Cell-permeable probes for identification and imaging of sialidases

Patent metadata

Field	Value
Publication number	US-9914956-B2
Application number	US-201314422310-A
Country	US
Kind code	B2
Filing date	Aug 16, 2013
Priority date	Aug 18, 2012
Publication date	Mar 13, 2018
Grant date	Mar 13, 2018

Abstract

Provided herein are novel irreversible sialidase inhibitors. These compounds can be conjugated with a detectable tagging moiety such as azide-annexed biotin via CuAAC for isolation and identification of sialidases. The provided compounds and the corresponding detectable conjugates are useful for detecting sialidase-containing pathogens and imaging in situ sialidase activities under physiological conditions.

First claim

We claim:

1. A sialidase protein adduct having the formula:

wherein
R 2 is OR 2O ;
each instance of R 2O is independently hydrogen, C 1-6 alkyl, or acyl;
each instance of R 3a and R 3b is independently hydrogen, or —C(═O)—R 3r , or when taken together R 3a and R 3b form a C 1-6 alkyl substituted five-membered heterocyclyl ring;
each instance of R 3r is C 1-6 alkyl;
X is selected from the group consisting of —O—, —O(C═O)—, and —O(C═O)NH—,
R 4 is H, C 1-6 alkyl, or -L-Z;
Y is C 1-6 alkyl or -L-Z;
each instance of L is —(CH 2 ) n —,
each instance of n is an integer from 1 to 8, inclusive;
each instance of Z is an alkynyl;
wherein when R 4 is H or C 1-6 alkyl, then Y is -L-Z;
wherein when Y is C 1-6 alkyl, R 4 is -L-Z;
with the proviso that only one of R 4 or Y is -L-Z; and
with the proviso that the C-1 carboxyl is covalently conjugated to a tyrosine residue at the tyrosinyl phenolic hydroxyl oxygen, of a sialidase protein.

2. The sialidase protein adduct of claim 1 , wherein the sialidase is a human, viral or bacterial sialidase.

3. The sialidase protein adduct of claim 2 , wherein the bacterial sialidase is selected from the group consisting of nanA, nanB, nanC, nanJ, nanI, and nanH.

4. The sialidase protein adduct of claim 2 , wherein the human sialidase is selected from the group consisting of Neu1, Neu2, Neu3 and Neu4.

5. The sialidase protein adduct of claim 1 , wherein the adduct is:

6. The sialidase protein adduct of claim 1 , wherein the C-1 carboxyl is covalently linked to a tyrosine residue at the tyrosinyl phenolic hydroxyl oxygen, of any peptide of SEQ ID NOS: 1-6, wherein the peptide is a fragment of nanA, nanB, nanC, nanJ, nanI or nanH.

7. The sialidase protein adduct of claim 1 , wherein the sialidase is an influenza virus neuraminidase (NA).

8. A process for making a detectable conjugate, the process comprising the step of reacting a sialidase adduct of claim 1 with a reagent in the presence of a Cu(I) catalyst to form a five-membered heterocycle ring, wherein the reagent is

wherein L a is alkylenyl, alkenylenyl, heteroalkylenyl, heteroalkenylenyl, arylenyl, heteroarylenyl, or acylenyl.

9. A detectable conjugate having a formula selected from:

or a salt thereof,
wherein
F atom at the C3-position is axial or equatorial;
R K is H, C 1-6 alkyl, or a tyrosinyl phenolic hydroxyl oxygen linked sialidase protein;
R 2 is OR 2O ;
each instance of R 2O is independently hydrogen, C 1-6 alkyl, or acyl;
each instance of R 3a and R 3b is independently hydrogen, or —C(═O)—R 3r , or when taken together R 3a R 3b form a C 1-6 alkyl substituted five-membered heterocyclyl ring;
each instance of R 3r is C 1-6 alkyl;
X is selected from the group consisting of —O—, —O(C═O)—, and —O(C═O)NH—;
R 4a is H, C 1-6 alkyl, or acyl;
Y a is H or C 1-6 alkyl;
each instance of L is —(CH 2 ) n —;
each instance of n is an integer from 1 to 8, inclusive;
each instance of R p and R q is independently hydrogen, aliphatic; heteroaliphatic; substituted or unsubstituted aryl; heteroaryl; acyl; a dye-labelled streptavidin protein; a fluorescent protein; a reporter or a label optionally joined by a linker L a , wherein the linker L a is alkylenyl; alkenylenyl; heteroalkylenyl; heteroalkenylenyl; arylenyl; heteroarylenyl; or acylenyl;
with the proviso that at least one of R p or R q is a dye-labelled streptavidin protein, a fluorescent protein, a reporter or a label optionally joined by a linker L a .

10. The detectable conjugate of claim 9 , wherein the label is a fluorophore.

11. A method for detecting the presence of a sialidase in a sample, the method comprising:
(a) obtaining a sample suspected of comprising a sialidase from a subject,
(b) contacting said sample with a compound of formula (I) to form a sialidase adduct of claim 1 ;
(c) adding a reporter;
(d) reacting the reporter to the alkynyl group on the compound of the sialidase adduct of claim 1 with the azide moiety by the click reaction in the presence of a Cu(I) catalyst to form a five-membered nitrogen-containing heterocycle ring; and
(e) detecting a signal,
wherein presence of a signal indicates the presence of the sialidase in the sample,
wherein the sample is extracted from a subject, and
wherein the reporter is:

wherein the linker L a is alkylenyl, alkenylenyl, heteroalkylenyl, heteroalkenylenyl, arylenyl, heteroarylenyl, or acylenyl;
wherein formula (I) has the structure:

or a salt thereof,
wherein
the F atom at the C3-position is axial or equatorial;
R 1 is H or optionally substituted C 1-6 alkyl;
R 2 is OR 2O , N(R 2N ) 2 , or guanidinyl;
each instance of R 2O is independently hydrogen, optionally substituted C 1-6 alkyl, optionally substituted acyl, or a hydroxyl protecting group;
each instance of R 2N is independently hydrogen, optionally substituted C 1-6 alkyl, optionally substituted acyl, or an amine protecting group;
each instance of R 3a and R 3b is independently hydrogen, —C(═O)—R 3r , or a hydroxyl protecting group;
each instance of R 3r is optionally substituted C 1-6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl, optionally substituted alkylenyl, optionally substituted alkylheteroaryl, or optionally substituted alkylheterocyclyl;
X is selected from the group consisting of —O—, —O(C═O)—, —NH—, —NH(C═O)—, —(C═O)NH—, —O(C═O)NH—, —O(C═S)NH—, —NH(C═O)NH—, and —NH(C═S)NH—;
R 4 is H, or optionally substituted C 1-6 alkyl;
Y is H or optionally substituted C 1-6 alkyl;
wherein when R 4 is H or C 1-6 alkyl, then Y is -L-Z;
wherein when Y is C 1-6 alkyl, R 4 is -L-Z;
each instance of Z is an alkynyl;
with the proviso that only one of R 4 or Y is -L-Z;
each instance of L is independently selected from the group consisting of —(CH 2 ) n —, —(CH 2 ) n C═O—, —(CH 2 ) n NH—, —(C═O)(CH 2 ) n —, —(CH 2 ) n NH(C═O)—, —(C═O)(CH 2 ) n NH(C═O)—, —(CH 2 ) n SCH 2 (C═O)—, and —(CH 2 CH 2 O) n —; and
each instance of n is an integer from 1 to 8, inclusive.

12. The method of claim 11 , wherein the sialidase in the sample is intracellular.

13. The method of claim 11 , wherein the compound of formula (I) is PDFSA-5-yne (IV) or PDFSA-7-yne (VII):

14. The method of claim 11 , wherein the sample is from a mammal, fowl, or fish.

15. The method of claim 14 , wherein the mammal is human.

16. The method of claim 11 wherein the sample is tested to determine

Assignees

• Academia Sinica

Inventors

• Wong Chi-Huey
• Fang Jim-Min
• Cheng Yih-Shyun E
• Tsai Charng-Sheng

Classifications

• C12Q1/34 Primary

  involving hydrolase · CPC title

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C07H13/12

  by acids having the group -X-C(=X)-X-, or halides thereof, in which each X means nitrogen, oxygen, sulfur, selenium or tellurium, e.g. carbonic acid, carbamic acid · CPC title

• C12Q1/70

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title

• C07H5/00

  Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium · CPC title