{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T18:54:08Z","timestamp":1793300048494,"version":"4.5.0"},"reference-count":36,"publisher":"American Chemical Society (ACS)","issue":"32","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000997","name":"Arnold and Mabel Beckman Foundation","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/100000997","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Caltech Summer Undergraduate Research Fellowship","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Phosphorus is one of the six elements of life, yet enzymatic carbon\u2013phosphorus (C\u2013P) bond formation is exceedingly rare in nature. This gap in the biosynthetic toolkit is particularly consequential, given the prominence of stereogenic-at-phosphorus P(V) scaffolds in pharmaceuticals and agrochemicals, where the absolute configuration at phosphorus often directly governs biological activity. Despite significant advances in the asymmetric synthesis of P-stereogenic compounds, a general enzymatic platform based on direct C\u2013P bond formation has not been realized. Here, we describe the discovery and directed evolution of Aeropyrum pernix protoglobin (ApePgb) variants that catalyze carbene insertion into the P\u2013H bonds of secondary phosphine oxides and H\u2013phosphinates. Mechanistic investigations reveal that carbene P\u2013H insertion proceeds through a stereoretentive kinetic resolution. Directed evolution of a single protein scaffold produced an enantiodivergent enzyme platform that selectively delivers either P-configuration with high enantioselectivity across a broad substrate scope. The evolution campaign further revealed latent activity for nitrene P\u2013H insertion, opening a path toward P-stereogenic phosphinamides and phosphonamidates through future evolution.<\/jats:p>","DOI":"10.1021\/jacs.6c10375","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T03:01:34Z","timestamp":1786071694000},"page":"34791-34797","source":"Crossref","is-referenced-by-count":1,"title":["Enantiodivergent Carbon\u2013Phosphorus\nBond Formation\nvia Enzymatic Carbene Insertion"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3811-5375","authenticated-orcid":true,"given":"Hayden M.","family":"Carder","sequence":"first","affiliation":[{"name":"California Institute of Technology , , , ,","place":["Pasadena, California, United States, 91125"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ethan N.","family":"Lin","sequence":"additional","affiliation":[{"name":"California Institute of Technology , , , ,","place":["Pasadena, California, United States, 91125"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Roth","sequence":"additional","affiliation":[{"name":"California Institute of Technology , , , ,","place":["Pasadena, California, United States, 91125"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frances H.","family":"Arnold","sequence":"additional","affiliation":[{"name":"California Institute of Technology , , , ,","place":["Pasadena, California, United States, 91125"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"key":"2026081910232281400_cit1","doi-asserted-by":"crossref","DOI":"10.1039\/9781839162312","volume-title":"The Chemical Biology\nof Phosphorus","author":"Walsh","year":"2020"},{"issue":"5","key":"2026081910232281400_cit2","doi-asserted-by":"publisher","first-page":"889","DOI":"10.3390\/sym13050889","article-title":"Phosphorus\nCompounds of Natural Origin: Prebiotic, Stereochemistry, Application","volume":"13","author":"Kolodiazhnyi","year":"2021","journal-title":"Symmetry"},{"issue":"4","key":"2026081910232281400_cit3","doi-asserted-by":"publisher","first-page":"580","DOI":"10.1016\/j.cbpa.2013.06.018","article-title":"Phosphonate Biosynthesis and Catabolism: A Treasure\nTrove of Unusual Enzymology","volume":"17","author":"Peck","year":"2013","journal-title":"Curr. Opin. Chem.\nBiol."},{"key":"2026081910232281400_cit4","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1515\/9783110535839-003","article-title":"The Importance\nof Organophosphorus Compounds as Biologically Active Agents","volume-title":"Organophosphorus Chemistry","author":"Tajti","year":"2018"},{"issue":"8","key":"2026081910232281400_cit5","doi-asserted-by":"publisher","first-page":"5704","DOI":"10.1021\/acs.chemrev.6b00536","article-title":"Phosphonate Biochemistry","volume":"117","author":"Horsman","year":"2017","journal-title":"Chem.\nRev."},{"issue":"19","key":"2026081910232281400_cit6","doi-asserted-by":"publisher","first-page":"7202","DOI":"10.1021\/jm100863x","article-title":"Discovery\nof a \u03b2- d \u22122\u2032-Deoxy-2\u2032-\u03b1-Fluoro-2\u2032-\u03b2- C -Methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment\nof Hepatitis C Virus","volume":"53","author":"Sofia","year":"2010","journal-title":"J. Med. Chem."},{"issue":"20","key":"2026081910232281400_cit7","doi-asserted-by":"publisher","first-page":"8293","DOI":"10.1021\/jm500522y","article-title":"C -Glycoside\nMimetics Inhibit Glioma Stem Cell Proliferation, Migration, and Invasion","volume":"57","author":"Clarion","year":"2014","journal-title":"J. Med. Chem."},{"issue":"1","key":"2026081910232281400_cit8","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1021\/jm980142s","article-title":"Design\nand Synthesis of Phosphinamide-Based Hydroxamic Acids as Inhibitors\nof Matrix Metalloproteinases","volume":"42","author":"Pikul","year":"1999","journal-title":"J. Med. Chem."},{"issue":"24","key":"2026081910232281400_cit9","doi-asserted-by":"publisher","first-page":"5461","DOI":"10.1016\/j.bmc.2003.09.015","article-title":"Cyclic Phosphinamides\nand Phosphonamides, Novel Series of Potent Matrix Metalloproteinase\nInhibitors with Antitumour Activity","volume":"11","author":"S\u00f8rensen","year":"2003","journal-title":"Bioorg.\nMed. Chem."},{"issue":"5","key":"2026081910232281400_cit10","doi-asserted-by":"publisher","first-page":"1188","DOI":"10.1002\/chem.201805039","article-title":"Phosphorus versus\nSulfur: Discovery\nof Benzenephosphonamidates as Versatile Sulfonamide-Mimic Chemotypes\nActing as Carbonic Anhydrase Inhibitors","volume":"25","author":"Nocentini","year":"2019","journal-title":"Chem.\n\u2013 Eur. J."},{"issue":"14","key":"2026081910232281400_cit11","doi-asserted-by":"publisher","first-page":"8657","DOI":"10.1021\/acs.chemrev.3c00875","article-title":"P-Stereogenic\nPhosphorus Ligands in Asymmetric Catalysis","volume":"124","author":"Imamoto","year":"2024","journal-title":"Chem. Rev."},{"issue":"7","key":"2026081910232281400_cit12","doi-asserted-by":"publisher","first-page":"2474","DOI":"10.1021\/ja312352p","article-title":"Efficient Asymmetric Synthesis of P -Chiral Phosphine Oxides via Properly Designed and Activated\nBenzoxazaphosphinine-2-Oxide Agents","volume":"135","author":"Han","year":"2013","journal-title":"J. Am.\nChem. Soc."},{"issue":"6408","key":"2026081910232281400_cit13","doi-asserted-by":"publisher","first-page":"1234","DOI":"10.1126\/science.aau3369","article-title":"Unlocking P(V):\nReagents for Chiral Phosphorothioate Synthesis","volume":"361","author":"Knouse","year":"2018","journal-title":"Science"},{"issue":"22","key":"2026081910232281400_cit14","doi-asserted-by":"publisher","first-page":"7478","DOI":"10.1021\/jo015909u","article-title":"A Simple\nResolution Procedure Using the Staudinger Reaction for the Preparation\nof P -Stereogenic Phosphine Oxides","volume":"66","author":"Andersen","year":"2001","journal-title":"J. Org. Chem."},{"issue":"51","key":"2026081910232281400_cit15","doi-asserted-by":"publisher","first-page":"20556","DOI":"10.1021\/jacs.9b11938","article-title":"P-Chiral Phosphines Enabled by Palladium\/Xiao-Phos-Catalyzed\nAsymmetric P\u2013C Cross-Coupling of Secondary Phosphine Oxides\nand Aryl Bromides","volume":"141","author":"Dai","year":"2019","journal-title":"J. Am. Chem. Soc."},{"issue":"40","key":"2026081910232281400_cit16","doi-asserted-by":"publisher","first-page":"13183","DOI":"10.1021\/jacs.6b09334","article-title":"Enantioselective Cu-Catalyzed Arylation\nof Secondary Phosphine Oxides with Diaryliodonium Salts toward the\nSynthesis of P-Chiral Phosphines","volume":"138","author":"Beaud","year":"2016","journal-title":"J. Am. Chem.\nSoc."},{"issue":"42","key":"2026081910232281400_cit17","doi-asserted-by":"publisher","first-page":"16584","DOI":"10.1021\/jacs.9b08734","article-title":"Ni-Catalyzed\nAsymmetric Allylation of Secondary Phosphine Oxides","volume":"141","author":"Liu","year":"2019","journal-title":"J. Am. Chem. Soc."},{"issue":"6598","key":"2026081910232281400_cit18","doi-asserted-by":"publisher","first-page":"1230","DOI":"10.1126\/science.abp8488","article-title":"Enantioselective\nHydrogen-Bond-Donor Catalysis to Access\nDiverse Stereogenic-at-P(V) Compounds","volume":"376","author":"Forbes","year":"2022","journal-title":"Science"},{"issue":"5","key":"2026081910232281400_cit19","doi-asserted-by":"publisher","first-page":"714","DOI":"10.1038\/s41557-023-01165-6","article-title":"Catalytic Enantioselective Nucleophilic Desymmetrization\nof Phosphonate Esters","volume":"15","author":"Formica","year":"2023","journal-title":"Nat. Chem."},{"issue":"8027","key":"2026081910232281400_cit20","doi-asserted-by":"publisher","first-page":"1052","DOI":"10.1038\/s41586-024-07811-4","article-title":"Catalysis\nof an SN2 Pathway by Geometric\nPreorganization","volume":"632","author":"Lovinger","year":"2024","journal-title":"Nature"},{"issue":"32","key":"2026081910232281400_cit21","doi-asserted-by":"publisher","first-page":"6463","DOI":"10.1021\/bi300811t","article-title":"Enzymes for the\nHomeland Defense: Optimizing Phosphotriesterase\nfor the Hydrolysis of Organophosphate Nerve Agents","volume":"51","author":"Tsai","year":"2012","journal-title":"Biochemistry"},{"issue":"38","key":"2026081910232281400_cit22","doi-asserted-by":"publisher","first-page":"7081","DOI":"10.1016\/0040-4039(94)88231-2","article-title":"Enzymatic Resolution of Racemic Phosphinoylacetates\nHaving a Stereogenic Phosphorus Atom","volume":"35","author":"Kie\u0142basin\u0301ski","year":"1994","journal-title":"Tetrahedron\nLett."},{"issue":"15","key":"2026081910232281400_cit23","doi-asserted-by":"publisher","first-page":"2641","DOI":"10.1016\/S0957-4166(98)00258-4","article-title":"Enzyme-Promoted Kinetic Resolution of Racemic, P-Chiral\nPhosphonyl and Phosphorylacetates","volume":"9","author":"Kie\u0142basin\u0301ski","year":"1998","journal-title":"Tetrahedron:\nAsymmetry"},{"key":"2026081910232281400_cit24","doi-asserted-by":"publisher","DOI":"10.26434\/chemrxiv-2025-ssqg9","article-title":"Photoenzymatic\nStereodivergent Hydrophosphinylation to Access Diverse P-Chirogenic\nand P-\/C-Bichirogenic Phosphorus Compounds","author":"Mou","year":"2025"},{"issue":"5","key":"2026081910232281400_cit25","doi-asserted-by":"publisher","first-page":"1209","DOI":"10.1021\/acs.accounts.0c00591","article-title":"Navigating the Unnatural\nReaction Space: Directed Evolution\nof Heme Proteins for Selective Carbene and Nitrene Transfer","volume":"54","author":"Yang","year":"2021","journal-title":"Acc. Chem. Res."},{"key":"2026081910232281400_cit26","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1016\/j.copbio.2020.12.005","article-title":"New-to-Nature Chemistry\nfrom Old Protein Machinery:\nCarbene and Nitrene Transferases","volume":"69","author":"Liu","year":"2021","journal-title":"Curr. Opin.\nBiotechnol."},{"issue":"6315","key":"2026081910232281400_cit27","doi-asserted-by":"publisher","first-page":"1048","DOI":"10.1126\/science.aah6219","article-title":"Directed Evolution\nof Cytochrome c for Carbon\u2013Silicon Bond Formation: Bringing\nSilicon to Life","volume":"354","author":"Kan","year":"2016","journal-title":"Science"},{"issue":"7683","key":"2026081910232281400_cit28","doi-asserted-by":"publisher","first-page":"132","DOI":"10.1038\/nature24996","article-title":"Genetically\nProgrammed Chiral Organoborane Synthesis","volume":"552","author":"Kan","year":"2017","journal-title":"Nature"},{"issue":"2","key":"2026081910232281400_cit29","doi-asserted-by":"publisher","first-page":"598","DOI":"10.1039\/C3SC52535J","article-title":"Cytochrome P450-Catalyzed\nInsertion of Carbenoids into N\u2013H Bonds","volume":"5","author":"Wang","year":"2014","journal-title":"Chem. Sci."},{"issue":"23","key":"2026081910232281400_cit30","doi-asserted-by":"publisher","first-page":"10279","DOI":"10.1021\/jacs.0c03428","article-title":"Enzymatic Primary Amination of Benzylic and Allylic\nC(Sp3)\u2013H Bonds","volume":"142","author":"Jia","year":"2020","journal-title":"J. Am. Chem.\nSoc."},{"issue":"41","key":"2026081910232281400_cit31","doi-asserted-by":"publisher","first-page":"19097","DOI":"10.1021\/jacs.2c08285","article-title":"Enzymatic Nitrogen Insertion into\nUnactivated C\u2013H Bonds","volume":"144","author":"Athavale","year":"2022","journal-title":"J. Am. Chem. Soc."},{"issue":"40","key":"2026081910232281400_cit32","doi-asserted-by":"publisher","first-page":"27267","DOI":"10.1021\/jacs.4c09989","article-title":"Biocatalytic Synthesis of \u03b1-Amino Esters via\nNitrene C\u2013H Insertion","volume":"146","author":"Alfonzo","year":"2024","journal-title":"J. Am. Chem. Soc."},{"issue":"13","key":"2026081910232281400_cit33","doi-asserted-by":"publisher","first-page":"2338","DOI":"10.1021\/acs.orglett.3c00722","article-title":"Visible-Light-Induced\nCarbene Insertion into P\u2013H Bonds between\nAcylsilanes and H -Phosphorus Oxides","volume":"25","author":"Mo","year":"2023","journal-title":"Org. Lett."},{"issue":"7","key":"2026081910232281400_cit34","doi-asserted-by":"publisher","first-page":"1530","DOI":"10.1021\/acs.orglett.2c00198","article-title":"Visible-Light-Promoted\nAerobic Oxyphosphorylation of \u03b1-Diazoesters with H-Phosphine\nOxides","volume":"24","author":"Zhou","year":"2022","journal-title":"Org. Lett."},{"issue":"7","key":"2026081910232281400_cit35","doi-asserted-by":"publisher","first-page":"2258","DOI":"10.31635\/ccschem.021.202101056","article-title":"Nitrene-Mediated P\u2013N\nCoupling Under Iron Catalysis","volume":"4","author":"Bai","year":"2022","journal-title":"CCS Chem."},{"issue":"36","key":"2026081910232281400_cit36","doi-asserted-by":"publisher","first-page":"20031","DOI":"10.1021\/jacs.3c06906","article-title":"Catalytic\nAsymmetric P\u2013H Insertion Reactions","volume":"145","author":"Gu","year":"2023","journal-title":"J.\nAm. Chem. Soc."}],"container-title":["Journal of the American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c10375","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/32\/34791\/66452062\/jacs.6c10375.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/32\/34791\/66452062\/jacs.6c10375.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T14:23:31Z","timestamp":1787149411000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/32\/34791\/5249851\/Enantiodivergent-Carbon-Phosphorus-Bond-Formation"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,6]]},"references-count":36,"journal-issue":{"issue":"32","published-online":{"date-parts":[[2026,8,6]]},"published-print":{"date-parts":[[2026,8,19]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c10375","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,6]]}}}