Basic Studies on Heterobifunctional Biotin−PEG Conjugates with a 3-(4-Pyridyldithio)propionyl Marker on the Second Terminus

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Abstract
Heterobifunctional poly(ethylene glycol) (PEG) derivatives with a biotin terminus have been synthesized and characterized with respect to avidin binding. Unambiguous measurement of biotinyl and pyridyldithiopropionyl end groups was established by selecting suitable assays and introducing necessary modifications. Functional studies on the binding of biotin-PEG conjugates to avidin tetramers revealed much similarity to known biotin-spacer-peptide conjugates with 7-27 atom spacers: dissociation kinetics of the initially formed 4:1 complexes were multiexponential, the complex with 2 ligands per avidin dissociating rather slowly with half-times of approximately 2 days at 25 degrees C. The observed stability of 3:1 and 2:1 complexes with avidin is particularly significant since it allows exploitation of the additional advantages of PEG spacers, i.e. reduced steric strain in biotin-avidin-biotin bridges, reduced nonspecific adsorption of biotinylated probes and markers, and, especially, uncomparable fluorescence intensities of biotin-PEG-fluorophore conjugates as is demonstrated in the accompanying study (second of three papers in this issue).