1-{[(7-Methoxy-2-Oxo-2H-Chromen-4-Yl)Acetyl]Oxy}Pyrrolidine-2,5-Dione

The compound designated 1-{[(7-Methoxy-2-Oxo-2H-Chromen-4-Yl)Acetyl]Oxy}Pyrrolidine-2,5-Dione — systematically a succinimidyl ester of 7-methoxycoumarin-4-acetic acid — functions as a pre-activated, amine-reactive fluorescent probe with excitation and emission maxima in the near‑UV/visible boundary. Catalogued under the internal code F-100, the product is supplied as a lyophilised, off‑white powder in septum‑sealed amber vials containing 5 mg, 25 mg, or 100 mg under an argon atmosphere. The active ester operates through nucleophilic substitution at the carbonyl carbon of the acyloxy linkage, where the pyrrolidine-2,5-dione leaving group is displaced by primary aliphatic amines (ε‑lysine, N‑terminal amino groups, amine‑modified oligonucleotides) to yield a stable amide bond and the fluorophore 7-methoxy-2-oxo-2H-chromen-4-yl-acetyl conjugate. No carbodiimide activation step is required, streamlining derivatisation protocols.

Fluorescence excitation and emission maxima in aqueous buffer

Excitation and emission spectra obtained on a Horiba Fluorolog-3 spectrofluorometer equipped with a 450 W xenon lamp and double‑grating monochromators (bandpass set to 4 nm for excitation and 4 nm for emission) reveal an absorption cluster centred at 325 nm and a structureless emission band peaking at 400 nm in 0.1 M sodium phosphate, pH 7.4. Spectral correction for wavelength‑dependent detector response was performed using a calibrated tungsten‑halogen lamp according to ASTM E388-04, and all wavelength values are referenced to NIST‑traceable holmium oxide and didymium glass filters. The Stokes shift of 75 nm minimises inner‑filter effects and permits effective separation of excitation light from collected fluorescence using standard long‑pass filters with cut‑on wavelengths of 375 nm. Fluorescence quantum yield (Φ) determined by the comparative method of Williams et al. (1983) against quinine sulphate dihydrate in 0.1 M H₂SO₄ (Φ = 0.54) yields a value of 0.20 ± 0.02 (n = 3 independent batches).

Labelling of lysine ε‑amino groups in proteins at neutral to slightly alkaline pH exploits the rapid aminolysis of the NHS ester, yet competitive hydrolysis imposes a narrow processing window. In 0.1 M sodium bicarbonate buffer, pH 8.5, at 25 °C, the pseudo‑first‑order hydrolysis half‑life (t₁/₂) of the ester is 7.5 min (± 0.8 min, as monitored by the decrease in absorbance at 325 nm in a stopped‑flow accessory coupled to an Agilent Cary 60 UV‑Vis spectrophotometer; mixing dead time 2 ms, 1 cm path‑length quartz flow cell). The second‑order rate constant for aminolysis with glycine as a model nucleophile under identical conditions is 38 M⁻¹ s⁻¹, while the competing hydrolysis rate constant is 1.5 × 10⁻³ s⁻¹ (pH 8.5, 25 °C). This kinetic partitioning dictates that to achieve >85 % incorporation of fluorophore onto a target protein at 1 mg mL⁻¹ in a 100 µL reaction volume, a 10‑ to 20‑fold molar excess of the ester is typically added, with labelling quenched after 45 min by addition of Tris‑HCl, pH 7.0, to a final concentration of 50 mM. Prolonged exposure beyond 60 min results in net loss of active ester due to hydrolysis and can introduce heterogeneous labelling artefacts detected by matrix‑assisted laser desorption/ionisation time‑of‑flight (MALDI‑TOF) mass spectrometry as a broadened molecular envelope. A deeper examination of reaction vessel design reveals that batch‑to‑batch variance in labelling stoichiometry drops from ±12 % to ±4 % when the ester solution in anhydrous DMF is added dropwise to a gently vortexed protein solution at 4 °C, likely due to suppressed local pH excursions and mixing‑limited hydrolysis.

When the NHS leaving group confronts aqueous hydrolysis: kinetic partitioning

At pH values below 7.0, the amine group of lysine is largely protonated, reducing nucleophilicity and allowing hydrolysis to dominate; thus, the practical conjugation pH window is confined to 7.5–8.5, where the fraction of deprotonated amine is appreciable (~20 % at pH 8.0) and the spontaneous decomposition of the NHS ester is still manageable. Operation at pH 8.5 yields a kinetic selectivity factor (k_aminolysis/k_hydrolysis) of approximately 25 for a typical antibody, but this ratio collapses to ~5 when the target amine concentration drops below 10 µM. This concentration‑dependent attenuation is a primary differentiator from sulfonated indocyanine NHS esters, whose charged sulfonate groups weakly promote hydrophobic shielding of the ester linkage and extend hydrolysis half‑life to 20–30 min under the same conditions. Consequently, while the methoxycoumarin NHS ester is preferred for labelling in organic‑aqueous mixtures or solid‑phase contexts where local amine concentration remains high, in dilute solution format a careful cold‑start protocol with pre‑chilled buffers (2–4 °C) is mandatory to maintain label integrity.

For conjugation to solid‑phase‑synthesised peptides after TFA cleavage, the ester is dissolved in anhydrous N,N‑dimethylformamide (moisture content < 50 ppm by Karl Fischer titration) at a concentration of 50 mg mL⁻¹ and added to the peptide dissolved in 0.1 M sodium carbonate, pH 8.5, at a 2‑ to 3‑fold molar excess. Incubation proceeds at 4 °C for 2 h; reverse‑phase HPLC monitoring using a C18 column and a linear gradient of acetonitrile in 0.1 % TFA shows complete consumption of the NHS ester peak (retention time 12.3 min) and appearance of the fluorescent peptide adduct.

How does its Stokes shift compare to fluorescein isothiocyanate?

The Stokes shift of 75 nm distinguishes this coumarin derivative from the narrower shift of fluorescein isothiocyanate (FITC, ~28 nm, excitation 495 nm, emission 519 nm). This larger gap reduces self‑quenching in heavily labelled conjugates and allows excitation at 325 nm while collecting emission through a 400 nm band‑pass filter, effectively eliminating bleed‑through from the excitation beam. In multiplexed experiments, the blue fluorescence can be combined with red‑emitting dyes without spectral overlap, a feature exploited when the 7‑methoxy‑2‑oxo‑2H‑chromen‑4‑yl fluorophore is used alongside Cy5 NHS ester (647/665 nm) in dual‑colour protein microarrays. Additionally, because the absorbance maximum is distant from the aromatic protein absorbance envelope (280 nm), minimal inner‑filter correction is required when measuring fluorescence at protein concentrations up to 5 mg mL⁻¹. Under continuous illumination with a 100 W high‑pressure mercury lamp filtered to the 330–380 nm band (irradiance 25 mW cm⁻² at the sample plane), the photobleaching half‑life of a 10 µM solution of the fluorophore (as the free acid) in 0.1 M phosphate, pH 7.4, measured on a BMG LABTECH FLUOstar plate reader, averages 22 min (n = 6), contrasting with 6 min for FITC under analogous conditions. This enhanced photostability, although lower than that of Alexa Fluor dyes, is sufficient for confocal laser scanning microscopy acquisitions up to 60 frames without noticeable signal degradation.

Batch consistency evaluated via reverse‑phase HPLC and relative quantum yield

Each manufactured lot is subjected to analytical HPLC on a Phenomenex Luna C18(2) column (5 µm, 250 × 4.6 mm) using a gradient of acetonitrile in 0.1 % (v/v) trifluoroacetic acid/water. Detection at 325 nm reveals a single major peak at retention time 12.3 min (± 0.2 min) that integrates to ≥ 95.0 % peak area. Free 7‑methoxy‑2‑oxo‑2H‑chromen‑4‑yl‑acetic acid (retention time 9.8 min) is the sole quantifiable impurity and is limited to ≤ 3.0 %. The absorbance ratio A₃₂₅/A₂₈₀ is additionally monitored and must fall between 2.8 and 3.1 as an indicator of chromophore integrity. Relative quantum yield is measured against the quinine sulphate reference for every tenth batch and must fall within 0.18–0.22 to pass release testing. The test methodology comports with ISO 17025:2017 quality management requirements, and a lot‑specific Certificate of Analysis enumerates identity (¹H NMR, 400 MHz, DMSO‑d₆), purity, excitation/emission maxima, solubility in DMF, residual moisture by coulometric Karl Fischer titration, and microbial limits (≤ 10 CFU g⁻¹).

Preparation of stock solutions requires anhydrous DMF previously dried over activated 3 Å molecular sieves for at least 24 h. Contact with amine‑containing buffers (Tris, glycine, ethanolamine) must be avoided until the conjugation quenching step. Residual moisture in DMSO or DMF leads to rapid ester cleavage; a control hydrolysis curve can be generated by monitoring absorbance at 325 nm after addition of 10 µL of stock solution to 1 mL of 0.1 M phosphate buffer, pH 7.4 — the absorbance at 325 nm decays with a half‑life ≤ 10 min if solvent moisture is < 100 ppm. Use of desiccated containers and septum‑piercing needles under nitrogen overpressure is recommended for multi‑draw situations. The lyophilised powder is incompatible with strong reducing agents, metal chelators that may strip the fluorophore from its conjugate, and repeated freeze‑thaw cycles after reconstitution; single‑use aliquots prepared under inert gas are consequently advised. No published degradation data exist for storage excursions above −10 °C, and exposure to ambient humidity (RH > 60 %) for more than 30 min during vial opening has been observed to increase the hydrolysed acid fraction by 2–3 % in subsequent HPLC analysis. These operational boundaries define the product’s practical handling envelope in production‑scale labelling campaigns.