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.

In our study, Prodrug nucleoTide (ProTide) technology was applied to cordycepin to enhance its antiviral activities and metabolic stability. Using cordycepin as starting material, we developed a synthetic method to access a series of stereospecific-phosphoramidate derivatives with various ester groups. We also successfully synthesized halogenated cordycepin derivatives via stannylation. Our 17 ProTide-cordycepin derivatives were pharmacologically evaluated for their antiviral activities. Phosphorus diastereomers 22 S and 22 R showed moderate inhibitory activity against corona and influenza viruses, while these compounds and derivatives (25 S, 27 S, and 27 R) demonstrated promising antiviral efficacy against dengue virus. Pharmacological screening indicated that Sp-isomers generally exhibited slightly greater inhibitory activity than their Rp-isomer counterparts against the dengue virus. The selected ProTides were assessed for their metabolic mechanism and stability via carboxypeptidase and microsomes. The hydrolysis rate of the Rp-isomers was observed to be slightly higher than that of the Sp-isomers, and the addition of a fluorine group also modestly increased this rate and fluorinated 39 S extended its half-life compared to nonfluorinated counterparts. These findings suggested not only structure-activity relationships of cordycepin ProTide but also the comprehensive synthetic route to access cordycepin derivatives for further antiviral development.

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.