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Engineered enzyme does asymmetric C–N bond twofer

Enzyme delivers chiral anilines with high selectivity

by Leigh Krietsch Boerner
October 20, 2021 | A version of this story appeared in Volume 99, Issue 39


The scheme shows how a common enzyme is catalyzed into forming an enantioselective carbon-nitrogen bond.

Chemists have coaxed a common enzyme into catalyzing the formation of an enantioselective carbon-nitrogen bond. The approach can generate the preferred enantiomer of amines and anilines within molecules of interest (Nat. Chem., 2021, DOI: 10.1038/s41557-021-00794-z). Such chiral C–N bonds are practically ubiquitous in pharmaceuticals, from antibiotics to anticancer compounds.

Frances Arnold and other scientists from the California Institute of Technology and the University of Girona used directed evolution to engineer a dual-function enzyme that performs a two-step, asymmetric N–H insertion into a carbene intermediate. The enzyme can produce over 90% of the preferred enantiomer. Such selectivity is difficult using traditional organic synthesis methods.

Zhen Liu, a postdoctoral researcher in Arnold’s lab and the paper’s first author, says nonenzymatic carbene N–H insertion requires two different catalysts: one to form the carbene intermediate and a second to donate a proton and produce the final aniline product. The team’s enzyme can do both steps. It is adapted from a cytochrome P450 enzyme, which contains an iron-porphyrin cofactor. The Fe center on the cofactor catalyzes the carbene formation, and the adjacent amino acids aid in proton transfer. N–H insertion with a carbene is challenging to make enantioselective, Liu says, because any solvent or reagent that could donate a proton, including water, can react with the carbene, thwarting the desired reaction. In an enzyme, however, the pocket where the reaction takes place is protected from such wayward protons.

“This reaction is actually a bit of a serendipitous discovery,” Liu says. Looking through 40 enzyme variants that the group previously engineered to catalyze carbene and nitrene reactions, the team found one that had high enantiomeric selectivity for the N–H carbene insertion. To find out why it was so selective, the group swapped out a serine in the binding pocket for other amino acids. Without serine, the reaction flipped from highly to mildly selective. Computational studies revealed that the serine forms two H bonds with the carbene intermediate. These bonds anchor the compound into a specific orientation, leaving one side of the molecule more open for water to approach and make the proton transfer, and preferentially forming one enantiomer over the other, Liu says.

The researchers synthesized a variety of primary and secondary anilines and several aliphatic amines in moderate to high yields and with 50–98% enantiomeric selectivity. They also made over a gram of the precursor to the fungicide (S)-ofurace, demonstrating that the enzyme can produce large amounts of target compounds.

Enantioselective N–H carbene insertions are very hard to do, says Rudi Fasan, a biocatalytic chemist at the University of Rochester. “This work represents another great example of biocatalysis and directed evolution providing an efficient solution to a difficult synthetic problem,” he says.Liu says he hopes that reactions like this will show synthetic chemists that biocatalysis is something they can use to make valuable compounds. “You can use enzymes to do powerful transformations,” he says, which sometimes can work better than traditional synthetic pathways.


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