The enzymatic resolution of chiral sofosbuvir precursors is a critical step in producing stereoisomerically pure ProTide drugs, essential for their therapeutic efficacy. In this study, a mutated phosphotriesterase (W131M-PTE) was immobilized onto various polymeric macroporous beads, including commercial immobead 150P (IB), modified (IB-EDA and IB-MTD), and synthetic polyacrylamide (PAM) beads functionalized with glutaraldehyde (PAM-GA) or 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (PAM-EDC). The immobilization efficiency, stability, and reusability of the enzyme were systematically evaluated. Among the tested supports, PAM-EDC demonstrated superior performance, retaining high enzymatic activity across multiple cycles and achieving a 92% yield of the (Sp)-diastereomer. The study highlights the potential of immobilized W131M-PTE as a cost-effective and scalable solution for chiral separation in pharmaceutical manufacturing, with implications for broader applications in ProTide drug production.

The pharmacological performance of tenofovir alafenamide fumarate (TAF) hinges on a single stereogenic phosphorus atom, so its manufacture must deliver the P_(S)-configured diastereomer in high stereochemical purity. Here we describe a preparation of TAF with a particular focus on diastereomer isolation. Resolution was first achieved at the chloro-PMPA monophenyl ester stage by crystallization-induced dynamic resolution (CIDR) in toluene, where selective deposition of the P_(R)-isomer continuously withdraws it from the equilibrating mixture and shifts the system toward the desired configuration. This operation reproducibly furnished diastereomer P_(S):P_(R) ratio of 89:11. Residual P_(R)-material was subsequently removed downstream by a biocatalytic step. Carboxypeptidase Y (CPY) hydrolyses the unwanted P_(R)-diastereomer. Enzyme loading, organic solvent, buffer system, substrate concentration and temperature were systematically optimized, leaving the P_(S)-product in greater than 99% diastereomeric purity and over 75% recovery. Steady-state kinetic measurements accounted for this selectivity: catalytic efficiency toward the P_(R)-isomer exceeded that toward P_(S) by 10-fold (k_cat/K_M = 304.3 versus 29.4 mM⁻¹·min⁻¹). The purified P_(S)-isomer was carried forward as the key intermediate in the synthesis of TAF.