Bodipy fl nhs ester

Commercially available compounds were used without further purification unless otherwise stated. Bio Ultra Polyethylene Glycol (PEG) 300, Ethyl 4-nitrobenzoate, potassium carbonate (K₂CO₃), triethylamine (NEt₃) trifluoroacetic acid (TFA) and 4-nitroquinolone-1-oxide (4NQO) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC and Liquid Chromatography-Mass Spectrometry (LC–MS) grade MeCN were obtained from Fischer Scientific (Hampton, NH). Water (> 18.2 MΩ cm⁻¹ at 25 °C) was obtained from an Alpha-Q Ultrapure water system from Millipore (Bedford, MA). 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (PARP-NH precursor) was purchased from AA blocks (San Diego, CA) and purified by HPLC using the method described previously in general methods before its use and further synthesis. BODIPY-FL NHS-ester was purchased from Invitrogen, Carlsbad, CA without further purification. PARPi-FL was kept as a 1.5 mM stock solution in BioUltra PEG 300 and diluted to the final working concentration for the respective experiments.

The green fluorescent dye BODIPY-FL NHS-ester (Invitrogen, Carlsbad, CA) was conjugated to 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one and purified by preparative HPLC (Waters’ XTerra C-18 5 μm column, 7 mL/min, 5 to 95% of acetonitrile in 15 min) to afford PARPi-FL in 70–79% yield as a red solid. Analytical HPLC analysis (Waters’ Atlantis T3 C18 5 μm 4.6 × 250 mm column) showed high purity (>97%) of the imaging agent. The identity of PARPi-FL was confirmed using ESI-MS (MS(+) m/z = 663.4 [M + Na]+).

Synthesis of the optical imaging agent PARPi-FL was carried out analogous to what was previously described [32 (link)]. Briefly, the green fluorescent dye BODIPY-FL NHS-ester (Invitrogen, Carlsbad, CA) was conjugated to 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, followed by purification via preparative HPLC (Waters’ XTerra C-18 5 μm column, 7 ml/min, 5% to 95% of acetonitrile in 15 min). PARPi-FL was obtained in 70–79% yield as a red solid in >97% purity. The identity of PARPi-FL was confirmed using ESI-MS (MS(+) m/z = 663.4 [M+Na]+). For in vivo imaging studies, PBS (117 μl) was slowly added to an aliquot of PARPi-FL (50 μg, 75 nmol) in 50 μl of poly(ethylene glycol) (PEG300, Sigma-Aldrich, St. Louis, MO) to obtain a final injection volume of 167 μl.