Bioorthogonal tetrazine cycloadditions have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aqueous media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-infrared fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chemistry is suitable for in vitro labeling experiments, and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities.

We studied the selectivity of a functional model of cytochrome c oxidase's active site that mimics the coordination environment and relative locations of Fe(a3), Cu(B), and Tyr(244). To control electron flux, we covalently attached this model and analogs lacking copper and phenol onto self-assembled monolayer-coated gold electrodes. When the electron transfer rate was made rate limiting, both copper and phenol were required to enhance selective reduction of oxygen to water. This finding supports the hypothesis that, during steady-state turnover, the primary role of these redox centers is to rapidly provide all the electrons needed to reduce oxygen by four electrons, thus preventing the release of toxic partially reduced oxygen species.

We have prepared and characterized mixed self-assembled monolayers (SAM) on gold electrodes from azido alkane thiols and various omega-functionalized alkane thiols. In the presence of copper(I) catalysts, these azide-modified surfaces are shown to react rapidly and quantitatively with terminal acetylenes forming 1,2,3-triazoles, via "click" chemistry. The initial azide substituents can be identified and monitored using both grazing-angle infrared (IR) and X-ray photoelectron spectrosopies. Acetylenes possessing redox-active ferrocene substituents react with the azide-terminated mixed SAMs and electrochemical measurements of the ferrocene-modified SAM electrodes have been used to quantify the redox centers attached to these platforms. Time-resolved electrochemical measurements have enabled us to follow the formation of these ferrocene centers and thus to measure the rate of the surface "click" reaction. Under optimal conditions this well-behaved second-order reaction takes place with a rate constant of 1 x 10(3) M(-)(1) s(-)(1). Typical reaction times of several minutes were realized using micromolar concentrations of acetylene. These techniques have been used to construct well-characterized, covalently modified monolayers that can be employed as functional electrode surfaces.

We demonstrate the applicability of Sharpless "click" chemistry, specifically Huisgen 1,3-dipolar cycloadditions, as a general methodology for functionalizing surfaces coated with self-assembled monolayers. Ferrocene immobilization was used as our model, and the resulting monolayers were analyzed using traditional surface analytical techniques. Our preliminary results indicate that this reaction proceeds to completion at room temperature in aqueous solvent. The triazole group is a thermally and hydrolytically stable, conjugated linkage. The reactants, acetylenes and azides, are independently stable; they do not react with common organic reagents or with themselves. Thus the potential for this reaction to immobilize a wide range of functionally complex substances on metal surfaces is significant. To our knowledge this is the first report of the use of "click" chemistry to modify a well-defined electrode surface.

SUMMARY Parkinson disease is characterized by loss of dopamine neurons in the substantia nigra 1 . Similar to other major neurodegenerative disorders, no disease-modifying treatment exists. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits 2 . Herein we report an efficient single-step conversion of isolated mouse and human astrocytes into functional neurons by depleting the RNA binding protein PTB. Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes into new neurons that can innervate into endogenous neural circuits. Astrocytes in different brain regions are found to convert into different neuronal subtypes. Using a chemically induced model of Parkinson’s disease, we show conversion of midbrain astrocytes into dopaminergic neurons whose axons reconstruct the nigro-striatal circuit. Significantly, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. Similar disease phenotype reversal is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible new approach to treating neurodegeneration by replacing lost neurons.

Nanoparticles have emerged as key materials for biomedical applications because of their unique and tunable physical properties, multivalent targeting capability, and high cargo capacity1,2. Motivated by these properties and by current clinical needs, numerous diagnostic3–10 and therapeutic11–13 nanomaterials have recently emerged. Here we describe a novel nanoparticle targeting platform that uses a rapid, catalyst-free cycloaddition as the coupling mechanism. Antibodies against biomarkers of interest were modified with trans-cyclooctene and used as scaffolds to couple tetrazine-modified nanoparticles onto live cells. We show that the technique is fast, chemoselective, adaptable to metal nanomaterials, and scalable for biomedical use. This method also supports amplification of biomarker signals, making it superior to alternative targeting techniques including avidin/biotin.

Conspectus Disease mechanisms are increasingly being resolved at the molecular level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 103 M−1 sec−1) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-infrared-emitting fluorophores to dienophile-modifed extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control experiments showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concentrations of labeling agent over a timescale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chemical reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogs, inside living cells. Because the coupling partners are small molecules (<300 daltons), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chemistry can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin–avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clinical samples for biomarker profiling to predict malignancy and patient outcome. Finally, we discuss recent results with tetrazine reactions used for in vivo molecular imaging applications. Rapid tetrazine cycloadditions allow modular labeling of small molecules with the most commonly used positron emission tomography isotope, 18F. Additionally, in recent work we have begun to apply this reaction directly in vivo for the pre-targeted imaging of solid tumors. Future work with tetrazine cycloadditions will undoubtedly lead to optimized protocols, improved probes, and additional biomedical applications.

Bioorthogonal reactions have found widespread use in applications ranging from glycan engineering to in vivo imaging. Researchers have devised numerous reactions that can be predictably performed in a biological setting. Depending on the requirements of the intended application, one or more reactions from the available toolkit can be readily deployed. As an increasing number of investigators explore and apply chemical reactions in living systems, it is clear that there are a myriad of ways in which the field may advance. This article presents an outlook on the future of bioorthogonal chemistry. I discuss currently emerging opportunities and speculate on how bioorthogonal reactions might be applied in research and translational settings. I also outline hurdles that must be cleared if progress toward these goals is to be made. Given the incredible past successes of bioorthogonal chemistry and the rapid pace of innovations in the field, the future is undoubtedly very bright.