On-demand synthesis of phosphoramidites

Abstract

Automated chemical synthesis of oligonucleotides is of fundamental importance for the production of primers for the polymerase chain reaction (PCR), for oligonucleotide-based drugs, and for numerous other medical and biotechnological applications. The highly optimised automised chemical oligonucleotide synthesis relies upon phosphoramidites as the phosphate precursors and one of the drawbacks of this technology is the poor bench stability of phosphoramidites. Here, we report on the development of an on-demand flow synthesis of phosphoramidites from their corresponding alcohols, which is accomplished with short reaction times, near-quantitative yields and without the need of purification before being submitted directly to automated oligonucleotide synthesis. Sterically hindered as well as redox unstable phosphoramidites are synthesised using this methodology and the subsequent couplings are near-quantitative for all substrates. The vision for this technology is direct integration into DNA synthesisers thereby omitting manual synthesis and storage of phosphoramidites.

Synthesis of Phosphatidyl Glycerol Containing Unsymmetric Acyl Chains Using H-Phosphonate Methodology

“…Initial efforts to improve the synthetic route involved substituting fluoride sources [ 25 , 26 , 27 ] in the deprotection step and substituting diphenylmethylsilyl ethers (DPMS) [ 28 ] on the phosphoglycerol headgroup instead of TBS ethers. Through a series of high-throughput experiments using desorption electrospray ionization mass spectrometry [ 29 , 30 ], and the modification of solketal protection from p-methoxybenzyl ether (PMB) [ 31 , 32 ] to a phenyl acyl ester, followed by translation to flow chemistry [ 33 ], we were able to successfully phosphorylate the protected phosphoglycerol head group on the gram scale, allowing us to upscale the synthesis of phosphoramidite intermediate in higher yields compared to the batch methods. Unfortunately, subsequent acyl chain migration and deprotection of the labile DPMS groups prevented us from moving forward with the phosphoramidite approach.…”

Biomanufacturing for Space-Exploration – What to Take and When to Make

“…To this end, even the accommodation of a "space biofoundry" (i.e., automated infrastructure for engineering and analytics of biological systems 45 ) in the mission architecture is within scope. Eventually, this will also entail the ISM of specialty chemicals and reagents like e.g., phosphoramidites for DNA synthesis, supporting on-site bioengineering 46 , in addition to the total inventories of foods, therapeutics and materials.…”

Ferrocene as a potential electrochemical reporting surrogate of abasic sites in DNA

“…[1][2][3][4] The need for synthesizing oligonucleotides equipped with chemical alterations has been spurred further by the recent advent of mRNA-based vaccines. 5,6 In this context, besides solid-phase synthesis, [7][8][9] modified oligonucleotides can be constructed using polymerase-catalysed incorporation of unnatural nucleoside triphosphates ((d)N*TPs). 10 This method is particularly alluring since it is compatible with Darwinian evolution methods to identify functional nucleic acids and it is not limited in terms of size or nature of the functional groups that can be incorporated.…”