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Processes and reagents for oligonucleotide synthesis and purification

a technology of oligonucleotide and reagents, applied in the field of process and reagents for oligonucleotide synthesis and purification, can solve the problems of time-consuming deprotection, purification and analysis procedures, and many research efforts hampered

Inactive Publication Date: 2005-12-01
ALNYLAM PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] The present invention relates to processes and reagents for oligonucleotide synthesis and purification. One aspect of the present invention relates to compounds useful for activating phosphoramidites in oligonucleotide synthesis. Another aspect of the present invention relates to a method of preparing oligonucleotides via the phosphoramidite method using an activator of the invention. Another aspect of the present invention relates to sulfur-transfer agents. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to a method of preparing a phosphorothioate by treating a phosphite with a sulfur-transfer reagent of the invention. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present

Problems solved by technology

However, many research efforts are hampered by the small quantities of oligonucleotides that are available for study.
However, the synthesis of oligonucleotides and their analogs is often a tedious and costly process.
Unfortunately, the aforementioned chemical synthesis, deprotection, purification and analysis procedures are time consuming (10-15 min. coupling times), subject to inefficient activation of the RNA amidites by tetrazole, incomplete deprotection of the exocyclic amino protecting groups by NH4OH, limited by the low capacity of RNA purification using gel electrophoresis, and further limited by low resolution analysis of the RNA by gel electrophoresis.
In particular, the oligonucleotide sequences have poor serum solubility, poor cellular distribution and uptake, and are rapidly excreted through the kidneys.

Method used

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  • Processes and reagents for oligonucleotide synthesis and purification
  • Processes and reagents for oligonucleotide synthesis and purification
  • Processes and reagents for oligonucleotide synthesis and purification

Examples

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Effect test

example 1

Oligonucleotide Synthesis Using Phosphoramidite Activators 35-48 (see FIGS. 1-3)

[0534] In certain instances the strength of the activator is increased by forming the activated salt resulting in decreased coupling time for RNA Synthesis.

[0535] A decamer RNA molecules (49, 5′-CAUCGCTGAdT-3′) was synthesized on a 394 ABI machine (ALN 0208) using the standard 98 step cycle written by the manufacturer with modifications to a few wait steps as described below. The solid support was controlled pore glass (CPG, prepacked, 1 μmole, 500, Proligo Biochemie GmbH) and the monomers were RNA phosphoramidites with fast deprotecting groups obtained from Pierce Nucleic Acid Technologies used at concentrations of 0.15 M in acetonitrile (CH3CN) unless otherwise stated. Specifically the RNA phosphoramidites were 5′-O-Dimethoxytrityl-N6-phenoxyacetyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite, 5′-O-Dimethoxytrityl-N2-p-isopropylphenoxyacetyl-2′-O-tbutyldimeth...

example 2

Synthesis of compound 1 (R′=H and R″=C(S)OEt or R′,R″=H)

[0538]

[0539] A solution of chlorocarbonyl sulfenyl chloride (8.4 mL, 0.1 mol) in dry ether (50 mL) was added dropwise to a cold solution of thiourea (7.62 g, 0.1 mol) in dry ether (500 mL) and triethylamine (14 mL, 0.1 mol) cooled with ice-bath in 3 h under an argon atmosphere. The reaction mixture was stirred at the same temperature for total of 6 h. The solids were filtered off and the filtration was concentrated into a crude residue which was further crystallized with dichlorometrhane-hexanes to give a pure compound (2.5 g). The mother liquid was then concentrated into a crude residue which was applied to a column of silica gel eluted with dichloromethane-metahnol (40:1) to give a pure compound (180 mg). The total yield is about 30%. 1H-NMR (CDCl3, 400 MHz): δ 10.46 (br, 1H), 4.38 (q, 2H, J=6.8, 14.4 Hz, CH2), 1.39 (t, 3H, J=7.2 Hz, CH3). 3C-NMR (CDCl3, 100 MHz): 181.01, 177.00, 153.75, 64.68, 14.32.

example 3

Phosphorothioation of Di- and POLY-Oligothymidine Using Sulfur Transfer Reagent 1 (R′=H and R″=C(S)OEt or R′,R″=H):

[0540] Dinucleotide 2 and hexamer 3 were synthesized on a 394 ABI machine using the standard 93 step cycle written by the manufacturer with modifications to a few wait steps as described below. Activator used was 5-(ethylthio)-1H-tetrazole (0.25 M), and for PS-oxidation, 0.05 M 1 in anhydrous acetonitrile was used. The sulfurization time was about 4 min. After completion of the synthesis, 2 and 3 were deprotected from support by aqueous ammonia treatment at 55° C. for 1 h. After HPLC purification, the compound were analysed by LC-MS.

[0541] The results of phosphorothioation of oligothymdine using 1 as the sulfur-transfer agent are shown below.

Sequence,MassMassCompoundall P═SCalc.Found25′ TT 3′562.46562.2235′ TTTTTT 3′1843.521842.05

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Abstract

The present invention relates to processes and reagents for oligonucleotide synthesis and purification. One aspect of the present invention relates to compounds useful for activating phosphoramidites in oligonucleotide synthesis. Another aspect of the present invention relates to a method of preparing oligonucleotides via the phosphoramidite method using an activator of the invention. Another aspect of the present invention relates to sulfur-transfer agents. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to a method of preparing a phosphorothioate by treating a phosphite with a sulfur-transfer reagent of the invention. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to compounds that scavenge acrylonitrile produced during the deprotection of phosphate groups bearing ethylnitrile protecting groups. In a preferred embodiment, the acrylonitrile scavenger is a polymer-bound thiol. Another aspect of the present invention relates to agents used to oxidize a phosphite to a phosphate. In a preferred embodiment, the oxidizing agent is sodium chlorite, chloroamine, or pyridine-N-oxide. Another aspect of the present invention relates to methods of purifying an oligonucleotide by annealing a first single-stranded oligonucleotide and second single-stranded oligonucleotide to form a double-stranded oligonucleotide; and subjecting the double-stranded oligonucleotide to chromatographic purification. In a preferred embodiment, the chromatographic purification is high-performance liquid chromatography.

Description

RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 60 / 559,782, filed Apr. 5, 2004; the entirety of which is hereby incorporated by reference.BACKGROUND OF THE INVENTION [0002] The study of oligonucleotides is a key area of research for many academic and industrial laboratories. See S. Agrawal Trends in Biotechnology 1996, 14, 375-382; J. Marr Drug Discovery Today 1996, 1, 94-102; and W. Rush Science 1997, 276, 1192-1193. The therapeutic and diagnostic potential of oligonucleotides has sparked a substantial amount of research activity. One important application of oligonucleotides is the ability to modulate gene and protein function in a sequence-specific manner. However, many research efforts are hampered by the small quantities of oligonucleotides that are available for study. A method to produce large quantities of oligonucleotide compounds having high purity would greatly facilitate oligonucleotide research....

Claims

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Application Information

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IPC IPC(8): C07D285/00C07D285/01C07H21/00C07H21/04
CPCC07H21/04C07H21/00Y02P20/55
Inventor MANOHARAN, MUTHIAHJUNG, MICHAELRAJEEV, KALLANTHOTTATHILPANDEY, RAJENDRAWANG, GANG
Owner ALNYLAM PHARM INC
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