PMID: 22553995;PMCID: PMC3368396;Abstract:
The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARACs aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARACs aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARACs central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry. © 2012 American Chemical Society.
PMID: 22029411;PMCID: PMC3219546;Abstract:
Cyclooctyne-based probes that become fluorescent upon reaction with azides are important targets for real-time imaging of azide-labeled biomolecules. The concise synthesis of a coumarin-conjugated cyclooctyne, coumBARAC, that undergoes a 10-fold enhancement in fluorescence quantum yield upon triazole formation with organic azides is reported. The design principles embodied in coumBARAC establish a platform for generating fluorogenic cyclooctynes suited for biological imaging. © 2011 American Chemical Society.
Restrain and release: A one-pot MukaiyamaMichael/epoxidation sequence introduced three stereocenters, an intramolecular isocyanate trapping produced a rigid 10-membered cyclic carbamate, and the selective opening of the cyclic carbamate was used to reveal the fully constructed natural product. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis.
PMID: 24059816;PMCID: PMC3857746;Abstract:
A triarylphosphine reagent that reacts with organic azides to install amide-linked diazirines is reported. This traceless Staudinger reagent reacts with complex organic azides to yield amide-linked diazirines, thus expanding the scope of the utility of both azide and diazirine chemistry. © 2013 American Chemical Society.