A Dual Fluorescent-Raman Bioorthogonal Probe for Specific Biosynthetic Labeling of Gangliosides

A Dual Fluorescent-Raman Bioorthogonal Probe for Specific Biosynthetic Labeling of Gangliosides | 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 A Dual Fluorescent-Raman Bioorthogonal Probe for Specific Biosynthetic Labeling of Gangliosides John Hanover, Mana Mukherjee, Matthew Watson, Devin Biesbrock, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5611903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Oct, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract Gangliosides are sialic acid-containing glycosphingolipids integral to the cell membrane and are particularly abundant in the nervous system. Aberrant ganglioside metabolism contributes to pathological conditions including neurodegenerative diseases, lysosomal storage disorders, and cancer. Currently, tools to visualize and detect gangliosides are very limited and non-specific. Here, we describe a dual fluorescent and Raman-active ManNAlk derivative, phenanthrene-9-Pr4ManNAlk (MM-JH-2), capable of one-step selective labeling of gangliosides in cells. This modified ManNAlk derivative produces a biologically unique Raman spectral signature, which arise from the carbon-carbon triple bond augmented by conjugation to a fluorescent phenanthrene moiety. By using this dual probe, unambiguous identification is achieved within cells. Raman maps generated using the alkyne stretching frequency indicate a distribution of MM-JH-2 overlapping with intracellular membrane lipids. Using confocal fluorescence imaging, the cellular transport of labeled gangliosides was tracked from synthesis to recycling in the acidic compartments. Notably, MM-JH-2 can differentiate between cells that differ in ganglioside biosynthetic flux, such as malignant and nonmalignant cells, as well as distinguish between B cells and T cells. Thus, MM-JH-2 is a novel next-generation metabolic chemical reporter (MCR) that is Raman-active, fluorescent, and can be broadly applied to cellular studies investigating ganglioside biosynthetic flux. Biological sciences/Chemical biology/Glycobiology Biological sciences/Chemical biology/Chemical tools Biological sciences/Chemical biology/Lipids/Glycolipids Bioorthogonal chemistry Phenanthrene-9-Pr4ManNAlk Dual fluorescent and Raman probe One-step enzymatic labeling GM3 ganglioside Ex vivo labeling of lymphocytes Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFigures.pdf Supplementary Figures 1-18 Cite Share Download PDF Status: Published Journal Publication published 03 Oct, 2025 Read the published version in Communications Chemistry → 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-5611903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":458685595,"identity":"12c4632d-e397-4535-9739-32e468bdc42d","order_by":0,"name":"John 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Aberrant ganglioside metabolism contributes to pathological conditions including neurodegenerative diseases, lysosomal storage disorders, and cancer. Currently, tools to visualize and detect gangliosides are very limited and non-specific. Here, we describe a dual fluorescent and Raman-active ManNAlk derivative, phenanthrene-9-Pr4ManNAlk (MM-JH-2), capable of one-step selective labeling of gangliosides in cells. This modified ManNAlk derivative produces a biologically unique Raman spectral signature, which arise from the carbon-carbon triple bond augmented by conjugation to a fluorescent phenanthrene moiety. By using this dual probe, unambiguous identification is achieved within cells. Raman maps generated using the alkyne stretching frequency indicate a distribution of MM-JH-2 overlapping with intracellular membrane lipids. Using confocal fluorescence imaging, the cellular transport of labeled gangliosides was tracked from synthesis to recycling in the acidic compartments. Notably, MM-JH-2 can differentiate between cells that differ in ganglioside biosynthetic flux, such as malignant and nonmalignant cells, as well as distinguish between B cells and T cells. Thus, MM-JH-2 is a novel next-generation metabolic chemical reporter (MCR) that is Raman-active, fluorescent, and can be broadly applied to cellular studies investigating ganglioside biosynthetic flux.","manuscriptTitle":"A Dual Fluorescent-Raman Bioorthogonal Probe for Specific Biosynthetic Labeling of Gangliosides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-23 06:57:57","doi":"10.21203/rs.3.rs-5611903/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b4b8f57d-1e41-4c3e-8405-7b59cbbc6dc5","owner":[],"postedDate":"May 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48726796,"name":"Biological sciences/Chemical biology/Glycobiology"},{"id":48726797,"name":"Biological sciences/Chemical biology/Chemical tools"},{"id":48726798,"name":"Biological sciences/Chemical biology/Lipids/Glycolipids"}],"tags":[],"updatedAt":"2025-10-04T07:08:58+00:00","versionOfRecord":{"articleIdentity":"rs-5611903","link":"https://doi.org/10.1038/s42004-025-01685-x","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2025-10-03 04:00:00","publishedOnDateReadable":"October 3rd, 2025"},"versionCreatedAt":"2025-05-23 06:57:57","video":"","vorDoi":"10.1038/s42004-025-01685-x","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01685-x","workflowStages":[]},"version":"v1","identity":"rs-5611903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5611903","identity":"rs-5611903","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}