Biotin reagents for antibody pretargeting. 5. Additional studies of biotinconjugate design to provide biotinidase stability

An investigation was conducted in which the stabilities of four structurall y different biotin derivatives were assessed with regard to biotinamide bon d hydrolysis by the enzyme biotinidase. The biotin derivatives studied cont ained an extra methylene in the valeric acid chain of biotin (i.e., homobio tin), or contained conjugated amino acids having hydroxymethylene, carboxyl ate, or acetate functionalities on a methylene alpha to the biotinamide bon d. The biotinidase hydrolysis assay was conducted on biotin derivatives tha t were radioiodinated at high specific activity, and then subjected to dilu ted human serum at 37 degreesC for 2 h. After incubation, assessment of bio tinamide bond hydrolysis by biotinidase was readily achieved by measuring t he percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidas e cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not bl ock it. The data obtained also indicate that placing a hydroxymethylene, ca rboxylate, or acetate alpha to the biotinamide bond is effective in blockin g the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hyd roxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjug ate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applicati ons.