Thermodynamic Constraints on Glycerol-Based Proto-Nucleotides: Phosphate versus Arsenate in Early Backbone Evolution

Thermodynamic Constraints on Glycerol-Based Proto-Nucleotides: Phosphate versus Arsenate in Early Backbone Evolution | 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 Research Article Thermodynamic Constraints on Glycerol-Based Proto-Nucleotides: Phosphate versus Arsenate in Early Backbone Evolution Lázaro A. M. Castanedo, Chérif F. Matta This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9195456/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The emergence of the first nucleic acids required the prebiotic formation of nucleosides and nucleotides under chemically challenging conditions. Because assembly of the canonical ribose–phosphate framework is disfavored in water, simpler ancestral backbones may have preceded RNA and DNA. Here, semiempirical prescreening and density functional theory (DFT) calculations were used to evaluate the thermodynamics of glycerol-based nucleosides and nucleotides as candidate proto-nucleic-acid building blocks. Two assembly routes were examined: a classic pathway, in which glycerol first condenses with a recognition unit and then with an ionized linker, and an alternative pathway, in which glycerol first condenses with the ionized linker and only then with the recognition unit. Although Gibbs free energy is a state function, the two pathways access different regions of the potential energy surface and converge to distinct local minima, leading to pathway-dependent differences in the computed thermodynamic quantities. Glycerol-derived nucleosides are predicted to form favorably in both vacuum and implicit aqueous solution when combined with either canonical or the studied non-canonical bases. Glycerol-based nucleotides are, likewise, thermodynamically accessible by both pathways, although the classic route is consistently more favorable than the alternative route. Among the most stabilized products are derivatives containing adenine, uracil, and C-glycosylated barbituric acid. Replacement of phosphate by arsenate produces only modest energetic and structural changes, indicating that arsenate-containing analogues cannot be excluded on thermodynamic grounds alone. However, the consistently longer As–OC3 distances relative to P–OC3 suggest weaker backbone bond and therefore a plausible hydrolytic disadvantage for arsenate. These results support the view that glycerol-based backbones could have participated in early proto-nucleic-acid chemistry and suggest that phosphate may have been selected not because arsenate analogues were thermodynamically inaccessible, but because phosphate-containing backbones were more kinetically and hydrolytically persistent. prebiotic chemistry glycol nucleic acid proto-RNA arsenate substitution thermodynamic selection origin of life Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Supplementary Information Additional data supporting this work are provided in the Supporting Information. This includes ( i ) detailed conformational sampling statistics for all molecular components (Table S1), ( ii ) full Gibbs free energies for all canonical and non-canonical nucleotides across both pathways and environments (Table S2), and ( iii ) complete structural metrics, including P–OC3 and As–OC3 bond distances and RMSD comparisons between phosphate and arsenate analogues (Table S3). The Supporting Information also contains auxiliary figures: reaction schemes for the classic and alternative pathways (Fig. S1), benchmarking of the DFT protocol against the S66 dataset (Fig. S2), representative optimized geometries of glycerol nucleotides (Fig. S3), and torsional sampling distributions from the conformational search (Fig. S4). All scripts used for structure generation, analysis, and visualization are available at: https://github.com/mattas-research-group/scripts_PhD_thesis_Lazaro/ Cite Share Download PDF Status: Posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9195456","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611579862,"identity":"85d0b95b-f9f8-4757-b510-812abfe41c8d","order_by":0,"name":"Lázaro A. M. Castanedo","email":"","orcid":"","institution":"Saint Mary's University","correspondingAuthor":false,"prefix":"","firstName":"Lázaro","middleName":"A. M.","lastName":"Castanedo","suffix":""},{"id":611579863,"identity":"315cd7ab-b14b-4fd8-bb14-3274faf302ef","order_by":1,"name":"Chérif F. Matta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3OsQqCQBzH8b/84SbJ9ULoGQRBEupJWk6Epo7eQALnaBWCegVbblaE6xUcGgpfIHAxWrqioOlybLgvnCjch58AJtM/RtVhk/fHtT+ZqwdBsLLeBN4E7T7C2abyemYwc/absp0ukigv8KL9QXqSccYY8EwiulxUihCf6ohHFz5EHfCVRFCk8L3Chh9k2cJzZa9W7qFIngS7Hyv4IrlE4loCR4oQ7Qqt574ilB9kHIRrUY2GFQnGOuJkcWN1bMJ3VdnUN5HYg2Pa1DryGft6xx73TSaTyaTvAVjoRJz6VesqAAAAAElFTkSuQmCC","orcid":"","institution":"Mount Saint Vincent University","correspondingAuthor":true,"prefix":"","firstName":"Chérif","middleName":"F.","lastName":"Matta","suffix":""}],"badges":[],"createdAt":"2026-03-23 04:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9195456/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9195456/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105566703,"identity":"75a6813d-e59a-44ca-a71b-93251c3afa48","added_by":"auto","created_at":"2026-03-27 12:57:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":841531,"visible":true,"origin":"","legend":"","description":"","filename":"glycerolNtsver16.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9195456/v1_covered_b09570d1-9949-4c91-934f-6c1364983cd9.pdf"},{"id":105508722,"identity":"4568c37e-6311-480e-bac6-58c7a8d3bc12","added_by":"auto","created_at":"2026-03-26 19:54:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14554540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional data supporting this work are provided in the Supporting Information. 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Because assembly of the canonical ribose–phosphate framework is disfavored in water, simpler ancestral backbones may have preceded RNA and DNA. Here, semiempirical prescreening and density functional theory (DFT) calculations were used to evaluate the thermodynamics of glycerol-based nucleosides and nucleotides as candidate proto-nucleic-acid building blocks. Two assembly routes were examined: a classic pathway, in which glycerol first condenses with a recognition unit and then with an ionized linker, and an alternative pathway, in which glycerol first condenses with the ionized linker and only then with the recognition unit. Although Gibbs free energy is a state function, the two pathways access different regions of the potential energy surface and converge to distinct local minima, leading to pathway-dependent differences in the computed thermodynamic quantities. \n\nGlycerol-derived nucleosides are predicted to form favorably in both vacuum and implicit aqueous solution when combined with either canonical or the studied non-canonical bases. Glycerol-based nucleotides are, likewise, thermodynamically accessible by both pathways, although the classic route is consistently more favorable than the alternative route. Among the most stabilized products are derivatives containing adenine, uracil, and C-glycosylated barbituric acid. Replacement of phosphate by arsenate produces only modest energetic and structural changes, indicating that arsenate-containing analogues cannot be excluded on thermodynamic grounds alone. 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