Biotin reagents for antibody pretargeting. 5. Additional studies of biotin conjugate design to provide biotinidase stability.
An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 degrees C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the 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 biotinidase 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 block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.
Wilbur
D S
DS
Department of Radiation Oncology, University of Washington, Seattle, Washington 98195, USA. dswilbur@u.washington.edu
Hamlin
D K
DK
Chyan
M K
MK
Kegley
B B
BB
Pathare
P M
PM
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000097:blood; D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D043523:Biotinidase; D002264:Carboxylic Acids / Q000737:chemistry; D004355:Drug Stability; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes / Q000737:chemistry*
D002264:Carboxylic Acids; D007457:Iodine Radioisotopes; D001360:Avidin; C093086:biotinamide; D001710:Biotin; D000581:Amidohydrolases; D043523:Biotinidase