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.

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]

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].

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/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.

Absorption
The absolute bioavailability of orally administered thymidine has not been determined. The median time to peak plasma concentration (Tmax) is approximately 4 hours.
Elimination Pathway
Following oral administration, the amount of intact thymidine excreted in the urine of healthy subjects is <1% (dose).
Volume of Distribution
The transport rate of thymidine across the blood-brain barrier is generally considered extremely low. Studies have shown that an active transport system in the choroid plexus transports it to the cerebrospinal fluid (CSF) before entering brain cells.
Protein Binding
In vitro plasma protein binding is less than 10% in the concentration range of 0.23 mcg/mL to 23 mcg/mL.
Metabolism/Metabolites
Thymidine is primarily degraded (catabolized) by thymidine phosphorylase to the nucleotide and 2-deoxy-α-D-ribose-1-phosphate moiety. The pyrimidine nucleotide of docelibutrimidine is subsequently catabolized to dihydrothymidine, which is ultimately metabolized to γ-aminoisobutyric acid and carbon dioxide.
Biological half-life
The average half-life of a single oral dose of 133 mg/kg in healthy adults under food conditions is approximately 5 hours.

---

Metabolism/Incorporation: The tritium labeling in the synthesized thymidine is localized only to the pyrimidine (thymine) moiety. Upon addition to cell cultures, the labeled thymidine is incorporated into the insoluble compounds, primarily deoxyribonucleic acid (DNA), as evidenced by its localization in the cell nucleus and chromosomes. [2]

About KYGEVVI
KYGEVVI is a combination of doxexicetin and docelibitine, both pyrimidine nucleosides, indicated for the treatment of thymidine kinase 2 deficiency (TK2d) in adults and children 12 years and younger. KYGEVVI is administered to incorporate pyrimidine nucleosides (deoxycytidine and deoxythymidine) into skeletal muscle mitochondrial DNA.¹This action restores mitochondrial DNA copy number in TK2d mutant mice.
Important Safety Information for KYGEVVI¹
Elevated Liver Transaminases
Elevated levels of liver transaminases [alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST)] have been reported in patients treated with KYGEVVI. Baseline liver transaminase (ALT, AST) and total bilirubin levels should be obtained before initiating KYGEVVI treatment. If signs or symptoms consistent with liver injury are observed, KYGEVVI treatment should be discontinued until liver transaminase (ALT, AST) and total bilirubin levels return to baseline or stabilize at a new baseline value. If signs or symptoms consistent with liver injury persist or worsen, permanent discontinuation of KYGEVVI should be considered. Liver transaminase and total bilirubin levels should be monitored annually and as clinically necessary.
Gastrointestinal Adverse Reactions
Patients receiving KYGEVVI treatment have reported diarrhea and vomiting, leading to hospitalization, dose reduction, and permanent discontinuation. Depending on the severity of diarrhea and/or vomiting, the dose of KYGEVVI should be reduced or treatment should be discontinued until diarrhea and/or vomiting improve or return to baseline levels. Consider restarting KYGEVVI at the previously tolerated dose and increasing the dose as tolerated. For persistent or recurrent diarrhea and/or vomiting, permanent discontinuation of KYGEVVI should be considered, and supportive care such as electrolyte supplementation should be provided as clinically necessary.

---

Mice (intraperitoneal injection): LD50 2512 mg/kg
Toxicity Data
Mice (intraperitoneal injection): LD50 2512 mg/kg

---

Safety Note: Propidium iodide (PI) used in the accompanying cell cycle analysis protocol is a mutagen. Appropriate personal protective equipment (gloves, protective clothing, goggles) should be worn when handling propidium iodide. Specific toxicity data for thymidine itself are not provided in this protocol. [1]

[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.

Thymidine is a pyrimidine 2'-deoxynucleoside with the nucleobase thymine. It is a metabolite found in humans, E. coli, and mice. Its function is related to thymine. It is the enantiomer of telbivudine. Thymidine is a pyrimidine deoxynucleoside. Thymidine is a DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA. In cell biology, thymidine is used to synchronize cells to the S phase. Thymidine is found in or produced by E. coli (K12 strain, MG1655 strain). Thymidine has also been reported in Streptomyces piomogenus, Pseudomonas japonicum, and other organisms with relevant data. Thymidine is a pyrimidine nucleoside composed of the pyrimidine base thymine linked to a deoxyribose sugar. As a component of DNA, thymidine pairs with adenine in the DNA double helix structure. (NCI04)
Thymidine is a metabolite found or produced in Saccharomyces cerevisiae.
It is a nucleoside in which thymine is linked to deoxyribose.

---

The primary molecular function of thymidine is as a precursor to the DNA rescue pathway, which it utilizes as a potent biochemical regulator of antimetabolite chemotherapy. Unlike conventional drugs, thymidine works by regulating the balance of the intracellular deoxynucleoside triphosphate (dNTP) pool, which is crucial for DNA synthesis and repair. Upon administration, thymidine is rapidly rescued and phosphorylated by thymidine kinase to form dTTP (deoxythymidine triphosphate). This replenishment significantly increases the concentration of intracellular dTTP and dGTP pools. The alteration of dNTP balance by thymidine is the direct cause of its therapeutic effect, which in turn depends on combination therapy. Increased dTTP levels can affect the metabolism of cytotoxic drugs. For example, it is thought to enhance the activation of the active cytotoxic form of 5-fluorouracil (FU) and incorporate it into DNA and RNA. Increased dTTP and dGTP are associated with accelerated DNA replication fork progression observed in multiple cell types. Thymidine, as a biochemical regulator, acts systemically to optimize the efficacy and reduce the toxicity of standard antimetabolite chemotherapy drugs. Its core role is to regulate the metabolism of key enzymes in the DNA salvage pathway, thereby enhancing antitumor effects (e.g., when used in combination with 5-fluorouracil) or protecting the host from toxic damage (e.g., protecting host cells from methotrexate toxicity). Furthermore, TdR significantly accelerates DNA replication fork elongation by converting to dTTP and helps alleviate replication stress in rapidly proliferating cells; this fundamental role contributes to its role in maintaining genomic stability and altering drug selectivity. Thymidine is a DNA synthesis inhibitor. Its mechanism of action is to arrest cells at the G1/S phase junction of the cell cycle, prior to DNA replication. This characteristic makes it a valuable biochemical tool for synchronizing cell populations in vitro to study cell cycle regulatory events. The protocol involves dissolving thymidine in PBS to prepare a 100 mM stock solution. [1]

C10H14N2O5

242.2286

242.09

C, 49.58; H, 5.83; N, 11.56; O, 33.02

50-89-5

33430-62-5 (Thymidine-5-monophosphate disodium salt)

5789

White to off-white solid powder

1.6±0.1 g/cm3

510.1±60.0 °C at 760 mmHg

187-189ºC

262.3±32.9 °C

0.0±3.0 mmHg at 25°C

1.674

-0.9

3

5

2

17

381

3

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]

IQFYYKKMVGJFEH-XLPZGREQSA-N

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

1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione

DThyd; Deoxyribothymidine; Deoxythymidine; NSC 21548; NSC-21548; NSC21548; Thymidin; deoxythymidine; 2'-Deoxythymidine; 5-Methyldeoxyuridine; Thymidin; Beta-Thymidine; DThyd; AI3-52267; AI3 52267; AI352267

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 48~50 mg/mL (198.2~206.4 mM)
H2O: ~33.3 mg/mL (~137.6 mM)

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.)