# US 10,119,972 B2 · Reactive labelling compounds and uses thereof

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- Number: US 10,119,972 B2
- Document ID: US10119972B2
- Status: Granted
- Kind: B2
- Application number: 15/666,242
- Filed: 2017-08-01
- Published: 2018-01-11
- Granted: 2018-11-06
- Earliest claimed: 2014-03-27
- Assignees: Academia Sinica
- Applicants: Academia Sinica
- Inventors: Chi-Huey Wong; Jim-Min Fang; Jiun-Jie Shie
- Web page: https://patentgrep.com/patent/US10119972B2

## CPC

- G01N 33/582: {with fluorescent label}
- C07D 309/14: Nitrogen atoms not forming part of a nitro radical
- C07D 311/02: ortho- or peri-condensed with carbocyclic rings or ring systems
- C07F 5/027: {Organoboranes and organoborohydrides}
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C09B 23/04: one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
- C09B 57/02: Coumarine dyes
- G01N 21/64: Fluorescence; Phosphorescence
- G01N 2333/924: acting on glycosyl compounds (3.2)
- G01N 2458/30: Electrochemically active labels
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## 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

### 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.

### Claim 2 (depends on 1)

The compound of claim 1, wherein L is umbelliferyl.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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 G1, G2, G3, G5, G6, G7 and G8 is independently hydrogen, optionally substituted C1-6alkyl, optionally substituted C1-6alkenyl, optionally substituted C1-6alkynyl, optionally substituted aryl, optionally substituted acyl, —ORA, —CH2ORA, —OC(O)RA, —SRA, —N(RB)2, —N(RA)C(O)RA, —C(O)N(RB)2, —CN, —NO2, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO3RA, —SO2N(RB)2, and —NHSO2RB;
each RA is independently selected from hydrogen, optionally substituted C1-C6alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl;
each RB is independently selected from hydrogen, optionally substituted C1-C6alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl, or two RB taken together with the intervening nitrogen form a heterocycle;
each instance of G4a and G4b 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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 2)

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.

### Claim 7 (depends on 3)

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.