Nucleotide analogues comprising a reporter moiety and a polymerase enzyme blocking moiety

• US 7,078,499 B2
• Filed: 05/31/2001
• Issued: 07/18/2006
• Est. Priority Date: 06/01/2000
• Status: Expired due to Fees

• Current Assignee: Amersham Biosciences (General Electric Company)
• Original Assignee: GE Healthcare UK Limited (Danaher Corporation)
• Inventors: Simmonds, Adrian, Pickering, Lee, Odedra, Raj
• Primary Examiner(s): Jiang, Shaojia Anna
• Assistant Examiner(s): Khare, Devesh
• Application Number:
  US10/276,759
• Publication Number:
  US 20030194722A1
• Time in Patent Office: 1,874 Days
• Field of Search: 536/22.1, 536/25.32, 536/25.31, 536/26.6, 536/27.6, 536/28.4, 536/27.22, 536/124, 544/277, 514/45, 435/6, 435/7.92, 435/7.94
• US Class Current: 536/22.1
• 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: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

Nucleotides comprising a reporter moiety and a polymerase enzyme blocking moiety in which the reporter moiety does not also act as a polymerase enzyme blocking moiety are described. Also described are compounds of Formula (I): wherein W is a phosphate group, B is a base, Y is a linker comprising an enzyme-cleavable group, R² is a reporter moiety, R³ is selected from H or OH, Z and Z′ are selected from H, OH, or a group X—R¹, wherein X is a linker comprising an enzyme-cleavable group and R¹ is a polymerase enzyme blocking group, provided that at least one of Z and Z′ is X—R¹.

Claims

1. A compound of Formula I

2. The compound of claim 1 wherein W is a triphosphate.

3. The compound of claim 1 wherein B is selected from the bases A, C, G and T or their analogues.

4. The compound of claim 1 wherein the enzyme-cleavable groups, X and Y, are the same.

5. The compound of claim 1 wherein X and Y are groups cleavable by enzymes selected from esterases, phosphatases, peptidases, amidases, glycosidases or phosphorylases.

6. The compound of claim 1 wherein X and/or Y is a group cleavable by an amidase.

7. The compound of claim 1 wherein R² is a fluorophore selected from the group consisting of fluoresceins, rhodamines, coumarins, BODIPY™

dyes, cyanine dyes and squarate dyes.

8. The compound of claim 1 wherein R¹ is selected from the group consisting of CH₃, glycyl and leucyl groups.

9. The compound of claim 1 wherein Z′

is X—

R¹ and Z is OH.

10. The compound of claim 1 wherein R¹ is not a reporter moiety.

11. A chemical intermediate selected from the group consisting of:

4′

-C-(Glycylaminomethyl)thymidine triphosphate;

4′

-C-(N-trifluoroacetylaminomethyl)thymidine triphosphate;

4′

-C-(Aminomethyl)thymidine triphosphate;

5-(N-Trifluoroacetyl)propargylamino-4′

-C-(acetylaminomethyl)-2 ′

-deoxyuridine;

4′

-C-(N-Acetylglycylaminomethyl)thymidine;

4′

-C-(N-Leucylaminomethyl)thymidine;

4′

-C-(N-Glycylaminomethyl)thymidine (shown in FIG. 8);

N- {α

-[4′

-methyloxythymidyl]phenyl}phenylacetamide triphosphate;

N- [α

-(3 ′

-O -thymidyl)-phenyl)]-phenylacetamide triphosphate, N- {α

-[3′

-O-(5 -N-(α

-methyloxy-N′

-trifluoroacetylaminopropyl benzamide) phenylacetamide-2′

-deoxyuridyl]-phenyl }-phenylacetamide and a compound of formula;

12. A set of nucleotides containing at least one compound of Formula I.

13. The set of nucleotides of claim 12 comprising each of the four natural bases A, G, C and T (or their analogue).

14. The set of nucleotides of claim 12 containing at least two compounds of Formula I having different bases, B, and wherein each compound of Formula I has a different reporter moiety, R².

15. The set of nucleotides of claim 12 containing four compounds of Formula I wherein each compound has a different base, B, such that each of the bases A, G, C and T, or analogues thereof, are present and each of the four compounds of Formula I has a reporter moiety which is distinguishable from the reporter moieties of each of the other three compounds of Formula I.