Method of synthesizing oligonucleotides labeled with ammonia-labile groups on solid phase supports

• US 4,965,349 A
• Filed: 12/24/1987
• Issued: 10/23/1990
• Est. Priority Date: 12/24/1987
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven M., Woo, Sam L., Fung, Steven
• Primary Examiner(s): Rollins, John W.
• Assistant Examiner(s): Wilson, James O.
• Application Number:
  US07/138,287
• Time in Patent Office: 1,034 Days
• Field of Search: 536/23, 536/27, 536/28, 536/29, 525/54.2
• US Class Current: 536/25.3
• CPC Class Codes: C07F 9/24 Esteramides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

The invention provides a novel cleavage reagent for hydrolysing base-labile linking groups between a solid phase support and oligonucleotides. The cleavage reagent comprises a lower alkyl alcohol, water, and a non-nuccleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:1:1 to about 1:3:1, respectively. An important property of the cleavage reagent is that it preserves the fluorescent characteristics of rhodamine dyes during cleavage, thereby making it possible to completely synthesize rhodamine-labeled oligonucleotides by solid phase methods. Rhodamine phosphoramidites are provided to further enhance the efficiency of synthesizing rhodamine-labeled oligonucleotides by such methods.

Claims

1. A process of cleaving a base-labile linking group between an oligonucleotide and a solid phase support comprising the step of exposing the linking group to a cleavage reagent until the linking group is hydrolyzed, the cleavage reagent comprising a lower alkyl alcohol, water, and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:

1;

1 to about 1;

3;

1 by volume, respectively.

2. The process of claim 1 wherein said lower alkyl alcohol is selected from the group consisting of methanol, ethanol, and propanol, and wherein said nonnucleophilic hindered alkylamine is selected from the group consisting of isopropylamine, t-butylamine, diethylamine, piperidine, t-amylamine, diisopropylamine, and dipropylamine.

3. The process of claim 2 wherein said linking group is a succinate ester.

4. The process of claim 3 wherein said cleavage reagent comprises methanol, water and t-butylamine in a ratio of about 1:

2;

1, respectively.

5. A process of synthesizing oligonucleotides labeled with an ammonia-labile group on a solid phase support, the method comprising the steps of:

providing an oligonucleotide attached to a solid phase support by a base-labile linking group;

attaching one or more ammonia-labile groups to the oligonucleotide to form an oligonucleotide labeled with an ammonia-labile group; and

cleaving the linking group with a cleavage reagent so that the oligonucleotide labeled with an ammonia-labile group is freed from the solid phase support, the cleavage reagent comprising a lower alkyl alcohol, water, and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1;

1;

1 to about 1;

3;

1 by volume, respectively.

6. The process of claim 5 wherein said linking group forms a covalent bond between the 3'"'"' hydroxyl group of said oligonucleotide and said solid phase support.

7. The process of claim 6 wherein (1) said lower alkyl alcohol is selected from the group consisting of methanol, ethanol, and propanol, (2) said non-nucleophilic hindered alkylamine is selected from the group consisting of isopropylamine, t-butylamine, diethylamine, piperidine, t-amylamine, diisopropylamine, and dipropylamine, and (3) said base-labile linking group is a succinate ester.

8. The process of claim 7 wherein said ammonia-labile group is a rhodamine dye molecule.

9. The process of claim 8 wherein said cleavage reagent comprises methanol, water, and t-butylamine in a ratio of about 1:

2;

1, respectively.

10. The process of claim 8 wherein said step of attaching one or more rhodamine dye molecules includes reacting a rhodamine phosphoramidite with the 5'"'"' hydroxyl of said oligonucleotide.

11. The process of claim 5 wherein said ammonia-labile group is a rhodamine dye molecule.