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New enzyme catalyzes biaryl cross-coupling reactions

Researchers engineered an artificial P450 enzyme to selectively catalyse the formation of a key motif

by Fernando Gomollón-Bel, special to C&EN
March 7, 2022


Scheme showing the production of an engineered enzyme.

Using directed evolution, researchers have developed an enzyme that catalyses the formation of biaryl bonds (Nature 2022, DOI: 10.1038/s41586-021-04365-7). This new enzyme makes carbon-carbon bonds between aromatic moieties in a very selective manner, providing a new tool for the preparation of chiral ligands, pharmaceuticals, and materials.

Traditionally, chemists have synthesized biaryl bonds using metal-catalyzed reactions, such as Suzuki and Negishi cross-couplings. However robust, these processes require additional prefunctionalization steps to yield the target molecule. “It’s still challenging to make sterically hindered biaryl bonds,” explains lead author Alison R. H. Narayan, a chemist at the University of Michigan. Other structures, like electron deficient aromatic rings, also pose problems. “We envisioned a biocatalytic alternative could solve both issues and become a viable alternative for synthetic chemists,” she adds. The team used directed evolution, a technique that intentionally mutates the genes for a parent enzyme in successive rounds to evolve new catalytic functions.

The team explored different enzymes from secondary metabolic pathways involved in natural product formation to find a starting point. “Sometimes, these proteins already forge molecules that resemble our targets,” explains Narayan. “It’s all about finding a hint of reactivity, even 0.1%. Then it’s often possible to further optimize,” she adds. In this case, the team identified a cytochrome P450 enzyme that naturally catalyzes dimerization of coumarin into biaryls in Aspergillus fungi, and they engineered it to couple a broader range of substrates.

The team created over 2,000 variants of the natural enzyme and screened for those that could catalyze a cross-coupling reaction to produce chiral biaryl compounds. They used the best-yielding enzymes for successive rounds of engineering to improve yield, site selectivity and stereoselectivity. The team’s final enzyme gave a 92-fold improvement in yield.

“The catalytic and selective construction of carbon-carbon bonds is one of the most important tasks in organic chemistry,” says Ania Fryszkowska, a biocatalysis expert at Merck & Co. “Nature generates molecular complexity with ease and elegance, avoiding protecting groups and oxidative state readjustments, which is usually unachievable using traditional chemistry,” she adds.

Fryszkowska highlights how forming certain biaryl bonds is hard with the currently available tools: “It needs chiral ligands, protecting groups, auxiliary moieties,” she says. Biocatalysis offers a simple synthetic approach, which is typically safer and greener, too, since it minimizes the number of purifications and isolations along the way.

Further engineering resulted in an enzyme that offers unprecedented atroposelectivity—a preference between stereoisomers with axial chirality—in their target compound (shown). “This is a unique case in biocatalysis,” says Fryszkowska.

Atroposelectivity is key to preparing chiral ligands, which are used in asymmetric catalysis, and to synthesize certain commercial drugs like the antibiotic vancomycin and the antimalarial ancistrocladine.

By comparing the sequence of the engineered enzyme with other natural proteins, Narayan’s team has already found other promising leads: “We discovered a treasure trove of enzymes that have impressive cross-coupling activity on different classes of substrates, some with sufficient activity that engineering is not required,” says Narayan. “This panel of enzymes offers a solid starting point for others interested in planning this transformation into a synthesis, as biaryl bond formation is a bread-and-butter transformation.”

UPDATE: This story was updated on March 17, 2022, to add Alison R. H. Narayan's affiliation. She is at the University of Michigan.


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