Frédéric Friscourt

Metal-Free Sequential [3 + 2]-Dipolar Cycloadditions using Cyclooctynes and 1,3-Dipoles of Different Reactivity

Although metal-free cycloadditions of cyclooctynes and azides to give stable 1,2,3-triazoles have found wide utility in chemical biology and material sciences, there is an urgent need for faster and more versatile bioorthogonal reactions. We have found that nitrile oxides and diazocarbonyl derivatives undergo facile 1,3-dipolar cycloadditions with cyclooctynes. Cycloadditions with diazocarbonyl derivatives exhibited similar kinetics as compared to azides, whereas the reaction rates of cycloadditions with nitrile oxides were much faster. Nitrile oxides could conveniently be prepared by direct oxidation of the corresponding oximes with BAIB, and these conditions made it possible to perform oxime formation, oxidation, and cycloaddition as a one-pot procedure. The methodology was employed to functionalize the anomeric center of carbohydrates with various tags. Furthermore, oximes and azides provide an orthogonal pair of functional groups for sequential metal-free click reactions, and this feature makes it possible to multifunctionalize biomolecules and materials by a simple synthetic procedure that does not require toxic metal catalysts.

A Fluorogenic Probe for the Catalyst-Free Detection of Azide-Tagged Molecules

Fluorogenic reactions in which non- or weakly fluorescent reagents produce highly fluorescent products can be exploited to detect a broad range of compounds including biomolecules and materials. We describe a modified dibenzocyclooctyne that under catalyst-free conditions undergoes fast strain-promoted cycloadditions with azides to yield strongly fluorescent triazoles. The cycloaddition products are more than 1000-fold brighter compared to the starting cyclooctyne, exhibit large Stokes shift, and can be excited above 350 nm, which is required for many applications. Quantum mechanical calculations indicate that the fluorescence increase upon triazole formation is due to large differences in oscillator strengths of the S(0) ↔ S(1) transitions in the planar C(2v)-symmetric starting material compared to the symmetry-broken and nonplanar cycloaddition products. The new fluorogenic probe was successfully employed for labeling of proteins modified by an azide moiety.

Polar Dibenzocyclooctynes for Selective Labeling of Extracellular Glycoconjugates of Living Cells

Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represent a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location, and in particular it has been shown that DIBO derivative 4 can enter cells, thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.

One-Pot Three-Step Synthesis of 1,2,3-Triazoles by Copper-Catalyzed Cycloaddition of Azides with Alkynes formed by a Sonogashira Cross-Coupling and Desilylation

A microwave-assisted, one-pot, three-step Sonogashira cross-coupling-desilylation-cycloaddition sequence was developed for the convenient preparation of 1,4-disubstituted 1,2,3-triazoles starting from a range of halides, acyl chlorides, ethynyltrimethylsilane, and azides.

Convergent Assembly and Surface Modification of Multifunctional Dendrimers by Three Consecutive Click Reactions

Multifunctional dendrimers bearing two or more surface functionalities have the promise to provide smart drug delivery devices that can for example combine tissue targeting and imaging or be directed more precisely to a specific tissue or cell type. We have developed a concise synthetic methodology for efficient dendrimer assembly and heterobifunctionalization based on three sequential azide-alkyne cycloadditions. The methodology is compatible with biologically important compounds rich in chemical functionalities such as peptides, carbohydrates, and fluorescent tags. In the approach, a strain-promoted azide-alkyne cycloaddition (SPAAC) between polyester dendrons modified at the focal point with an azido and 4-dibenzocyclooctynol (DIBO) moiety provided dendrimers bearing terminal and TMS-protected (TMS=trimethylsilyl) alkynes at the periphery. The terminal alkynes were outfitted with azido-modified polyethylene glycol (PEG) chains or galactosyl residues by using Cu(I) -catalyzed azide-alkyne cycloadditions (CuAAC). Next, a one-pot TMS deprotection and second click reaction of the resulting terminal alkyne with azido-containing compounds gave multifunctional dendrimers bearing complex biologically active moieties at the periphery.

Fluorogenic Strain-Promoted Alkyne-Diazo Cycloadditions

Fluorogenic reactions, in which non- or weakly fluorescent reagents produce highly fluorescent products, are attractive for detecting a broad range of compounds in the fields of bioconjugation and material sciences. Herein, we report that a dibenzocyclooctyne derivative modified with a cyclopropenone moiety (Fl-DIBO) can undergo fast strain-promoted cycloaddition reactions under catalyst-free conditions with azides, nitrones, nitrile oxides, as well as mono- and disubstituted diazo-derivatives. Although the reaction with nitrile oxides, nitrones, and disubstituted diazo compounds gave cycloadducts with low quantum yield, monosubstituted diazo reagents produced 1H-pyrazole derivatives that exhibited an approximately 160-fold fluorescence enhancement over Fl-DIBO combined with a greater than 10,000-fold increase in brightness. Concluding from quantum chemical calculations, fluorescence quenching of 3H-pyrazoles, which are formed by reaction with disubstituted diazo-derivatives, is likely due to the presence of energetically low-lying (n,π*) states. The fluorogenic probe Fl-DIBO was successfully employed for the labeling of diazo-tagged proteins without detectable background signal. Diazo-derivatives are emerging as attractive reporters for the labeling of biomolecules, and the studies presented herein demonstrate that Fl-DIBO can be employed for visualizing such biomolecules without the need for probe washout.

Selective and reversible photochemical derivatization of cysteine residues in peptides and proteins

Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 μM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.

Branched Polyhedral Oligomeric Silsesquioxane Nanoparticles Prepared via Strain-Promoted 1,3-Dipolar Cycloadditions

Conjugation of small organic molecules and polymers to polyhedral oligosilsesquioxane (POSS) cores results in novel hybrid materials with unique physical characteristics. We report here an approach in which star-shaped organic-inorganic scaffolds bearing eight cyclooctyne moieties can be rapidly functionalized via strain-promoted azide-alkyne cycloaddition (SPAAC) to synthesize a series of nearly monodisperse branched core-shell nanoparticles with hydrophobic POSS cores and hydrophilic arms. We established that SPAAC is a robust method for POSS core octafunctionalization with the reaction rate constant of 1.9 × 10(-2) M(-1) s(-1). Functionalization with poly(ethylene glycol) (PEG) azide, fluorescein azide, and unprotected lactose azide gave conjugates which represent different classes of compounds: polymer conjugates, fluorescent dots, and bioconjugates. These resulting hybrid compounds were preliminarily tested for their ability to self-assemble in solution and at the air-water interface. We observed the formation of robust smooth Langmuir monolayers with diverse morphologies. We found that polar lactose moieties are completely submerged into the subphase whereas the relatively hydrophobic fluorescein arms had extended conformation at the interface, and PEG arms were partially submerged. Finally, we observed the formation of stable micelles with sizes between 70 and 160 nm in aqueous solutions with size and morphology of the structures dependent on the molecular weight and the type of the peripheral hydrophilic moieties.

A metal-free turn-on fluorescent probe for the fast and sensitive detection of inorganic azides.

Sodium azide is toxic and widely used in agricultural, commercial products, and research laboratories. Thus it is of a significant environmental concern and there is a need for the development of a rapid detection method. A fluorogenic dibenzylcyclooctyne derivative (Fl-DIBO) is herein described as a fluorescent probe for the rapid detection of inorganic azide via Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC). Fl-DIBO was found to be highly selective toward NaN3 in comparison to other common anions with good sensitivity and detection limit of 10μM.