Study On The Synthesis And Hydrolytic Stability Of Peptide-conjugated Antisense Oligonucleotides

Antisense oligonucleotide is a kind of important molecular biological research tool and potential therapeutic agents. Many classes of oligonucleotides are polyanions and can not pass through cell membrance. Desired improvement of such property can be achieved by covalent association to peptides. Over the past two decades, various synthetic approaches have been developed for peptide-oligonucleotide conjugates (POCs) synthesis. However, there remains a need for highly efficient methods that are widely applicable to varied types of peptides. A liquid-phase fragment coupling approach in peptide oligonucleotide conjugation is developed and is successfully applied to synthesize mimetic signal peptide-conjugated oligonucleotide. The hydrolytic stability of the synthesized 1,2,3-triazole linkage was studied as a function of pH.This method applies copper(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition. For that purpose, alkyne group was introduced into peptides following the standard procedures of Fmoc solid-phase peptide synthesis at the last coupling step with propynoic acid. After cleavage and purification, alkynyl-peptide was obtained. 5′-Bromohexyl oligonucleotide was synthesized on an ABI 3400 DNA Synthesizer using a standard phosphoramidite elongation cycle for coupling of commercial nucleoside phosphoramidites and new prepared 6-bromohexyl 2-cyanoethyl dissopropylphosphoramidite, followed by azidation using sodium azide and ammonolysis to generate 5′-azidohexyl-oligonucleotide. The corresponding 5′-azidohexyl oligonucleotide was used for'Click'conjugation with peptide-alkyne under aqueous conditions to produce peptide-oligonucleotide conjugates in high purity and yield.