Thymidine (also known as Doxribtimine; deoxythymidine; other names deoxyribosylthymine, thymine deoxyriboside) is a pyrimidine deoxynucleoside used as a cell synchronizing agent. The nucleoside T in double-stranded DNA is deoxythymidine, and it pairs with deoxyadenosine (A). It synchronizes the cells in the G1/early S phase, which is useful in cell biology.

On November 3, 2025 – The Muscular Dystrophy Association (MDA) marks another milestone today in our legacy of progress with the FDA approval of KYGEVVI® (doxecitine and doxribtimine), the first and only treatment for adults and children living with thymidine kinase 2 deficiency (TK2d), a rare and life-threatening mitochondrial myopathy.

Physicochemical Properties

Molecular Formula	C10H14N2O5
Molecular Weight	242.2286
Exact Mass	242.09
Elemental Analysis	C, 49.58; H, 5.83; N, 11.56; O, 33.02
CAS #	50-89-5
Related CAS #	33430-62-5 (Thymidine-5-monophosphate disodium salt)
PubChem CID	5789
Appearance	White to off-white solid powder
Density	1.6±0.1 g/cm3
Boiling Point	510.1±60.0 °C at 760 mmHg
Melting Point	187-189ºC
Flash Point	262.3±32.9 °C
Vapour Pressure	0.0±3.0 mmHg at 25°C
Index of Refraction	1.674
LogP	-0.9
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	2
Heavy Atom Count	17
Complexity	381
Defined Atom Stereocenter Count	3
SMILES	O1[C@]([H])(C([H])([H])O[H])[C@]([H])(C([H])([H])[C@]1([H])N1C(N([H])C(C(C([H])([H])[H])=C1[H])=O)=O)O[H]
InChi Key	IQFYYKKMVGJFEH-XLPZGREQSA-N
InChi Code	InChI=1S/C10H14N2O5/c1-5-3-12(10(16)11-9(5)15)8-2-6(14)7(4-13)17-8/h3,6-8,13-14H,2,4H2,1H3,(H,11,15,16)/t6-,7+,8+/m0/s1
Chemical Name	1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione
Synonyms	DThyd; Deoxyribothymidine; Deoxythymidine; NSC 21548; NSC-21548; NSC21548; Thymidin; deoxythymidine; 2'-Deoxythymidine; 5-Methyldeoxyuridine; Thymidin; Beta-Thymidine; DThyd; AI3-52267; AI3 52267; AI352267
HS Tariff Code	2934.99.03.00
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Microbial Metabolite; DNA Synthesis; Human Endogenous Metabolite Thymidine is involved in the process of DNA synthesis, serving as a precursor for deoxyribonucleotide synthesis, which is essential for DNA replication. It does not act as an inhibitor of specific enzymes but rather participates in nucleotide metabolism as a substrate.
ln Vitro	Thymidine (NSC 21548), a pyrimidine nucleoside that pairs with adenine during DNA synthesis. It is made up of the pyrimidine base thymine attached to the sugar deoxyribose.[1] - In cell synchronization experiments using the double thymidine block method, treatment with thymidine leads to the accumulation of cells at the G1/S phase boundary. After releasing the block, cells proceed through the cell cycle synchronously, allowing for the study of cell cycle progression. [1] - Tritium-labelled thymidine is incorporated into newly synthesized DNA in tissue culture cells, enabling the visualization and localization of DNA synthesis sites via autoradiography. This demonstrates that thymidine is a key precursor for DNA synthesis in vitro. [2] Tritium-labelled thymidine was used to visualize DNA synthesis in chick fibroblast tissue cultures. When added to the culture medium, the labelled thymidine was incorporated into the DNA of cells. Autoradiographs showed silver grains (indicating tritium presence) only over resting cell nuclei and on chromosomes in dividing cells, confirming its specific incorporation into nuclear DNA. The cytoplasm was never labelled. After 2 hours of exposure, only a few nuclei were labelled. With longer exposure times, the number of labelled nuclei and mitotic figures increased. However, even after three days, up to 30% of nuclei (mainly in the inner part of the growth zone) remained unlabelled, suggesting these cells were not actively synthesizing DNA or undergoing mitosis. The ratio of labelled to total mitotic figures was determined over time, indicating that DNA synthesis (incorporation of thymidine) in chick fibroblasts in vitro occurs mainly between the sixth and eleventh hours before the onset of mitosis. Grain counts on the two chromosome groups during anaphase and telophase showed approximately equal numbers, supporting the theory of equal DNA division during cell mitosis. [2]
ln Vivo	The loss of CD4+Vβ8+ and CD8+Vβ8+ T cells induced by thymidine and Tomudex is totally restored by thymidine (500 mg/kg; i.p.; twice daily) [3].
Cell Assay	72 hours following the infection of shRNA lentiviruses, 4 mM thymidine is applied to MDA-MB-231 cells for 18 hours. At 0, 6, and 18 hours, release and harvest cells. Use FACS analysis to ascertain the cell cycle distribution. - For double thymidine block: Cells are first treated with a certain concentration of thymidine for a period sufficient to block cells in S phase, then washed to remove thymidine and incubated in fresh medium to allow cells to progress through the cycle. After a specific interval, a second thymidine treatment is applied to synchronize cells at the G1/S boundary. [1] - For autoradiographic visualization: Tissue culture cells are incubated with tritium-labelled thymidine for a specified duration. Cells are then fixed, and autoradiographic procedures (including coating with emulsion, exposure, development, and staining) are performed to detect the incorporation of labelled thymidine into DNA. [2] A protocol for cell cycle synchronization at the G1/S boundary using a double thymidine block is described. Human H1299 tumor cells are seeded in culture dishes and incubated overnight. Cells are first treated with thymidine at a final concentration of 2 mM for 18 hours. The thymidine is then removed by washing the cells with PBS, and fresh medium is added for a 9-hour release incubation. A second round of thymidine treatment (2 mM) is applied for another 18 hours to achieve stringent synchronization. Finally, cells are released into fresh medium by washing, and collected at various time points (e.g., 0, 2, 6, 8, 10, 12, 14, 24 hours) for downstream analysis such as cell cycle profiling by propidium iodide staining and flow cytometry, or protein expression analysis (e.g., Cyclin A, Cyclin D) by Western blot. [1]
Animal Protocol	Animal/Disease Models: 8-12 weeks balb/c (Bagg ALBino) mouse [3] Doses: 500 mg/kg Route of Administration: intraperitoneal (ip) injection; twice (two times) daily Experimental Results: After injection of SEB, methotrexate and Tomudex-induced Vβ8+ T were completely eliminated Cell loss.
ADME/Pharmacokinetics	Absorption The absolute bioavailability of thymidine following oral administration has not been determined. The median time to peak plasma concentration (Tmax) was approximately four hours. Route of Elimination Urinary excretion of intact thymidine was <1% of the dose in healthy subjects following an oral administration. Volume of Distribution Thymidine's transfer across the blood-brain barrier is generally considered extremely low, with studies suggesting an active transport system in the choroid plexus transfers it into the cerebrospinal fluid (CSF) before it enters brain cells. Protein Binding In vitro plasma protein binding of thymidine was less than 10% over the concentration range between 0.23 mcg/mL and 23 mcg/mL. Metabolism / Metabolites Thymidine is primarily degraded (catabolized) by thymidine phosphorylase to the nucleobases and the 2-deoxy-α-D-ribose 1-phosphate moiety. Thymine, the pyrimidine nucleobase of doxribtimine, is subsequently catabolized to dihydrothymine and ultimately to γ-amino-isobutyric acid and CO2. Biological Half-Life The mean half-life was approximately five hours following a single oral administration of 133 mg/kg dose under fed conditions in healthy adults. Metabolism/Incorporation: The tritium label in the synthesized thymidine was exclusively localized in the pyrimidine (thymine) moiety. After addition to cell culture, the labelled thymidine is incorporated into insoluble compounds, primarily deoxyribonucleic acid (DNA), as evidenced by its localization in nuclei and chromosomes. [2]
Toxicity/Toxicokinetics	About KYGEVVI KYGEVVI is a combination of doxecitine and doxribtimine, both pyrimidine nucleosides, indicated for the treatment of thymidine kinase 2 deficiency (TK2d) in adults and pediatric patients with an age of symptom onset on or before 12 years. Administration of KYGEVVI is intended to incorporate the pyrimidine nucleosides, deoxycytidine and deoxythymidine, into skeletal muscle mitochondrial DNA.1 This action restores mitochondrial DNA copy number in TK2d mutant mice. Important safety information for KYGEVVI1 Increase in Liver Transaminases Elevated liver transaminase [alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST)] levels were reported in patients treated with KYGEVVI. Obtain baseline liver transaminase (ALT, AST) and total bilirubin levels in patients prior to treatment initiation with KYGEVVI. If signs or symptoms consistent with liver injury are observed, interrupt treatment with KYGEVVI until liver transaminase (ALT, AST) and total bilirubin levels have either returned to baseline or stabilized at a new baseline value. Consider permanently discontinuing KYGEVVI if signs or symptoms consistent with liver injury persist or worsen. Monitor liver transaminases and total bilirubin levels yearly and as clinically indicated. Gastrointestinal Adverse Reactions Diarrhea and vomiting leading to hospitalization, dose reduction, and permanent discontinuation were reported in patients treated with KYGEVVI. Based on the severity of the diarrhea and/or vomiting, reduce the dosage of KYGEVVI or interrupt treatment until diarrhea and/or vomiting improves or returns to baseline. Consider restarting KYGEVVI at the last tolerated dose, and increase the dose as tolerated. For persistent or recurring diarrhea and/or vomiting, consider discontinuing KYGEVVI permanently and provide supportive care with electrolyte repletion as clinically indicated. Mouse(ip): LD50 2512 mg/kg Toxicity Data Mouse(ip): LD50 2512 mg/kg A safety note is provided: Propidium Iodide (PI), used in the accompanying cell cycle analysis protocol, is a mutagen. Appropriate personal protective equipment (gloves, protective clothing, eyewear) should be worn when handling it. No specific toxicity data for thymidine itself is provided in this protocol. [1]
References	[1]. Cell Synchronization by Double Thymidine Block. Bio Protoc. 2018 Sep 5;8(17). [2]. Autoradiographic visualization of synthesis of deoxyribonucleic acid in tissue culture with tritium-labelled thymidine. Nature. 1958 Jan 24;181(4604):274-5. [3]. Inhibition of thymidine synthesis by folate analogues induces a Fas-Fas ligand-independentdeletion of superantigen-reactive peripheral T cells. Int Immunol. 2001 Jan;13(1):85-93.
Additional Infomation	Thymidine is a pyrimidine 2'-deoxyribonucleoside having thymine as the nucleobase. It has a role as a metabolite, a human metabolite, an Escherichia coli metabolite and a mouse metabolite. It is functionally related to a thymine. It is an enantiomer of a telbivudine. Thymidine is a pyrimidine deoxynucleoside. Thymidine is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA. In cell biology it is used to synchronize the cells in S phase. Thymidine is a metabolite found in or produced by Escherichia coli (strain K12, MG1655). Thymidine has been reported in Streptomyces piomogenus, Peucedanum japonicum, and other organisms with data available. Thymidine is a pyrimidine nucleoside that is composed of the pyrimidine base thymine attached to the sugar deoxyribose. As a constituent of DNA, thymidine pairs with adenine in the DNA double helix. (NCI04) Thymidine is a metabolite found in or produced by Saccharomyces cerevisiae. A nucleoside in which THYMINE is linked to DEOXYRIBOSE. The primary molecular function of Thymidine is its role as a precursor in the DNA salvage pathway, which it exploits to act as a potent biochemical modulator of anti-metabolite chemotherapy. Unlike its use as a traditional drug, thymidine's effect is achieved by manipulating the intracellular balance of deoxyribonucleotide triphosphate (dNTP) pools, which are essential for DNA synthesis and repair. Upon administration, thymidine is rapidly salvaged and phosphorylated by thymidine kinases to form dTTP (deoxythymidine triphosphate). This supplementation significantly increases the intracellular concentration of both dTTP and dGTP pools. This alteration of dNTP balance is directly responsible for thymidine's observed therapeutic effects, which are dependent on the co-administered drug. The elevated dTTP pools influence the metabolism of cytotoxic drugs. For example, it is thought to enhance the activation and incorporation of the active cytotoxic form of 5-Fluorouracil (FU) into DNA and RNA. The increase in dTTP and dGTP is linked to the observed acceleration of DNA replication fork progression in various cell types. Thymidine acts systemically as a biochemical modulator to optimize the efficacy and reduce the toxicity of standard anti-metabolite chemotherapeutic agents. Its core action is the metabolic modulation of key enzymes in the DNA salvage pathway, which results in the potentiation of anti-tumor effects (e.g., when combined with 5-Fluorouracil) or protection against host toxicity (e.g., host cell rescue from Methotrexate toxicity). Furthermore, TdR, through its conversion to dTTP, significantly accelerates DNA replication fork progression and helps alleviate replication stress in rapidly proliferating cells, a fundamental effect that contributes to its role in preserving genomic stability and altering the selectivity of drug therapy. Thymidine is a DNA synthesis inhibitor. It functions by arresting cells at the G1/S boundary of the cell cycle, prior to DNA replication. This property makes it a valuable biochemical tool for synchronizing cell populations in vitro to study cell cycle-regulated events. The protocol involves creating a 100 mM stock solution of thymidine by dissolving it in PBS. [1]

Solubility Data

Solubility (In Vitro)	DMSO: 48~50 mg/mL (198.2~206.4 mM) H2O: ~33.3 mg/mL (~137.6 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 20 mg/mL (82.57 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.1283 mL	20.6415 mL	41.2831 mL
	5 mM	0.8257 mL	4.1283 mL	8.2566 mL
	10 mM	0.4128 mL	2.0642 mL	4.1283 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.