An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir

Abstract

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Figure 1

Proposed biocatalytic route to Molnupiravir. (a) Wild-type cytidine deaminase (CD) catalyzes the hydrolysis of 2 to 3. (b) The active site of CD with uridine bound (PDB code: 1AF2). The Zn²⁺ ion is shown in gray. His102, Cys129, Cys132, and catalytic Glu104 are shown as atom-colored sticks with blue carbons. Uridine ligand is shown as atom-colored sticks with black carbons. (c) Proposed synthetic route to Molnupiravir 1. 2 is converted to 4 by an engineered CD followed by acylation using Novozym 435.^,

Figure 2

Characterization of wild-type cytidine deaminase (CD). (a) Pathways for conversion of 2 to 4 catalyzed by CD. Pathway A involves direct conversion of 2 to 4 using NH₂OH as a nucleophile. Pathway B involves initial hydrolysis of 2 to uridine 3, which is then transformed to 4 through condensation with NH₂OH. Pathway B is the dominant pathway when using the wild-type enzyme, leading to an equilibrium distribution of 3 and 4. (b) The conversion of 2 (1 mM) and 3 (1 mM) to 4 by CD (5 μM) in the presence of 1% NH₂OH (∼300 mM, pH 7) is monitored by increasing absorbance at 310 nm. Similar final concentrations of 4 are formed using either 2 (red) or 3 (blue) as a starting material, or in reactions starting from 4 (green). (c) The conversion of 2 (750 mM) by CD (25 μM) to 3 and 4 is monitored by HPLC analysis in the presence of 10% NH₂OH (∼3 M, pH 7). The time course of the reaction indicates CD operates via pathway B.

Figure 3

Directed evolution of a cytidine deaminase (CD). (a) Directed evolution of CD to CD1.3 showing mutations installed during each round. (b) Time course for the formation of 4 from 2 (50 mM) catalyzed by CD1.3 (2.5 μM, red), CD1.2 (2.5 μM, green), CD1.1 (2.5 μM, orange), wild-type CD (2.5 μM, blue), and no enzyme (black) in the presence of 1% (∼300 mM) NH₂OH, pH 7. (c) HPLC traces showing 2 (500 mM) conversion to 4 and 3 catalyzed by CD1.3 (25 μM, red) and wild-type CD (25 μM, blue) in the presence of 10% (∼3 M) NH₂OH, pH 7. (d) The active site of CD with uridine bound (PDB code: 1AF2). Mutations installed in rounds 1, 2, and 3 are shown as orange, green, and red spheres, respectively. The uridine ligand is shown as atom-colored sticks with black carbons, and the Zn²⁺ cofactor is shown in gray. His102, Cys129, Cys132, and catalytic Glu104 are shown as atom-colored sticks with blue carbons.

Figure 4

Biocatalytic process for N-hydroxy-cytidine synthesis. (a) In situ crystallization of 4 in reactions catalyzed by CD1.3 leads to product enrichment. Reaction conditions: 750 mM 2, 7.5 μM CD1.3, 10% NH₂OH (∼3 M, pH 7), 4 °C. (b) HPLC trace of the product isolated from the biotransformation described in (a). (c) Stacked ¹H NMR traces of 4, commercial standard (top), and the product isolated from the biotransformation described in (a) (bottom).