Biotin Reagents for Antibody Pretargeting. 4. Selection of Biotin Conjugates for in Vivo Application Based on Their Dissociation Rate from Avidin and Streptavidin

• Published in: Bioconjugate chemistry 2000-07, Vol.11 (4), p.569-583
• Main Authors: Wilbur, D. Scott, Chyan, Ming-Kuan, Pathare, Pradip M, Hamlin, Donald K, Frownfelter, Milah B, Kegley, Brian B
• Format: Article
• Language: English
• Subjects: Antibodies - chemistry; Avidin - chemistry; Biotin - chemistry; Chromatography, High Pressure Liquid; Kinetics; Magnetic Resonance Spectroscopy; Spectrometry, Mass, Fast Atom Bombardment; Streptavidin - chemistry
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc000024v

An investigation was conducted to determine the affect of structural variation of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in vivo applications. Fifteen biotin derivatives were conjugated with a cyanocobalamin (CN−Cbl) derivative for evaluation of their “relative” dissociation rates by size exclusion HPLC analysis. Two biotin-CN−Cbl conjugates, one containing unaltered biotin and the other containing iminobiotin, were prepared as reference compounds for comparison purposes. The first structural variations studied involved modification of the biotinamide bond with a N-methyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side chain 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 biotin derivatives containing those structural features. Nine additional biotin conjugates were obtained by coupling amino acids or functional group protected amino acids to the biotin moiety. In the conjugates, the biotin moiety and biotinamide bond were not altered, but substituents of various sizes were introduced α to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAv was not affected by small substituents α to the biotinamide (e.g., methyl, hydroxymethyl, 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 moiety and have a linker molecule conjugated to it that has a small functional group (e.g., hydroxymethylene or carboxylate) α to the biotinamide bond are excellent candidates for in vivo applications. These structural features are obtained in the biotin amino acid conjugates:  biotin-serine, biotin-aspartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin derivatives can be readily conjugated with other molecules for specific in vivo applications. In our studies, these derivatives will be used in the design of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.