Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylamino)toluic Acid] (PATA) for Efficient Biotinylation of Peptides and Oligonucleotides

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.

Copper‐Catalyzed Huisgen 1,3‐Dipolar Cycloaddition Tailored for Phosphorothioate Oligonucleotides

“…Various methods have been developed for conjugation of specific ligands to ONs, either by in‐line conjugation (Zaramella, Yeheskiely, & Stromberg, ) or by post‐conjugation, either in solution (Juliano, Ming, & Nakagawa, ) or on solid phase (Jezowska, Honcharenko, Ghidini, Stromberg, & Honcharenko, ; Wenska et al., ). Use of Cu(I)‐catalyzed Huisgen azide‐alkyne 1,3‐dipolar cycloaddition (Honcharenko, Honcharenko, & Stromberg, ; Verma, ), known as “click reaction,” has been proven to be a highly efficient approach for conjugation of ONs to peptides, other ONs, the m 3 G‐CAP (Honcharenko et al., ; Honcharenko et al., ; Verma, ), small molecules like biotin (Jezowska, Romanowska, Bestas, Tedebark, & Honcharenko, ), or fluorophores and even for preparation of multiple conjugates (Jezowska et al., ).…”

Synthetic m3G-CAP attachment necessitates a minimum trinucleotide constituent to be recognised as a nuclear import signal

“…In 9 the same methylene modification of the triphosphate as in 4 is included. 35 Thus, biotin constructs with different linkers attached to an activated triple bond donor, p-(N-propynoylamino)toluic acid PATS, 35 were synthesized (11, 12 36 and 13, Figure 2). Synthesis of 9 and 10 was performed as reported in the synthesis of 8 with the exception that for 10 the capping step was performed with the methylene(bisphosphonate) analog of N 2 , N 2 , N 7 -trimethylguanosinepyrophosphoryl imidazolide.…”

Fast and efficient synthesis of Zorro-LNA type 3′-5′-5′-3′ oligonucleotide conjugates via parallel in situ stepwise conjugation

“…This so called "fragment" is equipped with a functional group, like an amine or carboxylic acid that reacts with a preattached linker on the ON to create the covalent linkage. For amide formation, coupling reagents typically used are reagents commonly used in peptide synthesis, such as HBTU/HOBt or HBTU/NMM [47][48][49] . Interestingly, copper catalyzed 1,3-dipolar cycloaddition reactions are also utilized for fragment conjugations 50 and microwave assisted conditions has been used to speed up the process 51 .…”

“…The other one is biotin, which is not fluorescent on its own but possess exclusive affinity to fluorescent streptavidin/avidin proteins. The complex they create is a basis for many important biotechnological applications including studies of cellular transport 49 .…”

“…The newly prepared complexes were than transfected into U2OS cells by PULS in protein transfection agent and after six hours the cells were analyzed by fluorescent microscopy. Figure 19 depicts the extensive variety of biotinylated cap structures ranging from the adenosine based ones with different linker length and modifications on the triphosphate bridge (39)(40)(41)(42)(43)(44)(45)(46) to more complex molecules with an AUA sequence (47)(48)(49). Additionally, the noncapped controls are also presented (Figure 20, 50-51).…”