A New and Efficient Method for Synthesis of 5′-Conjugates of Oligonucleotides through Amide-Bond Formation on Solid Phase

Abstract

Dedicated to Prof. Dr. Wolfgang Pfleiderer on the occasion of his 75th birthday An efficient method for synthesis of oligonucleotide 5'-conjugates through amide-bond formation on solid phase is described. Protected oligonucleotides containing a 5'-carboxylic acid function were obtained by use of a novel non-nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2-chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid-phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5'-Carboxylate-modified oligonucleotides were efficiently conjugated on solid support under normal peptide-coupling conditions to various amines or to the N-termini of small peptides to yield products of high purity. The method is well-suited in principle for the synthesis of peptide-oligonucleotide conjugates containing an amide linkage between the 5'-end of an oligonucleotide and the N-terminus of a peptide.

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

“…We have developed previously a number of methods for such conjugation, e.g. amide bond fragment condensation on solid support (28,29), native ligation of a cysteine-containing oligonucleotide with a thioester peptide (30), 2′-aldehyde oligonucleotide conjugations to form thiazolidine, oxime and hydrazine linkages (31) and total stepwise solid-phase synthesis on a single solid support (32). All of these methods give rise to linkages expected to be stable within cells.…”

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 .…”

Self-neutralizing oligonucleotides with enhanced cellular uptake

“…As with the case of the polyamine conjugates, the linker is typically attached to the 5′-end of the ON upon the completion of the automated synthesis. 27, 28 Attachment of amino linkers to the internucleoside phosphoroamidate groups has been performed using H-phosphonate chemistry, 29, 30 but this approach limits the number of amino linkers that can be incorporated.…”