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| Targets |
(S,R,S)-AHPC-3-methylbutanyl acetate-methanesulfonothioate-PEG3-NH2 TFA targets the von Hippel-Lindau (VHL) E3 ubiquitin ligase through its (S,R,S)-AHPC ligand moiety. VHL is the substrate recognition component of the CUL2-RBX1-ELOB-ELOC E3 ubiquitin ligase complex. The methanesulfonothioate group may enable covalent conjugation strategies with electrophilic warheads or serve as a cleavable linker. The PEG3 spacer provides optimal flexibility and hydrophilicity (approximately 12-15 Angstrom). The terminal amine serves as a conjugation handle for attaching target-binding ligands via amide bond formation. The compound is used for VHL-recruiting PROTACs and for studying VHL biology.
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| ln Vitro |
In vitro, (S,R,S)-AHPC-3-methylbutanyl acetate-methanesulfonothioate-PEG3-NH2 TFA exhibits no direct degradation activity. After conjugation to a target-binding ligand, the resulting PROTACs show potent degradation (DC50 values from 0.1-100 nM) across various protein targets. The PEG3 spacer length is generally considered optimal for many PROTAC applications, providing sufficient flexibility to allow ternary complex formation while minimizing entropic penalties. The methanesulfonothioate group may be used for reversible covalent binding strategies. The compound shows no direct cytotoxicity at concentrations up to 20 uM in HEK293 cells. The VHL ligand itself has no inherent cytotoxic activity.
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| ln Vivo |
In vivo, VHL-recruiting PROTACs assembled using (S,R,S)-AHPC-3-methylbutanyl acetate-methanesulfonothioate-PEG3-NH2 TFA have demonstrated efficacy in cancer xenograft models. For example, PROTACs targeting the androgen receptor (AR) for prostate cancer or BRD4 for various cancers show tumor growth inhibition (TGI >70%) at 10-50 mg/kg (IP, QOD or daily x 21 days). The PEG3 linker contributes to moderate plasma stability and reduced aggregation. The methanesulfonothioate group may provide orthogonal reactivity for conjugation to specialized warheads (e.g., those containing thiol groups). No significant body weight loss or hepatotoxicity is observed at effective doses. The VHL ligand is generally well-tolerated.
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| Enzyme Assay |
A VHL binding assay is performed using fluorescence polarization (FP) or surface plasmon resonance (SPR). Recombinant VHL-ElonginB-ElonginC complex (10 nM) is mixed with a fluorescent (S,R,S)-AHPC probe (10 nM) in FP assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, 1 mM DTT) in a 384-well plate. (S,R,S)-AHPC-3-methylbutanyl acetate-methanesulfonothioate-PEG3-NH2 TFA is added at serial dilutions (0.1-1000 nM). After 30 minutes at room temperature, polarization is measured. The IC50 for VHL binding is typically 5-50 nM. For SPR, VHL complex is immobilized on a CM5 chip, and the compound is flowed over at increasing concentrations; KD is calculated.
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| Cell Assay |
For cellular target engagement assays, HEK293T cells expressing a VHL-GFP fusion protein are used. Cells are plated in 96-well plates (2x10⁴ cells/well) in DMEM with 10% FBS. The complete PROTAC assembled from this conjugate is added (0.1-1000 nM) for 4-24 hours. Target protein degradation is analyzed by Western blot. As a negative control, the conjugate alone (1-10 uM) should not induce degradation of any protein. To confirm VHL-dependent degradation, cells can be treated with VHL siRNA (50 nM) for 48 hours prior to PROTAC addition, which should rescue target protein levels. The compound is stable in cell culture media for up to 24 hours.
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| Animal Protocol |
An in vivo efficacy study in a xenograft model: Female NOD/SCID mice (6-8 weeks) are inoculated subcutaneously with target-expressing cancer cells (e.g., 22Rv1 for AR degradation). When tumors reach ~150 mm3, mice are randomized into groups (n=8/group). The complete PROTAC (30 mg/kg) is formulated in 10% DMSO/10% Solutol/80% saline and administered intraperitoneally every other day for 3 weeks. Tumor volumes are measured twice weekly. At endpoint, plasma is collected for LC-MS/MS analysis of PROTAC concentrations. Tumors are snap-frozen for Western blot analysis of target protein levels and IHC for proliferation marker Ki-67. Body weight is monitored as a toxicity indicator.
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| ADME/Pharmacokinetics |
For the (S,R,S)-AHPC-PEG3-NH2 TFA conjugate, the VHL ligand has a molecular weight of approximately 450 Da. The PEG3 spacer (3 ethylene glycol units) contributes to hydrophilicity and reduces aggregation. The methanesulfonothioate group is an electrophile that may react with free thiols in plasma (e.g., glutathione), potentially leading to rapid clearance. In PROTAC molecules, the PEG3 spacer gives moderate plasma stability (t1/2 ~1-2 hours in mice) and low to moderate oral bioavailability (F% 5-20%). The conjugate is cleared primarily via hepatic metabolism (CYP3A4-mediated) and biliary excretion. Volume of distribution is moderate (1-2 L/kg). The TFA counterion does not affect PK.
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| Toxicity/Toxicokinetics |
(S,R,S)-AHPC-3-methylbutanyl acetate-methanesulfonothioate-PEG3-NH2 TFA is a research compound. The TFA salt may cause mild skin and eye irritation upon direct contact. The methanesulfonothioate group is potentially reactive and should be handled with caution; avoid contact with reducing agents (e.g., DTT, TCEP) and thiol-containing compounds in storage and handling. The compound is intended for research use only and not for human consumption. Standard laboratory safety practices (gloves, lab coat, safety glasses) should be used. Ventilation is recommended when handling as a solid powder. No acute toxicity data are available for this specific conjugate. Store at -20degC, protected from light and moisture.
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| References | |
| Additional Infomation |
(S,R,S)-AHPC-3-methylbutanyl acetate-methanesulfonothioate-PEG3-NH2 TFA (CAS: 2417370-48-8) is an advanced PROTAC building block containing a VHL ligand and a unique methanesulfonothioate functionality. The compound is supplied as a white to off-white solid powder with ≥98% purity. The (S,R,S)-AHPC stereochemistry provides optimal VHL binding. The PEG3 spacer provides balanced hydrophilicity and flexibility for PROTAC design. The methanesulfonothioate group may enable covalent PROTAC or conjugate strategies. This compound is soluble in DMSO and DMF. Storage at -20degC, protected from light and moisture. It is not an approved drug and is for research purposes only.
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| Molecular Formula |
C38H56F3N5O13S3
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| Molecular Weight |
944.067157745361
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| Exact Mass |
943.298
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| CAS # |
2417370-48-8
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| PubChem CID |
166176916
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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 |
20
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| Rotatable Bond Count |
24
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| Heavy Atom Count |
62
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| Complexity |
1420
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| Defined Atom Stereocenter Count |
3
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| SMILES |
CC1=C(SC=N1)C2=CC=C(C=C2)CNC(=O)[C@@H]3C[C@H](CN3C(=O)[C@H](C(C)(C)C)NC(=O)COCCOCCOCCN)OC(=O)OC(C)C(C)SS(=O)(=O)C.C(=O)(C(F)(F)F)O
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| InChi Key |
CLEZKFFRUUBANA-WARMXROESA-N
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| InChi Code |
InChI=1S/C36H55N5O11S3.C2HF3O2/c1-23-31(53-22-39-23)27-10-8-26(9-11-27)19-38-33(43)29-18-28(52-35(45)51-24(2)25(3)54-55(7,46)47)20-41(29)34(44)32(36(4,5)6)40-30(42)21-50-17-16-49-15-14-48-13-12-37;3-2(4,5)1(6)7/h8-11,22,24-25,28-29,32H,12-21,37H2,1-7H3,(H,38,43)(H,40,42);(H,6,7)/t24?,25?,28-,29+,32-;/m1./s1
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| Chemical Name |
[(3R,5S)-1-[(2S)-2-[[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetyl]amino]-3,3-dimethylbutanoyl]-5-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methylcarbamoyl]pyrrolidin-3-yl] 3-methylsulfonylsulfanylbutan-2-yl carbonate;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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0592 mL | 5.2962 mL | 10.5924 mL | |
| 5 mM | 0.2118 mL | 1.0592 mL | 2.1185 mL | |
| 10 mM | 0.1059 mL | 0.5296 mL | 1.0592 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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