Reductive release probes containing a chemoselectively cleavable α-azidoether linker and methods of use thereof
View Patent ↗Probes comprising one or more selectively cleavable α-azidoether moieties are provided; and linkers comprising the one or more selectively cleavable α-azidoether moieties. The α-azidoether moiety will undergo a Staudinger reaction with a suitable reducing agent, resulting in cleavage. The probes find use in a variety of detection assays, e.g. specific polynucleotide binding assays, polypeptide binding assays, etc. The cleavable linkers are suitable for synthetic reactions, e.g. to prepare probes of the invention; in the synthesis of cleavable peptide conjugates; and the like.
1. A selectively cleavable probe of structure (I):
wherein A is an aromatic ring;
T 1 and T 2 are independently a tether; and
one of Z 1 and Z 2 is a a specific binding moiety and the other of Z 1 and Z 2 is a detection moiety.
2. The selectively cleavable probe of claim 1 , wherein the compound is of structure (III):
3. A selectively cleavable probe of one of structures (VIIIa) and (VIIIb):
wherein T 14 and T 15 are independently a tether;
Q 6 is a detection moiety; and
M 2 is a specific binding moiety.
4. The selectively cleavable probe of claim 3 , wherein the probe is of structure (IX):
wherein Q 7 is a quencher;
M 3 is a polynucleotide; and
T 16 and T 17 are independently a tether.
5. The selectively cleavable probe of claim 4 , wherein:
M 3 comprises a fluorophore that is quenched by Q 7 .
6. The selectively cleavable probe of claim 1 , wherein the detection moiety is selected from a fluorophore and a quencher.
7. The selectively cleavable probe of claim 1 , wherein the specific binding moiety is selected from an antibody or fragment thereof, a polypeptide and a polynucleotide.
8. The selectively cleavable probe of claim 7 , wherein the specific binding moiety is a polynucleotide of at least 6 bases in length and not more than about 100 bases in length.
9. The selectively cleavable probe of claim 8 , wherein the detection moiety is a quencher, and said polynucleotide further comprises at least one fluorophore quenched by the detection moiety.
10. The selectively cleavable probe of claim 9 , wherein the quencher is a dabsyl quencher.
11. The selectively cleavable probe of claim 3 , wherein T 14 and T 15 are independently selected from one of the following structures, or a substituted version thereof:
wherein:
n is an integer from 1 to 20;
n 1 and n 2 are independently selected integers from 1 to 20; where n 1 +n 2 is about 20 or less;
m is an integer from 1 to 7; and
R 7 is hydrogen or an alkyl.
12. The selectively cleavable probe of claim 3 , wherein Q 6 is selected from a fluorophore and a quencher.
13. The selectively cleavable probe of claim 3 , wherein M 2 is selected from an antibody or fragment thereof, a polypeptide and a polynucleotide.
14. The selectively cleavable probe of claim 13 , wherein M 1 is a polynucleotide of at least 6 bases in length and not more than about 100 bases in length.
15. The selectively cleavable probe of claim 14 , wherein Q 6 is a quencher, and said polynucleotide further comprises at least one fluorophore quenched by Q 6 .
16. The selectively cleavable probe of claim 15 , wherein Q 6 is a dabsyl quencher.