Activation of Ammonia by a Carbene-Stabilized Dithiolene Zwitterion.

A carbene-stabilized dithiolene zwitterion (3) activates ammonia, affording 4• and 5, through both single-electron transfer (SET) and hydrogen atom transfer (HAT). Reaction products were characterized spectroscopically and by single-crystal X-ray diffraction. The mechanism of the formation of 4• and 5 was probed by experimental and computational methods. This discovery is the first example of metal-free ammonia activation via HAT.

Modular PET Agent Construction Strategy through Strain-Promoted Double-Click Reagent with Efficient Photoclick Step.

An efficient modular strategy for rapid assembly of positron emission tomography (PET) agents has been developed. The use of a sequential, rapid, and selective double-click reaction allows for a combinatorial approach to the cross-linking of positron emitter-bearing prosthetic groups with various ligands. The strain-promoted azide alkyne cyclization (SPAAC) coupling of 18F-labeled azide synthon with MC-DIBOD, a cyclooctadiyne with one of the triple bonds caged as a cyclopropenone moiety, produces a stable intermediate. A brief exposure of the latter to 350-420 nm light removes protection of the second triple bond allowing for the addition of an azide-tagged biomolecule. The utility of this strategy has been demonstrated by the construction of several PET agents. The value of modularity was demonstrated in the preparation of PSMA PET agents, where the hydrophilicity was easily modified to improve tumor to background contrast.

Photo-Click-Facilitated Screening Platform for the Development of Hetero-Bivalent Agents with High Potency.

A novel photo-click-based platform has been developed for rapid screening and affinity optimization of heterobivalent agents. This method allows for the efficient selection of high-affinity dual receptor-targeting agents via streamlining tedious organic synthesis and biological evaluation procedures required by traditional approaches. The high-avidity heterobivalent agents targeting both integrin αvß3 and urokinase-type plasminogen activator receptors have been developed using this photo-click-facilitated screening platform. The affinity screening results were further validated by traditional in vitro and in vivo evaluation techniques, reaffirming the reliability of the method. The convenience, rapidity, universality, and robustness of the screening platform, discussed in this report, can greatly facilitate the development of new heterobivalent agents for research and/or clinical applications.

Dual-Bioorthogonal Molecular Tool: "Click-to-Release" and "Double-Click" Reactivity on Small Molecules and Material Surfaces.

The development of reactive moieties that enable molecular control of bond-forming and bond-breaking reactions within complex media is highly important in materials and biomaterials research as it provides opportunities to carefully manipulate small molecules and material surfaces in a reliable manner. Despite recent advances in the realization of new ligation strategies and "click-and-release" systems, there has been little development of multifunctional moieties that feature a broad range of chemical capabilities. To address this challenge, we designed a molecular tool that can utilize four well-defined bioorthogonal chemistries interchangeably for the attachment, replacement, and release of molecules within a system: the Staudinger-Bertozzi ligation (SBL), perfluoroaryl azide Staudinger reaction (PFAA-SR), strain-promoted alkyne-azide cycloaddition (SPAAC), and strain-promoted alkyne-nitrone cycloaddition (SPANC). We demonstrate "click-to-release" and "double-click" reactivity on small molecules and gold nanoparticles (AuNPs) as a model material substrate. As a proof of concept for material derivatization, we employed 5 nm AuNPs-functionalized with a Rhodamine B derivative and biotin through the double-click strategy-and showed their potential as a pretargeted delivery nanocarrier. This multifunctional molecular tool enables the design and production of molecular and material systems with unique, modular, and tunable dynamic properties that can be altered under mild and bioorthogonal conditions.

Development of Bispecific NT-PSMA Heterodimer for Prostate Cancer Imaging: A Potential Approach to Address Tumor Heterogeneity.

Prostate cancer is a heterogeneous disease with a poor survival rate at late stage. In this report, a dual targeting PET agent was developed to partially address the tumor heterogeneity issue. The heterodimer F-BCN-PSMA-NT was designed to target PSMA and neurotensin receptor1 (NTR1), both of which have demonstrated great potential in prostate cancer management. The heterodimer was synthesized through the conjugation of Glu-urea-lys(Ahx) (PSMA targeting motif) and NT20.3 (NTR1 targeting motif) to a symmetric trifunctional linker, bearing an azide group for further modification. Radio-labeling was performed using strain promoted azide-alkyne click reaction with high yield. Cell based assays suggested that F-BCN-PSMA-NT has comparable or only slightly reduced binding affinity with the corresponding monomers. Small animal PET clearly demonstrated that the heterodimer probe has prominent uptake not only in NTR1 positive/PSMA negative PC-3 tumors (1.4 ± 0.3%ID/g), but also in the PSMA positive/NTR1 negative LnCap tumors (1.3 ± 0.2%ID/g). The tracer showed comparable tumor to background ratio with each monomer. In summary, prostate cancer is a heterogeneous disease in need of improved diagnostics and treatments. The PSMA-NT heterodimer represents a new class of molecules that can be used to target two distinct antigens related to prostate cancer. In addition to the imaging applications demonstrated in this study, the agent also holds great potential on the treatment of heterogeneous prostate cancer.

[18F]ODIBO: a prosthetic group for bioorthogonal radiolabeling of macromolecules via strain-promoted alkyne-azide cycloaddition.

A novel prosthetic group for the efficient radiolabeling of macromolecules has been developed. [18F]oxadibenzocyclooctyne ([18F]ODIBO) is synthesized in high radiochemical yield and applied for nearly quantitative conjugation to azide-tagged peptides and proteins at room temperature and low substrate concentrations. The resulting bioconjugates are chemically and radiochemically pure and free of toxic solvents and catalysts.

Multiphoton Activation of Photo-Strain-Promoted Azide Alkyne Cycloaddition "Click" Reagents Enables in Situ Labeling with Submicrometer Resolution.

Irradiation of cyclopropenone-masked dibenzocyclooctynes with near-infrared pulses from a femtosecond laser triggers photodecarbonylation via nonresonant two- or three-photon excitation. Multiphoton-generated cyclooctynes undergo a SPAAC reaction with organic azides, yielding the expected triazoles. Multiphoton-triggered SPAAC (MP-SPAAC) enables high resolution 3-D photoclick derivatization of hydrogels and tissues.

Temporal Labeling of Nascent RNA Using Photoclick Chemistry in Live Cells.

We report the first cellular application of a photoclick SPAAC reagent to label azide-functionalized RNA. 350 nm irradiation of a cyclopropenone caged oxo-dibenzocyclooctyne (photo-ODIBO) biotin yields formation of the SPAAC reactive species, which rapidly forms adducts with RNA containing 2'-azidoadenosine (2'N3-A). Photo-ODIBO was found to be highly stable in the presence of thiols, conferring greater stability relative to ODIBO. Light activated photo-ODIBO enabled tagging of cellular RNA, in addition to fluorescent imaging as well as enrichment of RNA in cell subpopulations via selective irradiation.

Facile Quenching and Spatial Patterning of Cylooctynes via Strain-Promoted Alkyne-Azide Cycloaddition of Inorganic Azides.

Little is known about the reactivity of strain-promoted alkyne-azide cycloaddition (SPAAC) reagents with inorganic azides. We explore the reactions of a variety of popular SPAAC reagents with sodium azide and hydrozoic acid. We find that the reactions proceed in water at rates comparable to those with organic azides, yielding in all cases a triazole adduct. The azide ion's utility as a cyclooctyne quenching reagent is demonstrated by using it to spatially pattern uniformly doped hydrogels. The facile quenching of cyclooctynes demonstrated here should be useful in other bioorthogonal ligation techniques in which cyclooctynes are employed, including SPANC, Diels-Alder, and thiol-yne.

Artificial Membrane Fusion Triggered by Strain-Promoted Alkyne-Azide Cycloaddition.

Artificial systems for controlled membrane fusion applicable for drug delivery would ideally use triggers that are orthogonal to biology. To apply the strain-promoted alkyne-azide cycloaddition (SPAAC) to drive membrane fusion, oxo-dibenzocyclooctyne (ODIBO)-lipid 1 was designed, synthesized, and studied alongside azadibenzocyclooctyne (ADIBO)-lipids 2-4 to assess fusion with liposomes containing azido-lipid 5. Lipids 1-2 were first shown to be effective for liposome derivatization. Next, fusion was evaluated using liposomes containing 1 and varying ratios of PC and PE via a FRET dilution fusion assay, and a 1:1 PC-to-PE ratio yielded the greatest signal change attributed to fusion. Finally, lipids 1-4 were compared, and 1 yielded the greatest triggering of fusion, while 2-4 yielded varying efficacies depending on the structural features of each lipid. Fusion was further validated through STEM studies showing larger multilamellar assemblies after liposome mixing, and FRET assay results supporting the mixing of liposome aqueous contents. This work provides a platform for triggered fusion toward drug delivery applications and an understanding of the effects of lipid structure and membrane composition on fusion.

"Shine & Click" Photo-Induced Interfacial Unmasking of Strained Alkynes on Small Water-Soluble Gold Nanoparticles.

In this study, we report the design, synthesis, and characterization of small 3 nm water soluble gold nanoparticles (AuNPs) that feature cyclopropenone-masked strained alkyne moieties capable of undergoing interfacial strain-promoted cycloaddition (i-SPAAC) with azides after exposure to UV-A light. A strained alkyne precursor was incorporated onto AuNPs by direct ligand exchange of a thiol-modified cyclopropenone-masked dibenzocyclooctyne (photoDIBO) ligand. These photoDIBO-AuNPs were characterized by 1 H NMR, IR, and UV/Vis spectroscopy, as well as transmission electron microscopy (TEM) and thermogravimetric analysis (TGA), and the extent of modification was quantified. Upon irradiation with UV-A light, photoDIBO-AuNPs underwent efficient and quantitative regeneration of the parent strained alkyne by photochemical decarbonylation to afford DIBO-derivatized AuNPs. DIBO-AuNPs were found to react cleanly and rapidly (k=5.3×10-2 m-1 s-1 ) by an interfacial strain-promoted alkyne-azide cycloadditon (i-SPAAC) with benzyl azide, which served as a simple model system. Furthermore, DIBO-AuNPs were reacted with various azides and a nitrone (interfacial strain-promoted alkyne-nitrone cycloaddition, i-SPANC) to showcase the generality of this approach for the facile modification of AuNP surfaces and their properties. The cyclopropenone-based photo-triggered click chemistry at the interface of water-soluble AuNPs offers exciting opportunities for the atom-by-atom control and assembly of functional materials for applications in materials and biomaterials science as well as in chemical biology.

Multifunctional Surface Manipulation Using Orthogonal Click Chemistry.

Polymer brushes are excellent substrates for the covalent immobilization of a wide variety of molecules due to their unique physicochemical properties and high functional group density. By using reactive microcapillary printing, poly(pentafluorophenyl acrylate) brushes with rapid kinetic rates toward aminolysis can be partially patterned with other click functionalities such as strained cyclooctyne derivatives and sulfonyl fluorides. This trireactive surface can then react locally and selectively in a one pot reaction via three orthogonal chemistries at room temperature: activated ester aminolysis, strain promoted azide-alkyne cycloaddition, and sulfur(VI) fluoride exchange, all of which are tolerant of ambient moisture and oxygen. Furthermore, we demonstrate that these reactions can also be used to create areas of morphologically distinct surface features on the nanoscale, by inducing buckling instabilities in the films and the grafting of nanoparticles. This approach is modular, and allows for the development of highly complex surface motifs patterned with different chemistry and morphology.

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

An order of magnitude difference in photoreactivity between bis- (photo-DIBOD, 1) and mono-cyclopropenone-caged dibenzocyclooctadiynes (MC-DIBOD, 5) allows for selective monodecarbonylation of 1. Alternatively, 5 is prepared by selective mono-cyclopropanation of dibenzo[a,e]cyclooctadiyne (DIBOD). MC-DIBOD (5) permits efficient sequential SPAAC cross-linking of azide-derivatized substrates. Cycloaddition to 5 converts an azide moiety into a photocaged form of triazole-fused dibenzo[a,e]cyclooctyne (3). While the azide reactivity of MC-DIBOD (5) and DIBOD is similar to that of other dibenzocyclooctynes, fusion of triazole to the dibenzocyclooctyne system in 3 results in a 3 orders of magnitude enhancement in SPAAC rates. In methanol, 3 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 (5) was utilized in the preparation of mixed bis-triazoles and derivatization of the protein BSA with fluorescent dye and polyethylene glycol.

A clickable and photocleavable lipid analogue for cell membrane delivery and release.

For drug delivery purposes, the ability to conveniently attach a targeting moiety that will deliver drugs to cells and then enable controlled release of the active molecule after localization is desirable. Toward this end, we designed and synthesized clickable and photocleavable lipid analogue 1 to maximize the efficiency of bioconjugation and triggered release. This compound contains a dibenzocyclooctyne group for bioorthogonal derivatization linked via a photocleavable 2-nitrobenzyl moiety at the headgroup of a synthetic lipid backbone for targeting to cell membranes. To assess delivery and release using this system, we report fluorescence-based assays for liposomal modification and photocleavage in solution as well as through surface immobilization to demonstrate successful liposome functionalization and photoinduced release. In addition, fluorophore delivery to and release from live cells was confirmed and characterized using fluorescence microscopy and flow cytometry analysis in which 1 was delivered to cells, derivatized, and photocleaved. Finally, drug delivery studies were performed using an azide-tagged analogue of camptothecin, a potent anticancer drug that is challenging to deliver due to poor solubility. In this case, the ester attachment of the azide tag acted as a caging group for release by intracellular esterases rather than through photocleavage. This resulted in a dose-dependent response in the presence of liposomes containing delivery agent 1, confirming the ability of this compound to stimulate delivery to the cytoplasm of cells.

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.

Copper-free click-chemistry platform to functionalize cisplatin prodrugs.

The ability to rationally design and construct a platform technology to develop new platinum(IV) [Pt(IV)] prodrugs with functionalities for installation of targeting moieties, delivery systems, fluorescent reporters from a single precursor with the ability to release biologically active cisplatin by using well-defined chemistry is critical for discovering new platinum-based therapeutics. With limited numbers of possibilities considering the sensitivity of Pt(IV) centers, we used a strain-promoted azide-alkyne cycloaddition approach to provide a platform, in which new functionalities can easily be installed on cisplatin prodrugs from a single Pt(IV) precursor. The ability of this platform to be incorporated in nanodelivery vehicle and conjugation to fluorescent reporters were also investigated.

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 (k = 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.

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.

Mutation of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at Trp-110 affects stereoselectivity of aromatic ketone reduction.

Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP(+))-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.

Attach, remove, or replace: reversible surface functionalization using thiol-quinone methide photoclick chemistry.

A very facile reaction between photochemically generated o-naphthoquinone methides (oNQMs) 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 oNQM species in the exposed areas. The latter react with thiol groups on the surface, producing thioether links between the substrate and the surface. Unreacted oNQM groups are rapidly hydrated to regenerate NQMP. The short lifetime of oNQM 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 oNQM-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 oNQM 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.