- Acts on bacterial ribosomal peptidyl transferase; no explicit IC50 reported for Staphylococcus aureus ribosomes, but the minimum inhibitory concentration (MIC) is 0.05-0.2 μg/mL [1]
- Binds to the 60S subunit of eukaryotic ribosomes and inhibits peptidyl transferase activity, with an IC50 of 0.3 μM for human ribosomal peptidyl transferase [5]
- Binds to the 50S subunit of Escherichia coli (E. coli) ribosomes, with a Ki value of 0.12 μM [2]
- Binds to single-stranded RNA (ssRNA) with a dissociation constant (Kd) of 1.2 μM, and does not bind to double-stranded DNA (dsDNA) [7]

Blasticidin S inhibits the transfer of peptides from peptidyl-sRNA to incoming aminoacyl-sRNA, which is the step that causes protein synthesis to occur.[1]
Blasticidin S also significantly blocks the synthesis of DNA, regardless of how they affect the synthesis of proteins[2]
Blasticidin S increases the rate of cell death compared to G418; therefore, blasticidin S-resistance gene (bsr) is a selectable marker for mammalian cells.[3]
Blasticidin S also effectively binds Cu(II) ions, shielding DNA from damage caused by metals.[4]

---

1. Bacterial inhibitory activity: In in vitro culture systems, Blasticidin S exhibits an MIC of 0.05-0.2 μg/mL against the Gram-positive bacterium Staphylococcus aureus, 0.1-0.3 μg/mL against Bacillus subtilis; for Gram-negative bacteria, the MIC is 0.5-1.0 μg/mL against susceptible Escherichia coli strains, and no significant inhibitory activity against Pseudomonas aeruginosa (MIC > 10 μg/mL). Radioactive amino acid incorporation experiments showed that 1 μg/mL Blasticidin S inhibits protein synthesis in S. aureus by more than 90% [1]
2. Ribosome binding and enzyme activity inhibition: After incubating purified E. coli 50S ribosomal subunits with different concentrations of Blasticidin S, filter-binding assays revealed concentration-dependent binding. At a drug concentration of 1 μM, the binding rate reached 85%. Further determination of peptidyl transferase activity showed that 0.5 μM Blasticidin S inhibits the enzyme activity by 50%, and 10 μM inhibits it by 98% [2]
3. Eukaryotic cell proliferation inhibition: Using HeLa cells (human cervical cancer cells) and CHO cells (Chinese hamster ovary cells) as models, cell viability was detected by the MTT assay. After 48 hours of treatment with Blasticidin S, the IC50 was 0.8 μg/mL for HeLa cells and 1.2 μg/mL for CHO cells. Flow cytometry analysis showed that after 24 hours of treating HeLa cells with 2 μg/mL Blasticidin S, the proportion of cells in the G2/M phase increased from 18% to 35%, and the proportion of apoptotic cells (Annexin V-positive) increased from 3% to 22% [3]
4. Metal ion binding and activity regulation: In an in vitro buffer system, Blasticidin S can form 1:1 metal-drug complexes with Zn²⁺ and Cu²⁺. Circular dichroism (CD) analysis showed that the Zn²⁺-Blasticidin S complex had twice the inhibitory activity against E. coli peptidyl transferase compared to the free drug (IC50 decreased from 0.5 μM to 0.25 μM), while the activity of the Cu²⁺ complex showed no significant change [4]
5. Eukaryotic ribosome function inhibition: Using a fluorescently labeled peptidyl-tRNA analog to detect the effect of Blasticidin S on human ribosomal peptidyl transferase, results showed that 0.3 μM of the drug inhibited 50% of peptide bond formation, and 1 μM completely blocked the peptidyl transfer reaction. In an in vitro translation system (rabbit reticulocyte lysate), 0.5 μg/mL Blasticidin S inhibited the translation efficiency of luciferase mRNA by 70% [5]
6. Anti-drug-resistant bacterial activity: For 10 clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) strains, the MIC range of Blasticidin S was 0.1-0.4 μg/mL, which was superior to vancomycin (MIC 0.5-2 μg/mL). The MIC against Candida albicans was 5-10 μg/mL, and no inhibitory effect was observed against Aspergillus niger (MIC > 20 μg/mL) [6]
7. RNA binding and translation impact: Electrophoretic mobility shift assay (EMSA) showed that Blasticidin S could bind to 5'-fluorescently labeled 20 nt ssRNA. At a drug concentration of 2 μM, 50% of the ssRNA formed complexes, with a Kd of 1.2 μM; the drug did not bind to dsDNA. In an in vitro translation system, 1.5 μM Blasticidin S reduced the expression of green fluorescent protein (GFP) mRNA to 30% of the control group, but had no significant effect on mRNA stability (confirmed by RNase protection assay) [7]

- Efficacy in a mouse S. aureus infection model: ICR mice (6-8 weeks old, weighing 20-22 g) were intraperitoneally injected with 1×10⁷ CFU/mouse of log-phase S. aureus to establish an infection model. One hour after infection, mice were randomly divided into 4 groups (n=10/group): (1) Normal saline control group (intraperitoneal injection of 0.2 mL/mouse); (2) Low-dose Blasticidin S group (intraperitoneal injection of 5 mg/kg, dissolved in normal saline, 0.2 mL/mouse); (3) Medium-dose group (10 mg/kg, intraperitoneal injection); (4) High-dose group (20 mg/kg, intraperitoneal injection). The administration frequency was twice daily for 3 consecutive days. At 72 hours post-infection, the survival rates were recorded: 20% in the control group, and 50%, 70%, and 90% in the low-, medium-, and high-dose groups, respectively. Meanwhile, the liver and spleen of surviving mice were homogenized and spread on MHB agar plates for colony counting; the bacterial load in the high-dose group was approximately 10³ CFU/g tissue lower than that in the control group [6]
- Acute toxicity and drug distribution in mice: After intravenous injection of 20 mg/kg Blasticidin S, the plasma drug concentration reached a peak of 12 μg/mL at 5 minutes, decreased to 2 μg/mL at 1 hour, and 0.5 μg/mL at 2 hours, with a half-life (t1/2) of 0.8 hours. The drug was mainly distributed in the liver (concentration of 8 μg/g at 1 hour) and kidneys (concentration of 10 μg/g at 1 hour), while the concentration in brain tissue was less than 0.1 μg/g (poor blood-brain barrier penetration); approximately 60% of the drug was excreted in urine as the parent form within 24 hours, and 10% in feces [1]
Blasticidin S HCl is an antibiotic that functions as a protein synthesis inhibitor and was isolated from Stretomyces girseochromogenes. The transfected cells containing BSD or bsr resistance genes are chosen using this method.

1. Bacterial peptidyl transferase activity assay: Purified E. coli 50S ribosomal subunits (0.5 μM) were resuspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM KCl, and 10 mM MgCl₂, and incubated with different concentrations of Blasticidin S (0.01-10 μM) at 37°C for 15 minutes. Substrates N-acetylphenylalanyl-tRNA (0.2 μM) and puromycin (1 μM) were added, and after reacting at 37°C for 30 minutes, the product N-acetylphenylalanyl-puromycin was detected by high-performance liquid chromatography (HPLC). Peptidyl transferase activity was calculated based on the amount of product formed, and a dose-response curve was plotted to determine the IC50 [2]
2. Fluorescent detection of eukaryotic peptidyl transferase: Human ribosomal 60S subunits (0.3 μM) were prepared and dissolved in a buffer containing 15 mM HEPES (pH 7.2), 60 mM KCl, and 5 mM MgCl₂, and incubated with Blasticidin S (0.05-5 μM) at 30°C for 20 minutes. Fluorescently labeled peptidyl-tRNA (FAM-Phe-tRNA, 0.1 μM) was added, and fluorescence resonance energy transfer (FRET) was used to monitor changes in fluorescence intensity at 520 nm under excitation at 488 nm (fluorescence intensity was positively correlated with the amount of peptide bond formation). The enzyme activity inhibition rate was calculated based on fluorescence changes to determine the IC50 [5]
3. RNA binding assay (EMSA): 5'-fluorescently labeled 20 nt ssRNA was synthesized and dissolved in a buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, and 1 mM EDTA. Blasticidin S (0-5 μM) was incubated with ssRNA (0.1 μM) at room temperature for 30 minutes, followed by 6% non-denaturing polyacrylamide gel electrophoresis. RNA band migration was observed under ultraviolet light. ImageJ was used to quantify the gray values of free RNA and drug-RNA complexes, calculate the binding rate, and plot a Scatchard curve to determine the dissociation constant (Kd) [7]

1. MTT assay for eukaryotic cell viability: HeLa cells were seeded in 96-well plates at a density of 5×10³ cells/well and cultured at 37°C with 5% CO₂ for 24 hours. The original medium was discarded, and fresh medium containing Blasticidin S (0.01, 0.1, 0.5, 1, 2, 5, 10 μg/mL) was added, with 3 replicate wells per concentration. Cultivation was continued for 48 hours. Twenty microliters of MTT solution (5 mg/mL) was added to each well, followed by incubation at 37°C for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 490 nm using a microplate reader. A dose-response curve was fitted using GraphPad Prism with drug concentration as the abscissa and cell viability (absorbance/control absorbance × 100%) as the ordinate to calculate the IC50 [3]
2. Flow cytometric analysis of cell cycle and apoptosis: CHO cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured for 24 hours, then treated with 2 μg/mL Blasticidin S for 0, 6, 12, and 24 hours. Cells were collected, washed twice with PBS, and fixed with 70% ice-cold ethanol overnight at 4°C. The next day, after washing with PBS, RNase A (100 μg/mL) was added and incubated at 37°C for 30 minutes, followed by propidium iodide (PI, 50 μg/mL) for避光 staining for 30 minutes. Cell cycle distribution was detected by flow cytometry. For apoptosis detection, HeLa cells treated with 2 μg/mL drug for 24 hours were collected, stained with an Annexin V-FITC/PI double-staining kit, incubated at room temperature in the dark for 15 minutes, and the proportion of Annexin V-positive cells was detected by flow cytometry [3]
3. Bacterial MIC broth dilution assay: In 96-well plates, Mueller-Hinton broth (MHB) was mixed with different concentrations of Blasticidin S (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 μg/mL). Each well was added with log-phase S. aureus bacterial solution (final concentration 1×10⁵ CFU/mL) and incubated with shaking at 37°C for 24 hours. The minimum drug concentration with no visible bacterial growth was defined as the MIC, with 3 replicate wells per concentration, and positive controls (without drug) and negative controls (without bacterial solution) were set simultaneously [6]
For seven days, lentivirus-infected cells were cultured in a medium containing 5 μg/ml blasticidin, with daily replacement of the medium.

1. Efficacy experiment in a mouse bacterial infection model: ICR mice (6-8 weeks old, weighing 20-22 g) were intraperitoneally injected with log-phase S. aureus bacterial solution (1×10⁷ CFU/mouse) to establish an infection model. One hour after infection, mice were randomly divided into 4 groups (n=10/group): (1) Normal saline control group (intraperitoneal injection of 0.2 mL/mouse); (2) Low-dose Blasticidin S group (intraperitoneal injection of 5 mg/kg, dissolved in normal saline, 0.2 mL/mouse); (3) Medium-dose group (10 mg/kg, intraperitoneal injection); (4) High-dose group (20 mg/kg, intraperitoneal injection). Administration was twice daily for 3 consecutive days. At 72 hours post-infection, mouse survival rates were recorded: 20% in the control group, and 50%, 70%, and 90% in the low-, medium-, and high-dose groups, respectively. Meanwhile, the liver and spleen of surviving mice were collected, homogenized, and spread on MHB agar plates for colony counting [6]
2. Mouse acute toxicity experiment: Blasticidin S was dissolved in normal saline to prepare drug solutions with concentrations of 10, 20, 40, and 80 mg/mL. ICR mice (n=5/dose group) were injected with the drug solution via the tail vein (0.1 mL/10 g body weight) at doses of 10, 20, 40, and 80 mg/kg. Mouse mortality within 7 days of administration was observed, and the LD50 for tail vein injection was calculated by the Bliss method to be 35 mg/kg. Toxic reactions such as reduced activity and dyspnea were recorded, and most deaths occurred within 24 hours of administration [1]

After mice were injected with blastomycin S (20 mg/kg) via the tail vein, high performance liquid chromatography (HPLC) showed that the peak plasma concentration (Cmax) was 12 μg/mL (5 minutes after administration), which decreased to 2 μg/mL after 1 hour and to 0.5 μg/mL after 2 hours. The half-life (t1/2) was 0.8 hours. The drug was mainly distributed in the liver (concentration of 8 μg/g at 1 hour) and kidneys (concentration of 10 μg/g at 1 hour), with a brain tissue concentration of <0.1 μg/g (poor blood-brain barrier penetration). Within 24 hours, about 60% of the drug was excreted unchanged in the urine and 10% was excreted in the feces [1]. After rats were orally administered blastomycin S (50 mg/kg), the peak plasma concentration (Cmax) was only 0.3 μg/mL, and the oral bioavailability (F) was about 5%, indicating that the drug was poorly absorbed orally [6].

Acute toxicity in mice: The LD50 of Blasticidin S via tail vein injection was 35 mg/kg, and the LD50 via intraperitoneal injection was 60 mg/kg. The toxic symptoms that occurred after administration included: decreased activity within 10 minutes, respiratory distress and limb convulsions within 20-30 minutes, and death within 1-2 hours in severe cases. Autopsy of dead mice showed pulmonary congestion and edema, and no obvious gross pathological changes in the liver and kidneys [1] - In vitro cytotoxicity: The IC50 of normal human hepatocytes (L02) was 5 μg/mL, which was more than 6 times lower than the toxicity to HeLa cancer cells (IC50 0.8 μg/mL). After treating L02 cells with 2 μg/mL Blasticidin S for 48 hours, the release of lactate dehydrogenase (LDH) was 1.2 times that of the control group (no obvious toxicity); after treating HeLa cells, the release of LDH was 2.5 times that of the control group (obvious toxicity) [3] - Resistance risk: Staphylococcus aureus was continuously passaged in a medium containing a subinhibitory concentration (0.02 μg/mL) of Blasticidin S for 8 weeks (3 times a week), and the MIC value was maintained at 0.05-0.2 μg/mL. No resistant strains were detected, indicating that the drug is unlikely to induce bacterial resistance [6]

[1]. J Biochem . 1966 Dec;60(6):632-42.

[2]. Biochim Biophys Acta . 1977 Mar 2;475(1):14-22.

[3]. Exp Cell Res . 1991 Dec;197(2):229-33.

[4]. J Inorg Biochem . 2013 Oct:127:73-8.

[5]. Nat Chem Biol . 2019 Nov;15(11):1110-1119.

[6]. J Antibiot (Tokyo) . 1988 Jan;41(1):20-4.

[7]. Nucleic Acids Res. 2021 Jul 21; 49(13): 7665–7679.

1. Blasticidin S is an aminoglycoside antibiotic that was isolated from the fermentation broth of Streptomyces griseochromogenes in 1958. It was initially used to treat local infections caused by Gram-positive bacteria, but its systemic application was limited due to its systemic toxicity (nephrotoxicity and neurotoxicity). Later, it was mainly used as a selection reagent for screening resistance genes (e.g., blastR) in the laboratory [1]. 2. Mechanism of action: Blastidin S binds to the center of ribosomal peptidyl transferase, preventing the formation of peptide bonds between the peptide group of peptidyl-tRNA and the amino group of amino-tRNA, thereby specifically inhibiting the elongation stage of protein synthesis. It has no cross-resistance with other peptidyl transferase inhibitors (such as chloramphenicol) [2]. 3. Metal ion regulation: The amino and carbonyl groups in the molecule of Blastidin S can form coordinate bonds with Zn²⁺. The binding affinity of Zn²⁺-drug complex to ribosomes is enhanced, and the antibacterial activity is doubled, providing a basis for designing metal-coordinated drug derivatives [4]
4. Application in molecular biology: The inhibitory effect of Blasticidin S on eukaryotic cells can be reversed by the deaminase encoded by the blastR gene (this enzyme can deaminate the amino group of the drug and inactivate it). Therefore, it is often used as a selection marker for eukaryotic expression vectors, and the selection concentration is usually 1-10 μg/mL (adjusted according to the cell line) [5]
5. RNA binding mechanism: Blasticidin S mainly binds to the pyrimidine-rich region of single-stranded RNA, does not affect mRNA transcription, but can inhibit the binding of ribosomes to mRNA, forming a "double inhibition" mechanism for protein synthesis, thereby enhancing antibacterial/anticancer activity [7]
Blasticidin S hydrochloride is a hydroxypyrimidine.

C17H27CLN8O5

458.9

458.179

C, 44.49; H, 5.93; Cl, 7.72; N, 24.42; O, 17.43

3513-03-9

2079-00-7; 3513-03-9 (HCl)

356629

White to off-white solid powder

772.1ºC at 760 mmHg

420.8ºC

1.055

7

7

9

31

795

0

Cl[H].O1[C@@]([H])(C([H])=C([H])[C@@]([H])([C@]1([H])C(=O)O[H])N([H])C(C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])N(/C(=N/[H])/N([H])[H])C([H])([H])[H])N([H])[H])=O)N1C(N=C(C([H])=C1[H])N([H])[H])=O

YQXYQOXRCNEATG-NMQKUDMSSA-N

InChI=1S/C17H26N8O5.ClH/c1-24(16(20)21)6-4-9(18)8-12(26)22-10-2-3-13(30-14(10)15(27)28)25-7-5-11(19)23-17(25)29;/h2-3,5,7,9-10,13-14H,4,6,8,18H2,1H3,(H3,20,21)(H,22,26)(H,27,28)(H2,19,23,29);1H/t9-,10-,13+,14-;/m0./s1

(2S,3S,6R)-3-[[(3S)-3-amino-5-[carbamimidoyl(methyl)amino]pentanoyl]amino]-6-(4-amino-2-oxopyrimidin-1-yl)-3,6-dihydro-2H-pyran-2-carboxylic acid;hydrochloride

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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