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.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer.

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.

Internal Oligoguanidinium Transporter: Mercury-Free Scalable Synthesis, Improvement of Cellular Localization, Endosomal Escape, Mitochondrial Localization, and Conjugation with Antisense Morpholino for NANOG Inhibition to Induce Chemosensitization of Taxol in MCF-7 Cells.

A nontoxic delivery vehicle is essential for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs). Though guanidinium-rich or arginine-rich cellular transporter conjugated Vivo-PMO or PPMO has been developed for in vivo application, however, either their toxicity or stability has become an issue. Previously, we reported nonpeptidic internal guanidinium transporter (IGT) mediated delivery of PMO for gene silencing and got encouraging results. In this paper, we report the synthesis of IGT using a Hg-free method for scale up and N-terminal modification of IGT with a suitable hydrophobic or lipophilic group to improve the cell permeability, endosomal escape, and mitochondrial localization and to reduce toxicity in the MTT assay. For the delivery of PMO, IGT-PMO conjugate was synthesized to target NANOG in cells, a transcription factor required for cancer stem cell proliferation and embryonic development and is involved in many cancers. Our data shows IGT-PMO-facilitated NANOG inhibition, and thereby the prevention of EpCAM-N-Cadherin-Vimentin axis mediated epithelial to mesenchymal transition (EMT) in MCF-7 cells. Moreover, unlike taxol, NANOG inhibition influences the expression of stemness factor c-Myc, Hh-Gli signaling proteins, other cancer related factors, and their respective phenotypes in cancer cells. To the best of our knowledge, this is the first report to illustrate that the IGT-PMO-mediated NANOG inhibition increases the therapeutic potential of taxol and induces G0-G1 arrest in cancer cells to prevent cancer progression. However, it warrants further investigation in other cancer cells and preclinical platforms.

Synthesis of Chlorophosphoramidate Monomer Morpholinos and PMOs.

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a successful class of antisense reagents that efficiently modulate gene expression. Because PMOs do not follow standard phosphoramidite chemistry, optimized synthetic protocols for these compounds are relatively scarce in the literature. This paper presents detailed protocols for synthesizing full-length PMOs using chlorophosphoramidate chemistry by manual solid-phase synthesis. We first describe the synthesis of Fmoc-protected morpholino hydroxyl monomers, and the corresponding chlorophosphoramidate monomers, from commercially available protected ribonucleosides. The new Fmoc chemistry necessitates the use of a milder base, such as N-ethylmorpholine (NEM), and coupling reagent, such as 5-(ethylthio)-1H-tetrazole (ETT), which are also tolerated for acid-sensitive trityl chemistry. These chlorophosphoramidate monomers are then employed for PMO synthesis in a manual solid-phase procedure using four sequential steps. The synthetic cycle for each nucleotide incorporation consists of (a) deblocking of the 3'-N protecting group using an acidic deblocking cocktail for trityl and base deblocking for Fmoc, (b) neutralization, (c) coupling in the presence of ETT and NEM, and (d) capping of the unreacted morpholine ring-amine. The method uses safe, stable, and inexpensive reagents, and the process is expected to be scalable. After full-length PMO synthesis and ammonia-mediated cleavage from the solid support and deprotection, a range of PMOs with different lengths can be obtained conveniently and efficiently with reproducible good yields. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the novel Fmoc-protected morpholino monomers Basic Protocol 2: Synthesis of the phosphorylating reagent (N,N-dimethylphosphoramic dichloride) required for chlorophosphoramidate monomer synthesis Basic Protocol 3: Synthesis of chlorophosphoramidate monomers of Fmoc-protected morpholino monomers Basic Protocol 4: Solution-phase standardization of dimer and trimer PMO synthesis using Fmoc chemistry Basic Protocol 5: Solid-phase synthesis, purification, and characterization of full-length (25-mer) no-tail PMO using both trityl and Fmoc chemistry.