Arch, including the burgeoning use of siRNA, have led to an explosion in growth of oligoribonucleotide production. Instead of being carried out in a few specialist labs, RNA synthesis has now grown to a level requiring high-throughput synthesis and demands the use of a universal support. The first category of universal support defined above is incompatible with RNA deprotection since it relies on procedures known to degrade RNA. However, universal supports in the second category may be compatible with RNA synthesis. DISCUSSION A number of universal supports have been introduced recently and representative structures are shown in Figures 1 and 2. All of these solid supports function similarly: regular detritylation (although universal support 3 has no DMT group, the regular deblock step generates the hydroxyl group), the addition of the first nucleoside monomer, and then the remaining oligonucleotide preparation steps proceed without any changes from standard procedures, as shown in Scheme 1. 2
Figure 2. Universal Supports – Dephosphorylation during Cleavage
Cleavage THEN Deprotection and Dephosphorylation In the case of the supports in Figure 1, the elimination of the terminal phosphodiester group utilizes the same reagents (ammonium hydroxide, aqueous methylamine, a mixture of the first two (AMA), aqueous sodium hydroxide, etc.), as needed for routine deprotection of oligonucleotides, as shown in Scheme 2. However, much more aggressive and lengthy conditions are typically required. Upon the completion of oligonucleotide assembly, the 3′-terminal phosphotriester group is first converted into the phosphodiester function by elimination of the cyanoethyl protection group, as shown in Scheme 2. Only upon the release of the 3′-hydroxyl of the tether nucleoside and hydrolysis of the linker to the CPG does the intramolecular nucleophilic attack on the phosphorous atom of the phosphodiester group take place to effect dephosphorylation. This dephosphorylation reaction is a relatively slow process, requiring lengthy aggressive treatment with ammonium hydroxide if the presence of some 3′-tethered product along with the target oligonucleotide in the final mixture is to be avoided.500579-04-4 custom synthesis Thus, in the case of these supports, the process of oligonucleotide cleavage, base deprotection, 3′-dephosphorylation at elevated temperature and evaporation of aqueous ammonia normally require 8-10 h. Another universal support 6 was also tested in this study but its structure was not revealed by the manufacturer. However, its behavior during testing indicated that it fell into this first category.98327-87-8 web Indeed, using the same tests as outlined in Table 1, this support also generated mixtures of the target oligomer and the 3′-tethered oligomer in ratios similar to those found for universal supports 1-3.PMID:30725745 The product profile can be improved by adding metal ions to the mix, and Li+, Na+ and Zn2+ have all been used to speed up the elimination reaction, presumably by stabilizing the 5-membered transition state. However, the speed and simplicity of evaporation of the deprotection solution to give the crude oligonucleotide with no need for desalting is not possible with these ionic additives. All of these facts make these solid matrices unattractive for high throughput oligonucleotide manufacturing. These ionic additives would also assist in the degradation of RNA linkages and this makes their use to
Scheme 1. Oligonucleotide Assembly on Universal Supports
Scheme 2. Cleavage/Dephosphoryl.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com