Nucleoside analogs with polycyclic aromatic groups attached, methods of synthesis and uses therefor

• US 6,218,108 B1
• Filed: 05/16/1997
• Issued: 04/17/2001
• Est. Priority Date: 05/16/1997
• Status: Expired due to Term

• Current Assignee: University of Rochester
• Original Assignee: Research Corporation Technologies
• Inventors: Kool, Eric T.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/857,721
• Time in Patent Office: 1,432 Days
• Field of Search: 536/1.11, 536/4.1, 536/17.2, 536/17.3, 536/17.4, 536/29.2, 536/25.34, 536/18.5, 536/18.6, 536/23.1, 536/25.3, 536/25.32, 435/6
• US Class Current: 435/6.11
• 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 7/04 Carbocyclic radicals

Abstract

The present invention provides fluorescent nucleosides carrying polycyclic aromatic hydrocarbons such as anthracene, phenanthrene, pyrene stilbene, tetracene or pentacene. The subject nucleosides may be synthesized using a C-glycosidic bond formation method employing organocadmium or organozinc derivatives of the aromatic compounds and coupling with a 1-α-chlororibose or deoxyribose synthon. The α-anomers of the coupling reaction may be epimerized to the β-anomers by acid-catalyzed equilibration. The fluorescent nucleosides act as DNA or RNA base analogs and can be incorporated into nucleic acids. Resultant fluorescently tagged nucleic acids are useful as probes for target nucleic acids in tissues, solutions or immobilized on membranes.

Claims

1. A nucleoside derivative comprising a pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene joined to the C-1 atom of a sugar moiety through a carbon-carbon bond in either an α

or β

configuration wherein said sugar moiety is ribose or deoxyribose.

6. A phosphoramidite derivative of the nucleoside derivative of claim 1 wherein the phosphoramidite moiety is jointed to the sugar moiety at the 3′

position.

7. The nucleoside derivative of claim 1 wherein the pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene is derivatized at an available carbon position with a substituent selected from the group consisting of methoxy, ethoxy, dimethylamino, diethylamino, nitro, methyl, cyano, carboxy, chloro, bromo, iodo and amino.

8. A nucleic acid comprising at least one nucleoside derivative according to any one of claims 1-6.

9. A method of synthesizing the nucleoside analog of claim 1 which comprises:

a. coupling an organocadmium or organozinc derivative of pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene to the C1 position of Hoffer'"'"'s α

-chlorosugar; and

b. removing the protecting groups with a methanolic base.

10. A method of synthesizing a phosphoramidite derivative of the nucleoside of claim 1 which comprises:

a. coupling an organocadmium or organozinc derivative of pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene to the C1 position of Hoffer'"'"'s α

-chlorosugar;

b. removing the protecting groups with a methanolic base;

c. tritilating the 5′

-OH with dimeoxytritylchloride in the presence of a base; and

d. phosphytilating the 3′

-OH with a phosphytilating agent.

11. A method of preparing a fluorescently labeled nucleic acid which comprises incorporating a nucleoside analog of any one of claims 1-6 into an RNA or DNA molecule under conditions sufficient to incorporate said nucleoside.

12. A method of detecting a target nucleic acid in a sample to be tested which comprises contacting the target nucleic acid with a nucleic acid probe comprising a nucleoside derivative of any one of claims 1-6 for a time and under conditions sufficient to permit hybridization between said target and said probe;

and detecting said hybridization.

15. The phosphoramidite derivative of claim 6 wherein the phosphoramidite derivative is N,N-diisopropyl-O-cyanoethyl phosphoramidite derivatized at the 3′

alcohol of the pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene derivatized nucleoside.

16. The nucleic acid of claim 8 wherein the nucleoside derivative is incorporated adjacent to an adenine base.

18. The nucleic acid of claim 8 comprising at an odd number of nucleoside derivatives.

19. The nucleic acid according to claim 8 comprising at least three, five, or seven nucleoside derivatives.

20. The method of claim 12 wherein the method of detecting a target nucleic acid is by fluorescence in situ hybridization.

2. α

-9-phenanthrenyl deoxynucleoside.

3. α

-1-pyrenyl deoxynucleoside.

4. β

-9-phenanthrenyl deoxynucleoside.

5. β

-1-pyrenyl deoxynucleoside.

13. α

-1-anthracenyl deoxynucleoside having the following structural formula;

14. β

-1-anthracenyl deoxynucleoside having the following structural formula;

17. A nucleic acid comprising at least three to five nucleoside derivatives selected from the group consisting of a pyrene, anthracene, phenanthrene, stilbene, tetracene and pentacene joined to the C-1 atom of a sugar moiety through a carbon-carbon bond in either an α

or β

configuration wherein said sugar moiety is ribose or deoxyribose and wherein said nucleoside derivative is incorporated adjacent to an adenine base.