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.

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.

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+.