Unless noted, materials and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and used without further purification. GdCl3·6H2O and 1,4,7,10-tetraazacyclododecane (cyclen) were purchased from Strem Chemicals (Newburyport, MA) and used without further purification. Unless noted, all reactions were performed under a nitrogen atmosphere. THF, acetonitrile, and dichloromethane were purified using a Glass Contour Solvent system. Deionized water was obtained from a Millipore Q-Guard System equipped with a quantum Ex cartridge (Billerica, MA). Thin-layer chromatography (TLC) was performed on EMD 60F 254 silca gel plates. Visualization of compounds was accomplished using either an iodoplatinate or UV light. Standard grade 60 Å 230–400 mesh silca gel (Sorbent Technologies) was used for flash column chromatography.
1H and 13C NMR spectra were obtained on a Bruker 500 MHz Avance III NMR Spectrometer or a Varian Inova 400 MHz NMR Spectrometer with deuterated solvent as noted. Electrospray ionization mass spectrometry (ESI-MS) spectra were taken on a Varian 1200 L single-quadrupole mass spectrometer. High resolution mass spectrometry data was aquired on an Agilent 6210 LC-TOF (ESI, APCI, APPI). Analytical reverse-phase HPLC-MS was performed on a Varian Prostar 500 system with a Waters 4.6 × 250 mm 5 μM Atlantis C18 column. This system is equipped with a Varian 380 LC ELSD system, a Varian 363 fluorescence detector, and a Varian 335 UV-Vis detector. Preparative runs were performed on a Water 19 × 250 mm Atlantis C18 Column. The mobile phases consisted of Millipore water (A) and HPLC-grade acetonitrile (B). HPLC method 1: 0–5 min 100% A, 5–24:08 min 57.5% A, 24:08–30 min 0% A, 30–35 min 0% A, 35–40 min 100% A.
Determination of r1 was accomplished using a Bruker minispec 60 MHz (1.41 T) magnet and a Varian Inova 400 MHz (9.4 T) NMR Spectrometer. At 1.41 T the T1 relaxation times were determined using an inversion recovery method while at 9.4 T a saturation recovery method was used. The saturation recovery method utilized a 2 second presaturation pulse centered on the water frequency. All measurements were done at 37 °C at an approximate 1 mM concentration of CA in 10 mM DPBS purchased from Invitrogen.
NMRD measurements were performed with a Stelar Spinmaster FFC-2000-1T fast field cycling relaxometer in the 0.01–40 MHz proton Larmor frequency range at 298 and 310 K. Standard field cycling protocol was used. Longitudinal water proton relaxation rates were obtained with an error smaller than 1%. Proton nuclear magnetic relaxation dispersion (NMRD) profiles were obtained by plotting proton relaxation rates as a function of applied magnetic field after subtraction of the diamagnetic contribution of buffer alone and normalization to 1 mM Gd(III) concentration.
(6) 1-azido-3-chloropropan-2-ol was synthesized as described by Ingham et al with the following modifications.63 Diethyl ether was used in the place of dichloromethane in the procedure. The final product was not distilled as in the literature procedure but simply extracted into ether and evaporated. The product was used directly in subsequent reactions. Caution: Safe handling procedures for perchlorates and small molecule azides should be reviewed before performing this reaction, as there is a danger of explosion if heat, friction, or shock is applied.
Tri-tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate hydrobromide (trist-butyl-DO3A HBr) was synthesized according to the procedure of Oskar with the following modifications.64 To a 500 mL RB flask was added 10.279 grams (59.8 mmoles) of cyclen to this was added 14.8405 grams (179.2 mmoles) of NaOAc. The solids were dissolved in 180 mL of dimethylacetamide (DMA). The reaction was cooled to 0 °C with ice and 26.5 mL (179.3 mmol) of tert-butyl bromoacetate dissolved in 70 mL of DMA was added drop wise over 40 minutes at 0 °C. The reaction was allowed to warm to RT and stirred for two days and was poured into a solution of 16.6 grams of KBr in 1000 mL of H2O. The solution was brought to a basic pH with 17.7g (3.5 eq) of NaHCO3. (Caution: A large amount of gas is produced.) Add 10 mL of ether to initiate precipitation of the HBr salt of tris-t-butyl-DO3A. The final white to off white powder was filtered and dried under vacuum to give a yield of 21.4976 grams (60% yield). 1H NMR (500 MHz, CDCl3) δ 3.56 – 2.57 (m, 21H), 1.46 (d, J = 3.6 Hz, 27H). 13C NMR (126 MHz, CDCl3) δ 170.53, 169.63, 81.86, 81.71, 77.29, 77.04, 76.78, 58.22, 51.31, 51.12, 49.14, 47.53, 28.23, 28.19, 28.03, 0.00.