Luminescent Peptide/Lanthanide(III) Complex Conjugates with Push-Pull Antennas: Application to One- and Two-Photon Microscopy Imaging.

Lanthanide(III) (Ln3+) complexes feature desirable luminescence properties for cell microscopy imaging, but cytosolic delivery of Ln3+ complexes and their use for 2P imaging of live cells are challenging. In this article, we describe the synthesis and spectroscopic characterizations of a series of Ln3+ complexes based on two ligands, L1 and L2, featuring extended picolinate push-pull antennas for longer wavelength absorption and 2P absorption properties as well as a free carboxylate function for conjugation to peptides. Several cell penetrating peptide/Ln3+ complex conjugates were then prepared with the most interesting luminescent complexes, Tb(L1) and Eu(L2), and with two cell penetrating peptides (CPPs), ZF5.3 and TP2. A spectroscopic analysis demonstrates that the luminescence properties of the complexes are not affected by conjugation to the peptide. The conjugates were evaluated for one-photon (1P) time-gated microscopy imaging, which suppresses biological background fluorescence, and 2P confocal microscopy. Whereas TP2-based conjugates were unable to enter cells, successful 1P and 2P imaging was performed with ZF5.3[Tb(L1)]. 2P confocal imaging suggests proper internalization and cytosolic delivery as expected for this CPP. Noteworthy, 2P confocal microscopy also allowed characterization of the luminescence properties of the complex (spectrum, lifetime) within the cell, opening the way to functional luminescent probes for 2P confocal imaging of live cells.

Ratiometric Luminescence Detection of Copper(I) by a Resonant System Comprising Two Antenna/Lanthanide Pairs.

Selective and sensitive detection of Cu(I) is an ongoing challenge due to its important role in biological systems, for example. Herein, we describe a photoluminescent molecular chemosensor integrating two lanthanide ions (Tb3+ and Eu3+) and respective tryptophan and naphthalene antennas onto a polypeptide backbone. The latter was structurally inspired from copper-regulating biomacromolecules in Gram-negative bacteria and was found to bind Cu+ effectively under pseudobiological conditions (log KCu+ = 9.7 ± 0.2). Ion regulated modulation of lanthanide luminescence in terms of intensity and long, millisecond lifetime offers perspectives in terms of ratiometric and time-gated detection of Cu+. The role of the bound ion in determining the photophysical properties is discussed with the aid of additional model compounds.

Bioinspired Luminescent Europium-Based Probe Capable of Discrimination between Ag+ and Cu.

Due to their similar coordination properties, discrimination of Cu+ and Ag+ by water-soluble luminescent probes is challenging. We have synthesized LCC4Eu, an 18 amino acid cyclic peptide bearing a europium complex, which is able to bind one Cu+ or Ag+ ion by the side chains of two methionines, a histidine and a 3-(1-naphthyl)-l-alanine. In this system, the naphthyl moiety establishes a cation-π interaction with these cations. It also acts as an antenna for the sensitization of Eu3+ luminescence. Interestingly, when excited at 280 nm, LCC4Eu behaves as a turn-on probe for Ag+ (+150% Eu emission) and as a turn-off probe for Cu+ (-50% Eu3+ emission). Shifting the excitation wavelength to 305 nm makes the probe responsive to Ag+ (+380% Eu3+ emission) but not to Cu+ or other physiological cations. Thus, LCC4Eu is uniquely capable of discriminating Ag+ from Cu+. A detailed spectroscopic characterization based on steady-state and time-resolved measurements clearly demonstrates that Eu3+ sensitization relies on electronic energy transfer from the naphthalene triplet state to the Eu3+ excited states and that the cation-π interaction lowers the energy of this triplet state by 700 and 2400 cm-1 for Ag+ and Cu+, respectively. Spectroscopic data point to a modulation of the efficiency of the electronic energy transfer caused by the differential red shift of the naphthalene triplet, deciphering the differential luminescence response of LCC4Eu toward Ag+ and Cu+.

Using Native Chemical Ligation for Site-Specific Synthesis of Hetero-bis-lanthanide Peptide Conjugates: Application to Ratiometric Visible or Near-Infrared Detection of Zn2.

The interest in ratiometric luminescent probes that detect and quantify a specific analyte is growing. Owing to their special luminescence properties, lanthanide(III) cations offer attractive opportunities for the design of dual-color ratiometric probes. Here, the design principle of hetero-bis-lanthanide peptide conjugates by using native chemical ligation is described for perfect control of the localization of each lanthanide cation within the molecule. Two zinc-responsive probes, r-LZF1Tb|Cs124|Eu and r-LZF1Eu|Cs124|Tb are described on the basis of a zinc finger peptide and two DOTA (DOTA=1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) complexes of terbium and europium. Both display dual-color ratiometric emission in response to the presence of Zn2+ . By using a screening approach, anthracene was identified for the sensitization of the luminescence of two near-infrared-emitting lanthanides, Yb3+ and Nd3+ . Thus, two novel zinc-responsive hetero-bis-lanthanide probes, r-LZF3Yb|Anthra|Nd and r-LZF3Nd|Anthra|Yb were assembled, the former offering a neat ratiometric response to Zn2+ with emission in the near-infrared around 1000 nm, which is unprecedented.

Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle.

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet-visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

Mercury Trithiolate Binding (HgS3) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains.

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS3 coordination). The model cyclodecapeptide P3C, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS3 coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P2C cyclodecapeptide validated previously as a model for d10 metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP3C complex with a HgS3 coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and 199Hg NMR. Hg LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP3C complex was refined based on 1H-1H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgP3CH complex has a p Ka of 4.3, indicating that the HgS3 coordination mode is stable over a large range of pH. This low p Ka value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P3C.

Luminescent Zinc Fingers: Zn-Responsive Neodymium Near-Infrared Emission in Water.

Responsive luminescent probes emitting in the near-infrared (NIR) are in high demand today for biological applications as they allow for the easy and unambiguous discrimination of autofluorescence. Due to their luminescence properties, lanthanide ions offer an interesting alternative to classical organic fluorescent dyes. This has stimulated the development of lanthanide-based responsive probes. Nevertheless, responsive probes that can operate in water with NIR-emitting lanthanide ions are scarce. In this communication, zinc fingers are shown to be versatile scaffolds to elaborate a variety of Zn2+ -responsive probes based on lanthanide emission and featuring desirable properties for the selective detection of Zn2+ in experimental conditions close to cellular. Of special interest is a NIR-emitting probe relying on Nd3+ emission.

Reactivity of a Zn(Cys)2(His)2 Zinc Finger with Singlet Oxygen: Oxidation Directed toward Cysteines but not Histidines.

Singlet oxygen ((1)O2) is an important reactive oxygen species in biology that has deleterious effects. Proteins constitute the main target of (1)O2 in cells. Several organisms are able to mount a transcriptional defense against (1)O2. ChrR and MBS are two proteins with Zn(Cys)2(His)2 zinc finger sites that are involved in the regulation of the defense against (1)O2. In this article, we investigate the reactivity of Zn⋅CPF, a Zn(Cys)2(His)2 classical ßßα zinc finger, with (1)O2. We show that Zn⋅CPF interacts with (1)O2 mainly by physical quenching using a combination of (1)O2 luminescence quenching and kinetic competition experiments. The chemical reaction, which accounts for 5% of the interaction, leads to oxidation of cysteines but not histidines. Primary photooxidation products, identified by HPLC and mass spectrometry, are sulfinate (75±5%) and disulfides (25±5%). The peptides that have a single cysteine thiolate oxidized into a sulfinate are still able to bind one equivalent Zn(2+) but with a dramatic reduction of the binding constant compared to Zn⋅CPF despite the preservation of the ßßα fold, as shown by NMR and CD titrations. Finally, Zn⋅CPF is compared to Zn⋅LTC, a treble clef Zn(Cys)4 zinc finger, to gain further insight into the behavior of zinc fingers toward (1)O2.

Reactivity of Cys4 zinc finger domains with gold(III) complexes: insights into the formation of "gold fingers".

Gold(I) complexes such as auranofin or aurothiomalate have been used as therapeutic agents for the treatment of rheumatoid arthritis for several decades. Several gold(I) and gold(III) complexes have also shown in vitro anticancer properties against human cancer cell lines, including cell lines resistant to cisplatin. Because of the thiophilicity of gold, cysteine-containing proteins appear as likely targets for gold complexes. Among them, zinc finger proteins have attracted attention and, recently, gold(I) and gold(III) complexes have been shown to inhibit poly(adenosine diphosphate ribose)polymerase-1 (PARP-1), which is an essential protein involved in DNA repair and in cancer resistance to chemotherapies. In this Article, we characterize the reactivity of the gold(III) complex [Au(III)(terpy)Cl]Cl2 (Auterpy) with a model of Zn(Cys)4 "zinc ribbon" zinc finger by a combination of absorption spectroscopy, circular dichroism, mass spectrometry, high-performance liquid chromatography analysis, and X-ray absorption spectroscopy. We show that the Zn(Cys)4 site of Zn·LZR is rapidly oxidized by Auterpy to form a disulfide bond. The Zn(2+) ion is released, and the two remaining cysteines coordinate the Au(+) ion that is produced during the redox reaction. Subsequent oxidation of these cysteines can take place in conditions of excess gold(III) complex. In the presence of excess free thiols mimicking the presence of glutathione in cells, mixing of the zinc finger model and gold(III) complex yields a different product: complex (Au(I))2·LZR with two Au(+) ions bound to cysteines is formed. Thus, on the basis of detailed speciation and kinetic measurements, we demonstrate herein that the destruction of Zn(Cys)4 zinc fingers by gold(III) complexes to achieve the formation of "gold fingers" is worth consideration, either directly or mediated by reducing agents.

Lanthanide Luminescence Modulation by Cation-π Interaction in a Bioinspired Scaffold: Selective Detection of Copper(I).

A prototype luminescent turn-on probe for Cu(+) (and Ag(+)) is described, harnessing a selective binding site (log Kass = 9.4 and 7.3 for Cu(+) and Ag(+), respectively) based on the coordinating environment of the bacterial metallo-chaperone CusF, integrated with a terbium-ion-signaling moiety. Cation-π interactions were shown to enhance tryptophan triplet population, which subsequently sensitized, on the microsecond timescale, the long-lived terbium emission, offering a novel approach in bioinspired chemosensor design.

Efficient oxidation and destabilization of Zn(Cys)4 zinc fingers by singlet oxygen.

Singlet oxygen ((1)O2) plays an important role in oxidative stress in all types of organisms, most of them being able to mount a defense against this oxidant. Recently, zinc finger proteins have been proposed to be involved in its cellular detection but the molecular basis of this process still remains unknown. We have studied the reactivity of a Zn(Cys)4 zinc finger with (1)O2 by combinations of spectroscopic and analytical techniques, focusing on the products formed and the kinetics of the reaction. We report that the cysteines of this zinc finger are oxidized to sulfinates by (1)O2. The reaction of the ZnS4 core with (1)O2 is very fast and efficient with almost no physical quenching of (1)O2. A drastic (ca. five orders of magnitude) decrease of the Zn(2+) binding constant was observed upon oxidation. This suggests that the Zn(Cys)4 zinc finger proteins would release their Zn(2+) ion and unfold upon reaction with (1)O2 under cellular conditions and that zinc finger sites are likely targets for (1)O2.

A diiron(III,IV) imido species very active in nitrene-transfer reactions.

Metal-catalyzed nitrene transfer reactions arouse intense interest as clean and efficient procedures for amine synthesis. Efficient Rh- and Ru-based catalysts exist but Fe alternatives are actively pursued. However, reactive iron imido species can be very short-lived and getting evidence of their occurrence in efficient nitrene-transfer reactions is an important challenge. We recently reported that a diiron(III,II) complex is a very efficient nitrene-transfer catalyst to various substrates. We describe herein how, by combining desorption electrospray ionization mass spectrometry, quantitative chemical quench experiments, and DFT calculations, we obtained conclusive evidence for the occurrence of an {Fe(III) Fe(IV) NTosyl} intermediate that is very active in H-abstraction and nitrene-transfer reactions. DFT calculations revealed a strong radical character of the tosyl nitrogen atom in very low-lying electronic configurations of the Fe(IV) ion which are likely to confer its high reactivity.

Efficient cytosolic delivery of luminescent lanthanide bioprobes in live cells for two-photon microscopy.

Lanthanide(iii) (Ln3+) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb3+ and Eu3+ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln3+ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln3+ complex. The cytotoxicity of these three probes was evaluated and the IC50 ranged from 7 µM to >50 µM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.

On the design of zinc-finger models with cyclic peptides bearing a linear tail.

Cyclic peptides with a linear tail (CPLT) have been successfully used to model two zinc fingers (ZFs) adopting the treble-clef- and loosened zinc-ribbon folds. In this article, we examine the factors that may influence the design of such ZF models: mutations in the sequence, size of the cycle, and size of the tail. For this purpose, several peptides derived from the CPLT-based models of the treble-clef- and loosened zinc-ribbon ZF were synthesized and studied. CPLT-based models appear to be robust toward mutations, accommodate various cycle sizes, and are sensible to the size of the linking region of the tail located between the cycle and the coordinating amino acids. Based on these criteria, we describe the design of a new CPLT-based model for the zinc-ribbon ZFs, LZR , and compare it to a linear analogue, LZR(lin) . The model complex Zn⋅LZR is able to fold correctly around the metal ion contrary to Zn⋅LZR(lin) , suggesting that CPLT-based models are more likely to yield structurally meaningful models of ZF sites than linear peptide models. Finally, we draw some rules that could allow the design of new CPLT-based metallopeptides with a controlled fold.

A phosphorylated zinc finger peptide bearing a gadolinium complex for zinc detection by MRI.

Two zinc finger peptides, namely ZFQDLn and ZFQELn (Ln = Tb or Gd), with an appended Ln3+ chelate and a phosphoserine able to coordinate the Ln3+ ion are presented. The two peptides differ by the amino acid anchorage of the chelate, either aspartate (D) or glutamate (E). Both peptides are able to bind Zn2+ and adopt the ßßα fold. Interestingly, ZFQETb shows a decrease in sensitized Tb3+ luminescence upon Zn2+ binding whereas ZFQDTb does not. The luminescence change upon Zn2+ binding is attributed to a change in hydration number (q) of the Tb3+ ion due to the decoordination of the phosphoserine from the Ln3+ ion upon Zn2+ binding and peptide folding. This process is highly sensitive to the length of the linker between the Ln chelate and the peptidic backbone. The magnetic properties of the gadolinium analogue ZFQEGd were studied. An impressive relaxivity increase of 140% is observed at 60 MHz and 25 °C upon Zn2+ binding. These changes can be attributed to a combined increase effect of the hydration number of Gd3+ and of the rigidity of the system upon Zn2+ binding. Phantom MR images at 9.4 T show a clear signal enhancement in the presence of Zn2+. These zinc finger peptides offer a unique platform to design such Zn-responsive probes.

Copper Induces Protein Aggregation, a Toxic Process Compensated by Molecular Chaperones.

Copper is well known for its antimicrobial and antiviral properties. Under aerobic conditions, copper toxicity relies in part on the production of reactive oxygen species (ROS), especially in the periplasmic compartment. However, copper is significantly more toxic under anaerobic conditions, in which ROS cannot be produced. This toxicity has been proposed to arise from the inactivation of proteins through mismetallations. Here, using the bacterium Escherichia coli, we discovered that copper treatment under anaerobic conditions leads to a significant increase in protein aggregation. In vitro experiments using E. coli lysates and tightly controlled redox conditions confirmed that treatment with Cu+ under anaerobic conditions leads to severe ROS-independent protein aggregation. Proteomic analysis of aggregated proteins revealed an enrichment of cysteine- and histidine-containing proteins in the Cu+-treated samples, suggesting that nonspecific interactions of Cu+ with these residues are likely responsible for the observed protein aggregation. In addition, E. coli strains lacking the cytosolic chaperone DnaK or trigger factor are highly sensitive to copper stress. These results reveal that bacteria rely on these chaperone systems to protect themselves against Cu-mediated protein aggregation and further support our finding that Cu toxicity is related to Cu-induced protein aggregation. Overall, our work provides new insights into the mechanism of Cu toxicity and the defense mechanisms that bacteria employ to survive. IMPORTANCE With the increase of antibiotic drug resistance, alternative antibacterial treatment strategies are needed. Copper is a well-known antimicrobial and antiviral agent; however, the underlying molecular mechanisms by which copper causes cell death are not yet fully understood. Herein, we report the finding that Cu+, the physiologically relevant copper species in bacteria, causes widespread protein aggregation. We demonstrate that the molecular chaperones DnaK and trigger factor protect bacteria against Cu-induced cell death, highlighting, for the first time, the central role of these chaperones under Cu+ stress. Our studies reveal Cu-induced protein aggregation to be a central mechanism of Cu toxicity, a finding that will serve to guide future mechanistic studies and drug development.

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms.

The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.

Oxidation of Zn(Cys)4 zinc finger peptides by O2 and H2O2: products, mechanism and kinetics.

The reactivity of a series of Zn(Cys)(4) zinc finger model peptides towards H(2)O(2) and O(2) has been investigated. The oxidation products were identified by HPLC and ESI-MS analysis. At pH<7.5, the zinc complexes and the free peptides are oxidised to bis-disulfide-containing peptides. Above pH 7.5, the oxidation of the zinc complexes by H(2)O(2) also yields sulfinate- and sulfonate-containing overoxidised peptides. At pH 7.0, monitoring of the reactions between the zinc complexes and H(2)O(2) by HPLC revealed the sequential formation of two disulfides. Several techniques for the determination of the rate constant for the first oxidation step corresponding to the attack of H(2)O(2) by the Zn(Cys)(4) site have been compared. This rate constant can be reliably determined by monitoring the oxidation by HPLC, fluorescence, circular dichroism or absorption spectroscopy in the presence of excess ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid. In contrast, monitoring of the release of zinc with 4-(2-pyridylazo)resorcinol or of the thiol content with 5,5'-dithiobis(2-nitrobenzoate) did not yield reliable values of this rate constant for the case in which the formation of the second disulfide is slower than the formation of the first. The kinetic measurements clearly evidence a protective effect of zinc on the oxidation of the cysteines by both H(2)O(2) and O(2), which points to the fact that zinc binding diminishes the nucleophilicity of the thiolates. In addition, the reaction between the zinc finger and H(2)O(2) is too slow to consider zinc fingers as potential sensors for H(2)O(2) in cells.

Coordination properties of zinc finger peptides revisited: ligand competition studies reveal higher affinities for zinc and cobalt.

Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)(4-x)(His)(x) site. They possess great diversity in their structure and amino acid composition. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal association and dissociation. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ßßα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding constants, and structure of these peptides have been investigated as a function of pH. The zinc binding constants of peptides, which adopt a well-defined structure, were found to be around 10(15) at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding constant and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability constant by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-containing proteins.

A novel DOTA-like building block with a picolinate arm for the synthesis of lanthanide complex-peptide conjugates with improved luminescence properties.

Combination of complexes of lanthanide cations (Ln3+) for their luminescent properties and peptides for their recognition properties is interesting in view of designing responsive luminescent probes. The octadentate DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelate is the most popular chelate to design Ln3+ complex-peptide conjugates. We describe a novel building block, DO3Apic-tris(allyl)ester, which provides access to peptides with a conjugated nonadentate chelate, namely DO3Apic, featuring a picolinate arm in place of one of the acetate arms compared to DOTA, for improved luminescence properties. This building block, with allyl protecting groups, is readily obtained by solid phase synthesis. We show that it is superior to its analogue with tBu protecting groups for the preparation of peptide conjugates because of the difficult removal of the tBu protecting groups for the latter. Then, we compare two luminescent zinc fingers (LZF) comprising (i) a zinc finger peptide for selective Zn2+ binding, (ii) a Eu3+ complex and (iii) an acridone antenna (ACD) for long-wavelength sensitization of Eu3+ luminescence. The first one, LZF3ACD|Eu, incorporates a DOTA chelate for Eu3+ whereas the other, LZF4ACD|Eu, incorporates a DO3Apic chelate. Both act as Zn2+-responsive luminescent probes but we show that changing DOTA for DO3Apic results in a higher Eu3+ luminescence lifetime and in a doubling of the quantum yield, confirming the interest of the DO3Apic chelate and the DO3Apic(tris(allyl)ester building block for the preparation of Ln3+ complex-peptide conjugates. Additionally, the DO3Apic chelate provides self-calibration for LZF4ACD|Eu luminescence upon excitation of its picolinamide chromophore, making LZF4ACD|Eu a ratiometric sensor for Zn2+.