“The glory of the reaction is that it is significantly faster than other types of reactions in this class,” Fox explained.
Fox’ discovery comes in the wake of the 2008 Nobel Prize in Chemistry, awarded to the scientists who discovered the green fluorescent protein (GFP). Fox’ reaction, a variant on the Diels-Alder reaction, does not produce toxic byproducts and is potentially applicable to experiments that track the activity of proteins in live organisms. His results were published in the Journal of the American Chemical Society in September.
The tagging of biomolecules has become common practice in chemistry and molecular biology as a way to track the activity of proteins in a cell. One limitation of previously used tagging molecules, however, is that they must be incorporated into the protein when it is synthesized and thus cannot be used to tag molecules at different stages in the life of the protein.
GFP, for example, must be genetically linked to the protein that it tags when the DNA for that protein is transcribed.
“[GFP is] a great way to tag proteins, but you have to make a fusion at the gene level between your protein and GFP,” chemistry professor Michael Hecht explained. “However, there could be situations where you want to tag proteins chemically because you want to tag at a particular location or time.”
The reaction developed by Fox is an alternative to “click chemistry,” a relatively young method of organic synthesis that focuses on linking large molecules. The concept can be likened to stapling, Hecht said.
A persistent problem with popular click-chemistry reactions, though, has been that they necessitated the use of toxic catalysts, like copper, which prevented their use in live proteins. The only byproduct of the reaction is nitrogen gas, which is non-toxic and naturally occurring.
In addition to the issue of toxicity, controlled reactions in organisms must be bioorthogonal, which severely restricts the range of mechanisms one can use. A bioorthogonal reaction is one that does not normally occur in the cell and can therefore be easily directed.
“You wouldn’t want to use a type of chemistry that is the same or similar to the chemistry that goes on in cells,” Hecht said. “You want to use an orthogonal chemistry [so that] you can choose when you want to activate it and not have the cells make nonspecific reactions.”
Fox’ reaction, which involves the cycloaddition of tetrazine and cyclooctene, solves several of the problems of in vivo biomolecule tagging.
“The reaction is unique because it is faster than existing bioorthogonal reactions,” Melissa Blackman, the doctoral student in the Fox group who performed the experiments, said in an e-mail.
Currently used reactions also require higher concentrations of substrates, which are impractical for many experimental applications. That Fox’ reaction will proceed readily at very low concentrations is particularly useful.
“The enhanced reactivity of the reaction makes it suitable for applications that require low concentrations of substrates, such as protein modification,” Blackman said.
The applications of the reaction have already struck the scientific community. “Other scientists have contacted me so that’s been really interesting, being contacted by people who are interested in using it,” Fox said.
The Fox group was founded at the University of Delaware in 2001, and its research focuses on developing new types of organic reactions, specifically mechanisms of organic synthesis.
Chemistry professor emeritus Maitland Jones was Fox’ thesis adviser in the field of mechanistic organic chemistry.
“[Fox] wrote what I thought was a great thesis on a problem I loved,” he said in an e-mail. “[He] showed that conventional wisdom was dead wrong.”
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