An expanded framework toward improving the detritylation reaction in solid-phase oligonucleotide syntheses - filling the gap.

A few interactions should be considered during the detritylation reaction of solid-phase oligonucleotide synthesis (SPOS): (i) interaction of solvent with acid; (ii) interaction (or reaction) of solvent with trityl cation, and (iii) interaction of scavenger with acid, with the last one as the focus of this work. Using a stopped-flow setup, commonly used trityl cation scavengers (methanol, thioanisole, 1-dodecanethiol, triisopropylsilane, triethylsilane, and trihexylsilane) were evaluated for their reactivity toward tritylium hexafluorophosphate. Among the scavengers screened, methanol and thioanisole were found to be the most and least reactive, respectively; however, methanol does interact and react with trichloroacetic acid, thus it should not be pre-mixed and stored with acid as deblock solutions. Overall, all aspects of interactions must be taken into consideration while optimizing the detritylation reaction, especially for large scale SPOS.

An improved synthesis of guanosine TNA phosphoramidite for oligonucleotide synthesis.

The chemical synthesis of guanosine nucleosides generates both the N9 and N7 regioisomers, which require careful separation to obtain the desired N9 isomer. To preferentially obtain the N9 isomer, a bulky diphenylcarbamoyl (DPC) group can be installed at the O6 position of guanine. However, installation of the DPC group presents a challenging task due to low solubility of the N-acetyl protected guanine. Here we report the usage of commercially available 2-amino-6-chloro purine as a new strategy that offers a more efficient route to the synthesis of the guanine phosphoramidite of threose nucleic acid (TNA).

Loading Determination of DMTr-substituted Resins for Large-scale Oligonucleotide Synthesis.

The loading (i.e., substitution) of solid supports for oligonucleotide synthesis is an important parameter in large-scale manufacturing of oligonucleotides. Several key process parameters are dependent on the substitution of the solid support, including the number of phosphoramidite nucleoside equivalents used in the coupling step. For dimethoxytrityl (DMTr)-loaded solid supports, the substitution of the resin is determined by quantitatively cleaving the DMTr protecting group from the resin under acidic conditions and then analyzing the DMTr cation extinction by UV/vis spectroscopy. The spectrometric measurement can be performed at 409 nm or the global extinction maximum of 510 nm. The substitution is then calculated based on the Lambert-Beer law analogously to the substitution determination of Fmoc-substituted resins. Below, the determination of the molar extinction coefficient at 510 nm in a solution of 10% dichloroacetic acid in toluene and subsequent determination of the DMTr loading of DMTr-substituted resins is reported. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Determination of the molar extinction coefficient at 510 nm in DCA Deblock solution Basic Protocol 2: Substitution determination of DMTr-substituted resins by cleavage of the DMTr cation.

Synthesis of triazole-nucleoside phosphoramidites and their use in solid-phase oligonucleotide synthesis.

Triazole-backbone oligonucleotides are macromolecules that have one or more triazole units that are acting as a backbone mimic. Triazoles within the backbone have been used within oligonucleotides for a variety of applications. This unit describes the preparation and synthesis of two triazole-nucleoside phosphoramidites [uracil-triazole-uracil (UtU) and cytosine-triazole-uracil (CtU)] based on a PNA-like scaffold, and their incorporation within oligonucleotides.