RESUMEN
Galactose oxidase (GOase) is a Cu-dependent metalloenzyme that catalyzes the oxidation of alcohols to aldehydes. An evolved GOase variant was recently shown to catalyze a desymmetrizing oxidation as the first enzymatic step in the biocatalytic synthesis of islatravir. Horseradish peroxidase (HRP) is required to activate the GOase, introducing cost and protein burden to the process. Herein we describe that complexes of earth-abundant Mn(iii) (e.g. Mn(OAc)3) can be used at low loadings (2 mol%) as small molecule alternatives to HRP, providing similar yields and purity profiles. While an induction period is observed when using Mn(OAc)3 as the activator, employment of alternative Mn(iii) sources, such as Mn(acac)3 and K3[Mn(C2O4)3], eliminates the induction period and provides higher conversions to product. We demonstrate that use of the Mn(OAc)3 additive is also compatible with subsequent biocatalytic steps in the islatravir-forming cascade. Finally, to exhibit the wider utility of Mn(OAc)3, we show that Mn(OAc)3 functions as a suitable activator for several commercially available variants of GOase with a series of alcohol substrates.
RESUMEN
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.