Protected fluorescent reagent compounds

• US 9,957,291 B2
• Filed: 08/05/2014
• Issued: 05/01/2018
• Est. Priority Date: 08/05/2013
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sebo, Lubomir, Hanes, Jeremiah, Shen, Gene, Brogley, Louis, Yue, Stephen, Zheng, Frank, Lapin, Yuri, Lyle, John, Osuna, Honey, Fedorov, Andrei
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/452,497
• Publication Number:
  US 20150050659A1
• Time in Patent Office: 1,365 Days
• Field of Search: 536 231, 544194
• CPC Class Codes: C07F 9/65586 at least one of the hetero rings does not contain nitrogen as ring hetero atom
• CPC Class Codes: C07F 9/65616 containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 69/105 containing a methine or polymethine dye
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.

Claims

1. A compound of structural formula (III):

2. The compound of claim 1, wherein X comprises a polyamine.

3. The compound of claim 2, wherein the polyamine has the structure:

4. The compound of claim 1, wherein X comprises a substituted cyclohexane.

5. The compound of claim 4, wherein the substituted cyclohexane is a 1,3,5-triamino-cyclohexane.

6. The compound of claim 1, wherein X comprises a substituted 1,3,5-triazine.

7. The compound of claim 1, wherein X comprises a substituted benzene.

8. The compound of claim 7, wherein the substituted benzene is

9. The compound of claim 1, comprising at least one donor fluorophore and at least one acceptor fluorophore.

10. The compound of claim 1, wherein at least one side chain has a molecular weight of at least 300.

11. The compound of claim 1, wherein all of the side chains have a molecular weight of at least 300.

12. The compound of claim 1, wherein at least one side chain comprises a dendrimer.

13. The compound of claim 1, wherein at least one side chain comprises a polyethylene glycol.

14. The compound of claim 1, wherein at least one side chain comprises a negatively-charged component.

15. The compound of claim 14, wherein the negatively-charged component comprises a sulfonic acid.

16. The compound of claim 1, wherein at least one side chain comprises a substituted phenyl group.

17. The compound of claim 16, wherein the at least one side chain comprises the structure:

18. The compound of claim 17, wherein each x is independently an integer from 1 to 4.

19. The compound of claim 1, wherein at least one side chain comprises a triazole.

20. The compound of claim 1, wherein at least one side chain comprises the structure:

21. The compound of claim 1, wherein at least one side chain comprises the structure:

22. The compound of claim 1, wherein the shield element comprises the structure:

23. The compound of claim 1, wherein the shield element comprises an inner layer and an outer layer.

24. The compound of claim 1, wherein at least one S′

-B′

group comprises the structure;

25. The compound of claim 1, wherein the binding element comprises a nucleotide.

26. The compound of claim 1, wherein the binding element comprises biotin.

27. The compound of claim 1, wherein the binding element comprises a polyphosphate.

28. The compound of claim 1, wherein the branching element comprises a substituted phenyl group.

29. The compound of claim 1, wherein the branching element comprises the structure:

30. The compound of claim 29, wherein each z′

is independently an integer from 1 to 4.

31. The compound of claim 1, wherein n is an integer from 2 to 4.

32. The compound of claim 1, wherein each p′

is independently an integer from 1 to 3.

33. The compound of claim 1, wherein each p″

is independently an integer from 1 to 3.