Protected fluorescent reagent compounds

Patent metadata

Field	Value
Publication number	US-12060383-B2
Application number	US-202318167729-A
Country	US
Kind code	B2
Filing date	Feb 10, 2023
Priority date	Aug 5, 2013
Publication date	Aug 13, 2024
Grant date	Aug 13, 2024

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 (I):
Z—[S′—B′] m   (I); wherein

Z is a multivalent central core element comprising a non-fluorescent multivalent central core element and a fluorescent dye element;
each S′ is independently an intermediate chemical group, wherein at least one S′ comprises a shield element, wherein the shield element comprises a shield core element and a side chain;
each B′ is independently a terminal chemical group, wherein at least one B′ comprises a binding element; and
m is an integer from 2 to 24.

2. The compound of claim 1 , wherein the shield element decreases photodamage of the compound or of a biomolecule associated with the binding element.

3. The compound of claim 1 , wherein the shield element decreases contact between the fluorescent dye element and the binding element.

4. The compound of claim 1 , wherein the shield element comprises a plurality of side chains.

5. The compound of claim 4 , wherein at least one side chain has a molecular weight of at least 300.

6. The compound of claim 4 , wherein at least one side chain comprises a dendrimer.

7. The compound of claim 4 , wherein at least one side chain comprises a negatively-charged component.

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

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

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

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

12. The compound of claim 1 , wherein Z comprises a branching element.

13. The compound of claim 1 , wherein m is an integer from 2 to 12.

14. The compound of claim 1 , wherein the fluorescent dye element is a cyanine dye.

15. The compound of claim 1 , wherein Z comprises a linker group.

16. The compound of claim 1 having structural formula (IIa) or (IIb):

wherein
X is the non-fluorescent multivalent central core element;
at least one D is the fluorescent dye element;
at least one W, if present, is a branching element;
n is an integer from 2 to 6;
each o is independently an integer from 1 to 4; and
each p is independently an integer from 1 to 4.

17. The compound of claim 16 , wherein X comprises a polyamine.

18. The compound of claim 16 , comprising at least one donor fluorophore and at least one acceptor fluorophore.

19. The compound of claim 16 , wherein the shield element comprises a plurality of side chains.

20. The compound of claim 16 , wherein the shield element comprises an inner layer and an outer layer.

21. The compound of claim 16 , wherein n is an integer from 2 to 4.

22. The compound of claim 16 , wherein each o is independently an integer from 1 to 3.

23. The compound of claim 16 , wherein each p is independently an integer from 1 to 3.

24. The compound of claim 1 having structural formula (III):

wherein
X is the non-fluorescent multivalent central core element;
at least one D is the fluorescent dye element;
at least one W is a branching 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.

25. The compound of claim 24 , wherein X comprises a polyamine.

26. The compound of claim 24 , comprising at least one donor fluorophore and at least one acceptor fluorophore.

27. The compound of claim 24 , wherein the shield element comprises a plurality of side chains.

28. The compound of claim 24 , wherein the shield element comprises an inner layer and an outer layer.

29. The compound of claim 24 , wherein n is an integer from 2 to 4.

30. The compound of claim 24 , wherein each p′ is independently an integer from 1 to 3.

31. The compound of claim 24 , 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

• C12Q1/6869

  Methods for sequencing · CPC title

• C09B69/105

  containing a methine or polymethine dye · CPC title

• C09B23/083

  five >CH- groups · CPC title

• C09B23/06

  three >CH- groups, e.g. carbocyanines · CPC title

• 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