Design of an in vitro biocatalytic cascade for the manufacture of islatravir

Abstract

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Organic solvent stability and long‐term storage of myoglobin‐based carbene transfer biocatalysts

“…Attractive features of enzymes in this regard include their potentially high degrees of chemo-, regio-, and stereoselectivity [1][2][3], which can be altered and tuned by protein engineering and directed evolution [7][8][9]. As a prominent example, a multienzyme cascade process involving a total of nine enzymes, five of which were modified via protein engineering, was recently implemented by Merck for the large-scale synthesis of the anti-HIV drug islatravir, providing a much shorter and more efficient route for the production of this molecule compared with previously reported synthetic routes [10]. In addition to the desired activity and selectivity, an important requirement for the application of enzymes in industrial and large-scale processes is an inherent (or acquired) stability in the presence of organic solvents, since high concentration of organic co-solvents are often required to solubilize lipophilic substrates and/or achieve high substrate loadings [2,4,[11][12][13][14].…”

Natural Heterogeneous Catalysis with Immobilised Oxidase Biocatalysts

“…GOase was also immobilised in the multi-enzyme cascade synthesis of islatravir, using affinity resin to improve downstream processing. 21 A summary of the improvements for the immobilised GOase M1 versus the free enzyme is summarised in Table 2. In general, biocatalysis adheres to 10 of the 12 principles of green chemistry.…”

“…[13][14][15] Previous engineering efforts for GOase and choline oxidase have developed a number of variants capable of oxidising a myriad of alcohols and have been demonstrated in both batch and flow reactor configurations, 1,[16][17][18][19][20] including the implementation of GOase in the gram scale biocatalytic manufacture of the investigational anti-HIV drug Islatravir. 21 Additionally, MAO-N versatility has been extensively shown in amine resolution and functionalization. 15,22 Here, we demonstrate a general immobilisation strategy that can be applied to a number of oxidase enzymes, enabling multifunctional catalysis that could be readily implemented in industrial processes.…”

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

“…Besides the well-established metal-and organocatalytic approaches [1], biocatalytic strategies offer an interesting alternative to install chirality into small molecules. To date, industrial biocatalysis has mastered a range of enzyme families including ketoreductases [2], transaminases [3], and imine reductases [4]. Looking forward, the increasing power of genomic mining and enzyme engineering will allow industrial access to even more enzyme families leading to an expansion of the available biocatalytic toolbox [5].…”