Benzoic Acid, 5-[(2-Aminoethyl)dithio]-2-nitro- (2010)

[71899-86-0] C9H10N2O4S2 (MW 274.32) InChI = 1S/C9H10N2O4S2/c10-3-4-16-17-6-1-2-8(11(14)15)7(5-6)9(12)13/h1-2,5H,3-4,10H2,(H,12,13) InChIKey = DSNFVSIOSWJQBV-UHFFFAOYSA-N (reagent is used for the quantitation of sulfhydryl groups in proteins and other organic compounds1, 5, 7; reagent can also be exploited to facilitate synthesis of heterobifunctional disulfide-bridged conjugates2-4, 6, 8, 9 and to protect thiol, amine, or hydroxyl groups) Alternate Names: 5-[(2-aminoethyl)dithio]-2-nitrobenzoate (zwitterionic form), ADNB, 5-[(2-aminoethyl)dithio]-2-nitrobenzoic acid. Physical Data: mp 215–221 °C (decomposition), bp 481.4 ± 45.0 °C, d 1.54 ± 0.1 g cm−3, fp 245.0 ± 28.7 °C, log P 2.856 ± 0.541 (at 25 °C), and log D 0.34 (at pH 7 and 25 °C). Solubility: soluble in aqueous and mixed solvent systems such as water at pH 7, water (pH 7)/CH3CN, MOPS buffer [3-(N-morpholino)propanesulfonic acid (pH 8.5)]/DMF, CHES buffer [2-(N-cyclohexylamino)ethane sulfonic acid (pH 8.6)]/DMSO, and absolute ethanol/TEA. Form Supplied in: light yellow crystals (typical impurity can be 5-thio-2-nitrobenzoate, TNB). Analysis of Reagent Purity: mp (215–221 °C); single peak upon C18 analytical reverse-phase (RP)-HPLC at wavelengths (λ) = 328 and 412 nm; 1H NMR (200 MHz, D2O): δ 7.95 (d, 1H, J = 8.7 Hz), 7.61 (dd, 1H, J = 8.7, 2.2 Hz), 7.58 (d, 1H, J = 2.2 Hz), 3.18 (t, 2H, J = 6.5 Hz), 2.92 (t, 2H, J = 6.7 Hz); 13C NMR data are not available; FAB-LRMS (m/z): (M + H)+ calcd. for C9H10N2O4S2, 275.34; found, 275.0; UV/Vis: λmax = 328 nm (ɛ∼8730 M−1 cm−1, 45 μM ADNB in 50 mM PBS at pH 7.4). Purity can be confirmed by the absence of typical UV/Vis spectrum of TNB (λmax = 412 nm and ɛ = 14,150 M−1 cm−1). Preparative Methods: 5-[(2-aminoethyl)dithio]-2-nitro-benzoic acid (ADNB) has been synthesized according to a variety of related protocols from 2-mercaptoethylamine/HCl (MEA) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB or Ellman's reagent) as starting materials, but with different bases (NaHCO3, Na2CO3, NaOH, and TEA), solvent systems (water, MOPS buffer/DMF, CHES buffer/DMSO, and water/CH3CN), temperatures (20 °C to rt), and durations of reaction (2–14 h) (eq 1).1-9 In general, MEA (1 equiv) and DTNB (1 equiv) are dissolved in argon-purged water (minimum 15 min before use) at room temperature and adjusted to pH 7.4 (to pH 8) with argon-purged saturated NaHCO3 (minimum 15 min before use) while bubbling with argon. The reaction mixture is stirred under argon at room temperature for 2 h. The progress of the reaction is monitored using analytical RP-HPLC at λ = 328 and 412 nm (A = 0.1% TFA in water pH 2; B = 90% CH3CN/10% water containing 0.09% TFA; C18 column, flow rate = 1 mL min−1). After placing the flask in an ice bath, the solid precipitate is collected by filtration and washed with cold water, methanol, CHCl3, and finally cold water again. (1) Purification: ADNB can be purified by preparative RP-HPLC (A = 0.1% TFA in water, pH 2; B = 90% CH3CN/10% water containing 0.09% TFA; C18 column, flow rate = 26 mL min−1; monitored at λ = 328 nm) and lyophilized to yield light yellow powder. It can also be purified by crystallization in water to yield light yellow crystals. Handling, Storage, and Precautions: handle with adequate ventilation in a fume hood. Store under argon atmosphere at −20 °C in order to prevent autoxidation. Avoid breathing dust and contact with eyes, skin, and clothing, since it may cause respiratory tract, eye, and skin irritation. Carcinogenic and mutagenic data are not available.