Photoactive fluorescent compounds and use thereof for labeling of proteins

Abstract

Provided herein are novel photoactive fluorescent compounds and their use in labeling proteins, for example, tagged proteins, and their use for visualizing the location and dynamics of proteins in living cells.

Description

This article presents novel photoactive fluorescent compounds and their uses in labeling proteins, such as tagged proteins, as well as their applications in visualizing the location and dynamics of proteins in living cells. Abstract

Claims

What is claimed is:

1. A compound of Formula (VIII): or a salt, a single form thereof,wherein: R1is H, or –C(=O)N(C1-3 alkyl)2, each L1is independently: –O-C1-6-alkylene–; –C(O)NH–, –C1-6-alkylene-N(C1-3-alkyl)(C1-6-alkylene)–, or –C1-6- alkylene-O-C1-6-alkylene–; each L2is –(OCH2CH2)p–; p is an integer of 1 to 3; a is an integer of 1 or 2; b is an integer of 0 to 2; andwhen R1is H and a is 1, then L1is not , wherein the wavy line ( ) represents a point of attachment to the rest of the compound.

2. The compound of claim 1, wherein: p is 1, a is 1, and b is an integer of 0 to 1. 154 NAI-1539154927v13. The compound of claim 1 or claim 2, wherein: R1is H, each L1is independently: –O-C1-6-alkylene–, –C1-6-alkylene-N(C1-3-alkyl)(C1-6-alkylene)–, or –C1-6-alkylene-O-C1-6-alkylene–; each L2is –(OCH2CH2)p–; p is an integer of 1 to 3; a is an integer of 1 or 2; b is an integer of 0 to 2; and .claim 1 or claim 2, wherein: R1is –C(=O)N(C1-3 alkyl)2; each L1is –C(O)NH– each L2is –(OCH2CH2)p–; p is an integer of 1 to 3; a is an integer of 1 or 2; b is an integer of 0 to 2; and .Formula (IX): 155 NAI-1539154927v1or a salt, a single rm thereof, wherein LBis (i) –O-C1-6-alkylene–, –C1-6-alkylene-N(C1-3-alkyl)–, or –C1-6-alkylene-O–.

6. The compound of claim 5, wherein LB is a linker comprising: –OCH2–, –CH2N(CH3)–, or –CH2O–.

7. A compound of Formula (X): or a salt, a singlethereof, wherein: R1is H or –C(=O)N(C1-3 alkyl)2.

8. The compound of claim 7, wherein R1is H.

9. The compound of claim 7, wherein R1is –C(=O)N(C1-3 alkyl)2. 156 NAI-1539154927v1O N 10. The compound of claim 7, wherein R1.

11. The compound of claim 5, having the structure: .

12. The compound of claim 5, having the structure: .

13. The compound of claim 5, having the structure: 157 NAI-1539154927v1.

14. The compound of claim 7, having the structure: O O .

15. A method of labeling a protein, comprising contacting a sample comprising a tagged protein with the compound of any one of claims 1-14, so as to yield a labeled protein.

16. The method of claim 15, wherein the tagged protein is a kinase, a transcription factor, a chromatin modulator, an adapter, a transporter, or a pathogenic aggregator.

17. The method of claim 15, wherein the tagged protein is a histone.

18. The method of claim 17, wherein the histone is H2B. 158 NAI-1539154927v119. The method of any one of claims 15-18, wherein the labeled protein fluoresces when exposed to light.

20. The method of claim 19, wherein the light is a laser.

21. The method of claim 20, wherein the light wavelength (λ) is about 405 nm.

22. A method for preparing a compound of Formula (VIII-a ): wherein:;alkyl)2, each L1is independently: –O-C1-6-alkylene–; –C(O)NH–, –C(O)NH(C1-3-alkylene)–, –C1-6-alkylene-N(C1-3- alkyl)(C1-6-alkylene)–, or –C1-6-alkylene-O-C1-6-alkylene–; each L2is –(OCH2CH2)p–; p is an integer of 1 to 3; a is an integer of 1 or 2; b is an integer of 0 to 2; NAI-when R1is H and a is 1, then L1is not , wherein the wavy line ( ) represents a point of attachment to the rest of the compound, the process comprising: converting a compound of the following Formula (a-1): wherein:;2, R is –C(=O)OCH3 or –C(=O)NH(C1-3 alkylene)–(OCH2CH2)–Z; and wherein ,the presence of a diazomethane agent and a solvent, to prepare the compound of Formula (VIII-a).

23. A method for preparing a compound of Formula (XI): wherein: R1is H, or –C(=O)N(C1-3alkyl)2, each L1is: –C(O)NH(C1-3-alkylene)–; 160 NAI-1539154927v1each L2is –(OCH2CH2)p–; p is an integer of 1 to 3; a is an integer of 1 or 2; b is an integer of 0 to 2; Z ; the process comprising: the following Formula (a-2):wherein:R1is H, or –C(=O)N(C1-3alkyl)2, R is –C(=O)OCH3or –C(=O)NH(C1-3alkylene)–(OCH2CH2)–Z; and wherein ,the presence of a diazomethane agent and a solvent, to prepare the compound of Formula (XI).

24. The method of claim 22 or 23, wherein the diazomethane agent is trimethylsilyldiazomethane diethyl ether.

25. The method of claim 22 or 23, wherein the solvent comprises dichloromethane.

26. The method of any one of claims 22-25, further comprising using a drying agent and a metal oxide. 161 NAI-1539154927v127. The method of claim 26, wherein the drying agent is a molecular sieve and the metal oxide is calcium oxide.

28. A compound of Formula (VIII-a) made by the process of claim 22.

29. A compound of Formula (XI) made by the process of claim 23.

30. The compound of claim 28 or 29, which is substantially chemically pure.

31. The compound of claim 28 or 29, which is substantially free of chemical impurities.

32. The compound of claim 1, which is substantially chemically pure.

33. The compound of claim 1, which is substantially free of chemical impurities. 162 NAI-1539154927v1

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