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Bolstering Biocatalysis

Advance in chiral amine synthesis positions engineered enzymes for an enhanced role in organic synthesis

by Stephen K. Ritter
July 29, 2013 | A version of this story appeared in Volume 91, Issue 30

From a racemic mixture, an engineered enzyme, monoamine oxidase, selectively oxidizes the (S)-enantiomer to an imine. The imine is recycled back to a mixture of (R)- and (S)-enantiomers. Eventually, only the desired (R)-enantiomer remains. It is then used in the synthesis of the antihistamine levocetirizine.
A reaction scheme shows the way a new bioenzymatic approach makes enantiomerically pure substances by selectively recycling the undesired enantiomer.
From a racemic mixture, an engineered enzyme, monoamine oxidase, selectively oxidizes the (S)-enantiomer to an imine. The imine is recycled back to a mixture of (R)- and (S)-enantiomers. Eventually, only the desired (R)-enantiomer remains. It is then used in the synthesis of the antihistamine levocetirizine.

When you pop a prescription medicine into your mouth, chances are pretty good that the active ingredient derives its healing power from a chiral amine unit in the molecular structure. This fact may not interest you because you’re probably focused on getting well or preventing getting sick. But to the chemists behind the scenes, the fact that up to 45% of all the molecules they are developing into drugs contain a chiral amine is a big deal.

Chiral molecules are like gloves. They come in a pair and look the same but are asymmetrical, nonsuperimposable mirror images of each other. Try putting a left-handed glove on your right hand and you see it doesn’t work. It’s the same with left- and right-handed molecules. Each enantiomer, as each right- or left-handed molecule is called, can produce different biological effects. For instance, one enantiomer of a drug may alleviate symptoms, whereas its mirror image might not show any bioactivity or even cause side effects.

These differences and the policies of the Food & Drug Administration stemming from them put pressure on chemists to identify the enantiomer they need and to figure out the most efficient synthetic pathway to that molecule. To do that, researchers are increasingly turning to enzyme-based processes as an alternative to traditional chemical syntheses. Enzymes are catalytic proteins with exquisite ability to selectively make one enantiomer over the other.

“We are entering an era where the use of biocatalysts should sit alongside more traditional methodologies in the forefront of chemists’ minds when designing synthetic routes,” says enzyme expert Nicholas J. Turner of the University of Manchester, in England. Compared with classical chemical methods, Turner says, enzymatic methods typically are shorter, more efficient, and safer. Overall they tend to be greener and cheaper. Companies that manufacture pharmaceuticals, agricultural chemicals, flavors and fragrances, and biofuels are salivating over the potential. They just need the enzymes.

To that end, Turner’s group has developed a toolbox of monoamine oxidase enzymes that are generally applicable to commercial-scale organic synthesis of chiral amines, both simple and complex (J. Am. Chem. Soc. 2013, DOI: 10.1021/ja4051235).

Monoamine oxidases use oxygen to catalyze the oxidation of amines to imines. Because the enzymes selectively act on only one of the amine enantiomers, they allow scientists to deracemize a racemic mixture, that is, a 1:1 mixture of the mirror-image molecules. Through deracemization, scientists isolate the enantiomer they need and use it as such or to complete the synthesis of the active chiral amine they are after.

Nature has provided a rich array of enzymes to synthesize the biomolecules necessary for life, Turner explains. And chemists have successfully exploited these natural enzymes in chemical synthesis. But natural biocatalysts act upon only a limited range of molecule types and don’t always perform optimally in large-scale reactions. To unleash the full power of enzymes, scientists are modifying them.

Turner’s team started with a natural monoamine oxidase from the fungus Aspergillus niger. The researchers introduced random errors in the genes coding for the enzyme to alter the size and shape of its active site. This site is a snug pocket where reactant molecules bind and are chemically altered by the enzyme. The researchers then screened the altered enzymes for their ability to oxidize reactant molecules of different complexity. The team then stitched the revised genetic code for optimized enzymes into the DNA of an Escherichia coli bacterium, which served as a surrogate to mass-produce the engineered enzymes.

Pharmaceutical companies are starting to use biocatalytic methods in redesigning the chemical syntheses of drugs already on the market to help drive down cost, Turner says. For example, Codexis and Merck & Co. scientists used one of Turner’s variants to redo the manufacture of a chiral amine intermediate for making boceprevir, a Merck drug to treat hepatitis C (J. Am. Chem. Soc. 2012, DOI: 10.1021/ja3010495).

“This report from the Turner lab is a beautiful example of how enzymes can be tuned for activity on substrates of increasing complexity,” says Gjalt W. Huisman, vice president of pharmaceutical R&D at Codexis. A key advantage of this class of enzymes over traditional amine-producing enzymes is that they make accessible not only primary chiral amines but also secondary and tertiary ones, Huisman points out.

Turner’s group has devised “a particularly clever system for biocatalytic deracemization,” adds Manfred T. Reetz of Philipps University, in Marburg, Germany, a pioneer in developing engineered enzymes. “It’s an excellent example of directed evolution of stereoselective enzymes as catalysts in organic chemistry.”

The challenge now remains in getting more of these enzymes into the hands of industrial chemists who can use them to build molecules, Turner says.

To that end, in 2012 Turner and his collaborators launched a nonprofit enzyme clearinghouse called Discovery Biocatalysts. The advantages of being nonprofit, explains Gareth A. DeBoos, the company’s director, are that the university gets recognition for its development and that its intellectual property is not being used by Discovery to generate profit. The benefits for Discovery’s industrial customers, he adds, are that they get access to new enzymes that are difficult to source elsewhere and they work with a company that is not competing with them at any level. Discovery has one product offering so far, a monoamine oxidase screening kit containing 10 enzyme variants, which goes for about $940.

What Turner would ultimately like to happen is to bring into focus the need to rethink the way target molecules are constructed, like in the case of Merck’s boceprevir. To aid chemists in identifying where biocatalysts might best be applied, Turner and his Manchester colleague Elaine O’Reilly have proposed that the concept of “biocatalytic retrosynthesis” be further developed and integrated into the training of future organic chemists (Nat. Chem. Biol. 2013, DOI:10.1038/nchembio.1235).

Retrosynthesis is a thought experiment in which a chemist identifies a target molecule to synthesize and then works backward on paper to disassemble the molecule. By pulling the molecule apart bond by bond, it’s easier to see the building blocks needed and chart the most efficient path to making the molecule from available starting materials.

Turner and O’Reilly suggest that the best way to design molecules in the future will be to combine traditional and biocatalytic retro­synthesis in a single tool. Turner thinks his collection of enzyme variants that chemists can pull off a shelf and use when needed is a stepping-stone in that direction.


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