Bovine carbonic anhydrase 2

Bovine carbonic anhydrase II and bovine ubiquitin were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. LC/MS grade water and methanol were obtained from Fisher Chemical (Hampton, NH, USA). For electrospray ionization, aqueous solutions containing 10–20 μM protein, 49.5% water, 49.5% methanol, and 1% formic acid (v/v) were prepared.

All synthetic reagents were purchased from commercial sources and used without further purification unless otherwise noted. All reactions were run under N₂ atmosphere unless otherwise noted. All reverse-phase purification was performed using a Biotage Isolera One Flash Chromatography Instrument. NDM-1, VIM-2, and IMP-1 were expressed and purified as described previously. [20 –22 (link)] Bovine carbonic anhydrase II and alkaline phosphatase were purchased from Sigma Aldrich. Phosphotriesterase was kindly provided by Prof. Frank Raushel from Texas A&M University. For all spectroscopic studies, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0 containing 10 μM ZnSO₄ was prepared as the buffer. ¹H and ¹³C NMR spectra were recorded on a 400 MHz Agilent MR Spectrometer or a 400 MHz Bruker AVANCE NEO400 Spectrometer, prepared in deuterated acetonitrile, acetone, or methanol. The residual solvent peaks were used as an internal standard. Spectroscopic studies were performed using an Agilent Cary 60 UV–Vis spectrophotometer. Fluorescence spectroscopic measurements were made using an Agilent Cary Eclipse fluorescence spectrofluorometer. Confocal imaging was performed on a Zeiss 710 Laser Scanning Confocal Microscope. Data analysis was performed using GraphPad Prism Version 8.4.3.