Modular Multifunctional 5HP2O Linkers for the Versatile Assembly of Protein-Oligonucleotide Conjugates

Modular Multifunctional 5HP2O Linkers for the Versatile Assembly of Protein-Oligonucleotide Conjugates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Modular Multifunctional 5HP2O Linkers for the Versatile Assembly of Protein-Oligonucleotide Conjugates Annemieke Madder, Jan Meffert, Alexandros Kostakis, Joshua McHale, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9441903/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract As complex biotherapeutics continue to evolve, the demand for single-site, multi-functionalized protein conjugates intensifies. Current methods often suffer from maleimide-related hydrolytic instability or require multi-step invasive protein modifications. Here, we establish 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) as modular, hydrolytically stable hubs for assembling multi-functional protein-oligonucleotide conjugates (POCs). Synthesized via one-pot photooxidation of 2-substituted furans, these linkers incorporate tunable spacers, a 5HP2O Michael acceptor, a NHS active ester and secondary cargoes. We demonstrate that 5HP2O-functionalized amino-oligonucleotides (AON) maintain complete hydrolytic resistance across a broad pH range, streamlining intermediate storage. Subsequent bioconjugation yields constructs with superior retro-Michael stability compared to aliphatic maleimide analogues. Expanding this platform, we generated diverse heterobifunctional, trifunctional (click-reactive and fluorescent), and tetrafunctional (enzymatically cleavable) linkers. We validated these tools through in vitro conjugate tracking and the assembly of a dual-antisense oligonucleotide conjugate. Physical sciences/Chemistry/Chemical biology/Chemical modification Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology Biological sciences/Chemical biology/Proteins Biological sciences/Chemical biology/Nucleic acids bioconjugation protein modification protein-oligonucleotide conjugates 5HP2O maleimide alternative Full Text Additional Declarations Yes there is potential Competing Interest. Annemieke Madder has a pending patent application relating to 5HP2O applications for bioconjugation (Bioconjugation reagent and methods, WO2020174086A2, Annemieke MADDER, Ewout DE GEYTER, Eirini ANTONATOU, Sabina SMOLEN, Dimitris KALAITZAKIS, Georgios VASSILIKOGIANNAKIS, Europe&US). Alexandros Kostakis is an employee of Strike Pharma AB that owns intellectual property related to STRIKE2001 and the pTag technology. Sara Mangsbo is the co-founder and board member of Strike Pharma AB and is also the inventor of the intellectual property related to STRIKE2001 and the pTag technology. All other authors declare no competing interest. Supplementary Files MeffertMadderNatCommChem2026SupportingInformationfinalsubmitted.docx Supplementary Information to "Modular Multifunctional 5HP2O Linkers for the Versatile Assembly of Protein-Oligonucleotide Conjugates " Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9441903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":624940233,"identity":"c958083d-4c4c-47d0-9947-0d720db5607e","order_by":0,"name":"Annemieke Madder","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-0179-7608","institution":"Ghent University","correspondingAuthor":true,"prefix":"","firstName":"Annemieke","middleName":"","lastName":"Madder","suffix":""},{"id":624940234,"identity":"99d0dfd6-6a28-4bcd-80ed-c7ec68ae41a2","order_by":1,"name":"Jan Meffert","email":"","orcid":"https://orcid.org/0000-0003-4342-5618","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Meffert","suffix":""},{"id":624940235,"identity":"60acc9c4-1058-46ec-a0aa-8d7872b80799","order_by":2,"name":"Alexandros Kostakis","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alexandros","middleName":"","lastName":"Kostakis","suffix":""},{"id":624940236,"identity":"9ebbddf9-c584-470f-88d4-86eb25de462c","order_by":3,"name":"Joshua McHale","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"McHale","suffix":""},{"id":624940237,"identity":"92a7a4c5-e4f3-40c7-ba10-f73a8d597dfd","order_by":4,"name":"Sara Mangsbo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Mangsbo","suffix":""},{"id":624940238,"identity":"54fb2ca4-507d-455f-844a-38da73d1869c","order_by":5,"name":"Carlo Vascotto","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Carlo","middleName":"","lastName":"Vascotto","suffix":""},{"id":624940239,"identity":"2bacd24f-62c0-4c5b-b3bd-d85202923222","order_by":6,"name":"Martin Bollmark","email":"","orcid":"https://orcid.org/0000-0002-3715-1959","institution":"Research Institutes of Sweden","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Bollmark","suffix":""}],"badges":[],"createdAt":"2026-04-16 20:35:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9441903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9441903/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107707197,"identity":"715c74ad-36e5-417a-9ae9-c8eac8f357aa","added_by":"auto","created_at":"2026-04-24 09:19:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1688037,"visible":true,"origin":"","legend":"","description":"","filename":"MeffertMadderNatCommChem2026manuscriptfinalsubmitted.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9441903/v1_covered_eefd8c69-24c2-48dc-80dc-cfea801a71d7.pdf"},{"id":107690330,"identity":"3b7a8f64-efb2-43ee-b501-dd021a0b7fce","added_by":"auto","created_at":"2026-04-24 05:46:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28736278,"visible":true,"origin":"","legend":"Supplementary Information to \"Modular Multifunctional 5HP2O Linkers for the Versatile Assembly of Protein-Oligonucleotide Conjugates \"","description":"","filename":"MeffertMadderNatCommChem2026SupportingInformationfinalsubmitted.docx","url":"https://assets-eu.researchsquare.com/files/rs-9441903/v1/b409bec5eb5094516edb7d00.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nAnnemieke Madder has a pending patent application relating to 5HP2O applications for bioconjugation (Bioconjugation reagent and methods, WO2020174086A2, Annemieke MADDER, Ewout DE GEYTER, Eirini ANTONATOU, Sabina SMOLEN, Dimitris KALAITZAKIS, Georgios VASSILIKOGIANNAKIS, Europe\u0026US). 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