Product Overview

5-Fluorouridine (5-FUrd, CAS 1531-85-7, C₉H₁₁FN₂O₆, MW 262.19 g/mol) is a fluorinated pyrimidine nucleoside analog consisting of the antimetabolite 5-fluorouracil (5-FU) N1-glycosidically linked to a β-D-ribofuranose sugar moiety. The defining structural feature is the fluorine atom at the C5 position of the uracil ring (replacing the native C5 hydrogen), which is nearly isosteric with hydrogen (van der Waals radius: F = 1.47 Å vs. H = 1.20 Å) yet dramatically alters the electronic structure of the pyrimidine ring due to fluorine’s strong electron-withdrawing inductive effect. This C5-F substitution perturbs the pKa of the adjacent N3-H (decreased by ~0.5-1.0 pH units), modifies the tautomeric equilibrium of the uracil ring, and stabilizes the enolate transition state in the rate-limiting step of thymidylate synthase-catalyzed dUMP methylation — the molecular basis of fluorine’s potent TS inhibition. Unlike 5-FU (the free base), 5-fluorouridine is a pre-formed ribonucleoside that bypasses the rate-limiting phosphoribosyltransferase (OPRT) activation step. It enters cells efficiently via equilibrative (ENT1/SLC29A1, ENT2/SLC29A2) and concentrative (CNT1/SLC28A1, CNT2/SLC28A2, CNT3/SLC28A3) nucleoside transporters, and is rapidly phosphorylated by uridine-cytidine kinase 1/2 (UCK1/2) to 5-fluorouridine-5′-monophosphate (FUMP), committing the molecule to the intracellular nucleotide pool. FUMP is then sequentially phosphorylated to 5-fluorouridine-5′-triphosphate (FUTP), the active RNA-directed metabolite that competes with UTP for incorporation by all three eukaryotic RNA polymerases (Pol I, Pol II, Pol III). The resulting fluorine-substituted RNA transcripts exhibit impaired codon-anticodon recognition, defective pre-mRNA splicing (particularly U2 snRNA-dependent intron recognition), disrupted pre-rRNA processing leading to nucleolar fragmentation, and activation of the p53-dependent nucleolar stress checkpoint — collectively producing RNA-directed cytotoxicity that complements, and is mechanistically distinct from, the DNA-directed thymidylate synthase inhibition by the parallel FdUMP metabolite pathway. The molecule contains four chiral centers (C1′, C2′, C3′, C4′ of ribofuranose, all in the natural D-ribo configuration), a logP of ~-1.34 (reflecting the polar ribose hydroxyls and pyrimidine carbonyls), and a molar volume of ~148 cm³/mol. The ¹⁹F NMR spectrum is diagnostic: a single sharp resonance at δ -165 to -170 ppm (relative to CFCl₃) confirms the C5-F substitution and provides a convenient, non-destructive method for verifying compound identity, quantifying purity, and tracking metabolic fate in biological samples without the interference from endogenous fluorine-containing metabolites.

UPOR Biotech supplies 5-Fluorouridine at Research Grade with a minimum assay of ≥98.0% by HPLC, manufactured under ISO 9001:2015 and c-GMP quality management systems. The product appears as a white to off-white crystalline powder with a melting point of 178-182°C (with decomposition) and a specific rotation of [α]D²⁰ = +18° to +22° (c=0.5, H₂O) — characteristic of the natural β-D-ribonucleoside configuration. Comprehensive analytical certification includes HPLC purity, ¹H/¹⁹F/¹³C NMR, high-resolution mass spectrometry, water content (Karl Fischer), residual solvent analysis (ICH Q3C), heavy metals, and microbiological testing. The compound is soluble in DMSO, DMF, and warm methanol; slightly soluble in water and PBS — facilitating preparation of concentrated stock solutions for cell culture and in vivo studies. We serve academic, pharmaceutical, and biotechnology researchers investigating fluoropyrimidine pharmacology, RNA metabolism, nucleoside transporter biology, and anticancer drug mechanisms. Research Use Only (RUO) — not for human diagnostic or therapeutic use.

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5-Fluorouridine — The Ribonucleoside Key to Understanding RNA-Directed Fluoropyrimidine Cytotoxicity

5-Fluorouridine is not merely a metabolite of 5-FU — it is the molecular probe that unlocked the RNA-targeting dimension of fluoropyrimidine pharmacology. For decades, the anticancer activity of 5-fluorouracil was attributed almost exclusively to thymidylate synthase inhibition and DNA-directed effects (the “dTMP depletion” model). However, mechanistic studies using 5-fluorouridine — which selectively enters the ribonucleotide pool and is incorporated into RNA without requiring prior conversion to the deoxyribonucleotide — demonstrated unequivocally that RNA incorporation of fluorinated uracil is a major, independent contributor to cytotoxicity. This RNA-directed mechanism explains several clinical observations that the DNA-directed model alone could not: (1) the efficacy of 5-FU in tumors with wild-type p53 and intact DNA damage checkpoints, (2) the time-dependent cytotoxicity kinetics that do not correlate with TS inhibition kinetics, and (3) the lack of complete cross-resistance between 5-FU and TS-specific antifolates (raltitrexed, pemetrexed). For researchers investigating fluoropyrimidine mechanisms, nucleoside transporter pharmacology, or nucleolar stress biology, 5-fluorouridine is an indispensable chemical tool — delivering the fluorine label directly into RNA with predictable, well-characterized metabolic activation, bypassing the confounding variables of prodrug activation and transport that complicate 5-FU studies.

Technical Specifications

PropertySpecification
Product Name5-Fluorouridine — Fluorinated Uridine Nucleoside Analog, Antimetabolite, ≥98% HPLC, Research Grade
Synonyms / Common Names5-Fluorouridine; 5-FUrd; 1-β-D-Ribofuranosyl-5-fluorouracil; 5-Fluoro-1-β-D-ribofuranosyluracil; 5-Fluorouracil ribonucleoside; 1-(β-D-Ribofuranosyl)-5-fluoropyrimidine-2,4(1H,3H)-dione; NSC 529469
CAS Number1531-85-7
Molecular FormulaC₉H₁₁FN₂O₆
Molecular Weight262.19 g/mol
AppearanceWhite to off-white crystalline powder
Key FeatureFluorinated pyrimidine nucleoside — C5-F substitution on uracil creates a potent antimetabolite that is incorporated into RNA via FUTP (disrupting mRNA translation, pre-mRNA splicing, and rRNA processing) and also converted to FdUMP for thymidylate synthase inhibition; the pre-formed ribonucleoside bypasses the rate-limiting OPRT activation step required by 5-FU
Mechanism of ActionDual RNA/DNA antimetabolite — (a) phosphorylated by UCK1/2 to FUMP → FUDP → FUTP, then incorporated into all RNA classes by RNA polymerases I/II/III in place of UTP, causing defective mRNA translation, aberrant pre-mRNA splicing, and nucleolar stress; (b) FUDP converted to FdUDP by ribonucleotide reductase → FdUMP, which forms a stable ternary complex with thymidylate synthase and 5,10-CH₂-THF, irreversibly inhibiting TS and depleting dTMP
Assay (HPLC-UV)≥98.0%
Melting Point178-182°C (with decomposition)
Specific Rotation[α]D²⁰ = +18° to +22° (c=0.5, H₂O)
Identification (¹H-NMR)Characteristic H6 doublet at δ 8.0-8.2 ppm, J(H-F) ~6-7 Hz (confirming C5-F substitution); ribose H1′ doublet at δ 5.8-6.0 ppm, J~4-5 Hz (β-configuration); ribose H2′-H5′ at δ 3.7-4.3 ppm
Identification (¹⁹F-NMR)Singlet at δ -165 to -170 ppm (relative to CFCl₃) — diagnostic for C5-F substitution on the uracil ring; absence of other ¹⁹F signals confirms purity
Identification (MS-ESI)[M+H]⁺ at m/z 263.1; [M+Na]⁺ at m/z 285.1; [M-H]⁻ at m/z 261.0
Related Substances (HPLC)Individual impurity ≤1.0%; total impurities ≤2.0%
Water Content (Karl Fischer)≤1.0%
SolubilitySoluble in DMSO (~50 mg/mL with sonication), DMF, and warm methanol (~10 mg/mL); slightly soluble in water (~5 mg/mL at 25°C) and PBS (pH 7.4); practically insoluble in chloroform, ethyl acetate, hexane
Calculated logP~-1.34 (ACD/Labs) — hydrophilic, consistent with poly-hydroxylated ribonucleoside structure
pH (1% aqueous solution)5.5-7.0
Loss on Drying≤1.0% (105°C, 2 hours)
Ash Content≤0.1%
Heavy MetalsTotal ≤10 ppm; Pb ≤2.0 ppm; As ≤2.0 ppm; Cd ≤1.0 ppm; Hg ≤0.1 ppm
Total Plate Count≤100 CFU/g
Yeast & Mold≤10 CFU/g
E. coli / Salmonella / S. aureusAbsent in 1 g
Residual Solvents (ICH Q3C)Complies with ICH Q3C limits; Class 2 and Class 3 solvents controlled to ≤0.5% individually by GC-HS
Recommended StorageStore at -20°C ± 5°C, tightly sealed in amber glass vial, protected from light and moisture, under inert atmosphere (argon). Allow vial to equilibrate to room temperature before opening to prevent moisture condensation. Prepare stock solutions in anhydrous DMSO and aliquot into single-use portions. Avoid repeated freeze-thaw cycles.
GradeResearch Grade (RUO — Research Use Only)
CertificationsISO 9001:2015, c-GMP
Packaging10 mg / 25 mg / 50 mg / 100 mg / 250 mg / 500 mg / 1 g in amber glass vials under argon atmosphere; custom packaging available upon request
Shelf Life2 years from date of manufacture when stored under recommended conditions; retest date on CoA

Key Benefits — 5-Fluorouridine

Pre-Formed Ribonucleoside — Bypasses Rate-Limiting 5-FU Activation Step

Unlike 5-fluorouracil (5-FU), which requires conversion to FUMP by the often-rate-limiting enzyme orotate phosphoribosyltransferase (OPRT), 5-fluorouridine is a pre-formed ribonucleoside that enters cells directly via nucleoside transporters and is phosphorylated by uridine-cytidine kinase (UCK1/2) — a more widely expressed and higher-capacity kinase. This bypass ensures efficient, predictable intracellular activation to FUTP and FdUMP, eliminating the variable OPRT expression that confounds 5-FU studies and making 5-fluorouridine the preferred tool compound for mechanistic investigations of fluoropyrimidine pharmacology where reproducible intracellular nucleotide levels are critical.

Direct Activation

RNA-Directed Cytotoxicity — FU Incorporation Disrupts Multiple RNA Processes

The fluorine at C5 is nearly isosteric with hydrogen (F = 1.47 Å vs. H = 1.20 Å), allowing FUTP to be efficiently incorporated by RNA polymerases as a UTP surrogate. However, the strongly electron-withdrawing fluorine alters the electronic structure of the uracil ring, perturbing the Watson-Crick base-pairing properties and the critical N3-H hydrogen bond that defines U:A Watson-Crick recognition. Fluorine-substituted mRNAs exhibit impaired ribosomal decoding fidelity and reduced translation elongation rates; fluorine-substituted pre-mRNAs show defective U2 snRNA-dependent intron branch-point recognition; and fluorine-substituted pre-rRNAs undergo aberrant processing, nucleolar fragmentation, and p53-dependent nucleolar stress — a mechanistically distinct cytotoxicity pathway from TS inhibition that is uniquely addressable with 5-fluorouridine as the research probe.

RNA Targeting

Dual-Mechanism Antimetabolite — Simultaneous RNA + DNA Pathway Disruption

5-Fluorouridine is metabolically channeled into two parallel cytotoxic pathways: (a) phosphorylation to FUTP for RNA incorporation (RNA-directed toxicity), and (b) conversion via ribonucleotide reductase to FdUDP → FdUMP for thymidylate synthase ternary complex formation (DNA-directed toxicity). This dual mechanism makes 5-fluorouridine a more comprehensive antimetabolite than either 5-FU (inconsistent activation) or 5-fluoro-2′-deoxyuridine (DNA-selective, minimal RNA incorporation). For researchers, this dual pathway enables experimental designs that dissect the relative contributions of RNA vs. DNA damage to the overall cytotoxicity phenotype by using specific rescue agents: uridine (reverses RNA-directed toxicity by competing with FUTP for RNA incorporation) vs. thymidine (reverses DNA-directed toxicity by bypassing TS blockade via thymidine kinase-mediated dTMP salvage).

Dual Pathway

¹⁹F NMR Handle — Direct, Non-Destructive Quantification in Complex Biological Matrices

The single C5-F fluorine atom provides an ideal ¹⁹F NMR spectroscopic handle for studying 5-fluorouridine metabolism without interference from endogenous compounds (biological tissues contain essentially no fluorine). ¹⁹F NMR enables: (a) direct quantification of 5-fluorouridine and its nucleotide metabolites (FUTP, FUDP, FUMP, FdUMP) in cell extracts and tissues without chromatographic separation, (b) real-time monitoring of 5-fluorouridine uptake and phosphorylation kinetics in perfused cells or organs, (c) non-invasive in vivo ¹⁹F MRS measurement of fluoropyrimidine tumor pharmacokinetics and anabolism in animal models, and (d) pH mapping of the intracellular environment using the pH-dependent ¹⁹F chemical shift of 5-fluorouridine (the N3-H ionization shifts the ¹⁹F resonance by ~0.5-1.0 ppm across the physiological pH range). This built-in spectroscopic reporter — requiring no additional labeling, derivatization, or antibody-based detection — makes 5-fluorouridine an exceptionally powerful tool compound for quantitative pharmacology and drug metabolism studies.

¹⁹F NMR Probe

Applications

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RNA Metabolism & Transcription Studies

Metabolic labeling of nascent RNA with 5-fluorouridine (10-100 μM, 15-60 min pulse) followed by anti-BrdU/FU immunoprecipitation or ¹⁹F NMR detection. Used to measure transcription rates, RNA polymerase processivity, and mRNA half-life. Compatible with dual-label experiments (5-bromouridine + 5-fluorouridine) for pulse-chase RNA dynamics studies.

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Nucleolar Stress & Ribosome Biogenesis Research

5-Fluorouridine at 0.1-10 μM induces pre-rRNA processing defects and nucleolar fragmentation within 2-6 hours. Classic chemical probe for activating the p53-MDM2 nucleolar stress checkpoint. Used to study ribosome biogenesis surveillance, nucleolar protein redistribution (NPM1/B23, nucleolin), and the role of nucleolar stress in senescence and apoptosis.

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Anticancer Drug Mechanism & Resistance Research

5-Fluorouridine at 0.01-100 μM to establish concentration-response curves in fluoropyrimidine-sensitive vs. -resistant cancer cell lines. Used with uridine (RNA rescue) and thymidine (DNA rescue) to dissect RNA vs. DNA contributions to cytotoxicity. Essential tool for studying UCK1/2 downregulation, ENT1/CNT1 transporter loss, and TS overexpression as fluoropyrimidine resistance mechanisms.

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Nucleoside Transporter Pharmacology

5-Fluorouridine is a pan-substrate for all human nucleoside transporters (ENT1/2, CNT1/2/3), making it an ideal reporter substrate for characterizing nucleoside transport kinetics, inhibitor potency (e.g., nitrobenzylmercaptopurine ribonucleoside / NBMPR for ENT1, phloridzin for CNTs), and transporter expression profiling in cancer vs. normal tissues.

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¹⁹F NMR/MRS Metabolism & Pharmacokinetic Studies

Non-invasive ¹⁹F MRS of 5-fluorouridine tumor pharmacokinetics in xenograft models. ¹⁹F NMR of tissue and biofluid extracts quantifies FUTP, FUDP, FUMP, FdUMP, and FUPA/F-β-alanine catabolites simultaneously without chromatography. pH-sensitive ¹⁹F chemical shift enables intracellular pH mapping in tumors and isolated perfused organs.

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Antiviral Nucleoside Analog Screening

5-Fluorouridine serves as a reference fluorinated nucleoside for benchmarking novel antiviral nucleoside analogs. Used at 0.1-100 μM in viral replication assays (plaque reduction, RT-qPCR, luciferase reporter) against RNA viruses. Its well-characterized cytotoxicity profile provides a critical specificity control for distinguishing antiviral activity from non-specific nucleoside toxicity.

Frequently Asked Questions

5-Fluorouridine (5-FUrd, CAS 1531-85-7) is a fluorinated pyrimidine nucleoside analog in which the hydrogen at the C5 position of the uracil ring is replaced by a fluorine atom. It consists of 5-fluorouracil (5-FU) N1-glycosidically linked to a D-ribofuranose sugar. 5-Fluorouridine is the ribonucleoside metabolite of 5-fluorouracil and the prodrug doxifluridine (5′-deoxy-5-fluorouridine). Its antimetabolite mechanism operates through two primary pathways: (1) Incorporation into RNA — 5-fluorouridine is converted intracellularly to 5-fluorouridine-5′-triphosphate (FUTP) by uridine-cytidine kinase and nucleotide kinases. FUTP is then incorporated into all classes of RNA (mRNA, rRNA, tRNA, snRNA) by RNA polymerases in place of UTP. Fluorine-substituted RNA exhibits altered secondary structure, impaired mRNA translation, defective pre-mRNA splicing (particularly of U2 snRNA-dependent introns), and disrupted tRNA maturation — collectively producing profound cytotoxicity. (2) Inhibition of thymidylate synthase — 5-fluorouridine is also metabolized to 5-fluoro-2′-deoxyuridine-5′-monophosphate (FdUMP), which forms a stable ternary complex with thymidylate synthase (TS) and 5,10-methylenetetrahydrofolate, irreversibly inhibiting TS and depleting dTMP pools required for DNA synthesis. This dual RNA/DNA-directed mechanism distinguishes 5-fluorouridine from 5-FU itself, as the ribonucleoside form preferentially targets RNA metabolism. 5-Fluorouridine is also a valuable research tool for studying RNA processing, ribosome biogenesis, and the cellular responses to nucleoside analog-induced nucleolar stress.

5-Fluorouridine is the ribonucleoside form of 5-fluorouracil — the same fluorinated pyrimidine base linked to D-ribose rather than existing as the free base (5-FU) or the deoxyribonucleoside (5-fluoro-2′-deoxyuridine, FdUrd). The key differences: (1) 5-FU (CAS 51-21-8) is the parent fluoropyrimidine base — it must be converted intracellularly to active nucleotide metabolites (FUTP, FdUMP) by the pyrimidine salvage pathway, a process that is inefficient in many tumor types lacking high uridine phosphorylase/kinase activity. 5-Fluorouridine bypasses the rate-limiting first step (conversion of 5-FU to 5-fluorouridine-5′-monophosphate by uridine phosphorylase + uridine kinase), delivering the pre-formed ribonucleoside directly to the kinase step. (2) Doxifluridine (5′-deoxy-5-fluorouridine, CAS 3094-09-5) is the oral prodrug that is converted to 5-fluorouracil by pyrimidine nucleoside phosphorylase — an enzyme preferentially expressed in tumor tissue — providing tumor-selective 5-FU delivery. 5-Fluorouridine is the intermediate metabolite in this activation pathway. (3) Research significance: 5-Fluorouridine is preferred over 5-FU for mechanistic studies of RNA-directed fluoropyrimidine toxicity because it enters cells efficiently via nucleoside transporters (ENT1/2, CNT1/2/3) and is rapidly phosphorylated, ensuring high intracellular FUTP levels for RNA incorporation studies without the confounding variable of variable prodrug activation efficiency. For thymidylate synthase-directed studies, 5-fluoro-2′-deoxyuridine (FdUrd) is usually the preferred tool compound, as it more selectively targets the dTMP pathway with less RNA incorporation.

5-Fluorouridine is a versatile research tool with applications spanning nucleic acid biochemistry, cancer biology, and antiviral research: (1) RNA metabolism and processing studies — 5-fluorouridine incorporation into nascent RNA serves as a metabolic labeling tool (coupled with 5-bromouridine for dual-label experiments) to study transcription rates, RNA polymerase processivity, and RNA half-life by immunoprecipitation (anti-BrdU/anti-FU antibodies) or LC-MS/MS detection of fluorine-labeled RNA. (2) Nucleolar stress and ribosome biogenesis — 5-fluorouridine incorporation into pre-rRNA disrupts pre-rRNA processing, causing nucleolar fragmentation and activation of the p53-MDM2 nucleolar stress checkpoint. This makes 5-FUrd a valuable chemical probe for studying ribosome biogenesis surveillance mechanisms. (3) Anticancer drug mechanism studies — 5-fluorouridine is used to dissect the RNA-directed vs. DNA-directed contributions to fluoropyrimidine cytotoxicity, a longstanding question in cancer pharmacology. (4) Prodrug activation studies — as the metabolic intermediate of doxifluridine and capecitabine activation, 5-fluorouridine is used to calibrate pyrimidine nucleoside phosphorylase activity assays and to validate tumor-selective prodrug activation strategies. (5) Nucleoside transporter research — 5-fluorouridine is a substrate for all human equilibrative (ENT1, ENT2) and concentrative (CNT1, CNT2, CNT3) nucleoside transporters, making it a useful tool for characterizing nucleoside transport kinetics and inhibitor pharmacology. (6) Antiviral research — fluorinated nucleosides have shown activity against RNA viruses, and 5-fluorouridine serves as a reference compound for screening novel fluorinated nucleoside analogs with improved antiviral selectivity indices.

5-Fluorouridine should be stored at -20°C ± 5°C in a tightly sealed, light-protected container under an inert atmosphere (argon or nitrogen). The compound is stable for at least 2 years under these conditions. Solubility: 5-Fluorouridine is soluble in DMSO (~50 mg/mL with sonication), DMF, and warm methanol (~10 mg/mL). It is slightly soluble in water (~5 mg/mL at 25°C) and PBS (pH 7.4). For cell culture experiments, prepare stock solutions in DMSO at 100-500 mM and dilute into culture medium immediately before use (final DMSO concentration should not exceed 0.1% v/v). For in vivo studies, 5-fluorouridine can be formulated in saline or PBS with gentle heating and sonication. The compound is hygroscopic — always allow the storage vial to equilibrate to room temperature before opening to prevent moisture condensation. 5-Fluorouridine is classified as a mutagen and should be handled with appropriate personal protective equipment (nitrile gloves, safety goggles, lab coat) in a chemical fume hood. Avoid inhalation of powder and contact with skin. Waste should be disposed of as hazardous chemical waste in accordance with institutional guidelines. A Safety Data Sheet (SDS) and Certificate of Analysis (CoA) are provided with every shipment.

UPOR Biotech supplies 5-Fluorouridine at Research Grade with a minimum HPLC purity of ≥98.0%, accompanied by a comprehensive Certificate of Analysis (CoA) detailing: assay (HPLC-UV, ≥98.0%), identity confirmation (¹H-NMR — characteristic H6 doublet at δ 8.0-8.2 ppm, J(H-F) ~6-7 Hz confirming C5 fluorine; ¹⁹F-NMR — singlet at δ -165 to -170 ppm confirming C5-F substitution; ESI-MS — [M+H]⁺ at m/z 263.1, [M+Na]⁺ at m/z 285.1), melting point (178-182°C with decomposition), specific rotation ([α]D²⁰ = +18° to +22°, c=0.5, H₂O), water content (Karl Fischer, ≤1.0%), residual solvents (ICH Q3C compliant, GC-HS), heavy metals panel (total ≤10 ppm; Pb ≤2.0 ppm; As ≤2.0 ppm; Cd ≤1.0 ppm; Hg ≤0.1 ppm), and microbiological limits (TPC ≤100 CFU/g, yeast & mold ≤10 CFU/g, E. coli/Salmonella/S. aureus absent). Additional documentation includes: Material Safety Data Sheet (MSDS/SDS), HPLC Chromatogram, ¹H/¹⁹F/¹³C NMR Spectra, Mass Spectrum, Stability Data (real-time and accelerated), and Complete Lot Traceability. Our manufacturing operations are ISO 9001:2015 certified and conducted under c-GMP guidelines. Standard packaging is amber glass vials under argon atmosphere. All documents are available in English, with translations upon request. Custom packaging, larger bulk quantities, and additional analytical testing (elemental impurities per ICH Q3D, bacterial endotoxins, residual solvent profile) are available upon request.