{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T21:01:06Z","timestamp":1793048466872,"version":"4.5.0"},"reference-count":41,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,7,30]],"date-time":"2024-07-30T00:00:00Z","timestamp":1722297600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioeng. Biotechnol."],"abstract":"<jats:p>Specific stereoisomer is paramount as it is vital for optimizing drug efficacy and safety. The quest for the isolation of desired stereoisomer of active pharmaceutical ingredients or key intermediates drives innovation in drug synthetic and biocatalytic methods. Chiral phosphoramidate is an important building block for the synthesis of antiviral drugs such as remdesivir and sofosbuvir. Given the clinical potency of the (<jats:italic>S<\/jats:italic>p)-diastereomer of the drugs, an enzyme capable of completely hydrolyzing the (<jats:italic>R<\/jats:italic>p)-diastereomer is needed to achieve the purified diastereomers <jats:italic>via<\/jats:italic> biocatalytic reaction. In this study, protein engineering of phosphotriesterase (PTE) was aimed to improve the specificity. Employing rational design and site-directed mutagenesis, we generated a small library comprising 24 variants for activity screening. Notably, W131M and I106A\/W131M variants demonstrated successful preparation of pure (<jats:italic>S<\/jats:italic>p)-diastereomer of remdesivir and sofosbuvir precursors within a remarkably short hydrolysis time (&amp;lt;20\u00a0min). Our work unveils a promising methodology for producing pure stereoisomeric compounds, utilizing novel biocatalysts to enable the chemoenzymatic synthesis of phosphoramidate nucleoside prodrugs.<\/jats:p>","DOI":"10.3389\/fbioe.2024.1446566","type":"journal-article","created":{"date-parts":[[2024,7,30]],"date-time":"2024-07-30T04:25:50Z","timestamp":1722313550000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["Improving stereoselectivity of phosphotriesterase (PTE) for kinetic resolution of chiral phosphates"],"prefix":"10.3389","volume":"12","author":[{"given":"Nongluck","family":"Jaito","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suthathip","family":"Phetlum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Titiporn","family":"Saeoung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thanat","family":"Tiyasakulchai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nitipol","family":"Srimongkolpithak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanaporn","family":"Uengwetwanit","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,7,30]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"2421","DOI":"10.1002\/adsc.201900424","article-title":"Biocatalysis: a pharma perspective","volume":"361","author":"Adams","year":"2019","journal-title":"Adv. Synthesis Catal."},{"key":"B2","doi-asserted-by":"publisher","first-page":"e202200709","DOI":"10.1002\/cssc.202200709","article-title":"Biocatalysis as key to sustainable industrial chemistry","volume":"15","author":"Alc\u00e1ntara","year":"2022","journal-title":"ChemSusChem"},{"key":"B3","doi-asserted-by":"publisher","first-page":"5707","DOI":"10.1021\/bi0497805","article-title":"Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase","volume":"43","author":"Aubert","year":"2004","journal-title":"Biochemistry"},{"key":"B4","first-page":"51","article-title":"Synthetic routes to sofosbuvir","volume-title":"Synthesis of heterocycles in contemporary medicinal chemistry","author":"Barth","year":"2016"},{"key":"B5","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The protein data bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"B6","doi-asserted-by":"publisher","first-page":"3038","DOI":"10.1021\/acs.biochem.0c00591","article-title":"A chemoenzymatic synthesis of the (RP)-Isomer of the antiviral prodrug remdesivir","volume":"59","author":"Bigley","year":"2020","journal-title":"Biochemistry"},{"key":"B7","doi-asserted-by":"publisher","first-page":"443","DOI":"10.1016\/j.bbapap.2012.04.004","article-title":"Catalytic mechanisms for phosphotriesterases","volume":"1834","author":"Bigley","year":"2013","journal-title":"Biochim. Biophys. Acta"},{"key":"B8","doi-asserted-by":"publisher","first-page":"80","DOI":"10.1016\/j.cbi.2019.05.023","article-title":"The evolution of phosphotriesterase for decontamination and detoxification of organophosphorus chemical warfare agents","volume":"308","author":"Bigley","year":"2019","journal-title":"Chem. Biol. Interact."},{"key":"B9","doi-asserted-by":"publisher","first-page":"10426","DOI":"10.1021\/ja402832z","article-title":"Enzymatic neutralization of the chemical warfare agent VX: evolution of phosphotriesterase for phosphorothiolate hydrolysis","volume":"135","author":"Bigley","year":"2013","journal-title":"J. Am. Chem. Soc."},{"key":"B10","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1093\/protein\/gzq046","article-title":"Highest paraoxonase turnover rate found in a bacterial phosphotriesterase variant","volume":"24","author":"Brise\u00f1o-Roa","year":"2011","journal-title":"Protein Eng. Des. Sel."},{"key":"B11","volume-title":"Molecular operating environment (MOE), 2022.02\u201d. 1010 sherbooke st. West, suite #910","year":"2023"},{"key":"B12","doi-asserted-by":"publisher","first-page":"1325","DOI":"10.1021\/bi001548l","article-title":"Structural determinants of the substrate and stereochemical specificity of phosphotriesterase","volume":"40","author":"Chen-Goodspeed","year":"","journal-title":"Biochemistry"},{"key":"B13","doi-asserted-by":"publisher","first-page":"1332","DOI":"10.1021\/bi001549d","article-title":"Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues","volume":"40","author":"Chen-Goodspeed","year":"","journal-title":"Biochemistry"},{"key":"B14","doi-asserted-by":"publisher","first-page":"1812","DOI":"10.1021\/jm400201a","article-title":"Discovery of the first C-nucleoside HCV polymerase inhibitor (GS-6620) with demonstrated antiviral response in HCV infected patients","volume":"57","author":"Cho","year":"2014","journal-title":"J. Med. Chem."},{"key":"B15","doi-asserted-by":"publisher","first-page":"204020661875643","DOI":"10.1177\/2040206618756430","article-title":"Current and future use of nucleo(s)tide prodrugs in the treatment of hepatitis C virus infection","volume":"26","author":"Dousson","year":"2018","journal-title":"Antivir. Chem. Chemother."},{"key":"B16","doi-asserted-by":"publisher","first-page":"338","DOI":"10.1002\/chir.530040513","article-title":"FDA'S policy statement for the development of new stereoisomeric drugs","volume":"4","year":"1992","journal-title":"Chirality"},{"key":"B17","doi-asserted-by":"publisher","first-page":"10904","DOI":"10.1021\/bi960663m","article-title":"Metal\u2212Substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase","volume":"35","author":"Hong","year":"1996","journal-title":"Biochemistry"},{"key":"B18","doi-asserted-by":"publisher","first-page":"1159","DOI":"10.1021\/bi982204m","article-title":"Stereochemical constraints on the substrate specificity of phosphotriesterase","volume":"38","author":"Hong","year":"1999","journal-title":"Biochemistry"},{"key":"B19","doi-asserted-by":"publisher","first-page":"27516","DOI":"10.1021\/acsomega.2c02835","article-title":"Practical and highly efficient synthesis of remdesivir from GS-441524","volume":"7","author":"Hu","year":"2022","journal-title":"ACS Omega"},{"key":"B20","doi-asserted-by":"publisher","first-page":"3847","DOI":"10.1038\/s41467-021-24075-y","article-title":"Physical constraints and functional plasticity of cellulases","volume":"12","author":"Kari","year":"2021","journal-title":"Nat. Commun."},{"key":"B21","doi-asserted-by":"publisher","first-page":"e202300263","DOI":"10.1002\/open.202300263","article-title":"Computational design of phosphotriesterase improves V-agent degradation efficiency","volume":"13","author":"Kronenberg","year":"2024","journal-title":"ChemistryOpen"},{"key":"B22","doi-asserted-by":"publisher","first-page":"153590","DOI":"10.1016\/j.tetlet.2021.153590","article-title":"Total synthesis of remdesivir","volume":"88","author":"Kumar Palli","year":"2022","journal-title":"Tetrahedron Lett."},{"key":"B23","doi-asserted-by":"publisher","first-page":"5571","DOI":"10.1021\/acscatal.3c00812","article-title":"Emerging technologies for biocatalysis in the pharmaceutical industry","volume":"13","author":"Lewis","year":"2023","journal-title":"ACS Catal."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1111","DOI":"10.3390\/molecules13051111","article-title":"A convenient synthesis of amino acid methyl esters","volume":"13","author":"Li","year":"2008","journal-title":"Molecules"},{"key":"B25","doi-asserted-by":"publisher","first-page":"112527","DOI":"10.1016\/j.ejmech.2020.112527","article-title":"A promising antiviral candidate drug for the COVID-19 pandemic: a mini-review of remdesivir","volume":"201","author":"Liang","year":"2020","journal-title":"Eur. J. Med. Chem."},{"key":"B26","doi-asserted-by":"publisher","first-page":"2211","DOI":"10.1021\/acs.jmedchem.7b00734","article-title":"The ProTide prodrug technology: from the concept to the clinic","volume":"61","author":"Mehellou","year":"2018","journal-title":"J. Med. Chem."},{"key":"B27","doi-asserted-by":"publisher","first-page":"e94177","DOI":"10.1371\/journal.pone.0094177","article-title":"A 5000-fold increase in the specificity of a bacterial phosphotriesterase for malathion through combinatorial active site mutagenesis","volume":"9","author":"Naqvi","year":"2014","journal-title":"PLOS ONE"},{"key":"B28","doi-asserted-by":"publisher","first-page":"5900","DOI":"10.1021\/ja502205q","article-title":"A general and enantioselective approach to pentoses: a rapid synthesis of PSI-6130, the nucleoside core of sofosbuvir","volume":"136","author":"Peifer","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"B29","doi-asserted-by":"publisher","first-page":"e49589","DOI":"10.1371\/journal.pone.0049589","article-title":"The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated","volume":"7","author":"Raran-Kurussi","year":"2012","journal-title":"PLOS ONE"},{"key":"B30","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1093\/protein\/gzi005","article-title":"Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state","volume":"18","author":"Roodveldt","year":"2005","journal-title":"Protein Eng. Des. Sel."},{"key":"B31","doi-asserted-by":"publisher","first-page":"1403","DOI":"10.1002\/chir.23498","article-title":"Biocatalysis: a smart and green tool for the preparation of chiral drugs","volume":"34","author":"Rossino","year":"2022","journal-title":"Chirality"},{"key":"B32","doi-asserted-by":"publisher","first-page":"1809","DOI":"10.1021\/acs.oprd.5b00141","article-title":"Application of continuous preferential crystallization to efficiently access enantiopure chemicals","volume":"19","author":"Rougeot","year":"2015","journal-title":"Org. Process Res. Dev."},{"key":"B33","doi-asserted-by":"publisher","first-page":"105723","DOI":"10.1016\/j.bioorg.2022.105723","article-title":"Synthesis, antiviral and antitumor activities investigations of a series of Ribavirin C-nucleoside analogue prodrugs","volume":"122","author":"Sabat","year":"2022","journal-title":"Bioorg. Chem."},{"key":"B34","doi-asserted-by":"publisher","first-page":"2587","DOI":"10.1039\/c9sc05746c","article-title":"The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis","volume":"11","author":"Sheldon","year":"2020","journal-title":"Chem. Sci."},{"key":"B35","doi-asserted-by":"publisher","first-page":"204020661877524","DOI":"10.1177\/2040206618775243","article-title":"Phosphoramidates and phosphonamidates (ProTides) with antiviral activity","volume":"26","author":"Slusarczyk","year":"2018","journal-title":"Antivir. Chem. Chemother."},{"key":"B36","doi-asserted-by":"publisher","first-page":"7202","DOI":"10.1021\/jm100863x","article-title":"Discovery of a \u03b2-d-2\u2032-Deoxy-2\u2032-\u03b1-fluoro-2\u2032-\u03b2-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus","volume":"53","author":"Sofia","year":"2010","journal-title":"J. Med. Chem."},{"key":"B37","doi-asserted-by":"publisher","first-page":"11955","DOI":"10.1039\/d3sc01630g","article-title":"Strategies for chiral separation: from racemate to enantiomer","volume":"14","author":"Sui","year":"2023","journal-title":"Chem. Sci."},{"key":"B38","doi-asserted-by":"publisher","first-page":"7978","DOI":"10.1021\/bi101056m","article-title":"Stereoselective hydrolysis of organophosphate nerve agents by the bacterial phosphotriesterase","volume":"49","author":"Tsai","year":"2010","journal-title":"Biochemistry"},{"key":"B39","doi-asserted-by":"publisher","first-page":"6020","DOI":"10.1021\/bi960325l","article-title":"Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate","volume":"35","author":"Vanhooke","year":"1996","journal-title":"Biochemistry"},{"key":"B40","doi-asserted-by":"publisher","first-page":"381","DOI":"10.1038\/nature17180","article-title":"Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys","volume":"531","author":"Warren","year":"2016","journal-title":"Nature"},{"key":"B41","doi-asserted-by":"publisher","first-page":"3204","DOI":"10.1021\/acs.biochem.9b00530","article-title":"Enzyme-catalyzed kinetic resolution of chiral precursors to antiviral prodrugs","volume":"58","author":"Xiang","year":"2019","journal-title":"Biochemistry"}],"container-title":["Frontiers in Bioengineering and Biotechnology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fbioe.2024.1446566\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,7,30]],"date-time":"2024-07-30T04:25:54Z","timestamp":1722313554000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fbioe.2024.1446566\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,7,30]]},"references-count":41,"alternative-id":["10.3389\/fbioe.2024.1446566"],"URL":"https:\/\/doi.org\/10.3389\/fbioe.2024.1446566","relation":{},"ISSN":["2296-4185"],"issn-type":[{"value":"2296-4185","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,7,30]]},"article-number":"1446566"}}