Bigley Andrew N, Raushel Frank M
Department of Chemistry and Physics, Southwestern Oklahoma State University, Weatherford, OK, 73096, USA.
Department of Chemistry, Texas A&M University, College Station, TX, 77843, USA.
Chem Biol Interact. 2025 Sep 5;418:111597. doi: 10.1016/j.cbi.2025.111597. Epub 2025 Jun 6.
Outbreaks of viral diseases, such as COVID-19, and chronic viral diseases, such as HIV and hepatitis, have highlighted the need to develop antiviral medications. ProTide nucleotide analogs such as Remdesivir and Sofosbuvir have become an important class of antivirals. The ProTides are phosphonamidate prodrugs, which contain an alanine ester and a phenyl group esterified to a chiral phosphorus of a nucleotide analog. The resulting triester effectively masks the charge on the phosphate moiety to facilitate entry into the cell and are much more effective than the corresponding nucleoside analogs. Once in the cell, the ProTides require activation by cellular enzymes to remove the masking groups on the phosphorus. The activation in the cell is dependent on the stereochemistry of the phosphorus center with the effectiveness of a given isomer differing between tissue types. The ProTides are produced as single isomers at the phosphorus center by chiral chromatography or selective crystallization, but in many cases only a single isomer can be produced, potentially limiting the effectiveness of the ProTides. The phosphotriesterase (PTE) from Brevundimonas diminuta is well known for its ability to selectively hydrolyze chiral phosphotriesters. The extensive directed evolution of PTE has led to the identification of variants that can selectively hydrolyze the phosphonamidate precursor of the ProTides, allowing the preparation of optically pure ProTides. Importantly, the variant In1W-PTE allows the isolation of the pure R-isomer while G60A-PTE and W131M-PTE allow the isolation of the pure S-isomer, thereby facilitating the efficient preparation of either isomer of the final ProTide.
诸如新冠病毒病等病毒性疾病的爆发，以及诸如艾滋病毒和肝炎等慢性病毒性疾病，凸显了开发抗病毒药物的必要性。瑞德西韦和索磷布韦等前药型核苷酸类似物已成为一类重要的抗病毒药物。前药型核苷酸类似物是膦酰胺酯前体药物，其含有一个丙氨酸酯和一个苯基，它们被酯化到核苷酸类似物的手性磷原子上。由此产生的三酯有效地掩盖了磷酸部分的电荷，便于进入细胞，并且比相应的核苷类似物更有效。一旦进入细胞，前药型核苷酸类似物需要细胞内的酶激活，以去除磷原子上的掩蔽基团。细胞内的激活取决于磷中心的立体化学，给定异构体的有效性在不同组织类型之间存在差异。前药型核苷酸类似物通过手性色谱法或选择性结晶法在磷中心以单一异构体形式产生，但在许多情况下只能产生单一异构体，这可能会限制前药型核苷酸类似物的有效性。来自纤细短杆菌的磷酸三酯酶（PTE）以其选择性水解手性磷酸三酯的能力而闻名。对PTE进行的广泛定向进化已导致鉴定出能够选择性水解前药型核苷酸类似物的膦酰胺酯前体的变体，从而能够制备光学纯的前药型核苷酸类似物。重要的是，变体In1W-PTE能够分离出纯R-异构体，而G60A-PTE和W131M-PTE能够分离出纯S-异构体，从而有助于高效制备最终前药型核苷酸类似物的任何一种异构体。
