Puromycin 2HCl (CL13900), an analog of aminoacyl-tRNA (anaminonucleoside), is a potent antibiotic which acts as a protein synthesis inhibitor. It inhibits the incorporation of aminoacyl-tRNA into the C-terminal on a synthesizing polypeptide, resulting in the premature termination of the polypeptide chain. Puromycin is toxic to the growth of various eukaryote cells including mammalian cells. Concentrations of puromycin sufficient to inhibit the cell growth of mammalian cells range from 0.5-10 μg/ml. IC90s for puromycin to inhibit the growth of Plasmodium falciparum and Giardia lamblia are 60 ng/ml and 54 μg/ml, respectively.

Physicochemical Properties

Molecular Formula	C22H29N7O5.2HC
Molecular Weight	544.43
Exact Mass	543.176372
Elemental Analysis	C, 48.54; H, 5.74; Cl, 13.02; N, 18.01; O, 14.69
CAS #	58-58-2
Related CAS #	Puromycin-d3 dihydrochloride;53-79-2;58-60-6;
PubChem CID	439530
Appearance	White to light yellow solid powder
Density	1.5±0.1 g/cm3
Melting Point	168-170℃
Index of Refraction	1.701
LogP	0.93
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	8
Heavy Atom Count	34
Complexity	680
Defined Atom Stereocenter Count	5
SMILES	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
InChi Key	MKSVFGKWZLUTTO-FZFAUISWSA-N
InChi Code	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
Chemical Name	(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
Synonyms	Puromycin Dihydrochloride; Puromycin 2HCl; CL13900 2HCl; CL-13900 dihydrochloride; CL 13900 dihydrochloride; Puromycine; NSC-3055; NSC3055; Puromycin hydrochloride; Puromycin 2HCl; Puromycin (Dihydrochloride); CL 16,536; DTXSID7045861; PGN54228S5; NSC 3055
HS Tariff Code	2934.99.9001
Storage	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.
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	Aminoglycoside
ln Vitro	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].
ln Vivo	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.
Enzyme Assay	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].
Cell Assay	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.
Animal Protocol
Toxicity/Toxicokinetics	mouse LD50 oral 720 mg/kg guinea pig LD50 oral 600 mg/kg
References	[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.
Additional Infomation	Puromycin dihydrochloride is a white powder. (NTP, 1992) Puromycin is an aminonucleoside antibiotic, derived from the Streptomyces alboniger bacterium, that causes premature chain termination during translation taking place in the ribosome. It has a role as a nucleoside antibiotic, an antiinfective agent, an antineoplastic agent, a protein synthesis inhibitor, an antimicrobial agent, an EC 3.4.11.14 (cytosol alanyl aminopeptidase) inhibitor and an EC 3.4.14.2 (dipeptidyl-peptidase II) inhibitor. It is a conjugate base of a puromycin(1+). Puromycin is an antibiotic that prevents bacterial protein translation. It is utilized as a selective agent in laboratory cell cultures. Puromycin is toxic to both prokaryotic and eukaryotic cells, resulting in significant cell death at appropriate doses. Puromycin has been reported in Streptomyces anthocyanicus, Apis cerana, and other organisms with data available. Puromycin is an aminoglycoside antibiotic isolated from the bacterium Streptomyces alboniger. Acting as an analog of the 3' terminal end of aminoacyl-tRNA, puromycin incorporates itself into a growing polypeptide chain and causes its premature termination, thereby inhibiting protein synthesis. This agent has antimicrobial, antitrypanosomal, and antineoplastic properties; it is used as an antibiotic in cell culture. (NCI04) A cinnamamido ADENOSINE found in STREPTOMYCES alboniger. It inhibits protein synthesis by binding to RNA. It is an antineoplastic and antitrypanosomal agent and is used in research as an inhibitor of protein synthesis.

Solubility Data

Solubility (In Vitro)

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 Vivo)

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. 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8368 mL	9.1839 mL	18.3678 mL
	5 mM	0.3674 mL	1.8368 mL	3.6736 mL
	10 mM	0.1837 mL	0.9184 mL	1.8368 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.