Uridine | CAS 58-96-8 | ≥99.0%

Uridine is a naturally occurring pyrimidine ribonucleoside composed of uracil linked to D-ribofuranose through a β-N1-glycosidic bond. The pyrimidine glycosidic linkage is markedly more resistant to acid hydrolysis than the corresponding purine N9-glycosidic bonds of adenosine and guanosine, supporting uridine’s routine compatibility with the mildly acidic mobile-phase conditions commonly used in reversed-phase HPLC and LC-MS of nucleosides; absorbance maxima near 262 nm and a molar extinction coefficient near 10,100 M−1 cm−1 in neutral aqueous solution underpin routine spectrophotometric and chromatographic quantification. As one of the four canonical RNA nucleosides, uridine is a fundamental building block of RNA and a central entry point into pyrimidine nucleotide metabolism, with cellular phosphorylation by uridine-cytidine kinase (UCK1/UCK2; EC 2.7.1.48) generating UMP, which is further phosphorylated to UDP and UTP by nucleoside-monophosphate and nucleoside-diphosphate kinases. UTP and CTP supply RNA polymerase substrates, regulate pyrimidine-pool feedback, and feed UDP-sugar formation for glycoconjugate biosynthesis. Cellular uptake is mediated by the SLC28 concentrative, Na+-dependent CNT and SLC29 equilibrative ENT nucleoside-transporter families, while uridine phosphorylase (UPP1/UPP2; EC 2.4.2.3) catalyses the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. In research workflows, uridine is used as a mammalian cell-culture supplement, a substrate or reference compound in pyrimidine-salvage and nucleoside-transporter studies, a starting material for modified uridine derivatives in synthetic RNA chemistry, and a metabolic building block in glycoengineering. It is supplied as a white to off-white crystalline powder that is soluble in water.

Cell Culture Supplementation and Pyrimidine Salvage in Mitochondrial Biology

Uridine is the standard supplement that supports mammalian cells with impaired de novo pyrimidine biosynthesis. The de novo pathway is architecturally split between cytosol and mitochondrion: the trifunctional cytosolic CAD complex (carbamoyl phosphate synthetase II, aspartate transcarbamoylase, dihydroorotase) generates dihydroorotate from glutamine, ATP, bicarbonate, and aspartate; the mitochondrial inner-membrane enzyme dihydroorotate dehydrogenase (DHODH; EC 1.3.5.2) oxidises dihydroorotate to orotate while transferring electrons to ubiquinone; and the cytosolic bifunctional UMP synthase (UMPS) finally couples orotate to PRPP and decarboxylates the product to UMP. Because DHODH ubiquinol must be reoxidised by Complex III, de novo pyrimidine biosynthesis is hard-wired into a functional electron-transport chain — and disruption of mitochondrial respiration (mitochondrial DNA depletion in ρ0 cells, Complex III inhibition by antimycin A or myxothiazol, OXPHOS dysfunction, or loss of cytochrome bc1 assembly) collapses pyrimidine output even though the cytosolic CAD and UMPS arms remain intact. Exogenous uridine bypasses the mitochondrial bottleneck entirely by entering through SLC28/SLC29 transporters and feeding the UCK1/UCK2 salvage step directly to UMP, restoring pyrimidine pools independently of mitochondrial respiration. In the canonical ρ0 cell-culture framework, uridine is co-supplemented with pyruvate, which serves a redox-support role: regeneration of cytosolic NAD+ via lactate dehydrogenase, thereby restoring the electron-acceptor availability needed to sustain aspartate biosynthesis when respiration is compromised. This combination is foundational in mitochondrial biology, OXPHOS research, electron-transport-chain mutant analysis, mtDNA-depleted cell-line maintenance, and metabolic-flux studies of respiration-deficient model systems.

Nascent-RNA Click-Chemistry Labelling with 5-Ethynyluridine

5-Ethynyluridine (EU), the C5-alkynyl analogue of uridine, is the most widely used non-radioactive metabolic label for nascent RNA. EU enters cells through the same SLC28/SLC29 transporters that import the parent nucleoside, is phosphorylated by UCK1/UCK2 and onward kinases to EUTP, and is incorporated by RNA polymerases I, II, and III into newly transcribed RNA at an average frequency of one EU residue per 35 uridine positions in total RNA. The terminal alkyne handle is then ligated to fluorescent or biotinylated azides by the copper-catalysed azide–alkyne cycloaddition (CuAAC), the canonical click reaction, providing a non-denaturing, fluorophore-flexible alternative to 3H-uridine autoradiography and BrU/anti-BrU immunolabelling for transcription-rate measurement, RNA-turnover kinetics, single-cell transcription imaging, and whole-mount visualisation of newly synthesised RNA in tissues and embryos.

4-Thiouridine Photocrosslinking and Metabolic Sequencing Chemistries

4-Thiouridine (s4U), the C4-thiocarbonyl analogue of uridine, supports a different chemistry portfolio anchored on the nucleophilicity and photoreactivity of the thiocarbonyl group. UV-365 nm irradiation generates a covalent zero-distance crosslink between s4U-labelled RNA and contacting RNA-binding proteins, which underpins PAR-CLIP-class transcriptome-wide RBP target mapping. The same thiocarbonyl supports several orthogonal RNA-recoding chemistries that read s4U incorporation as a sequencing-detectable T-to-C substitution, allowing nascent and steady-state transcripts to be distinguished at single-nucleotide resolution: iodoacetamide alkylation (SLAM-seq), osmium-tetroxide-mediated thiouridine-to-cytidine conversion (TUC-seq), and oxidative trifluoroethylamine-mediated recoding (TimeLapse-seq). Together with EU, s4U has redefined transcriptomic measurement of RNA synthesis, processing, and decay kinetics. Uridine itself remains the standard substrate and reference compound for studies of uridine kinase activity, uridine phosphorylase kinetics, and SLC28/SLC29-mediated nucleoside transport, and is the entry-point reference compound for metabolic-labelling studies of pyrimidine pool regulation, nucleotide turnover, and pyrimidine-pathway flux.

Modified Nucleoside Synthesis and RNA Chemistry

Uridine is a key starting material and reference nucleoside for the synthesis and study of modified uridine derivatives across synthetic RNA chemistry. Selective protection and functionalisation of the 2′-, 3′-, and 5′-hydroxyl groups of the ribose, and of the C5 and C4 positions of the uracil base, provides access to scaffolds including 2′-O-methyluridine, 2′-fluoro-2′-deoxyuridine, 5-bromouridine, 5-iodouridine, 5-aminoallyluridine, 5-ethynyluridine, and 4-thiouridine, each of which finds use in distinct downstream applications. Pseudouridine and N1-methylpseudouridine are structurally related as the C-5 glycoside isomer of uridine and the N1-methyl derivative of that isomer, respectively, but are best treated as a separate modified-nucleoside class because their preparation typically proceeds through dedicated chemical or biocatalytic routes rather than direct uridine functionalisation. These modified nucleosides are central to modified-mRNA chemistry: N1-methylpseudouridine substitution alters RNA hydrogen-bonding, decoding, and innate RNA-sensing behaviour relative to unmodified uridine, making it a distinct benchmark modification in synthetic mRNA research. These materials support synthetic RNA chemistry, antisense-oligonucleotide and siRNA development, modified-mRNA studies, RNA structure analysis, RNA–protein interaction work, and analytical comparison of natural and modified pyrimidine ribosides by HPLC, LC-MS, and NMR.

UDP-Sugar Biosynthesis and Glycoconjugate Research

Uridine nucleotides are central to glycosylation chemistry through their conversion into UDP-activated sugar donors. UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-xylose, and UDP-rhamnose serve glycosyltransferases that construct N- and O-linked glycoproteins, glycolipids, proteoglycans, glycosaminoglycans, plant cell-wall polysaccharides, and bacterial capsular polysaccharides. Uridine availability is therefore relevant to glycoengineering, carbohydrate metabolism, and cellular biosynthesis studies where UDP-sugar pool size, glycosyltransferase activity, hexosamine biosynthetic pathway flux to UDP-GlcNAc, and overall glycan composition are central. The same chemistry underpins chemoenzymatic UDP-sugar synthesis, where uridine or UMP is converted enzymatically through pyrophosphorylase or kinase cascades to provide isotopically labelled or non-natural UDP-sugars for in vitro glycosylation reactions.

Other Applications

• Reference compound in HPLC, LC-MS, and capillary electrophoresis analysis of nucleosides and nucleoside metabolites, with absorbance near 262 nm enabling routine spectrophotometric quantification
• Substrate in nucleoside-transporter (CNT/ENT) and pyrimidine salvage-pathway enzyme assays
• Precursor and reference material for chemoenzymatic UDP-sugar synthesis and glycosyltransferase substrate preparation
• Standard pyrimidine ribonucleoside in NMR, X-ray, and computational structural studies of nucleic-acid–protein and nucleic-acid–small-molecule interactions
• Reference standard for quantitative analysis of pyrimidine pools in metabolomics and isotope-tracing workflows.