Exocyclic amine triaryl methyl protecting groups in two-step polynucleotide synthesis

• US 7,193,077 B2
• Filed: 08/30/2003
• Issued: 03/20/2007
• Est. Priority Date: 08/30/2003
• Status: Active Grant

• 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., Dellinger, Douglas J., Sierzchala, Agnieszka B.
• Primary Examiner(s): Lewis; Patrick
• Application Number:
  US10/652,064
• Publication Number:
  US 20050049411A1
• Time in Patent Office: 1,298 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.33, 536/25.34
• US Class Current: 536/25.3
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Precursors for use in the synthesis of polynucleotides and methods of using the precursors in synthesizing polynucleotides are disclosed. The precursors include a heterocyclic base having an exocyclic amine group and a substituted or unsubstituted triaryl methyl protecting group bound to the exocyclic amine group.

Claims

1. A method of forming an intemucleotide bond, the method comprising

contacting a nucleoside moiety having a reactive site hydroxyl with a precursor having an exocyclic amine triaryl methyl protecting group under conditions and for a time sufficient to result in internucleotide bond formation, said precursor having the structure (I),

Rag-Sugar-Base-Tram 

 

(I)

wherein;

Rag is a reactive group capable of reacting with the reactive site hydroxyl,

Sugar is a sugar group having a hydroxyl protecting group,

Base is a heterocyclic base having an exocyclic amine group

Tram is the exocyclic amine triaryl methyl protecting group

wherein the reactive group has the structure (IV);

2. The method of claim 1, wherein the exocyclic amine triaryl methyl protecting group has the structure (V)

3. The method of claim 2, wherein R4, R5, and R6 are independently selected from unsubstituted or substituted phenyl groups.

4. The method of claim 2, wherein R4, R5, and R6 are optionally substituted aryl groups independently selected from phenyl, biphenyl, naphthanyl, indolyl, pyridinyl, pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, and anthracenyl.

5. The method of claim 2, wherein R4, R5, and R6 are independently selected from phenyl, methoxyphenyl, trimethoxyphenyl, and furanyl.

6. The method of claim 1, wherein the sugar group is a monosaccharide characterized as being one or more of the group consisting of a ketose, an aldose, a pentose, and a hexose.

7. The method of claim 1, wherein the sugar group is selected from ribose, 2′

-deoxyribose, arabinose, xylose, and lyxose.

8. The method of claim 1, wherein the sugar group is based on the structure

H—

[CH(OH)]_n—

C(═

O)—

[CH(OH)]_m—

H 

 

(IIa)

in which n is an integer from 1 to about 5 and m is an integer from 1 to about 5;

provided that one of the hydrogens or hydroxyls in structure (IIa) is replaced by the reactive group; and

provided that the heterocyclic base is directly bound to one of the carbons of structure (IIa), thereby replacing a hydrogen or hydroxyl of structure (IIa) or adding to the carbonyl carbon of structure (IIa).

9. The method of claim 8 wherein the sugar group is a cyclic hemiacetal.

10. The method of claim 1, wherein the sugar group is based on the structure

H—

[CH(OH)]_n—

C(═

O)H 

 

(IIb)

in which n is an integer from 2 to about 8;

provided one of the hydrogens or hydroxyls in structure (IIb) is replaced by the reactive group; and

provided that the heterocyclic base is directly bound to one of the carbons of structure (IIb), thereby replacing a hydrogen or hydroxyl of structure (IIb) or adding to the carbonyl carbon of structure (IIb).

11. The method of claim 10 wherein the sugar group is a cyclic hemiacetal.

17. The method of claim 1, wherein the exocyclic amine triaryl methyl protecting group has the structure (V)

18. The method of claim 17, wherein for each of R4, R5 and R6 the lower alkyl group is substituted with one or more groups selected from:

a lower alkyl group, an alkoxy group, a thioalkyl group, a hydroxyl thio group, a mercapto group, an amino group, an imino group, a halo group, a cyano group, a nitro group, a nitroso group, an azido group, a carboxy group, a sulfide group, a sulfone group, a sulfoxy group, a phosphoryl group, a silyl group, a silyloxy group, and a boronyl group.

21. The method of claim 1, wherein Y is an electron-withdrawing β

-substituted ethyl.

22. The method of claim 21, wherein Y is selected from the group consisting of:

β

-trihalomethyl ethyl, β

-cyanoethyl, β

-sulfoethyl, and β

-nitro-substituted ethyl.

23. The method of claim 1, wherein Y is selected from the group consisting of:

halo-substituted phenyl, sulfo-substituted phenyl, cyano-substituted phenyl, and nitro-substituted phenyl.

24. The method of claim 1, wherein Y is selected from the group consisting of:

β

-cyanoethyl, methyl-β

-cyanoethyl, dimethyl-β

-cyanoethyl, phenylsulfonylethyl, methyl-sulfonylethyl, p-nitrophenylsulfonylethyl, 2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl, 2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol, β

-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, 2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl, 9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl, 2-(diphenylphosphino)-ethyl, 1-methyl-1-phenylethyl, 3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl, α

-methylcinnamyl, and 8-quinolyl.

25. The method of claim 1, wherein X is NQ1Q2, wherein Q1 and Q2 are independently selected from the group consisting of:

alkyl, aiyl, aralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, and cycloalkynyl.

26. The method of claim 25, wherein Q1 and Q2 are independently selected from the group consisting of:

t-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, and sec-pentyl.

27. The method of claim 25, wherein NQ1Q2 is selected from the group consisting of:

dimethylamine, diethylamine, diisopropylamine, dibutylamine, methyipropylamine, methylhexylamine, methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, butylcyclohexylamine, thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine, and piperazine.

12. A method of forming an intemucleotide bond, the method comprising contacting a nucleoside moiety having a reactive site hydroxyl with a precursor having an exocyclic amine triaryl methyl protecting group to result in intemucleotide bond formation, said precursor having the structure (III)

13. The method of claim 12, wherein for each of R4, R5 and R6 the lower alkyl group is substituted with one or more groups selected from:

a lower alkyl group, an alkoxy group, a thioalkyl group, a hydroxyl thio group, a mercapto group, amino group, an imino group, a halo group, a cyano group, a nitro group, a nitroso group, an azido group, a carboxy group, a sulfide group, a sulfone group, a sulfoxy group, a phosphoryl group, a silyl group, a silyloxy group, and a boronyl group.

14. The method of claim 12, wherein R4, R5, and R6 are optionally substituted aryl groups independently selected from phenyl, biphenyl, naphthanyl, indolyl, pyridinyl, pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, and anthracenyl.

15. The method of claim 12, wherein R4, R5, and R6 are independently selected from phenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, and furanyl.

30. The method of claim 12, wherein R4, R5, and R6 are optionally substituted aryl groups independently selected from:

naphthanyl, indolyl, pyridinyl, pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, and anthracenyl.

16. A method of forming an intemucleotide bond, the method comprising

contacting a nucleoside moiety having a reactive site hydroxyl with a precursor having an exocyclic amine triaryl methyl protecting group under conditions and for a time sufficient to result in intemucleotide bond formation, said precursor having the structure (I),

Rag-Sugar-Base-Tram 

 

(I)

wherein;

Rag is a reactive group capable of reacting with the reactive site hydroxyl,

Sugar is a sugar group having a hydroxyl protecting group,

Base is a heterocyclic base having an exocyclic amine group

Tram is the exocyclic amine triaryl methyl protecting, group

wherein the reactive group has the structure (IV);

19. The method of claim 16, wherein the exocyclic amine triaiyl methyl protecting group has the structure (V)

20. The method of claim 19, wherein for each R4, R5 and R6 the lower aikyl group is substituted with one or more groups selected from:

a lower alkyl group, an alkoxy group, a thioalkyl group, a hydroxyl thio group, a mercapto group, an amino group, an imino group, a halo group, a cyano group, a nitro group, a nitroso group, an azido group, a carboxy group, a sulfide group, a sulfone group, a sulfoxy group, a phosphorvi group, a silyl group, a silyloxy group, and a boronyl group.

28. The method of claim 16, wherein Q1 and Q2 are independently selected from the group consisting:

t-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, and sec-pentyl.

29. The method of claim 16, wherein NQ1Q2 is selected from the group consisting of:

dimethylamine, diethylamine, diisopropylamine, dibutylamine, methyipropylamine, methyihexylamine, methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, butylcyclohexylamine, thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine, and piperazine.