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.

Deeper insight into protease-sensitive "covalent-assembly" fluorescent probes for practical biosensing applications.

We report a rational and systematic study devoted to the structural optimisation of a novel class of protease-sensitive fluorescent probes that we recently reported (S. Debieu and A. Romieu, Org. Biomol. Chem., 2017, 15, 2575-2584), based on the "covalent-assembly" strategy and using the targeted enzyme penicillin G acylase as a model protease to build a fluorescent pyronin dye by triggering a biocompatible domino cyclisation-aromatisation reaction. The aim is to identify ad hoc probe candidate(s) that might combine fast/reliable fluorogenic "turn-on" response, full stability in complex biological media and ability to release a second molecule of interest (drug or second fluorescent reporter), for applications in disease diagnosis and therapy. We base our strategy on screening a set of active methylene compounds (C-nucleophiles) to convert the parent probe to various pyronin caged precursors bearing Michael acceptor moieties of differing reactivities. In vitro stability and fluorescent enzymatic assays combined with HPLC-fluorescence analyses provide data useful for defining the most appropriate structural features for these fluorogenic scaffolds depending on the specifications inherent to biological application (from biosensing to theranostics) for which they will be used.

Divergent synthesis of 5',7'-difluorinated dihydroxanthene-hemicyanine fused near-infrared fluorophores.

We describe an expedient access to a 5',6',7'-trifluoro dihydroxanthene-hemicyanine fused scaffold in 2 steps and 54% overall yield from the corresponding salicylic aldehyde. A 6'-regioselective nucleophilic aromatic substitution (SNAr) reaction with a wide range of nitrogen, sulfur or selenium nucleophiles then gives access to 16 near-infrared (NIR) fluorophores emitting in the 710-750 nm range. We also report the experimental and theoretical photophysical investigations of these unique optical agents that include the first series of 6'-heavy atom substituted dihydroxanthenes, extending the pool of polyfluorinated markers for biomedical and material applications.

Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer.

BACKGROUND: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM). METHODS: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 µM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 µM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment. RESULTS: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. CONCLUSION: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

Site-specific near-infrared fluorescent labelling of proteins on cysteine residues with meso-chloro-substituted heptamethine cyanine dyes.

Near-infrared (NIR) fluorescence imaging is a promising new medical imaging modality. Associated with a targeting molecule, NIR fluorophores can accumulate selectively in tissues of interest and become valuable tools for the diagnosis and therapy of various pathologies. To facilitate the design of targeted NIR imaging agents, it is important to identify simple and affordable fluorescent probes, allowing rapid labelling of biovectors such as proteins, ideally in a site-specific manner. Here, we demonstrate that heptamethine cyanine based fluorophores, such as IR-783, that contain a chloro-cyclohexyl moiety within their polymethine chain can react selectively, at neutral pH, with cysteine residues in proteins to give stable, site-specifically labelled conjugates, that emit in the NIR spectral window. This reaction is exemplified with the labelling of peptides and two protein models: albumin and a Fab' antibody fragment. The resulting fluorescent proteins are stable and suitable for in vivo NIR imaging applications, as shown on a mice model. This straightforward one-step procedure, that does not require the prior derivatisation of the fluorophore with a bioconjugatable handle, should facilitate the production and use of near-infrared labelled proteins in life sciences.

Rationalisation of the optical signatures of nor-dihydroxanthene-hemicyanine fused near-infrared fluorophores by first-principle tools.

Using a computational approach combining the Time-Dependent Density Functional Theory (TD-DFT) and the second-order Coupled Cluster (CC2) approaches, we investigate the spectral properties of a large panel of nor-dihydroxanthene (DHX)-hemicyanine fused dyes. First we compare the theoretical and experimental 0-0 energies for a set of 14 known synthetic compounds and show that a remarkable agreement between theory and experiment is obtained when a suitable environmental model is selected. In addition, we obtain vibrationally-resolved spectra for several compounds and theory also accurately reproduces the experimental band shapes. We show that the electronic transitions in nor-DHX-based fluorophores are associated with small variations of the dipole moments but large oscillator strengths. Using various chemical strategies, we design a series of compounds with red-shifted 0-0 energies.

In situ formation of pyronin dyes for fluorescence protease sensing.

We report a reaction-based strategy for the fluorogenic detection of protease activity. Based on the "covalent-assembly" probe design principle recently put forward by the Yang group for detection of Sarin related threats (J. Am. Chem. Soc., 2014, 136, 6594-6597), we have designed two unusual non-fluorescent caged precursors (mixed bis-aryl ethers) which are readily converted into a fluorescent unsymmetrical pyronin dye through a domino cyclisation-aromatisation reaction triggered by penicillin G acylase (PGA) or leucine aminopeptidase (LAP). Fluorescence-based in vitro assays and HPLC-fluorescence/-MS analyses support the claimed activation mechanism whose the further implementation to "smart" imaging agents for the study of protease function in vivo is expected.

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

New 3-(heteroaryl)-2-iminocoumarin-based borate complexes: synthesis, photophysical properties, and rational functionalization for biosensing/biolabeling applications.

Members of a series of boron difluoride complexes with 3-(heteroaryl)-2-iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N-aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two-step method. These novel fluorogenic dyes belong to the family of "Boricos" (D. Frath et al., Chem. Commun.- 2013, 49, 4908-4910) and are the first examples of phenol-based fluorophores of which the photophysical properties in the green-yellow spectral range are dramatically improved by N,N-chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4-dinitrophenyl (DNP) ethers, which led to a dramatic "OFF-ON" fluorescence response upon reaction with thiols. Additionally, to expand the scope of these "7-hydroxy-Borico" derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and "Borico"-DOTA-like scaffolds in an effort to design novel monomolecular multimodal fluorescence- radioisotope imaging agents.

"AND" luminescent "reactive" molecular logic gates: a gateway to multi-analyte bioimaging and biosensing.

This review outlines examples that illustrate a recent and highly innovative concept in the field of (bio)molecular sensing, namely the simultaneous multi-analyte detection using "reactive" luminescent probes that are able to produce an optical signal only in response to multiple (bio)chemical inputs and through covalent chemical reactions with target (bio)analytes. Unlike conventional "AND" molecular logic gates based on supramolecular photochemical mechanisms, these unusual "smart" optical (bio)probes are suitable tools to track the rise and fall of a wider range of biologically relevant analytes, in complex media and with higher selectivity. The potential utility of this concept for in vivo molecular imaging and possible solutions to adapt the described luminogenic processes to far-red or NIR emitters are also discussed.

Dual enzyme-responsive "turn-on" fluorescence sensing systems based on in situ formation of 7-hydroxy-2-iminocoumarin scaffolds.

A new strategy for the simultaneous fluorogenic detection of two distinct enzyme activities namely hydrolase (amidase or esterase) and reductase is described. This innovative biosensing method is based on the powerful "covalent-assembly" principle that involves in situ synthesis of a fluorophore from a non-fluorescent caged precursor and through domino reactions triggered by the two analytes of interest. To establish this approach, penicillin G acylase (PGA) (or pig liver esterase (PLE)) and nitroreductase (NTR) were chosen as model enzymes, and original bis-O-protected 2,4-dihydroxycinnamonitrile derivatives acting as dual-reactive probes readily convertible to highly fluorescent 7-hydroxy-2-iminocoumarin scaffolds upon reacting with the two selected enzymes were synthesised. The two phenolic groups available within the core structure of these probes play a pivotal role in generating iminocoumarin scaffold through an intramolecular cyclisation reaction (hydroxyl group in C-2 position) and in enhancing its push-pull character (hydroxyl group in C-4 position). Their orthogonal and temporary protection with two different enzyme-labile masking groups is the cornerstone in the design of this novel class of fluorogenic "turn-on" probes. Their evaluation using fluorescence-based in vitro assays and HPLC-fluorescence/-MS analyses have enabled us both to demonstrate the claimed activation mechanism (in particular the specific order in which the two enzymes react with the probe) and to highlight the potential utility of these advanced chemical tools in multi-analyte sensing applications.

Highly fluorescent and water-soluble diketopyrrolopyrrole dyes for bioconjugation.

The preparation of highly water-soluble and strongly fluorescent diketopyrrolopyrrole (DPP) dyes using an unusual taurine-like sulfonated linker has been achieved. Exchanging a phenyl for a thienyl substituent shifts the emission wavelength to near λ=600 nm. The free carboxylic acid group present in these new derivatives was readily activated and the dyes were subsequently covalently linked to a model protein (bovine serum albumin; BSA). The bioconjugates were characterized by electronic absorption, fluorescence spectroscopy and MALDI-TOF mass spectrometry, thus enabling precise determination of the labeling density (ratio DPP/BSA about 3 to 8). Outstanding values of fluorescence quantum yield (30% to 59%) for these bioconjugates are obtained. The photostability of these DPP dyes is considerably greater than that of fluorescein under the same irradiation conditions. Remarkably low detection limits between 80 and 300 molecules/µm(2) were found for the BSA bioconjugates by fluorescence imaging with a epifluorescence microscope.

Thiocarbamate-linked polysulfonate-peptide conjugates as selective hepatocyte growth factor receptor binders.

The capacity of many proteins to interact with natural or synthetic polyanions has been exploited for modulating their biological action. However, the polydispersity of these macromolecular polyanions as well as their poor specificity is a severe limitation to their use as drugs. An emerging trend in this field is the synthesis of homogeneous and well-defined polyanion-peptide conjugates, which act as bivalent ligands, with the peptide part bringing the selectivity of the scaffold. Alternately, this strategy can be used for improving the binding of short peptides to polyanion-binding protein targets. This work describes the design and first synthesis of homogeneous polysulfonate-peptide conjugates using thiocarbamate ligation for binding to the extracellular domain of MET tyrosine kinase receptor for hepatocyte growth factor.

Straightforward access to water-soluble unsymmetrical sulfoxanthene dyes: application to the preparation of far-red fluorescent dyes with large stokes' shifts.

An efficient synthesis of water-soluble unsymmetrical sulforhodamine/sulforhodol fluorophores containing a single julolidine fragment is presented. Owing to their valuable spectral properties in aqueous buffers, these dyes, especially those bearing a free aniline or phenol moiety, are valuable components of fluorogenic probes for a variety of biosensing applications. A further extension of this synthetic methodology to unusual phenols, namely 7-N,N-dialkylamino-4-hydroxy coumarins has enabled us to provide a new family water-soluble dyes of large Stokes' shift with far-red spectral features.

Kondrat'eva ligation: Diels-Alder-based irreversible reaction for bioconjugation.

Diversification of existing chemoselective ligations is required to efficiently access complex and well-defined biomolecular assemblies with unique and valuable properties. The development and bioconjugation applications of a novel Diels-Alder-based irreversible site-specific ligation are reported. The strategy is based on a Kondrat'eva cycloaddition between bioinert and readily functionalizable 5-alkoxyoxazoles and maleimides that readily react together under mild and easily tunable reaction conditions to afford a fully stable pyridine scaffold. The potential of this novel bioconjugation is demonstrated through the preparation of fluorescent conjugates of biomolecules and a novel Förster resonance energy transfer (FRET)-based probe suitable for the in vivo detection and imaging of urokinase-like plasminogen activator (uPA), which is a key protease involved in cancer invasion and metastasis.

Reaction site-driven regioselective synthesis of AChE inhibitors.

The enzyme-directed synthesis is an emerging fragment-based lead discovery approach in which the biological target is able to assemble its own multidentate ligands from a pool of building blocks. Here, we report for the first time the use of the human acetylcholinesterase (AChE) as an enzyme for the design and synthesis of new potent heterodimeric huprine-based inhibitors. Both the specific click chemistry site within the protein and the regioselectivity of the Huisgen cycloaddition observed suggest promising alternatives in the design of efficient mono- and dimeric ligands of AChE. Finally, a detailed computational modelling of the click reaction was conducted to further understand the origin of this TGS selectivity.

Synthesis, biological evaluation, and in vivo imaging of the first camptothecin-fluorescein conjugate.

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a highly convergent and flexible approach which enables the rapid chemical modification of the AB ring system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first green-emitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetics of cell endocytosis, as well as the efficacy of CPT-FI were compared to those of CPT. CPT-FI fluorescence was measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained detectable until 48 h. As CPT, CPT-FI drastically inhibited glioma growth and cell cycle but exhibited a reduced affinity as compared to the native CPT. In vivo and ex vivo imaging studies of CPT-FI intratumoraly injected into a model of NIH-3T3 murine tumor xenografts in nude mice, showed accumulation around the injected site area, which is very promising to target tumors and follow biodistribution in vivo.

Bioconjugatable azo-based dark-quencher dyes: synthesis and application to protease-activatable far-red fluorescent probes.

We describe the efficient synthesis and one-step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher-3 (BHQ-3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α-iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water-soluble (sulfonated derivative) and aldehyde-modified BHQ-3 dyes, the direct preparation of which failed by means of conventional azo-coupling reactions. The aldehyde- and α-iodoacetyl-containing fluorescence quenchers were readily conjugated to aminooxy- and cysteine-containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red- or far-red-emitting rhodamine or cyanine dyes through sequential and/or one-pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis.

The first "ready-to-use" benzene-based heterotrifunctional cross-linker for multiple bioconjugation.

The synthesis and applications of the first water-soluble benzene derivative bearing a set of three different and orthogonal bioconjugatable groups (aminooxy, azido and thiol) are described. The combined use of a 5-amino isophthalic acid scaffold and unusual acid-labile protecting groups for temporarily masking aminooxy and thiol moieties has enabled the development of a highly convergent approach towards the synthesis of such a trivalent bioconjugation platform in good yields. The potential utility of this "ready-to-use" cross-linking reagent for creating complex and fragile tri-component (bio)molecular systems was illustrated through (1) the rapid preparation of a three-colour FRET cascade with valuable spectral properties and (2) the luminescent/fluorescent labelling of peptides and peptide-oligonucleotide conjugates. Thus, such (bio)molecular assemblies were readily obtained via a three-step process or in a "one-pot" manner, both involving oxime ligation, thiol-alkylation (S(N)2 or Michael addition) and copper-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions.

A novel sulfonated prosthetic group for [18F]-radiolabelling and imparting water solubility of biomolecules and cyanine fluorophores.

Synthesis and some applications of a novel [(18)F]-fluorinated prosthetic group based on the promising sultone radiochemistry and suitable for the labelling of amine-containing (bio)chemical compounds are described. The combined sequential use of two easy and efficient conjugation reactions namely the fluoride ring-opening of a 1,3-propanesultone moiety and the aminolysis of an N-hydroxysuccinimidyl ester is the key component of this original radiolabelling strategy. The mild reaction conditions and the release of a free sulfonic acid moiety as a result of the [(18)F]-induced sultone ring-opening reaction, both make this [(18)F]-conjugation method suitable for the radiofluorination of fragile and hydrophobic biomolecules and fluorophores, particularly by making the separation of the targeted [(18)F]-tagged sulfonated compound from its starting precursor easier and thus faster. The ability of this unusual prosthetic group to readily introduce the radioisotope within complex (bio)molecular architectures has been demonstrated by (1) the preparation of the first [(18)F]-labelled cyanine 5.5 (Cy 5.5) dye, a suitable precursor for the construction of hybrid positron emission tomography/near-infrared fluorescence (PET/NIRF) imaging probes and (2) the radiolabelling of a biologically relevant peptide bearing a single lysine residue.