Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids

Article abstract of DOI:10.1021/ic300448y

The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P²², which incorporates two unnatural amino acids Ada₂ with aminodiacetate chelating arms, showed picomolar affinity for Tb³⁺. To design peptides with higher denticity, expected to show higher affinity for Ln³⁺, we synthesized the novel unnatural amino acid Ed3a₂ which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a₂(tBu)₃-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P^HD2 (Ac-Trp-Ed3a ₂-Pro-Gly-Ada₂-Gly-NH₂) and P^HD5 (Ac-Trp-Ada₂-Pro-Gly-Ed3a₂-Gly-NH₂) led to octadentate Tb³⁺ complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a₂ in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P^HD5 promotes the formation of polymetallic species in excess of Ln³⁺, P^HD2 forms exclusively the mononuclear complex. The octadentate coordination of Tb³⁺ by both P^HD leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P^HD2 forms a unique octadentate Tb³⁺ complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP²² complex by a factor of 4.

Full text of DOI:10.1021/ic300448y

Article
pubs.acs.org/IC
Femtomolar Ln(III) Affinity in Peptide-Based Ligands Containing
Unnatural Chelating Amino Acids
Agnieszka Niedzwiecka, Federico Cisnetti,† Colette Lebrun, and Pascale Delangle*
́
Service de Chimie Inorganique et Biologique (UMR_E 3 CEA UJF), INAC, Commissariat a
38054 Grenoble Cedex, France
̀
l′Energie Atomique, 17 rue des martyrs,
S
* Supporting Information
ABSTRACT: The incorporation of unnatural chelating amino acids in
short peptide sequences leads to lanthanide-binding peptides with a higher
stability than sequences built exclusively from natural residues. In particular,
the hexadentate peptide P22, which incorporates two unnatural amino acids
Ada2 with aminodiacetate chelating arms, showed picomolar affinity for
Tb3+. To design peptides with higher denticity, expected to show higher
affinity for Ln3+, we synthesized the novel unnatural amino acid Ed3a2
which carries an ethylenediamine triacetate side-chain and affords a
pentadentate coordination site. The synthesis of the derivative Fmoc-
Ed3a2(tBu)3-OH, with appropriate protecting groups for direct use in the
solid phase peptide synthesis (Fmoc strategy), is described. The two high
denticity peptides PHD2 (Ac-Trp-Ed3a2-Pro-Gly-Ada2-Gly-NH2) and PHD5
(Ac-Trp-Ada2-Pro-Gly-Ed3a2-Gly-NH2) led to octadentate Tb3+ complexes with femtomolar stability in water. The position of
the high denticity amino acid Ed3a2 in the hexapeptide sequence appears to be critical for the control of the metal complex
speciation. Whereas PHD5 promotes the formation of polymetallic species in excess of Ln3+, PHD2 forms exclusively the
mononuclear complex. The octadentate coordination of Tb3+ by both PHD leads to total dehydration of the metal ion in the
mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying
polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the
novel peptide PHD2 forms a unique octadentate Tb3+ complex with femtomolar stability in water and an improvement of the
luminescence properties with respect to the trisaquo TbP22 complex by a factor of 4.
ics.1,20,21 Other native sequences may be modified to generate
INTRODUCTION
■
Ln-binding peptides. For instance a “lanthanide-finger” was
recently designed by replacing the cysteine and histidine
residues involved in the metal coordination of a zinc finger
(Cys2His2) by aspartic and glutamic acids to generate a
tetracarboxylate peptide ligand (Asp2Glu2).22 Another strategy
consists in preorganizing the side-chains of coordinating amino
acids in de novo designed peptides to afford well-defined Ln3+
coordination sites. This methodology was applied to the design
of a cyclodecapeptide with a β-sheet structure which allows the
preorientation of four amino acid side-chains of glutamic or
aspartic acids to coordinate the Ln3+ ion. Interestingly, the
corresponding Gd3+ complex combines the relaxation proper-
ties of Gd3+ with the high hydrophilicity of the peptide scaffold
to provide large second-sphere contributions to water
relaxation.23,24 Natural amino acids with carboxylates, pheno-
lates, or amides as metal-binding groups, show only a moderate
affinity for Ln3+ ions. Therefore, peptide sequences built
exclusively from these natural residues exhibit an affinity for
Ln3+ in the micromolar range16,22,24,25 or for the best Ln-
binding tags, in the low nanomolar range.19 For either in vitro
Lanthanide-binding peptides are tremendously powerful tools
to investigate biologically relevant species such as proteins or
DNA.1 Indeed, they combine the unique spectroscopic and
magnetic properties of lanthanide trivalent cations with the
characteristics of the peptide scaffold, such as high solubility in
water, versatile insertion in proteins by standard peptide
synthesis or molecular biology techniques, and most
importantly selective recognition of biological partners. The
lanthanide series offers a variety of metal ions with diverse
physical properties: for instance Eu3+ and Tb3+ complexes find
applications as luminescent sensors because of their long-lived
luminescence2−5 and Gd3+ complexes are commonly used as
magnetic resonance imaging contrast agents, thanks to the high
spin value and long electronic relaxation time of the Gd3+
ion.6−8
Ln3+ cations bind to proteins in Ca2+-binding sites,9,10
therefore native protein sequences such as the Ca-binding EF-
hand motifs were first selected to generate Ln-binding
peptides.11−16 These motifs were subsequently optimized by
screening methods to select those combining both stability and
efficient luminescence sensitization in the Tb-peptide com-
plexes.17−19 These optimized sequences have been appended to
proteins to investigate their structure, function and dynam-
Received: February 28, 2012
Published: April 10, 2012
© 2012 American Chemical Society
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Article
methyloxycarbonyl), HEDTA (2-[2-[bis(carboxymethyl)amino]ethyl-
(2-hydroxyethyl)amino] acetic acid), tBu (tert-butyl), TFA (trifluoro-
acetic acid), TLC (Thin Layer Chromatography).
General Remarks. Reagents and solvents were purchased from
Aldrich and Novabiochem and used without further purification unless
specified. Organic products were characterized by NMR using a Varian
Mercury 400 MHz spectrometer at 298 K. Peptides PHD2 and PHD5
were characterized in light water containing 10% D2O by NMR using a
Bruker Avance 500 MHz. Analytical and preparative peptide RP-
HPLC were performed with LaChrom and LaPrep systems.
The 3 mM aqueous solutions of lanthanides were prepared by
dissolving corresponding salts (TbCl3·6H2O, Tb(OTf)3, EuCl3·6H2O)
in deionized water. The precise concentrations were determined by
colorimetric titration with 5 mM 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
use. The precise concentrations were established by UV absorption at
or in vivo applications, these short Ln-binding sequences are
inserted in more complex architectures for targeting or
detection, and therefore the affinity for the lanthanide ion
must be sufficient to avoid leaching of the cation and its
coordination in other metal binding-sites such as Ca2+-binding
EF motifs.
Therefore, the design of high affinity Ln-binding peptides is a
challenging question, which we have chosen to address by using
unnatural amino acids carrying high affinity chelating groups.
Recently, we developed short peptide sequences containing two
unnatural amino acids bearing tridentate aminodiacetate
chelating groups (Adan). For instance the peptide P22 (see
Scheme 1) formed stable and well-defined lanthanide
Scheme 1. Design of Lanthanide-Binding Peptides
Incorporating Non Natural Chelating Amino Acids
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-ethanesul-
fonic acid (Fluka) and potassium chloride in deionized H2O (or D2O).
The pH (or pD) was adjusted to 7.0 with KOH (or NaOD). A 5 mM
HEDTA solution to be used in competition experiments was prepared
in pure water from solid HEDTA (Riedel-de-Haen), and its precise
̈
concentration was determined pH-metrically.
S y n t h e s i s . T h e a l d e h y d e
2
a n d N , N , N ′ - t r i s -
(tertbutyloxycarbonylmethyl)ethylenediamine 1 were synthesized
following published procedures.27,29
Compound (3). The aldehyde 2 (0.496 mmol, 1 equiv, 220 mg)
and N,N,N′-tris(tertbutyloxycarbonylmethyl)ethylenediamine (0.893
mmol, 1.8 equiv, 359.5 mg) were dissolved in 1 mL of 1,2-
dichloroethane under Ar atmosphere. After addition of NaBH(OAc)3
(183.3 mg, 0.893 mmol, 1.8 equiv), the resulting suspension was
vigorously stirred. The progress of coupling was monitored by TLC
analysis (CH2Cl2/AcOEt v/v = 9/1, Rf = 0.2), which indicated
complete conversion after 4 h. Dichloromethane (20 mL) and
saturated aqueous NaHCO3 (20 mL) were added, and the organic
phase was decanted. The aqueous phase was further extracted with
CH2Cl2 (2 × 20 mL). The combined organic phases were dried over
Na2SO4 and evaporated. The resulting crude product was purified by
silica-gel column chromatography (elution gradient: CH2Cl2/AcOEt,
v/v = 19/1 to 9/1) to yield Fmoc-Ed3a2(tBu)3-OBn as a colorless oil
20
1
(353 mg, 87% yield). [α]D = +10.8 (c = 1.13 in CHCl3). H NMR
(400 MHz, CDCl3, 298 K): δ(ppm) = 7.75 (2H, d, 3J(H,H) = 7.5 Hz,
Ar); 7.63 (2H, t, J(H,H) = 7.6 Hz, Ar); 7.39−7.35 (4H, m, ArFmoc);
3
7.29 (5H, m, ArBn); 7.03 (1H, d, 3J(H,H) = 8.0 Hz, NH(Fmoc)); 5.17,
5.14 (2H, AB system, JAB = 12.4 Hz, CH2(Bn)); [4.47 (2H, m), 4.30−
4.21 (2H, m), Hα, CH2 (Fmoc) and CH(Fmoc)]; 3.43, 3.31 (4H, AB
system, JAB = 17.5 Hz, NηCH2COOtBu); 3.26, 3.18 (2H, AB system,
JAB = 17.0 Hz, NδCH2COOtBu); 2.87−2.71 (4H, m, Hε, Hζ)); 2.76−
2.54 (2H, m, Hγ)); 2.05−1.97 (2H, m, Hβ); 1.45 (9H, s,
NδCH2COOtBu); 1.43 (18H, s, NηCH2COOtBu). 13C NMR (100
MHz, CDCl3, 298 K): δ(ppm) = 172.5 (COOBn); 170.9 (1C), 170.8
(2C) (COOtBu); 156.8 (CO(Fmoc)); 144.3 (2C), 141.7 (2C) (Cq
(Fmoc)); 136.0 (Cq(Bn)); 128.7 (2C), 128.5 (2C), 128.4 (CAr-
H(Bn)); 127.8 (2C), 127.3 (2C), 125.6, 125.4, 120.2 (2C) (CAr-
H(Fmoc)); 81.1 (3C) (Cq(tBu)); 67.1, 66.9 (CH2(Bn),
(CH2(Fmoc)); 56.2 (NδCH2COOtBu); 55.9 (2C) (NηCH2COOtBu);
53.1 (Cγ); 52.2, 51.3 (Cε, Cξ); 49.9 (Cα); 47.3 (CH(Fmoc)); 28.6
(Cβ); 28.4 (9C) (tBu). ES+-MS (intensity %): m/z = 816.2 (100) [M
+H]+, 838.3 (96) [M+Na]+.
complexes with stabilizing peptide secondary structure
interactions: the stability of TbP22 was found in the picomolar
range.26−28 In this compound, the sequence between the two
tridentate chelating aminoacids was chosen to favor the
formation of a β-turn, which leads to the hexadentate TbP22
complex with 3 Tb-bound water molecules (qTb = 3) in water.27
In this paper, we report the design of peptides forming totally
dehydrated Tb3+ complexes with a femtomolar stability in
water. The two high denticity hexapeptides PHD2 and PHD5
were obtained by replacing one tridentate Ada2 residue in P22
with the new pentadentate unnatural amino acid Ed3a2, which
carries an ethylenediamine triacetate side-chain (Scheme 1).
The synthesis of the novel amino acid derivative Fmoc-
Ed3a2(tBu)3-OH with appropriate protecting groups for direct
use during the solid phase peptide synthesis is described, as well
as the properties of the Tb3+ complexes in water.
Fmoc-Ed3a2(tBu)3-OH. Compound 3 (350 mg, 0.43 mmol) was
dissolved in 50 mL of absolute ethanol and hydrogenated under
pressure (3 bar, overnight) over 50 mg of 10% Pd/C. 296 mg of
resulting Fmoc-Ed3a2(tBu)3-OH (95% yield) were recovered after
filtration of the reaction mixture through a Celite pad and solvent
evaporation. [α]D20 = +4.3 (c = 1.03 in CHCl3). 1H NMR (400 MHz,
3
EXPERIMENTAL SECTION
CDCl3, 298 K): δ(ppm) = 7.76 (2H, d, J(H,H) = 7.5 Hz, Ar); 7.63
■
3
3
Abbreviations. AcOEt (ethyl acetate), AcONH4 (ammonium
acetate), Ar (aromatic), Bn (benzyl), Bu (butyl); Fmoc (9-fluorenyl-
(2H, t, J(H,H) = 7.5 Hz, Ar); 7.40 (2H, t, J(H,H) = 7.5 Hz, Ar);
3
3
7.32 (2H,d, J(H,H) = 7.5 Hz, Ar); 6.53 (1H, d, J(H,H) = 5.7 Hz,
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NH(Fmoc)); [4.38−4.28 (2H, m), 4.22 (1H, m) CH(Fmoc),
CH2(Fmoc)]; 4.14, 4.09 (2H, AB system, JAB = 17.3 Hz,
NδCH2COOtBu); 4.40 (1H, m, Hα); 3.44 (4H, s, NηCH2COOtBu);
3.42−3.34 (4H, m, Hγ, Hε or Hζ); 3,14 (2H, m, Hε or Hζ); 2.31
(2H, m, Hβ); 1.49 (9H, s, NδCH2COOtBu); 1.45 (18H, s,
NηCH2COOtBu). 13C NMR (100 MHz, CDCl3, 298 K): δ(ppm) =
173.5 (COOH); 171.0 (2C), 166.4 (1C) (COOtBu); 156.6 (CO-
(Fmoc)); 144.4, 144.2, 141.7 (2C) (Cq (Fmoc)); 128.1 (2C), 127.6
(2C), 125.8, 125.7, 120.3 (2C) (CAr-H(Fmoc)); 84.3 (1C), 82.4 (2C)
(Cq(tBu)); 67.5, (CH2(Fmoc)); 56.2 (3C) (NCH2COOtBu); 53.8
(Cα); 54.4, 50.5, 52.5 (Cγ, Cε, Cξ); 47.5 (CH(Fmoc)); 28.5 (6C),
28.4 (4C) (tBu, Cβ). ES+-MS (intensity %): m/z = 726.3 (40) [M
+H]+, 748.3 (100) [M+Na]+.
The enantiopurity of Fmoc-Ed3a2(tBu)2-OH was checked by
analyzing the NMR spectrum of the coupling product with the chiral
amine α-methylbenzylamine either in enantiopure (S) or in racemic
form, as described previously for Adan amino acids.27,28 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-Ed3a2(tBu)2-OH occurred during the
synthesis (assuming that 2% diastereomeric product would be detected
by NMR, ee > 96%).
intense Tb3+ emission band were recorded after excitation 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 >4τ.
The quantum yield measurements were performed according to
Chauvin et al.30,31 in a 1 cm path length luminescence cell at 25 °C on
aqueous solutions of TbPHD (50 μM, A280 = 0.3), [Tb(dpa)3]3− (0.107
mM, A280 = 0.295) in HEPES buffer (10 mM, 0.1 M KCl, pH = 7.0).
The standard solutions of [Tb(dpa)3]3− was prepared by mixing Tb3+
with dipicolinic 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 intensity, and A is
the absorbance of the solution at the excitation wavelength λ. The
value of the quantum yields ΦTb(dpa)3 = 18.4 2.5% (0.107 mM, A280
= 0.295) was applied to the calculations assuming that the change of a
buffer from Tris (0.1 M, pH = 7.45) to HEPES (10 mM, 0.1 M KCl,
pH = 7.0) has affected the reference values in the error range.30,31
The conditional stability constants of TbPHD2 and TbPHD5 were
measured by recording the Tb-centered luminescence of these
complexes (16 μM) in HEPES buffer (10 mM, 1 M KCl, pH = 7.0)
upon addition of aliquots of a HEDTA solution. Forty minutes waiting
between two successive points ensured reaching the thermodynamic
equilibrium. The data obtained from these competitions were fitted
with a fixed value of the conditional stability constant of the reference
Tb(HEDTA), log β11pH 7= 12.6(5), determined by taking into account
the known equilibrium constants of HEDTA and global stability
constant of Tb(HEDTA).32
Peptide Synthesis and Purification. These were performed as
previously described for Ada2-based peptides.27
PHD2. Ac-Trp-Ed3a2-Pro-Gly-Ada2-Gly-NH2, Yield of the on-resin
synthesis (UV): 68%, Isolated mass: 49.0 mg (isolated yield assuming
that the solid is PHD2·3TFA: 50%). ES+-MS (AcONH4, pH = 7.0): m/
z = 990.3 [M+H]+, ES−-MS (AcONH4, pH = 7): m/z = 988.3 [M-
H]−; RP-HPLC: tR = 8.4 min, >95% purity (NMR).
PHD5. Ac-Trp-Ada2-Pro-Gly-Ed3a2-Gly-NH2, Yield of the on-resin
synthesis (UV): 24%, Isolated mass: 17.2 mg (isolated yield assuming
that the solid is PHD5·3TFA: 17%). ES+-MS (AcONH4, pH = 7.0): m/
z = 990.5 [M+H]+, ES−-MS (AcONH4, pH 7): m/z = 988.5 [M-H]−;
RP-HPLC: tR = 8.4 min, >95% purity (NMR).
RESULTS
■
Synthesis of the Octadentate Hexapeptides. The novel
chelating unnatural amino acid Ed3a2 was synthesized with
protections compatible with Fmoc/tBu strategy in solid phase
peptide synthesis, that is, in the form Fmoc-Ed3a2(tBu)3-OH
(Scheme 2). The synthesis of Fmoc-Ed3a2(tBu)3-OH consists
Electrospray Mass Spectrometry of Eu 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 EuPHD
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 were injected into the spectrometer at a flow rate of
5 μL min−1. 2 kV voltage and 250 °C capillary temperature were
applied.
Scheme 2. Synthesis of the High Denticity Peptides PHD2 and
PHD5
Circular Dichroism Measurements. The CD spectra were
collected on an Applied Photophysics Chirascan spectrometer in a 1
cm path length quartz cell at 25 °C. Twenty μM solutions of free
peptides and Tb-peptide complexes (0 to 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 1 nm interval, 2 s time constant, 1 nm bandwidth,
and three scans. The CD signal was reported in molar ellipticity per α-
amino acid residue ([θ] in units of deg cm2 mol−1; [θ]= θobs/(10lcn),
where θobs is the observed ellipticity in m°, l is the optical path length
of the cell in cm, c is the peptide concentration in mol L−1, and n is the
number of residues in the peptide (n = 6 in this case)).
Luminescence Measurements. The luminescence measure-
ments were performed on a LS50B spectrofluorimeter connected to
a computer equipped with 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) or 545 nm (Tb-centered lumines-
cence). For fluorescence measurements, 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/emission slit: 1 ms/10 nm/5 nm and 0.5 ms/10 nm/15
nm for Tb3+ and Eu3+, respectively. The 430 nm cutoff filter was used
during all tryptophan-sensitized luminescence measurements.
Lifetime measurements were performed on peptide samples
containing 0.9 equiv of TbCl3 to ensure exclusive contribution from
the mononuclear complexes. The emission intensities of the most
in two steps from the aldehyde 2, which we previously
described.27 Compound 1 was first prepared according to
Micklitsch et al.29 and then coupled to aldehyde 2 to afford the
benzylated amino acid 3, which was deprotected by catalytic
hydrogenation. High denticity peptides were then manually
assembled by solid phase peptide synthesis starting from Fmoc-
Ada2(tBu)2-OH, Fmoc-Ed3a2(tBu)3-OH and commercial Fmoc
derivatives of natural amino acids, on Rink amide MBHA resin
using standard conditions as performed previously for P22.27
No significant byproducts were detected by 1H NMR, showing
that the unnatural amino acid Fmoc-Ed3a2(tBu)3-OH displayed
normal reactivity in the on-resin synthetic steps. The pure
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peptides were obtained after preparative HPLC purification.
Their identity and purity were confirmed by electrospray
ionization mass spectrometry and 1H NMR (Supporting
Information, Table S1 and S2).
Tb-centered 5D4→7FJ emission bands (J = 3−6) build up upon
addition of Tb3+ to the free peptide in water at pH 7 as seen in
Supporting Information, Figure S5. The luminescence spectra
are not sensitive to the presence of water as seen in Figure 2,
Speciation of the Ln Complexes of PHD2 and PHD5
.
Several analytical techniques were used to investigate the
speciation of metal complexes formed with the two octadentate
peptides PHD2 and PHD5. Electrospray ionization mass
spectrometry (ES-MS) gives a qualitative insight into the
speciation of metal-peptide complexes in solution. Circular
dichroism (CD) is sensitive to peptide conformational changes
upon complexation. The tryptophan (Trp) residue in position
1 is a Tb3+ sensitizer, and we demonstrated that the
fluorescence evolution of the Trp indole moiety upon
Tb3+complexation was dominated in our systems by the
modification of the indole environment.27,28 These three
techniques point to different behaviors of the two peptides.
Compound PHD2 forms exclusively a monometallic complex
LnPHD2, which is the only species detected in the ES-MS
spectra of the Eu3+ complexes either as a monocation
Figure 2. Tb-centered luminescence of 30 μM TbPHD2 in D2O/H2O
mixtures HEPES buffer (10 mM, pH = 7.0).
([EuPHD2-2H]+, m/z = 1140.3) or as a dication ([EuPHD2
-
H]2+, m/z = 570.8) (see Supporting Information, Table S3).
The formation of the Tb3+ complex induces a decrease of Trp
fluorescence which stabilizes after one metal equivalent (Figure
1) as the evolution of the CD signal (Supporting Information,
which shows the perfect superimposition of spectra acquired in
light or heavy water, in which there is no deactivation by the
potentially coordinated O−D vibrators
The luminescence lifetimes of Tb3+ in the mononuclear
complexes TbPHD in water at pH 7 have very similar values
(τH2O = 2.3 ms). These values compare well with luminescence
lifetimes measured for totally dehydrated octadentate Tb3+
complexes, for instance Barbara Imperiali’s Tb-binding peptides
(τH2O = 2.6 ms)19 or octadentate cages of Tb3+ 2-
hydroxyisophthalamides (2.45 ms < τH2O < 2.6 ms).33
The corresponding values in D2O were determined by the
extrapolated limit of the luminescence decay rates in solutions
of increasing D2O molar fractions tending to a H2O-free
solution (Supporting Information, Figure S6). The hydration
numbers were calculated from the lifetimes in heavy and light
water according to Parker’s equation (Table 1).34 They are very
close to 0 and confirm the total dehydration of the TbPHD
complexes and therefore the absence of deactivation because of
inner-sphere water molecules. The octadentate nature of the
peptide ligands in the complexes is confirmed by these
measurements.
Figure 1. Fluorescence of PHD2 (45 μM) in HEPES buffer (10 mM,
KCl 0.1 M, pH = 7.0) upon excitation at 280 nm, with addition of
TbCl3 (0 to 2 equiv).
Figure S1). Whereas all these data demonstrate the formation
of a unique LnPHD2 monometallic complex, the same
experiments performed with PHD5 show the transformation of
the monometallic species TbPHD5 into polymetallic adducts in
excess of metal. Indeed Trp fluorescence (Supporting
Information, Figure S2) and CD spectra (Supporting
Information, Figure S3) clearly evolve in excess of metal ion,
and polymetallic complexes are also detected in the ES-MS
spectra in particular the bimetallic adduct ([Eu2PHD5-4H]2+, m/
z = 644.6) (Supporting Information, Figure S4). This indicates
that PHD5 sequence allows an independent coordination by the
two chelating amino acids, which leads to polymetallic species,
at the expense of the mononuclear complex formation.
Interestingly, the sequence of PHD2, which differs only from
PHD5 by the positions of the two chelating amino acids with
respect to the Pro-Gly sequence, promotes exclusively an
octadentate monometallic complex which does not evolve to
polymetallic species in excess of Ln3+ to give bimetallic
complexes.
The quantum yields were measured as previously described28
following the procedure of Bunzli et al., which uses the Tb
̈
tris(dipicolinate) complex as a secondary standard, which
excitation band perfectly overlaps with the excitation band of
tryptophan in our peptides.30,31 The quantum yields are 1% for
both TbPHD complexes which are significantly enhanced with
respect to the TbP22 complex (Φ = 0.24%). Of course these
values are still low because the energy transfer from Trp to
Tb3+ is not the most efficient. Anyway, Tb3+ is sufficiently
sensitized upon binding to the peptides as previously described
in other Ln-binding peptides or proteins to allow detection in
the nM range.10,17−19,35 The second-generation peptides PHD
provide a gain in luminescence efficiency of a factor 4 with
respect to the first generation of Ada2-based peptides since they
promote total dehydration of the Tb ion.
Stability Constant Determination. The pKa's of PHD5
were measured by potentiometry (see Supporting Information,
Figure S7 ). Six acidity constants could be determined in our
experimental conditions (2.5 < pH < 11). The three higher
values (pKa = 9.1, 8.4, and 5.6) correspond to the
Luminescence Properties of the Tb Complexes. Owing
to the antenna effect, that is, Tb3+ luminescence sensitization by
energy transfer through Trp indole absorption at 280 nm, the
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Table 1. Comparison between PHD2 and PHD5 Properties
a
a
b
c
d
pH = 7
11
complex
τH2O (ms)
τD2O (ms)
q
log β
log β110
polymetallic Ln complexes
TbPHD2
TbPHD5
2.35(2)
2.30(2)
2.86(2)
2.81(2)
0.08(3)
0.09(3)
12.7(5)
12.7(5)
16.2(5)
16.2(5)
no
yes
a
τH2O and τD2O are the luminescence lifetimes of Tb3+ in the complexes in HEPES buffer (10 mM, KCl 0.1 M, pH = 7.0) in 100% H2O and
extrapolated to 100% D2O, respectively. q is the hydration number calculated from the lifetimes with Parker’s equation.34 β1p1H = 7 is the conditional
b
c
d
stability constant of TbPHD at pH 7, 298 K in 0.1 M KCl. β110 is the global stability constant of TbPHD calculated from β1p1H = 7 and the peptide pKa's.
deprotonation of the 3 ammonium functions of the chelating
amino acids and the three lower values to the deprotonation of
three carboxylic acid moieties. Potentiometry requires large
amounts of material (mM concentration experiments),
especially for reliable determination of metal-complex stability
constants.36,37 Therefore the affinity of the two peptides for
Tb3+ was investigated by luminescence (16 μM concentration)
using competition experiments.
The two octadentate peptides demonstrate significantly
improved affinities for Tb3+ in comparison to the hexadentate
peptide P22, as shown by competition experiments with
HEDTA. As seen in Figure 3, the Tb-centered luminescence
was very powerful to design high affinity Ln-binding
peptides.26−28 Indeed, peptides built exclusively from natural
amino acids, even in very well-defined structures, show at most
a nanomolar affinity for Ln3+ ions, which need high
coordination numbers.1,19 The incorporation of phosphorylated
amino acids such as phosphotyrosine or phosphoserine, with
more efficient coordinating side-chains than other natural
amino acids, also lead to μM affinity for Tb3+, in α-synuclein
peptide fragments.38,39 On the contrary, hexapeptides built with
two Adan residues show up to picomolar affinity for TbP22 in
water.27 To further enhance the denticity of these peptides, we
used the shorter side-chain analogue Ada1, which may act as a
tetradentate donor thanks to the coordination of its backbone
carbonyl in a six-membered chelate ring. This attempt was
successful in decreasing the hydration state of the Ln3+ ion (qTb
= 0−1)26,28 but did not lead to greater stability of the Ln3+
complexes. Even though the carbonyl function of Ada1 was
involved in the metal coordination by the two peptides P12 and
P11,26,28 no significant stabilization was observed because of this
additional donor. Indeed, supplementary carbonyl binding
groups are neutral donors, which do not provide significant
stabilization of Ln3+ complexes in water, as evidenced before
with neutral tripodal N,O ligands.40
On the contrary, the novel chelating amino acid Ed3a2
developed in the present study bears a pentadentate
polyaminocarboxylate side-chain and therefore an extra amino-
acetate group which has a significant affinity for Ln3+ and thus
contributes to both dehydration and stability. The protected
derivative Fmoc-Ed3a2(tBu)3-OH could be introduced in short
peptide sequences with standard solid phase peptide synthesis
protocols. Ed3a2 was inserted in place of one of the two
tridentate residues in P22, to obtain two second-generation
hexapeptides PHD2 and PHD5, differing in the position of the
pentadentate residue. The two latter high denticity peptides are
able to bind Ln3+ in an octadentate coordination mode and the
mononuclear Tb3+ complexes demonstrate femtomolar stability
in water, that is, a gain of 4 orders of magnitude with respect to
the first generation hexapeptide P22.
Figure 3. Tb-centered emission at selected wavelengths during the
titration of 16 μM TbPHD2 with HEDTA in HEPES buffer (10 mM,
0.1 M KCl, pH = 7.0). Dashed lines represent calculated data.
of TbPHD2 decreases by nearly 50% upon addition of 1 equiv of
HEDTA, which points to similar stabilities for TbPHD2 and
Tb(HEDTA) at pH 7. The conditional stability constant of
Tb(HEDTA) at pH 7 was calculated from published data:32 log
β11pH 7 = 12.6, and the fit of the competition experiments led to
The octadentate coordination of Tb3+ by PHD leads to total
dehydration of the metal ion in the mononuclear complexes
with long luminescence lifetimes (>2 ms). This results in an
improvement of the luminescence properties with respect to
the trisaquo TbP22 complex of a factor 4. The quantum yield
values remain quite low (∼1%) because the energy transfer
from the indole group of tryptophan to Tb3+ is not very
efficient. Nevertheless, as previously described by other groups
working with Ln-binding peptides or proteins, Tb3+ is
sufficiently sensitized by Trp upon binding to the peptides to
allow detection in the nanomolar range.10,17−19,35 In the future,
the replacement of Trp in the peptide sequence by more
efficient Ln3+ sensitizers could be performed to enhance the
energy transfer to the Ln3+ ion. For instance, non natural amino
acids bearing carbostyril 124 or acridone fluorophores, which
are excited at lower energy than tryptophan, have been inserted
pH 7
conditional stability constants log β11
= 12.7 for the two
octadentate peptide PHD
.
The global stability constants β110, independent of the
basicity of the ligand and the pH, were calculated by taking into
account the three protonation constants, which significantly
contribute at pH 7, that is, pKa = 9.1, 8.4, and 5.6. The
calculation leads to logβ110 = 16.0(5). The octadentate peptides
PHD2 and PHD5 provide thus totally dehydrated Tb3+ complexes
with a femtomolar stability in water.
DISCUSSION
■
Recent work in our laboratory has been focused on the
development of Ln-binding peptides, which incorporate
unnatural chelating amino acids to enhance the affinity for
Ln3+ cations. We demonstrated that the introduction of two
chelating Adan residues, with tridentate aminodiacetate groups,
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Notes
in lanthanide binding tags without modifying the coordination
properties of the peptide scaffold.41 The chromophore 1,8-
naphtalimide has also been appended at the N-terminus of a
Ca-binding loop from paravalbumin to obtain luminescent Eu3+
peptide complexes.42,43 These strategies could thus be applied
to our hexapeptides to improve the luminescence properties.
The position of the high denticity amino acid Ed3a2 in the
hexapeptide sequence appears to be critical for the control of
the metal complex speciation. The two peptides PHD2 and PHD5
only differ by the positions of the two chelating amino acids
with respect to the Pro-Gly spacer and show different
behaviors. Whereas PHD5 promotes the formation of poly-
metallic species in excess of Ln3+, PHD2 forms exclusively the
mononuclear complex. Incidentally, the introduction of the
tetradentate aminoacid Ada1 leads to similar observation: Ada1
acts as a tetradentate donor only in position 2 and induces the
formation of polymetallic species if in position 5.26,28 It appears
then that positioning the high denticity amino acid, either Ada1
or Ed3a2 in position 5 after the Pro-Gly spacer favors an
independent coordination of the two chelating moieties. Hence,
to control speciation and stabilize the octadentate coordination
mode that involves the two chelating arms of the hexapeptides,
the higher denticity amino acid has to be introduced in position
2, prior to the cyclic proline residue, which probably constrains
more efficiently the peptide backbone in a β-turn structure.
To conclude, the incorporation of two unnatural amino acids
with polyaminocarboxylate side-chains in short peptide
sequences leads to high stability Ln3+ complexes. In particular,
the design of the novel pentadentate chelating amino acid
Ed3a2 and its insertion in P22 sequence in place of one Ada2
was demonstrated to afford octadentate Tb3+ complexes with
femtomolar stability in water. The octadentate coordination
provides totally dehydrated complexes with better lumines-
cence properties than the previously described TbP22 complex.
The pentadentate amino acid position is demonstrated to be
critical: it has to be inserted in position 2, to control the
speciation and avoid the formation of polymetallic complexes.
Overall, PHD2 is a short Ln-binding peptide which gives a
unique octadentate Tb3+ complex with femtomolar stability,
which could be inserted in more complex peptide or protein
sequences to investigate biologically relevant functions. In
particular, the greater stability of these Ln-peptide complexes
will prevent the decoordination of the Ln3+ ion from the probe
when interacting with biological molecules, which bear
potential Ln3+-binding sites such as proteins with metal-binding
EF hands or DNA sequences.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors want to thank Yves Chenavier for the synthesis of
the unnatural amino acids.
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Corresponding Author
*Phone: +33(0) 4 38 78 98 22. Fax: +33(0) 4 38 78 50 90. E-
mail: pascale.delangle@cea.fr.
Present Address
†Clermont Universite,
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