Conjugatable and Bioreduction Cleavable Linker for the 5′-Functionalization of Oligonucleotides

Abstract

An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5'-terminal ends of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and incorporated at the 5'-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides.

Development of Bioreduction Labile Protecting Groups for the 2′-Hydroxyl Group of RNA

“…After cellular uptake, the protecting groups are deprotected by esterase or GSH to release active RNA species. − However, special synthetic procedures are needed for the preparation of these RNAs. In our laboratory, bioreduction labile protecting groups have been used for the development of reduction-activated DNA type oligonucleotides − that can be activated by the hypoxic conditions found in advanced solid tumors. , These oligonucleotides were also amenable to solid-phase DNA synthesis and purification. To expand our study into reduction-activated RNA type molecules, development of bioreduction labile 2′-hydroxyl protecting groups is critical.…”

A Bioreductive Prodrug of Cucurbitacin B Significantly Inhibits Tumor Growth in the 4T1 Xenograft Mice Model

“…Embedment of an additional molecular device, which is cleavable by a specific enzyme expressed predominantly in tumor cells, is a commonly applied method in contemporary drug design and development . A bioreductive prodrug can be designed to target a specific tumor followed by in situ release of the therapeutic drugs with the reductase (NAD­(P)­H:quinone oxidoreductase 1, NQO1) overexpressed in some tumor cells. − Fortunately, the reductases are often reported to be overexpressed in a variety of cancer cells, such as breast cancer, ovarian cancer, thyroid cancer, adrenal cancer, and colon cancer. ,− …”

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

“…For most of the conjugates (except rhodamine and cyanine dyes), we performed on-column deprotection with 1,2-ethylenediamine in toluene in the synthesizer to make a straightforward automated procedure for conjugate preparation. The solid-phase click chemistry of coumarin 343 to oligonucleotides was recently reported; however, we failed to find deprotection conditions for hetero oligonucleotides because dye degraded up to 80% faster than during 2 h of exposure to concentrated aqueous ammonia at room temperature (conditions for ultramild protected phosphoramidites). In the case of coumarin 343 azide, we also performed common solution CuAAC reaction with 5′-alkyne T 20 oligonucleotide to obtain the desired reference conjugate.…”

“…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).…”