The interest in ratiometric luminescentp robes that detecta nd quantify as pecific analyte is growing. Owing to their special luminescence properties, lanthanide(III) cationso ffer attractive opportunities for the design of dual-color ratiometric probes. Here, the design principle of hetero-bis-lanthanide peptidec onjugates 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-LZF1 Tb j Cs124 j Eu and r-LZF1 Eu j Cs124 j Tb are described on the basis of az inc finger peptide andt wo DOTA(DOTA = 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetra-acetic acid) complexes of terbium and europium.B oth display dual-color ratiometric emission in response to the presence of Zn 2 + .B yu sing as creening approach, anthracene was identified for the sensitization of the luminescence of two near-infrared-emitting lanthanides, Yb 3 + and Nd 3 +. Thus, two novel zinc-responsive hetero-bis-lanthanide probes, r-LZF3 Yb j Anthra j Nd and r-LZF3 Nd j Anthra j Yb were assembled, the former offeringaneat ratiometric response to Zn 2 + with emission in the near-infrared around1 000 nm, which is unprecedented.

Combination of complexes of trivalent lanthanide cations (Ln 3+) for their luminescent properties and peptides for their recognition properties or folding abilities is interesting in view of designing responsive luminescent probes. The octadentate DOTA chelate is the most popular chelate to design luminescent Ln 3+ complex-peptide conjugates. In this article, we describe a novel building block, DO3Apic-tris(allyl)ester, which provides access to peptides with a conjugated nonadentate chelate, namely DO3Apic, featuring a cyclen macrocycle functionalized by three acetate and one picolinamide arms, 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, two Zn 2+-responsive luminescent probes, which rely on (i) a zinc finger scaffold for selective Zn 2+ binding, (ii) a Eu 3+ complex and (iii) an acridone antenna for long-wavelength sensitization of Eu 3+ luminescence, are compared. One of these probes, LZF3 ACD|Eu , incorporates a DOTA chelate whereas the other, LZF4 ACD|Eu , incorporates a DO3Apic chelate. We show that changing the octadentate DOTA for the nonadentate DO3Apic ligand results in a higher Eu 3+ 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 Ln 3+ complex-peptide conjugates. Additionally, the DO3Apic chelate provides self-calibration for LZF4 ACD|Eu luminescence upon excitation of its picolinamide chromophore, making LZF4 ACD|Eu a ratiometric sensor for Zn 2+ detection.

Lanthanide(III) (Ln 3+ ) complexes feature desirable luminescence properties for cell microscopy imaging, but cytosolic delivery of Ln 3+ 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 Ln 3+ 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/ Ln 3+ 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.