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Orthoester compositions for affinity purification of oligonucleotides

An oligonucleotide and orthoester technology, applied in sugar derivatives, organic chemistry, sugar derivatives, etc., can solve the problems of difficult removal of DMT groups, long reaction time under strong acid conditions, and decreased purity.

Pending Publication Date: 2020-04-17
AGILENT TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, HPLC purification using DMT has several disadvantages
Although the DMT group is easily removed while the nucleotide is on the solid support due to favorable equilibrium conditions, the DMT group is significantly more difficult to remove after removal of the oligonucleotide from the solid support, requiring strong acidic conditions and long reaction times
Unfortunately, the conditions required to remove the DMT group in solution can degrade the purified oligonucleotides, ultimately resulting in a significant loss of purity

Method used

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  • Orthoester compositions for affinity purification of oligonucleotides
  • Orthoester compositions for affinity purification of oligonucleotides
  • Orthoester compositions for affinity purification of oligonucleotides

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[0133] In some embodiments, methods of oligonucleotide synthesis include support-bound nucleosides with a 3'-DMT protecting group. In some embodiments, methods of oligonucleotide synthesis include support-bound nucleosides with 5'-silyl protecting groups. In some embodiments, methods of oligonucleotide synthesis include support-bound nucleosides with oxidatively removable protecting groups. In some embodiments, oligonucleotide synthesis includes detritylation, coupling of support-bound nucleosides to nucleoside phosphoramidite monomers, capping of unreacted 5'-hydroxyl and phosphoramidite monomers oxidation step. In some embodiments, oligonucleotide synthesis is automated. In some embodiments, the oligonucleotides are detritylated prior to performing the methods of the invention.

[0134]In one embodiment, the invention provides a method of purifying oligonucleotides. The method comprises synthesizing an oligonucleotide on a solid support; reacting the oligonucle...

Embodiment 1

[0384] Synthesis of 2-methoxy-4-(((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)methyl) -1,3-dioxolane. 3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy]-1,2-propanediol (1 equivalent, BOC Sciences) was dissolved in trifluorotoluene (Aldrich) at a concentration of 2M. Trimethylorthoformate (3 eq., Aldrich) was dissolved in anhydrous cyclohexane (Aldrich) at a concentration of 3.5M in a round bottom flask. A catalytic amount of Amberlyst 15 (Type H, Aldrich) was added to the flask along with a Teflon-coated magnetic stir bar. Add 3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy]-1,2-propanediol under stirring trifluorotoluene solution and the flask was fitted with a Dean-Stark distillation head. The reaction was slowly heated to reflux and methanol was removed by azeotropic distillation at bp 45°C. After 3 hours, the reaction was complete and the flask was cooled to room temperature. The Amberlyst resin was removed by filtration, and the filtrate was concentra...

Embodiment 2

[0387] 4-(((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)methyl)-2-(2,2 ,2-trifluoroethoxy)-1,3-dioxolane. Tris(2,2,2-trifluoroethyl) orthoformate (50 g, 161 mmol, SynQuest Laboratories) and 3-[(3,3,4,4,5,5,6,6 ,7,7,8,8,8-Tridecafluorooctyl)oxy]-1,2-propanediol (25 g, 57 mmol, Wako Chemicals) was dissolved in 800 mL of anhydrous cyclohexane (Aldrich). Trifluorotoluene (Aldrich) was added in 5 mL portions until the solution was clear (30 mL). A catalytic amount of p-toluenesulfonic acid (Aldrich) was added to the flask along with a Teflon-coated magnetic stir bar. The flask was fitted with a Dean Stark distillation head. The reaction was slowly heated to reflux and trifluoroethanol was removed by azeotropic distillation at bp 65°C. After 3 hours, the reaction was complete and the flask was cooled to room temperature. The reaction mixture was concentrated under reduced pressure on a rotary evaporator. The residue was purified by fractional distillation under reduced pr...

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Abstract

Compounds and methods for purifying oligonucleotides such as RNA and DNA. A target oligonucleotide is reacted with an orthoester linker comprising an affinity tag to form an orthoester oligonucleotide-orthoester linker conjugate which is subjected to a purification technique to separate the target oligonucleotide from impurities such as truncated oligonucleotides. The orthoester linker can be thenremoved under mild conditions to generate the target oligonucleotide in high purity.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 547,687, filed August 18, 2017, which is incorporated by reference in its entirety. technical field [0003] The present application relates to compounds and methods for the purification of biopolymers such as RNA and DNA. Background technique [0004] Solid phase synthesis is an invaluable tool that can be used to prepare custom oligonucleotide sequences (eg, custom RNA and DNA), custom peptides, custom oligosaccharides, and various metabolites. The chemical synthesis of oligonucleotides is usually performed in a sequential manner, in which one end of a growing chain is attached to a solid surface, and then the reactive nucleoside monomers are sequentially condensed using repeated synthesis cycles. These nucleomonomers contain reactive phosphorous groups such as phosphoramidites, H-phosphates, or other reactive phosphorous or modifying groups ...

Claims

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Application Information

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IPC IPC(8): C07H1/06C07H21/02C07H21/04
CPCC07H1/06C07H21/02C07H21/04C07D317/34C07D403/06C07D405/12
Inventor D·J·德林杰J·迈尔森B·斯马特
Owner AGILENT TECH INC
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