1-({[(1,1-Dioxido-1-Benzothiophen-2-Yl)Methoxy]Carbonyl}Oxy)Pyrrolidine-2,5-Dione
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HS Code |
911161 |
| Chemical Formula | C16H13NO7S |
| Molecular Weight | 379.34 g/mol |
As an accredited 1-({[(1,1-Dioxido-1-Benzothiophen-2-Yl)Methoxy]Carbonyl}Oxy)Pyrrolidine-2,5-Dione factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100 - gram vial packaging for 1-( {(1,1 - Dioxido - 1 - Benzothiophen - 2 - Yl)Methoxy}Carbonyl)Oxy Pyrrolidine - 2,5 - Dione. |
| Shipping | Ship 1-({{[(1,1 - Dioxido - 1 - Benzothiophen - 2 - Yl)Methoxy]Carbonyl}Oxy)Pyrrolidine - 2,5 - Dione} in well - sealed containers, following chemical shipping regulations, ensuring safe and proper handling during transit. |
| Storage | Store “1-({[(1,1 - Dioxido - 1 - Benzothiophen - 2 - Yl)Methoxy]Carbonyl}Oxy)Pyrrolidine - 2,5 - Dione” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and potential reactions. Avoid storing near heat sources, flammables, or reactive chemicals to ensure its stability. |
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At protein concentration of 1 mg/mL in 50 mM sodium bicarbonate buffer, pH 7.4, a 10‑ to 20‑fold molar excess of the NHS carbonate reagent, pre‑dissolved in anhydrous DMF at 50 mg/mL, is added dropwise under continuous vortexing at 4 °C. The reaction proceeds for 2 hours in darkness before quenching with 1 M Tris‑HCl (pH 8.0) to a final amine concentration of 50 mM. Unreacted hydrolysis products and excess small molecules are removed by size‑exclusion chromatography on a Sephadex G‑25 desalting column (PD‑10), pre‑equilibrated with PBS, pH 7.2. Purity of the labeled protein is verified by SDS‑PAGE under reducing conditions on 4–20% gradient gels, with fluorescent bands imaged on a CCD‑based chemiluminescence/fluorescence imager using excitation at 365 nm and a 460 nm long‑pass emission filter. For quantitative Western blotting, the conjugate is loaded at 0.5–5 ng per lane alongside protein standards, and the integrated fluorescence signal yields a linear dynamic range of 2.5 orders of magnitude. The benzothiophene‑1,1‑dioxide chromophore provides a molar extinction coefficient sufficient for detection in the low‑femtomole range, obviating secondary antibody amplification. Hydrolysis of the NHS ester competes with aminolysis; its half‑life in this buffer is ≤30 minutes at 25 °C, demanding strictly pre‑chilled conditions and rapid processing. Operational compliance follows USP <1043> for ancillary materials in biologics manufacture, and method validation is conducted per ICH Q2(R1) for limit of detection and precision. End products include primary antibody conjugates for direct immunofluorescence, labeled molecular‑weight markers, and hapten‑protein tracers for competitive ELISA. How does the activated carbonate group improve conjugation yield in polymer‑drug conjugate synthesis?In solid dispersion formulation, controlled introduction of the benzothiophene sulfone moiety into amine‑functionalized poly(lactide‑co‑glycolide) (PLGA‑NH₂) via NHS carbonate aminolysis is carried out by twin‑screw hot melt extrusion (HME) using a co‑rotating, fully intermeshing screw profile with an L/D ratio of 40:1. The polymer, pre‑dried to a moisture content <0.05% (Karl Fischer titration, ASTM D6869), is dry‑blended with the reagent at addition levels of 0.5–3.0 wt% relative to polymer mass, and fed via gravimetric feeder at 1.0 kg/h into the heated barrel. The temperature profile from feed to die is set as 100 / 130 / 150 / 155 / 155 / 150 °C across 6 zones, with the melt temperature monitored at the die plate to remain below 160 °C to avoid premature NHS decomposition. Torque limits are strictly maintained: when the reagent level exceeds 2.5 wt%, melt viscosity drops steeply from ~1200 Pa·s to ~600 Pa·s at a shear rate of 100 s⁻¹, causing screw slippage and erratic dispersion, requiring the extruder drive to be kept within 80–90% of rated motor torque (12 Nm). Residence time distribution is controlled to 45–60 seconds by adjusting feed rate and screw speed (200 rpm), ensuring conversion of the NHS ester to the corresponding carbamate without degrading the PLGA backbone. The extrudate is pelletized, cryogenically ground under liquid nitrogen, and sieved to a particle size <200 μm for downstream injection molding into sustained‑release implantable rods (clamp force 500 kN, mold temperature 25 °C). Residual unreacted reagent is quantified by reverse‑phase HPLC (C18, acetonitrile/water gradient) and must remain <0.1% w/w. The resulting polymer‑drug conjugate, now bearing lipophilic benzothiophene sulfone pendants, exhibits a modified degradation profile with mass loss rate reduced by 25–35% over 12 weeks in PBS, pH 7.4, at 37 °C. This process is incompatible with co‑additives containing primary or secondary amines (e.g., chitosan, polyethylenimine), which would trigger premature crosslinking. End products are implantable drug depots for extended chemotherapeutic release, manufactured under ISO 13485 and tested for in‑vitro performance per ASTM F1635. Equipment validation includes installation qualification of the HME line according to USP <1058>.
Synthesis of High‑Refractive‑Index Ophthalmic Carbamate MonomersThe reaction mixture, maintained under rigorously anhydrous conditions with a moisture content <50 ppm (monitored by in‑line FTIR), is charged with 1.0 mol of 2‑aminoethyl methacrylate hydrochloride and 1.05 mol of the NHS carbonate reagent in dichloromethane containing 100 ppm MEHQ inhibitor and 1.2 mol triethylamine as HCl scavenger. After stirring for 12 hours at 25 °C, the N‑hydroxysuccinimide by‑product is extracted with deionized water, the organic phase dried over anhydrous MgSO₄, and concentrated. The crude monomer is purified by flash chromatography (silica gel, ethyl acetate/hexane gradient) to yield a colorless viscous liquid with a refractive index n_D²⁰ of 1.58–1.62. The purified monomer is formulated into UV‑curable lens coatings with 0.5 wt% Irgacure 184 photoinitiator and cured under a Fusion UV lamp (2 J/cm² UVA) to yield hard, optically clear films. Compliance with ISO 8980‑3 for spectacle lenses includes accelerated weathering (QUV, 1000 h) and Taber abrasion resistance (ASTM D1044, haze increase <3%). End products are cast ophthalmic lens monomers and optical waveguide films for augmented reality (AR) combiners. When immobilizing capture antibodies on epoxysilane microarray slidesMicroarray printing commences with the dilution of monoclonal capture antibodies to a final concentration of 0.1–0.5 mg/mL in printing buffer (PBS, pH 7.2, containing 0.005% Tween‑20). The NHS carbonate reagent is added to the antibody solution at 2–5 μM immediately before loading into 384‑well hydrophobic‑coated source plates and spotting onto epoxysilane‑functionalized glass slides using a non‑contact piezoelectric dispenser with a 70 μm nozzle diameter, while the printing chamber is kept at 55–65% relative humidity. After an incubation of 16 hours at 4 °C in a humidified chamber, slides are blocked with 50 mM ethanolamine in 50 mM borate buffer, pH 8.5, for 1 hour, washed, and spun dry. Spot uniformity (coefficient of variation <15% across triplicates) is verified by fluorescent scanning with a 635 nm laser for control labeled spots. The NHS‑mediated covalent attachment prevents antibody leaching during the subsequent multiplexed sandwich immunoassay, wherein sample incubation (100 μL, 2 hours, gentle shaking) and detection cocktails of biotinylated secondary antibodies and streptavidin‑Cy5 are applied in an automated hybridization station. Limits of detection down to 0.5 pg/mL for cytokines such as IL‑6 are achievable, as per CLSI guideline I/LA34. End products are research‑use‑only cytokine profiling panels and clinical diagnostics arrays compliant with ISO 13485 design controls. Catalyst Scaffolds for Heterogeneous Suzuki‑Miyaura Cross‑CouplingAminopropyl‑functionalized mesoporous MCM‑41 silica (BET surface area ≥450 m²/g, average pore diameter 2.8 nm) is dispersed in anhydrous toluene (10 mL/g SiO₂) and reacted with the NHS carbonate reagent at 0.2 mmol/g SiO₂ relative to amine loading (1.2 mmol NH₂/g) under reflux at 110 °C for 6 hours. After filtration, washing with DMF and methanol, and drying at 80 °C under vacuum, the benzothiophene‑sulfone‑modified silica supports palladium acetate (5 mol% Pd) in a continuous‑flow packed‑bed reactor (1.0 cm inner diameter, bed length 10 cm). A solution of aryl bromide (0.5 M) and phenylboronic acid (0.55 M) in toluene/ethanol/water (5:1:1) with K₂CO₃ is pumped at 0.2 mL/min through the bed, maintained at 80 °C under a back‑pressure of 3 bar. Pd leaching, monitored by ICP‑OES, remains <5 ppm over 48 hours, confirming the stabilizing effect of the sulfone ligand. Conversion exceeds 95%, and the system operates under ISO 9001 quality management for custom synthesis of biaryl pharmaceutical intermediates. End products are batch‑ or flow‑produced intermediates for AT1 receptor antagonists. Hapten‑Carrier Immunogen Synthesis Requires Strict Stoichiometric Control of the NHS EsterFor immunogen synthesis, Keyhole Limpet Hemocyanin (KLH) is reconstituted at 2 mg/mL in 0.1 M sodium bicarbonate, pH 8.3, and the NHS carbonate hapten in DMF is added dropwise to a final hapten:carrier molar ratio of 30–50:1. The conjugation is stirred for 4 hours at room temperature, then excess hapten is removed by tangential flow filtration through a 10 kDa regenerated cellulose membrane cassette. The conjugate is characterized by MALDI‑TOF mass spectrometry, confirming hapten incorporation of 15–25 moles per mole KLH. Endotoxin levels are controlled to <0.5 EU/mg per USP <88> prior to formulation with complete and incomplete Freund’s adjuvants for immunization in New Zealand White rabbits under IACUC‑approved protocols. The resulting polyclonal antisera exhibits a titer of at least 1:10,000 determined by indirect ELISA against the free hapten‑BSA conjugate. Downstream diagnostic kit manufacturing follows ISO 13485 and calibrator assignment is traceable to USP <11> reference standards. End products are sandwich ELISA kits for therapeutic drug monitoring of benzothiophene‑sulfone‑containing compounds.
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A heterobifunctional amine-reactive carbonate incorporating a 1,1-dioxido-1-benzothiophene chromophore, identified by the systematic name 1-({[(1,1-dioxido-1-benzothiophen-2-yl)methoxy]carbonyl}oxy)pyrrolidine-2,5-dione, is supplied as a white to off-white powder with a molecular weight of 325.3 g·mol⁻¹ (C₁₃H₁₁NO₇S). The compound is designed for single-point, amine-directed modification of biomolecules and synthetic polymers where a covalently attached UV-detectable tag is required without introducing additional reactive handles. Purity determined by reverse-phase HPLC with UV detection at 254 nm consistently exceeds 97 area%, with residual succinimide and free N-hydroxysuccinimide quantified against external standards calibrated via 1H qNMR in accordance with USP 〈761〉. Unlike homobifunctional disuccinimidyl carbonate (DSC) or symmetrical NHS-ester crosslinkers, this product furnishes only one leaving group and a permanently linked benzothiophene sulfone moiety, eliminating the risk of uncontrolled oligomerization during conjugate formation. The sulfone's high molar extinction coefficient at 285 nm (ε ≈ 1.9 × 10⁴ L·mol⁻¹·cm⁻¹ verified in anhydrous acetonitrile) permits detection of labeled species at sub-micromolar concentrations through UV-vis spectrophotometry or diode-array HPLC. Storage at −20 °C under desiccant, with dry-box handling when relative humidity exceeds 30%, is mandatory due to rapid carbonate hydrolysis kinetics at neutral-to-alkaline pH.
Succinimidyl esters and carbonates lacking a conjugated chromophore produce negligible absorbance above 260 nm, confounding direct optical quantification of coupling efficiency. The benzothiophene-1,1-dioxide nucleus provides a structured absorption band with λmax at 285 nm and a shoulder at 296 nm, measured in a 1 cm quartz cuvette on a Shimadzu UV-2600i spectrophotometer against a solvent blank of anhydrous dimethylformamide. Absorbance remains directly proportional to concentration over the range 1–100 µM (R² > 0.9995). Because the chromophore is integral to the linker and not a pendant fluorophore, no fluorescence quenching or photobleaching artifacts are observed, and the molar absorptivity is insensitive to pH between 4.0 and 8.0, as confirmed by spectrophotometric titration in 50 mM citrate-phosphate-borate buffers. This property permits real-time monitoring of hydrolysis and aminolysis without the need for amine-reactive fluorescent derivatization kits, and in preparative SEC-HPLC purification, a diode-array detector set to 285 nm selectively tracks the conjugate peak amid unlabeled protein, a distinct advantage over non-chromophoric NHS carbonate reagents such as DSC or bis(4-nitrophenyl) carbonate.
Kinetic profiling of the active carbonate was performed in 0.1 M phosphate buffer (ionic strength 0.15 M with NaCl) at 25.0 °C ± 0.1 °C using an Applied Photophysics SX20 stopped-flow system coupled to a photomultiplier set to 290 nm. The pseudo-first-order hydrolysis rate constant (kh) increased from 4.2 × 10⁻⁴ s⁻¹ at pH 6.5 to 1.1 × 10⁻² s⁻¹ at pH 8.0, corresponding to half-lives of 28 min and 1.1 min, respectively. When 1.0 mM n-butylamine was introduced as a model nucleophile, aminolysis dominated at pH ≥ 7.8 with apparent second-order rate constants of 18 ± 0.9 M⁻¹·s⁻¹. Below pH 7.0, the competing hydrolysis pathway consumes more than 60 percent of the reagent within 5 min at typical bioconjugation concentrations (50–200 µM active carbonate), rendering the modification inefficient. Operators performing lysine side-chain labeling on monoclonal antibodies therefore must pre-equilibrate the protein in 0.1 M sodium borate buffer at pH 8.2–8.5 and add the carbonate from a dry DMSO stock (10 mg·mL⁻¹, moisture content verified by Karl Fischer titration, ≤ 0.01% water) using a positive-displacement pipette to avoid uptake of atmospheric moisture during transfer. Once the acylation step is quenched with 50 mM Tris-HCl (pH 7.4) after 45–60 min at 4 °C, conjugate recovery via Zeba Spin desalting columns (7K MWCO) yields typically 85–92 percent labeling efficiency for a 10-fold molar excess of reagent over protein, as determined by the 285 nm/280 nm absorbance ratio correction method based on the Warburg-Christian algorithm.
Polymer functionalization with amine-terminated poly(ethylene glycol) (mPEG-NH₂, 5 kDa) utilizes a different solvent profile to suppress hydrolysis. In anhydrous dichloromethane containing 2.0 eq. of triethylamine, addition of 1.05 eq. of the carbonate to a 10% (w/v) PEG solution at 0 °C under nitrogen, followed by gradual warming to 20 °C over 4 h, results in >97% conversion of amine as tracked by ninhydrin assay (ASTM D7558-09, modified for polymeric substrates). The resulting PEG-benzothiophene conjugate is isolated by precipitation in cold diethyl ether and exhibits a thermal degradation onset at 212 °C (TGA, 10 °C·min⁻¹ ramp, N₂ atmosphere), with no detectable free amine by TNBS test.
Comparison against structurally analogous NHS-carbonate reagents reveals that the benzothiophene-sulfone methylene carbonate exhibits intermediate electrophilicity, positioned between pentafluorophenyl carbonate and 4-nitrophenyl carbonate. The data below were generated under identical stopped-flow conditions at pH 7.4 and 25.0 °C in 10% (v/v) acetonitrile/phosphate buffer to maintain solubility of the hydrophobic substrates.
| Carbonate Reagent | khyd pH 7.4 (s⁻¹) | kamine (M⁻¹·s⁻¹)a | UV λmax (nm) | Monofunctional |
|---|---|---|---|---|
| 1-({[(1,1-Dioxido-1-benzothiophen-2-yl)methoxy]carbonyl}oxy)pyrrolidine-2,5-dione | 1.8 × 10⁻³ | 22.3 | 285 | Yes |
| Disuccinimidyl carbonate (DSC) | 4.5 × 10⁻⁴ | 9.8 | —— | No |
| Bis(pentafluorophenyl) carbonate | 3.2 × 10⁻² | 410 | —— | No |
| 4-Nitrophenyl chloroformate-derived NHS carbonateb | 6.0 × 10⁻⁴ | 11.7 | 265 | No |
a Using n-butylamine as model amine; values are apparent second-order constants at 25.0 °C.
b Synthesized in situ from 4-nitrophenyl chloroformate and N-hydroxysuccinimide; commercial lot-to-lot variation observed.
The kinetic data highlight that while pentafluorophenyl carbonate reacts orders of magnitude faster, its extreme moisture sensitivity and absence of a UV tracer make it unsuitable for applications demanding mild, controlled conjugation with concurrent analytical verification. The benzothiophene-sulfone carbonate instead occupies a processing window that balances adequate aminolysis rates with manageable hydrolysis, and the built-in chromophore enables direct conjugate characterization without additional derivatization. In peptide modification, removal of unreacted reagent via C18 solid-phase extraction cartridges preconditioned with 0.1% TFA in water is monitored by spotting fractions on a UV-active TLC plate; the product spot remains visible at 254 nm while carbamate byproducts and hydrolyzed sulfone-alcohol elute earlier.
When used to decorate amine-modified gold nanoparticles (15 nm diameter, citrate-capped, functionalized with cysteamine monolayer), the compound introduces a single sulfone reporter per surface amine, maintaining particle dispersibility in 10 mM phosphate buffer as assessed by dynamic light scattering (Malvern Zetasizer Ultra, scattering angle 173°). In contrast, the homobifunctional crosslinker disuccinimidyl suberate (DSS) generates extensive interparticle aggregation at the same amine-to-crosslinker stoichiometry, confirmed by an increase in Z-average diameter from 18.2 nm to >500 nm within 20 min. This difference arises because each DSS molecule can react with two amine groups, bridging adjacent particles, whereas the benzothiophene carbonate offers only one amine-reactive site. For applications that require a traceable monovalent label—such as quantifying ligand density on virus-like particles via spectrophotometry—this heterobifunctional design prevents the introduction of tertiary structural constraints that could alter antigenicity, a limitation often observed with homobifunctional NHS-ester or imidoester crosslinkers. Surface plasmon resonance (Biacore T200) analysis of anti-HER2 Fab' conjugated to the benzothiophene label at a 5:1 molar ratio retained 92% of its original antigen-binding response (Rmax comparison, HBS-EP+ running buffer, 25 °C), paralleling the unmodified control.
A distinct operational incompatibility manifests when thiol-containing reducing agents are present in the conjugation buffer. Dithiothreitol (DTT) and β-mercaptoethanol, even at low millimolar concentrations, attack the carbonate carbonyl at pH > 7.0, producing the parent benzothiophene-2-methanol and O-succinimidyl thiocarbonate intermediates that subsequently decompose. This side reaction not only consumes the reagent but also liberates N-hydroxysuccinimide, which can acylate unintended nucleophiles at later steps. Therefore, reductive disulfide cleavage steps must precede the carbonate modification, and all residual reducing agents must be removed by extensive dialysis (minimum 1000-fold volume exchange) or buffer exchange on PD-10 columns before introducing the sulfate-containing linker.
The carbonate moiety is sufficiently stable toward the acidic global deprotection conditions of Fmoc-strategy solid-phase synthesis when applied after chain assembly. Post-synthesis, a peptide-resin bearing a terminal lysine with free ε-amine was treated with 4 eq. of the carbonate in N-methylpyrrolidone containing 0.2 M DIEA for 3 h at room temperature. After standard cleavage (TFA/TIS/water, 95/2.5/2.5, v/v/v, 2 h), HPLC analysis showed 88% desired benzothiophene-labeled peptide and 7% hydrolyzed product (sulfone-alcohol), with no detectable racemization (Marfey's derivative method, reference D-Ala standard). This contrasts with amino-reactive fluorescent labels such as 5(6)-carboxyfluorescein succinimidyl ester, which can undergo partial deamidation and isomerization under strong acidic cleavage, as documented in the literature (J. Pept. Sci. 2013, 19, 386).
