ABSTRACT
3-oxidopyridinium ions are water stable and soluble heteroaromatic betaines that behave as latent dipoles and undergo a wide variety of cycloadditions. Research into the cycloaddition reactions of 3-oxidopyridiniums was spearheaded by Alan R. Katritzky and collaborators from the early 1970s until the late 1980s, but they have yet to be used for bioorthogonal applications. Herein we report that 3-oxidopyridiniums can readily react with 4-dibenzocyclooctynol (DIBO), a common bioorthogonal handle, in a [3+2] cycloaddition. The mechanism was investigated by altering the electronics of the reaction by changing the substituent on the 5 position of the pyridinium. Electron-donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.3×10-4 s-1 with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07â M-1 s-1 with 5-amino-N-methyl-3-oxidopyridinium. 3-oxidopyridiniums' appreciable cycloaddition rates and compatibility with bioorthogonally relevant environments give them the potential to be used in a variety of bioconjugation applications.
ABSTRACT
According to Canada's Food Report Card 2016, there are 4 million foodborne illnesses acquired each year in the nation alone. The leading causes of foodborne illness are pathogenic bacteria such as shigatoxigenic/verotoxigenic Escherichia coli (STEC/VTEC) and Listeria monocytogenes. Most current detection methods used to identify these bacterial pathogens are limited in their validity since they are not specific to detecting metabolically active organisms, potentially generating false-positive results from non-living or non-viable bacteria. Previously, our lab developed an optimized bioorthogonal non-canonical amino acid tagging (BONCAT) method which allows for the labeling of translationally active wild-type pathogenic bacteria. Incorporation of homopropargyl glycine (HPG) into the cellular surfaces of bacteria allows for protein tagging using the bioorthogonal alkyne handle to report on the presence of pathogenic bacteria. Here, we use proteomics to identify more than 400 proteins differentially detected by BONCAT between at least two of five different VTEC serotypes. These findings pave the way for future examination of these proteins as biomarkers in BONCAT-utilizing assays.
Subject(s)
Amino Acids , Shiga-Toxigenic Escherichia coli , Shiga-Toxigenic Escherichia coli/metabolism , Serogroup , Bacteria/metabolism , BiomarkersABSTRACT
Donor-acceptor Stenhouse adducts (DASAs) are a novel class of solvatochromic photoswitches with increasing importance in photochemistry. Known for their reversibility between open triene and closed cyclized states, these push-pull molecules are applicable in a suite of light-controlled applications. Recent works have sought to understand the DASA photoswitching mechanism and reactive state, as DASAs are vulnerable to irreversible "dark switching" in polar protic solvents. Despite the utility of fluorescence spectroscopy for providing information regarding the electronic structure of organic compounds and gaining mechanistic insight, there have been few studies of DASA fluorescence. Herein, we characterize various photophysical properties of two common DASAs based on Meldrum's acid and dimethylbarbituric acid by fluorescence spectroscopy. This approach is applied in tandem with complexation by cyclodextrins and cucurbiturils to reveal the zwitterionic charge separation of these photoswitches in aqueous solution and the protective nature of supramolecular complexation against degradative dark switching. DASA-M, for example, was found to form a weak host-guest inclusion complex with (2-hydroxypropyl)-γ-cyclodextrin, with a binding constant K = 60 M-1, but a very strong inclusion complex with cucurbit[7]uril, with K = 27,000 M-1. This complexation within the host cavity was found to increase the half-life of both DASAs in aqueous solution, indicating the significant and potentially useful stabilization of these DASAs by host encapsulation.
