Reactive labelling compounds and uses thereof

• US 10,119,972 B2
• Filed: 08/01/2017
• Issued: 11/06/2018
• Est. Priority Date: 03/27/2014
• Status: Active Grant

• Current Assignee: Academia Sinica (Government of The Republic of China)
• Original Assignee: Academia Sinica (Government of The Republic of China)
• Inventors: Wong, Chi-Huey, Fang, Jim-Min, Shie, Jiun-Jie
• Primary Examiner(s): Ward, Paul V
• Application Number:
  US15/666,242
• Publication Number:
  US 20180011104A1
• Time in Patent Office: 462 Days
• CPC Class Codes: C07D 309/14 Nitrogen atoms not forming part of a nitro radical
• CPC Class Codes: C07D 311/02 ortho- or peri-condensed with carbocyclic rings or ring systems
• CPC Class Codes: C07F 5/027 Organoboranes and organoborohydrides
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)
• CPC Class Codes: G01N 2458/30 Electrochemically active labels
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Provided are azido-BODIPY compounds of formula (I), cyclooctyne-based fluorogenic probes of formula (IV), and activity-based probes of formula (VI). These compounds undergo azide-alkyne cycloadditions (AAC) with to form triazolyl products. The provided compounds are useful for detection and imaging of alkyne-, or azide-containing molecules. Methods for detection and imaging biomolecules using compounds of the present disclosure are disclosed.

Claims

1. A compound of formula (V) or (VI):

2. The compound of claim 1, wherein L is umbelliferyl.

3. The compound of claim 2, wherein the compound is compound 601:

4. A method for imaging the active site of a sialidase enzyme comprising:

(a) contacting a compound of claim 1 with a sample suspected of comprising the sialidase enzyme under conditions for ligation of the compound to an active site of the sialidase enzyme to form a covalent bond product,

(b) contacting the covalent bond product with an azide-containing fluorogenic probe of formula (I) to form a fluorogenic triazole product,

(c) measuring a fluorescent signal released from the triazole product;

wherein the azide-containing fluorogenic probe of formula (I) has the formula;

5. A method for detecting the active site of a sialidase enzyme in a sample, comprising:

(a) contacting the compound of claim 1, wherein L is coumarin oxide, with a sample suspected of having an sialidase enzyme molecule to release coumarin;

(b) measuring the level of a fluorescent signal released from the released coumarin in the sample mixture, and

(c) determining the presence of the sialidase enzyme molecule in the sample, wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of said compound indicates presence of the azide-containing molecule.

6. A method for detecting the active site of a sialidase enzyme in a sample, comprising:

(a) contacting the compound of claim 2 with a sample suspected of having an sialidase enzyme molecule to release an umbelliferyl moiety;

(b) measuring the level of a fluorescent signal released from the released umbelliferyl moiety in the sample mixture, and

(c) determining the presence of the sialidase enzyme molecule in the sample, wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of said compound indicates presence of the azide-containing molecule.

7. A method for detecting the active site of a sialidase enzyme in a sample, comprising:

(a) contacting the compound of claim 3 with a sample suspected of having an sialidase enzyme molecule to release coumarin;

(b) measuring the level of a fluorescent signal released from the released coumarin in the sample mixture, and

(c) determining the presence of the sialidase enzyme molecule in the sample, wherein an enhanced fluorescent signal as compared to a level of the fluorescent signal in the absence of said compound indicates presence of the azide-containing molecule.