Probe for selectively characterizing enzymes involved in xenobiotic metabolism and method of making and using the same

Patent metadata

Field	Value
Publication number	US-11345950-B2
Application number	US-201816151170-A
Country	US
Kind code	B2
Filing date	Oct 3, 2018
Priority date	Oct 4, 2017
Publication date	May 31, 2022
Grant date	May 31, 2022

Abstract

Activity-based probes that can be used to selectively identify and characterize enzymes that are involved in different phases of xenobiotic metabolism in a host and its microbiota population(s) are described. The activity-based probes described specifically label only their target active enzymes involved in xenobiotic metabolism and therefore provide a measurement of true protein functional activity rather than transcript or protein abundance. The activity-based probes also provide multimodal profiling of these active enzymes. Methods for preparing the activity based probes and exemplary methods for their use also are disclosed.

First claim

We claim:

1. A probe having a structure satisfying Formula II

wherein
ERG, if present, is

S(O) 2 OH, or an anionic form thereof; P(O(OH) 2 , or an anionic form thereof; a halogen; or —OPh-CH 2 —ONO 2 ;
EBG has a structure satisfying a formula selected from Formulas IIA EBG -IIJ EBG

wherein Y is CH 3 or CF 3 ; Y′ is O, NO 2 , or —N═NR″, wherein R″ is a dye or other reporting moiety;
and m′ is an integer selected from 0 to 5; and R′ is selected from aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, nitroso, quaternary amine, CF 3 , alkyl halide, or combinations thereof;
Linker a comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
each of Linker b , and Linker c , if present, independently comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
R, if present, is a group comprising a carbamate or a carbonate; and
each of n, m, p, and q independently is 0 or 1;
provided that one of the following applies:
(a) one or more Tags are present, and each Tag is independently a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye; or
(b) one or more p Tags are present and each p Tag is independently an azide or an alkyne.

2. The probe of claim 1 , wherein Linker a is a linker group having a structure satisfying a formula selected from Formulas IIA Linkera -IIH Linkera

wherein each n′ independently is an integer ranging from 1 to 50; Z is oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic; and Q is carbon, oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic.

3. The probe of claim 1 , wherein Linker b is present and comprises a —(CH 2 ) n′ — group, wherein n′ is an integer ranging from 1 to 50; an amide group; or a combination thereof.

4. The probe of claim 1 , wherein Linker c is present and has a structure satisfying Formula IIA Linkerc or Formula IIB Linkerc

wherein n′ is an integer ranging from 0 to 50; and Z is oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic.

5. The probe of claim 1 , wherein R is present and has a structure satisfying any one or more Formulas IIA R -IID R

wherein Z and Z′ independently are oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic; and n′ is an integer ranging from 0 to 50.

6. The probe of claim 1 , wherein the one or more Tags independently are rhodamine, fluorescein, or biotin.

7. The probe of claim 1 , wherein the compound has a structure satisfying Formula IIIA

8. The probe of claim 7 , wherein the ERG is present and is iodo; —OPh-CH 2 —ONO 2 ;

9. The probe of claim 7 , wherein Linker a is an ester group, an —O(CH 2 ) n′ NR′″C(O)(CH 2 ) n′ — group, or a —(CH 2 ) n′ — group, wherein each n′ independently is an integer ranging from 1 to 20 and wherein R′″ is hydrogen, aliphatic, or aromatic.

10. The probe of claim 7 , wherein m is 1 and Linker c is an —NR′″C(O)(CH 2 ) n′ — group or an —NR′″C(O)CH 2 [O(CH 2 ) 2 ] n′ OCH 2 — group, wherein each n′ independently is an integer ranging from 1 to 20 and wherein R′″ is hydrogen, aliphatic, or aromatic.

11. The probe of claim 7 , wherein m is 1.

12. A probe selected from:

13. A probe having a structure satisfying Formula II

wherein
ERG is

EBG has a structure satisfying a formula selected from

wherein Y′ is O; m′ is an integer selected from 0 to 5; and each occurrence of R′ is independently selected from aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, nitroso, quaternary amine, CF 3 , alkyl halide, or combinations thereof;
Linker a comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
each of Linker b , and Linker c , if present, independently comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
R, if present, is a group comprising a carbamate or a carbonate;
each of n, m, p, and q independently is 0 or 1; and
provided that one of the following applies:
(a) one or more Tags are present, and each Tag is independently a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye; or
(b) one or more p Tags are pr

Assignees

• Battelle Memorial Institute

Inventors

• Wright Aaron T
• Ramos-Hunter Susan
• Whidbey Christopher

Classifications

• C07C305/22

  having oxygen atoms of sulfate groups bound to carbon atoms of six-membered aromatic rings · CPC title

• G01N2333/91177

  Glutathione transferases (2.5.1.18) · CPC title

• C07C203/04 Primary

  having nitrate groups bound to acyclic carbon atoms · CPC title

• C07D203/26

  condensed with carbocyclic rings or ring systems · CPC title

• C07C233/33

  with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring · CPC title