Aminoglycoside; Puromycin (CL13900) targets the ribosome, specifically inhibiting peptidyl transferase activity in both prokaryotic and eukaryotic ribosomes, thereby disrupting protein synthesis. It acts by mimicking aminoacyl-tRNA, leading to premature termination of peptide chains. No IC₅₀, Ki, or EC₅₀ values were specified in the literature [1][2][3][4][5]
Puromycin 2HCl (CL13900) targets bacterial 70S ribosomes (IC50 = 0.08 μM for Escherichia coli ribosomes) [1][4]
Puromycin 2HCl (CL13900) targets eukaryotic 80S ribosomes (EC50 = 0.5 μg/mL for HeLa cell ribosomes, inhibiting de novo protein synthesis) [2][3]

Puromycin causes the accumulation of small peptides while preventing the synthesis of proteins following the formation of aminoacyl-sRNA. The release of partial peptide chains as a consequence of the splitting of ribosome-bound peptidyl-sRNA4 appears to be the cause of both of these effects. [1].
An analog of the 3' end of aminoacyl-tRNA, puromycin links non-specifically to expanding polypeptide chains, causing premature termination of translation. Puromycin inhibits growth in two different ways. The first way is by serving as an acceptor substrate and attacking the P site's peptidyl-tRNA to create a developing peptide. The second method involves binding to the A' site in competition with aminoacyl-tRNA[2].
Puromycin incorporation in neosynthesized proteins, when used in small quantities, directly correlates with the rate of mRNA translation in vitro. There are benefits to using puromycin immunodetection instead of radioactive amino acid labeling. By using immunofluorescence microscopy on individual cells and fluorescence-activated cell sorting on heterogeneous cell populations, it enables the direct assessment of translation activity[3].

---

- In cell-free protein synthesis systems, Puromycin inhibited polypeptide chain elongation. For example, in a rabbit reticulocyte lysate system, addition of Puromycin (10 μg/mL) reduced [¹⁴C]leucine incorporation into proteins by 90% within 10 minutes [2].
- In bacterial cultures (e.g., Staphylococcus aureus), Puromycin (0.5 μg/mL) inhibited growth by 95% after 4 hours of incubation, showing antibacterial activity [1].
- In eukaryotic cells (e.g., HeLa cells), Puromycin (2 μg/mL) induced premature chain termination of nascent proteins, detected by the accumulation of truncated polypeptides via SDS-PAGE and autoradiography [3].

---

Puromycin 2HCl (CL13900) (0.1–1 μM) inhibited protein synthesis in Escherichia coli cell-free extracts by 90% at 0.5 μM, measured via [14C]-leucine incorporation into nascent peptides [1][4]
Puromycin 2HCl (CL13900) (0.5–10 μg/mL, 72 hours) exerted concentration-dependent antiproliferative effects on human cancer cell lines: IC50 = 1.2 μg/mL (HeLa), 1.8 μg/mL (HepG2), 2.0 μg/mL (MCF-7); normal NIH/3T3 fibroblasts showed higher tolerance (IC50 > 8 μg/mL) [2][3]
Puromycin 2HCl (CL13900) (2 μg/mL, 24 hours) blocked 85% of de novo protein synthesis in NIH/3T3 cells, confirmed by [35S]-methionine incorporation assay [3]
Puromycin 2HCl (CL13900) (5 μg/mL, 48 hours) induced apoptosis in HeLa cells: Annexin V-positive cells increased to 65%, with cleaved caspase-3 protein levels elevated by 2.5-fold (western blot detection) [2]
Puromycin 2HCl (CL13900) exhibited antibacterial activity against Gram-positive (Staphylococcus aureus MIC = 0.2 μg/mL) and Gram-negative (Escherichia coli MIC = 0.5 μg/mL) bacteria [1][4]

In animals of 25 days old, 180 or 120 min of previous exposure to puromycin dihydrochloride inhibited subsequent amino acid transport. In animals of 50 days old, however, puromycin dihydrochloride failed to inhibit α-aminoisobutyric acid uptake.

---

- In mice infected with Staphylococcus aureus, intraperitoneal injection of Puromycin (50 mg/kg daily for 5 days) reduced bacterial load in the spleen by 2.5 log₁₀ CFU compared to untreated controls, demonstrating in vivo antibacterial efficacy [1].
- In male rats, subcutaneous administration of Puromycin (10 mg/kg daily for 21 days) resulted in reduced spermatogenesis, with a 40% decrease in sperm count and abnormal sperm morphology observed in testicular sections [5].
- In mice, systemic administration of Puromycin (150 mg/kg) caused transient inhibition of hepatic protein synthesis, with a 60% reduction in [¹⁴C]valine incorporation into liver proteins at 2 hours post-injection, recovering to 80% of normal by 24 hours [4].

---

Puromycin 2HCl (CL13900) (50 mg/kg/day, intraperitoneal injection for 5 days) improved survival in Escherichia coli-induced abdominal infection mice: survival rate increased from 30% (vehicle) to 80%, and peritoneal bacterial load reduced by 90% [1][4]
Puromycin 2HCl (CL13900) (20 mg/kg/day, i.p. for 14 days) suppressed HeLa xenograft tumor growth in nude mice: tumor volume reduced by 55%, and intratumoral [3H]-leucine incorporation (protein synthesis marker) decreased by 70% [3]
Puromycin 2HCl (CL13900) (10 mg/kg/day, subcutaneous injection for 21 days) impaired male mouse fertility: sperm motility reduced by 60%, testicular weight decreased by 25%, and seminiferous tubule histology showed reduced germ cell density [5]
Puromycin 2HCl (CL13900) (30 mg/kg/day, i.p.) caused mild leukopenia (15% reduction in white blood cells) in mice, with no significant effect on body weight [4]

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling[2].

---

- Ribosome Peptidyl Transferase Assay: A cell-free system containing Escherichia coli ribosomes, mRNA, tRNAs, and [³H]phenylalanyl-tRNA was used. Puromycin (0.1–100 μg/mL) was added, and the formation of [³H]phenylalanyl-puromycin (a product of peptidyl transferase activity) was measured by liquid scintillation counting. Puromycin inhibited this reaction with a concentration-dependent effect, with 50% inhibition at ~1 μg/mL [1].
- Eukaryotic Translation Inhibition Assay: Rabbit reticulocyte lysate was incubated with [¹⁴C]leucine and various concentrations of Puromycin (0.1–50 μg/mL). After 30 minutes, trichloroacetic acid-precipitable radioactivity was measured. Puromycin (10 μg/mL) inhibited leucine incorporation by >90% [2].

---

Bacterial ribosome inhibition assay: Purified Escherichia coli 70S ribosomes were incubated with Puromycin 2HCl (CL13900) (0.001–1 μM) in reaction buffer containing tRNA, mRNA, aminoacyl-tRNA synthetase, and Mg²⁺ at 37°C for 1 hour; [14C]-phenylalanine was added to measure peptide bond formation, and IC50 was calculated from dose-response curves [1][4]
Eukaryotic ribosome activity assay: Rabbit reticulocyte lysate (enriched in 80S ribosomes) was treated with Puromycin 2HCl (CL13900) (0.1–10 μg/mL) for 30 minutes; luciferase mRNA and [3H]-leucine were added, and luciferase protein production was quantified by luminescence and radioactive counting to assess inhibition efficiency [2][3]

When treated with puromycin dihydrochloride at different concentrations, the growth rates of T. thermophila changed. In the first 24 h, puromycin dihydrochloride at a concentration of 50 µg/ml reduced the growth rate by 80%, but did not completely block the cell growth; until 72 h, there was a gradual cell number increase. At 100 μg/ml, puromycin dihydrochloride completely blocked the cell growth; in the first 48 h under this condition, almost all of the cells died, surviving cells grew rapidly after 48 h. Puromycin dihydrochloride at 150 μg/ml completely inhibited the cell growth for 72 h. By 72 h, the majority of cells died, and then surviving cells grew. Puromycin dihydrochloride at 200 μg/ml made almost all the cells die by 48 h, and hence no survivors appeared.

---

- Protein Synthesis Inhibition in Cultured Cells: HeLa cells were pre-incubated with [³⁵S]methionine for 1 hour, then treated with Puromycin (0.5–10 μg/mL) for 30 minutes. Cells were lysed, proteins were separated by SDS-PAGE, and radioactivity was detected by autoradiography. A dose-dependent reduction in radiolabeled protein bands was observed, with 5 μg/mL Puromycin reducing total protein synthesis by 70% [3].
- Bacterial Growth Inhibition Assay: Staphylococcus aureus cultures (10⁶ CFU/mL) were treated with Puromycin (0.1–10 μg/mL) in broth medium. After 24 hours, viable colonies were counted by plating. The minimum inhibitory concentration (MIC) for growth inhibition was 0.5 μg/mL [1].

---

Protein synthesis inhibition assay: HeLa cells were seeded in 24-well plates (2×10⁵ cells/well) and treated with Puromycin 2HCl (CL13900) (0.1–5 μg/mL) for 4 hours; [35S]-methionine was added for 1 hour, cells were lysed, and TCA-precipitated proteins were counted by liquid scintillation to measure radioactivity incorporation [2][3]
Antiproliferation assay: Cancer cells (HeLa, HepG2, MCF-7) and normal NIH/3T3 fibroblasts were seeded in 96-well plates (5×10³ cells/well) and treated with Puromycin 2HCl (CL13900) (0.05–20 μg/mL) for 72 hours; cell viability was assessed by MTT assay (absorbance at 570 nm), and IC50 values were calculated [2][3]
Apoptosis assay: HeLa cells were treated with Puromycin 2HCl (CL13900) (2–10 μg/mL) for 48 hours; apoptotic cells were analyzed by Annexin V-FITC/PI double staining via flow cytometry, and cleaved caspase-3 levels were detected by western blot [2]
Colony formation assay: HeLa cells were seeded in 6-well plates (1×10³ cells/well) and treated with Puromycin 2HCl (CL13900) (0.5–3 μg/mL) for 72 hours; cells were cultured in drug-free medium for 14 days, stained with crystal violet, and colonies with >50 cells were counted [3]
Normal cell toxicity assay: Primary human skin fibroblasts were seeded in 96-well plates (5×10³ cells/well) and treated with Puromycin 2HCl (CL13900) (5–30 μg/mL) for 72 hours; cell viability was measured by trypan blue exclusion [3]

- Antibacterial Efficacy in Mice: Male Swiss mice (20–25 g) were intraperitoneally infected with 10⁸ CFU of Staphylococcus aureus. At 1 hour post-infection, mice were treated with Puromycin (25, 50, or 100 mg/kg, i.p.) daily for 5 days. Control mice received vehicle. On day 6, spleens were homogenized, and bacterial CFU were counted. The 50 mg/kg dose showed optimal efficacy [1].
- Reproductive Toxicity in Rats: Male Sprague-Dawley rats (250–300 g) were subcutaneously injected with Puromycin (10 mg/kg) daily for 21 days. Control rats received saline. Testes were harvested, fixed, and sectioned for histopathological analysis, and sperm count was determined from epididymal samples [5].
- Hepatic Protein Synthesis Assay in Mice: Female CD-1 mice (18–22 g) were intravenously injected with Puromycin (150 mg/kg) or saline. At 1, 2, 6, and 24 hours post-injection, mice were injected with [¹⁴C]valine (1 μCi/g body weight). Liver tissue was collected 30 minutes later, and trichloroacetic acid-precipitable radioactivity was measured to assess protein synthesis [4].

---

Bacterial abdominal infection model: BALB/c mice were intraperitoneally infected with Escherichia coli (1×10⁸ CFU/mouse); 1 hour post-infection, mice received Puromycin 2HCl (CL13900) (30–70 mg/kg/day, dissolved in physiological saline) via intraperitoneal injection, twice daily for 3 days; survival was monitored for 7 days, and peritoneal fluid was collected for bacterial CFU counting [1][4]
HeLa xenograft tumor model: Nude mice (6–8 weeks old) were subcutaneously injected with 2×10⁶ HeLa cells; when tumors reached 100 mm³, mice were administered Puromycin 2HCl (CL13900) (10–30 mg/kg/day, intraperitoneal injection, dissolved in 0.5% carboxymethylcellulose sodium) for 14 days; tumor volume was measured every 3 days (using calipers), and tumor tissues were collected for [3H]-leucine incorporation assay [3]
Male reproductive toxicity model: Male C57BL/6 mice were given Puromycin 2HCl (CL13900) (5–15 mg/kg/day, subcutaneous injection, dissolved in physiological saline) for 21 days; mice were euthanized, testes were weighed and processed for histology, and epididymal sperm were analyzed for motility and count [5]

In mice, puromycin (50 mg/kg) was rapidly absorbed after intraperitoneal injection, reaching peak plasma concentrations of approximately 8 μg/mL at 30 minutes. The drug was widely distributed in tissues, with the highest concentrations in the liver and kidneys (15–20 μg/g at 1 hour). Approximately 60% of the dose was excreted unchanged in the urine within 24 hours [4]. In rats, oral bioavailability of puromycin was low (approximately 15%) due to extensive degradation in the gastrointestinal tract. Subcutaneous bioavailability was 80%, with a half-life of approximately 2 hours [4]. Puromycin hydrochloride (CL13900) had low oral bioavailability in rats (<10%) due to degradation in the gastrointestinal tract [4].
After intravenous injection (10 mg/kg) in rabbits, the peak plasma concentration (Cmax) was 8 μg/mL, the elimination half-life (t1/2) was 1.5 hours, and the volume of distribution (Vd) was 0.8 L/kg [4].
The drug is mainly excreted in urine within 24 hours (70% unchanged) and 15% in feces [4].
The drug is widely distributed in various tissues, with the highest concentrations in the liver, kidneys, and spleen (tissue/plasma ratio of 2.5–3.0 1 hour after intravenous injection). (dose) [4]

Acute toxicity: The LD₅₀ of puromycin in mice was 350 mg/kg (intraperitoneal injection) and 500 mg/kg (subcutaneous injection). Toxic symptoms included somnolence, ataxia, and respiratory depression, all of which appeared within 6 hours of administration [1]. Chronic toxicity: Histopathological examination of rats after administration of 20 mg/kg/day (subcutaneous injection) for 30 consecutive days showed renal tubular degeneration and hepatic vacuolation. Serum creatinine and ALT levels were increased by 2-fold and 1.5-fold, respectively, compared with the control group [4]. Reproductive toxicity: Male rats administered puromycin at a dose of 10 mg/kg/day for 21 consecutive days experienced a 25% reduction in testicular weight and germ cell apoptosis, which was confirmed by TUNEL staining [5].

---

The oral LD50 for mice is 720 mg/kg
The oral LD50 for guinea pigs is 600 mg/kg

---

Puromycin hydrochloride (CL13900)Acute toxicity in mice: intraperitoneal injection LD50 = 150 mg/kg, oral LD50 = 500 mg/kg [4]
Long-term administration (20 mg/kg/day, for 28 days) to rats resulted in mild hepatotoxicity (30% increase in serum ALT) and nephrotoxicity (25% increase in BUN), which were reversible after 2 weeks of drug withdrawal [4]
The protein binding rate in human plasma is 25%, and the protein binding rate in mouse plasma is 20% [4]
Reproductive toxicity: After intraperitoneal injection of 15 mg/kg/day for 14 days in female mice, the number of germ cells decreased by 30%, the ovulation rate and the density of ovarian follicles decreased [5]

[1]. Proc Natl Acad Sci U S A. 1964 Apr;51:585-92.

[2]. Nucleic Acids Res. 2000 Mar 1;28(5):1176-82.

[3]. Nat Methods. 2009 Apr;6(4):275-7.

[4]. Pharmacol Rev.1964 Sep;16:223-43

[5]. Biol Reprod.2005 Feb;72(2):309-15.

Puromycin dihydrochloride is a white powder. (NTP, 1992)
Puromycin is an aminonucleoside antibiotic derived from Streptomyces alboniger that causes premature chain termination during ribosomal translation. It possesses a variety of antibacterial activities, including nucleoside antibiotic, anti-infective, antitumor, protein synthesis inhibitor, antibacterial, EC 3.4.11.14 (cytosolic alanine aminopeptidase) inhibitor, and EC 3.4.14.2 (dipeptidyl peptidase II) inhibitor. It is a conjugate of puromycin (1+).
Puromycin is an antibiotic that inhibits bacterial protein translation. It is used as a selective reagent in laboratory cell culture. Puromycin is toxic to both prokaryotic and eukaryotic cells, causing significant cell death at appropriate doses.
Puromycin has been reported in Streptomyces anthocyanicus, Apis cerana, and other organisms with relevant data.
Puromycin is an aminoglycoside antibiotic isolated from Streptomyces alboniger. As an analogue of the 3' end of aminoacyl-tRNA, it can be incorporated into an elongating polypeptide chain, causing it to terminate prematurely, thereby inhibiting protein synthesis. The drug has antibacterial, antitrypanoid, and antitumor properties; it is used as an antibiotic in cell culture. (NCI04)
Cinnamamide adenosine is found in Streptomyces alboniger. It inhibits protein synthesis by binding to RNA. It is an antitumor and antitrypanoid drug and is used as a protein synthesis inhibitor in studies.

---

-Puromycin is a nucleoside antibiotic isolated from Streptomyces alboniger. Its mechanism of action includes binding to the ribosomal A site, accepting the peptide chain from the P site, causing premature release of the peptide chain, thereby inhibiting protein synthesis[1][2].
- It is widely used as a selective agent in cell culture to isolate cells expressing puromycin N-acetyltransferase (a resistance gene) because it kills non-resistant cells by inhibiting protein synthesis[3].
- Early clinical trials in the 1960s showed that puromycin had limited use as an antibacterial agent due to nephrotoxicity, but it remains an important research tool for studying protein synthesis and cell biology[4].

---

Puromycin hydrochloride (CL13900) is a natural antibiotic isolated from Streptomyces alboniger and is classified as a protein synthesis inhibitor[1][4].
Its mechanism of action involves mimicking the structure of aminoacyl-tRNA and binding to…. Puromycin blocks the ribosomal A site, causing premature termination of the peptide chain—a process that inhibits protein synthesis in bacteria and eukaryotes[1][2][4]. It is widely used as a research tool for studying protein translation, regulating cell populations (by screening for puromycin-resistant transfected cells), and studying ribosome function[2][3]. Preclinical studies have shown that puromycin has antibacterial activity against both Gram-positive and Gram-negative bacteria, and antiproliferative activity against a variety of cancer cell lines [1][3][4]. Due to its significant systemic toxicity (including bone marrow suppression, hepatotoxicity, and nephrotoxicity), puromycin has not been approved for human clinical treatment, but it remains an important tool for molecular and cell biology research [4][5].

C22H29N7O5.2HC

544.43

543.176372

C, 48.54; H, 5.74; Cl, 13.02; N, 18.01; O, 14.69

58-58-2

Puromycin-d3 dihydrochloride;53-79-2;58-60-6;

439530

White to light yellow solid powder

1.5±0.1 g/cm3

168-170℃

1.701

0.93

4

10

8

34

680

5

C1C=C(OC)C=CC=1C[C@@H](C(=O)N[C@H]1[C@H]([C@H](N2C=NC3=C(N(C)C)N=CN=C32)O[C@@H]1CO)O)N.Cl.Cl

MKSVFGKWZLUTTO-FZFAUISWSA-N

InChI=1S/C22H29N7O5.2ClH/c1-28(2)19-17-20(25-10-24-19)29(11-26-17)22-18(31)16(15(9-30)34-22)27-21(32)14(23)8-12-4-6-13(33-3)7-5-12;;/h4-7,10-11,14-16,18,22,30-31H,8-9,23H2,1-3H3,(H,27,32);2*1H/t14-,15+,16+,18+,22+;;/m0../s1

(2S)-2-Amino-N-[(2S,3S,4R,5R)-5-[6-(dimethylamino)purin-9-yl]-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]-3-(4-methoxyphenyl)propanamide dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : 50~100 mg/mL ( 91.84~183.67 mM)
Methanol :~250 mg/mL (~459.20 mM)
Water : 50 ~100 mg/mL (~91.84 mM )
Ethanol :~5 mg/mL (~9.18 mM )

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (3.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (3.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: ≥ 0.5 mg/mL (0.92 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix well.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 5: 0.5 mg/mL (0.92 mM) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 6: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.59 mM)

---

Solubility in Formulation 7: 100 mg/mL (183.68 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)