Christopher D. McNitt

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.

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.

Photo-click chemistry strategies for spatiotemporal control of metal-free ligation, labeling, and surface derivatization

Three photo-click ligation strategies described in this account provide scientists with efficient and selective tools for derivatization of various molecules, polymers, and surfaces. Fast photochemical reactions that are utilized in these techniques permit spatiotemporal control of the process. The absence of activating reagents and catalysts, as well as compatibility with aqueous media, makes photo-click ligations suitable for biomedical applications. The first of these approaches relies on the photochemical decarbonylation of cyclopropenones to produce cyclooctynes. The latter undergo rapid catalyst-free strain-promoted azide–alkyne cycloaddition (SPAAC) to azide-tagged substrates. The second method is based on a very fast (>104 M–1 s–1) light-triggered hetero-Diels–Alder reaction and permits efficient derivatization of substrates bearing vinyl ether moiety. An even faster reaction between photochemically generated naphthoquinone methides (oNQMs) and thiols (~2 × 105 M–1 s–1) serves as a basis for a third method. This thiol photo-click chemistry allows for the selective derivatization of thiol-functionalized substrates or labeling of free cysteine residues in proteins. The thioether linkage produced by the reaction of oNQMs and a thiol is stable under ambient conditions, but can be cleaved by UV irradiation, regenerating free thiol. This feature permits the removal or replacement of immobilized compounds, as well as traceless substrate release.

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.

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.

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.

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.

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 ions 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.