Scalable One-Pot-Liquid-Phase Oligonucleotide Synthesis for Model Network Hydrogels

Abstract

Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis, but suffers from scalability limitations and high reagent consumption. Liquid-phase oligonucleotide synthesis (LPOS) uses soluble polymer supports and has the potential of being scalable. However, at present, LPOS requires 3 separate reaction steps and 4-5 precipitation steps per nucleotide addition. Moreover, long acid exposure times during the deprotection step degrade sequences with high A-content (adenine) due to depurination and chain cleavage. In this work, we present the first one-pot liquid-phase DNA synthesis technique, which allows the addition of one nucleotide in a one-pot reaction of sequential coupling, oxidation and deprotection, followed by a single precipitation step. Furthermore, we demonstrate how to suppress depurination during the addition of adenine nucleotides. We showcase the potential of this technique to prepare high-purity 4-arm PEG-T 20 (T = thymine) and 4-arm PEG-A 20 building blocks in multi-gram scale. Such complementary 4-arm PEG-DNA building blocks reversibly self-assemble into supramolecular model network hydrogels, and facilitate the elucidation of bond lifetimes. These model network hydrogels exhibit new levels of mechanical properties, high stability at room temperature (melting at 44 °C), and thus open up pathways to next-generation, scalable DNA-materials programmable through sequence recognition and available for macroscale applications. File list (2) download file view on ChemRxiv MS final.pdf (7.23 MiB) download file view on ChemRxiv ESI final.pdf (12.53 MiB)

Putting DNA to Work as Generic Polymeric Materials

“…By the same method, they also prepared oligo(dC)‐modified four‐arm PEG to create hydrogels crosslinked by proton‐induced i‐motif structures [40] . The Walther group developed a one‐pot liquid‐phase oligonucleotide synthesis to prepare two different four‐arm PEGs for producing the hydrogels [41] …”

Solution-phase synthesis of oligodeoxyribonucleotides using the H-phosphonate method with N-unprotected 5′-phosphite monomers

“…The organic layers were combined, dried over Na 2 SO 4 , ltered, and concentrated under reduced pressure to afford 5b as a colorless foam (414 mg, 0.594 mmol, 97%). Triethylammonium 5 0 -O-dimethoxytrityl-thymidine 3 0 -succinate (10). 5 0 -O-Dimethoxytrityl-thymidine (2.72 g, 5.00 mmol) was dried by repeated coevaporation with dry pyridine and dry toluene, and then dissolved in dry CH 2 Cl 2 (15 mL).…”

“…S13 in the ESI †); (7) the internucleotidic Hphosphonates of the trimer 8 were oxidized by iodine and water, then, the excess iodine was quenched with diethyl phosphonate; (8) the residue of reagents was removed in the aqueous layer using LLE. The trimer 9 was distributed to the organic layer as a triethylammonium salt using triethylammonium bicarbonate (TEAB) buffer (pH 8) as an aqueous layer; (9) the TBDPS group at the 3 0 -end and the DMTr group at the 5 0 -end were removed; (10) the desired trimer TTT was extracted to the aqueous layer by using LLE and the aqueous layer was dried by lyophilization to afford the crude mixture. The result of reversed-phase high performance liquid chromatography (RP-HPLC) analysis for the crude mixture is shown in Fig.…”

“…Some kinds of soluble supports for the solution-phase synthesis of oligonucleotides or peptides have been reported to date. 8,10,17,30,31 Among them, we selected the gallate derivative, which is one of the most commonly used soluble supports and easily precipitated by the addition of polar solvents owing to its high hydrophobicity. Thymidine 5 0 -H-phosphonate bearing a soluble support was synthesized as shown in Scheme 6.…”

“…Thymidine 5 0 -H-phosphonate bearing a soluble support was synthesized as shown in Scheme 6. Then, we attempted solution-phase synthesis of two sequences of precipitation using CH 3 CN; (10) the soluble support at the 3 0end and the DMTr group at the 5 0 -end were removed; (11) the desired tetramer was extracted to the aqueous layer using LLE and the aqueous layer was dried by lyophilization to afford the crude mixture. The results of RP-HPLC analysis for the crude mixtures are shown in Fig.…”