Biotin Reagents for Antibody Pretargeting. 2. Synthesis and in Vitro Evaluation of Biotin Dimers and Trimers for Cross-Linking of Streptavidin

Abstract

Polymerization and/or cross-linking of recombinant streptavidin (r-SAv) with biotin derivatives containing two biotin moieties (biotin dimers) or three biotin moieties (biotin trimers) has been investigated as a model for reagents to be used to increase the amount of radioactivity on cancer cells in tumor pretargeting protocols. In the investigation, six biotin dimers and three biotin trimers were synthesized. Most biotin derivatives synthesized had ether containing linker molecules incorporated to improve their aqueous solubility. The synthesized biotin dimers contained linker moieties which provided distances (when fully extended) of 13-49 A between biotin carboxylate carbon atoms, and the biotin trimers contained linker moieties which provided distances of 31-53 A between any two biotin carboxylate atoms. All of the biotin derivatives were evaluated for their ability to polymerize r-SAv in solution. When the biotin derivatives were mixed with r-SAv, none of the biotin dimers caused polymerization, but all of the biotin trimers resulted in complete polymerization. Some of the biotin dimers did cross-link r-SAv (to form r-SAv dimers, trimers, etc.), but the percentage of cross-linking was low (< or = 40%). The length of the linker molecule was important in cross-linking of biotin dimers. While linkers which provided distances of 13 and 19 A between biotin carboxylate carbon atoms did not result in cross-linking, a linker which provided a 17 A distance resulted in a small (< or = 10%) amount of cross-linking. Also cross-linking was increased in biotin dimers with linkers which provided distances between biotin carboxylate carbon atoms of > or = 23 A. Cross-linking of streptavidin bound in polystyrene wells with biotin dimers and trimers was also examined. In those experiments, an excess of each biotin derivative was incubated at 37 degrees C for 10-30 min in polystyrene wells containing bound SAv. After the excess biotin derivative was rinsed from the wells, an excess of r-[125I]SAv was incubated for another 10-30 min. The amount of r-[125I]SAv bound after rinsing the excess from the wells was an indicator of the extent of cross-linking that occurred. The process of alternating additions of reagents was repeated four times to demonstrate that bound radioactivity could be increased with each addition of [125I]SAv. The results of cross-linking r-SAv in polystyrene wells paralleled results from cross-linking in solution.

Target Visualization by MRI Using the Avidin/Biotin Amplification Route: Synthesis and Testing of a Biotin–Gd‐DOTA Monoamide Trimer

“…[8] Many derivatives have been synthesized by Wilbur and coworkers containing two and three biotin moieties with varying distances between the carbon atoms of the biotin amide. [9] Only tris-biotinylated compounds completely polymerized in SAv solution and similar results were obtained in the surface-bound SAv cross-linking experiment.…”

“…Only linkers in which the distance between biotin moieties (between amide carbon atoms) is longer than 20 , result in the intermolecular binding of the second biotin. [9] Moreover, in the case of well-spaced bisbiotinylated compounds the formation of long chain polymers is, in part, hampered by the occurrence of intramolecular linkages, as previously shown in the case of biotin-DTPA dimers. [10] Therefore, molecules with three biotin moieties have to be designed to allow the formation of polymeric chains.…”

Hierarchical Self‐Assembly of One‐Dimensional Streptavidin Bundles as a Collagen Mimetic for the Biomineralization of Calcite

“…[42,43] For this proof-ofprinciple study, we selected a preorganized bis-biotinylated terpyridine ligand Biot 2 -terpy that binds to two cis-related sites in streptavidin. [44][45][46][47] A ferrous ion was chosen as terpyridine binds in a cooperative fashion (K 1 = 1.26 10 7 m À1 , K 2 = 6.31 10 13 m À1 ), [48] thus ensuring the exclusive formation of a linear connector bearing four biotin anchors ( Figure 2). Experimental details for the synthesis and the characterization of the [Fe(Biot 2 -terpy) 2 ] 2+ complex are described in the Supporting Information.…”

Monovalent Streptavidin that Senses Oligonucleotides

“…This is in contrast to previously reported designs, in which intramolecular cyclization was disfavored by using very short trisbiotinylated ligands. [8,9] We first tested the tris-biotinylated oligonucleotide 5-/52-Bio/TTT TTT TTT TTT TTT TTT TTT TTT T-/3BioTEG/-3 (1): mixing this oligonucleotide with one equivalent of streptavidin and then heating for 15mins at 70°C and cooling, resulted in estimated yields around 55% for the major product STV-(1) as shown in Figure 1b Figure S3). The yield could be increased to around 70% by using two equivalents of streptavidin with heating the mixture to 70°C and allowing to cool, see Supporting Information Figure S4.…”