{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T13:58:10Z","timestamp":1791381490013,"version":"4.3.3"},"reference-count":40,"publisher":"American Chemical Society (ACS)","issue":"5","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100031931","name":"Shanghai Artificial Intelligence Laboratory","doi-asserted-by":"publisher","award":["Climber Initiative"],"award-info":[{"award-number":["Climber Initiative"]}],"id":[{"id":"10.13039\/100031931","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Guangxi Basic Research Program key project","award":["2025GXNSFDA04240006"],"award-info":[{"award-number":["2025GXNSFDA04240006"]}]},{"name":"Shanghai Science and Technology Innovation Action Plan","award":["23HC1400500"],"award-info":[{"award-number":["23HC1400500"]}]},{"name":"Agricultural Science and Technology Innovation System of Shanghai, China","award":["T2023217"],"award-info":[{"award-number":["T2023217"]}]},{"name":"Key R&D Plan of Ningxia Hui Autonomous Region","award":["2024BEE02021"],"award-info":[{"award-number":["2024BEE02021"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Peptides have broad applications in fields such as food, medicine, and functional materials. Conventional solid-phase peptide synthesis (SPPS) constructs diverse peptide sequences through stepwise amino acid elongation but relies on stoichiometric coupling reagents and organic solvents, thereby generating considerable chemical waste. Aqualigase, an engineered subtilisin, offers a green alternative that directly condenses free carboxylic acids and amines in pure aqueous media. However, whether this system can support stepwise elongation using amino acids as building blocks remains unclear. Here, using the Aqualigase-catalyzed ligation of Ala-Ala-Pro-Phe and Ala-Phe as a model reaction, hydrogen-bond analysis revealed that a stable interaction between the P1\u2032 residue of the acyl acceptor and Asn155 in the substrate-binding pocket is critical for effective binding of the acyl acceptor. Guided by this finding, we demonstrated that Aqualigase ligates peptides ranging from dipeptides to pentapeptides with amino acids as acyl acceptors, expanding its catalytic mode from peptide-fragment ligation to fragment-amino acid ligation. RMSF analysis further revealed pronounced flexibility in the loops surrounding the catalytic pocket, particularly residues 95 to 106 and 155 to 164, providing a structural basis for substrate accommodation. Substrate scope analysis showed that, under Aqualigase catalysis, amino acids are more broadly compatible as acyl acceptors than as acyl donors, supporting an N-to-C-terminal stepwise elongation strategy. Using this approach, the functional tripeptide Gly-Leu-Phe was synthesized in pure aqueous media through sequential amino acid elongation. This study establishes an engineered subtilisin-catalyzed strategy for stepwise peptide synthesis in pure aqueous media, providing a green route for flexible peptide construction.<\/jats:p>","DOI":"10.1021\/acsorginorgau.6c00067","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T10:48:46Z","timestamp":1788259726000},"page":"687-696","source":"Crossref","is-referenced-by-count":0,"title":["Aqueous-Phase\nPeptide Synthesis via Stepwise Amino\nAcid Elongation Catalyzed by Aqualigase"],"prefix":"10.1021","volume":"6","author":[{"given":"Xin","family":"Yan","sequence":"first","affiliation":[{"name":"East China Normal University , , ,","place":["Shanghai, China, 200062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihan","family":"Lin","sequence":"additional","affiliation":[{"name":"East China Normal University , , ,","place":["Shanghai, China, 200062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruyi","family":"Chen","sequence":"additional","affiliation":[{"name":"East China Normal University , , ,","place":["Shanghai, China, 200062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luobin","family":"Huang","sequence":"additional","affiliation":[{"name":"East China Normal University , , ,","place":["Shanghai, China, 200062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lige","family":"Tong","sequence":"additional","affiliation":[{"name":"East China Normal University , , ,","place":["Shanghai, China, 200062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1506-6198","authenticated-orcid":true,"given":"Bei","family":"Gao","sequence":"additional","affiliation":[{"name":"East China University of Science and Technology , , ,","place":["Shanghai, China, 200237"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3566-917X","authenticated-orcid":true,"given":"Lujia","family":"Zhang","sequence":"additional","affiliation":[{"name":"East China Normal University , , ,","place":["Shanghai, China, 200062"]},{"name":"NYU-ECNU Center for Computational Chemistry at NYU Shanghai , ,","place":["Shanghai, China, 200062"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"issue":"6","key":"2026100709062749800_cit1","doi-asserted-by":"publisher","first-page":"527","DOI":"10.1631\/jzus.B2400029","article-title":"Applications\nof Bioactive Peptides in Cosmeceuticals: A Review","volume":"26","author":"Ashaolu","year":"2025","journal-title":"J. Zhejiang Univ. Sci. B"},{"issue":"1","key":"2026100709062749800_cit2","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1038\/s41392-022-00904-4","article-title":"Therapeutic\nPeptides: Current Applications and Future Directions","volume":"7","author":"Wang","year":"2022","journal-title":"Signal Transduction Targeted Ther."},{"issue":"29","key":"2026100709062749800_cit3","doi-asserted-by":"publisher","first-page":"18420","DOI":"10.1021\/acs.jafc.5c02065","article-title":"Modifying Antifungal Peptides as Safe Food Preservatives","volume":"73","author":"Simons","year":"2025","journal-title":"J. Agric. Food Chem."},{"issue":"31","key":"2026100709062749800_cit4","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202501775","article-title":"Peptide-Based\nBiomaterials as a Promising Tool for\nCancer Radiotherapy","volume":"12","author":"Wang","year":"2025","journal-title":"Adv. Sci."},{"issue":"1","key":"2026100709062749800_cit5","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1038\/s41392-024-02107-5","article-title":"Advance in Peptide-Based\nDrug Development: Delivery Platforms, Therapeutics and Vaccines","volume":"10","author":"Xiao","year":"2025","journal-title":"Signal Transduction Targeted Ther."},{"issue":"15","key":"2026100709062749800_cit6","doi-asserted-by":"publisher","first-page":"5917","DOI":"10.1021\/acs.jafc.2c08835","article-title":"Production\nof Food-Derived Bioactive Peptides with Potential Application in the\nManagement of Diabetes and Obesity: A Review","volume":"71","author":"Wang","year":"2023","journal-title":"J. Agric. Food Chem."},{"key":"2026100709062749800_cit7","doi-asserted-by":"publisher","first-page":"88","DOI":"10.3390\/biom15010088","article-title":"Peptides: Emerging\nCandidates for the Prevention and Treatment of Skin Senescence: A\nReview","volume":"15","author":"Pintea","year":"2025","journal-title":"Biomolecules"},{"key":"2026100709062749800_cit8","doi-asserted-by":"publisher","first-page":"6445","DOI":"10.1039\/D2CS00991A","article-title":"Material-Specific\nBinding Peptides Empower Sustainable Innovations in Plant Health,\nBiocatalysis, Medicine and Microplastic Quantification","volume":"53","author":"Mao","year":"2024","journal-title":"Chem. Soc. Rev."},{"issue":"2","key":"2026100709062749800_cit9","doi-asserted-by":"publisher","first-page":"606","DOI":"10.1039\/B701677H","article-title":"Amide Bond Formation:\nBeyond the Myth of Coupling Reagents","volume":"38","author":"Valeur","year":"2009","journal-title":"Chem.\nSoc. Rev."},{"key":"2026100709062749800_cit10","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202505167","article-title":"Br\u00f8nsted\nAcid-Catalyzed Atroposelective Coupling of Carboxylic Acids with Amines\nand Alcohols via Ynamide Mediation","volume":"64","author":"Chen","year":"2025","journal-title":"Angew. Chem.,\nInt. Ed."},{"issue":"5","key":"2026100709062749800_cit11","doi-asserted-by":"publisher","first-page":"464","DOI":"10.1038\/nchembio.2318","article-title":"A Fully\nAutomated Flow-Based Approach\nfor Accelerated Peptide Synthesis","volume":"13","author":"Mijalis","year":"2017","journal-title":"Nat. Chem.\nBiol."},{"issue":"7","key":"2026100709062749800_cit12","doi-asserted-by":"publisher","first-page":"587","DOI":"10.1038\/nrd1133","article-title":"Large-Scale\nManufacture of Peptide Therapeutics by Chemical Synthesis","volume":"2","author":"Bray","year":"2003","journal-title":"Nat. Rev. Drug Discovery"},{"issue":"5","key":"2026100709062749800_cit13","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1016\/j.trechm.2025.03.003","article-title":"Site-Selective\nModification of Native Proteins","volume":"7","author":"Kim","year":"2025","journal-title":"Trends Chem."},{"issue":"3","key":"2026100709062749800_cit14","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202308251","article-title":"Cyclic Peptides for Drug Development","volume":"63","author":"Ji","year":"2024","journal-title":"Angew. Chem., Int. Ed."},{"issue":"3","key":"2026100709062749800_cit15","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1038\/s41571-023-00850-2","article-title":"Exploring the next\nGeneration of Antibody\u2013Drug Conjugates","volume":"21","author":"Tsuchikama","year":"2024","journal-title":"Nat. Rev. Clin. Oncol."},{"issue":"1","key":"2026100709062749800_cit16","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1038\/s41392-022-01285-4","article-title":"Ribosome Biogenesis\nin Disease: New\nPlayers and Therapeutic Targets","volume":"8","author":"Jiao","year":"2023","journal-title":"Signal Transduction\nTargeted Ther."},{"issue":"10","key":"2026100709062749800_cit17","doi-asserted-by":"publisher","first-page":"6444","DOI":"10.1021\/acs.chemrev.3c00894","article-title":"Engineering\ntRNAs for the Ribosomal Translation of Non-Proteinogenic Monomers","volume":"124","author":"Sigal","year":"2024","journal-title":"Chem. Rev."},{"issue":"1","key":"2026100709062749800_cit18","doi-asserted-by":"publisher","first-page":"476","DOI":"10.1038\/s41467-022-27967-9","article-title":"High-Resolution\nStructures of a Thermophilic Eukaryotic 80S Ribosome Reveal Atomistic\nDetails of Translocation","volume":"13","author":"Kis\u030conaite\u0307","year":"2022","journal-title":"Nat. Commun."},{"key":"2026100709062749800_cit19","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.cbpa.2017.01.017","article-title":"Enzyme-Mediated Ligation\nTechnologies for Peptides and Proteins","volume":"38","author":"Schmidt","year":"2017","journal-title":"Curr.\nOpin. Chem. Biol."},{"issue":"22","key":"2026100709062749800_cit20","doi-asserted-by":"publisher","first-page":"12424","DOI":"10.1073\/pnas.96.22.12424","article-title":"Purification\nand Characterization of Sortase, the Transpeptidase That Cleaves Surface\nProteins of Staphylococcus Aureus at the LPXTG Motif","volume":"96","author":"Ton-That","year":"1999","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"9","key":"2026100709062749800_cit21","doi-asserted-by":"publisher","first-page":"732","DOI":"10.1038\/nchembio.1586","article-title":"Butelase 1 Is an\nAsx-Specific Ligase Enabling Peptide\nMacrocyclization and Synthesis","volume":"10","author":"Nguyen","year":"2014","journal-title":"Nat. Chem. Biol."},{"issue":"10","key":"2026100709062749800_cit22","doi-asserted-by":"publisher","first-page":"1977","DOI":"10.1038\/nprot.2016.118","article-title":"Butelase-Mediated Cyclization and Ligation of Peptides\nand Proteins","volume":"11","author":"Nguyen","year":"2016","journal-title":"Nat. Protoc."},{"issue":"6","key":"2026100709062749800_cit23","doi-asserted-by":"publisher","first-page":"3127","DOI":"10.1021\/acs.chemrev.9b00372","article-title":"Subtiligase-Catalyzed\nPeptide Ligation","volume":"120","author":"Weeks","year":"2020","journal-title":"Chem. Rev."},{"issue":"26","key":"2026100709062749800_cit24","doi-asserted-by":"publisher","first-page":"4157","DOI":"10.1021\/ol0167614","article-title":"Subtilisin-Catalyzed\nSynthesis of Amino Acid and Peptide\nEsters. Application in a Two-Step Enzymatic Ligation Strategy","volume":"3","author":"Liu","year":"2001","journal-title":"Org. Lett."},{"key":"2026100709062749800_cit25","doi-asserted-by":"publisher","first-page":"6767","DOI":"10.1039\/c2ob25662b","article-title":"Improving the Carboxyamidomethyl\nEster for Subtilisin\nA-Catalysed Peptide Synthesis","volume":"10","author":"Beer","year":"2012","journal-title":"Org. Biomol.\nChem."},{"issue":"3","key":"2026100709062749800_cit26","doi-asserted-by":"publisher","first-page":"779","DOI":"10.1042\/bj1750779","article-title":"Generalized\nMicroscopic Reversibility, Kinetic Co-Operativity of Enzymes and Evolution","volume":"175","author":"Ricard","year":"1978","journal-title":"Biochem. J."},{"issue":"2","key":"2026100709062749800_cit27","doi-asserted-by":"publisher","first-page":"610","DOI":"10.1021\/bi00854a032","article-title":"The Reactivity of Thiol-Subtilisin, an Enzyme Containing\na Synthetic Functional Group*","volume":"6","author":"Polgar","year":"1967","journal-title":"Biochemistry"},{"issue":"17","key":"2026100709062749800_cit28","doi-asserted-by":"publisher","first-page":"4151","DOI":"10.1021\/bi00231a007","article-title":"Engineering Subtilisin and Its Substrates for Efficient\nLigation of Peptide Bonds in Aqueous Solution","volume":"30","author":"Abrahmsen","year":"1991","journal-title":"Biochemistry"},{"issue":"26","key":"2026100709062749800_cit29","doi-asserted-by":"publisher","first-page":"12544","DOI":"10.1073\/pnas.91.26.12544","article-title":"Subtiligase: A Tool\nfor Semisynthesis of Proteins","volume":"91","author":"Chang","year":"1994","journal-title":"Proc. Natl. Acad.\nSci. U. S. A."},{"issue":"11","key":"2026100709062749800_cit30","doi-asserted-by":"publisher","first-page":"925","DOI":"10.1038\/nmeth.4004","article-title":"Enzyme-Catalyzed\nExpressed Protein\nLigation","volume":"13","author":"Henager","year":"2016","journal-title":"Nat. Methods"},{"issue":"1","key":"2026100709062749800_cit31","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1038\/nchembio.2521","article-title":"Engineering Peptide\nLigase Specificity by Proteomic\nIdentification of Ligation Sites","volume":"14","author":"Weeks","year":"2018","journal-title":"Nat. Chem.\nBiol."},{"issue":"8","key":"2026100709062749800_cit32","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2018809118","article-title":"Mapping Proteolytic\nNeo-N Termini at the Surface of Living Cells","volume":"118","author":"Weeks","year":"2021","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"13","key":"2026100709062749800_cit33","doi-asserted-by":"publisher","first-page":"2140","DOI":"10.1002\/adsc.201600017","article-title":"Peptiligase,\nan Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization\nin Water","volume":"358","author":"Toplak","year":"2016","journal-title":"Adv. Synth. Catal."},{"issue":"4","key":"2026100709062749800_cit34","doi-asserted-by":"publisher","first-page":"609","DOI":"10.1039\/C7OB02812A","article-title":"Design of\na Substrate-Tailored Peptiligase Variant for the Efficient Synthesis\nof Thymosin-\u03911","volume":"16","author":"Schmidt","year":"2018","journal-title":"Org. Biomol. Chem."},{"key":"2026100709062749800_cit35","doi-asserted-by":"publisher","first-page":"1277","DOI":"10.1016\/j.csbj.2021.02.002","article-title":"From Thiol-Subtilisin\nto Omniligase:\nDesign and Structure of a Broadly Applicable Peptide Ligase","volume":"19","author":"Toplak","year":"2021","journal-title":"Comput. Struct. Biotechnol. J."},{"issue":"5","key":"2026100709062749800_cit36","doi-asserted-by":"publisher","first-page":"nwab158","DOI":"10.1093\/nsr\/nwab158","article-title":"Traceless\nEnzymatic Protein Synthesis without Ligation Sites Constraint","volume":"9","author":"Li","year":"2022","journal-title":"Natl. Sci. Rev."},{"issue":"13","key":"2026100709062749800_cit37","doi-asserted-by":"publisher","first-page":"11594","DOI":"10.1021\/acscatal.5c01532","article-title":"Aqualigase: A Star Enzyme for One-Step\nPeptide Bond Dehydration Condensation in a Nature Aqueous Phase","volume":"15","author":"Feng","year":"2025","journal-title":"ACS Catal."},{"issue":"1","key":"2026100709062749800_cit38","doi-asserted-by":"publisher","first-page":"309","DOI":"10.1016\/0022-2836(91)80221-F","article-title":"Refined crystal structure\nof the complex of subtilisin BPN\u2032 and Streptomyces subtilisin\ninhibitor at 1\u00b78\u00c5resolution","volume":"221","author":"Takeuchi","year":"1991","journal-title":"J. Mol.\nBiol."},{"issue":"8","key":"2026100709062749800_cit39","doi-asserted-by":"publisher","first-page":"1633","DOI":"10.1271\/bbb.69.1633","article-title":"Anti-Alopecia\nEffect of Gly-Leu-Phe, an Immunostimulating\nPeptide Derived from \u03b1-Lactalbumin","volume":"69","author":"Tsuruki","year":"2005","journal-title":"Biosci.,\nBiotechnol., Biochem."},{"key":"2026100709062749800_cit40","doi-asserted-by":"publisher","first-page":"169856","DOI":"10.1016\/j.cej.2025.169856","article-title":"AI-FRET-Guided\nEngineering of Subtilisin for Green Aqueous-Phase Amide Bond Formation\nwith Enhanced Catalytic Activity","volume":"525","author":"Yan","year":"2025","journal-title":"Chem. Eng.\nJ."}],"container-title":["ACS Organic &amp; Inorganic\nAu"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsorginorgau.6c00067","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/aoiab5\/article-pdf\/6\/5\/687\/67420284\/acsorginorgau.6c00067.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/aoiab5\/article-pdf\/6\/5\/687\/67420284\/acsorginorgau.6c00067.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T13:29:54Z","timestamp":1791379794000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/aoiab5\/article\/6\/5\/687\/5329787\/Aqueous-Phase-Peptide-Synthesis-via-Stepwise-Amino"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,26]]},"references-count":40,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,8,26]]},"published-print":{"date-parts":[[2026,10,7]]}},"URL":"https:\/\/doi.org\/10.1021\/acsorginorgau.6c00067","relation":{},"ISSN":["2694-247X"],"issn-type":[{"value":"2694-247X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,26]]}}}