Part 1 laid out the strategy. Now, it’s time to make the molecule. Four enzymes, one vessel, and plenty of competing amines. What could possibly go wrong? Dani and LC return with Stephanie Galanie, Director in Biocatalysis, and Artis Klapars, Distinguished Scientist in Process Chemistry, for a closer look at the chemistry behind MK-0616 manufacturing.

We start with the northern fragment, where making three bonds selectively means getting four engineered enzymes to play well together, ATP recycling included. Then we tackle fragment ligation with an evolved thioesterase and a hydrogen-borrowing cascade that closes a macrocycle. How do you drive an uphill reaction toward completion? And why would you start engineering an enzyme with the “wrong” substrate? Stephanie and Artis explain the solutions behind the synthesis.

Finally, traditional coupling chemistry gets its moment alongside enzymatic macrocyclization, and a “counterion dance” gets MK-0616 out of solution in the right purity and salt form. We wrap up with what it takes to turn ambitious chemistry into a manufacturing process, and why protecting-group-free fragment coupling, concentrated macrocyclizations, and crystallization could change how we make complex peptides.

Read some of the papers we discussed today:

Biocatalytic cascades enable manufacture of the macrocyclic peptide MK-0616 - ⁠⁠Science⁠⁠

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