Phenyl glyoxal probes

Patent metadata

Field	Value
Publication number	US-9921225-B2
Application number	US-201615160079-A
Country	US
Kind code	B2
Filing date	May 20, 2016
Priority date	Jun 4, 2012
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

Novel phenyl-glyoxal based anti-citrulline probes and methods of synthesis are provided. Methods of use, such as, the development of methods for monitoring substrate citrullination over time; for identifying citrullinated proteins from cells are described.

First claim

What is claimed:

1. A phenylglyoxal probe for detection of a citrullinated protein or protein fragment in a biological sample, the probe comprising a fluorophore labeled phenylglyoxal probe, or a biotin labeled phenylglyoxal probe,
wherein the probe is a product of a copper-catalyzed click condensation reaction of an alkyne-bearing reporter tag comprising a fluorophore tag or a biotin tag, and an azide-bearing reagent comprising a phenylglyoxal group, in the presence of an effective amount of a copper catalyst, to provide as the phenylglyoxal probe a conjugate of the phenylglyoxal group and the reporter tag linked by a triazole ring resulting from the click condensation reaction.

2. The phenylglyoxal probe of claim 1 , wherein the fluorophore labeled phenylglyoxal comprises: rhodamine labeled phenylglyoxal (Rh-PG) or fluorescein labeled phenylglyoxal.

3. The phenylglyoxal probe of claim 1 , wherein the phenylglyoxal probe is a biotin labeled phenylglyoxal (biotin-PG) probe.

4. A method of labeling a citrullinated protein or a fragment thereof in a biological sample, with a phenylglyoxal probe comprising a conjugate of a phenylglyoxal group and a reporter tag linked by a triazole ring, comprising:
contacting an effective amount of an alkyne-bearing reporter tag comprising a fluorophore reporter tag or a biotin reporter tag, an effective amount of an azide-bearing reagent comprising a phenylglyoxal group, in the presence of an effective concentration of a copper catalyst for a click condensation reaction, and a biological sample comprising a citrullinated protein or protein fragment, to provide a labeled citrullinated protein or protein fragment.

5. The method of claim 4 , wherein the fluorophore reporter tag comprises: rhodamine or fluorescein.

6. The method of claim 4 , wherein the step of contacting the biological sample is conducted in a medium having a pH value of about 0.001 to about 13.9.

7. The method of claim 4 , wherein the step of contacting the biological sample is conducted in a medium haying a pH value of about 0.001 to about 6.9.

8. The method of claim 4 , wherein the step of contacting the biological sample is conducted in a medium having a pH value of about 6.9 to about 13.9.

9. The method of claim 4 , wherein the labeled citrullinated protein or fragment is detectable by assay means comprising: gels, blots, chromatography, fluorescence, chemiluminesence, or immunoassays.

10. A method of determining the rate of citrullination and amount of citrullination of proteins in a biological sample, with a phenylglyoxal probe comprising a conjugate of a phenylglyoxal group and a reporter tag linked by a triazole ring, comprising:
contacting an effective amount of an alkyne-bearing reporter tag comprising a fluorophore reporter tag or a biotin reporter tag, an effective amount of an azide-bearing reagent comprising a phenylglyoxal group, in the presence of an effective concentration of a copper catalyst for a click condensation reaction, and a biological sample comprising a citrullinated protein or protein fragment, to provide a labeled citrullinated protein or protein fragment wherein the phenylglyoxal probe covalently bonds to a citrullinated protein or fragments thereof in the biological sample; wherein the step of contacting is conducted under varying acidic and basic pH values, thereby selectively labeling arginine and citrulline residues of the proteins in the biological sample; and,
determining the rate and amount of citrullination of proteins in the sample.

11. The method of claim 10 , wherein the fluorophore comprises: rhodamine or fluorescein.

12. The method of claim 10 , wherein the labeled citrullinated protein is detectable by assay means comprising gels, blots, chromatography, fluorescence, chemiluminesence, pull-down assays, immunoassays, high-throughput assays or combinations thereof.

13. A method of identifying bio markers of a disease
in biological sample, with a phenylglyoxal probe comprising a conjugate of a phenylglyoxal group and a reporter tag linked by a triazole ring, comprising
contacting an effective amount of an alkyne-bearing reporter tag comprising a fluorophore reporter tag or a biotin reporter tag, an effective amount of an azide-bearing reagent comprising a phenylglyoxal group, in the presence of an effective concentration of a copper catalyst for a click condensation reaction, and a biological sample comprising a citrullinated protein or protein fragment, to provide a labeled citrullinated protein or protein fragment, wherein the phenylglyoxal probe specifically binds to a citrullinated protein or fragments thereof, in the biological sample;
comparing protein citrullination patterns of proteins detected in the sample;
thereby identifying biomarkers of a disease.

14. The method of claim 13 , wherein the protein citrullination patterns comprise one or more biomarkers which discriminate between diseased and healthy cells, serum, and tissues.

15. A biomarker identified by the method of claim 13 .

16. A method of identifying biomarkers of a disease, with a phenylglyoxal probe comprising a conjugate of a phenylglyoxal group and a reporter tag linked by a triazole ring, comprising
contacting an effective amount of an alkyne-bearing reporter tag comprising a fluorophore reporter tag or a biotin reporter tag, an effective amount of an azide-bearing reagent comprising a phenylglyoxal group, in the presence of an effective concentration of a copper catalyst for a click condensation reaction, and a biological sample comprising a citrullinated protein or protein fragment, to provide a labeled citrullinated protein or protein fragment,
wherein the phenylglyoxal probe specifically binds via a covalent bond to a citrullinated protein or fragments thereof in the biological sample; thereby,
labeling the citrullinated protein or fragments thereof in the biological sample; and, identifying a citrullination profile of citrullinated proteins or fragments thereof;
comparing the citrullination profile of citrullinated proteins or fragments thereof in control samples as compared to a patient sample; thereby,
identifying biomarkers of a disease.

17. The method of claim 16 , wherein the biomarkers of a disease comprise one or more citrullinated proteins or fragments thereof.

18. The method of claim 16 , wherein the biomarkers of a disease comprise a citrullination profile of citrullinated proteins or fragments thereof, wherein the citrullination profile comprises at least two citrullinated proteins or fragments thereof having varying concentrations of citrullination and/or citrullinated proteins.

19. The method of claim 16 , wherein the fluorophore comprises: rhodamine or fluorescein.

20. The method of claim 16 , wherein the labeled citrullinated protein is detectable by assay means comprising: gels, blots, chromatography, fluorescence, chemiluminesence, pull-down assays, immunoassays, high-throughput assays or combinations thereof.

21. A biomarker identified by claim 16 .

22. A method of binding and identifying citrullinated proteins from serum or cellular extracts, with phenylglyoxal probe comprising a conjugate of a phenylglyoxal group and a reporter tag linked by a triazole ring comprising:
contacting an effective amount of an alkyne-bearing reporter tag comprising a fluorophore reporter tag or a biotin reporter tag, an effective amount of an azide-bearing reagent comprising a phenylglyoxal group, in the presence of an effective concentration of a copper cataly

Assignees

• Scripps Research Inst

Inventors

• Thompson Paul R
• Bicker Kevin L
• Subramanian Venkataraman

Classifications

• C07D311/90

  with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9 · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title

• C07D493/10

  Spiro-condensed systems · CPC title

• G01N33/583

  with non-fluorescent dye label · CPC title