WO2009140125A2

WO2009140125A2 - Deprotection of synthetic oligonucleotides using aqueous ethanolamine

Info

Publication number: WO2009140125A2
Application number: PCT/US2009/043064
Authority: WO
Inventors: Francis Gosselin; Amran Gowani
Original assignee: Merck & Co., Inc.
Priority date: 2008-05-15
Filing date: 2009-05-07
Publication date: 2009-11-19
Also published as: WO2009140125A3

Abstract

This invention concerns an efficient and safer (easy to handle and environmentally less hazardous) protocol for cleavage and deprotection of synthetic oligonucleotides utilizing ethanolamine. Specifically, the present invention features a method for 1) the removal of amide protecting groups from nucleobases and 2) cleavage of the synthetic oligonucleotides from their solid support allowing the isolation and subsequent purification of said oligonucleotides.

Description

TITLE QF THE INVENTION

DEPROTECTION OF SYNTHETIC OLIGONUCLEOTIDES USING AQUEOUS

ETHANOLAMINE

BACKGROUND OF THE INVENTION

This invention relates to the use of aqueous ethanolamine for the cleavage and deprotection of synthetic oligonucleotides for therapeutic purposes.

Currently, cleavage and deprotection of synthetic oligonucleotides is accomplished utilizing aqueous methylamine or aqueous ammonium hydroxide solutions. The drawbacks to using either of these reagents, alone or in concert, are that they are volatile, caustic and generally difficult to handle.

As such there exists an unmet need for safer (easy to handle and less hazardous), fast and efficient protocols which allow for cleavage and deprotection of modified and unmodified oligonucleotides. Such a method will simplify and facilitate large scale synthesis of such oligonucleotides for use as therapeutic agents and the small scale synthesis of such molecules for combinatorial screening as well as other uses.

SUMMARY OF THE INVENTION

DETAILED DESCRIPTION OF THE INVENTION

Synthesis of oligonucleotides including modified, unmodified or abasic nucleotides (and also including oligonucleotides derived from all commercially available phosphoramidites), comprises the steps of: (a) solid phase, solution phase, and/or hybrid phase, (e.g.; phosphoramidite-based or H-phosphonate~based) oligonucleotide synthesis comprising the steps of detritylation, activation, coupling, capping, and oxidation or the equivalent thereof, in any suitable order, followed by (b) deprotection comprising contacting the oligonucleotide with a base, from temperatures ranging from 23 to 65 ⁰C for times ranging from 10 minutes to 24 hours, followed by, where appropriate, (c) contacting the partially deprotected oligonucleotide comprising one or more nucleotides with fluoride salts in the absence or presence of a co- solvent. The synthesis of oligonucleotides is well known in the art.

The instant invention features a process, utilizing ethanolamine, for the rapid deprotection of oligonucleotides comprising modified or unmodified oligonucleotides which are protected with amide protecting groups. Additionally, the invention provides a process, utilizing ethanolamine, for the deprotection of oligonucleotides comprising 2'-deoxy- riboraranose and T- modified-ribofuranose moieties whose bases are protected with benzoate (Bz), acetate (Ac), isobutyrate (i-Bu) protecting groups, modifications of these amides or other similar amide protecting groups. The instant invention also features the use of an aqueous ethanolamine solution to partially deprotect oligonucleotides followed by treatment with fluoride salts (for example, potassium fluoride) in the absence or presence of a co-solvent (for example, DMSO, DMAC, DMF, NMP) for the complete removal of T-O- silyl protecting groups.

In an embodiment, the invention features a deprotection process, comprising (a) contacting a solid phase, solution phase, and/or hybrid phase oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100°C to fully deprotect the oligonucleotide and cleave the oligonucleotide and (b) where appropriate, contacting a partially deprotected oligonucleotide with a fluoride salt in the absence or presence of a co-solvent at a pH range of 4 - 13, and (c) heating at about 10 to 100 ⁰C for about 5 to 240 minutes to remove 2 '-hydroxy! protecting groups.

In another embodiment, the invention features a deprotection process, comprising (a) contacting a solid phase, solution phase, and/or hybrid phase oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100⁰C or 20⁰C to 8O⁰C or 3O⁰C to 65°C or preferably 23⁰C to fully deprotect the oligonucleotide and cleave the oligonucleotide from a resin and (b) where appropriate, contacting a partially deprotected oligonucleotide with fluoride salts in the absence or presence of a co-solvent at a pH range of 4 - 13, and (c) heating at about 10 to 100 ⁰C, preferably at about 55 ⁰C, for about 5 to 240 minutes, preferably about 60 minutes, to remove 2'-hydroxyl protecting groups.

In another embodiment, the invention features a deprotection process, comprising (a) contacting a solid-support bound oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100⁰C or 20⁰C to 80⁰C or 30⁰C to 65°C or preferably 23⁰C to fully deprotect the oligonucleotide and cleave the oligonucleotide from a resin and (b) where appropriate, contacting a partially deprotected oligonucleotide with triethylamine trihydrofluoride (or other fluoride salts including but not limited to potassium fluoride, sodium fluoride, calcium fluoride, magnesium fluoride etc.) in the absence or presence of a co-solvent (for example DMSO, DMAC, DMF, HMPA, ethanol, methanol, isopropanol, N-methylpyrrolidinone and others) at a pH range of 4 - 13 , and (c) heating at about 10 to 100 ⁰C, preferably at about 55 ⁰C, for about 5 to 240 minutes, preferably about 60 minutes, to remove 2'-hydroxyl protecting groups (for example, t-butyldimethylsilyl).

In another embodiment, the invention features a deprotection process, comprising (a) contacting a solid-support bound oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100⁰C or 20⁰C to 8O⁰C or 30⁰C to 65°C or preferably 23°C to fully deprotect the oligonucleotide and cleave the oligonucleotide from a resin and (b) where appropriate, contacting a partially deprotected oligonucleotide with triethylamine trihydrofluoride in the absence or presence of a co-solvent (for example DMSO, DMAC, DMF₅ HMPA, ethanol, methanol, isopropanol, N-methylpyrrolidinone and others) at a pH range of 4 - 13, and (c) heating at about 10 to 100 ⁰C, preferably at about 55 ⁰C, for about 5 to 240 minutes, preferably about 60 minutes, to remove 2'-hydroxyl protecting groups (for example, t-butyldimethylsilyl).

In another embodiment, the invention features a deprotection process, comprising (a) contacting a solid-support bound oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100⁰C or 2O⁰C to 8O⁰C or 30⁰C to 65°C or preferably 23°C to fully deprotect the oligonucleotide and cleave the oligonucleotide from a resin and (b) where appropriate, contacting a partially deprotected oligonucleotide with triethylamine trihydrofluoride in the absence or presence of a co-solvent selected from DMSO, DMAC, DMF, HMPA, ethanol, methanol, isopropanol, and N-methylpyrrolidinone at a pH range of 4 - 13, and (c) heating at about 10 to 100 ⁰C, preferably at about 55 ⁰C, for about 5 to 240 minutes, preferably about 60 minutes, to remove 2'-hydroxyl protecting groups (for example, t-butyldimethylsilyl). In another embodiment, the invention features a deprotection process, comprising

(a) contacting a solid-support bound oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100⁰C or 20⁰C to 80⁰C or 3O⁰C to 65⁰C or preferably 23°C to fully deprotect the oligonucleotide and cleave the oligonucleotide from a resin and (b) where appropriate, contacting a partially deprotected oligonucleotide with triethylamine trihydrofluoride in the absence or presence of a co-solvent selected from DMSO, DMAC, DMF, HMPA, ethanol, methanol, isopropanol, and N-methylpyrrolidinone at a pH range of 4 - 13, and (c) heating at about 10 to 100 ⁰C, preferably at about 55 ⁰C, for about 5 to 240 minutes, preferably about 60 minutes, to remove the 2 '-hydroxy! protecting group, t-butyldimethylsilyl. By "oligonucleotide" is meant a nucleic acid molecule. The oligonucleotide can be single, double or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof, which are well known in the art. The oligonucleotide may be RNA (modified or unmodified, enzymatic or nonenzymatic) or DNA (modified or unmodified). The oligonucleotide may be an antisense or enzymatic RNA molecule including siRNA and mϊRNA. Modified oligonucleotides are well known in the art.

Oligonucleotides can be used for purposes including but not limited to use as therapeutic agents, diagnostic reagents, and research reagents.

Procedure- 1 Schematic representation of the deprotection of oligonucleotides

Liberated, Deprotected Oligonucleotide

Schematic showing deprotection of oligonucleotides with ethanolamine

Liberated, Deprotected Oligonucleotide

Materials MW Amount Moles

Ohgonucelotide 100 mg 25 μmol

Ethanolamine (12.9M in water) 61.08 1O mL 129 mmol

3.36M aqueous AcOH 35-40 mL 117-134 mmol

Process Description: To a 10 mL reaction flask/bottle/vial was added solid CPG protected oligonucleotide, followed by 12.9M ethanolamine (10 mL), and the reaction mixture was aged at rt. Aliquots were taken during 30 minute intervals and analyzed by HPLC. After oh, the reaction was complete and the mixture was cooled to 5 ⁰C and neutralized with 3.36M aqueous AcOH. The mixture was then filtered through a 0.2 micron filter. Analysis by m/s, SAX-HPLC and UPLC ensued.

Sample preparation: 20 μL of the reaction mixture is diluted 100 μL of 3.36M AcOH and 100 μL of water, and then passed through a syringe filter to remove the CPG. SAX-HPLC conditions: Dionex BioLC DNA Pac PAlOO, 4 * 250 mm <part# 043010), 1 mL / min, 10 μL injection, detection: 260 nm, T = 80⁰C

Buffer A: 10 mM NaClO₄, 10 mM Tris-HCl, 20% EtOH

Buffer B: 300 mM NaClO₄, 10 mM Tris-HCl, 20% EtOH Gradient: 0-83% B over 25 min, 100% B to 26 min, 100% A to 27 min, hold 3 min.

Procedure-2

Process Description: To a 10 mL reaction flask/bottle/vial was added solid CPG protected oligonucleotide, followed by 5.0M ethanolamine (10 mL), and the reaction mixture was aged at rt. Aliquots were taken during 30 minute intervals and analyzed by HPLC. After 24h, the reaction was complete and the mixture was cooled to 5 ⁰C and neutralized with 3.36M aqueous

AcOH. The mixture was then filtered through a 0.2 micron filter. Analysis by m/s_> SAX-HPLC and UPLC followed.

Sample preparation: 20 μL of the reaction mixture is diluted with 20 μL of 3.36M AcOH and

100 μL of water, and then passed through a syringe filter to remove the CPG. SAX-HPLC conditions: Dionex BioLC DNA Pac PAlOO, 4 x 250 mm (ρart# 043010), 1 mL / min, 10 μL injection, detection: 260 nm, T = 80⁰C

Buffer A: 10 mM NaClO₄, 10 mM Tris-HCl, 20% EtOH

Buffer B: 300 mM NaClO₄, 10 mM Tris-HCl, 20% EtOH

Gradient: 0-83% B over 25 min, 100% B to 26 min, 100% A to 27 min, hold 3 min. Procedure-3

Process Description: To a 10 mL reaction flask/bottle/vial was added solid CPG protected oligonucleotide, followed by 5.0M ethanolamine (10 mL), and the reaction mixture was aged at

55 ⁰C for 100 min. After removing from the heating source, the mixture was cooled to 5 ⁰C and neutralized with 3.36M aqueous AcOH. The mixture was then filtered through a 0.2 micron filter. Analysis by m/s, S AX-HPLC and UPLC ensued.

100 μL of water, and then passed through a syringe filter to remove the CPG.

SAX-HPLC conditions: Dionex BioLC DNA Pac PAlOO, 4 x 250 mm (part# 043010), 1 mL / min, 10 μL injection, detection: 260 nm, T = 80⁰C Buffer A: 10 mM NaClO₄, 10 mM Tris-HCl, 20% EtOH

Buffer B: 300 mM NaClO₄, 10 mM Tris-HCl, 20% EtOH

Gradient: 0-83% B over 25 min, 100% B to 26 min, 100% A to 27 min, hold 3 min.

EXAMPLE l

Deprotection of an oligonucleotide, 23mer, trityl-off, Procedure 1. Oligonucleotide is comprised of standard phosporamidites including but not limited to dA, dC, dG, T, rA, rC, rG, rU, iB, OMeA, OMeC, OMeG and OMeU.

Reverse phase UPLC results, wavelength = 260 nm, LCMS results, desired mass = 6887.20 amu.

EXAMPLE 2 Deprotection of an oligonucleotide, 23mer, trityl-off, Procedure 1.

Oligonucleotide is comprised of standard phosporamidites including but not limited to dA, dC, dG, T, rA, rC, rG_? rU, iB, OMeA, OMeC, OMeG and OMeU. Reverse phase UPLC results, wavelength = 260 nm.

EXAMPLE 3 Deprotection of an oligonucleotide, 23mer, trityl-off, Procedure 2,

Oligonucleotide is comprised of standard phosporamidites including but not limited to dA, dC, dG, T, rA, rC, rG, rU, iB, OMeA, OMeC, OMeG and OMeU. Reverse phase UPLC results, wavelength = 260 nm.

EXAMPLE 4 Deprotection of an oligonucleotide, 23mer, trityl-off, Procedure 3.

Oligonucleotide is comprised of standard phosporamidites including but not limited to dA, dC, dG, T, rA, rC, rG, rU_} iB, OMeA, OMeC, OMeG and OMeU.

Reverse phase UPLC results, wavelength = 260 nm. SAX-HPLC results, wavelength = 260 nm. LCMS results, desired mass = 6846,22 amu.

Claims

WHAT IS CLAIMED IS:

1. A deprotection process, comprising (a) contacting a solid phase oligonucleotide with ethanolamine (neat or aqueous) at about 10 to 100⁰C to folly deprotect the oligonucleotide and cleave the oligonucleotide and (b) where appropriate, contacting a partially deprotected oligonucleotide with a fluoride salt in the absence or presence of a co-solvent at a pH range of 4 - 13, and (c) heating at about 10 to 100 ⁰C for about 5 to 240 minutes to remove 2' -hydroxy 1 protecting groups.