Probes for rapid and specific detection of mycobacteria

Patent metadata

Field	Value
Publication number	US-10370538-B2
Application number	US-201615001591-A
Country	US
Kind code	B2
Filing date	Jan 20, 2016
Priority date	Aug 11, 2015
Publication date	Aug 6, 2019
Grant date	Aug 6, 2019

Abstract

The compositions of the present disclosure provide novel fluorogenic probes for use in the specific imaging and detection of mycobacteria species, and in particular β-lactam-antibiotic resistant. Specificity for mycobacteria is conferred on these probes by incorporating a moiety that specifically targets the unique trapping mechanism of the DprE1 found in in mycobacteria. Accordingly, only Mycobacteria species that express both a β-lactamase and DprE1 enable both the activation of the caged fluorescent probe, and the affixing of the released fluorescent probes to the bacteria cells through the functioning reduction-covalent binding mechanism. Advantageously, such a probe is able, at its most sensitive, to allow single mycobacterium detection.

First claim

The invention claimed is:

1. A probe specific for a mycobacterium, said probe comprising a β-lactamase substrate group covalently attached to a detectable label group, wherein the β-lactamase substrate group has the formula VI:

wherein, in the β-lactamase substrate group:
n=0 or 1;
R 4 is selected from the group consisting of: a hydrogen, an alkyl, a substituted alkyl, an alkoxy, an alkoxycarbonyl, an alkoxyl, an alkoxyalkyl, an alkylene, an aralkoxycarbonyl, an aralkyl, an aralkyloxyl, an aroyl, an aroylamino, an aryl, a substituted aryl group, a carbamoyl, a carbonyl, a carboxamide, a carboxyl, a heteroaryl, a heterocyclic, a hydroxyalkyl, a substituted alkenyl, a substituted cycloaliphatic, a substituted aliphatic, a thiol, a thioalkoxy, a thioalkyl, a thioaryl, a thiophene, or a substituted thiophene;
R 5 is Me, —CH 2 CH 3 , —CF 3 , —CH 2 CF 3 , —CF 2 CH 3 , —CHF 2 , —CH 2 F, —OCH 2 F, or OCHF 2 ;
R 6 and R 7 are each H or

 and
R 8 is O or S,

and wherein the detectable label has a caging group covalently attached thereto by an esterase-cleavable ester bond, wherein the detectable label is a fluorophore or a dye having the caging group covalently attached thereto by an esterase-cleavable ester bond, wherein said caging group is an acetoxymethyl group.

2. A probe specific for a mycobacterium, said probe comprising a β-lactamase substrate group covalently attached to a detectable label group, wherein the β-lactamase substrate group has the formula VI:

wherein, in the β-lactamase substrate group:
n=0 or 1;
R 4 is selected from the group consisting of: a hydrogen, an alkyl, a substituted alkyl, an alkoxy, an alkoxycarbonyl, an alkoxyl, an alkoxyalkyl, an alkylene, an aralkoxycarbonyl, an aralkyl, an aralkyloxyl, an aroyl, an aroylamino, an aryl, a substituted aryl group, a carbamoyl, a carbonyl, a carboxamide, a carboxyl, a heteroaryl, a heterocyclic, a hydroxyalkyl, a substituted alkenyl, a substituted cycloaliphatic, a substituted aliphatic, a thiol, a thioalkoxy, a thioalkyl, a thioaryl, a thiophene, or a substituted thiophene;
R 5 is Me, —CH 2 CH 3 , —CF 3 , —CH 2 CF 3 , —CF 2 CH 3 , —CHF 2 , —CH 2 F, —OCH 2 F, or —OCHF 2 ;
R 6 and R 7 are each H or

 and
R 8 is O or S,

and wherein the detectable label has a caging group covalently attached thereto by an esterase-cleavable ester bond, said probe further comprising a mycobacterium -specific decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1)-trapping moiety, or an ester or salt thereof, said DprE1-trapping moiety covalently attached to the detectable label.

3. The probe of claim 2 , wherein said mycobacterium -specific DprE1-trapping moiety is covalently attached to the detectable label by a first linker.

4. The probe of claim 2 , wherein said mycobacterium -specific DprE1-trapping moiety has the formula:

and
wherein R 1 is H, —NO 2 , or CF 3 .

5. The probe of claim 2 , wherein the first linker is selected from the group consisting of: ethoxyphenyl, —CH 2 —CH 2 —, -1-ethyl 3-methyl 1,2,3-triazole, and —CH 2 —CH 2 —NR 2 —CS—NH 2 —, wherein R 2 is H or —CH 3 .

6. A probe specific for a mycobacterium, wherein the probe is selected from the group consisting of:

wherein R 9 is each independently Me, —CH 2 CH 3 , —CF 3 , —CH 2 CF 3 , —CF 2 CH 3 , —CHF 2 , —CH 2 F, —OCH 2 F, or —OCHF 2 .

Assignees

• Univ Leland Stanford Junior

Inventors

• Rao Jianghong
• Cheng Yunfeng
• Xie Jinghang

Classifications

• C07D501/36

  Methylene radicals, substituted by sulfur atoms · CPC title

• C07D501/32

  with the 7-amino radical acylated by an araliphatic carboxylic acid, which is substituted on the aliphatic radical by hetero atoms · CPC title

• C09B11/24 Primary

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title