenous Ln3+ in
biological media, Ln3+ chelates are gaining increasing im-
portance in biological studies and biomedical applications
due to their unique magnetic and photophysical properties.
For instance, Gd3+ complexes are used as magnetic reso-
nance imaging contrast agents,[2,3] other paramagnetic lan-
number of donor atoms close to or equal to the high coordi-
[2]
nation numbers (8 to 10) preferred by Ln3+
.
Complexation
by such ligands avoids deleterious decoordination in vivo.
The use of peptides as Ln3+ ligands already adapted to
the in vivo environment is an attractive idea. Small peptides
derived from Ca2+-binding proteins have been used as the
starting point for the studies of Ln3+–peptide complexes.[7]
Ln3+ complexes of Ca2+-binding EF-hand loops (as in calm-
odulin), designed by Franklin and co-workers to obtain arti-
ficial endonucleases and 33-mer peptides binding Ln3+ with
micromolar affinities, have been investigated.[8] Imperiali
and co-workers optimized the sequence of lanthanide-bind-
ing tags (LBTs) by using screening methods starting from
Ca2+-binding sites to obtain the highest possible affinity.[9,10]
[a] Dr. F. Cisnetti, Dr. C. Gateau, C. Lebrun, Dr. P. Delangle
INAC, Service de Chimie Inorganique et Biologique
(UMR E 3 CEA UJF, FRE CNRS 3200)
Commissariat ꢀ l’Energie Atomique
17 rue des martyrs, 38054 Grenoble Cedex (France)
Fax : (+33)438785090
E-mail: pascale.delangle@cea.fr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900747.
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FULL PAPER
For instance, a 17-mer peptide has been demonstrated to
bind Tb3+ in an eight-coordinate complex without any
In this paper we report the synthesis and solution studies
of Ln3+ complexes with two hexapeptides (Pn) that incorpo-
rate two metal-binding amino acids Adan (n=2, 3). The two
hexapeptides Pn can be viewed as models of poly(aminocar-
boxylate) ligands like EDTA or TMDTA with a peptide
spacer instead of an alkyl spacer (see Scheme 1). In such
inner-sphere water molecules with a stability constant of
pH 7
logb =7.2.[10] We recently reported a de novo designed
11
cyclic decapeptide bearing four carboxyl side-chains, namely
c(DREPGEWDPG). Although the solution structure of the
apopeptide highlights a conformation suitable for complex-
ing Ln3+ ions, with the four carboxyl side-chains oriented on
the same face of the peptide scaffold, the stability of the lan-
pH 7
11
thanide complexes is also in the micromolar range (logb
ꢀ6.5).[11]
The above-mentioned examples show that there is an in-
trinsic limitation in the stability of lanthanide–peptide com-
plexes if only natural amino acids are used. Indeed, the
latter bear only simple binding groups, like carboxylates
(Asp, Glu), phenolates (Tyr), or amides (Asn, Gln, back-
bone peptide linkage), which have a low affinity for Ln3+ in
comparison with synthetic multidentate ligands of the poly-
(aminocarboxylate) family. Therefore, nonstandard amino
acids are increasingly being used in the design of peptide li-
gands to introduce strong donor groups for metal complexa-
tion or to improve the sensitization of metal-ion emission.[12]
Chelating moieties such as bipyridines, phenanthrolines, or
hydroxyquinolines have been included in peptide sequences
to obtain high-affinity metal binding sites for Zn2+ or other
divalent d-block metals, either to design metal sensors[13,14]
or to promote metal-assisted peptide self-assembly.[15]
Amino acids bearing chelating side-chains adapted to hard
cations, like g-carboxyglutamate, have been inserted into
peptides to induce folding through Ln3+ complexation.[16]
Among metal-chelating amino acid side-chains, aminodiace-
tate groups have attracted our attention.[17–19] The corre-
sponding residues are referred to as Adan (n being the
length of the alkyl chain separating the peptide backbone
from the aminodiacetate nitrogen). They have been shown
to stabilize helical structures in synthetic peptides through
divalent-metal-ion coordination.[20] The relationship between
Adan-containing peptides and poly(aminocarboxylate) li-
gands is clear as Adan structures bear a tridentate chelating
group analogous to the simple N-methyliminodiacetate
(MIDA) ligand.
To design lanthanide–peptide complexes of enhanced sta-
bility with a ligating site embedded in the peptide structure,
we decided to insert Adan unnatural amino acids into pep-
tide sequences. As Ln3+ ions require high coordination
numbers, several of these chelating units have to be used.
However, the mere inclusion of chelating amino acids into
an arbitrary peptide sequence is insufficient to achieve
highly stable mononuclear complexes because the chelating
side-chains show independent behavior. To overcome this
difficulty, the propensity of peptide chains to adopt defined
conformations in solution can be an asset to the design of
coordinating sites involving two or more chelating side-
chains. An analogy may be seen with the definition of
metal-binding sites in metalloproteins by the spatial disposi-
tion of coordinating site-chains due to secondary and terti-
ary structure elements.[21]
Scheme 1. Ln3+ chelates with poly(aminocarboxylate) hexadentate li-
gands and peptide ligands bearing two aminodiacetate groups.
systems the peptide backbone is used as a non-innocent
spacer between the coordinating groups and was designed to
favor a b-turn-containing structure. In such a turn, a hydro-
gen bond is usually present and the residues in positions i
and i+3 are brought into close proximity.[22] This would
bring the aminodiacetate side-chains closer into a disposi-
tion suitable for metal chelation. We demonstrate herein
that the Adan side-chain length has a dramatic effect on the
Ln3+ complexation properties. Whereas the two aminodiace-
tate moieties in the peptide P3 behave as nearly independent
subunits, the more compact ligand P2 acts as an efficient
hexadentate ligand of Ln3+ ions. Indeed, the formation of
the stable mononuclear LnP2 complex benefits from peptide
secondary structure stabilizing interactions.
Results and Discussion
Metal-chelating amino acid synthesis: The synthesis of
Adan-containing peptides (n=1–4) was first reported by
Hopkins and co-workers using N-Boc-Adan-OH synthons.[17]
However, undesired side-reactions such as trifluoroacetate
capping of the peptide were reported to occur using this
Boc strategy. Other authors subsequently published the pro-
cedure for the synthesis of Adan-containing peptides using
Fmoc-Adan-OH protected synthons.[18,19] However, the
Fmoc group was introduced at the end of the synthetic se-
quence by using a temporary protection of the amine group.
As a result, lengthy procedures involving multiple protec-
tion/deprotection steps were reported. The desired Fmoc-
AdanACTHNUTRGNE(NUG tBu)2-OH synthons were obtained in less than 10%
overall yield from commercial products. Finally, no proof of
the optical integrity of the protected synthetic amino acids
was provided. We thus decided to develop new synthetic
pathways to obtain Fmoc-AdanACHTNUGTRENUNG(tBu)2-OH (n=2, 3) by using
Fmoc-protected intermediates in the first steps of the syn-
thetic pathways.
The key steps in the synthesis of Fmoc-Ada
2ACHTUNGTERN(NUNG tBu)2-OH
are a straightforward reductive amination/hydrogenolytic
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P. Delangle et al.
debenzylation sequence starting from aldehyde 2. The re-
ductive amination procedure chosen ensures synthetic versa-
tility as the nature of the amine to be coupled with aldehyde
2 could be easily varied. Protection of the carboxylic acid is
necessary to facilitate purification of the synthetic products.
Benzyl is an appropriate protecting group because of its
ease of removal under catalytic hydrogenation conditions in
the last step of the synthesis. The three-step synthetic path-
way to 2 starting from commercially available l-homoserine
(l-Hse) was reported by Rocchi and co-workers.[23] Howev-
er, in the second step, Fmoc-Hse-OBn was isolated in only
48% yield probably because of the prompt lactonization of
this intermediate. The synthesis of Fmoc-Hse-OBn was im-
proved by conducting the benzylation in anhydrous DMSO
using the conditions reported for the synthesis of Fmoc-Ser-
OBn.[24] The crude Fmoc-Hse-OBn was oxidized, as pub-
lished, under Swern conditions to give 2.[23] Aldehyde 2 was
then subjected to a reductive amination with di-tert-butyl
Scheme 3. Synthetic pathway to Fmoc-Ada3ACTHNUTRGNEU(GN tBu)2-OH.
need to use an excess of electrophile, and led to an improve-
ment in the yield of 5. After final deprotection, Fmoc-Ada3-
AHCTUNGTRENNUNG
iminodiacetate using NaBH
resulting intermediate 3 was debenzylated to obtain the de-
sired Fmoc-Ada2A(tBu)2-OH. The synthetic procedure is sum-
N
H
CHTUNGTRENNUNG
marized in Scheme 2.
tions likely to preserve the enantiomeric integrity through-
out the synthesis. However, en-
antiomeric purity is essential
for peptide synthesis and must
be confirmed. Coupling with a
chiral amine (a-methylbenzyla-
mine) either in an enantiopure
or racemic form was performed
and the coupling product was
1
examined by H NMR spectros-
copy. Splitting of some signals
(especially NH doublets) oc-
curred only with the coupling
product formed with the race-
mic amine, which indicates that
the Fmoc-AdanACHTNUTRGNENUG(tBu)2-OH syn-
thons were enantiopure (see
the Supporting Information for
details).
Scheme 2. Synthetic pathway to Fmoc-Ada2ACTHNUTRGNEU(GN tBu)2-OH.
Peptide design and synthesis:
Two hexapeptides that incorporate two Adan chelating
amino acids were designed with a peptide spacer (aa2aa3 =
PG in Scheme 1) favoring a b-turn conformation. The
design of short peptides natively forming b-turns is a diffi-
cult task.[27] Research efforts in that direction are motivated
by the assumption that reverse turns are nucleating sites in
protein-folding.[28] Even though it is not usually considered
as a strong turn-inducer in linear peptides, the XPGX se-
quence is frequently encountered in type II b-turns in pro-
teins.[22] Experimental data in solution[29] as well as molecu-
lar dynamics simulations[30] show that the turn structures are
accessible in terms of conformational energy. Moreover,
turns formed by l-prolylglycine spacers between coordinat-
ing amino acids have been found to tune metal-monitoring
and induce some selectivity in fluorescent peptide probes in-
Protected derivatives of l-ornithine (Orn), which already
bear the Ne amine, are commercially available and the syn-
thesis of Fmoc-Ada3ACHTUNGTRENNUNG(tBu)2-OH has previously been reported
starting from this amino acid.[18] This synthesis was based on
the difunctionalization of the Ne amine by a standard alky-
lation procedure. Again, the Fmoc group was introduced at
the end of the synthetic sequence. We report herein a syn-
thesis based on the same sequence of reactions that was
conducted directly from Fmoc-OrnACHTUNGTRNEUNG
(Boc)-OH 4[26] sparing te-
dious protection/deprotection steps (Scheme 3).
After quantitative Boc deprotection of the primary amine
in a strong acid medium, nucleophilic substitution of the
amine could be performed in moderate yield in mild basic
conditions. The use of NBu4I as a catalyst instead of poorly
soluble NaI[18] greatly reduced the reaction time and the
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LanthanideACHTUNGTRENNUNG(III) Complexes with Two Hexapeptides
FULL PAPER
Table 1. Equilibrium constants of P2 and P3.[a]
cluding artificial fluorophores.[31] Being cyclic, l-proline is
unique among amino acids in promoting changes in protein
backbone direction and in restricting the conformational
space accessible to the peptide. In contrast, glycine possesses
enough conformational freedom to comply with the F and
Y backbone dihedral angle requirements of the type II b-
turn structure and should also provide sufficient conforma-
tional elasticity for efficient metal coordination. A trypto-
phan residue was also inserted into the sequence of P2 and
P3 to benefit from the antenna effect to sensitize Tb3+ lumi-
nescence. P2 and P3 may be viewed as minimal models to as-
certain whether the Adan-PG-Adan sequences are compati-
ble with the turn formation and to investigate the tuning of
the complexation behavior by the l-prolylglycine spacer.
Equilibrium constants
P2
P3
MIDA[b]
pKa1
pKa2
pKa3
pKa4
logb
8.8(1)
8.2(1)
2.8(1)
ꢀ2
9.6(2)
8.9(2)
2.9(1)
2.0(1)
5.4(3)
ꢀ9
9.59
2.32
ꢀ1.9
–
4.5
–
pH 7
(TbL)
(Tb2L)
9.1(5)
–
11
pH 7
21
Logb
logb110 (TbL)
12.1(5)
9.9(3)
7.11
pH 7
x1
[a] I=0.1m (KCl) at 298 K. b
represents the conditional stability con-
stants at pH 7 and b110 the stability constants. [b] See ref. [34].
could be detected due to sample dilution. The values of
pKa1 and pKa2 are important as they will be used later in
this paper to calculate the stability constants from the condi-
tional stability constants measured at pH 7. The first pKa of
P3 has the same value as the first pKa of MIDA, which indi-
cates no effect of the peptide backbone. Chain-shortening
from three to two carbon atoms lowers the amine pKa
values by 0.8 log units. This can be attributed to the with-
drawing effect of the peptide backbone amide functions
through the short side-chains of Ada2 in P2.
Starting from Fmoc-AdanACTHNUTRGNE(UNG tBu)2-OH and natural commer-
cial amino acid derivatives, peptides P2 and P3 (see
Scheme 4) were manually assembled by solid-phase peptide
Potentiometry requires large amounts of material, espe-
cially for the reliable determination of metal-complex stabil-
ity constants. Therefore, it is not the technique of choice for
studying the metal complexes of P2 and P3. We report here-
after the characterization of the Pn–Ln interaction in solu-
tion by other physicochemical techniques.
Scheme 4. Peptides P2 (n=2) and P3 (n=3) with sequence numbering.
synthesis (SSPS)[32] on the Rink amide MBHA resin using
standard conditions (see the Experimental Section).
No significant byproducts were detected, which shows
that the unnatural amino acids Fmoc-AdanACTHNUGTRNE(UNG tBu)2-OH dis-
played normal reactivity in the on-resin synthetic steps.
Before resin cleavage, N-terminal acetylation was per-
formed. As the choice of resin ensured the isolation of pep-
tides as C-terminal amides, peptides were recovered after si-
multaneous resin cleavage and side-chain deprotection with
both extremities protected. The deprotected peptides were
obtained on a ~50 mg scale after preparative HPLC purifi-
cation. Electrospray ionization mass spectrometry confirmed
the identity of the purified peptides. P2 and P3 were further
characterized by the standard combination of 1D and 2D
1H NMR techniques:[33] COSY and TOCSY were used to es-
tablish intra-residue spin systems and ROESY allowed the
assignment of signals. The spectra were assigned to only one
species, which proves that no large-scale racemization pro-
cesses occur in solution even though the Adan side-chains
carry basic functions, that is, unprotected tertiary amines.
The ROESY spectra of both peptides (500 MHz, 298 K)
show no though-space correlations other than sequential
ones. This, along with little dispersion of the NH amide and
glycine Ha resonances, is indicative of flexible conforma-
tions in solution, as expected for linear oligopeptides (see
Tables S1 and S2).
Mass spectrometry of EuPn: Electrospray mass spectrometry
(ES-MS) provides a qualitative insight into the speciation in
solution of the Ln3+ complexes of P2 and P3. This method is
the most suitable of the MS techniques for the study of
metal–ligand complexes in solution.[35]
Mass spectra were recorded for P2 and P3 in the presence
of EuCl3. The most significant peaks observed in the posi-
tive and negative ionization experiments are reported in
Table 2. For P2 only signals corresponding to mononuclear
Table 2. Significant peaks observed in the (+)- or (ꢁ)ESI mass spectra
of 60 mm peptide solutions in AcONH4 buffer (20 mm, pH 7) in the pres-
ence of EuCl3.[a]
(+)ESI
(ꢁ)ESI
P2 +0.5 Eu3+ 889.4 [P2+H]+
445.2 [P2+2H]2+
887.3 [P2ꢁH]ꢁ
443.2 [P2ꢁ2H]2ꢁ
1039.3 [P2ꢁ2H+Eu]+
1037.3 [P2ꢁ4H+Eu]ꢁ
518.2 [P2ꢁ5H+Eu]2ꢁ
1037.3 [P2ꢁ4H+Eu]ꢁ
520.3 [P2ꢁH+Eu]2+
+1.0 Eu3+ 1039.3 [P2ꢁ2H+Eu]+
520.3 [P2ꢁH+Eu]2+[b]
518.2 [P2ꢁ5H+Eu]2ꢁ[b]
[c]
[c]
+2.0 Eu3+
P3 +0.5 Eu3+ 917.6 [P3+H]+
459.5 [P3+2H]2+
915.5 [P3ꢁH]ꢁ
457.3 [P3ꢁ2H]2ꢁ
1067.5 [P3ꢁ2H+Eu]+
1065.3 [P3ꢁ4H+Eu]ꢁ
ACHTUNGTRENNUNG(weak)
534.4 [P3ꢁH+Eu]2+
532.3 [P3ꢁ5H+Eu]2ꢁ (weak)
Peptide protonation: P2 and P3 were subjected to pH-metric
titrations. They allowed the determination of two pKa values
>8 (amine functions) as well as two pKa values <5 (acidic
functions). The pKa values are reported in Table 1 (see the
Supporting Information for the experimental curves and de-
tails). Not all the carboxylic acids (pKa5 and pKa6 are <2)
[d]
[d]
+1.0 Eu3+
+2.0 Eu3+ 608.3 [P3ꢁ4H+2Eu]2+[c]
[c]
[a] Sodium (+22/z) and potassium (+38/z) adducts were also detected.
[b] Nearly total disappearance of the signals of the free peptide. [c] The
same peaks as 1.0 equiv. [d] The same peaks as 0.5 equiv; no disappear-
ance of the signals of the free peptide.
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P. Delangle et al.
species were detected. With 1 equiv or excess Eu3+, signals
associated with the uncomplexed peptide disappeared from
the spectra. P3 showed different behavior. In comparison
with P2, the intensity ratio of the complexed/uncomplexed
peptide signals is smaller in each experiment. Signals of the
free peptide were detected even with a two-fold excess of
EuCl3. More interestingly, a Eu2P3 species was detected in
the presence of 2 equiv EuCl3.
with that of a carbonyl amide in a random coil environment
(strong negative band at 195 nm).[37] Furthermore, aromatic
side-chains such as Trp indoles could contribute to far-UV
CD and thus complicate spectral interpretation.[38]
As anticipated from the results of the ES-MS experi-
ments, the two peptides show different behavior. For P2, the
addition of up to 1 equiv Tb3+ caused a change in the CD
signal clearly associated with an isodichroic point at 208 nm
(Figure 2, top). No further ellipticity changes were observed
Strikingly, the major peaks observed in a competition ex-
periment ([P2]=[P3]=[Eu]) correspond to complexed P2
and uncomplexed P3 (Figure 1). Mass spectrometry is not a
Figure 1. (ꢁ)-ESI mass spectrum of a solution containing P2, P3, and
EuCl3 (30 mm) in AcONH4 buffer (20 mm, pH 7). See Table 2 for peak as-
signments (m/z=547.9 [P2ꢁ4H+Eu+AcO]2ꢁ, molecular association).
quantitative analysis technique. However, in the case of sim-
ilar molecules like the peptide ligands P2 and P3, it is likely
that the relative intensities of signals belonging to species of
the same charge reflect abundances in solution. The results
of this competition experiment thus indicate that P2 is a
Figure 2. Changes in the far-UV CD signals of P2 (top) and P3 (bottom)
upon addition of TbCl3 in water (pH 7). Peptide concentration about
20 mm. P2: titration with 0.33 equiv aliquots of Tb3+ until 1 equiv. P3: ti-
tration with 0.33 equiv aliquots of Tb3+ until 2 equiv, then 3, 5, 7 equiv.
1) Spectral traces up to 1 equiv (highlighted). 2) Spectral traces for 1.33–
7 equiv.
stronger ligand than P3 towards Eu3+
.
Circular dichroism studies: Circular dichroism (CD) titra-
tions were performed to monitor the modification in the di-
chroic signal in the region of the amide bands upon the ad-
dition of Tb3+ to P2 and P3 solutions. The use of Tb3+ was
chosen for consistency with luminescence studies reported
hereafter. CD spectra can reveal the presence of secondary
structure elements in peptides or proteins. However, in con-
trast with the well-defined CD signature of b-sheets and a-
helices, the identification of turns by their CD signature
alone is usually not reliable.[36,37] Indeed, the CD signature
of type II b-turns (type B spectrum in Woodyꢂs theory) may
not be seen in linear peptides: The mean molar ellipticity
per residue in such a turn (including a positive band at
around 200 nm) is lower by a factor of four in comparison
upon the addition of excess Tb3+. In contrast, a two-phase
change in the dichroic signal was detected for the P3–Tb
system (Figure 2, bottom). Up to 1 equiv of Tb3+, a decrease
in the negative ellipticity at 199 nm was observed associated
with an isodichroic point at 214 nm. Above 1 equiv, the for-
mation of a second species was detected with a slow de-
crease in the negative ellipticity at 195 nm. For this second
process, no plateau was reached even with a large excess of
Tb3+, which indicates that the second process is not com-
pleted even with a 10-fold excess of the metal ion. These re-
sults are in agreement with the speciation model suggested
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LanthanideACHTUNGTRENNUNG(III) Complexes with Two Hexapeptides
FULL PAPER
by the ES-MS experiments: TbP2 is the only metallic species
formed in water (pH 7) whereas the mononuclear complex
TbP3 is transformed into a second complex, Tb2P3, in the
presence of an excess of the metal ion.
The build-up of the Tb-centered luminescence through
excitation of the tryptophan moiety shows a sharp end-point
for the P2 +Tb3+ titration, which is characteristic of a large
stability constant (Figure 4). The binding curves recorded
Tryptophan fluorescence and Tb3+ phosphorescence: Owing
to the antenna effect, in the present case Tb3+ luminescence
sensitization by energy transfer through Trp indole absorp-
tion at around 280 nm, the Tb–peptide complexes could be
detected at concentrations as low as 0.5 mm. Reliable titra-
tions could be performed at concentrations as low as around
10 mm peptide. TbCl3 titrations of solutions of peptides dis-
solved in HEPES buffer (10 mm, pH 7) were performed.
7
The build-up of the Tb-centered 5D4! FJ bands (J=3–6)
and the modification of the Trp fluorescence were moni-
tored.
The intensity of the Trp fluorescence signal increased sig-
nificantly up to 1 metal equiv during the Tb titration
(+34%) with P2. A modest redshift in the emission maxima
(from 349 to 351 nm) was also observed. Titrations conduct-
ed with the nonluminescent lanthanum ion La3+ instead of
Tb3+ gave very similar results: +40% intensity, 2 nm red-
shift (see Figure 3 for La and Figure S4 for Tb). Even
Figure 4. Build-up of the Tb-centered emission during the titration of P2
(49.5 mm) with TbCl3 in HEPES buffer (10 mm, pH 7, 0.1m KCl). Inset:
&
^
Variation of the intensities of the peak maxima with Tb ( : 490 nm;
:
~
545 nm; : 587 nm).
Figure 3. Increase in the Trp-centered emission of P2 (76.4 mm) upon addi-
tion of LaCl3 in HEPES buffer (10 mm, pH 7, 0.1m KCl). The spectral
traces with 0 and 1 equiv Tb are shown as bold lines. Inset: Variation of
the intensity of the peak maximum (average intensity 346–352 nm) with
La.
Figure 5. Build-up of the Tb-centered emission at selected wavelengths
during the titration of P3 (19.7 mm) with TbCl3 in HEPES buffer (10 mm,
pH 7
pH 7, 0.1m KCl). Dashed lines represent calculated data with logb
=
11
~
&
^
5.4 ( : 490 nm; : 545 nm; : 587 nm).
though the photophysics of tryptophan side-chains in pep-
tides is a rather complex subject,[39] this enhancement of the
Trp luminescence may indicate that the Trp side-chain is in
a more rigid environment in the complex in comparison
with the free peptide and/or that the tryptophan is less ac-
cessible to water in the complexes. This effect largely ex-
ceeds in magnitude the Trp-to-Tb3+ energy transfer, which
is expected to lead to a decrease in the Trp luminescence in-
tensity. Indeed, the Trp-to-Tb3+ transfer is not an efficient
process.[40] The intensity of the Trp emission also increased
during the Tb titration with P3 (+15%), as it did in titra-
tions with nonluminescent La3+ (Figure S6, +16%). The in-
crease is less marked than for P2, which may indicate differ-
ences in the tryptophan environment in the two complexes.
with P3 are much smoother (Figure 5), which indicates a
lower affinity of this latter peptide for Tb3+. In contrast to
the CD titrations, the binuclear complex Tb2P3 is not direct-
ly viewed in the spectral evolution above 1 equiv Tb3+ be-
cause the luminescence spectra of TbP3 and Tb2P3 may be
very similar.
The luminescence lifetimes of Tb3+ in the TbPn com-
plexes in H2O and D2O were measured to obtain the hydra-
tion number qTb of these complexed ions using the empirical
equations of Horrocks and Sudnick[41] or of Parker and co-
workers.[42] To avoid underestimating the luminescence life-
times in D2O because the peptides are accompanied by H2O
hydration molecules, tD O was determined as the extrapolat-
2
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7461

P. Delangle et al.
ed limit of the luminescent decay rates in solutions of in-
creasing D2O molar fractions tending to a H2O-free solution
(Figure S7). The experimental values of tH O and tD O
2
2
(Table 3) for TbP2 and TbP3 give a hydration number close
Table 3. Luminescence lifetimes and calculated hydration numbers for
the TbPn complexes.
Peptide
tH O/ms
tD O/ms
q[a]
q[b]
2
2
P2
P3
1.11(1)
1.01(1)
3.71(3)
2.75(3)
2.9(1)
2.8(1)
2.8(1)
2.6(1)
[a] Calculated according to ref. [42]. [b] Calculated according to ref. [42].
to 3 for both peptide complexes. These values compare well
(H2O)q]ꢁ complex, which were
with those of the [TbACTHNUTRGENNUG(EDTA)AHCTUNGTRENNGUN
Figure 6. Decline of the Tb-centered emission at selected wavelengths
during the titration of a solution containing P2 (91.1 mm) and TbCl3
(91.1 mm) with nitrilotriacetic acid in HEPES buffer (10 mm, pH 7, 0.1m
found to be 2.8.[42,43] They are thus consistent with both pep-
tides behaving as hexadentate ligands. Different denticity is
thus not the origin of the different stabilities of the mononu-
clear TbPn complexes.
KCl). Dashed lines represent calculated data with a conditional stability
pH 7
constant logb11 =9.1 for the TbP2 complex ( : 490 nm; : 545 nm;
~
:
&
^
587 nm).
Stability constants: Tb3+ luminescence titrations are particu-
larly suitable for obtaining quantitative information about
the stability constants of the complexes in solution. For P3,
titrations conducted in diluted peptide solutions ([P3]
ꢀ20 mm) at pH 7 could be interpreted as the formation of
only one complex, TbP3, or of the two species, TbP3 and
Tb2P3. These two models give the same conditional stability
constant for the mononuclear complex within experimental
error: logbp11H 7 =5.4(3). Figure 5 shows the good agreement
between experimental and calculated data for the most in-
tense terbium emission bands. In contrast, titrations con-
ducted at higher P3 concentrations (>100 mm) could not be
fitted with only one metallic complex and the formation of
ity constant of TbP2 was determined to be logb1p1H 7 =9.1(5).
Uncertainties arise in approximately equal parts from exper-
imental errors in the competition experiments and from the
uncertainties in the published Tb–NTA stability constants.
The values of the conditional stability constants at pH 7
are reported in Table 1. These values determined at a specif-
ic pH depend on the basicity of the ligands. Therefore, to
compare the stabilities of the two TbPn complexes, the sta-
bility constants (Table 1) were calculated from the condi-
tional values at pH 7 and the pKas of the two peptide li-
gands. The TbP2 complex is found to be two orders of mag-
nitude more stable than the TbP3 complex (Dlogb110 ꢀ2.2),
although the two peptides act as hexadentate ligands of lan-
thanide ions. Indeed, the Tb3+ ion has the same hydration
number in the two mononuclear complexes. Furthermore,
the formation of the binuclear adduct with P3 clearly eviden-
ces the tendency of P3 to act as a bis(tridentate) ligand and
to complex two metal ions. The TbP3 complex shows nearly
the same stability as hexadentate poly(aminocarboxylate) li-
the binuclear complex Tb2P3 had to be taken into account in
pH 7
21
the fitting procedure, with logb ꢀ9. Owing to the limited
change in the observed signal, precise determination of
pH 7
21
logb
from the experimental data was not possible.
The analysis of the titration data obtained with P2 concen-
trations in the range 20–200 mm revealed that the P2–terbium
system could be completely described by the total formation
of TbP2 between 0 and 1.0 equiv of added metal (Figure 4).
Direct titrations did not allow the stability constants for
TbP2 to be determined because the signal-to-noise ratio was
not sufficiently favorable for dilute samples to detect signifi-
cant amounts of the free peptide in the presence of a stoi-
chiometric quantity of Tb3+. Nitrilotriacetic acid (NTA) was
chosen as a competitive ligand for P2 because the pKas and
gands with long alkyl chains (Scheme 1, n=2–4, logb110
=
9.5–10.8).[34] The latter do not provide significant chelate ef-
fects between the two aminodiacetate moieties and benefit
only from the entropic stabilization because of the presence
of the two tridentate units in the same molecule.
In contrast, [TbACTHUNTGRENNUG(EDTA)] or [TbACHUTNGTRENN(GUN TMDTA)] complexes
(Scheme 1, n=0, 1, respectively) show very large chelate ef-
fects due to the formation of five- or six-membered chelate
rings through the coordination of the two nitrogen atoms.
Although not as marked as for these ligands, TbP2 shows a
significant stabilization of two orders of magnitude in com-
parison with ligands bearing independent aminodiacetate
moieties, even though the coordinating nitrogen atoms are
separated by 14 atoms. This effect is a result of peptide sec-
ondary structure elements, such as, b-turn formation, as
demonstrated by solution NMR studies and presented in the
following paragraphs.
the stability constants of the mononuclear Tb
ACHTUNGTRENUN(NG NTA) and bi-
nuclear Tb(NTA)2 complexes are known. Conditional stabil-
ACHTUNGTRENNUNG
ity constants at 298 K, pH 7.0, and 0.1m KCl can be calculat-
ed from published data: logbp11H 7 =8.8(2) and logbpH 7
=
12
15.6(3).[34] Tb3+ luminescence spectra were recorded during
the titration of TbP2 with NTA. The data could be fitted
with a simple model that involves the displacement of Tb
from the peptide ligand and the formation of nonlumines-
cent Tb
ACHTUNGTRENNUNG(NTA) and TbACHTUNGTERN(NUGN NTA)2 (Figure 6). Knowing the con-
ditional stability constants of the latter complexes, the stabil-
7462
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LanthanideACHTUNGTRENNUNG(III) Complexes with Two Hexapeptides
FULL PAPER
1
NMR signature of LnPn: H NMR spectroscopy was used to
investigate the effect of Ln3+-binding on the backbones of
peptides P2 and P3. To detect the changes in the CH region
(1–5 ppm) clearly, the study was performed in D2O. The pD
was adjusted to a value of 7 for consistency with the lumi-
nescence and CD studies. Attempts to use lanthanide-in-
duced shifts and relaxation[4,44] to investigate the solution
structure of paramagnetic lanthanide complexes of P2 and
P3 were unsuccessful. The NMR spectra of Eu3+ and Yb3+
complexes show very broad proton resonances because of
intramolecular dynamics, as already observed for tetrapodal
ligands with soft donor nitrogen groups.[45] More details on
the structures of the complexes were obtained from the
NMR data of the diamagnetic lanthanum complexes.
The addition of LaACTHNUTRGNEUNG
(OTf)3 to a solution containing P3 re-
sulted in general line-broadening of the spectrum as well as
small modifications in the chemical shifts of the observed
signals (Figure 7). The signals that experienced the greatest
Figure 8. Partial 1H NMR spectra of P2 +La
298 K, pD 7, [P2]=1.05 mm): bottom) 0 equiv La3+
La3+, and top) 1.0 equiv La3+. The following signals are marked: Open
ACHTUNGTRENNUNG
,
*
^
symbols for Ada2 residues (Ha: &; Hg: ; He: ); filled symbols for
*
^
other residues (Pro3 Ha: ; Gly4 Ha: ; Pro3 Hd (pro-R): ꢀ; Pro3 Hd
(pro-S): +).
coordination site showed significant modifications. In partic-
ular, the signals of the Pro3 Ha and Hd atoms experience
changes in chemical shifts of up to 0.36 ppm. Moreover, the
glycine Ha region is clearly modified. A poorly resolved
multiplet accounting for all four Gly Ha atoms is observed
in the spectrum of the apopeptide, whereas the protons of
one of the glycines in the LaP2 complex give rise to a pair
of doublets.
However, at this point it was not possible to attribute the
split signals to one of the glycines in particular because of
the overlapping of all the glycine Ha atoms in the apopep-
tide spectrum. The 1H WATERGATE NMR spectrum of
LaP2 in H2O/D2O (9:1, v/v) at pH 7 shows the signals of the
NH amide protons at 278 K (Figure S8). 2D NOESY and
ROESY spectra recorded at this temperature allowed us to
confirm the signal assignment and to assign unambiguously
the glycine resonances in the complex. The Gly4 Ha atoms
in LaP2 correspond to a pair of well-separated (0.32 ppm)
doublets, which indicates that this glycine is in a well-de-
fined environment. The most significant changes in the
NMR signal are thus in the Pro3-Gly4 region of the spec-
trum, that is, the spacer between coordinating residues.
These observations could indicate that a transition from an
open to a turn-containing structure occurs upon complexa-
tion. Such changes prompted us to determine the structure
of the LaP2 complex by an NMR study
Figure 7. Partial 1H NMR spectra of P3 +La
298 K, pD 7, [P3]=0.99 mm): bottom) 0 equiv La3+, top) 1.0 equiv La3+
The following signals are marked: Open symbols for Ada3 residues (Ha:
ACHTUNGTREN(UNGN OTf)3 (D2O, 500 MHz,
.
*
^
*
&; Hd: ; Hz: ); filled symbols for other residues (Pro3 Ha: ; Gly4
^
and Gly6 Ha: ; Pro3 Hd ꢀ and + symbols).
shift and the most severe line-broadening were the side-
chains of the Ada3 residues (CzH2 @CdH2 >CgH2). Howev-
er, the peptide backbone signals are virtually unmodified
upon lanthanum coordination (Figure 7 and Tables S3 and
S4). This can be interpreted as an indication of limited con-
formational rearrangement upon coordination.
In contrast to P3, the addition of La
containing P2 resulted in marked changes in the NMR spec-
tra. The addition of less than 1 equiv of La(OTf)3 resulted in
ACHTUNGRTEN(NGNU OTf)3 to a solution
AHCTUNGTRENNUNG
the observation of two sets of signals: Those of the unreact-
ed apopeptide and a new set of signals belonging to the
mononuclear complex LaP2. The signals of the complex
were unambiguously assigned with a 2D EXSY spectrum of
1
a sample containing 0.5 equiv La
N
NMR structure of LaP2: The objective of this structural
analysis was to determine if the lanthanum complexation by
P2 is associated with characteristic elements of peptide sec-
ondary structure. Indeed the presence or absence of secon-
dary structure elements has been invoked to explain the rel-
ative stabilities of metal complexes.[13]
tra are shown in Figure 8 and the full NMR assignment is
given in Tables S5 and S6. Line-broadening was observed in
the side-chains of the coordinating amino acids (CeH2 @
CgH2 >CbH2), but this effect was less pronounced than for
P3. More interestingly, the signals of protons far from the
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7463

P. Delangle et al.
The solution structure of the LaP2 complex (3.53 mm) was
investigated by 1D and 2D H NMR experiments in H2O/
as the Ada2 side-chains, especially that of Ada25, are more
flexible (Figure 9, right).
1
D2O (9:1, v/v) at pH 7 and 278 K. The temperature of 278 K
was chosen for the structural study for two reasons. First,
the 1H NMR WATERGATE spectrum in H2O/D2O (9:1,
v/v) displayed only low intensity residual amide NH signals
at 298 K. These signals partly recovered their intensity at
278 K. Secondly, preliminary experiments showed that sig-
nals of very low intensity were detected in NOESY experi-
ments at 298 K due to an unfavorable w0tc product, as is
common for medium-sized molecules.[46] The product w0tc
can be increased by lowering the temperature and therefore
the solvent viscosity and large negative NOE correlations
could be obtained at 278 K. A total of 92 NOE restraints
obtained from the 2D-NOESY spectrum were used in struc-
tural calculations with 23 intra-residue NOEs and 47 se-
quential NOEs as well as 22 medium-range NOEs (i.e., be-
tween residues i and iꢂ2 or iꢂ3). Correlations of the
barely assignable and very broad signals of He (CH2COOꢁ)
were not used in the calculations. Of the medium-range
NOEs, 16 correlations were ascribed to contacts between
the Pro pyrrolidine ring and the indole aromatic side-chain
of Trp. The trans conformation of the peptide bond with
proline is evidenced by the presence of short contacts:
d(Ada22 Ha, Pro3 Hd1)=d(Ada22 Ha, Pro3 Hd2)=2.2 ꢃ.
The Ada2ProGlyAda2 motif shows two characteristic fea-
tures of a type II b-turn structure: A cis relationship be-
tween Gly4 NH and Pro3 Ha (2.3 ꢃ) and weak NOE cross-
peaks between Gly4 NH and Pro4 Hd1 (4.7 ꢃ) and Hd2
(4.8 ꢃ). Unfortunately, no strong correlations between the i
and i+3 side-chains could be detected because the identical
nature of the Ada2 residues results in signal overlap. The
turn structure was, however, deduced by the observation of
a number of short- and medium-range NOE correlations in
the Ada2GPAda2 region.
The efficiency of the energy transfer between the donor
(tryptophan) and the acceptor (Tb3+) is described by a
dipole–dipole mechanism and is strongly related to the dis-
tance that separates the donor and the acceptor.[40] In the
LBT structures reported in the literature this distance is ap-
proximately 7 ꢃ because the backbone carbonyl of the tryp-
tophan residue is coordinated to the terbium ion.[10,48] In the
10 lowest-energy structures of the complex LaP2, the La3+
ion is located 8.7
G
distance between the acceptor and the donor is slightly
larger in LaP2 than in the LBTs, mainly because the trypto-
phan carbonyl is not coordinated to the metal center. Never-
theless the complex LaP2 was clearly detected in the micro-
molar range under our experimental conditions.
The backbone adopts a U-shaped conformation and the
Trp–Pro hydrophobic interaction is clearly apparent in the
calculated structures: The average separation between the
centroids of the Trp benzene and the Pro five-membered
ring is 4.36(1) ꢃ with a tilt angle of 9.0(5)8. This hydropho-
bic interaction is likely responsible for the significant en-
hancement of the Trp fluorescence upon lanthanide com-
plexation mentioned in a previous section. Trp–Pro stacking
has been recognized as a stabilizing interaction in peptides
and the interaction observed in LaP2 between the nearly co-
planar proline and benzenic rings is the most frequently en-
countered case for Trp-X-Pro sequences.[49] In summary, the
LaP2 complex adopts a U-shaped structure with an “upper
face” devoted to lanthanum coordination and a “lower
face” involved in the hydrophobic interaction.
Solution structures of peptide–metal complexes with cat-
ions displaying strong and directional metal coordination
bonds (for instance, mercury[50] and palladium[51]) have been
obtained by NMR studies. For these types of metal cations
secondary structure elements are enforced by metal coordi-
nation. In contrast to these metals, Ln3+ coordination bonds
are weaker and not directional. This is in agreement with
the flexible behavior of the Ada2 side-chains in the complex.
Fifty structures were calculated by using CNSsolve 1.1[47]
with standard refinement protocols using custom topologies
and parameter files for the Ada2 residues and for the lantha-
num coordination sphere (see the Experimental Section). A
superimposition of the 10
lowest-energy
structures
is
shown in Figure 9. In these
structures the Ada2ProGlyAda2
motif forms a type II b-turn.
The carbonyl group of Ada22
and the NH group of Ada25 are
linked by a hydrogen bond with
an angle between the donor
proton, the donor atom, and
the acceptor atom of 170(7)8
ꢁ
and an average O N distance
of 3.01(7) ꢃ, which is close to
the optimum for such a bond.[22]
As seen in Figure 9 (middle),
the peptide backbone and the
Trp side-chain are rather well-
Figure 9. NMR structure of LaP2. Left: lowest energy ball-and-stick structure, stabilizing interactions repre-
sented as orange lines; Middle: superimposition of the backbones and Trp side-chains of the 10 lowest-energy
structures; Right: superimposition of the 10 lowest-energy structures (backbone and Trp side-chain: sticks, co-
defined except for Gly6, where- ordinating side-chain: lines).
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Chem. Eur. J. 2009, 15, 7456 – 7469

LanthanideACHTUNGTRENNUNG(III) Complexes with Two Hexapeptides
FULL PAPER
It is also for this reason that lanthanide–peptide complexes
are usually poorly defined structurally and indeed only one
NMR structure of a La–peptide complex has been pub-
lished.[48] Lanthanum coordination by P2 and the establish-
ment of the Ada25 NH···OC Ada22 hydrogen bond as well
as the Trp–Pro hydrophobic interaction may thus better be
viewed as occurring synergistically. These interactions can
reduce the unfavorable conformational energy terms associ-
ated with peptide backbone reorganization upon complexa-
tion. Turn formation and hydrophobic Trp–Pro contact
occur only in LnP2 and may be considered as the structural
origin of the marked difference in stability between the
Ln3+ complexes of P2 and P3.
independent. Although the solution NMR spectra indicate
no structure elements for LaP3, a well-defined structure is
clearly observed in the 1D and 2D 1H NMR spectra of
LaP2. Indeed, the features characteristic of a peptide secon-
dary structure are apparent in the NOESY and ROESY
spectra of LaP2: The Ada2PGAda2 motif adopts a conforma-
tion with a type II b-turn showing a perfect hydrogen bond
between the carbonyl of Ada22 and the NH group of Ada25
and a hydrophobic interaction between the aromatic group
of the tryptophan residue and the aliphatic proline ring.
This complex adopts a U-shaped structure with an “upper
face” devoted to lanthanide coordination and a “lower face”
involved in the hydrophobic interaction. These elements of
peptide secondary structure contribute to the stability of the
LaP2 complex and explain the marked difference in the sta-
bilities of LnP2 and LnP3. Although the two aminodiacetate
moieties act nearly like independent tridentate binding
groups in P3, synergistic effects of Ln3+ complexation, b-
turn formation, and the Trp–Pro hydrophobic contact for
the more compact peptide P2 provide an efficient lantha-
nide-binding peptide. In addition, the withdrawing effect of
the amide groups of the peptide backbone on the amine ni-
trogen atoms of P2 lowers the two tertiary amine pKa values,
which leads to a conditional stability constant of 109.1 at
pH 7. This value can be compared with previously reported
values for peptides based exclusively on natural amino acids
that are in the range of 106–107.
Conclusions
Metal-chelating unnatural amino acids bearing aminodiace-
tate moieties (Adan) have been introduced into peptide se-
quences to obtain peptide ligands with enhanced affinity for
trivalent lanthanide ions. The two AcWAdanPGAdanGNH2
hexapeptides differ only in the length of the Adan side-
chains (two carbon atoms for P2 and three for P3). More-
over, these peptides contain an XPGX sequence as a non-in-
nocent spacer between the two metal-chelating amino acids.
This motif is known to favor a type II b-turn, which should
bring closer the two Adan subunits to allow hexadentate co-
ordination of Ln3+ ions. These two peptides were satisfacto-
rily obtained by solid-phase peptide synthesis using the clas-
sic Fmoc/tBu strategy from commercially available protect-
ed natural amino acids and the unnatural amino acid deriva-
To conclude, this study demonstrates that a subtle inter-
play between the basicity, the compactness of the ligand
(lengths of the side-chains bearing the chelating groups),
and the secondary structure of the peptide backbone can
lead to lanthanide–peptide complexes of enhanced stability
in comparison with peptides containing only natural amino
acids. In these complexes, the ligating site is embedded in
the peptide structure. Systems with higher denticity with
scaffolds similar to P2 should lead to even more stable Ln3+
peptide complexes.
tives Fmoc-AdanACHTUNGTRENNUNG(tBu)2-OH. The latter were synthesized in
the enantiopure form with overall yields of 32 and 50% for
n=2 and n=3, respectively.
The complexation of Ln3+ by P2 and P3 was studied by
complementary analytical methods like ES-MS, CD, lumi-
nescence, and solution NMR spectroscopy. These studies
demonstrate that the Adan side-chain length has a dramatic
effect on the complexation properties of the peptides to-
wards Ln3+ ions. Indeed, the more flexible P3 forms a mon-
onuclear complex of low stability, which tends to transform
into a binuclear species in the presence of an excess of the
metal ion. In this latter complex, the two binding aminodia-
cetate moieties act as independent chelating groups. The sta-
bility constant of TbP3 is 109.9, which is similar to the con-
stants obtained with poly(aminocarboxylate) ligands with
long alkyl spacers between two aminodiacetate groups (nꢃ2
in Scheme 1). In the complexes of these ligands, the large
chelate rings formed through the coordination of the two ni-
trogen atoms do not significantly stabilize the complexes.
Therefore we do not expect a peptide secondary structure to
contribute significantly to the stability of the LnP3 complex.
Interestingly, the more compact peptide P2 provides more
stable Ln3+ complexes with the exclusive formation of the
mononuclear LnP2 adduct. The stability constant is two
orders of magnitude higher (1012.1) than that measured for
P3, which demonstrates that the two Adan subunits are not
Experimental Section
General: Reagents and solvents were purchased from Aldrich and Nova-
biochem and used without further purification unless specified. The or-
ganic products were characterized by NMR spectroscopy by using a
Varian Mercury 400 MHz spectrometer at 298 K. Analytical and prepara-
tive peptide RP-HPLC were performed with LaChrom and LaPrep sys-
tems (see text for details). Specific rotations [a] are given in
10ꢁ1 degcm2 gꢁ1, with the concentration c in g per 100 mL.
Abbreviations: Ac2O, acetic anhydride; DMF, N,N-dimethylformamide;
DIEA, N,N-diisopropylethylamine; DTT, dithiothreitol; Et2O, diethyl
ether; Fmoc, 9-fluorenylmethoxycarbonyl; PyBOP, (benzotriazol-1-yloxy)-
tris(pyrrolidino)phosphonium hexafluorophosphate; TFA, trifluoroacetic
acid; TIS, triisopropylsilane; TNBS, 2,4,6-trinitrobenzenesulfonic acid.
Aldehyde 2: Compound 1[23] (3.128 g, 9.16 mmol), KHCO3 (1.377 g,
13.76 mmol, 1.5 equiv) and tetrabutylammonium iodide (0.3385 g,
0.916 mmol, 0.1 equiv) were dissolved in dry DMSO (18 mL) under
argon. The resulting white suspension was stirred at room temperature
for 10 min to obtain a clear solution. Benzyl bromide (3.25 mL, 4.67 g,
27.33 mmol, 2.99 equiv) was added to this solution and the colorless reac-
tion mixture was stirred for 36 h at room temperature. The reaction was
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7465

P. Delangle et al.
quenched by the addition of water (100 mL) and the DMSO/water layer
was extracted with EtOAc (3ꢄ150 mL). The combined organic layers
were washed with saturated aq. NaHCO3 (2ꢄ100 mL), saturated aq.
Na2S2O3 (1ꢄ100 mL), and finally with brine (1ꢄ100 mL). The organic
layer was dried (Na2SO4) and concentrated by rotary evaporation. The
resulting yellowish oil was triturated in cold (08C) light petroleum ether
(b.p. 35—608C) until a white solid was obtained. The solid (3.128 g) was
recovered by filtration. 1H NMR and ES-MS analysis data for the crude
solid were compatible with published data for the purified product[23]
except for the presence of trace amounts of DMSO. The solid was sub-
jected without further purification to Swern oxidation following the pro-
cedure reported by Rocchi et al.[23] Aldehyde 2 (1.782 g) was obtained
after SiO2 column chromatography (eluent: cyclohexane/EtOAc, 2:1,
v/v). Yield: 45% over two steps (lit.:[23] 30% over two steps). 1H NMR
and ES-MS analysis data were identical to published data. [a]2D5 =+9.2
(c=1.13 in CHCl3) (lit.: [a]2D5 =+9.3).[23]
The aqueous phase was decanted and extracted with Et2O (3ꢄ50 mL).
The joint organic phases were washed with saturated Na2S2O3 (50 mL)
and water (50 mL), dried (Na2SO4), and the solvents were evaporated in
vacuo. The resulting oil was purified by SiO2 column chromatography
(eluent: toluene/Et2O, 4:1, v/v) to give 5 (1.047 g, 56% yield) as a color-
less oil. [a]2D0 =+8.3 (c=1.00 in CHCl3); 1H NMR (400 MHz, CDCl3,
258C): d=7.76 (d, 3J
2H; Ar), 7.41–7.27 (m, 9H; Ar), 5.85 (d, 3J
5.18 (m, 2H; CH2Bn), 4.38 (m, 3H; Ha and CH2 (Fmoc)), 4.22 (dd (app.
t), 3J
(H,H)ꢀ6.7 Hz, 1H; CH (Fmoc)), 3.36 (s, 4H; CH2COOtBu), 2.70
A
ACHTUNGTREN(NUNG H,H)=7.1 Hz,
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
(m, 2H; Hd), 1.98–1.78 (m, 2H; Hb), 1.50 (m, 2H; Hg), 1.44 ppm (s,
18H; tBu); 13C NMR (100 MHz, CDCl3, 258C): d=172.6 (COOBn),
170.1 (COOtBu), 156.4 (CO (Fmoc)), 144.2, 144.1, 141.5 (2C) (Cq
(Fmoc)), 135.7 (Cq (Bn)), 128.8, 128.6, 128.5 (Ph), [127.9, 127.3, 125.4,
120.2 (CAr-H (Fmoc), 81.3 (Cq (tBu)), 67.3, 67.2 (CH2 (Fmoc) and CH2
(Bn)), 56.1 (CH2COOtBu), 54.3 (Ca), 53.7 (Cd), 47.4 (CH (Fmoc)), 30.0
(Cb), 28.4 (tBu), 24.1 ppm (Cg); ES-MS: m/z (%): 673.3 (100) [M+H+].
Fmoc-Ada
butyl iminodiacetate (1.317 g, 5.37 mmol, 1.5 equiv) were dissolved in an-
hydrous 1,2-dichloroethane (15 mL). NaBH(OAc)3 (1.366 g, 6.44 mmol,
2ACHTUNGTRENNUNG(tBu)2-OBn (3): Compound 2 (1.536 g, 3.58 mmol) and di-tert-
Fmoc-Ada
3A
AHCTUNGTRENNUNG
ated following the same protocol as used for the debenzylation of 3.
1.8 equiv) was added to this solution, which was stirred overnight under
argon. Then saturated aq. NaHCO3 (30 mL) and dichloromethane
(30 mL) were added and the organic phase was decanted. The aqueous
phase was again extracted with dichloromethane (2ꢄ30 mL). The joint
organic phases were dried (Na2SO4) and evaporated to yield a colorless
oil, which was purified by SiO2 column chromatography (eluent: CH2Cl2/
AcOEt/NEt3, 20:1:0.1, v/v/v) to give 3 as an oil (1.794 g, 77% yield).
[a]2D0 =ꢁ8.4 (c=1.13 in CHCl3); 1H NMR (400 MHz, CDCl3, 258C): d=
Fmoc-Ada
(tBu)2-OH was obtained as a hygroscopic glassy solid (0.833 g,
100% yield). [a]2D0 =ꢁ3.2 (c=1.00 in CHCl3); 1H NMR (400 MHz,
CDCl3, 258C): d=7.77 (d, 3J(H,H)=7.5 Hz, 2H; Ar), 7.62 (d, 3J
ACTHNUTRGENNUG ACHTUNGTRENNUNG(H,H)=
6.9 Hz, 2H; Ar), 7.42–7.38 (m, 2H; Ar), 7.34–7.23 (m, 2H; Ar), 5.89 (d,
3J
(H,H)=6.1 Hz, 1H; NH), 4.55–4.32 (m, 3H; Ha and CH2 (Fmoc)),
AHCTUNGTRENNUNG
4.24–4.20 (m, 1H; CH (Fmoc)), 3.58–3.44 (m, 4H; CH2COOtBu), 2.80
(m, 2H; Hd), 2.01–1.77 (m, 2H; Hb), 1.56–1.49 (m, 2H; Hg), 1.45 ppm
(s, 18H; tBu); 13C NMR (100 MHz, CDCl3, 258C): d=174.4 (COOH),
169.6 (COOtBu), 156.4 (CO (Fmoc)), 144.2, 144.1, 141.5 (2) (Cq
(Fmoc)], 127.9, 127.3, 125.4, 120.2 (CAr-H (Fmoc)), 82.2 (Cq (tBu)), 67.1
(CH2 (Fmoc), 54.9 (CH2COOtBu), 53.9, 53.8 (Ca and Cd), 47.4 (CH
(Fmoc)), 30.4 (Cb), 28.3 (tBu), 22.4 ppm (Cg); ES-MS: m/z (%): 583.3
(100) [M+H+]; elemental analysis calcd (%) for C32H42N2O8·H2O: C
63.98, H 7.38, N 4.66; found: C 63.94, H 7.37, N 4.64.
7.76 (d, 3J(H,H)=7.5 Hz, 2H; Ar, NH), 7.70 (d, 3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
=
12.4 Hz, 2H; CH2 (Bn)), 4.58 (m, 1H; Ha), 4.39 (m, 1H; CH2 (Fmoc)),
4.23 (m, 1H; CH2 (Fmoc)), 4.29 (m, 1H; CH (Fmoc)), 3.36, 3.27 (AB
system, JAB =17.5 Hz, 2H; CH2COOtBu), 2.86 (m, 1H; Hg); 2.71 (m,
1H; Hg), 2.08 (m, 1H; Hb), 1.95 (m, 1H; Hb), 1.46 ppm (s, 18H; tBu);
13C NMR (100 MHz, CDCl3, 258C): d=172.4 (COOBn), 170.9
(COOtBu), 156.9 (CO (Fmoc)), 144.5, 144.3, 141.5 (2C) (Cq (Fmoc)),
136.0 (Cq (Bn)), 128.7, 128.5, 128.4 (CAr-H (Bn)), 127.8, 127.3, 125.7,
120.1 (CAr-H (Fmoc)), 81.5 (Cq (tBu)), 67.3, 67.2 (CH2 (Fmoc) and CH2
(Bn)), 56.7 (CH2COOtBu), 53.1 (Cg), 50.4 (Ca), 47.4 (CH (Fmoc)), 28.9
(Cb), 28.4 ppm (tBu); ES-MS: m/z (%): 657.2 (100) [M+H+].
Peptide synthesis and purification: Both peptides were assembled man-
ually by solid-phase peptide synthesis on a Rink Amide MBHA resin
(substitution 0.57 mmolgꢁ1, 200 mg) by using Fmoc chemistry. The syn-
thesis was started by an initial deprotection of the commercial resin-
bound Fmoc with DMF/piperidine (4:1, v/v). Couplings were performed
with Na-Fmoc-protected amino acids (2 equiv), PyBOP (2 equiv), and
Fmoc-Ada
(tBu)2-OH: Compound 3 (1.75 g, 2.66 mmol) was dissolved in
DIEA (6 equiv) in DMF for 30 min. In the case of Fmoc-Ada
the coupling reaction was monitored by using the TNBS test.[52] For in-
complete reactions, second coupling with Fmoc-AdanA(tBu)2-OH
nACHTUNGTRENNUNG(tBu)2-OH
absolute ethanol (50 mL). This solution was hydrogenated overnight at
room temperature and atmospheric pressure with 10% Pd/C catalyst
(175 mg). The catalyst was filtered through Celite and the solvent was
a
CHTUNGTRENNUNG
(0.5 equiv), PyBOP (1 equiv), and DIEA (4 equiv) was performed. After
each coupling, the resin was treated with DMF/pyridine/Ac2O (7:2:1, v/v/
v) to acetylate unreacted amino groups (2ꢄ2 min). Fmoc deprotection
was achieved with DMF/piperidine (4:1, v/v) (3ꢄ3 min). The yield of
each peptide coupling reaction was determined by UV/Vis spectroscopy
(e300 =7800 Lmolꢁ1 cmꢁ1 for the piperidine adduct of dibenzofulvene).
After the final Fmoc deprotection, the peptide was acetylated as de-
scribed above. The peptide was cleaved from the resin and the side-chain
protections were removed by treatment with a cleavage cocktail consist-
ing of DTT (200 mg) dissolved in TFA/TIS/H2O (20 mL, 92:4:4, v/v/v).
After stirring for 2.5 h, the solution was evaporated to yield a yellow oil,
which was triturated several times in Et2O to yield a white powder. The
solid residue was dissolved in water/acetonitrile (9:1, v/v) and purified by
reversed-phase high-performance liquid chromatography (RP-HPLC,
Merck Purospher, 250ꢄ40 mm, 10 mm C18 particles, solvent A=H2O/
TFA (99.925:0.075, v/v), solvent B=CH3CN/H2O/TFA (90:10:0.1, v/v/v),
elution gradient from 10% A/90% B to 40% A/60% B in 15 min, flow
rate 75 mLminꢁ1, to yield the desired peptides as white powders. The
purity was checked by analytical RP-HPLC (Merck Purospher STAR
endcapped, 4.6ꢄ250 mm, 5 mm C18 particles, solvent A=H2O/TFA
(99.925:0.075, v/v), solvent B=CH3CN/H2O/TFA (90:10:0.1, v/v/v, elu-
tion gradient from 10% A/90% B to 40% A/60% B in 15 min, flow rate
1 mLminꢁ1, UV monitoring at 280 nm, see Figure S3).
evaporated to obtain Fmoc-Ada2ACTHNUTRGNE(NUG tBu)2-OH as a hygroscopic glassy solid
(1.55 g, 99% yield). [a]2D0 =+13.9 (c=1.10 in CHCl3); 1H NMR
3
(400 MHz, CDCl3, 258C): d=7.76 (d, J
A
3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(H,H)=7.1 Hz, 2H; Ar), 7.39 (dd (app. t), 3J
7.31 (dd (app. t), 3J
G
ACHTUNGTRENNUNG
1H; NH), 4.47 (m, 1H; Ha), 4.35 (m, 2H; CH2 (Fmoc)), 4.22 (m, 1H;
CH (Fmoc)), 3.45 (s, 4H; CH2COOtBu), 2.98 (m, 2H; Hg), 2.02 (m, 2H;
Hb), 1.47 ppm (s, 18H; tBu); 13C NMR (100 MHz, CDCl3, 258C): d=
173.5 (COOH), 169.2 (COOtBu), 156.3 (CO (Fmoc)), 144.2, 141.5 (Cq
(Fmoc)), 127.9, 127.3, 125.4, 120.2 (CAr-H (Fmoc)), 82.7 (Cq (tBu)), 67.3
(CH2 (Fmoc)), 55.3 (CH2COOtBu), 54.5(Cg), 52.7 (Ca), 47.4 (CH
(Fmoc)), 29.7 (Cb), 28.3 ppm (tBu); ES-MS: m/z (%): 569.08 (100)
[M+H+]; 591.2 (92) [M+Na+]; elemental analysis calcd (%) for
C31H40N2O8·1= H2O: C 64.16, H 7.11, N 4.77; found: C 64.45, H 7.15, N
2
4.93.
Fmoc-Ada3ACHTUNGTRENNUNG
(tBu)2-OBn (5): Compound 4[26] (1.593 g, 2.92 mmol) was dis-
solved in CH2Cl2 (40 mL). Trifluoroacetic acid (20 mL) was added drop-
wise to this stirred solution. After 1.5 h, TLC analysis showed the disap-
pearance of 4. The solvents were evaporated in vacuo. ES-MS analysis of
the resulting crude oil showed the molecular peak belonging to Fmoc-
Orn-OBn (m/z=445.3 [M+H+]). This oil was dissolved in DMF
(20 mL). tert-Butyl bromoacetate (1.3 mL, 2.27 g, 11.69 mmol, 4 equiv),
KHCO3 (5.85 g, 58.6 mmol, 20 equiv), and NBu4I (1.08 g, 2.92 mmol,
1 equiv) were added. The resulting mixture was stirred for 7 h at 358C.
Water (100 mL) and Et2O (100 mL) were added to the reaction mixture.
Ac-WAda2PGAda2G-NH2 (P2): Yield of the on-resin synthesis (UV):
92%. Isolated mass: 58.5 mg (isolated yield assuming that the solid is
P2·2TFA: 48%). (+)ES-MS: m/z: 889.3 [M+H+], 445.3 [M+H22+];
7466
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 7456 – 7469

LanthanideACHTUNGTRENNUNG(III) Complexes with Two Hexapeptides
FULL PAPER
1
(ꢁ)ES-MS: m/z: 887.3 [MꢁHꢁ], 443.2 [Mꢁ2H2ꢁ]; RP-HPLC: tr =
points in the time domain. 2D H NMR spectra were recorded at various
temperatures in H2O/D2O (9:1, v/v) using WATERGATE or presatura-
tion solvent suppression or in D2O with presaturation suppression of re-
sidual HOD. Spectra were acquired in phase-sensitive mode with TPPI
for quadrature detection in the indirect dimension using 2048ꢄ512 matri-
ces over a 5000 Hz spectral width. TOCSY experiments were performed
by using a MLEV-17 spin-lock sequence with a mixing time of 70 ms.
ROESY and NOESY/EXSY experiments were recorded with a mixing
time of 300 ms (3300 Hz spin lock for ROESY). 2D NOESY NMR spec-
10.8 min, 97% purity.
Ac-WAda3PGAda3G-NH2 ACHTNURTGENUNG
(P3:): Yield of the on-resin synthesis (UV):
77%. Isolated mass: 43.5 mg (isolated yield assuming that the solid is
P3·2TFA: 35%). (+)ES-MS: m/z: 917.4 [M+H+], 459.4 [M+H22+];
(ꢁ)ES-MS: m/z: 915.5 [MꢁHꢁ], 457.3 [Mꢁ2H2ꢁ]; RP-HPLC: tr =
10.6 min, 97% purity.
Preparation of aqueous solutions: 5 mm metal solutions, used as stock
solutions in the luminescence, CD, and ES-MS experiments, were pre-
pared from the corresponding chloride salts (LaCl3·7H2O, EuCl3·6H2O,
and TbCl3·6H2O) in pure H2O. Their precise concentration was obtained
tra for structure determination were recorded on
800 MHz spectrometer equipped with a H/2H/15N/13C cryogenic probe. A
a Varian Vnmrs
1
by titration with
(Fisher Chemicals) in the presence of a colorimetric indicator. For NMR
analysis in D2O, a solution of anhydrous La(OTf)3 in pure D2O was pre-
a 5 mm volumetric ethylenediaminetetraacetic acid
mixing time of 250 ms was used.
NMR structure refinement: NMR solution structures were obtained with
CNSsolve[47] (version 1.1) following standard refinement protocols and
using the “protein-allhdg” forcefield for natural amino acids. For unnatu-
ral amino acid side-chains and the lanthanum coordination sphere
custom topology and parameter files were generated. The parameters
were chosen as follows. For Ada2 side-chains, the parameters for Ca, Cb,
Cg, and bound hydrogen atoms were set identical to those of Ca, Cb,
and Ce of l-lysine. Parameters for the bond lengths of the Nd, Ce, and
Cz atoms were adapted from glycine parameters. Parameters for the La–
AHCTUNGTRENNUNG
pared and titrated similarly. Peptide solutions were prepared freshly
before use and the precise peptide concentration was determined by UV
analysis (e280 =5690 Lmolꢁ1 cmꢁ1 owing to the presence of the Trp resi-
due). A 5 mm NTA solution to be used in competition experiments was
prepared in pure water from solid nitriloacetic acid (Riedel-de-Haꢅn)
and its precise concentration was determined pH-metrically. HEPES
buffer was prepared by dissolving solid 4-(2-hydroxyethyl)-piperazine-1-
ethanesulfonic acid (Fluka) in H2O (or D2O) and by adjusting the pH (or
pD) to 7.0 with KOH (or NaOD).
ꢁ
ꢁ
ꢁ
ꢁ
N and La–O distances, as well as the Cg–Nd Ce, Cz Oh1 La, and Nd
ꢁ
ꢁ
ꢁ
ꢁ
Ce Cz angles, and the Ce Cz Oh1 La torsion angle were obtained
from high-resolution X-ray data of La3+ complexes containing the N-al-
kylaminodiacetate moiety referenced in the Cambridge Structural Data-
base (CSD). The structure search and visualization were performed with
CSD ConQuest 1.10.[54] Ten relevant structures were found in the data-
base and the average structural parameters were extracted with CSD
Vista 2.1. CSD references: ASEMUG, ASEMUG01, GEQFOX,
HETALA, HETALA01, KIBZUR, SOHNOS, TISQOB, XALSOS,
Circular dichroism: CD spectra were recorded at 258C on a Applied
Photophysics Chirascan spectrometer in a cell with a path length of 1 cm.
The peptide concentration was ~20 mm in water and the pH was adjusted
with KOH. All the spectra were obtained from 320 to 190 nm with a
1 nm interval, a time constant of 2 s, a bandwidth of 1 nm, and three
scans. The CD spectra are reported in molar ellipticity per a-amino acid
residue ([V] in units of degcm2 molꢁ1; [V]=qobs
/AHCUTNGERNUN(G 10lcn), with qobs the ob-
served ellipticity in m8, l the optical path length of the cell in cm, c the
peptide concentration in molLꢁ1, and n the number of residues in the
peptide (n=6 in the present case)).
ꢁ
ZAMHEA. Average parameters (standard deviation) (units: ꢃ,8): La N
ꢁ
ꢁ ꢁ
ꢁ ꢁ
2.830 (0.038), La O 2.522 (0.042),C N C 110.52 (1.47), C O La 126.68
ꢁ ꢁ
ꢁ ꢁ ꢁ
(2.08), N C C 112.37 (2.06), C C O La 1.5 (25) (was set to 0). Similar-
Luminescence: Luminescence spectra were recorded on a LS50B spectro-
fluorimeter connected to a computer equipped with FLWINLAB 2.0.
The measurements were performed at 298 K. Trp fluorescence titrations
were performed with 280 nm excitation (excitation slit: 3.0 nm). The
emission slit was adjusted (3.5–4.5 nm) to avoid signal saturation. Tb
phosphorescence spectra were recorded upon Trp excitation (280 nm)
after a 0.05 ms delay and with a 0.5 ms gate time. The excitation and
emission slits were 10.0 and 15.0 nm, respectively. A 430 nm cut-off filter
was used. Conditional stability constants were extracted from the spectral
data by using SPECFIT.[53] Experiments were performed in triplicate to
ensure reproducibility. The reported conditional stability constants are
averages of the three experimental values. Lifetime measurements were
performed on peptide samples containing 0.5 equiv TbCl3 to ensure ex-
clusive contribution from the mononuclear complexes. The emission in-
tensities of the most intense Tb3+ emission band were recorded after ex-
citation at 280 nm with a first delay of 0.05 ms, a delay increment of
0.05 ms, and the number of measurements adjusted to have a final delay
of >4t.
ꢁ
ꢁ
ly, Cz Oh1 and Cz Oh2 (distances between metal bound and metal un-
bound oxygen atoms in the Ada2 side-chain, respectively) were adapted
from the three published structures containing monodentate and not
ꢁ
bridging La carboxy moieties. CSD references: TISQOB, XALSOS,
ꢁ
ZAMHEA. Average parameters (standard deviation) (units: ꢃ,8): Cz
3+
ꢁ
Oh1 1.26 (0.09), Cz Oh2 1.245 (0.01). The La nonbonded radius was
determined from crystallographic data,[55] as for the other ions already
implemented in CNSsolve. Coordinated water molecules were not includ-
ed explicitly in the model. However, the ionic radius of La3+ for a coordi-
nation number of 9 was used.
As spin diffusion generated some artifacts in the NOESY spectrum (the
presence of antiphase components in the NOESY spectra reported in
Figures S9 and S10), a tROESY spectrum was recorded to determine
whether some signals were artifactual in nature.[46] Some Trp
ACTHNUGRTENUNG(indole)-Pro
correlations were indeed shown to be absent and thus the corresponding
restraints were removed from the calculations. Upper and lower limits
for distance constraints were set to ꢂ15% of the H–H distances obtained
by integration of the 2D NOESY spectra (250 ms). rꢁ6 averaging was
chosen for the NOE restraints. Geminal Pro Hd1/Hd2, Ada22 Hb1/Hb2,
Ada25 Hb1/Hb2, and Gly4 Ha1/Ha2 distances (taken as 1.8 ꢃ) as well as
Pro Ha/Hb1 (2.4 ꢃ) were used as references for distance calibration.
Mass spectrometry of the Eu3+ complexes: Eu3+ was chosen as a repre-
sentative Ln3+ ion because of its characteristic isotopic signature (151Eu
47.8%, 153Eu 52.2%). 60 mm peptide solutions were prepared in 20 mm
ammonium acetate buffer (pH 7). The 5 mm EuCl3 stock solution was
added to prepare aliquots containing 0, 0.5, 1, and 2 equiv of EuCl3 per
peptide. For competition between P2 and P3, a solution containing both
peptides (30 mm each) was prepared by mixing equal volumes of the origi-
nal peptide solutions and 1 equiv EuCl3 was added. Mass spectra were re-
corded on an LXQ-type THERMO SCIENTIFIC spectrometer equipped
with an electrospray ionization source and a linear trap detector. Solu-
Four 3J coupling constants (Gly4 JNH,Ha and Trp1 JHa,Hb) measured in
the 1H NMR spectra were included as well as restraints with standard
3
3
CNSsolve Karplus parameters (Gly) or published Karplus parameters for
[56]
3JHa,Hb
.
Pseudo-atom corrections were applied to methyl and overlap-
ping or nonstereospecifically assigned methylenes.[57] Dynamics experi-
ments were performed with a hot phase at 2000 K with 5000 0.003 ps
steps followed by a slow cooling phase with 5000 0.005 ps steps with 5 K
cooling per step. Final minimizations were performed with the Powell al-
gorithm (200 steps, 100 cycles). An ensemble of 50 structures was gener-
ated. None of the structures displayed NOE violations greater than
0.4 ꢃ. The 10 lowest-energy structures were visualized and aligned (back-
bone atoms) by using PyMol v. 0.99 (DeLano Scientific LLC).[58]
tions were injected into the spectrometer at a flow rate of 5 mLminꢁ1
.
The ionization voltage and capillary temperature were about 2 kV and
2508C, respectively.
Peptide NMR spectroscopy: NMR experiments for apopeptide and com-
plex characterization were recorded on a 500 MHz Bruker Avance spec-
trometer equipped with a BBI probe with a triple-axis gradient field.
1H NMR spectra were recorded with 12 ppm windows and 32k data
Chem. Eur. J. 2009, 15, 7456 – 7469
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7467

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