phosphoramidite and thiophosphoric-acid

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.

Topics: DNA; Indicators and Reagents; Nucleosides; Organophosphonates; Organophosphorus Compounds; Phosphates; Phosphites; RNA

Although high-throughput methods for solid-phase synthesis of DNA sequences are currently available for synthetic biology applications and technologies for large-scale production of nucleic acid-based drugs have been exploited for various therapeutic indications, little has been done to develop high-throughput procedures for the purification of synthetic nucleic acid sequences. An efficient process for purification of phosphorothioate and native DNA sequences is described herein. This process consists of functionalizing commercial aminopropylated silica gel with aminooxyalkyl functions to enable capture of DNA sequences carrying a 5'-siloxyl ether linker with a "keto" function through an oximation reaction. Deoxyribonucleoside phosphoramidites functionalized with the 5'-siloxyl ether linker were prepared in yields of 75-83% and incorporated last into the solid-phase assembly of DNA sequences. Capture of nucleobase- and phosphate-deprotected DNA sequences released from the synthesis support is demonstrated to proceed near quantitatively. After shorter than full-length DNA sequences were washed from the capture support, the purified DNA sequences were released from this support upon treatment with tetra-n-butylammonium fluoride in dry DMSO. The purity of released DNA sequences exceeds 98%. The scalability and high-throughput features of the purification process are demonstrated without sacrificing purity of the DNA sequences.

Topics: Animals; Base Sequence; Chromatography, High Pressure Liquid; DNA; Escherichia coli; Magnetic Resonance Spectroscopy; Nucleic Acids; Organophosphorus Compounds; Phosphates; Silicon Dioxide; Snake Venoms; Solid-Phase Synthesis Techniques

The detailed preparation of deoxyribonucleoside phosphoramidites functionalized with a 4-methylthio-1-butyl group for P(III) protection is described, along with the incorporation of these phosphoramidites into DNA oligonucleotides via solid-phase techniques. The versatility of the thermolabile 4-methylthio-1-butyl phosphate/thiophosphate-protecting group is exemplified through its facile removal from oligonucleotides under neutral conditions or under standard basic conditions. The sulfonium salt that is produced during the thermolytic deprotection of oligonucleotides did not alter DNA nucleobases or desulfurize phosphorothioate diesters to a significant extent.

Topics: Hot Temperature; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Oligodeoxyribonucleotides; Organophosphorus Compounds; Phosphates

This unit provides procedures for the preparation of deoxyribonucleoside phosphoramidites and appropriate phosphordiamidite precursors with P(III) protecting groups different than the standard 2-cyanoethyl group. Specifically, these phosphoramidites are functionalized with the 3-(N-tert-butylcarboxamido)-1-propyl or 4-oxopentyl groups. The usefulness of these novel deoxyribonucleoside phosphoramidites in the solid-phase synthesis of a 20-mer DNA oligonucleotide and its phosphorothioated analog is demonstrated. It is also shown that removal of the 3-(N-tert-butylcarboxamido)-1-propyl phosphate/thiophosphate-protecting group from these oligonucleotides is rapidly effected under thermolytic conditions at neutral pH, whereas the 4-oxopentyl group is preferably removed by treatment with pressurized ammonia gas or concentrated ammonium hydroxide at ambient temperature. These detailed methods constitute an economical and alkylation-free approach to large-scale preparations of therapeutic oligonucleotides.

Topics: Oligodeoxyribonucleotides; Organophosphates; Organophosphorus Compounds; Organothiophosphates; Phosphates