STRUCTURE-BASED DESIGN OF SYNTHETIC AZOBENZENE LIGANDS FOR STREPTAVIDIN

A structure-based design strategy was used to elaborate modifications of the dye 2-[(4'-hydroxyphenyl)-azo]benzoate (HABA) in order to increase its affinity for the biotin-binding site of Streptavidin. Analogs of HABA incorporating methyl, tert-butyl, and methoxy hydrophobic substituent groups, as well as the compound in which a naphthyl ring was substituted for the phenyl ring, bound to streptavidin with up to 165-fold greater affinity than unmodified HABA. Binding was most enhanced for HABA derivatives with methyl substituents at the 3'- and 5'-hydroxyphenyl ring positions, where the free energy of binding increased from -5.27 kcal/mol for HABA to -7.23 kcal/mol for 2-[(3'-methyl-4'-hydroxyphenyl)azo]benzoate (3'-methyl-HABA), and to -8.29 kcal/mol for 2-[(3',5'-dimethyl-4'-hydroxyphenyl) azo]benzoate (3',5'-dimethyl-HABA). Measurements of ligand binding energetics using isothermal titration calorimetry indicate that the increases in ligand affinity arise primarily from favorable entropy contributions to the free energy of ligand binding. High-resolution X-ray structural studies (3'-methyl-HABA, space group I222, a = 95.2 Angstrom, b = 106.5 Angstrom, c = 47.9 Angstrom, crystallographic R-value = 0.17 to 1.8-Angstrom resolution; 3',5'-dimethyl-HABA, R = 0.18 to 1.65-Angstrom resolution) suggest that both water displacement from the ligand binding site and retention of ligand flexibility in the bound state may contribute to overall favorable entropy changes on ligand binding. Comparative thermodynamic binding data, derived from differential scanning calorimetry measurements, together with high-resolution crystallographic structures, are also reported for streptavidin:ligand complexes of 2-[(3'-tert-butyl-4'-hydroxyphenyl)-azo]benzoate (K-a = 2.5 x 10(5) M(-1), R = 0.18 to 2.0-Angstrom resolution), 2-[(3',5'-dimethoxy-4'-hydroxyphenyl)azo] benzoate (K-a = 3.5 x 10(6) M(-1), R = 0.19 to 2.2-Angstrom resolution), and 2-[(4'-hydroxynaphthyl)azo]benzoate (K-a = 2.8 x 10(6) M(-1), R = 0.18 to 1.8-Angstrom resolution).