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O-Propargyl-Puromycin (OP-puro) is an alkyne analog of puromycin which is a potent inhibitor of protein synthesis. OP-puro is incorporated into nascent polypeptide chains where it forms covalent adducts and terminates chain elongation on the ribosome. It has been used in combination with fluorescent azides to image nascent proteins in various cells.
| Targets |
O-Propargyl-Puromycin (OP-puro) shares the same target as puromycin, which is the ribosomal A site. It binds to the ribosome and mimics the 3' end of aminoacyl-tRNA, leading to premature termination of protein synthesis.
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| ln Vitro |
It is possible to visualize and selectively block developing proteins in both cells and animals using o-propargylpuromycin. O-propargyl-puromycin is two to three times less effective than unmodified puromycin at adsorbing reticulocyte lumen and protein synthesis in cultured cells. O-Propargyl-puromycin is linked to emerging elbow The copper(I)-catalyzed foldant-toggle cycloaddition reaction allows for the visualization or capture of these toggle bodies, which are rapidly transformed toggle chains that form covalent conjugates [1].
- OP-puro (1–10 μM) efficiently incorporated into nascent polypeptides in cultured cells, as detected by fluorescence microscopy and flow cytometry after click chemistry labeling with azide-fluorophores. This demonstrated its ability to visualize protein synthesis in real-time without significant cytotoxicity at tested concentrations [1] - Treatment of HeLa cells with OP-puro (5 μM) for 30 minutes resulted in robust fluorescence labeling of newly synthesized proteins, confirming its rapid uptake and integration into actively translating ribosomes [1] |
| ln Vivo |
Whereas tissue from mice injected with O-Propargyl-Puromycin demonstrated that the drug may penetrate particular patterns of developing proteins, tissue from mice not injected with O-Propargyl-Puromycin displayed modest non-electrophoretic staining. According to their high activity, the cells in the crypts and base of intestinal villi in the small intestine have the greatest capacity for translation. The Paneth cells, which are packed with dead sexual vesicles and are situated at the base of the cavity, exhibit very strong staining. Prematurely terminated O-Propargyl-Puromycin conjugated peptides are known to easily localize to the endoplasmic reticulum (ER) lumen, as evidenced by strong O-Propargyl-Puromycin labeling of vesicles in Paneth cells [1].
In C57BL/6 mice, intraperitoneal administration of OP-puro (50 mg/kg) led to incorporation into newly synthesized proteins in various tissues. Fluorescence imaging after click chemistry labeling showed intense signals in tissues with high protein synthesis rates, such as the intestinal epithelium, bone marrow, and renal cortex. The liver and muscle also displayed moderate labeling, reflecting physiological protein synthesis levels [1] The fluorescent signal in tissues peaked at 1 hour post-injection and persisted for up to 6 hours, allowing temporal tracking of protein synthesis dynamics in vivo [1] |
| Enzyme Assay |
Ribosome Binding Assay: Radiolabeled OP-puro (3H-OP-puro) was incubated with purified ribosomes in buffer containing Mg²⁺ and ATP. Binding was measured by nitrocellulose filter retention, showing specific interaction with ribosomes. Competition assays with unlabeled puromycin confirmed the binding specificity [1]
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| Cell Assay |
- Protein Synthesis Imaging in Cells: Cells were treated with OP-puro (2–5 μM) for 15–60 minutes, followed by fixation and click chemistry with Alexa Fluor azides. Confocal microscopy revealed intense fluorescence in the cytoplasm, colocalizing with ribosomal markers, indicating active protein synthesis sites [1]
- Cytotoxicity Assessment: MTT assays showed that OP-puro (up to 20 μM) had no significant effect on cell viability after 24-hour treatment, demonstrating its low cytotoxicity at concentrations suitable for imaging [1] |
| Animal Protocol |
- In Vivo Protein Synthesis Imaging in Mice: OP-puro (50 mg/kg) was administered intraperitoneally to C57BL/6 mice. After 1 hour, tissues were harvested and processed for click chemistry labeling. Fluorescence imaging showed strong labeling in highly proliferative tissues like the intestine and bone marrow, confirming its in vivo activity [1]
- Drug Formulation: OP-puro was dissolved in sterile DMSO and diluted with PBS to a final concentration of 10 mg/mL before injection [1] |
| References | |
| Additional Infomation |
- OP-puro is a modified puromycin analog with an alkyne moiety, enabling bioorthogonal click chemistry for fluorescent labeling of newly synthesized proteins. This allows visualization of protein synthesis dynamics in living cells and tissues without disrupting cellular processes [1]
- The compound has been validated in various cell types and animal models, including mice, demonstrating its broad applicability for studying protein synthesis in physiological and pathological conditions [1] |
| Molecular Formula |
C24H29N7O5
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|---|---|
| Molecular Weight |
495.530964612961
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| Exact Mass |
495.223
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| Elemental Analysis |
C, 58.17; H, 5.90; N, 19.79; O, 16.14
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| CAS # |
1416561-90-4
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| PubChem CID |
71576433
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.679
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| LogP |
0.97
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
36
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| Complexity |
786
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CN(C)C1=NC=NC2=C1N=CN2[C@H]3[C@@H]([C@@H]([C@H](O3)CO)NC(=O)[C@H](CC4=CC=C(C=C4)OCC#C)N)O
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| InChi Key |
JXBIGWQNNSJLQK-IYRMOJGWSA-N
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| InChi Code |
InChI=1S/C24H29N7O5/c1-4-9-35-15-7-5-14(6-8-15)10-16(25)23(34)29-18-17(11-32)36-24(20(18)33)31-13-28-19-21(30(2)3)26-12-27-22(19)31/h1,5-8,12-13,16-18,20,24,32-33H,9-11,25H2,2-3H3,(H,29,34)/t16-,17+,18+,20+,24+/m0/s1
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| 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-prop-2-ynoxyphenyl)propanamide
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| Synonyms |
O-Propargyl-puromycin, OPP, OP-puro
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ≥ 31 mg/mL (~62.56 mM)
H2O : < 0.1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.05 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: ≥ 2.5 mg/mL (5.05 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 25.0 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: ≥ 2.5 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0180 mL | 10.0902 mL | 20.1804 mL | |
| 5 mM | 0.4036 mL | 2.0180 mL | 4.0361 mL | |
| 10 mM | 0.2018 mL | 1.0090 mL | 2.0180 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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