Photo-triggering of the metal-free azide to acetylene cycloaddition reaction was achieved by masking the triple bond of dibenzocyclooctynes as cyclopropenone. Such masked cyclooctynes do not react with azides in the dark. Irradiation of cyclopropenones results in the efficient (Φ355 = 0.33) and clean regeneration of the corresponding dibenzocyclooctynes, which then undergo facile catalyst-free cycloadditions with azides to give corresponding triazoles under ambient conditions. In-situ light activation of a cyclopropenone linked to biotin made it possible to label living cells expressing glycoproteins containing N-azidoacetyl-sialic acid. The cyclopropenone-based photo-triggered click chemistry offers exciting opportunities to label living organisms in a temporally and spatially controlled manner and may facilitate the preparation of microarrays.

It is generally accepted that the presentation of multiple ligands on a nanoparticle surface can improve cell targeting; however, little work has been done to determine whether an optimal ligand density exists. We have recently developed a site-specific bioconjugation strategy that allows for distinct control of ligand density on a nanoparticle through the combined utilization of expressed protein ligation (EPL) and copper-free click chemistry. This EPL-Click conjugation strategy was applied to create superparamagnetic iron oxide (SPIO) nanoparticles labeled with HER2/neu targeting affibodies at differing ligand densities. It was discovered that an intermediate ligand density provided statistically significant improvements in cell binding compared with higher and lower ligand densities. This intermediate optimal ligand density was conserved across nanoparticles with differing hydrodynamic diameters, different HER2/neu targeting ligands and also to cells with lower receptor densities. Additionally, an intermediate optimal ligand density was also evident when nanoparticles were labeled with folic acid.

The utility of catalyst-free azide-alkyne [3 + 2] cycloaddition for the immobilization of a variety of molecules onto a solid surface and microbeads was demonstrated. In this process, the surfaces are derivatized with aza-dibenzocyclooctyne (ADIBO) for the immobilization of azide-tagged substrates via a copper-free click reaction. Alternatively, ADIBO-conjugated molecules are anchored to the azide-derivatized surface. Both immobilization techniques work well in aqueous solutions and show excellent kinetics under ambient conditions. We report an efficient synthesis of aza-dibenzocyclooctyne (ADIBO), thus far the most reactive cyclooctyne in cycloaddition to azides. We also describe convenient methods for the conjugation of ADIBO with a variety of molecules directly or via a PEG linker.

Surfaces containing reactive ester polymer brushes were functionalized with cyclopropenone-masked dibenzocyclooctynes for the light activated immobilization of azides using catalyst-free click chemistry. The photodecarbonylation reaction in the amorphous brush layer is first order for the first 45 s with a rate constant of 0.022 s(-1). The catalyst-free cycloaddition of surface bound dibeznocyclooctynes proceeds rapidly in the presence of azides under ambient conditions. Photolithography using a shadow mask was used to demonstrate patterning with multiple azide containing molecules. This surface immobilization strategy provides a general and facile platform for the generation of multicomponent surfaces with spatially resolved chemical functionality.

Methods for fluorophore targeting to cellular proteins can allow imaging with dyes that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the E. coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 minutes. Second, we evaluated a panel of five different cyclooctyne structures, and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

Block copolymer-based vesicles have recently garnered a great deal of interest as nanoplatforms for drug delivery and molecular imaging applications due to their unique structural properties. These nanovesicles have been shown to direct their cargo to disease sites either through enhanced permeability and retention or even more efficiently via active targeting. Here we show that the efficacy of nanovesicle targeting can be significantly improved when prepared from polymer-lipid blends compared with block copolymer alone. Polymer-lipid hybrid nanovesicles were produced from the aqueous co-assembly of the diblock copolymer, poly(ethylene oxide)-block-polybutadiene (PEO-PBD), and the phospholipid, hydrogenated soy phosphatidylcholine (HSPC). The PEG-based vesicles, 117 nm in diameter, were functionalized with either folic acid or anti-HER2/neu affibodies as targeting ligands to confer specificity for cancer cells. Our results revealed that nanovesicles prepared from polymer-lipid blends led to significant improvement in cell binding compared to nanovesicles prepared from block copolymer alone in both in vitro cell studies and murine tumor models. Therefore, it is envisioned that nanovesicles composed of polymer-lipid blends may constitute a preferred embodiment for targeted drug delivery and molecular imaging applications.

UV irradiation of alkyl-, aryl-, and heteroatom-substituted cyclopropenones results in the loss of carbon monoxide and the formation of quantitative yields of corresponding alkynes. The quantum yield of the photochemical decarbonylation reaction ranges from 20% to 30% for alkyl-substituted cyclopropenones to above 70% for the diphenyl- and dinaphthylcyclorpopenones. Rapid formation (<5 ns) and then a somewhat slower decay (ca. 40 ns) of an intermediate in this reaction was observed by using laser flash photolysis. The DFT calculations allowed us to identify this intermediate as a zwitterionic species formed by a cleavage of one of the carbon-carbon bonds of the cyclopropenone ring. The latter then rapidly loses carbon monoxide to produce the ultimate acetylenic product. Despite their high photoreactivity, cyclopropenones were found to be thermally stable compounds with the exception of hydroxy- and methoxy-substituted cyclopropenones. The latter undergo rapid solvolysis in hydroxylic solvents even at room temperature. The application of this reaction to the in situ generation of the enediyne structure was illustrated by the photochemical preparation of benzannulated enediyne 12.

The density functional theory analysis predicts that the thermal decarbonylation of cyclopropenones proceeds by the sequential and regioselective cleavage of both single bonds in a three-membered ring. The initial ring-opening process results in the formation of a reactive zwitterionic intermediate 6, which is separated from the free alkyne and carbon monoxide by a very low energy barrier. Femtosecond pump-probe transient absorption spectroscopy experiments showed that light-induced decarbonylation is also a stepwise process but apparently proceeds on the excited-state surface. The lifetime of the intermediate in the photodecarbonylation reaction is very short and is dependent on substitution and solvent polarity. Thus, bis-p-anisyl-substituted species decays with tau = 0.6 ps, bis-alpha-naphthyl-substituted intermediate has a lifetime of tau = 11 ps, while the bis(2-methoxy-1-naphthyl)-substituted analogue survives for 83 ps in chloroform and for 168 ps in argon-saturated methanol. The loss of carbon monoxide from these intermediates results in the formation of corresponding acetylenes in an electronically ground state. The addition of triplet quenchers does not affect the dynamics or outcome of the reaction.