Methods of synthesis of halogen base-modified oligonucleotides and subsequent labeling with a metal-catalyzed reaction

• US 6,288,221 B1
• Filed: 08/19/1999
• Issued: 09/11/2001
• Est. Priority Date: 08/20/1998
• Status: Expired due to Fees

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Khan, Shoeb I., Beilstein, Amy E., Grinstaff, Mark W.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/377,612
• Time in Patent Office: 754 Days
• Field of Search: 536/25.3, 536/25.32, 435/6, 422/50, 422/68.1, 422/131
• 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 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 present invention provides metal-containing purines, pyrimidines, nucleosides, nucleotides and oligonucleotides; including phosphoramidite and photolabile derivatives thereof, including methods of making and method of using same. The present invention provides a method for detection of nucleic acid sequences via electrochemical or photochemical means.

Claims

1. An improved method of synthesizing a labeled oligonucleotide comprising:

attaching a nucleoside of the oligonucleotide to be synthesized to a solid support through a labile linker arm;

said nucleoside containing a removable protecting group attached to a 5′

hyroxyl of a sugar group of said nucleoside;

removing said protecting group;

adding a further nucleoside containing a linker arm and a protecting group to said solid support under conditions where said further nucleoside is coupled to said 5′

hydroxyl of said sugar group through said linker arm;

optionally capping any uncoupled nucleoside;

removing the protecting group of said further nucleoside;

repeating the above with further nucleoside derivatives wherein at least one of said further nucleoside contains a detectable label, until a desired oligonucleolide is formed; and

cleaving said oligonucleotide from said solid support;

wherein the improvement comprises adding at least one dimethoxytrityl, phosphorarridite halonucleoside derivative in place of one of said at least one nucleoside containing a detectable label, wherein said halonucleoside is selected from the group consisting of a C5-halo-pyrimidine, a C8-halo-puine and a C7-halo-7-deazapurine; and

labeling said halonucleoside derivative with a detectable label in place of said halogen during or subsequent to formation of said oligonucleotide.

2. The method of claim 1 wherein said cleaving comprising adding concentrated ammonium hydroxide.

3. The method of claim 1, further comprising treating said cleaved oligonucleotide with heat or light to remove any protecting group.

11. The improved method of claim 1, wherein said nucleoside comprises a sugar selected from the group consisting of a ribose, a deoxyribose and a dideoxyribose.

13. The improved method of claim 1, wherein said nucleoside further comprises a dimethoxytritylchloride, α

-methyl-6-nitropipronyloxy carbonyl or 2-cyanoethyl N,N′

-diisopropylcholoro phosphoramidite derivative.

15. The improved method of claim 1, wherein said nucleoside further comprises a compound of the following structure:

wherein R₁, R₂, R₃, and R₄ independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thio, thioether, amino, nitro, carboxyl, formate, foramino or phosphido group, or adjacent substituents R₁-R₄ are substituted oxygen groups that together form a cyclic acetal or ketal;

R₅ is hydrogen, alkoxyl. alkyl, halo, aryl or alkenyl group, n=0, 1, 2 or 3; and

Y is a hydroxyl group of the nucleoside.

17. The improved method of claim 1, wherein said nucleoside contains a uricil, adenine, thymine, cylosine, guanine or natural or synthetic analogs thereof.

4. An improved method of oligonucleotide synthesis comprising combining phosphoramidite nucleoside derivatives in combination with a solid support wherein the improvement comprises adding at least one phosphoramidite halonucleoside during the synthesis and subsequently labeling said halonucleoside with a marker in a metal catalyzed reaction in place of said halogen to form a labeled nucleoside, wherein said halonucleoside is selected from the group consisting of a C5-halo-pyrimidine, a C8-halopurine and a C7-halo7-deazapurine.

5. The improved method of claim 4 wherein said metal is palladium.

6. The improved method of claim 4 wherein said halonucleoside is selected from the group consisting of fluorouridine, fluoroadenosine, fluoroguanosine, fluorocytidine, fluorothymidine, chlorouridine, chloroadenosine, chloroguanosine, chlorocytidine, chlorothymidine, iodouridine, iodoadenosine, iodoguanosine, iodocytidine, iodothymidine, bromouridine, bromoadenosine, bromoguanosine, bromocytidine and bromothymidine.

7. The improved method of claim 4 wherein said oligonucleotide synthesis progresses in the 3′

to 5′

or the 5′

to 3′

direction.

8. The improved method of claim 4 wherein the marker is a metal-containing alkene or alkyne derivative.

9. The improved method of claim 4, wherein said labeled nucleoside comprises a detectable marker which is a metal complex containing at least one of a M(diimine)_x^y+ complex, M(terpyridine)_x^y+ complex or a metallocene wherein M is a transition metal, said diimine is selected from a bipyridine, phenanthroline or terpyridine derivative;

x is 1, 2, or 3 and y is 0, 1, 2, 3, or 4, said bipyridine, phenanthroline, terpyridine and metallocene being optionally substituted by any one of alkyl, alkene, alkyne, aryl, alkylaryl, and carboxyalkyl, amide, ester or ether.

10. The improved method of claim 9, wherein said detectable marker contains at least one of a M(diimine)₂(diimine)²⁺ complex wherein M is a metal selected from Fe⁺², Ru⁺², Os⁺², Co⁺², Rh⁺², and Cr⁺² , or a M(diimine)₂(diimine)³⁺ complex wherein M is a metal selected from Fe⁺³, Ru⁺³, OS⁺³, Co⁺³, Rh⁺³, and Cr⁺³, or a M(terpyridine)₂³⁺ complex wherein M is a selected from Fe⁺³, Ru⁺³, Os⁺³, Co⁺³, Rh⁺³, and Cr⁺³, or a M(diimine)₂²⁺ complex wherein M is a metal selected from Fe⁺², Ru⁺², Os⁺², Co⁺², Rh⁺², and Cr⁺².

12. The improved method of claim 4, wherein said nucleoside comprises a sugar selected from the group consisting of a ribose, a deoxyribose and a dideoxyribose.

14. The improved method of claim 4, wherein said nucleoside further comprises a dimethoxytritylchloride, α

-methyl-6-nitropipronyloxy carbonyl or 2-cyanoethyl N,N′

-diisopropylcholoro phosphoramidite derivative.

16. The improved method of claim 4, wherein said nucleoside further comprises a compound of the following structure:

wherein R₁, R₂, R₃, and R₄ independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl. thio, thioether, amino, nitro, carboxyl, formate, for amino or phosphido group, or adjacent substituents R₁-R₄ are substituted oxygen groups that together form a cyclic acetal or ketal;

R₅ is hydrogen, alkoxyl, alkyl, halo, aryl or alkenyl group, n=0, 1, 2 or 3; and

Y is a hydroxyl group of the nucleoside.

18. The improved method of claim 4, wherein said nucleoside contains a uricil, adenine, thymine, cytosine, guanine or natural or synthetic analogs thereof.

19. The improved method according to claim 9, wherein said detectable marker is selected from a bis(2,2′

-bipyridine)(4′

-methyl-2,2′

-bipyridine-4-carbonyl propargyl amine) ruthenium (II) substituent, a ferrocene substituent; and

a bis(2,2′

-bipyridine) (4′

-methyl-2,2′

-bipyridine-4-carbonyl propargyl amine) osmium (II) substituent.

20. The improved method according to claim 9, wherein said diimine ligands around said metal are all the same.

21. The improved method according to claim 9, wherein at least two of said diimine ligands around said metal are not the same.