Evaluation and Visualization of Pathway Efficiency based on Subcellular Protein Localizations (Workshop Abstract)

Popik OV, Sommer B, Hofestädt R, Ivanisenko VA. Evaluation and Visualization of Pathway Efficiency based on Subcellular Protein Localizations (Workshop Abstract). In: Sommer B, ed. Proceedings of the CELLmicrocosmos neXt workshop. Bielefeld: Bielefeld University; 2014: 9-10

Attach, Remove, or Replace: Reversible Surface Functionalization Using Thiol–Quinone Methide Photoclick Chemistry

A very facile reaction between photochemically generated <i>o</i>-naphthoquinone methides (<i>o</i>NQMs) and thiols is employed for reversible light-directed surface derivatization and patterning. A thiol-functionalized glass slide is covered with an aqueous solution of a substrate conjugated to 3-(hydroxymethyl)-2-naphthol (NQMP). Subsequent irradiation via shadow mask results in the efficient conversion of NQMP into reactive <i>o</i>NQM species in the exposed areas. The latter react with thiol groups on the surface, producing thioether links between the substrate and the surface. Unreacted <i>o</i>NQM groups are rapidly hydrated to regenerate NQMP. The short lifetime of <i>o</i>NQM in aqueous solution prevents its migration from the site of irradiation, thus allowing for the spatial control of the surface derivatization. A two-step procedure was employed for protein patterning: photobiotinylation of the surface with an NQMP–biotin conjugate followed by staining with FITC–avidin. The orthogonality of <i>o</i>NQM–thiol and azide click chemistry allowed for the development of a sequential click strategy, which might be useful for the immobilization of light-sensitive compounds. The thioether linkage produced by the reaction of <i>o</i>NQM and a thiol is stable under ambient conditions but can be cleaved by UV irradiation, regenerating the free thiol. This feature allows for the removal or replacement of immobilized substrates

Sequential “Click” – “Photo-Click” Cross-Linker for Catalyst-Free Ligation of Azide-Tagged Substrates

Heterobifunctional linker allows for selective catalyst-free ligation of two different azide-tagged substrates via strained-promoted azide–alkyne cycloaddition (SPAAC). The linker contains an azadibenzocyclooctyne (ADIBO) moiety on one end and a cyclopropenone-masked dibenzocyclooctyne (photo-DIBO) group on the other. The first azide-derivatized substrate reacts only at the ADIBO end of the linker as the photo-DIBO moiety is azide-inert. After the completion of the first SPAAC step, photo-DIBO is activated by brief exposure to 350 nm light from a fluorescent UV lamp. The unmasked DIBO group then reacts with the second azide-tagged substrate. Both click reactions are fast (<i>k =</i> 0.4 and 0.07 M^–1 s^–1, respectively) and produce quantitative yield of ligation in organic solvents or aqueous solutions. The utility of the new cross-linker has been demonstrated by conjugation of azide functionalized bovine serum albumin (azido-BSA) with azido-fluorescein and by the immobilization of the latter protein on azide-derivatized silica beads. The BSA–bead linker was designed to incorporate hydrolytically labile fragment, which permits release of protein under the action of dilute acid. UV activation of the second click reaction permits spatiotemporal control of the ligation process

Sequential Photochemistry of Dibenzo[<i>a</i>,<i>e</i>]dicyclopropa[<i>c</i>,<i>g</i>][8]annulene-1,6-dione: Selective Formation of Didehydrodibenzo[<i>a</i>,<i>e</i>][8]annulenes with Ultrafast SPAAC Reactivity

An order of magnitude difference in photoreactivity between bis- (photo-DIBOD, <b>1</b>) and mono-cyclopropenone-caged dibenzocyclooctadiynes (MC-DIBOD, <b>5</b>) allows for selective monodecarbonylation of <b>1</b>. Alternatively, <b>5</b> is prepared by selective mono-cyclopropanation of dibenzo­[<i>a</i>,<i>e</i>]­cyclooctadiyne (DIBOD). MC-DIBOD (<b>5</b>) permits efficient sequential SPAAC cross-linking of azide-derivatized substrates. Cycloaddition to <b>5</b> converts an azide moiety into a photocaged form of triazole-fused dibenzo­[<i>a</i>,<i>e</i>]­cyclooctyne (<b>3</b>). While the azide reactivity of MC-DIBOD (<b>5</b>) and DIBOD is similar to that of other dibenzocyclooctynes, fusion of triazole to the dibenzocyclooctyne system in <b>3</b> results in a 3 orders of magnitude enhancement in SPAAC rates. In methanol, <b>3</b> reacts with butyl azide at an astonishing rate of 34 M^–1 s^–1, thus representing the most reactive cyclooctyne analogue reported so far. MC-DIBOD (<b>5</b>) was utilized in the preparation of mixed bis-triazoles and derivatization of the protein BSA with fluorescent dye and polyethylene glycol

Photoactivatable Fluorescein Derivatives Caged with a (3-Hydroxy-2-naphthalenyl)methyl Group

The (3-hydroxy-2-naphthalenyl)­methyl (NQMP) group represents an efficient photocage for fluorescein-based dyes. Thus, irradiation of the 6-NQMP ether of 2′-hydroxymethylfluorescein with low-intensity UVA light results in a 4-fold increase in emission intensity. Photoactivation of nonfluorescent NQMP-caged 3-allyloxyfluorescein produces a highly emissive fluorescein monoether. To facilitate conjugation of the caged dye to the substrate of interest via click chemistry, the allyloxy appendage was functionalized with an azide moiety

A Dynamic Duo: Pairing Click Chemistry and Postpolymerization Modification To Design Complex Surfaces

Advances in key 21st century technologies such as biosensors, biomedical implants, and organic light-emitting diodes rely heavily on our ability to imagine, design, and understand spatially complex interfaces. Polymer-based thin films provide many advantages in this regard, but the direct synthesis of polymers with incompatible functional groups is extremely difficult. Using postpolymerization modification in conjunction with click chemistry can circumvent this limitation and result in multicomponent surfaces that are otherwise unattainable. The two methods used to form polymer thin films include physisorption and chemisorption. Physisorbed polymers suffer from instability because of the weak intermolecular forces between the film and the substrate, which can lead to dewetting, delamination, desorption, or displacement. Covalent immobilization of polymers to surfaces through either a “grafting to” or “grafting from” approach provides thin films that are more robust and less prone to degradation. The grafting to technique consists of adsorbing a polymer containing at least one reactive group along the backbone to form a covalent bond with a complementary surface functionality. Grafting from involves polymerization directly from the surface, in which the polymer chains deviate from their native conformation in solution and stretch away from the surface because of the high density of chains. Postpolymerization modification (PPM) is a strategy used by our groups over the past several years to immobilize two or more different chemical functionalities onto substrates that contain covalently grafted polymer films. PPM exploits monomers with reactive pendant groups that are stable under the polymerization conditions but are readily modified via covalent attachment of the desired functionality. “Click-like” reactions are the most common type of reactions used for PPM because they are orthogonal, high-yielding, and rapid. Some of these reactions include thiol-based additions, activated ester coupling, azide–alkyne cycloadditions, some Diels–Alder reactions, and non-aldol carbonyl chemistry such as oxime, hydrazone, and amide formation. In this Account, we highlight our research combining PPM and click chemistry to generate complexity in polymer thin films. For the purpose of this Account, we define a complex coating as a polymer film grafted to a planar surface that acts as a template for the patterning of two or more discrete chemical functionalities using PPM. After a brief introduction to grafting, the rest of the review is arranged in terms of the sequence in which PPM is performed. First, we describe sequential functionalization using iterations of the same click-type reaction. Next, we discuss the use of two or more different click-like reactions performed consecutively, and we conclude with examples of self-sorting reactions involving orthogonal chemistries used for one-pot surface patterning

Photoreactive Polymer Brushes for High-Density Patterned Surface Derivatization Using a Diels–Alder Photoclick Reaction

Reactive polymer brushes grown on silicon oxide surfaces were derivatized with photoreactive 3-(hydroxymethyl)­naphthalene-2-ol (NQMP) moieties. Upon 300 or 350 nm irradiation, NQMP efficiently produces <i>o</i>-naphthoquinone methide (<i>o</i>NQM), which in turn undergoes very rapid Diels–Alder addition to vinyl ether groups attached to a substrate, resulting in the covalent immobilization of the latter. Any unreacted <i>o</i>NQM groups rapidly add water to regenerate NQMP. High-resolution surface patterning is achieved by irradiating NQMP-derivatized surfaces using photolithographic methods. The Diels–Alder photoclick reaction is orthogonal to azide–alkyne click chemistry, enabling sequential photoclick/azide-click derivatizations to generate complex surface functionalities