Fig. 2. Synthetic route of GC4AOEG and fluorescence titrations. (A(a)) NaH, dry DMF, and 75 °C; (b) HNO3, AcOH, dry CH2Cl2, and r.t.; (c) SnCl2·2H2O, C2H5OH/AcOEt (1 : 1, v/v), and reflux; (d) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et3N, AgNO3, dry CH2Cl2, and r.t.; (e) SnCl4, AcOEt, and r.t. (B) Direct fluorescence titration of 0.5 μM EY with different concentrations of GC4AOEG (up to 13.8 μM) in HEPES buffer (10 mM, pH = 7.4), and λex = 517 nm. (Inset) The associated titration curve at λem = 537 nm and best fit according to a 1 : 1 binding stoichiometry. (C) Competitive fluorescence titration of GC4AOEG·EY (4.0/0.5 μM) with UFH (up to 8.4 μM in the concentration of monomer units of UFH), and λex = 517 nm. (Inset) The associated titration curve at λem = 537 nm and best fit according to a n : 1 competitive binding model, where n = 0.88.
