Self-immolative probes for enzyme activity detection

Patent metadata

Field	Value
Publication number	US-9574222-B2
Application number	US-201414447817-A
Country	US
Kind code	B2
Filing date	Jul 31, 2014
Priority date	Nov 16, 2010
Publication date	Feb 21, 2017
Grant date	Feb 21, 2017

Abstract

Provided is a compound comprising the structure:

(SIG)-(SI-MOD) m .

In this compound, SIG is a signaling molecule, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG without SI, MOD is a moiety bound to SI that is subject to modification by an activator, and m is an integer from 1 to about 10. With this compound, when MOD is modified by an activator, SI is destabilized and self-cleaved from SIG such that SIG generates an increased signal. Also provided is a method of determining whether a sample comprises an activator, using the above-described compound. Additionally provided is a method of determining whether a cell comprises a nitroreductase using the above-described compound where nitroreductase is the activator. Further provided is a method of determining whether a mammalian cell is hypoxic using the above-described compound where nitroreductase is the activator. A method of detecting a microorganism that comprises a nitroreductase, using the above-described compound where nitroreductase is the activator, is also provided. Also provided is a method of identifying nitroreductase in a sample, using the above-described compound where nitroreductase is the activator.

First claim

What is claimed is:

1. A composition, comprising:
a compound represented by the structure:

wherein SIG is a signaling moiety, SI is a self-immolative structure bound to SIG such that SIG has a reduced signal relative to the signal of SIG when cleaved from SI,
wherein each AA is independently an amino acid of an amino acid sequence, PTM is a prost-translational modification bound to the side chain of any of the amino acids of the amino acid sequence, n is an integer representing the total number of amino acids in the sequence, wherein n is from 1 to about 200, and m is an integer from 1 to about 10, and
where when the PTM is removed from the compound by an enzyme capable thereof, a protease or peptidase is then able to remove the amino acid sequence, allowing self-cleavage of SI and SIG; and
the protease or peptidase that is able to remove the amino acid sequence when PTM is removed by the enzyme.

2. The composition of claim 1 , wherein m>1.

3. The composition of claim 1 , wherein m=1.

4. The composition of claim 1 , wherein SIG is a fluorophore selected from a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin, an iminocoumarin, a xanthene, a rhodamine, a rhodamine derivative, a rosamine, a rosamine derivative, a rhodol or a rhodol deravitive.

5. The composition of claim 4 , wherein the xanthene has the structure:

wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), a carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphino group (PR 6 2 ), a silane group (SiR 6 3 ), a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an aromatic group; and
wherein each R 6 is independently hydrogen, a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, an aromatic group.

6. The composition of claim 4 , wherein the xanthene has the structure:

wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), a carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphino group (PR 6 2 ), a silane group (SiR 6 3 ), a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an aromatic group; and
wherein each R 6 is independently hydrogen, a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, an aromatic group.

7. The composition of claim 4 , wherein the fluorophore is a coumarin.

8. The composition of claim 7 , wherein the coumarin has the structure:

wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), a carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphino group (PR 6 2 ), a silane group (SiR 6 3 ), a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an aromatic group; and
wherein each R 6 is independently hydrogen, a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, an aromatic group.

9. The composition of claim 7 , wherein the coumarin is an iminocoumarin.

10. The composition of claim 9 , wherein the iminocoumarin has the structure:

wherein
each R 5 is independently hydrogen, a halogen (F, Cl, Br, I), a nitro group (NO 2 ), a nitroso group (NO), a hydroxylamino group (NHOH), a cyano group (CN), an isocyano group (NC), a thiocyano group (SCN), an isothiocyano group (SNC), an azido group (N 3 ), a trihalomethyl group (CX 3 , where X is a halogen); a sulfonate group (SO 3 R 6 ), a sulfate group (OSO 3 R 6 ), a carboxyl group (CO 2 H), a carbonyl group (COR 6 ), an ester group (CO 2 R 6 or OCOR 6 ), an amide group (CONR 6 2 or NR 6 COR 6 ), a carbamate group (NR 6 CO 2 R 6 or OCONR 6 2 ), a phosphate group (OPO 3 R 6 3 ), a phosphonate group (PO 3 R 6 2 ), an amino group (NR 6 2 ), an alkoxy group (OR 6 ), a thiol group (SR 6 ), a sulfoxy group (SOR 6 ), a sulfone group (SO 2 R 6 ), a sulfonamide group (SO 2 NR 6 2 ), a phosphino group (PR 6 2 ), a silane group (SiR 6 3 ), a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group, CONR 6 group, or an aromatic group; and
wherein each R 6 is independently hydrogen, a straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2 groups can be replaced with O atom, N atom, S atom, NH group, CO group, OCO group

Assignees

• Enzo Life Sciences Inc

Inventors

• Szczepanik Maciej
• Lebedeva Irina V
• Xiang Yuejun
• Pande Praveen
• Patton Wayne Forrest

Classifications

• C07D491/20

  Spiro-condensed systems · CPC title

• G01N2333/902

  Oxidoreductases (1.) · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• C09K11/06

  containing organic luminescent materials · CPC title

• C12Q1/37

  involving peptidase or proteinase · CPC title