Monomers for solid phase synthesis of oligonucleotides using carbonate protecting groups and alpha-effect nucleophile deprotection

• US 6,630,581 B2
• Filed: 01/08/2001
• Issued: 10/07/2003
• 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., Betley, Jason R., Dellinger, Douglas J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/756,991
• Publication Number:
  US 20020045221A1
• Time in Patent Office: 1,002 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.31, 536/25.33, 536/25.34
• US Class Current: 536/22.1
• 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 a method for synthesizing oligonucleotides using carbonate protection of hydroxyl groups and nucleophilic deprotection reagents. The deprotection reagents irreversibly cleave the carbonate protecting groups while simultaneously oxidizing the internucleotide phosphite triester linkage, and can be used 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. Reagents and kits for carrying out the aforementioned method are provided as well.

Claims

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

wherein:

B is a purine or pyrimidine base;

R is hydrido or hydroxyl;

R¹ is COOR³, wherein R³ is selected from the group consisting of o-nitrophenyl, phenyl, n-chlorophenyl, 5′

-(α

-methyl-2-nitropiperonyl), 1,1-dimethyl-2,2,2-trichloroethyl, and cyano-substituted lower alkyl moieties; and

R² has the structure (III)

wherein X is NQ¹Q² in which Q¹ and Q² are independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent, or wherein Q¹ and Q² are linked to form a mono- or polyheterocyclic ring, and Y is selected from the group consisting of hydrido, lower alkyl, electron-withdrawing β

-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.

2. The nucleoside monomer of claim 1, wherein X is di(lower alkyl)amino or morpholino.

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 comprises a moiety that becomes fluorescent or colored upon cleavage of the carbonate —

OCOOR³.

5. A kit for synthesizing an oligonucleotide on a solid support, comprising a hydroxyl-derivatized support surface, a protecting group for protecting hydroxyl moieties on said derivatized support surface, the nucleoside monomer of claim 1, least one nucleoside phosphoramidite, a nucleophile having a nucleophilic site and a lone electron pair on an atom adjacent to the nucleophilic site, and reagents suitable for establishing pH and for carrying out reactions of phosphoramidite coupling and simultaneous hydroxyl group deprotection and phosphite triester oxidation to form an internucleotide phosphotriester linkage.

33. The nucleoside monomer of claim 4, wherein R³ is selected from the group consisting of xanthenes, rhodamines, benzimidazoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicylic acids, benz-2-oxa-1-diazoles, fluorescamine, and 4-methylumbelliferone.

34. The nucleoside monomer of claim 33, wherein R³ is selected from the group consisting of 6-carboxy-fluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein, tetramethylrhodamine, 4-methyl-7-methoxycoumarin, dansyl, 5-dimethylamino-1-naphthalenesulfonyl, and 4-amino-7-nitrobenz-2-oxa-1,3-diazole.

36. The nucleoside monomer of claim 1, wherein R³ is substituted or unsubstituted alkyl.

37. The nucleoside monomer of claim 36, wherein R³ is substituted or unsubstituted C₁-C₁₂ alkyl.

38. The nucleoside monomer of claim 37, wherein R³ is fluorinated C₁-C₁₂ alkyl or cyano-substituted C₁-C₁₂ alkyl.

39. The nucleoside monomer of claim 1, wherein R³ is cyano-substituted lower alkyl.

40. The nucleoside monomer of claim 1, wherein R³ is 1,1-dimethyl-2,2,2-trichloroethyl or 1,1-dimethyl-2-cyanoethyl.

6. A nucleoside monomer having the structure of formula (X)

wherein:

B is a purine or pyrimidine base;

R is hydrido or hydroxyl;

R¹ is COOR³, wherein R³ is substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, and cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages selected from ether linkages, thioether linkages, oxo linkages, amine linkages, and imine linkages; and

R² has the structure (III)

wherein X is NQ¹Q² in which Q¹ and Q² are independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent, or wherein Q¹ and Q² are linked to form a mono- or polyheterocyclic ring, and Y is selected from the group consisting of hydrido, lower alkyl, electron-withdrawing β

-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.

7. The nucleoside monomer of claim 6, wherein X is di(lower alkyl)amino or morpholino.

8. The nucleoside monomer of claim 7, wherein Y is selected from the group consisting of methyl, β

-cyanoethyl and 4-nitrophenylethyl.

9. The nucleoside monomer of claim 6, wherein R³ is substituted or unsubstituted alkyl.

10. The nucleoside monomer of claim 9, wherein R³ is substituted or unsubstituted C₁-C₁₂ alkyl.

11. The nucleoside monomer of claim 10, wherein R³ is fluorinated C₁-C₁₂ alkyl or cyano-substituted C₁-C₁₂ alkyl.

12. The nucleoside monomer of claim 11, wherein R³ is cyano-substituted lower alkyl.

13. The nucleoside monomer of claim 6, wherein R³ is 1,1-dimethyl-2,2,2-trichloroethyl or 1,1-dimethyl-2-cyanoethyl.

14. The nucleoside monomer of claim 7, wherein R³ is substituted or unsubstituted alkyl.

15. The nucleoside monomer of claim 14, wherein R³ is substituted or unsubstituted C₁-C₁₂ alkyl.

16. The nucleoside monomer of claim 15, wherein R³ is fluorinated C₁-C₁₂ alkyl or cyano-substituted C₁-C₁₂ alkyl.

17. The nucleoside monomer of claim 16, wherein R³ is cyano-substituted lower alkyl.

18. The nucleoside monomer of claim 7, wherein R³ is 1,1-dimethyl-2,2,2-trichloroethyl or 1,1-dimethyl-2-cyanoethyl.

19. The nucleoside monomer of claim 8, wherein R³ is substituted or unsubstituted alkyl.

20. The nucleoside monomer of claim 19, wherein R³ is substituted or unsubstituted C₁-C₁₂ alkyl.

21. The nucleoside monomer of claim 20, wherein R³ is fluorinated C₁-C₁₂ alkyl or cyano-substituted C₁-C₁₂ alkyl.

22. The nucleoside monomer of claim 21, wherein R³ is cyano-substituted lower alkyl.

23. The nucleoside monomer of claim 8, wherein R³ is 2,2,2-trichloro-1,1-dimethylcarbonyl or 1,1-dimethyl-2-cyanoethyl.

35. A kit for synthesizing an oligonucleotide on a solid support, comprising a hydroxyl-derivatized support surface, a protecting group for protecting hydroxyl moieties on said derivatized support surface, the nucleoside monomer of claim 6, at least one nucleoside phosphoramidite, a nucleophile having a nucleophilic site and a lone electron pair on an atom adjacent to the nucleophilic site, and reagents suitable for establishing pH and for carrying out reactions of phosphoramidite coupling and simultaneous hydroxyl group deprotection and phosphite triester oxidation to form an internucleotide phosphotriester linkage.

24. A nucleoside monomer having the structure of formula (X)

wherein:

B is a purine or pyrimidine base;

R is hydrido or hydroxyl;

R¹ is COOR³, wherein R³ is selected from the group consisting of xanthenes, rhodamines, benzimidazoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicylic acids, benz-2-oxa-1-diazoles, fluorescamine, and 4-methylumbelliferone; and

R² has the structure (III)

wherein X is NQ¹Q² in which Q¹ and Q² are independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent, or wherein Q¹ and Q² are linked to form a mono- or polyheterocyclic ring, and Y is selected from the group consisting of hydrido, lower alkyl, electron-withdrawing p-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.

25. The nucleoside monomer of claim 24, wherein X is di(lower alkyl)amino or morpholino.

26. The nucleoside monomer of claim 25, wherein Y is selected from the group consisting of methyl, β

-cyanoethyl and 4-nitrophenylethyl.

27. The nucleoside monomer of claim 24, wherein R³ is selected from the group consisting of 6-carboxy-fluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein, tetramethylrhodamine, 4-methyl-7-methoxycoumarin, dansyl, 5-dimethylamino-1-naphthalenesulfonyl, and 4-amino-7-nitrobenz-2-oxa-1,3-diazole.

28. The nucleoside monomer of claim 25, wherein R³ is selected from the group consisting of 6-carboxy-fluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein, tetramethylrhodamine, 4-methyl-7-methoxycoumarin, dansyl, 5-dimethylamino-1-naphthalenesulfonyl, and 4-amino-7-nitrobenz-2-oxa-1,3-diazole.

29. The nucleoside monomer of claim 26, wherein R³ is selected from the group consisting of 6-carboxy-fluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein, tetramethylrhodamine, 4-methyl-7-methoxycoumarin, dansyl, 5-dimethylamino-1-naphthalenesulfonyl, and 4-amino-7-nitrobenz-2-oxa-1,3-diazole.

30. A nucleoside monomer having the structure of formula (X)

wherein:

B is a purine or pyrimidine base;

R is hydrido or hydroxyl;

R¹ is selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, and 9-fluorenylmethylcarbonyl (“

Fmoc”

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

TCBOC”

); and

R² has the structure (III)

wherein X is NQ¹Q² in which Q¹ and Q² are independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent, or wherein Q¹ and Q² are linked to form a mono- or polyheterocyclic ring, and Y is selected from the group consisting of hydrido, lower alkyl, electron-withdrawing B-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.

31. The nucleoside monomer of claim 30, wherein X is di(lower alkyl)amino or morpholino.

32. The nucleoside monomer of claim 31, wherein Y is selected from the group consisting of methyl, β

-cyanoethyl and 4-nitrophenylethyl.

41. A nucleoside monomer having the structure of formula (II)

wherein:

B is a purine or pyrimidine base;

R is hydrido or hydroxyl;

R¹ is COOR³, wherein R³ is a monocylic aryl, monocylic alkaryl, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl group, optionally substituted with a substituent selected from cyano, nitro, and halo, and optionally containing one or more nonhydrocarbyl linkages selected from ether, thioether, oxo, amine, and imine linkages; and

R² has the structure (III)

wherein X is NQ¹Q² in which Q¹ and Q² are independently selected from the group consisting of aryl, aralkyl, alkaryl, cycloalkyl, cycloalkenyl, or cycloalkynyl, optionally containing one or more nonhydrocaryl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocaryl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent, or wherein Q¹ and Q² are linked to form a mono- or polyheterocyclic ring, and Y is selected from the group consisting of hydrido, lower alkyl, electron-withdrawing β

-substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl hydrido or lower alkyl.

43. A kit for synthesizing an oligonucleotide on a solid support, comprising a hydroxyl-derivatized support surface, a protecting hydroxyl moieties on said derivatized support surface, the nucleoside monomer of one of claim 41 or 42, at least one nucleoside phosphoramidite, a nucleophile having a nucleophilic site and a lone electron pair on an atom adjacent to the nucleophilic site, and reagents suitable for establishing pH and for carrying out reactions of phosphoramidite coupling and simultaneous hydroxyl group deprotection and phosphate trimester oxidation to form an internucleotide phosphotriester linkage.

42. A nucleoside monomer having the structure of formula (II)

wherein:

B is a purine or pyrimidine base;

R is hydrido or hydroxyl;

R¹ is COOR³, wherein R³ is a monocylic aryl, monocylic alkaryl, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl group, optionally substituted with a substituent selected from cyano, nitro, and halo, and optionally containing one or more nonhydrocarbyl linkages selected from ether, thioether, oxo, amine, and imine linkages; and

R² has the structure (III)

wherein X is NQ¹Q² in which Q¹ and Q² are independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent, or wherein Q¹ and Q² are linked to form a mono- or polyheterocyclic ring, and Y is hydrido or lower alkyl.