Synthesis of 5′-Thiol Functionalized Morpholino Oligo-Nucleotide and Subsequent Conjugation with IGT to Improve Delivery and Antisense Efficacy In Vitro

Thiol functionalized oligonucleotides are useful intermediates for a wide range of applications including DNA nanobiotechnology field through conjugation with various types of probes and cargos. Due to the limitation of synthetic process, phosphorodiamidate morpholino oligonucleotides (PMOs) have not been explored like other oligonucleotides through SH conjugation as mentioned above. In this paper, we report the synthesis of 5′-SH functionalized PMO using a solid support synthesis protocol with an optimized cysteine derived linker so that loading and coupling efficiency of morpholino monomers were effective enough to get a 25-mer 5′-SH functionalized PMO against human Nanog. The PMO with SH functionality was subsequently conjugated with our previously reported Internal Oligo-guanidinium Transporter (IGT) in solution phase to obtain the IGT-PMO conjugate. Interestingly, 5′-conjugated PMO (IGT-PMO) showed 2.5 times better antisense efficacy than 3′-conjugated PMO with IGT (PMO-IGT). 5′-Conjugation enables us to use IGT-PMO for further conjugation at the 3′-N terminal of PMO which was not possible earlier with 5′-OH-PMO-IGT. PMO has become an important class of antisense reagents because four PMO-based drugs have been approved for the treatment of Duchenne muscular dystrophy; hence such an improved result with 5′-modified PMO could be useful for enhancing the therapeutic efficacy of DMD drugs. Similarly, thiol-modified PMO could also be explored like other thiol-containing oligonucleotides for various other applications.