Maleimide-based metal-free ligation with dienes: a comparative study.

A brief literature survey reveals that metal-free ligation such as the maleimide-based cycloaddition with electron-rich (hetero)dienes is a widespread tool for the assembly of (bio)molecular systems with applications in biotechnology, materials science, polymers and bio-organic chemistry. Despite their everyday use, only scattered data about their kinetics as well as the stabilities of corresponding products under physiological conditions, are accessible. These key parameters are yet, of paramount importance to ensure the rapid and effective preparation of stable compounds. Herein is reported a systematic study regarding the different classes of dienes used in chemoselective ligation, including their accessibility and stability, as well as comparative kinetic experiments and products stability assays. We took advantage of these data to develop a double labeling strategy from the combined use of cyclopentadiene and oxazole dienes.

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.

Investigation of tetrazine reactivity towards C-nucleophiles: pyrazolone-based modification of biomolecules.

Chemoselective, biocompatible ligation reactions are the key components for efficient and modular access to biomolecular scaffolds. Tetrazine ligation leads to the formation of a mixture of isomers, which makes reaction monitoring, purification and characterization of conjugates difficult. We report herein a modified tetrazine ligation strategy based on the use of a pyrazolone coupling partner, which provides a single molecule conjugate.

Covalent Modification of Biomolecules through Maleimide-Based Labeling Strategies.

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site-selective modification of proteins via thio-Michael addition of biothiols and, to a lesser extent, via Diels-Alder (DA) reactions with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris, which are used in cancer therapies. Among the key factors in the success of these groups is the availability of several maleimides that can be N-functionalized by fluorophores, affinity tags, spin labels, and pharmacophores, as well as their unique reactivities in terms of selectivity and kinetics. However, maleimide conjugate reactions have long been considered irreversible, and only recently have systematic studies regarding their reversibility and stability toward hydrolysis been reported. This review provides an overview of the diverse applications for maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being overcome by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged for the preparation of fluorogenic probes, which are mainly used for the specific detection of thiol analytes. A summary of the reported structures, their photophysical features, and their relative efficiencies is discussed in the last part of the review.

Correction: Metal-free oxidative ring contraction of benzodiazepinones: an entry to quinoxalinones.

Correction for 'Metal-free oxidative ring contraction of benzodiazepinones: an entry to quinoxalinones' by Hasan Mtiraoui, et al., Org. Biomol. Chem., 2017, 15, 3060-3068.

Metal-free oxidative ring contraction of benzodiazepinones: an entry to quinoxalinones.

A novel and practical synthesis of 3-benzoylquinoxalin-2(1H)-ones from benzodiazepin-2-ones in two steps from commercially available starting materials is reported. The reaction was achieved in the presence of N-bromosuccinimide in DMSO which served both as a solvent and an oxidant. Significantly, the yet unknown ketone to alcohol fluorescence turn-on of benzoylquinoxalinones was unveiled through the preparation of a fluorescently labelled cholesterol conjugate.

Fluorogenic Behaviour of the Hetero-Diels-Alder Ligation of 5-Alkoxyoxazoles with Maleimides and their Applications.

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero-Diels-Alder reaction of 5-alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions.

Valkyrie Probes: A Novel Class of Enzyme-Activatable Photosensitizers based on Sulfur- and Seleno-Rosamines with Pyridinium Unit.

The rational design of activatable photosensitizers (aPSs) uncaged by specific disease biomarkers is currently booming due to their positive attributes to achieve targeted photodynamic therapy (PDT). In this context, we present here the synthesis and detailed photophysical characterization of a novel class of hetero-rosamine dyes bearing sulfur or selenium as bridging heavy atom and 4-pyridyl meso-substituent as optically tunable group. The main feature of such photoactive platforms is the spectacular change of their spectral properties depending on the caging/decaging status of their 4-pyridyl moiety (cationic pyridinium vs. neutral pyridine). The preparation of two alkaline phosphatase (ALP)-responsive probes (named Valkyrie probes) was achieved through formal N-quaternarization with 4-phosphoryloxybenzyl, the traditional recognition moiety for this important diagnostic enzyme. Bio-analytical validations including fluorescence/singlet oxygen phosphorescence enzyme assays and RP-HPLC-fluorescence/-MS analyses have enabled us to demonstrate the viability and effectiveness of this novel photosensitizer activation strategy. Since sulfur-containing Valkyrie probe also retains high fluorogenicity in the orange-red spectral range, this study highlights meso-pyridyl-substituted S-pyronin scaffolds as valuable candidates for the rapid construction of molecular phototheranostic platforms suitable for combined fluorescence diagnosis and PDT.

Arylidene Meldrum's Acid: A Versatile Structural Motif for the Design of Enzyme-Responsive "Covalent-Assembly" Fluorescent Probes with Tailor-Made Properties.

Latent cyclic carbon-centered nucleophiles (latent C-nucleophiles) are recently proving their value in the field of reaction-based fluorescent probes, far beyond their primary utility in organic synthesis. They are typically used to introduce a Michael acceptor moiety acting as a recognition/reaction site for analyte to be detected or as a kinetic promoter of fluorogenic cascade reactions triggered by a reactive species. C-nucleophiles bearing a further reactive handle offer an additional opportunity for tuning the physicochemical/targeting properties or providing drug-releasing capabilities to these probes, through the covalent attachment of ad hoc chemical moiety. In order to implement such strategy to fluorogenic/chromogenic enzyme substrates based on the "covalent-assembly" principle, we have explored the potential of some functionalized derivatives of barbituric acid, piperidine-2,4-dione and Meldrum's acid. Our investigations based on the rational design and analytical validations of enzyme-responsive caged precursors of fluorescent pyronin dyes and 7-(diethylamino)coumarin-3-carboxylic acid, led to identify a versatile candidate suitable for this late-stage structural optimization approach. This Meldrum's acid derivative enables to either enhance water solubility or achieve the reversible conjugation of a targeting ligand, while promoting in situ formation of fluorophore upon enzymatic activation. This study opens the way to novel multifunctional fluorescence imaging probes and optically modulated small conjugate-based theranostics.

3-Benzoylquinoxalinone as a photoaffinity labelling derivative with fluorogenic properties allowing reaction monitoring under "no-wash" conditions.

Described herein is a quinoxalinone-based photoaffinity probe with caged fluorescence properties. Upon visible blue LED irradiation (λmax 450 nm), this photo-crosslinker is able to covalently capture proteins with concomitant fluorescence labelling. This process enables monitoring applications under "no wash" conditions.

Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes.

The ever-growing demand for fluorogenic dyes usable in the rapid construction of analyte-responsive fluorescent probes, has recently contributed to a revival of interest in the chemistry of diketopyrrolopyrrole (DPP) pigments. In this context, we have explored the potential of symmetrical and unsymmetrical DPP derivatives bearing two or one 4-pyridyl substituents acting as optically tunable group(s). The unique fluorogenic behavior of these molecules, closely linked to N-substitution/charge state of their pyridine unit (i.e., neutral pyridine or cationic pyridinium), has been used to design DPP-based fluorescent probes for detection of hypoxia-related redox enzymes and penicillin G acylase (PGA). In this paper, we describe synthesis, spectral characterization and bioanalytical validations of these probes. Dramatic differences in terms of aqueous stability and enzymatic fluorescence activation were observed. This systematic study enables to delineate the scope of application of pyridine-flanked DPP fluorophores in the field of enzyme biosensing.

Fluorogenic Enzyme-Triggered Domino Reactions Producing Quinoxalin-2(1H)-one-based Heterocycles.

A simple and effective biocompatible domino reaction triggered by a model protease and leading to the formation of strongly fluorescent quinoxalin-2(1H)-one N-heterocycles is described. Some positive attributes including versatility and the ability to provide outstanding fluorescence "OFF-ON" responses were revealed by this work. They open the way for practical applications of this novel type of "covalent-assembly"-based fluorescent probe in the fields of sensing and bioimaging.