Chemosensing strategies : utilizing the novel sulfonamidohydroxyquinoline amino acid Sox

Abstract

Modular peptide-based fluorescent chemosensors utilizing the chelation-sensitive fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline are powerful tools for sensing Zn²⁺ and for sensing protein kinase activity. This signaling component is prepared as the protected amino acid derivative Fmoc-Sox-OH, and integrated into peptide sequences. Selective and tunable chemosensors for Zn²⁺ can afford qualitative and quantitative information about the presence, distribution and concentration of this biologically-important metal ion. Nineteen synthetic peptides incorporating Sox exhibit a range of affinities for Zn²⁺ through variation of the type and number of Zn²⁺ ligands, ligand arrangement and the [beta]-turn sequence that acts as a preorganization element between the ligands. The binding stoichiometry and fluorescence response to pH changes and various relevant competing metal ions was carefully characterized. Eleven of these sequences form only 1:1 complexes with Zn²⁺ and their affinities range from 10 nM to nearly 1 [mu]M. When used in concert, the relative intensities of different chemosensor readouts can provide Zn²⁺ concentration information in a valuable range. This modular scaffold is useful for ratiometric sensing when an additional fluorophore is incorporated in the peptide sequence. New methods to quantify protein kinase activities are critical for understanding biological regulatory pathways. Fluorescent chemosensors of protein kinase activity utilizing Sox and physiological Mg²⁺ concentrations report phosphorylation with dramatic fluorescence changes in a continuous, high-throughput sensing format.

(cont.) The chemosensor comprises a small sensing module, containing Sox and a [beta]-turn sequence, appended to an optimized peptide substrate for the target kinase. The Mg²⁺ -binding affinity of the product phosphopeptide is much greater than the substrate peptide, which results in a large fluorescence increase upon phosphorylation. Notably, the reactivity of substrates is not affected by introduction of the sensing module on either side of the serine, threonine or tyrosine to be phosphorylated. Further, a homogeneous kinase assay utilizing these probes was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase in unfractionated cell lysates. These kinase chemosensors are powerful tools for studying the activity of recombinant kinases in vitro and endogenous kinases ex vivo.