Making small-molecule drugs usually goes something like this: set up a reaction, purify the intermediate, change a solvent, and repeat, repeat, repeat to get the final product. But there’s a lot of waste involved, which is why chemists stress the environmental benefits of an alternate approach: biocatalysis. Engineering enzymes to make reactions happen saves a lot of materials, minimizes chemical and hazardous waste, and even uses less plasticware and glassware. And not having to isolate intermediates saves time.
Some pharmaceutical companies are investigating biocatalysis at different points in their drug development pipelines, but mostly at one or two steps into the making of a small molecule. Scientists at Merck & Co. have taken this further—they are reporting an entire drug synthesis using a chain of nine enzymes, five of which had been engineered, to produce an experimental HIV drug at high yield in just a few steps (Science 2019, DOI: 10.1126/science.aay8484).
This biocatalytic cascade is turning heads. For the most part, scientists aren’t using biocatalysis to manufacture a compound so much as to develop it, says Princeton University chemist Todd Hyster. The Merck process stitches together nine enzymes to get good yields of the final product, which Hyster says is no small feat.
“I was blown away,” Hyster says of the first time he saw Merck scientists talk about this work. “It’s something that was very complicated.”
Mark Huffman, a chemist who led the work at Merck with Anna Fryszkowska, says they turned to biocatalysis in order to overcome a couple of key hurdles in synthesizing some molecules. One is stereochemistry. Islatravir is a nucleoside that blocks the HIV enzyme reverse transciptase and traditionally, in medicinal chemistry, it’s been hard to get the stereochemistry of nucleosides right, Huffman says. But this is something enzymes are designed by nature to do. The other is preventing unwanted side reactions. A number of steps in the traditional chemical synthesis of islatravir put the compound’s functional groups at risk of being lopped off, so they must be protected. Huffman says enzymes are specific in the types of reactions they catalyze, so there’s little to no risk of an unwanted side reaction.
On top of that, Huffman says, they are doing these reactions at neutral pH, in aqueous solvents, and at room temperature, which cuts down on electricity and the need for multiple bioreactors running under different conditions. Islatravir normally takes between 12 and 18 steps to make. With biocatalysis, the team has cut this down to three.
“You don’t have rigorous equipment requirements,” he says. “You’re usually running [these reactions] under much milder conditions.”
To run the cascade, the team started with 2-ethynylglycerol, and added a mixture of three enzymes to run one group of reactions. They then added more enzymes to drive a second set of reactions. Then, they remove the enzymes from the solution, which are immobilized and easy to filter out, and use four more enzymes to drive the final reactions that lead to islatravir. There are no intermediate purification steps. The overall yield is about 51% using biocatalysis, compared to yields of 7% and 15% using two more traditional syntheses.
To make their biocatalysts, the team surveyed natural enzymes, mostly from microbes, that interacted with the different intermediates in islatravir production. One of the reasons why Huffman says islatravir is an ideal small molecule to produce using biocatalysis is that most organisms have to make and break down nucleosides, so there are several natural enzymes found across multiple species. This gave the team a lot of starting material from which to alter amino acids and build the enzymes they needed to do their syntheses. By making adjustments to active sites and other areas of the enzymes, the team built five of the nine enzymes needed to make islatravir biochemically.
Huffman says that while islatravir is a good molecule to show that scientists can build large biocatalytic cascades, Merck is also looking at biocatalysis to make other small molecules and biologic drugs.
Alison Narayan, a biocatalysis chemist at the University of Michigan, calls Merck “bold” for putting the time, money, and people behind this change in production—it takes a lot of resources to try an entire synthesis via biocatalysis. And, she says, they’ve succeeded spectacularly. “It literally took my breath away,” Narayan says of her first exposure to this project in 2018. “I think it’s a huge accomplishment.”
She says that Merck’s islatravir work shows that industry is starting to appreciate what biocatalysis can do for their drug pipelines and their financial bottom lines. Alongside Merck, companies like GlaxoSmithKline and Pfizer are also exploring biocatalysis at different points in drug development and manufacturing.
“It’s an important proof of concept,” Narayan says. “This is a practical way to build molecules, and this will be the way that people will build molecules when you take into consideration efficiency, green-ness, and constructing an effective synthesis. Biocatalysis has a lot to offer.”