1-{6-[(2,5-Dioxopyrrolidin-1-Yl)Oxy]-6-Oxohexyl}-1H-Pyrrole-2,5-Dione
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HS Code |
990669 |
| Chemical Name | 1-{6-[(2,5-Dioxopyrrolidin-1-yl)Oxy]-6-Oxohexyl}-1H-Pyrrole-2,5-Dione |
As an accredited 1-{6-[(2,5-Dioxopyrrolidin-1-Yl)Oxy]-6-Oxohexyl}-1H-Pyrrole-2,5-Dione factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of 1-{6-[(2,5 - Dioxopyrrolidin - 1 - Yl)Oxy]-6 - Oxohexyl}-1H - Pyrrole - 2,5 - Dione in sealed chemical - grade packaging. |
| Shipping | Ship the chemical 1-{6-[(2,5 - Dioxopyrrolidin - 1 - Yl)Oxy]-6 - Oxohexyl}-1H - Pyrrole - 2,5 - Dione in well - sealed containers, following hazardous chemical shipping regulations. Ensure proper labeling and handling to prevent spills and damage during transit. |
| Storage | Store "1-{6-[(2,5 - Dioxopyrrolidin - 1 - Yl)Oxy]-6 - Oxohexyl}-1H - Pyrrole - 2,5 - Dione" in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially cause degradation. Avoid storing near heat sources or reactive chemicals. |
Controlled reduction of interchain disulfide bonds on a monoclonal antibody (mAb) IgG1 using 2–4 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline containing 5 mM EDTA at pH 7.4 and 25 °C for 2 h generates an average of 4–8 free sulfhydryl groups per antibody molecule. The heterobifunctional crosslinker 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (6-maleimidohexanoic acid N-hydroxysuccinimide ester) is first conjugated to an amine-containing cytotoxin—typically a maytansinoid derivative functionalized with a primary aliphatic amine—in anhydrous N,N-dimethylformamide containing 1.5 equivalents of N,N-diisopropylethylamine, using a linker-to-toxin molar ratio of 1.2–1.5:1. The reaction proceeds at 25 °C under argon for 1 h, after which the toxin–maleimide intermediate is purified by preparative reversed-phase HPLC on a C18 column with an acetonitrile/water gradient containing 0.1 % (v/v) trifluoroacetic acid. This dried intermediate is reconstituted in dimethylacetamide and added to the reduced antibody solution at a linker-toxin:antibody molar ratio of 5–10:1 in phosphate-buffered saline at pH 7.2 containing 10 % (v/v) N,N-dimethylformamide, and conjugation is allowed to proceed at 25 °C for 30–60 min. Unreacted maleimide groups are quenched with a 10-fold molar excess of N-ethylmaleimide, and the antibody–drug conjugate (ADC) is purified by tangential flow filtration on a 100 kDa MWCO regenerated cellulose membrane followed by size-exclusion chromatography (Superdex 200 prep grade). The mean drug-to-antibody ratio (DAR) is determined by hydrophobic interaction chromatography coupled with absorbance detection at 252 nm and 280 nm; the process is typically tuned to deliver a DAR of 3.2–4.0. The terminal product is a sterile lyophilized powder for injection, an antineoplastic immunoconjugate. Manufacturing must conform to ICH Q7 Good Manufacturing Practice for active pharmaceutical ingredients, FDA 21 CFR Part 211 for finished pharmaceuticals, and the stability testing framework of ICH Q5C. Because the maleimide ring undergoes irreversible ring-opening hydrolysis at pH >7.5, the conjugation and all subsequent processing steps are executed under mildly acidic conditions (pH 6.5–7.2) and with strict time control; published kinetic data (Hermanson, Bioconjugate Techniques, 3rd ed.) indicate a maleimide half-life of merely 8–12 h in 50 mM phosphate at pH 7.4 and 25 °C, dropping to 2–4 h at pH 8.3. This sensitivity constitutes the primary processing bottleneck that distinguishes 6-maleimidohexanoic acid NHS ester from hydrolysis-resistant maleimide analogs such as the phenyloxadiazole-methyl sulfone class, yet its C6 aliphatic spacer confers sufficient rotational freedom for efficient interchain crosslinking, a property that remains critical for achieving high-yield ADC constructs.
How Is Covalent Antibody Orientation Achieved on Superparamagnetic Microparticles?Superparamagnetic microparticles with a polyurethane surface layer presenting primary amines (typical particle diameter 2.8 µm, ∼1×10⁹ particles per mL) are washed and suspended in 50 mM MES, 150 mM NaCl, pH 6.0. A stock solution of 6-maleimidohexanoic acid NHS ester in anhydrous dimethyl sulfoxide (50 mg/mL) is added at a level of 0.05–0.2 mg of linker per milligram of microparticles, corresponding to a reactive NHS ester loading of approximately 0.15–0.6 µmol (mg beads)⁻¹. End-over-end mixing at 25 °C for 30 min yields maleimide-activated beads, which are separated magnetically and rinsed with MES buffer. The capture antibody is first reduced in 20 mM dithiothreitol in PBS-EDTA (pH 7.4) at 37 °C for 30 min to expose 2–4 hinge-region sulfhydryls per IgG molecule, then desalted into 50 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA, pH 7.2. The reduced antibody is incubated with the maleimide-functionalized beads at a ratio of 5–10 µg antibody per 10⁷ beads overnight at 4 °C. Residual maleimide sites are blocked with 1 mM cysteine (30 min), and the bead–antibody conjugate is washed and stored in PBS containing 0.1 % bovine serum albumin and 0.02 % sodium azide. Quality control metrics include antibody loading determined by bicinchoninic acid assay and residual maleimide quantification via Ellman’s reagent. Production of such immunomagnetic reagents for clinical diagnostics falls under ISO 13485:2016 and, for the European Union, Regulation (EU) 2017/746 (IVDR). The finished products are utilized as cell-enrichment magnetic beads, immunoprecipitation supports, and bacterial capture kits. Post-Insertion Maleimide Functionalization of Preformed Liposomes via NHS Ester-Amine CouplingUnilamellar liposomes composed of hydrogenated soy phosphatidylcholine, cholesterol (molar ratio 55:45), and 2 mol% distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-amine are prepared by thin-film hydration and extruded through 100 nm polycarbonate membranes at 60 °C. The liposome suspension (total lipid concentration 50 mM) is equilibrated in 10 mM HEPES, 150 mM NaCl, pH 7.5. A dimethylformamide solution of the heterobifunctional linker (50 mg/mL) is added dropwise under stirring to deliver a 5:1 molar excess of NHS ester over the amine-presenting phospholipid; the final organic solvent content is kept below 2 % (v/v). After 1 h at 25 °C in the dark, the mixture is passed through a Sephadex G-50 column pre-equilibrated with 10 mM HEPES, 150 mM NaCl, 5 mM EDTA, pH 6.5 to remove unincorporated linker while retarding maleimide hydrolysis. A cysteine-terminated targeting ligand—e.g., cyclic RGD peptide with a C-terminal spacer cysteine—is then introduced at a 2-fold molar excess relative to the calculated maleimide surface density, and coupling is allowed to proceed for 12 h at 4 °C. The targeted liposomes are sterile-filtered through a 0.22 µm membrane and characterized by dynamic light scattering for hydrodynamic diameter and zeta potential. Regulatory oversight for parenteral liposomal formulations invokes the FDA draft guidance on liposome drug products (2018) and USP ‹797› for sterile compounding; cytotoxic payloads such as doxorubicin additionally require stability evaluation under ICH Q1A. The terminal dosage form can be a ready-to-inject liposomal dispersion or a lyophilizate for reconstitution, providing prolonged vascular circulation and receptor-mediated tumor internalization. Oriented immobilization of capture proteins on surface plasmon resonance (SPR) chips typically exploits chelation of polyhistidine tags, yet many engineered binding fragments are now produced with a single C-terminal cysteine to permit uniform, site-specific attachment. A carboxymethylated dextran-coated gold sensor substrate is first activated by injection of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 100 mM N-hydroxysuccinimide, followed by 1 M ethylenediamine, generating an amine-terminated surface. The sensor slide is then incubated in a 10 mM solution of 6-maleimidohexanoic acid NHS ester in anhydrous dimethylformamide for 1 h at 25 °C, rinsed sequentially with dimethylformamide and deionized water, and dried under a nitrogen stream. The maleimide-activated surface is stable for several hours at 4 °C when desiccated. A recombinantly produced Fab fragment bearing a free C-terminal cysteine is dissolved at 50 µg/mL in 10 mM sodium acetate buffer, pH 5.0, and injected over the chip for 7 min at a flow rate of 10 µL min⁻¹. Real-time monitoring reveals a typical immobilization level of 2000–4000 RU; non-specific binding sites are subsequently blocked with 1 M ethanolamine (pH 8.5). The fabrication procedure adheres to ISO 10993-5:2009 for in vitro cytotoxicity when the functionalized chip is intended for implantable or invasive diagnostic systems, and may fall under the European IVD Regulation if integrated into a point-of-care platform. The resulting product is a dedicated SPR sensor chip configured for kinetic interaction screening on instruments requiring pre-immobilized ligand surfaces.When Native Lysine Residues Must Serve as the PEGylation AnchorA therapeutic protein, exemplified by a recombinant cytokine, is formulated at 5 mg/mL in 50 mM sodium phosphate, 150 mM NaCl, pH 7.2. The NHS ester crosslinker is introduced from a 100 mM stock in dimethyl sulfoxide at a 15–25-fold molar excess with respect to the protein, keeping the organic co-solvent concentration below 2 %. The mixture is held at 25 °C for 30 min, yielding an average incorporation of 2–4 maleimide groups per protein molecule as verified by electrospray ionization mass spectrometry. Excess low-molecular-weight reagent is removed by desalting on a PD-10 column equilibrated with 50 mM phosphate, 5 mM EDTA, pH 6.5. Methoxy-poly(ethylene glycol)-thiol (linear, 20 kDa) is subsequently added at a 2:1 molar ratio relative to the maleimide content, and the reaction is agitated gently at 4 °C for 16 h. The conjugation mixture is resolved by cation-exchange chromatography on SP Sepharose HP using a linear sodium chloride gradient to isolate the mono-PEGylated fraction. Specification setting follows ICH Q6B, and the drug substance is evaluated for purity and conjugation degree according to an adapted protocol drawn from Ph. Eur. monograph 2.6.21. The purified conjugate is formulated as a sterile subcutaneous injection, providing extended circulatory half-life and attenuated immunogenicity relative to the native polypeptide. Addressing NHS Ester Hydrolysis During Polysaccharide Activation at Alkaline pHHigh-molecular-weight hyaluronic acid (sodium salt, 200 kDa) is partially aminated by reaction with 1,3-diaminopropane via carbodiimide chemistry to achieve a free amine content of 80–120 µmol g⁻¹ polymer. The amino-functionalized biopolymer is dissolved at 10 mg/mL in 50 mM MES, pH 6.0, and the maleimide NHS ester is added at a 2:1 molar ratio with respect to the pendant amine groups. After 1 h at 25 °C, the maleimide-substituted hyaluronic acid is precipitated in chilled ethanol, redissolved in degassed water, and lyophilized. In parallel, native hyaluronic acid is thiolated using cysteine and catalytic 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to a free sulfhydryl content of 50–80 µmol g⁻¹. The two modified polymers are each reconstituted in 50 mM sodium phosphate, 5 mM EDTA, pH 7.0 at 20 mg/mL, mixed in a dual-barrel syringe, and extruded into a mold where covalent maleimide-thiol gelation occurs within 2–5 min at 37 °C. The resulting transparent hydrogel must pass ISO 10993-5 cytotoxicity testing on L-929 fibroblasts (extract dilution method) and meet the characterization requirements of ASTM F2150-19 for bio-derived tissue scaffolds. The finished construct is supplied as a pre-crosslinked hydrogel sheet or a sterile injectable filler for soft tissue augmentation and three-dimensional cell encapsulation.
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| Parameter | Method | Specification |
|---|---|---|
| Appearance | Visual inspection | White to off-white crystalline powder |
| Purity (HPLC) | C18, gradient MeCN/H2O + 0.1% TFA, UV 220 nm | ≥98.5 area% |
| Melting point | DSC, onset | 85–88°C |
| Moisture content | Karl Fischer coulometry | ≤0.5% w/w |
| Residual solvent (EtOAc) | Headspace GC-FID | ≤200 ppm |
| Heavy metals | ICP-MS | ≤10 ppm each |
| Storage condition | — | −20°C, under dry argon, desiccated |
| Crosslinker | Spacer arm (Å) | MW (g·mol−1) | Aqueous solubility | Maleimide t1/2 (pH 7.0, 25°C) |
|---|---|---|---|---|
| 6-Maleimidohexanoic acid NHS ester | 9.4 | 308.3 | Insoluble | 2.5 h |
| SMCC | 8.3 | 334.3 | Insoluble | 6.5 h |
| Sulfo-SMCC | 8.3 | 436.4 | > 50 mg·mL−1 | 6.5 h |
| MBS (m-maleimidobenzoyl-NHS) | 7.3 | 314.3 | Insoluble | 1.8 h |
