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Enzyme Makes Tough-To-Construct C–N Bonds

Chemical Biology: Halogenase directly adds nitrite or azide to unactivated aliphatic carbons

by Celia Henry Arnaud
January 30, 2014 | A version of this story appeared in Volume 92, Issue 5

Reaction scheme shows halogenase adding nitrite to unactivated aliphatic carbons.
Halogenase adds nitrite (shown here) or azide to unactivated aliphatic carbons.

The ability to add nitrogen-containing functional groups to unactivated aliphatic carbon atoms is a challenge for synthetic chemists. The search for enzymes to catalyze such reactions has likewise come up empty-handed. That luck, however, might be about to change.

Chemists at Pennsylvania State University have discovered that an enzyme that usually adds chloride ions to aliphatic carbons can be made to add nitrite or azide ions instead (Nat. Chem. Biol. 2014, DOI: 10.1038/nchembio.1438). Leading the research team were chemistry professor J. Martin Bollinger Jr. and graduate student Megan L. Matthews, now a postdoc at Scripps Research Institute, in La Jolla, Calif.

The enzyme, called SyrB2, was already known to halogenate or hydroxylate its substrate. SyrB2 has an Fe(IV) center that abstracts a hydrogen from an aliphatic carbon to form a carbon radical, which can then couple with either a halogen or a hydroxyl anion. Which reaction occurs is dictated by which anion is in striking distance. Because this effect is due just to positioning, team members thought they should be able to use other anions.

“We’re simply swapping out the ligands,” Matthews says. “We have this reactive intermediate. Substrate positioning allows us to transfer the one we want.”

But detecting the products proved to be a challenge and required that Matthews be a “bulldog about the analytical chemistry,” Bollinger says.

“Because we could detect binding, we knew the anions were getting into the pocket and behaving properly,” Matthews says. “That motivated us to push hard on the mass spec to detect these products.”

The team found that the natural enzyme can add nitrite or azide to unactivated carbons, but the reaction isn’t efficient. The researchers made a single mutation in the enzyme, which opened up the binding pocket to better accommodate the nitrogen-bearing ligands. The mutation also reduced the binding affinity of the usual halogen ligand. In this way, the researchers boosted the efficiency of C–N bond formation by about 30-fold. They hope to use directed evolution to improve the efficiency even more, Bollinger says.

The reported enzyme-catalyzed transformation is “amazing,” says Lawrence Que Jr., a chemistry professor at the University of Minnesota, Twin Cities, who studies iron-dependent enzymes. “It may also open the door to the development of parallel C–H bond functionalization reactions that may be catalyzed by synthetic iron complexes.”

M. Christina White, a chemistry professor at the University of Illinois, Urbana-Champaign, who is working on synthetic approaches to C–H bond activation, agrees. She says, “These exciting findings, which suggest anion promiscuity of halogenases, will surely inspire future studies to identify systems with useful yields and broad scope.”



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