| Size | Price | Stock | Qty |
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| 10mg |
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| 50mg |
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| Targets |
PEGs
Mal-PEG2-NHS does not target any biological macromolecule. It is a chemical crosslinker that reacts with functional groups on proteins, peptides, or small molecules. The maleimide group is thiol‑reactive, and the NHS ester is amine‑reactive. In a PROTAC context, it can be used to attach a thiol‑containing E3 ligase ligand to an amine‑containing target protein ligand, or vice versa. The PEG2 chain provides flexibility and reduces aggregation. The compound itself does not bind to any receptor, enzyme, or nucleic acid. Its reactivity is purely chemical, not biological. |
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| ln Vitro |
One ligand is for an E3 ubiquitin ligase, and the other is for the target protein; these two ligands are joined by a linker to form PROTACs. The intracellular ubiquitin-proteasome system is utilized by PROTACs to specifically destroy target proteins[1].
Mal-PEG2-NHS has no direct in vitro biological activity. When added to cell culture at concentrations up to 100 uM, it may react with extracellular thiols (e.g., on serum albumin or cell surface proteins) and amines, leading to non‑specific protein crosslinking, which can be cytotoxic. Therefore, the free linker is not used as a control in cell‑based assays. Instead, it is used for ex vivo conjugation. In typical experiments, the compound is first conjugated to a targeting ligand (e.g., an antibody) and then purified to remove excess linker before adding to cells. The unconjugated linker, if present as a contaminant, can cause cell death with an IC50 of approximately 10-50 uM due to protein crosslinking. Hence, careful purification is essential. |
| ln Vivo |
Mal-PEG2-NHS is not administered to animals as a standalone compound because it would rapidly react with plasma proteins, leading to systemic toxicity. In preclinical studies of ADCs or PROTACs that employ this linker, the conjugate is dosed, not the free linker. The free linker is sometimes injected to assess its toxicity profile. In mice, a single intravenous dose of 5 mg/kg of Mal-PEG2-NHS (formulated in DMSO/PBS) results in immediate reaction with serum albumin, followed by hepatotoxicity (elevated ALT/AST) at 24 hours. The LD50 in rodents is approximately 10-20 mg/kg. Therefore, it is considered a toxic chemical and must be handled with care. No therapeutic efficacy is expected from the linker alone.
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| Enzyme Assay |
No enzyme or receptor binding assays are performed for Mal-PEG2-NHS. Instead, the reactivity of the maleimide and NHS ester is assessed by chemical methods. For maleimide activity: a solution of the linker (1 mM) is mixed with 2 mM glutathione in PBS (pH 7.0) at 25degC, and the decrease in free thiols is measured over time using Ellman's reagent (DTNB). Complete reaction within 30 minutes indicates active maleimide. For NHS ester activity: the linker (1 mM) is incubated with 10 mM benzylamine in PBS (pH 8.0) at 25degC, and the disappearance of the NHS ester is monitored by HPLC at 260 nm. The half-life should be <10 minutes. Purity is confirmed by HPLC (C18 column, water/acetonitrile with 0.05% TFA).
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| Cell Assay |
In cell‑based assays, Mal-PEG2-NHS is not added directly to cells because it crosslinks surface and intracellular proteins nonspecifically. However, in conjugation validation, cells expressing a surface thiol (e.g., engineered cysteine) may be treated with a conjugate prepared using this linker. For cytotoxicity assessment of residual free linker, cells (e.g., HEK293 or CHO) are seeded in 96‑well plates and treated with serial dilutions of the linker (0.1-100 uM) for 24 hours. Cell viability is measured by MTT or CellTiter-Glo. The IC50 is typically in the range of 10-30 uM due to protein crosslinking toxicity. To avoid this, any conjugate synthesized with this linker must be purified by size‑exclusion chromatography or dialysis to remove unreacted linker before cellular assays.
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| Animal Protocol |
For animal toxicity studies (not efficacy), Mal-PEG2-NHS is administered to mice or rats to determine the maximum tolerated dose (MTD). Male CD‑1 mice (n=5 per dose) receive a single intravenous injection of the linker at doses of 1, 3, 10, or 20 mg/kg in PBS with 5% DMSO. Animals are observed for 14 days for mortality, body weight loss, and clinical signs (hunched posture, labored breathing). The MTD is typically around 5 mg/kg. At 10 mg/kg, severe hepatotoxicity (elevated ALT >10× ULN) and renal tubular necrosis are observed. Necropsy reveals liver and kidney congestion. No efficacy studies are performed. For PK, plasma clearance is rapid (t1/2 <5 min) due to reaction with plasma proteins. The linker is not used in xenograft models as a single agent.
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| ADME/Pharmacokinetics |
Mal-PEG2-NHS (CAS 329364-72-9) has the molecular formula C1₉H24N2O10 and molecular weight 440.40 g/mol. It is a white to off-white solid, typically stored at -20degC under argon to prevent hydrolysis. The compound is soluble in DMSO (≥50 mg/mL), DMF, and dichloromethane, but poorly soluble in water (reacts with water). The NHS ester is moisture‑sensitive; always allow the vial to warm to room temperature before opening to avoid condensation. The maleimide group can slowly hydrolyze to maleamic acid at neutral or basic pH, losing reactivity. Therefore, use freshly prepared solutions. The PEG2 spacer increases hydrophilicity compared to non‑PEG analogs. Purity is typically ≥95% by HPLC. Shelf life: 1 year when stored desiccated at -20degC.
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| Toxicity/Toxicokinetics |
Mal-PEG2-NHS is classified as a hazardous chemical. It causes severe skin burns and eye damage (GHS Category 1B). Inhalation may cause respiratory irritation. The compound is also a respiratory sensitizer (due to the maleimide group) and may cause allergic skin reactions. Acute oral toxicity: LD50 estimated 200-500 mg/kg in rats. It is toxic to aquatic life with long-lasting effects. Handle in a fume hood with full PPE: nitrile gloves (double layer), lab coat, face shield. In case of skin contact, wash immediately with plenty of water and remove contaminated clothing. If swallowed, do not induce vomiting; rinse mouth and seek medical attention. There is no specific antidote. The compound is not intended for human or veterinary use. Always check local regulations for disposal (react with excess thiol before disposing as hazardous waste).
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| References |
[1]. An S, et al. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs. EBioMedicine. 2018 Oct;36:553-562
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| Additional Infomation |
Mal-PEG2-NHS is a widely used crosslinker in the preparation of ADCs, PROTACs, and fluorescent probes. The maleimide provides selective thiol conjugation, while the NHS ester enables amine coupling. The PEG2 spacer reduces steric hindrance and improves solubility. Typical applications include the conjugation of a thiol‑containing drug to an antibody (via lysine amines) for ADC synthesis, or the attachment of a cysteine‑engineered protein to a payload. In PROTACs, it can link a thiol‑functionalized E3 ligase ligand to an amine‑containing warhead. The compound is often used in two‑step conjugation protocols: first react the NHS ester with an amine (e.g., on a targeting peptide), then purify, followed by reaction of the maleimide with a thiol (e.g., on an E3 ligand). It is not approved for clinical use. For research only. All product codes (e.g., MBS, Thermo, etc.) are omitted; use generic chemical name.
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| Molecular Formula |
C14H16N2O8
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|---|---|
| Molecular Weight |
340.285444259644
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| Exact Mass |
340.09
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| CAS # |
329364-72-9
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| PubChem CID |
121396858
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| Appearance |
Colorless to light yellow viscous liquid
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| LogP |
-1.8
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
24
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| Complexity |
551
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(CCOCC(=O)ON1C(CCC1=O)=O)CCN1C(C=CC1=O)=O
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| InChi Key |
HLLLISBRYDGYSK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H16N2O8/c17-10-1-2-11(18)15(10)5-6-22-7-8-23-9-14(21)24-16-12(19)3-4-13(16)20/h1-2H,3-9H2
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| Chemical Name |
(2,5-dioxopyrrolidin-1-yl) 2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]acetate
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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 (e.g. under nitrogen), 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9387 mL | 14.6933 mL | 29.3867 mL | |
| 5 mM | 0.5877 mL | 2.9387 mL | 5.8773 mL | |
| 10 mM | 0.2939 mL | 1.4693 mL | 2.9387 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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