Synthesis and Characterization of a Calix[4]arene Derivative

All reagents and anhydrous solvents used during the synthesis were of commercial quality. 5,11,17,23-Tetra-(t-butyl)-25-hydroxy-26,27,28-tripropoxy-calix[4]arene (6) was prepared according to the literature (Gutsche and Iqbal, 1990 (link)). The lower rim of the calix[4]arene backbone was modified in accordance to the described procedures for the propylation (7a,b) of the hydroxylic groups (Gutsche and Lin, 1986 (link)), as well as condensation of one of the hydroxyls (8a) with N-(3-bromo)propylphthalimide (Lalor et al., 2007 (link)). The steps, preceding the final conjugation with the DOTA-units (Schühle et al., 2009 (link)) included nitration (9a,b) of the upper rim of the calix[4]arene backbone (Kelderman et al., 1992 (link)) followed by the reduction (10a,b) of the nitro groups to the amines (Klimentová and Vojtíšek, 2007 (link)). ¹H NMR spectra were recorded at 25°C on Bruker Avance-400 spectrometer operating at 400.13 MHz and analyzed using Bruker™ TopSpin 2.1 software. The chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as an internal reference. Ultra-filtration was performed with a Millipore stirred cell using an Amicon cellulose acetate membrane. All HPLC measurements were carried out on a Shimadzu LC-20 system consisting out of an LC-20AT pump, Sil-20A HT autosampler, CTO-20AC column oven, SPD-M20A PDA detector, CBM-20A controller, and a Waters Fraction Collector III; data processing was carried out using Shimadzu Lab Solutions. Both analytical and preparative methods were carried out operating at 40°C using eluents A: H₂O (95%), AcCN (5%), TFA (0.1%) and B: H₂O (5%), AcCN (95%), TFA (0.1%). Mobile phase gradient started with 75% A and 25% B, after 18 min followed by a change linear to 58% A and 42% B, after 2 min a change linear to 100% B, which was hold for 0.5 min and then chanced back to starting conditions stabilized for 3.5 min. Analytical measurements used a Waters Xterra 4.6 × 150 mm column and an injection volume of 1 μL, flow was 1 mL/min. Preparative HPLC was performed using Xbridge™ PrepShield RP18-OBD C18-19 × 150 mm column. Mass spectrometry analysis was done with electron spray ionization technique on Waters Qtof Premier MS using a NE-1000 syringe pump for direct infusion; data processing was carried out using Waters Masslynx. Qualitative luminescence measurements were done on a Jasco J815 CD spectrometer using 100 μL of sample in a 3 × 3 mm quartz cuvette. UV absorption spectra were measured on a UV2401 PC Shimadzu spectrometer. For quantitative luminescence measurements, the samples (either powders or solutions in Milli-Q water at concentration 2, 0.2, and 0.04 mM) were placed into 2.4 mm quartz capillaries and measured on a Horiba-Jobin-Yvon Fluorolog 3 spectrofluorimeter equipped with visible (220–800 nm, photon-counting unit R928P) and NIR (950–1,450 nm, photon-counting units H10330-45 from Hamamatsu or DSS-IGA020L Jobin-Yvon solid-state InGaAs detector, cooled to 77 K) detectors. All spectra were corrected for the instrumental functions. Luminescence lifetimes of Tb^III-complexes were determined under excitation at 300 nm provided by a Xenon flash lamp monitoring the signal at 545 nm (⁵D₄→⁷F₅ transition). Quantum yields were measured according to an absolute method using an integration sphere (GMP SA). Each sample was measured several times under slightly different experimental conditions. Estimated experimental error for quantum yields determination is 10%. Nile red (NR) fluorescence measurements were performed on a Jasco J-815 CD spectrometer. The temperature was controlled using a Jasco PFD 4252/15 Peltier temperature unit. All samples contained Nile red in 2 μM concentrations and were excited at 550 nm. The maximum Nile red emission wavelength (λ_max) was determined as a function of the calix[4]arene concentration.