Preparation of pH-sensitive probes
Pyrazole-substituted NIR dyes were prepared according to the modified published procedure . Briefly, the precursor chloro dye IR-820 (20 mg) was dissolved in oxygen-free water (5 mL; the reaction is summarized in Eq. 1). An appropriate pyrazole derivative, such as pyrazole-substituted boronic acid (for dye 1) or boronic esters (for dye 2 and 3) in 1 mL of DMF was added along with Pd(PPh3)4 (3 mg). The reaction mixture was refluxed and the progress was monitored by TLC on RP C18 silica gel. The reaction was terminated when all IR820 dye was consumed (3 h for dye 2, and 16 h for dyes 1 and 3). The reaction mixture was then cooled to room temperature and filtered through Celite. Solvents were evaporated under vacuum and the residue was purified by column chromatography on RP C18 silica gel using water-methanol gradient elution. Fractions containing the desired product (determined by LCMS) were combined, the solvents were evaporated, and the residue was lyophilized. The resulting solid residue was redissolved in a small amount of methanol and triturated with diethyl ether. The precipitate was further washed with diethyl ether twice to afford pure dyes 1–3 as green powder.
Dye 1. Yield, 12%. ESI-MS, m/z: 949 (MH+); 1H NMR (400 MHz, CDCl3) δ ppm: 7.97–7.92 (7H, m), 7.66–7.62 (6H, m), 7.57–7.53 (5H, m), 7.45–7.42 (3H, m), 6.35 (2H, d, J = 14.4 Hz), 5.57 (2H, s), 4.19 (4H, m), 2.86 (4H, t, J = 6.8 Hz), 2.71 (4H, m), 2.01–1.91 (10H, m), 1.47 (12H, s); Abs/em (MeOH): λmax = 800 nm, λem = 821 nm.
Dye 2. Yield, 78%. ESI-MS, m/z: 914 (MH+); 1H NMR (400 MHz, CDCl3) δ ppm: 8.04 (2H, d, J = 8.8 Hz), 7.95–7.91 (4H, m), 7.74–7.66 (4H, m), 7.57–7.54 (4H, m), 7.41 (2H, t, J = 7.4 Hz), 6.26 (2H, d, J = 14 Hz), 4.21 (6H, m), 2.87 (4H, t, J = 7 Hz), 2.72 (4H, m), 2.41 (1H, m, J = 6.8Hz), 2.00–1.92 (10H, m), 1.67 (12H, s), 1.12 (6H, d, J = 6.8 Hz); Abs/em (MeOH): λmax = 802 nm, λem = 822 nm.
Dye 3. Yield, 97%. ESI-MS, m/z: 887 (MH+); 1H NMR (400 MHz, CDCl3) δ ppm: 8.53 (2H, d, J = 14 Hz), 8.24 (2H, d, J = 8.4 Hz), 8.01–7.96 (4H, m), 7.66–7.60 (4H, m), 7.47 (2H, t, J = 7.2 Hz), 6.35 (2H, d, J = 14.4 Hz), 4.32 (4H, m), 2.89 (4H, t, J = 6.8 Hz), 2.77 (4H, m), 2.17–1.92 (28H, m); Abs/em (MeOH): λmax = 760 nm, λem = 770 nm, pH < 5; λmax = 820 nm, λem = 824 nm, pH > 5.
Steady-state optical measurements
Absorption spectra were recorded on a DU 640 UV–Visible spectrophotometer (Beckman Coulter, Brea, CA), and fluorescence spectra were recorded on a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon, Inc., Edison, NJ). All fluorescence measurements were conducted at room temperature and were recorded at excitation of 700 nm and emission scan from 715 to 950 nm.
For titrations, the compounds were predissolved in MeOH maintaining the absorbance below 0.4 units at the maximum absorption. The methanolic solution was then either acidified with trifluoroacidic acid (TFA) or basified with triethylamine (TEA). The pH of the solution was measured after diluting with NaCl (0.1 m) solution (1:1 vol) to provide relatively constant ionic strength using Accumet pH meter AB15 (Fisher Sci. Pittsburgh, PA). At each pH point, absorption and fluorescence measurements were determined. The pKa value was calculated from the fluorescence intensity ratio between 770 and 820 nm emission peaks using sigmoidal dose–response curve fit implemented in Prism 5.0 software (GraphPad Software Inc., La Jolla, CA). The relative fluorescence quantum yields of the dye were determined at different pH using the equation:
where ΦF(X) is the fluorescence quantum yield, A is the absorbance, F is the area under the emission curve, n is the refractive index of the solvents used in the measurement and the subscripts s and x represent the reference and unknown parameters respectively. Indocyanine green (ICG; Akorn, Inc. Decatur, IL) was used as a reference standard, which has a quantum yield of 0.09 in MeOH at 700 nm excitation .