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

Abstract

Zorro-LNA is a new class of therapeutic anti-gene oligonucleotides (ONs) capable of invading supercoiled DNA. The synthesis of single stranded Zorro-LNA is typically complex and laborious, requiring reverse phosphoramidites and a chemical linker connecting the two separate ON arms. Here, a simplified synthesis strategy based on 'click chemistry' is presented with a high potential for screening Zorro-LNA ONs directed against new anti-gene targets. Four different Zorro type 3'-5' 5'-3' constructs were synthesized via parallel in situ Cu(i) [3 + 2] catalysed cycloaddition. They were prepared from commercially obtained ONs functionalized on solid support (one ON with the azide and the other ON with the activated triple bond linker N-propynoylamino)-p-toluic acid (PATA)) and after cleavage from resin, they were conjugated in solution. Our report shows the benefit of combining different approaches when developing anti-gene ONs, (1) the ability for rapid and robust screening of potential targets and (2) refining the hits with more anti-gene optimized constructs. We present as well the first report showing double-strand invasion (DSI) efficiency of two combined Zorro-LNAs.

Efficient Conjugation to Phosphorothioate Oligonucleotides by Cu-Catalyzed Huisgen 1,3-Dipolar Cycloaddition

“…At first, the conjugation of PS-ON with azido-containing peptide MIF-aza ( 3 ) was performed under previously described “click” reaction conditions for the solid-supported conjugation of phosphodiester ONs ,,,, using tert -butanol/water (1:1) mixture as solvent, 24 h reaction time, and 2 equiv of copper­(I) iodide (CuI) as a copper source (Figure ). The chromatogram of the crude deprotected phosphorothioate oligonucleotide conjugate (PS-ON1-MIF) showed good conversion of PS-ON1 to the peptide-conjugate product.…”

“…The conjugation of different uptake-promoting molecules to ONs is a promising approach to enhance their delivery, as exemplified by targeting the liver by use of N-acetylgalactosamine (GalNAc) conjugated ONs and to the pancreatic β-cells by conjugation of ONs to a glucagon-like peptide-1 (GLP-1) . A number of various methods for the conjugation of specific molecules, especially peptides, to ONs have been developed, both by in-line conjugation and by post-conjugation in solution and on solid phase. , The use of Cu­(I)-catalyzed Huisgen azide–alkyne 1,3-dipolar cycloaddition, 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, small molecules like biotin , or fluorophores, carbohydrates, − and even for the preparation of multiple conjugates …”

“…7 A number of various methods for the conjugation of specific molecules, especially peptides, to ONs have been developed, both by in-line conjugation 8 and by postconjugation in solution 9 and on solid phase. 10,11 The use of Cu(I)-catalyzed Huisgen azide−alkyne 1,3-dipolar cycloaddition, 12 known as "click" reaction, has been proven to be a highly efficient approach for conjugation of ONs to peptides, 11,13 other ONs, 14 the m 3 G-Cap, 15 small molecules like biotin 16,17 or fluorophores, 18 carbohydrates, 19−22 and even for the preparation of multiple conjugates. 10 Phosphorothioate (PS) linkage, where a nonbridging phosphodiester oxygen is replaced by sulfur, is one of the most widely used nucleic acid modifications.…”

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…Furthermore, a Zorro analog was conveniently synthesized by click reaction between a fragment bearing an azide at its terminus and another fragment bearing an alkyne (a direct synthesis of 3¤-5¤-5¤-3¤ strand, described above, is not very easy). 199 …”

Delivery, Effect on Cell Viability, and Plasticity of Modified Aptamer Constructs

“…One alternative to using reverse phosphoroamidites to connect two Zorro arms with different polarities is to link the arms post synthesis using 1,3-dipolar cycloaddition or "click" chemistry (see figure 4H) (95). This approach can efficiently join a terminal alkyne group to an azide group under aqueous conditions using Cu(I) as a catalyst, and has been widely used in different settings since it was reported in 2002 (96)(97)(98).…”