Development of Affinity-Based Chemical Probes for Fluorescence Detection of Human Carbonic Anhydrases

Abstract

The development of small molecule affinity-based chemical probes as research tools for studying the role of carbonic anhydrases (CAs) in their wider biological context has become an active field of research owing to an increasing awareness of the therapeutic relevance of this enzyme family, particularly in diseases such as glaucoma (CA II) and solid tumors (CA IX, CA XII). High CA isozyme selectivity, low nonspecific labeling, and efficient labeling yield are the characteristics of an ideal chemical probe, however achieving an effective balance of all three properties is challenging. A panel of chemical probes for two-step labeling of CA II or CA IX has been designed to study the impact of probe structural features on the efficiency and specificity of CA-selective labeling when in a challenging environment, including protein mixtures, cell lysates, or live cells. The panel comprised Generation 1 probes (P1 and novel probes P2–P5), Generation 2 linear PAL probes (P6 and novel probes P7–P15), and Generation 3 branched PAL probes (novel probes P16–P20).