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US 6,353,098 B1Granted
Synthesis, deprotection, analysis and purification of RNA and ribozymes
AssigneeRibozyme Pharmaceuticals, Inc.
InventorsNassim Usman, Francine Wincott
Life of this patent
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Abstract
A process for deprotecting RNA alkylsilyl groups comprising, contacting the groups with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at between 60° C.-70° C. for 0.25-24 h.
Claims22
A process for deprotecting and purifying chemically synthesized RNA, comprising the steps of:
- (a) deprotection, wherein said deprotection comprises the step of removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with an alkylamine or a mixture of ammonium hydroxide and alkylamine at a temperature of about 60-70 degrees centigrade for about 5-15 minutes, under conditions suitable for the removal of said exocyclic protecting groups, wherein said alkylamine is selected from the group consisting of ethylamine, propylamine and butylamine;
- and
- (b) purification, wherein said purification comprises passing the chemically synthesized RNA over a high performance liquid chromatography (HPLC) column or an anion exchange column or both, under conditions which allow for the purification of the RNA.
A process for deprotecting and purifying chemically synthesized RNA, comprising the steps of:
- (a) deprotection, wherein said deprotection comprises the step of removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises passing the chemically synthesized RNA over a high performance liquid chromatography (HPLC) column or an anion exchange column or both, under conditions which allow for the purification of the RNA.
A process for deprotecting and purifying chemically synthesized RNA, comprising the steps of:
- (a) deprotection, wherein said deprotection comprises the steps of:
- (i) removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with an alkylamine or a mixture of ammonium hydroxide and alkylamine at a temperature of about 60-70 degrees centigrade for about 5-15 minutes, under conditions suitable for the removal of said exocyclic protecting groups, wherein said alkylamine is selected from the group consisting of ethylamine, propylamine and butylamine;
- and (ii) removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises passing the chemically synthesized RNA over a high performance liquid chromatography (HPLC) column or an anion exchange column or both, under conditions which allow for the purification of the RNA.
2 more elements
A process for deprotecting and purifying chemically synthesized RNA, comprising the steps of:
- (a) deprotection, wherein said deprotection is a one pot deprotection process comprising the steps of:
- (i) removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with anhydrous alkylamine at a temperature of about 60-70 degrees centigrade for at least 5 minutes under conditions suitable for the removal of said exocyclic protecting groups;
- and (ii) removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises passing the chemically synthesized RNA over a high performance liquid chromatography (HPLC) column or an anion exchange column or both, under conditions which allow for the purification of the RNA.
2 more elements
Dependencies are inferred from the claim text.