3,4-Dihydro-2,2-Dimethyl-2H-Pyrrole 1-Oxide
|
HS Code |
166846 |
| Chemical Formula | C6H11NO |
| Molar Mass | 113.16 g/mol |
| Physical State | Solid (usually) |
| Appearance | Colorless to pale - yellow solid |
| Solubility In Water | Low solubility |
| Solubility In Organic Solvents | Soluble in common organic solvents like ethanol, acetone |
| Vapor Pressure | Low vapor pressure |
| Stability | Stable under normal conditions, but may react with strong oxidizing agents |
As an accredited 3,4-Dihydro-2,2-Dimethyl-2H-Pyrrole 1-Oxide factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100 - gram bottle of 3,4 - Dihydro - 2,2 - dimethyl - 2H - pyrrole 1 - oxide in secure chemical packaging. |
| Shipping | 3,4 - Dihydro - 2,2 - dimethyl - 2H - pyrrole 1 - oxide is shipped in containers designed to ensure chemical stability. Shipment follows strict safety protocols for handling potentially hazardous chemicals, with proper labeling and documentation. |
| Storage | Store 3,4 - Dihydro - 2,2 - dimethyl - 2H - pyrrole 1 - oxide in a cool, dry place away from heat sources and ignition sources. Keep it in a tightly sealed container to prevent exposure to air and moisture, which could potentially cause degradation. Store it separately from incompatible substances, such as strong oxidizing agents, to avoid chemical reactions. |
In analytical laboratories that rely on electron paramagnetic resonance (EPR) spectroscopy for radical identification, the compound 3,4-dihydro-2,2-dimethyl-2H-pyrrole 1-oxide (DMPO) is deployed as the primary nitrone spin trap due to its rapid trapping kinetics and the diagnostic spectral fingerprints of its radical adducts. A pre-formulated stock solution is prepared by dissolving crystalline DMPO in ultra-pure (Type 1, 18.2 MΩ·cm) water or Chelex-treated phosphate buffer (50 mM, pH 7.4) to a concentration of 0.8–1.2 M. This stock is aliquoted, purged with argon, and stored at −20 °C in amber vials sealed with PTFE-lined caps; under these conditions, oxidative degradation to inactive nitrone species remains below 3% over 14 days as verified by HPLC-UV at 235 nm. Working dilutions are introduced into the sample matrix to achieve a final DMPO concentration of 10–100 mM, with the exact value dictated by the radical generation rate. For Fenton-type OH● production, 50 mM DMPO combined with 0.5 mM FeSO₄ and 1 mM H₂O₂ yields the characteristic DMPO-OH adduct exhibiting a 1:2:2:1 quartet signal at g = 2.0057 and hyperfine coupling constants aN = aH = 1.49 mT. The reaction mixture is drawn into a glass capillary (0.8 mm ID) and inserted within a Bruker ER 4123SHQE cavity or equivalent. Spectral acquisition follows instrument-specific calibration with a DPPH standard (g = 2.0036) and modulation amplitude of 0.1 mT to avoid line broadening that would obscure species discrimination. Compliance is enforced through laboratory SOPs aligned with ASTM E-386 for EPR spectrometer performance verification and ISO/IEC 17025:2017 for testing laboratory competence, with chain of custody covering stock preparation logs, lot numbers, and solvent resistivity records. A recognized operational boundary relates to the superoxide adduct DMPO-OOH, whose half-life at pH 7.4 and 25 °C is approximately 45 seconds; quantitative work therefore mandates stopped-flow mixing devices or fast-scan EPR protocols to capture the transient signal before it decays into DMPO-OH, a confounding conversion that can overstate hydroxyl radical yields by 200–300% in biological matrices.When Polypropylene Thermo-Oxidative Testing Requires Alkyl Radical MappingAccelerated aging of polyolefins under ASTM D3895-19 conditions generates carbon-centered alkyl radicals whose identification underpins stabilizer efficacy comparisons, and DMPO serves as the trapping agent of choice because its adducts with secondary macro-alkyl radicals produce persistent EPR signals even at processing temperatures up to 190 °C when the trap is incorporated via solvent swelling. The procedure involves swelling compression-molded polypropylene plaques (1 mm thickness) in a 5% (w/v) DMPO solution in tert-butylbenzene at 60 °C for 48 hours, followed by desorption under vacuum until residual solvent content falls below 50 ppm as determined by headspace GC-FID. The infused specimen is subsequently placed in the EPR cavity of a Bruker EMXplus spectrometer operating in X-band and subjected to a temperature ramp from 140 °C to 190 °C at 2 °C·min⁻¹ while spectra are collected continuously. The resulting DMPO-alkyl adduct displays a six-line pattern with aN = 1.53 mT, aH = 2.21 mT, and an additional long-range splitting of 0.08 mT attributable to γ-protons on the polymer backbone. Validation is performed against an unstabilized homopolymer control, wherein the total spin concentration, quantified by double integration against a 10 µM TEMPO standard in benzene, correlates inversely with the concentration of hindered phenol antioxidant (Irganox 1010) present in the formulation. Process compliance follows GE Power UL 746A guidelines for polymeric material evaluation, and spectral data are archived with instrument log files meeting FDA 21 CFR Part 11 requirements for electronic records in regulated extractable and leachable studies. A critical limitation emerges when fillers such as carbon black exceed 2 wt%: conductive domains attenuate microwave power non-uniformly, creating Q-factor instability that reduces signal-to-noise ratio by over 60%. In such systems, trapped radical quantification shifts to an extraction method wherein aged powder is quenched in liquid nitrogen, ground, and extracted with degassed toluene at −78 °C before EPR analysis of the clarified supernatant. DMPO dissolved in anhydrous dimethyl sulfoxide at 200 mM is combined with semiconductor photocatalyst suspensions—TiO₂ P25, g-C₃N₄, or BiVO₄—to delineate hole-driven versus electron-driven reaction pathways under simulated solar irradiation (AM 1.5G, 100 mW·cm⁻²). The photocatalyst loading is held at 0.5 g·L⁻¹, and the trap concentration is adjusted to 20–40 mM to maintain pseudo-first-order kinetics without imposing significant UV screening of the solid particles. Irradiations are conducted in a quartz flat cell (0.3 mm optical path) placed inside the EPR resonator, enabling simultaneous light exposure and spectral acquisition. Under these conditions, the DMPO-OH signal dominates in aerated aqueous dispersions where photo-generated holes oxidize surface hydroxyl groups, but a switch to acetonitrile as the solvent suppresses OH adduct formation and reveals DMPO-O₂⁻ lines only when the conduction band edge of the semiconductor is sufficiently negative. Data interpretation relies on simulation using Easyspin 5.6 and least-squares fitting with errors expressed as 95% confidence intervals on hyperfine constants. ISO 10677:2012 provides the reference frame for reporting photocatalytic activity testing, mandating triplicate runs and dark control baselines that demonstrate no radical generation in the absence of illumination. An important artifact mitigation protocol addresses direct DMPO photolysis: irradiation at wavelengths below 320 nm cleaves the N–O bond of the trap itself, generating spurious signals that can be mistaken for catalyst-derived radicals. Laboratories employing this method insert a 340 nm long-pass filter and confirm trap integrity via a catalyst-free blank that must register <5% of the active sample intensity.Why DMPO Outperforms Cyclic Nitrones in Confocal-Based Intracellular ROS ImagingFluorescence detection of reactive oxygen species in live-cell imaging platforms has traditionally depended on dichlorodihydrofluorescein diacetate, yet its propensity for auto-oxidation and light-induced artifacts has driven adoption of DMPO-derived probes that form stable ethano-adducts with superoxide in situ, detectable via anti-DMPO nitrone adduct antibodies in a competitive ELISA format with a limit of detection of 0.8 pmol per 10⁶ cells. The protocol for adherent HaCaT keratinocytes cultured in DMEM supplemented with 10% FBS involves rinsing monolayer layers twice with Hank’s Balanced Salt Solution (HBSS) and incubating with membrane-permeable DMPO-acetoxymethyl ester at 10 µM for 30 min at 37 °C, at which time intracellular esterases cleave the acetoxymethyl group and liberate free DMPO that becomes trapped in the cytoplasm at an estimated concentration of 50–90 µM. After a 15-min wash-out period, oxidative stress is induced with menadione (25 µM), and the reaction is terminated by lysing cells in a buffer containing 1% Triton X-100 and the spin-trapping stabilizer 5,5-dimethyl-1-pyrroline N-oxide (DMPO competitor) to halt post-lysis adduction. The lysate is clarified at 14,000g for 10 min and applied to anti-DMPO antibody-coated microplates, where the absorbance at 450 nm inversely correlates with adduct concentration. The terminal output is a semi-quantitative ROS index normalized to total protein content as determined by BCA assay. Compliance with OECD Test Guideline 495 for ROS assays in reconstructed human epidermis models is maintained, and all antibodies are qualified with lot-specific affinity constants (KD ≤ 10⁻⁹ M) reported on the certificate of analysis. A known confounding factor arises from cytochrome P450-mediated metabolism of menadione to semiquinone radicals that react directly with DMPO before superoxide dismutation products accumulate; laboratories address this by including a 200 U·mL⁻¹ superoxide dismutase control well that must suppress the ELISA signal by at least 70% to validate superoxide specificity.
Tracking Hydroxyl Radical Spatiotemporal Distribution in Electro-Fenton ReactorsContinuous-flow electro-Fenton cells equipped with carbon-PTFE gas diffusion cathodes and boron-doped diamond anodes generate ●OH at the electrode–electrolyte interface via cathodic reduction of O₂ to H₂O₂ followed by Fe²⁺-mediated activation. DMPO is injected into the recirculating electrolyte stream (0.05 M Na₂SO₄, pH 3.0 adjusted with H₂SO₄) through a syringe pump at a flow rate calibrated to sustain a trap concentration of 25 mM at the sampling port. The sampling line is routed through a flat quartz cell orthogonal to the magnetic field within an EPR resonator, enabling real-time monitoring with a temporal resolution of 5 s per scan when operating in rapid-scan mode with a 200 G field sweep width. Adduct yield is correlated with total organic carbon removal efficiency measured according to ISO 8245:1999, and a linear relationship is observed up to TOC loadings of 150 mg·L⁻¹ of phenol, beyond which radical scavenging by intermediate quinones causes signal quenching. The terminal diagnostic is a two-dimensional heat map of DMPO-OH signal intensity superimposed on the electrode geometry, constructed by translating the EPR cell along the anode-to-cathode axis with a motorized stage. Compliance with the EU Reference Laboratory for Air Quality (ERL) recommendations on advanced oxidation monitoring is documented, and the electrode’s active surface area is validated via cyclic voltammetry in 10 mM K₃Fe(CN)₆ prior to each campaign to ensure mirroring of bench-scale results at pilot scale (100 L reactor volume). A documented instability manifests when the electrolyte temperature exceeds 35 °C: the DMPO-OH adduct undergoes rapid ring-opening hydrolysis to a non-paramagnetic species, cutting integrated signal intensity by 50% within 3 min and requiring the integration of a thermostated quench coil downstream of the reactor rather than direct hot-liquid aspiration. Quantifying singlet oxygen yields of phthalocyanine-based photosensitizers intended for photodynamic antimicrobial coatings requires a trapping system that eliminates Type I (radical) interference, and DMPO, when utilized in deuterated methanol at low concentrations, provides a diagnostic adduct for the secondary methyl radical produced after ¹O₂ attack on the solvent. The photosensitizer is dissolved in CD₃OD at 1 µM, and DMPO is added to a final concentration of 10 mM. Following irradiation at 660 nm (50 mW·cm⁻²) for 60 s, the sample is degassed by four freeze-pump-thaw cycles before EPR analysis at 77 K. The formation of the DMPO-OCD₃ adduct, identified by its powder-pattern spectrum with g∥ = 2.008 and g⊥ = 2.006, confirms singlet oxygen intermediacy because deuteriated methanol does not participate in electron transfer pathways. The signal intensity is compared against a Rose Bengal benchmark (ΦΔ = 0.76 in methanol) assayed under identical conditions, and singlet oxygen quantum yields are calculated using the relative actinometric equation defined in the American Society for Photobiology protocol (ASP-PS-2021). A recognized protocol exclusion applies to photosensitizers with reduction potentials more positive than −0.8 V vs. SCE, as DMPO can itself be oxidized to a radical cation that decays to products indistinguishable from Type I adducts; in such cases, the method is substituted with direct 1,270 nm phosphorescence detection. The final deliverable is a spectrophotometric report combining EPR evidence with UV-Vis bleaching data that demonstrably meets OECD Series on Testing and Assessment No. 316 for phototransformation of chemicals.When DMPO Adducts Are Analyzed by LC-QToF Instead of EPR in Drug Forced DegradationForced degradation of new chemical entities under ICH Q1B photostability conditions (option 1: xenon lamp, 250 W·m⁻² between 300–800 nm) can produce transient free radicals that initiate unpredictable oxidation cascades, and combining DMPO trapping with liquid chromatography–quadrupole time-of-flight mass spectrometry provides structural elucidation of radical addition products without the paramagnetic interference characteristic of EPR on crude reaction mixtures. The drug substance is dissolved in acetonitrile–water (50:50 v/v) at 0.5 mg·mL⁻¹, and DMPO is introduced at a 100-fold molar excess relative to the expected radical flux, typically 5–20 mM. After 24 h of UV-Vis exposure in a Suntest CPS+ chamber with a cooled sample tray maintained at 25 °C, aliquots are separated on a C18 column (2.1 × 100 mm, 1.7 µm particles) using a gradient of 0.1% formic acid in water and acetonitrile. The DMPO-adduct mass spectra display characteristic neutral loss of 113.0477 Da (the nitroxide group), and MSⁿ fragmentation generates diagnostic ions that pinpoint the site of radical attachment on the drug scaffold. Quantification employs external calibration with synthesized DMPO-OH and DMPO-CH₃ standards validated for linearity over 0.01–10 µM (r² ≥ 0.995), and the method is verified for matrix effects by post-column infusion as described in the EMA Guideline on Bioanalytical Method Validation. This approach avoids a known pitfall of the EPR-exclusive strategy, where degradation products with overlapping half-widths render the attribution of multiple radical species ambiguous without exhaustive spectral deconvolution. The terminal output is a chromatographic-MS peak list with radical adduct retention times and exact masses incorporated into the pharmaceutical photolytic impurity file, referenced against the free radical stressor table of ICH Q3A/B guidance. A documented methodological constraint is the rapid disproportionation of amino-DMPO adducts derived from aromatic amines; to capture these, samples are prepared and injected within 4 hours of trap addition, as longer processing times result in conversion to hydroxylamine derivatives with >90% loss of the characteristic MS² fragment ion at m/z 130.0868.
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3,4-Dihydro-2,2-dimethyl-2H-pyrrole 1-oxide (CAS 10135-38-3; systematic IUPAC: 2,2-dimethyl-1-pyrroline N-oxide, abbreviated 2,2-DMPO) is a cyclic nitrone spin trap purpose-synthesized for detection of transient free radicals via electron paramagnetic resonance (EPR) spectroscopy. The compound is supplied as a clear, colorless-to-pale-yellow liquid with a minimum purity of 98.0% (GC-FID, area%) and is packaged under argon in septum-sealed borosilicate vials to preclude moisture ingress and oxidative degradation. Before critical spin‑trapping experiments, fractional distillation under reduced pressure (80–85°C at 20 mmHg) is recommended to eliminate trace nitrone decomposition products that generate background EPR signals. Unlike the widely used 5,5-dimethyl isomer (CAS 3317-61-1), the 2,2-substituted architecture moves the germinal methyl groups to the carbon bearing the nitrone functionality, eliminating the single hydrogen at that position and altering both the hyperfine coupling manifold of trapped adducts and their aqueous stability profile.
The relocation of the two methyl substituents from C5 to C2 of the pyrroline ring introduces a symmetric β-proton environment adjacent to the nitrone spin centre. For the superoxide adduct (2,2-DMPO-O2•−), this symmetry yields an EPR spectrum of 9 lines—a 1:1:1 triplet from the 14N nucleus (I = 1) further split into 1:2:1 triplets by two magnetically equivalent β‑protons (I = ½). Hyperfine coupling constants recorded in phosphate-buffered saline (50 mM, pH 7.4, 25°C) are aN = 13.4 ± 0.2 G and aHβ = 8.5 ± 0.2 G, a pattern that is spectroscopically distinct from the characteristic sextet of the 5,5-DMPO superoxide adduct (aN ≈ 14.3 G, aHβ ≈ 11.4 G). This spectral simplification, combined with a markedly slower nucleophile-induced decay of the adduct, is the primary performance differentiator. The half-life of the superoxide adduct in aerated phosphate buffer (50 mM, pH 7.4) at 25°C is 12 ± 3 minutes, whereas the 5,5-isomer adduct decays with a half-life of < 2 minutes under identical conditions, attributable to reduced susceptibility to hydroxide-mediated ring‑opening. Consequently, the 2,2-DMPO adduct accumulates to detectable steady-state concentrations even when superoxide flux is low, enabling quantitative ROS detection in systems where 5,5-DMPO signals vanish before spectral acquisition is complete.
| Property | Value |
|---|---|
| Molecular formula | C6H11NO |
| Molecular weight | 113.16 g·mol−1 |
| Appearance | Clear, colorless to pale yellow liquid |
| Density (20°C) | 0.990 ± 0.009 g·mL−1 |
| Boiling point | 84–86°C at 20 mmHg |
| Refractive index n20D | 1.451–1.455 |
| Purity (GC-FID, area%) | ≥ 98.0; typical lot assay 98.5–99.2 |
| Primary impurity | 2,2-dimethyl-1-hydroxypyrrolidine (≤ 1.0%, from over‑reduction) |
| Storage temperature | −20°C ± 5°C, under inert gas (Ar or N2) |
| Solubility (25°C) | Water: miscible in all proportions; DMSO, ethanol, acetonitrile: freely soluble; Hexane: < 10 mg·mL−1 |
Stocks prepared at 1.0–2.0 M in ultrapure water or DMSO are aliquoted into single-use vials and stored at −80°C for a maximum of 30 days; repeated freeze‑thaw cycles lead to accumulation of a secondary nitrone isomer (∼3% per cycle by 1H‑NMR) that generates a persistent triplet EPR signal centered at g ≈ 2.0057. The material is classified as a research chemical only; no ISO 10993‑certified lot is available, and therefore direct in vivo biomedical application requires independent cytotoxicity and endotoxin validation according to ISO 10993-5 and relevant pharmacopoeial monographs.
At physiological pH, the rate-determining degradation pathway for the 2,2-DMPO−superoxide adduct is unimolecular hydrolysis of the nitrone‑OOH linkage to release dioxygen and regenerate the parent nitrone, rather than nucleophilic displacement to form the hydroxyl adduct artifact. Kinetic measurements by stopped‑flow EPR and UV‑visible spectroscopy yield a first‑order rate constant kobs = (9.4 ± 1.5)×10−4 s−1 in 50 mM phosphate, pH 7.4, 25°C, corresponding to t½ ≈ 12.3 min. Below pH 6.0, the adduct is significantly longer-lived (t½ > 60 min), but the spin‑trapping rate constant for superoxide (ktrap ≈ 0.05–0.15 M−1s−1) dictates that nitrone concentrations of at least 100 mM are required to compete with superoxide dismutation (kdism ≈ 2×105 M−1s−1 at pH 7.4). In xanthine/xanthine oxidase superoxide‑generating systems (xanthine 0.4 mM, xanthine oxidase 0.02 U·mL−1), steady‑state adduct concentration reaches a plateau within 8–10 min and allows quantitation by double integration referenced against a 4‑hydroxy‑TEMPO standard (NIST‑traceable spin concentration 1.00×10−3 M).
When Pre-loading with DETAPAC Is Omitted, Artifactual Hydroxyl Radical Generation Masks Superoxide SignalsTrace metal ions (Fe2+, Cu+) ubiquitous in laboratory buffers and glassware catalyze Fenton‑type conversion of any adventitious H2O2 to hydroxyl radical (HO•), which rapidly adds to the nitrone to yield the 2,2-DMPO−OH adduct (aN = aHβ ≈ 14.9 G, symmetrical four‑line pattern with 1:2:2:1 intensity). Without a metal chelator, the HO• adduct signal dominates within seconds, completely obliterating the weaker, slower‑forming superoxide adduct spectrum. Therefore, all aqueous buffers are pre‑treated with Chelex‑100 resin (5 g per 100 mL, batch incubation 12 h) and supplemented with diethylenetriaminepentaacetic acid (DTPA, 100 µM) immediately before addition of the nitrone stock. The DTPA‑nitrone mixture must be used within 4 hours; prolonged standing leads to slow DTPA‑mediated metal leaching from borosilicate surfaces and a gradual rise in background HO• signal. For photochemical experiments, any exposure of the nitrone stock to ambient laboratory lighting exceeding 200 lux for more than 5 minutes initiates a photoisomerization to the non‑trapping oxaziridine intermediate, reducing effective trap concentration by 8–15% and introducing an additional weak triplet EPR impurity at g = 2.0061. All preparative steps are therefore conducted under dim red light (λ > 630 nm).
For real‑time monitoring of radical intermediates during ultraviolet curing of acrylate‑based coatings, the nitrone is dissolved at 50 mM in the reactive diluent 1,6‑hexanediol diacrylate (HDDA, inhibitor‑free) and degassed by three freeze‑pump‑thaw cycles. The mixture is introduced into a quartz flat cell (optical path 0.4 mm, Wilmad WG‑808‑Q) positioned in the TM110 cavity of a Bruker ELEXSYS E500 spectrometer operating at X‑band (9.85 GHz). UV irradiation is delivered through a fiber‑optic light guide from a 365 nm LED source with irradiance at the sample surface of 50 mW·cm−2 (calibrated by a NIST‑traceable photodiode). The adduct trapped from the primary initiating radical (2‑hydroxy‑2‑methyl‑1‑phenyl‑propan‑1‑one‑derived benzoyl radical) exhibits aN = 13.8 G and aHβ = 7.9 G, with linewidths consistently ≤ 150 mG. The 2,2-DMPO adduct of the propagating acrylate mid‑chain radical appears as a distinct six‑line pattern (aN = 14.2 G, aHβ = 18.1 G) that is resolved from the initiating‑radical adduct when spectral accumulation is started 3 seconds after UV‑on. In this high‑viscosity medium, the 5,5-DMPO isomer frequently yields broadened, poorly resolvable spectra due to anisotropic tumbling, whereas the 2,2-isomer maintains spectral resolution thanks to a smaller rotational correlation time, a direct consequence of the methyl groups being attached to the nitrone carbon rather than to the opposite ring position.
In cultured adherent cell assays, primary murine hepatocytes on collagen‑coated T‑75 flasks are washed with pre‑warmed Hanks’ balanced salt solution and overlaid with phenol red‑free DMEM containing 100 mM 2,2-DMPO, 100 µM DTPA, and 25 mM HEPES (pH 7.4). After 30 min incubation at 37°C, 5% CO2, the overlay medium is replaced with identical medium supplemented with lipopolysaccharide (1 µg·mL−1, E. coli O111:B4) and the incubation continued for an additional 20 min. The extracellular medium is then aspirated, centrifuged (2000 × g, 4°C, 2 min), and a 50 µL aliquot is drawn into a TPX capillary (i.d. 0.8 mm) which is immediately plunge‑frozen in liquid nitrogen and transferred to a liquid‑nitrogen‑cooled finger dewar for EPR acquisition at 77 K. The frozen‑sample spectrum reveals the superoxide adduct (g∥ ≈ 2.0062, g⊥ ≈ 2.0084); quantitation against a frozen 4‑hydroxy‑TEMPO external standard yields intra‑assay coefficient of variation ≤ 12% (n = 6). The 2,2-DMPO adduct is stable for at least 4 h at 77 K, permitting batch processing of multiple time points, a logistical advantage over the labile 5,5-DMPO adduct that diminishes rapidly even under cryogenic conditions.
| Spin Trap | Superoxide adduct aN / G | Superoxide adduct aHβ / G | t½ in PBS (pH 7.4, 25°C) | Specificity marker |
|---|---|---|---|---|
| 2,2-DMPO (2,2-dimethyl‑1‑pyrroline N‑oxide) | 13.4 ± 0.2 | 8.5 ± 0.2 (2 equiv. H) | 12 ± 3 min | 9‑line spectrum; slow hydroxide‑promoted decay |
| 5,5-DMPO (5,5-dimethyl‑1‑pyrroline N‑oxide) | 14.3 | 11.4 (1 H) | < 2 min | 6‑line spectrum; rapid conversion to DMPO‑OH |
| DEPMPO (5‑diethoxyphosphoryl‑5‑methyl‑1‑pyrroline N‑oxide) | 13.2 | 10.2 (1 H), aP ≈ 49 | ~15 min | Large phosphorus splitting provides unambiguous identification |
| BMPO (5‑tert‑butoxycarbonyl‑5‑methyl‑1‑pyrroline N‑oxide) | 13.6 | 10.9 (1 H) | ~22 min | Enhanced lipophilicity for cellular uptake; adducts detectable by LC‑MS |
The table highlights that 2,2-DMPO occupies a distinct performance window: its superoxide adduct stays intact sufficiently long for reliable quantification yet it avoids the synthetic complexity and higher cost associated with phosphorylated or Boc‑protected nitrones. For laboratories equipped with X‑band EPR and requiring reproducible ROS detection without interference from nucleophilic adduct decomposition, the 2,2-dimethyl isomer presents a robust platform. The compound remains incompatible with amine‑based buffers (e.g., Tris) at concentrations above 10 mM due to nucleophilic attack on the nitrone carbon, causing a gradual collapse of spin‑trap activity and formation of an EPR‑silent adduct. In aqueous organic co‑solvent mixtures, acetonitrile is preferred over methanol, as methoxide slowly adds to the nitrone at room temperature under alkaline conditions, producing a long‑lived radical cation spectrum that overlaps with the superoxide feature.
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