Luminescent biAryl Phospholes for Peptide-Receptors Tracking – LAPPRET

We have described the synthesis of a new class of bisaryl P-hydroxyphospholes oxide or sulfide and P-aminophospholes and their coupling with amino acids and peptides, under the conditions required for synthesis in solution or on a solid support. This new method of coupling of phospholes to biomolecules was illustrated by the labeling of JMV2959, which is a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a). The affinity of the labeled ligands, for the GHS-R1a receptor was studied by Förster Resonance Energy Transfer (FRET). Finally, the competition tests implemented have demonstrated their applicability for the characterization of other GHS-R1a receptor ligands.

Two new classes of fluorophores, the P-hydroxyphospholes oxide or sulifde and the P-aminophospholes were developed, as well as their coupling to amino acids and peptides, through the phosphorus atom. Bisarylphospholes and the phospholamide derivatives were characterized by UV-visible absorption anf fluorescence spectroscopies and they exhibit emissions with a large Stokes shift (= 70nm). P-hydroxyphospholes oxide were used to label JMV2959, which is a potent antagonist of the growth hormone secretagogue receptor type 1a, and the obtained conjugates were used to characterize other ligands of this receptor.

The synthesis of P-hydroxyphospholes and P-aminophospholes with extended conjugated systems, such as phosphindoles and derivatives bearing electron-donor and acceptor substituents, is envisaged in order to obtain emissions towards the red/near infrared. The grafting strategy of these phospholes will be applied to the labeling of peptides of interest such as neurotensin, Ghrelin [1-8]-Cys9 and the GHS-R1a receptor, studied at IBMM. In connection with our researches at the laboratory, related to the radiolabeling of neurotensin analogues for Positron Emission Tomography (PET) imaging of cancer cells overexpressing the NTS1 receptor, the synthesis of boronated phospholes is also envisaged, to study bimodal labeling of this peptide by fluorescence and by PET using the radioelement [18F].

The researches related to the synthesis of P-hydroxyphosphole oxide or sulfide derivatives and their coupling to the amine groups of amino acids or peptides by formation of P-N bond, were published in the article entitled «Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation”, Emmanuelle Rémond, Jean-Alain Fehrentz, Laure Liénart, Sébastien Clément, Jean-Louis Banères, Florine Cavelier, Chem. Eur. J., 2022, 28, e202201526.

The development of fluorescence techniques is continually renewed by the instruments progresses, the discovery of new fluorophores and labelling methods. In a recent past, much efforts were dedicated to the development of optoelectronic devices, optical imaging for diagnosis and guided-treatment based on fluorescence emission in near-infrared (680-1300 nm) not interfering with the human tissues.
A large number of fluorophores have been described, because their uses depend on their physicochemical and optical properties (wavelength abs/em, photostability, molecular weight, solubility, pH and solvent sensitivity, ease of coupling…).
The development of new simple, stable and tunable dyes emitting toward the red, with large Stokes shift is of major interest for the labelling of small biomolecules, in order to minimize the reabsorption loss and to improve the sensibility and contrast for molecular imaging.
Phospholes based pi-conjugated materials were widely used for organic light-emitting diodes (OLEDs), due to their unique electroluminescent properties. The phosphole core is a weakly aromatic five membered heterocycle, having the characteristics of a dienic system bridged with a tetrahedral phosphorus atom allowing easy chemical modifications to afford suitable derivatives with tunable luminescent emission and high fluorescence quantum yields.
Very interestingly their emission of fluorescence is due to an unusual intramolecular charge transfer (ICT) of the excited state toward a hybrid LUMO, inducing a large Stokes shift. This is particularity favorable to preserve the large Stokes shift, even in the case of emission of lower energies and high wavelengths.
Despite their remarkable optical properties, and high photochemical stability phosphole compounds have not been used for tagging amino acids and peptides.
We have recently reported the stereoselective synthesis of phospholyl(borane) amino acids and their direct transformation into the corresponding free phospholyl, gold complex, oxide or sulfur derivatives as well as phospholinium salts, thus offering a variety of side chains. After selective deprotection of carboxylic function or amine, C- or N- peptide coupling proved their possible incorporation into peptides. Such phospholyl amino acid and peptide derivatives exhibit fluorescent properties with a large Stokes shift (160 nm) and fluorescence up to 535 nm (JACS 2018).
As grafting phosphole moiety to an amino acid side chain involves the use of phospholide anions, prepared by cleavage of the P-Phenyl bond with metal (Li, K), this is few compatible for the design of more aromatic and functionalized derivatives in order to enhance their emission wavelength toward the red region.
In this context, the aim of LAPPRET project is to develop luminescent properties of phospholes in amino acid and peptide chemistry:
• Developing the synthesis of aromatic and functionalized phospholes bearing a reactive substituent at the P-center (NR2 or OH), to allow new chemoselective coupling methods with hydroxyl-, amino- or thiol pendant groups of peptides under mild and neutral conditions. Borane will be used as protecting group of the P-center to isolate stable and easily handle complexes, that will be also be used for the preparation of derivatives.
• Developing new class of phosphole-based fluorophores emitting toward the near infrared (NIR) region, without interference between excitation source (excitation= 400 nm) and emission light (> 600 nm). Photophysical studies and TD-DFT calculations will be performed for understanding and planning the relation structure/molecular orbitals/emission properties.
• Proof of concept of using phosphole-based fluorophores with the development of new labelled neurotensin NT[8-13] analogues for specific tracking of NTs receptors.