Protected fluorescent reagent compounds

Patent metadata

Field	Value
Publication number	US-9957291-B2
Application number	US-201414452497-A
Country	US
Kind code	B2
Filing date	Aug 5, 2014
Priority date	Aug 5, 2013
Publication date	May 1, 2018
Grant date	May 1, 2018

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.

First claim

What is claimed is:

1. A compound of structural formula (III):

wherein
X is a non-fluorescent multivalent central core element;
at least one D is a fluorescent dye element;
at least one W is a branching element;
each S′ is independently an intermediate chemical group, wherein at least one S′ comprises a shield element, wherein the shield element comprises a plurality of side chains;
each B′ is independently a terminal chemical group, wherein at least one B′ comprises a binding element;
n is an integer from 2 to 6;
each p′ is independently an integer from 1 to 4; and
each p″ is independently an integer from 1 to 4.

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:

wherein each x is independently an integer from 1 to 6.

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:

wherein each y is independently an integer from 1 to 6.

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:

wherein R 1 and R 2 is each independently a side chain;
L is an alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl linker; and
Y is

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:

wherein each z′ is independently an integer from 1 to 6.

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.

Assignees

• Pacific Biosciences California Inc

Inventors

• Sebo Lubomir
• Hanes Jeremiah
• Shen Gene
• Brogley Louis
• Yue Stephen
• Zheng Frank
• Lapin Yuri
• Lyle John
• Osuna Honey
• Fedorov Andrei

Classifications

• C07F9/65616

  containing the ring system [IMAGE cpc-sch-C07F-1006.gif] 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 title

• C07F9/65586

  at least one of the hetero rings does not contain nitrogen as ring hetero atom · CPC title

• C07H19/10 Primary

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C09B69/105

  containing a methine or polymethine dye · CPC title