Methods of synthesizing oligonucleotides using carbonate protecting groups and alpha-effect nucleophile deprotection

• US 7,271,258 B2
• Filed: 06/15/2006
• Issued: 09/18/2007
• Est. Priority Date: 08/03/1998
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc., The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Caruthers, Marvin H., Dollinger, Douglas J., Betley, Jason R.
• Primary Examiner(s): Jiang; S. Anna
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US11/453,734
• Publication Number:
  US 20060252924A1
• Time in Patent Office: 460 Days
• Field of Search: 536/25.3, 536/25.33, 536/25.34, 536/26.7, 536/26.8, 536/27.6, 536/28.5, 536/27.81, 536/28.53
• US Class Current: 536/26.7
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides methods for synthesizing oligonucleotides using nucleoside monomers having carbonate protected hydroxyl groups that are deprotected with α-effect nucleophiles. The α-effect nucleophile irreversibly cleave the carbonate protecting groups while simultaneously oxidizing the internucleotide phosphite triester linkage to a phosphodiester linkage. The procedure may be carried out in aqueous solution at neutral to mildly basic pH. The method eliminates the need for separate deprotection and oxidation steps, and, since the use of acid to remove protecting groups is unnecessary, acid-induced depurination is avoided. Fluorescent or other readily detectable carbonate protecting groups can be used, enabling monitoring of individual reaction steps during oligonucleotide synthesis. The invention is particularly useful in the highly parallel, microscale synthesis of oligonucleotides.

Claims

1. A nucleoside monomer having the structure of formula (II) or formula (X)

2. The nucleoside monomer of claim 1, wherein X is di(lower alkyl)amino or morpholino and Y is selected from the group consisting of lower alkyl, electron-withdrawing β

-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl, wherein the substitution is on one or more carbon atoms and the substitution is by a group selected from a cyano group, a nitro group, a sulfo group, and a halo group.

3. The nucleoside monomer of claim 2, wherein Y is selected from the group consisting of methyl, β

-cyanoethyl and 4-nitrophenylethyl.

4. The nucleoside monomer of claim 1, wherein R³ is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′

-(α

-methyl-2-nitropiperonyl)oxycarbonyl (“

MeNPOC”

), 9-fluorenylmethylcarbonyl (“

Fmoc”

), 2,2,2-trichloro-1,1-dimethylcarbonyl (“

TCBOC”

) and cyano-substituted lower alkyl carbonates.

5. The nucleoside monomer of claim 2, wherein R³ is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′

-(α

-methyl-2-nitropiperonyl)oxycarbonyl (“

MeNPOC”

), 9-fluorenylmethylcarbonyl (“

Fmoc”

), 2,2,2-trichloro-1,1-dimethylcarbonyl (“

TCBOC”

) and cyano-substituted lower alkyl carbonates.

6. The nucleoside monomer of claim 3, wherein R³ is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′

-(α

-methyl-2-nitropiperonyl)oxycarbonyl (“

MeNPOC”

), 9-fluorenylmethylcarbonyl (“

Fmoc”

), 2,2,2-trichloro-1,1-dimethylcarbonyl (“

TCBOC”

) and cyano-substituted lower alkyl carbonates.

7. The nucleoside monomer of claim 1, wherein R³ comprises a moiety that becomes fluorescent or colored upon cleavage of the carbonate —

OCOOR³.

8. The nucleoside monomer of claim 1, having the structure of formula (II).

9. The nucleoside monomer of claim 1, having the structure of formula (X).

10. A kit for synthesizing a phosphoramidite-based oligonueleotide on a solid support, comprising a hydroxyl-derivatized support surface, a protecting group for protecting hydroxyi moieties on said derivatized support surface, at least one protected nucleoside, at least one nucleoside phosphoramidite, a nucleophile that exhibits an alpha effect at neutral to mildly basic pH, the exocyclic base amine groups are tritylated, the 5′

-hydroxyl group is carbonate protected, the 5′

-deprotection/P-oxidation step includes the use of a peroxide anion or alpha-effect oxidation capable anion, and reagents suitable for establishing pH and for carrying out reactions of deprotection, phosphoramidite coupling, and oxidation to form an intemucleotide phosphotriester linkage.