1-(3-{[(2,5-Dioxopyrrolidin-1-Yl)Oxy]Carbonyl}Phenyl)-1H-Pyrrole-2,5-Dione
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HS Code |
415622 |
| Chemical Formula | C16H8N2O6 |
| Molar Mass | 324.25 g/mol |
| Appearance | Solid (likely, based on similar compounds) |
| Solubility In Water | Low (due to non - polar and large organic structure) |
| Solubility In Organic Solvents | Soluble in common organic solvents like dichloromethane, chloroform (predicted from structure) |
| Stability | Stable under normal conditions (but may be reactive to strong oxidizing or reducing agents) |
As an accredited 1-(3-{[(2,5-Dioxopyrrolidin-1-Yl)Oxy]Carbonyl}Phenyl)-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-(3-{[(2,5 - Dioxopyrrolidin - 1 - Yl)Oxy]Carbonyl}Phenyl)-1H - Pyrrole - 2,5 - Dione in sealed vial. |
| Shipping | The chemical 1-(3-{[(2,5 - Dioxopyrrolidin - 1 - Yl)Oxy]Carbonyl}Phenyl)-1H - Pyrrole - 2,5 - Dione is shipped in sealed, corrosion - resistant containers. Special handling per safety protocols for chemicals is ensured during transportation to prevent spills and ensure safe delivery. |
| Storage | Store 1-(3-{[(2,5 - Dioxopyrrolidin - 1 - Yl)Oxy]Carbonyl}Phenyl)-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 degrade the chemical. Store it separately from incompatible substances to avoid reactions. |
In-site-specific bioconjugation protocols for maytansinoid-bearing antibody drug conjugates, the heterobifunctional linker 1-(3-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-1H-pyrrole-2,5-dione is employed to install a maleimido-functionalized intermediate on the immunoglobulin surface prior to thiol-based payload attachment. A typical reaction sequence initiates with an amine-reactive NHS ester acylation of accessible ε-amino groups on lysine residues of a humanized IgG1 monoclonal antibody at a stoichiometric feed ratio of 1:6 to 1:12 (antibody:linker) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. The conjugation is performed on a preparative scale using a tangential flow filtration system equipped with a 30 kDa MWCO polyethersulfone membrane to remove unreacted linker and NHS byproduct; the permeate is monitored for UV absorbance at 280 nm until baseline recovery. Excess linker is critical because maleimide hydrolysis to maleamic acid accelerates at this pH, with a half-life of approximately 8–12 h at 22 °C. Following buffer exchange into 10 mM sodium phosphate, 1 mM EDTA, pH 6.0, the purified linker-modified antibody is immediately reacted with a thiol-containing cytotoxic warhead, such as DM1 or MMAE, at a molar ratio of 1:1.2 (maleimide:thiol) for 90 min at ambient temperature. Quenching with 10 mM L-cysteine for 15 min eliminates residual maleimide sites. The average drug-to-antibody ratio is determined via hydrophobic interaction chromatography monitored at 248 nm and validated by liquid chromatography–mass spectrometry analysis of the intact conjugate. The process is conducted under GMP conditions aligned with ICH Q6B guidelines for chemical characterization and 21 CFR 211 for finished pharmaceuticals. Product storage at -80 °C in 20 mM histidine, 6 % trehalose, pH 5.8 limits deconjugation and aggregation. Operational boundaries include the requirement that the linker solution be prepared fresh in anhydrous DMSO (<50 µL per mL conjugation) immediately before use, as the NHS ester hydrolyses rapidly in aqueous buffers with a half-life of less than 5 min at pH 7.4. The aromatic benzamide spacer introduces minimal immunogenicity, but the short linker length between antibody and payload can restrict payload release into hydrophobic pockets, a factor evaluated in potency assays compliant with ICH S6. The resulting ADC product, when formulated, demonstrates target-dependent cytotoxicity against tumor cell lines at picomolar concentrations.What governs maleimide ring opening during antibody immobilization on lateral flow immunoassay nitrocellulose membranes?Immobilization of capture antibodies on activated polyester-backed nitrocellulose membranes for point-of-care troponin detection requires careful control of maleimide reactivity and competing hydrolysis. The NHS ester moiety of the compound is first used to graft heterofunctional handles onto carboxylated polystyrene latex particles, which are subsequently dispensed onto the conjugate pad. Latex particles (120 nm diameter, carboxyl-functionalized, surface charge density –45 µeq/g) are activated with EDC (2 mM) and sulfo-NHS (5 mM) in 50 mM MES, pH 5.5, for 20 min, then aminated by reaction with ethylenediamine to introduce terminal –NH₂ groups. After washing, the particles are resuspended in 0.1 M sodium borate, pH 8.0, and the heterobifunctional linker is added at a mass ratio of 1:0.5 (particles:linker). The NHS ester acylates the particle-tethered amines within 30 min, yielding a maleimide-decorated latex. Because maleimide hydrolysis has a reported activation energy of 48 kJ/mol, the entire reaction is performed at 4 °C, which retards ring opening and extends the functional maleimide lifetime beyond 24 h. The particle dispersion is then conjugated with an anti-cardiac troponin I monoclonal Fab' fragment that exposes a single hinge-region free cysteine via gentle reduction with 2 mM tris(2-carboxyethyl)phosphine at pH 7.0. Conjugation efficiency, monitored by dynamic light scattering and zeta potential shift (from –32 mV to –18 mV), routinely exceeds 85 % thiol-reactive coupling. The conjugated particles are striped onto glass fiber conjugate pads using automatic dispensers (BioDot XYZ platform) operating at a volumetric deposition of 0.8 µL/cm. Assembled lateral flow strips demonstrate a visual limit of detection of 0.01 ng/mL cTnI when paired with a gold nanoparticle signal label, evaluated according to CLSI EP17-A2 protocols. Regulatory compliance for in vitro diagnostic medical devices mandates adherence to ISO 13485:2016 quality management and ISO 23640:2021 for stability testing of in vitro diagnostic reagents. A documented processing bottleneck arises from batch-to-batch variation in maleimide surface density, which shifts the capture efficiency and must be controlled to ±7 % relative standard deviation by titrating linker input using a fluorometric fluorescamine assay. This manufacturing step is incompatible with phosphate-buffered saline above pH 7.2; intermediate washing steps employ 10 mM citrate, pH 6.2.
Thermoresponsive hydrogel injectable scaffolds for intervertebral disc repairWhen formulating injectable poly(NIPAM-co-glycidyl methacrylate) hydrogels capable of sol–gel transition at 33 °C, the compound serves as a covalent crosslinker between amine-tethered hyaluronic acid and cysteine-terminated PNIPAM chains. The hyaluronic acid backbone (Mw 740 kDa) is first aminolyzed with ethylenediamine via CDMT-mediated conjugation to yield a substitution degree of 15–20 mol% free amines. A solution of the heterobifunctional linker in dry DMF (10 mg/mL) is added dropwise to the hyaluronic acid solution in 50 mM HEPES, pH 7.6, at a linker-to-amine molar ratio of 0.6:1 to install pendant maleimide groups while avoiding cross-gelation. Unreacted NHS ester is quenched with 5 mM hydroxylamine. Separately, PNIPAM (Mn 22,000 g/mol) end-terminated with a free cysteine by RAFT polymerization using a cysteamine chain transfer agent is dissolved in cold PBS at 4 wt%. Mixing the two modified polymers at a volume ratio of 1:1 and at a thiol:maleimide stoichiometry of 1.05:1 triggers gel formation within 120 s at 37 °C. Oscillatory rheometry (AR-G2, cone-plate geometry, 40 mm diameter, 1° cone angle) reveals a storage modulus G' of 8.2 kPa at 1 Hz, which is sufficient to mimic the mechanical characteristics of the nucleus pulposus. The gel network is stabilized against rapid oxidative degradation by incorporating 1 mM methionine as a sacrificial oxidant during injection. Cytocompatibility evaluations according to ISO 10993-5:2009, using human mesenchymal stem cells in direct contact, confirm greater than 90 % viability by MTT assay at 24 h. The final injectable product is sterilized via 0.22 µm aseptic filtration of the cold precursor solutions and packaged in dual-barrel syringes with static mixing tips, complying with USP <797> for compounded sterile preparations. A critical process limit is the moisture content of the DMF solution: at water contents exceeding 0.1 %, the NHS ester hydrolyzes prematurely, reducing maleimide functionality below the gelation threshold and yielding non-elastic pastes.When photolithographic patterning requires covalent anchoring of cyclic RGD peptides on cyclic olefin copolymerMicropatterning of cell-adhesive peptides on cycloolefin copolymer slides for microfluidic cell culture devices utilizes this compound to achieve photo-triggered covalent linkage through a two-step sequence. First, the COC surface (188 µm thickness, Tg 78 °C) is activated by oxygen plasma treatment (40 W, 0.5 mbar, 180 s) to generate surface –OH and –COOH groups, confirmed by a water contact angle decrease from 89° to 28°. Amination is performed by immersing the treated slides in a 10 % (v/v) solution of 3-aminopropyltriethoxysilane in anhydrous toluene at 70 °C for 4 h. Following rinsing and curing at 110 °C for 30 min, a thin film of the heterobifunctional linker in 200 µL acetonitrile (2 mM, containing 0.1 % triethylamine) is applied via spin coating and left to react for 45 min at room temperature. The maleimide-terminated surface is then covered with a photomask and exposed to patterned UV light (365 nm, 45 mJ/cm²) in the presence of a photoinitiator-free thiol–ene solution containing thiolated cyclo-(RGDfK) peptide (50 µg/mL in 10 mM phosphate, pH 6.5). Thiol–maleimide click reaction occurs selectively in the irradiated regions, producing peptide features with 5 µm lateral resolution as verified by scanning electron microscopy and immunofluorescence staining with anti-RGD antibodies. Unbound peptide is removed by sonication in 0.1 % SDS for 10 min. Cell adhesion assays with human umbilical vein endothelial cells show focal adhesion formation limited strictly to patterned areas, exhibiting a 6-fold higher cell density than background unmodified regions. The patterned substrates are tested in accordance with ISO 10993-5 and exhibit no extractable cytotoxicity. The surface grafting density, quantified by X-ray photoelectron spectroscopy using the N 1s peak area, averages 5.8 ± 0.4 pmol/cm² maleimide groups. A notable failure mode is the radical-mediated maleimide homopolymerization if oxygen inhibition is not adequately controlled during UV exposure; hence the chamber is purged with argon (99.999 %) for 15 min prior to irradiation. This approach is utilized in the manufacture of single-use microphysiological system cartridges compliant with USP <88> Biological Reactivity Tests.For oriented ligand coupling in Protein A affinity resin preparation, agarose-based matrices (highly crosslinked 4 % beaded agarose, particle size 50–150 µm) are first derivatized with cysteamine to introduce pendant thiol groups through a divinyl sulfone activation route. The activated resin is then directly reslurried in 0.1 M sodium borate, 10 mM EDTA, pH 8.5, and the heterobifunctional linker is added at a concentration of 2 mM (approximately 5 mg linker per ml of drained resin). The NHS ester reacts with thiol groups on the resin with a conversion efficiency of 70–80 % in 60 min, yielding amine-reactive NHS moieties displayed on the solid support. Following a rapid wash with dry isopropanol to remove labile NHS esters, the resin is transferred to a coupling buffer of 0.2 M sodium phosphate, 0.5 M NaCl, pH 7.0, and recombinant Protein A (ligand concentration 12 mg/mL) is added. The ligand is immobilized predominantly through its N-terminal amine, preserving C-terminal immunoglobulin-binding domains in an oriented fashion. The dynamic binding capacity of the resulting resin, measured by frontal analysis at 4 min residence time using human polyclonal IgG at 1 mg/mL in 20 mM phosphate, 150 mM NaCl, pH 7.4 on an ÄKTA pure FPLC system, reaches 42 mg IgG/mL resin, which is 15–20 % higher than that obtained with random amine coupling via cyanogen bromide activation. Leakage of Protein A ligand, monitored by sandwich ELISA, remains below 10 ppm after 100 cleaning-in-place cycles with 0.1 M NaOH, demonstrating the robustness of the amide bond under alkaline sanitization conditions. Compliance with USP <1005> for chromatographic media and ICH Q7 GMP for active pharmaceutical ingredients is maintained. The short spacer arm inherently limits steric flexibility, which can lead to a 5–10 % loss of binding efficiency for certain IgG1 subtypes exhibiting hindered hinge region access; this is documented as a platform-specific constraint during resin lifetime qualification.Magnetic nanoparticle labeling of HER2-specific affibody molecules for circulating tumor cell isolation uses a reversed coupling sequence: amine-terminated iron oxide particles (core diameter 8 nm, with a 3-aminopropyl silane coating, hydrodynamic diameter 120 nm) are reacted directly with the NHS ester moiety of the linker at a nanoparticle:linker w/w ratio of 10:1 in anhydrous DMSO. After 45 min, the maleimide-decorated particles are precipitated by ultracentrifugation and redispersed in 50 mM PBS, pH 6.8, 5 mM TCEP. The affibody (ZHER2:342) engineered with a C-terminal cysteine residue is added at a molar ratio of 1:10 (particle:affibody) and the thiol–maleimide ligation proceeds for 2 h at 4 °C. Unreacted maleimides are capped with 1 mM 2-mercaptoethanol. Magnetophoretic mobility measured by an XSL-MP1 magnetophoresis analyzer increases by 4.8-fold after conjugation, confirming successful immobilization. The decorated particles achieve 78 % capture efficiency of SK-BR-3 cells spiked into whole blood at concentrations of 10 cells/mL, as validated by flow cytometry sorting in conjunction with anti-cytokeratin staining. The process is executed under an ISO 13485 quality system for investigational medical devices. Because trace amounts of DMSO interfere with subsequent PCR-based downstream assays, a final buffer exchange into 0.1 % BSA-Tris buffer (pH 7.4) is performed using a magnetic separator; failure to reduce residual DMSO below 0.01 % has been shown to inhibit DNA polymerase activity.
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With a molar mass of 314.25 g·mol⁻¹ and an NHS ester leaving group that at 4 °C in pH 7.0 phosphate buffer exhibits a hydrolysis half-life of approximately 4–5 h, the heterobifunctional crosslinker 3-maleimidobenzoic acid N-hydroxysuccinimide ester—routinely abbreviated MBS—introduces a 9.9 Å phenyl spacer between its maleimide and activated ester termini. Routine in‑process identity confirmation rests on HPLC retention time (8.2 min on a 150 × 4.6 mm C18 column, isocratic acetonitrile/water 60:40, detection at 254 nm) and the characteristic IR carbonyl stretch doublet at 1708 cm⁻¹ and 1738 cm⁻¹. The aromatic ring stamps a strong UV chromophore onto the molecule that is absent in cyclohexane‑bridged congeners, enabling direct monitoring of unreacted crosslinker removal by size‑exclusion chromatography with a absorbance detector set to 250 nm. All shipments compliant with ISO 9001:2015 quality management systems are analysed against a certificate-of-analysis template that enforces an area‑percent purity ≥ 97.0%, a melting‑range endpoint of 161.0–165.0 °C, and elemental C, H, N values within ±0.3% of theory.
The core operational divergence between MBS and the widely cited succinimidyl 4-(N-maleimidomethyl)cyclohexane‑1‑carboxylate (SMCC) originates in the aromatic spacer. Unlike the chair‑boat isomerisation that renders the cyclohexane ring of SMCC conformationally heterogeneous, the 1,3‑disubstituted phenyl core of MBS possesses a rotational barrier that locks the maleimide and NHS ester into a fixed relative orientation, yielding a narrower statistical distribution of end‑to‑end distances during protein surface mapping. Practical consequence: When β‑galactosidase tetramer crosslinking is followed by denaturing SDS‑PAGE under non‑reducing conditions, the MBS‑derived band pattern shows a 15–20% reduction in inter‑subunit bridge‑length variability compared with SMCC‑treatment under identical 10:1 crosslinker‑to‑protein ratio at pH 7.2, a difference quantified by densitometric peak width at half height on gels scanned via a Bio‑Rad Gel Doc XR+ system. Additionally, the phenyl ring contributes 0.8–1.0 log units of hydrophobicity to the conjugate surface, an effect exploited to transiently mask immunogenic epitopes when a downstream hydrophobic interaction chromatography (HIC) step with a 1.0 M ammonium sulphate gradient is part of the purification train.
In contrast to the water‑soluble Sulfo‑MBS derivative, unmodified MBS lacks the SO₃⁻ group and therefore partitions preferentially into organic phases. This property proves decisive when thiol‑containing payloads must be delivered across the plasma membrane of viable cells prior to capture with an amine‑tagged intracellular scaffold. Equilibration studies with HeLa suspension cultures maintained at 37 °C, 5% CO₂ demonstrate that a 50 µM bolus of MBS achieves cytoplasmic maleimide‑active pool concentrations above 12 µM within 90 s, while the sulfonated analogue remains > 98% excluded over the same interval—data obtained via LC‑MS/MS quantitation of the quenched N-acetylcysteine adduct in clarified lysates.
Because the NHS ester hydrolytically degrades with a measured rate constant of 1.4 × 10⁻³ s⁻¹ at 25 °C, pH 8.0, direct addition of solid MBS to an aqueous protein solution invariably leads to heterogeneous modification and elevated inter‑batch aggregate formation. Process records from 20 L‑scale cGMP antibody‑drug‑conjugate manufacturing campaigns show that pre‑dissolving the solid in anhydrous N,N‑dimethylformamide (DMF, water content < 50 ppm by Karl Fischer) to a stock concentration of 25–100 mM and injecting this solution into a vortex‑stirred protein pool held at 4 °C reduces the free‑NHS hydrolysis side‑product to ≤3% of total crosslinker at the end of the 2 h coupling window. The organic solvent fraction must be kept below 5% v/v (DMF) or 10% v/v (DMSO, purity ≥ 99.9%) to prevent solvent‑induced protein aggregation; excursions beyond those thresholds produce persistent sub‑visible particles detectable by micro‑flow imaging (MFI) with a particle count > 6000/mL for ≥ 2 µm size bins, exceeding the USP <788> alert threshold for injectable formulations.
Coupling buffer selection must exclude primary‑amine‑containing species. Tris, glycine, and ammonium bicarbonate buffers quench the activated ester, generating non‑reactive amide by‑products. Phosphate‑buffered saline (PBS, 100 mM phosphate, 150 mM NaCl, pH 7.2–7.4) or borate buffer (50 mM, pH 8.0–8.3) constitutes the standard reaction medium. When the target lysine residue resides in a sterically occluded pocket—typical of the CH2 domain of human IgG1—raising the MBS:IgG molar ratio to 20:1 and extending the incubation to 3 h at 4 °C improves the degree of conjugation to 3.8–4.2 crosslinker molecules per antibody as determined by intact‑mass deconvolution on a Waters Xevo G2‑XS QTof, compared with a base value of 1.5–2.0 at 10:1 ratio. After primary amine conjugation, unreacted NHS ester is quenched by adding 10 mM ethanolamine (pH 8.5) for 15 min before gel‑filtration desalting on Zeba Spin 7K MWCO columns or a HiPrep 26/10 desalting column plumbed into an ÄKTA pure system.
| Parameter | Specification | Method Reference |
|---|---|---|
| Appearance | White to off‑white crystalline powder | Visual, EP 2.2.2 |
| Molecular weight (anhydrous) | 314.25 g·mol⁻¹ | HRMS, ESI⁺ |
| Melting range | 161.0–165.0 °C | USP <741>, Class I |
| Purity (HPLC, 254 nm) | ≥ 97.0% area | USP <621> |
| Water content (KF) | ≤ 0.3% | USP <921> Method Ia |
| Elemental analysis (C, H, N) | Theory C 57.32%, H 3.37%, N 8.91%; allowed ± 0.3% | Combustion, thermal conductivity detection |
When intracellular thiol accessibility outweighs aqueous solubility constraints, MBS is applied without pre‑quenching organic cosolvent in a serum‑free medium pulse. Jurkat T‑cells activated with anti‑CD3/CD28 beads and suspended in RPMI‑1640 without phenol red receive a 20 µM MBS dose from a 10 mM stock in anhydrous DMSO. After 45 min at 37 °C, extracellular maleimide is quenched with 5 mM reduced glutathione, lysates are prepared by RIPA buffer extraction, and target capture proceeds via the NHS ester on protein A/G magnetic beads. This workflow yields > 70% capture efficiency for low‑abundance (10⁴ copies·cell⁻¹) adaptor proteins, whereas Sulfo‑MBS under identical conditions captures <5% due to membrane exclusion. Published data for this specific configuration in primary hepatocytes is limited; extrapolation must consider cell‑type‑dependent esterase activity that can regenerate the free acid and terminate conjugation competence.
Stability‑indicating storage is defined by accelerated degradation trials at 40 °C / 75% RH over 4 weeks in amber glass vials sealed under dry nitrogen. Loss of purity exceeds 1.2% when headspace moisture penetrates poorly crimped septa, detected by a new HPLC peak eluting at 3.4 min corresponding to 3‑maleimidobenzoic acid. Long‑term storage at −20 °C with desiccant maintains the purity above 96.8% for at least 24 months. Prior to each use, vials must be equilibrated to room temperature in a sealed desiccator to prevent condensation; if the relative humidity of the laboratory exceeds 60%, retrieval and aliquotting should be conducted inside a glove bag purged with dry nitrogen to preserve NHS ester integrity.
| Property | MBS | SMCC | Sulfo‑MBS | Sulfo‑SMCC |
|---|---|---|---|---|
| Spacer composition | Phenyl (9.9 Å) | Cyclohexane‑methylene (8.3 Å) | Phenyl + sulfonate (9.9 Å) | Cyclohexane‑methylene + sulfonate (8.3 Å) |
| Aqueous solubility (25 °C) | <0.1 mg/mL | <0.1 mg/mL | >25 mg/mL | >10 mg/mL |
| Membrane permeability (logP) | 0.9 | 1.4 | −2.1 | −1.8 |
| UV detection wavelength | 250 nm (strong) | End absorption only | 262 nm | End absorption only |
| Recommended amine‑reaction pH | 7.2–7.8 | 7.2–7.5 | 7.5–8.3 | 7.5–8.0 |
| Typical intra‑chain conjugate aggregation | Moderate (5–12%) | Low (<5%) | Low (<5%) | Very low (<3%) |
The removal of excess, unreacted crosslinker after the maleimide‑sulfhydryl step is most efficiently executed by size‑exclusion chromatography. For conjugates with hydrodynamic radii above 8 nm, a Superdex 200 Increase 10/300 GL column eluted with PBS, pH 7.4 at a flow rate of 0.5 mL·min⁻¹ resolves the conjugate peak at 12.0 mL from the residual small‑molecule peak at 20.5 mL. In‑line multi‑angle light scattering (MALS) with a DAWN HELEOS II detector verifies a monomer content > 92% and an oligomer fraction below 6%. If the oligomer fraction exceeds 8%, the protocol recommends reducing the crosslinker‑to‑target molar ratio by 25% and increasing the ionic strength of the coupling buffer to 300 mM NaCl to suppress electrostatic pre‑concentration effects at the protein surface.
