Abstract
Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylamino)toluic acid (PATA) gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.
Highlights
For various studies specially for labeling of proteins it seems valuable to have access to biotin linkers which are ready for cycloadditions at a low concentration, i.e., equipped with the activated triple bond donor PATA [12,13,14]
The streptavidin recognition pocket is relatively deep and biomolecules equipped with biotin typically require a spacer
Synthesis of compound 3 was commenced by activation of the triple bond donor PATA (2) with HBTU/N-methyl morpholine (NMM) followed by conjugation with compound 1 which served as an amine in this reaction
Summary
The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. Quite a few methodologies were reported in which biotin was connected to an attachable linker These methods rely on the synthesis of active esters [8], as well as propargyl and azide derivatives which allows use of “click chemistry” to perform conjugation [9]. From previous experiments in our own group and as well as by others, it is clear that conjugation by Cu catalyzed Hüisgen dipolar [3+2]-cycloaddition at a low concentration and using relatively large biomolecules for derivatization, like oligonucleotides or peptides, is very difficult and often virtually impossible This is provided that the triple bond donor is not in activated form as shown in a recent reports, where comparison of pentynoic acid as triple bond donor versus activated PATA bond donor is discussed [10,11]. For various studies specially for labeling of proteins it seems valuable to have access to biotin linkers which are ready for cycloadditions at a low concentration, i.e., equipped with the activated triple bond donor PATA [12,13,14]
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