Biotin reagents for antibody pretargeting. 4. Selection of biotin conjugates for in vivo application based on their dissociation rate from avidin andstreptavidin

An investigation was conducted to determine the affect of structural variat ion of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in v ivo applications. Fifteen biotin derivatives were conjugated with a cyanoco balamin (CN-Cbl) derivative for evaluation of their "relative" dissociation rates by size exclusion HPLC analysis. Two biotin-CN-Cbl conjugates, one c ontaining unaltered biotin and the other containing iminobiotin, were prepa red as reference compounds for comparison purposes. The first structural va riations studied involved modification of the biotinamide bond with a N-met hyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side c hain by a methylene unit (i.e., homobiotin), and replacing the biotinamide bond with thiourea bonds in two conjugates. The rate of dissociation of the biotin-CN-Cbl derivative from Av and SAv was significantly increased for b iotin derivatives containing those structural features. Nine additional bio tin conjugates were obtained by coupling amino acids or functional group pr otected amino acids to the biotin moiety. In the conjugates, the biotin moi ety and biotinamide bond were not altered, but substituents of various size s were introduced alpha to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAV was no t affected by small substituents alpha to the biotinamide (e.g., methyl, hy droxymethyl, and carboxylate groups), but was significantly increased when larger functional groups were present. On the basis of the results obtained , it appears that biotin conjugates which retain an unmodified biotin moiet y and have a linker molecule conjugated to it that has a small functional g roup (e.g., hydroxymethylene or carboxylate) alpha to the biotinamide bond are excellent candidates for in vivo applications. These structural feature s are obtained in the biotin amino acid conjugates: biotin-serine, biotin-a spartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin der ivatives can be readily conjugated with other molecules for specific in viv o applications. In our studies, these derivatives will be used in the desig n of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.