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Commercial Scale Manufacturing of Oligonucleotides Under Good Manufacturing Practices
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Jose E. Gonzalez, Richard G. Einig, Patricia Puma, Timothy P. Noonan, Paul E. Kennedy, Bruce G. Sturgeon, Bing H. Wang, Jin-yan Tang
After DMT removal, the resultant free 5'-hydroxyl group of the sugar moiety reacts in the coupling step with the protected nucleoside phosphoramidite, which is next in the oligonucleotide sequence. To obtain effective coupling, the amidite solution is activated with tetrazole before contacting the solid support. The molar stoichiometric excess ("molar equivalents") of phosphoramidite in this step is also used to drive the reaction towards completion. Efficient delivery of reagents and optimized design of process equipment permits use of only 1.5 molar equivalents of phosphoramidites, compared with 20 or more in a typical laboratory scale synthesizer.
Lipid Nanocarriers for Oligonucleotide Delivery to the Brain
Published in Carla Vitorino, Andreia Jorge, Alberto Pais, Nanoparticles for Brain Drug Delivery, 2021
Andreia F. Jorge, Santiago Grijalvo, Alberto Pais, Ramón Eritja
The clinical success of therapeutic ODNs in the CNS depends on their efficient and safe delivery to neuronal cells. Naked single-stranded ASOs, contrarily to double-stranded siRNAs, are able to enter into cells through gymnosis when administered in saline solution [100]. The gymnotic uptake poses great convenience in the delivery of ASOs, yet this strategy requires cost-prohibitive concentrations to reach an adequate efficacy. As an alternative, different approaches were appraised for increasing siRNA uptake, including either (i) the use of ssRNAs, these being still active for the RNAi pathway [101], or (ii) the conjugation of dsRNAs with hydrophobic modifications. The conjugation of lipophilic or amphiphilic molecules confers a hydrophobic character to ODNs, thereby facilitating the interaction of negatively charged ODNs with hydrophobic cell membranes. These moieties can be synthetically incorporated in three different positions in the ODN chain: (i) in the 3’-end by using a solid support holding the selected lipid, (ii) in the 5’-end by introducing a hydrophobic phosphoramidite and (iii) within the ODN sequence, where one nucleosidic building block bears itself the hydrophobic moiety. Nevertheless, the modification at the 5’-end is the most suitable to incorporate the lipid. In the case of siRNAs and miRNAs, due to steric reasons, the conjugation is usually introduced in the 3’- or 5’-overhangs of the sense strand since the antisense one is incorporated into RISC. Either presynthetic or postsynthetic approaches may be used to engineer the hybrid lipid-ODN conjugates. These two chemical routes have been reviewed in detail elsewhere [102].
Chemical Synthesis of Core Structures
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
A different donor for heptopyranosyl moieties, 7-dimethyl(phenyl)silyl heptopyranosyl chloride 53, was obtained in few steps via Grignard reaction from allyl 2,3,4-tri-O-benzyl-α-d-raaw2O-hexodialdo-1,5-pyrano-side. The advantage of the 7-dimethyl(phenyl)silyl group as a masked form of the hydroxy function was convincingly demonstrated for the assembly of the trisaccharide α-d-GlcpNAc-(2-aminoethyl)phosphate group. Following Agtriflate-mediated coupling of 53 with an appropriate acceptor to give an α-(1→3)-linked heptobioside intermediate, the 2-aminoethylphosphate moiety at the hydroxymethyl group of the distal heptose unit was obtained in two steps. Introduction of an intermediate 7′-O-phosphoramidite group and in situ condensation with 2-trifluoroacetamidoethanol followed by oxidation of the resulting phosphite triester furnished, after deprotection, the disaccharide phosphodiester 54. The N-tri-fluoroacetamido group was kept until the very last step of the synthesis, which comprised hydrogenolysis of 54 to afford 55. Further elongation of the aminopropyl spacer with a mercaptoacetamide unit and subsequent cleavage of the thioester gave the co-thiol derivative 57, which in turn was coupled with a synthetic bromoacetyl peptide. The peptide sequence Gly-Gly-TKISDFGSFIGF-Lys corresponds to a part of a meningococcal outer membrane protein. Ammonolysis of the trifluoroacetamide 58 followed by HPLC purification afforded the sugar-peptide conjugate containing T-helper cell epitopes for the generation of well-defined vaccines (54).
Drugs repurposing for SARS-CoV-2: new insight of COVID-19 druggability
Published in Expert Review of Anti-infective Therapy, 2022
Sujit Kumar Debnath, Monalisha Debnath, Rohit Srivastava, Abdelwahab Omri
Remdesivir is a nucleoside analog that inhibits coronaviruses’ RNA-dependent RNA polymerase (RdRp). Remdesivir is a phosphoramidite prodrug metabolized in cells to produce an active NTP analog known as remdesivir triphosphate. RdRp attributes the conversion from this triphosphate to monophosphate. This RdRp also extends RNA by nucleotides before exerting stalling mechanism to specific CoVs. It was observed that RdRp-RNA complexes were formed after adding remdesivir with RNA product 3’-end. Studies showed remdesivir monophosphate mimics adenosine monophosphate and forms standard [86]. This drug is effective against many viruses, like coronaviruses, pneumoviruses, paramyxoviruses, filoviruses, Ebola virus, etc. A placebo-controlled, randomized, double-blind trial was conducted with remdesivir [87]. The adverse effects were reported in 66% of patients, and an early stop of treatment was reported in 12% of patients. This study did not support the statistically significant improvement of clinical benefits.
Advances in biocatalytic and chemoenzymatic synthesis of nucleoside analogues
Published in Expert Opinion on Drug Discovery, 2022
Sebastian C. Cosgrove, Gavin J. Miller
It is sensible to consider this prospect of using enzymes to perform classical synthetic nucleoside-producing reactions in a broader context. For example, complex glycan synthesis has benefitted immeasurably from the development of glycosyltransferases to assemble (in part or in full) complex oligosaccharide targets for biological and biomedical applications [47–51]. In addition, and comparatively to the example reviewed earlier for the provision of ribose 1-phosphates, the provision of the key sugar nucleotide building blocks to achieve this has also flourished [52–56]. Similarly, looking to classical oligonucleotide synthesis [57], the phosphoramidite method of assembling these essential sequences has been challenged recently not only by new chemical approaches [58–60] but also by using enzyme platforms to achieve biopolymer synthesis [61,62]. Considering the enabling effect of introducing biocatalytic approaches into these fields, the future prospects for nucleoside analogue chemistry translate to one of significant potential.
Polymerization-sensitive switch-on monomer for terminal transferase activity assay
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Bhagwan S. Batule, Chang Yeol Lee, Ki Soo Park, Hyun Gyu Park
DNA molecules labelled with a fluorescent moiety, which is termed “fluorescence oligonucleotides”, have been utilized in various fields such as biotechnology, biomedicine, and molecular and cellular biology [1–3]. In general, the synthesis of fluorescence oligonucleotides is broadly classified into two types: (i) chemical DNA synthesis based on phosphoramidite method [4] and (ii) enzymatic DNA synthesis using modified mononucleotides [5,6]. In particular, the second one has gained momentum due to its simplicity, cost-effectiveness and especially, no limitation to form long DNA strands (up to thousands of bases) [5,6]. Many enzymes from DNA polymerase family that are capable of incorporating the modified dNTPs, have been exploited to synthesize the fluorescence oligonucleotides [5–7]. However, previous methods require extra, post-processing steps such as separation of unreacted dNTPs or secondary labelling with fluorophore, which makes procedures tedious, time-consuming and complicated, consequently limiting the widespread application.