Anna Fryszkowska

Noncanonical amino acids (ncAAs) are valuable building blocks for therapeutic design. Prolines are particularly useful because their cyclic cores can limit the conformational flexibility of a larger molecular framework, allowing such scaffolds to be further tuned to enhance engagement with their biological targets. Herein, we describe a convergent approach to synthesizing noncanonical prolines from readily available starting materials mediated by a tryptophan synthase β-subunit (TrpB)-imine reductase (IRED) cascade. In the first step, the TrpB catalyzes C−C bond formation between a ketone substrate and an l-serine- or l-threonine (l-Thr)-derived amino acrylate intermediate. In the second step, the IRED reduces the resulting cyclic imine, in some cases simultaneously setting two stereocenters via a dynamic kinetic resolution. With this one-pot, sequential cascade, we demonstrate the synthesis of mono-, bi-, and tricyclic prolines bearing as many as four chiral centers, including three examples that contain a remote desymmetrized stereocenter and one example featuring an l-Thr-derived, fully functionalized pyrrolidine ring. Furthermore, we show that a d-proline is formed from l-amino acid starting materials, which has not been observed in closed-shell tryptophan synthase catalysis previously. This cascade is a scalable, operationally simple method to synthesize new prolines, and is poised to expand the development of therapeutics featuring structurally complex ncAAs.

Historically, many compelling therapeutic targets have been accessible only by injectable biologic drugs. Macrocyclic peptides, such as the proprotein convertase subtilisin/kexin type 9 inhibitor enlicitide for the treatment of atherosclerotic cardiovascular disease, are beginning to unlock these targets to orally administered therapies to enable broader patient access. We report the convergent biocatalytic assembly of enlicitide from simple building blocks enabled by a suite of engineered enzymes to catalyze selective peptide fragment formation, coupling, and macrocyclization in a protecting group–free manner. Together with efficient crystallizations that obviate the need for chromatography, this approach reduces the number of steps by greater than half compared with prior state-of-the-art methods, addressing long-standing synthetic challenges and offering a sustainable blueprint for the scalable development of complex peptide therapeutics.

Noncanonical amino acids (ncAAs) are valuable building blocks for novel therapeutics. Prolines are particularly useful because their cyclic cores can limit the conformational flexibility of a larger molecular framework, allowing such scaffolds to be further tuned to enhance engagement with their biological targets. Herein, we describe a convergent approach to synthesizing noncanonical prolines from readily available starting materials mediated by a tryptophan synthase β-subunit (TrpB)-imine reductase (IRED) cascade. In the first step, the TrpB catalyzes ketone substrate activation and C–C bond formation with an L-serine- or L-threonine (L-Thr)-derived amino acrylate intermediate. In the second step, the IRED reduces the cyclic imine intermediate, in some cases simultaneously setting two stereocenters via a dynamic kinetic resolution. With this one-pot, sequential cascade, we demonstrate the synthesis of mono-, bi-, and tricyclic prolines bearing as many as four chiral centers, including three examples possessing a remote desymmetrized stereocenter and one example possessing an L-Thr-derived, fully functionalized pyrrolidine ring. Furthermore, we show that D-prolines are accessible from L-amino acid starting materials, which has not been observed in tryptophan synthase catalysis previously. This cascade is a scalable, operation-ally simple method to synthesize new prolines, and is poised to expand the development of novel therapeutics featuring structurally complex ncAAs.

Biocatalytic oxidations have the potential to address many synthetic chemistry challenges, enabling the selective synthesis of chiral intermediates such as carbonyl compounds, alcohols, or amines. The use of oxygen-dependent enzymes can dramatically reduce the environmental footprint of redox transformations at manufacturing scale. Here, as part of the biocatalytic cascade to an anti-HIV investigational drug islatravir 1, we describe the development of an aerobic oxidation process delivering (R)-ethynylglyceraldehyde 3-phosphate 3 using an evolved galactose oxidase enzyme. Integrated enzyme and reaction engineering were critical for achieving a robust, high-yielding oxidation performed at pilot plant scale (>20 kg, 90% yield).

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.