Methods for labeling nucleotides, labeled nucleotides and useful intermediates

• US 20020160396A1
• Filed: 01/14/2002
• Published: 10/31/2002
• Est. Priority Date: 10/08/1996
• Status: Active Grant

• Current Assignee: Leica Biosystems Newcastle Ltd. (Danaher Corporation)
• Original Assignee: Hendrick Jan Houthoff, Herman Hendricus Volkers, Jan Reedijk, Robert Jochem Heetebrij, Tinka Jelsma
• Inventors: Heetebrij, Robert Jochem, Volkers, Herman Hendricus, Jelsma, Tinka, Reedijk, Jan, Houthoff, Hendrick Jan
• Granted Patent: US 6,797,818 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention provides a labeled nucleotide having formula:

where X is an aliphatic diamine, and the labeling moiety comprises a label and a spacer, where the spacer is coupled at one end to a platinum atom and at the other end to the label, which spacer comprises a chain having at least four atoms. The invention further provides a method for labeling a nucleotide, kits for labeling a nucleotide, and kits containing labeled nucleotides.

Claims

1. A method for labeling nucleotides comprising the steps of:

reacting a reactive moiety of a linker of the formula

 

wherein X represents any stabilizing bridge and wherein A and B represent the same or different reactive moieties, with an electron donating moiety of a spacer, which spacer comprises a chain having at least four atoms and at least one heteroatom in the chain, which spacer further comprises said electron donating moiety at one end of the chain and a reactive moiety at the other end of the chain;

reacting the reactive moiety of said spacer with a label;

reacting the other reactive moiety of said linker with a nucleotide.

2. A method according to claim 1 wherein the label is biotin, avidin, streptavidin, digoxygenin or a functional equivalent thereof.

3. A method according to claim 1 or 2 wherein the nucleotide is adenine, thymidine, cytosine, guanine or uridine or a derivative thereof.

4. A method according to any of the preceding claims wherein the electron donating moiety is an amine or a thiolate anion.

5. A method according to claim 4 wherein the amine is an aromatic amine.

6. A method according to any of the preceding claims wherein at least one heteroatom is an oxygen atom.

7. A method according to any of the preceding claims wherein the spacer comprises no more than 20 carbon atoms in the chain and wherein the chain is essentially non-branched.

8. A method according to claim 7 wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

9. A method according to claim 7 wherein the spacer is an oligolysine or a polylysine.

10. A method according to claims 1-4 wherein the linker is reacted with a labeling moiety comprising a label and a spacer, which labeling moiety has the formula

11. A method according to claim 10, wherein Z and/or Z′

represent an NH₃, NH₂R, NHR₂, or NR₃ group, wherein R represents an alkyl group having from 1 to 6 carbon atoms.

12. A method according to any of the preceding claims, wherein X represents an aliphatic diamine.

13. A method according to claim 12 wherein X represents an aliphatic diamine having 2-6 carbon atoms.

14. A method according to claim 13 wherein X is an ethylene diamine group.

15. A method according to claims 12-14 wherein one or both of the nitrogen atoms of the aliphatic diamine are shielded.

16. A method according to claim 15 wherein one or both of the nitrogen atoms of the aliphatic diamine are substituted with an alkyl group of from 1 to 6 carbon atoms.

17. A method according to claim 16 wherein one or both of the nitrogen atoms of the aliphatic diamine are substituted with one or two methyl groups.

18. A method according to any of the preceding claims wherein A and/or B are selected from the group consisting of NO₃-, SO₃⁻

, Cl⁻

, I⁻

, or other halogens.

19. A method according to any of the preceding claims wherein A and B are the same.

20. A labeled nucleotide obtainable by a method according to any of the preceding claims.

21. A labeled nucleotide according to claim 20 wherein the spacer is an oligolysine or a polylysine.

22. A labeling substance having the formula