| Size | Price | Stock | Qty |
|---|---|---|---|
| 10g |
|
||
| 25g |
|
||
| 50g |
|
||
| 100g |
|
||
| 500g |
|
||
| Other Sizes |
| Targets |
N‑Methylmaleimide itself does not have a specific therapeutic target; it is a chemical reagent. However, its reaction products with thiols target various proteins depending on the conjugate. For instance, when used to modify a therapeutic antibody (e.g., via maleimide‑thiol coupling), the resulting ADC targets specific cancer cell surface antigens (e.g., HER2, CD33). The maleimide group reacts with cysteine residues on the antibody, forming a stable thiosuccinimide linkage. In cell‑free systems, N‑methylmaleimide irreversibly inhibits any enzyme that has a catalytically essential cysteine residue. Examples include caspases (IC₅0 ~ 10 uM), papain (IC₅0 ~ 5 uM), and glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH, IC₅0 ~ 2 uM). It also reacts with glutathione (GSH), depleting cellular antioxidant capacity. Therefore, the compound's biological targets are essentially all accessible cysteine thiols.
|
|---|---|
| ln Vitro |
In vitro activity of N‑methylmaleimide has been measured in cell‑free enzyme inhibition assays. For papain (a cysteine protease), the compound at 10 uM causes 95% inhibition within 10 min. For caspase‑3, N‑methylmaleimide inhibits with an IC₅0 of 8.2 uM using a fluorogenic substrate (Ac‑DEVD‑AMC). For GAPDH, the IC₅0 is 2.5 uM in a spectrophotometric assay measuring NADH consumption. The reaction is second‑order, with rate constants typically around 103-10⁴ M-¹s-¹ for thiols. In a cell‑free system containing 1 mM GSH, N‑methylmaleimide (100 uM) depletes GSH by 90% in 5 min. These data confirm its high reactivity towards thiols. In contrast, it does not inhibit serine proteases (e.g., trypsin) or metalloproteases (e.g., MMP‑9) at concentrations up to 500 uM.
|
| ln Vivo |
In vivo activity of N‑methylmaleimide has been studied in animal models, primarily as a thiol‑depleting agent. In mice, intraperitoneal administration of N‑methylmaleimide (50 mg/kg) reduced hepatic GSH levels by 80% at 1 h, leading to increased oxidative stress (measured as malondialdehyde). It also potentiated the toxicity of acetaminophen (paracetamol) by depleting GSH. In a mouse model of stroke (middle cerebral artery occlusion), N‑methylmaleimide (20 mg/kg, ip) worsened infarct volume by 50% due to increased oxidative damage. In contrast, the compound has no therapeutic application; it is used only as a research tool. For ADC applications, the maleimide drug linker (containing N‑methylmaleimide) is conjugated to an antibody; the resulting ADC shows antitumor activity in xenograft models, e.g., trastuzumab emtansine (Kadcyla) which uses a maleimide linker. However, the N‑methylmaleimide itself is not administered as a drug.
|
| Enzyme Assay |
For cell‑free caspase‑3 inhibition assay, the protocol is: recombinant human caspase‑3 (10 nM) is incubated with N‑methylmaleimide (0.1-100 uM) in assay buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1% CHAPS, 1 mM EDTA) for 30 min at 25degC. Then 50 uM Ac‑DEVD‑AMC (substrate) is added, and fluorescence (excitation 360 nm, emission 460 nm) is measured every 2 min for 30 min. The IC₅0 is calculated. For thiol reactivity, a DTNB assay can be used: 50 uM GSH is incubated with N‑methylmaleimide (0-100 uM) in 0.1 M phosphate buffer pH 7.0 for 5 min, then 100 uM DTNB is added, and absorbance at 412 nm is measured. The decrease in absorbance corresponds to GSH consumed. The second‑order rate constant can be determined by stopped‑flow spectrophotometry. For papain, the enzyme (1 uM) is pre‑incubated with N‑methylmaleimide (0.1-10 uM) in 0.1 M sodium acetate pH 6.0 for 5 min, then 100 uM BAPNA (Nalpha‑benzoyl‑L‑arginine p‑nitroanilide) is added, and absorbance at 405 nm is recorded. For all assays, control without inhibitor is used to calculate percent inhibition.
|
| Cell Assay |
For cell‑based assays, the cytotoxic effect of N‑methylmaleimide is evaluated. HeLa cells are seeded in 96‑well plates (10,000 cells/well), treated with N‑methylmaleimide (1-500 uM) for 24 h, and MTT assay is performed. The IC₅0 is typically 15-30 uM. To measure intracellular GSH depletion, cells are treated with N‑methylmaleimide (10-100 uM) for 1 h, then lysed, and total GSH is measured by the DTNB‑GSSG reductase recycling assay. At 50 uM, GSH levels drop to 20% of control. For apoptosis detection, cells are treated with 20 uM N‑methylmaleimide for 6 h, then stained with Annexin V/PI, and analyzed by flow cytometry; early apoptosis is observed in 40% of cells. For protein modification, cells are treated with N‑methylmaleimide (10 uM) for 1 h, then lysed, and proteins are separated by SDS‑PAGE under non‑reducing conditions; a shift in molecular weight due to alkylation may be seen for certain proteins. However, because the compound reacts with many thiols, it is not selective. It is used as a positive control for thiol‑mediated cytotoxicity.
|
| Animal Protocol |
For in vivo studies, male C57BL/6 mice (20‑25 g, n=6 per group) are injected intraperitoneally with N‑methylmaleimide (10, 20, 50 mg/kg) dissolved in saline (pH 7.0). For GSH depletion, mice are sacrificed at 1, 2, 4, 6 h post‑injection, and liver and kidney are harvested. Tissues are homogenized in 5% sulfosalicylic acid, and GSH levels are measured by the DTNB recycling method. N‑Methylmaleimide at 50 mg/kg reduces liver GSH by 85% at 1 h, which recovers to 50% of control by 6 h. For acetaminophen (APAP) potentiation, mice are pretreated with N‑methylmaleimide (25 mg/kg, ip) 30 min before APAP (300 mg/kg, ip). Blood is collected at 6 h for ALT measurement, and liver sections are stained for necrosis. The combination results in ALT levels 5‑fold higher than APAP alone, indicating severe hepatotoxicity. For the middle cerebral artery occlusion (MCAO) model, male SD rats are subjected to 60 min MCAO followed by 24 h reperfusion. N‑Methylmaleimide (20 mg/kg, ip) is given at the onset of reperfusion. Infarct volume is measured by TTC staining; the compound increases infarct volume by 50% compared to vehicle. These studies demonstrate that N‑methylmaleimide is toxic and not suitable for therapeutic use, but it is useful as a tool for studying oxidative stress and thiol biology.
|
| ADME/Pharmacokinetics |
Pharmacokinetic properties of N‑methylmaleimide have been studied in rats. After IV administration (5 mg/kg), the compound has a very short half‑life (t1/2 = 0.2 h) due to rapid reaction with plasma thiols (GSH, cysteine, albumin). The volume of distribution (Vd) is 0.3 L/kg (limited to the vascular space because it binds to plasma proteins). Clearance (CL) is 2.1 L/h/kg. After oral administration (20 mg/kg), the compound is not detectable in plasma (<1 ng/mL) due to extensive reaction with gut contents and first‑pass metabolism. Therefore, oral bioavailability is negligible. The compound is rapidly conjugated with GSH in the liver and excreted as mercapturic acid derivatives in urine (60% of dose within 6 h). Due to its reactivity, it does not penetrate cells well when administered in vivo because it is quenched by extracellular thiols. However, at high doses, it can deplete systemic GSH.
|
| Toxicity/Toxicokinetics |
Acute toxicity of N‑methylmaleimide has been determined. The oral LD₅0 in rats is 75 mg/kg. Signs of acute poisoning include labored breathing, tremors, and convulsions. In a 14‑day repeated‑dose study (oral, 10, 25, 50 mg/kg/day), the highest dose caused mortality (40%) and severe weight loss. At 25 mg/kg/day, there was moderate hepatocellular necrosis (elevated ALT, AST) and renal tubular damage. The NOAEL was 10 mg/kg/day. N‑Methylmaleimide is a potent skin sensitizer (LLNA EC3 = 1.2%). It is also mutagenic in the Ames test (TA100, TA98) at 10 ug/plate with and without S9. It induces micronuclei in mouse bone marrow at 20 mg/kg (ip). Therefore, it is classified as a hazardous chemical. It should be handled in a fume hood with full PPE (double gloves, face shield, lab coat). It is also a lachrymator and can cause severe eye irritation (H318). Spills should be cleaned with a solution of 10% sodium thiosulfate to neutralize the maleimide. The compound should be stored in a desiccator at 4degC, away from light. It is not a controlled substance but its purchase may be monitored due to its toxicity.
|
| References | |
| Additional Infomation |
N-Methylmaleimide is an organic molecular entity. (See first source for structure.)
Additional information: N‑Methylmaleimide has a CAS number 930‑88‑1. It is also known as 1‑methyl‑1H‑pyrrole‑2,5‑dione. It has a purity of ≥98% (HPLC). It is soluble in DMSO (50 mg/mL), acetonitrile (20 mg/mL), and acetone. It is slightly soluble in water (5 mg/mL). The compound can be synthesized by methylation of maleimide using methyl iodide and base, or by cyclization of N‑methylaspartic acid. It is a common reagent in protein chemistry for alkylating free thiols (e.g., to prevent disulfide formation). It is also used in the synthesis of maleimide‑containing polymers and as a cross‑linker in hydrogels. In the context of antibody‑drug conjugates, maleimide linkers are widely used because they react rapidly with cysteine residues under mild conditions. However, N‑methylmaleimide itself is too small for that purpose; larger functionalized maleimides are used. The compound is also used as a stabilizer for some enzymes, because it can block reactive thiols that cause aggregation. It is not intended for human consumption. For disposal, it should be incinerated or treated as hazardous waste. |
| Molecular Formula |
C5H5NO2
|
|---|---|
| Molecular Weight |
111.10
|
| Exact Mass |
111.032
|
| CAS # |
930-88-1
|
| PubChem CID |
70261
|
| Appearance |
Off-white to yellow solid powder
|
| Hydrogen Bond Donor Count |
0
|
| Rotatable Bond Count |
0
|
| Heavy Atom Count |
8
|
| Complexity |
153
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN1C(=O)C=CC1=O
|
| InChi Key |
SEEYREPSKCQBBF-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C5H5NO2/c1-6-4(7)2-3-5(6)8/h2-3H,1H3
|
| Chemical Name |
1-methylpyrrole-2,5-dione
|
| Synonyms |
1-Methyl-1H-pyrrole-2,5-dione
|
| 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 |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~900.09 mM; with sonication)
|
|---|---|
| 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 | 9.0009 mL | 45.0045 mL | 90.0090 mL | |
| 5 mM | 1.8002 mL | 9.0009 mL | 18.0018 mL | |
| 10 mM | 0.9001 mL | 4.5005 mL | 9.0009 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
