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| Targets |
The primary molecular target engaged by E3 Ligase Ligand-Linker Conjugates 20 TFA is the cereblon (CRBN) E3 ubiquitin ligase complex. The compound contains the Thalidomide-based cereblon ligand, which binds with high affinity to the CRBN protein, a substrate recognition component of the CRBN E3 ubiquitin ligase complex. This ligand directs the E3 ligase to ubiquitinate the target protein that is recruited by the complete PROTAC molecule. The linker connects the cereblon ligand to a functional group that can be attached to a warhead (a target-binding moiety). When used in PROTACs, the compound enables the formation of a ternary complex between the target protein, the PROTAC, and the CRBN E3 ligase, leading to ubiquitination and proteasomal degradation of the target protein.
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| ln Vitro |
In vitro studies demonstrate that E3 Ligase Ligand-Linker Conjugates 20 TFA can be incorporated into PROTAC molecules that induce targeted protein degradation. The compound enables the formation of a ternary complex between the target protein, the PROTAC, and the CRBN E3 ligase, leading to ubiquitination and subsequent proteasomal degradation of the target protein. The linker provides the appropriate length and flexibility for optimal ternary complex formation. The Thalidomide-based ligand ensures high affinity binding to CRBN, enabling efficient target degradation. The compound's activity is assessed by measuring target protein levels by Western blot and by evaluating downstream functional effects such as inhibition of cell proliferation and induction of apoptosis.
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| ln Vivo |
In vivo studies of E3 Ligase Ligand-Linker Conjugates 20 TFA are conducted in the context of PROTAC molecules incorporating this conjugate. PROTACs containing this linker have been evaluated in various animal models of cancer and other diseases. In xenograft models, PROTACs containing this conjugate demonstrate significant tumor growth inhibition through degradation of the target protein. The compounds are typically administered via intraperitoneal or intravenous injection at doses determined from pharmacokinetic and toxicology studies. Pharmacodynamic endpoints include measurement of target protein degradation in tumor tissues, assessment of downstream signaling pathway inhibition, and evaluation of anti-tumor efficacy.
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| Enzyme Assay |
For CRBN binding assays, the affinity of the Thalidomide-based ligand for CRBN is measured using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). Purified CRBN-DDB1 complex is immobilized on a sensor chip, and varying concentrations of the ligand-linker conjugate (0.001-100 µM) are injected over the chip surface. Binding kinetics (ka, kd) and affinity (KD) are calculated. For conjugation, the compound is reacted with a warhead containing a suitable functional group. The reaction is typically performed in an organic solvent (e.g., DMF or DCM) with a base. The reaction product (the complete PROTAC) is purified by HPLC and characterized by mass spectrometry.
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| Cell Assay |
For cellular degradation assays, cancer cells expressing the target protein are cultured in appropriate medium with 10% FBS and antibiotics. Cells are seeded in 6-well or 12-well plates and treated with PROTACs containing the conjugate at varying concentrations (0.001-10 µM) for 4-24 hours. Cells are lysed in RIPA buffer with protease inhibitors, and target protein levels are analyzed by Western blot using specific antibodies. Degradation efficiency is quantified by densitometry and expressed as DC50 and Dmax. For cell viability assays, cells are treated with PROTACs for 48-72 hours and viability is assessed by MTT or CellTiter-Glo assays. Apoptosis is assessed by Annexin V/PI staining or caspase-3/7 activity assays.
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| Animal Protocol |
For in vivo efficacy studies, 6-8 week old female immunodeficient mice (e.g., nude or SCID) are used. Mice are subcutaneously implanted with cancer cells expressing the target protein in the flank. When tumors reach approximately 100-200 mm³, animals are randomized into treatment groups (n = 6-10 per group). PROTACs containing the conjugate are formulated in vehicle (e.g., 10% DMSO, 40% PEG400, 50% PBS) and administered intravenously or intraperitoneally at doses of 1-50 mg/kg, typically on a q.d. or q.o.d. schedule for 2-3 weeks. Tumor volumes are measured twice weekly using calipers. Body weights are monitored for toxicity. At study termination, tumors are excised and processed for Western blot analysis of target protein degradation, histopathology, and biomarker analysis.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of E3 Ligase Ligand-Linker Conjugates 20 TFA are studied as part of the complete PROTAC molecule incorporating this conjugate. The linker contributes to the overall pharmacokinetic profile. PROTACs containing this conjugate typically show favorable pharmacokinetic properties, including moderate to good oral bioavailability, reasonable plasma half-life, and adequate tissue distribution. The conjugate's composition (Thalidomide-O-amido-C8-NH2) is designed to provide optimal properties for PROTAC drug development. However, specific pharmacokinetic data for the conjugate alone or for PROTACs containing this specific conjugate are not extensively reported.
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| Toxicity/Toxicokinetics |
Toxicological data for E3 Ligase Ligand-Linker Conjugates 20 TFA are derived from toxicology studies of PROTACs containing this conjugate, rather than studies of the conjugate alone. The conjugate is considered a non-toxic component of PROTAC molecules, as it is a synthetic chemical that is typically metabolized and cleared. The Thalidomide-based ligand may have immunomodulatory effects, which could contribute to the pharmacology and toxicity of the resulting PROTACs. The safety and tolerability of PROTACs containing this conjugate are evaluated in animal toxicology studies. Parameters assessed include clinical observations, body weight, food consumption, clinical pathology, organ weights, and histopathology.
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| References | |
| Additional Infomation |
E3 Ligase Ligand-Linker Conjugates 20 TFA (CAS 1950635-16-1), also known as Thalidomide-O-amido-C8-NH2 TFA, is a synthesized compound used in PROTAC technology. It incorporates the Thalidomide-based cereblon ligand and a linker. The compound has a molecular formula of C₂₅H₃₁F₃N₄O₈ and a molecular weight of 572.53. It enables targeted protein degradation by hijacking the CRBN E3 ubiquitin ligase. The TFA salt improves solubility and stability. The compound is an essential building block for developing PROTAC molecules and is strictly for research use only.
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| Molecular Formula |
C25H31F3N4O8
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| Molecular Weight |
572.5308573246
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| Exact Mass |
572.209
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| CAS # |
1950635-16-1
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| PubChem CID |
121411067
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
40
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| Complexity |
841
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C(=O)O)(F)F.O(CC(NCCCCCCCCN)=O)C1=CC=CC2=C1C(N(C2=O)C1C(NC(CC1)=O)=O)=O
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| InChi Key |
AJVLNIUPDHKOPS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H30N4O6.C2HF3O2/c24-12-5-3-1-2-4-6-13-25-19(29)14-33-17-9-7-8-15-20(17)23(32)27(22(15)31)16-10-11-18(28)26-21(16)30;3-2(4,5)1(6)7/h7-9,16H,1-6,10-14,24H2,(H,25,29)(H,26,28,30);(H,6,7)
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| Chemical Name |
N-(8-aminooctyl)-2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxyacetamide;2,2,2-trifluoroacetic acid
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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 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)
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| Solubility (In Vitro) |
DMSO : ≥ 131 mg/mL (~228.81 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.37 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 (4.37 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 (4.37 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 | 1.7466 mL | 8.7332 mL | 17.4663 mL | |
| 5 mM | 0.3493 mL | 1.7466 mL | 3.4933 mL | |
| 10 mM | 0.1747 mL | 0.8733 mL | 1.7466 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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