Enabling Multiple Conjugation to Oligonucleotides Using “Click Cycles”

Abstract

An efficient method for the synthesis of multiply functionalized oligonucleotides (ONs) utilizing a novel H-phosphonate alkyne-based linker for multiple functionalization (LMF) is developed. The strategy allows for the conjugation of various active entities to oligonucleotide through the postsynthetic attachment of LMF at the 5'-terminus of ONs using H-phosphonate chemistry followed by conjugation of various entities via [3 + 2] copper(I) catalyzed cycloaddition in a stepwise manner. Each cycle is composed of attachment of the LMF followed by a click reaction with azido-containing units. Sequential solid-phase synthesis of oligonucleotide conjugates containing three attached entities was performed using an acetylated form of MIF peptide conjugated to azido linker, achieving high conversions at each unit addition. In addition, to show the versatility of the method, oligonucleotide conjugates with several different classes of compounds were synthesized. Each conjugate containing three different entities, whose structure and function varied (e.g., sugars, peptides, fluorescent labels, and mG-Caps).

Modular Strategy To Expand the Chemical Diversity of DNA and Sequence-Controlled Polymers

“…Sequence-specific on-bead functionalization of PT1 in the synthesizer could also be an efficient method. Such a method has been reported with an alkyne-containing phosphonate reagent elsewhere . It was shown to be very modular with individual coupling yields between 63 and 89%.…”

“…Such a method has been reported with an alkyne-containing phosphonate reagent elsewhere. 54 It was shown to be very modular with individual coupling yields between 63 and 89%.…”

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

“…Conjugation of different uptake‐promoting molecules to ONs is a promising approach to enhance ON delivery, as exemplified by targeting hepatocytes by use of N ‐acetylgalactosamine (GalNAc)‐conjugated ONs (Miller et al., ) and targeting pancreatic β‐cells by conjugation of ONs to a glucagon‐like peptide‐1 (GLP‐1) (Ammala et al., ). 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., ).…”

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters

“…These techniques (for example, common acylation or aldehyde couplings) , were developed along with metal-catalyzed reactions . To date, a number of procedures for solid-phase oligonucleotide functionalization by Sonogashira, − Stille, , Glaser-Hey, , CuAAC, − and SPAAC and nitrile oxide and alkyne couplings − have been reported. Among them, only Sonogashira coupling was adopted for the automated DNA synthesizer in pioneer studies by Grinstaff and co-workers. , Most of the approaches mentioned above were based on off-synthesizer techniques (including synthesizer-incompatible microwave irradiation or heating) or required a prolonged reaction time (up to 24 h).…”