Reactive labelling compounds and uses thereof

Patent metadata

Field	Value
Publication number	US-10119972-B2
Application number	US-201715666242-A
Country	US
Kind code	B2
Filing date	Aug 1, 2017
Priority date	Mar 27, 2014
Publication date	Nov 6, 2018
Grant date	Nov 6, 2018

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.

First claim

We claim:

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

wherein L is selected from the group consisting of umbelliferyl, coumarin oxide, triflate, mesylate, tosylate, alkoxy, phenoxy, benzoate, pentafluorophenoxy, or 4-nitrophenoxy.

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:

wherein each instance of G 1 , G 2 , G 3 , G 5 , G 6 , G 7 and G 8 is independently hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 1-6 alkenyl, optionally substituted C 1-6 alkynyl, optionally substituted aryl, optionally substituted acyl, —OR A , —CH 2 OR A , —OC(O)R A , —SR A , —N(R B ) 2 , —N(R A )C(O)R A , —C(O)N(R B ) 2 , —CN, —NO 2 , —C(O)R A , —C(O)OR A , —S(O)R A , —SO 2 R A , —SO 3 R A , —SO 2 N(R B ) 2 , and —NHSO 2 R B ;
each R A is independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl;
each R B is independently selected from hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two R B taken together with the intervening nitrogen form a heterocycle;
each instance of G 4a and G 4b is fluoro, alkyl, alkoxy, aryloxy, or alkynyl,
wherein alkyl and alkoxy groups are unbranched, saturated, and have 1-4 carbon atoms; aryl groups and aryl groups of aryloxy can be either carbocyclic aryl or heterocyclic aryl; carbocyclic aryl groups have a total of 6-20 carbon atoms, including carbon atoms of substituents; heterocyclic aryl groups have a total of 5-20 carbon atoms, including carbon atoms of substituents; carboalkoxy groups are alkyl esters of a carboxylic acid wherein alkyl groups are as defined above; each alkyl, aryl, alkoxy, aryloxy, benzo, and carboalkoxy, independently, may be unsubstituted or substituted with one or more substituent; alkyl substituents are halo, hydroxyl, amino, or aryl; aryl substituents are halo, hydroxyl, amino, alkyl, aryl, nitro, or carboxyl; and halo substituents are fluoro or chloro; and
n is 0, 1, 2, 3, or 4.

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.

Assignees

• Academia Sinica

Inventors

• Wong Chi-Huey
• Fang Jim-Min
• Shie Jiun-Jie

Classifications

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N2458/30

  Electrochemically active labels · CPC title

• C07D309/14

  Nitrogen atoms not forming part of a nitro radical · CPC title

• C07K7/08 Primary

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• G01N21/64

  Fluorescence; Phosphorescence · CPC title