Enhanced deposition of chromogens utilizing pyrimidine analogs

Patent metadata

Field	Value
Publication number	US-9435795-B2
Application number	US-201414507447-A
Country	US
Kind code	B2
Filing date	Oct 6, 2014
Priority date	Dec 30, 2010
Publication date	Sep 6, 2016
Grant date	Sep 6, 2016

Abstract

This disclosure relates to compositions that enhance the deposition of detectable moieties on tissue samples, methods utilizing these compositions and kits including these compositions. The compositions include a deposition enhancer having a formula

where R 1 , R 2 , R 3 , and R 4 are independently selected from aliphatic, aryl, halogen, a heteroatom-containing moiety, and hydrogen; R 1 and/or R 3 can be bound to R 2 to form a fused, aromatic ring system; R 5 is selected from a heteroatom-containing moiety; A is selected from, a carbon atom, a heteroatom, other than sulfur, and any combination thereof; n is 1-5, an enzyme, a specific binding moiety and a detectable moiety.

First claim

We claim:

1. A composition for detecting a target in a sample by proximally depositing a detectable moiety, comprising:
a pyrimidine and/or pyridine deposition enhancer having a formula

wherein R 1 , R 2 , R 3 , and R 4 are independently selected from aliphatic, aryl, halogen, a heteroatom-containing moiety, and hydrogen; R 1 and/or R 3 can be bound to R 2 to form a fused, aromatic ring system; R 5 is a heteroatom-containing moiety; A is carbon, a heteroatom other than sulfur, and any combination thereof and n is 1-5;
a second deposition enhancer; and
an enzyme substrate.

2. The composition according to claim 1 where the second deposition enhancer has a formula

where:
A is carbon, a heteroatom other than sulfur, or any combination thereof;
B is carbon, nitrogen, or oxygen; and
R 8 , R 9 , R 10 and R 11 independently are aliphatic, aryl, halogen, a heteroatom-containing moiety, hydrogen, or any combination thereof.

3. The composition according to claim 2 where the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR 6 R 7 where R 6 and R 7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, or any combination thereof.

4. The composition according to claim 2 where A is carbon and B is nitrogen.

5. The composition according to claim 1 where the second deposition enhancer is selected from imidazole, L-histidine, thiazole, oxazole, or any combination thereof.

6. The composition according to claim 1 where the second deposition enhancer has a formula

where:
A is carbon, nitrogen, oxygen, or any combination thereof; and
R 12 -R 23 independently are aliphatic, aryl, halogen, a heteroatom-containing moiety, hydrogen, or any combination thereof.

7. The composition according to claim 6 where the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR 6 R 7 where R 6 and R 7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, or any combination thereof.

8. The composition according to claim 6 where R 22 is [O]. or [O] − .

9. The composition according to claim 6 where the second deposition enhancer is pyrimidine N-oxide, pyridine N-oxide, N-methyl morpholine (NMO), and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO).

10. The composition according to claim 1 where the second deposition enhancer is an inorganic or organic boronic acid.

11. The composition according to claim 10 where the second deposition enhancer has a formula

where R 24 , R 25 , and R 26 independently are aliphatic, aryl, heteroaliphatic, heteroaryl, or any combination thereof.

12. The composition according to claim 11 where two or more of R 24 , R 25 , and R 26 are hydroxyl, with any remaining R 24 ,R 25 , or R 26 being aliphatic, aryl, heteroaliphatic, or heteroaryl.

13. The composition according to claim 10 where the boronic acid is boric acid, phenyl boronic acid, 4-AcHN-phenyl boronic acid, or any combination thereof.

14. The composition according to claim 1 where the second deposition enhancer is a phenolic compound having a formula

where R 27 , R 28 , R 29 , R 30 , and R 31 independently are hydrogen, aliphatic, aryl, a heteroatom-containing moiety, halogen, or any combination thereof.

15. The composition according to claim 14 where the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR 6 R 7 where R 6 and R 7 independently are aliphatic, aryl, heteroaliphatic, heteroaryl, hydrogen, or any combination thereof.

16. The composition according to claim 14 where two adjacent groups selected from R 27 , R 28 , R 29 , R 30 , and R 31 form a fused aromatic or non-aromatic ring system.

17. The composition according to claim 14 where at least one of R 27 , R 28 , R 29 , R 30 , and R 31 is hydroxyl.

18. The composition according to claim 14 where the phenolic compound is pyrocatechol.

19. The composition according to claim 1 where the second deposition enhancer has a formula

where:
A is carbon, a heteroatom other than sulfur, and combinations thereof; and
R 1 , R 2 , R 3 , and R 4 are selected from aliphatic, aryl, halogen, a heteroatom-containing moiety, and hydrogen, or R 1 and/or R 3 can be bound to R 2 to form a fused aromatic ring system.

20. The composition according to claim 1 where the enzyme substrate is selected from 1,3-diaminobenzidine, 3-amino-9-ethylcarbazole, tetramethylbenzidine, a fluorescein, a luminophore, a coumarin, a BODIPY dye, a resorufin, a rhodamine, a tyramide, or a derivative thereof.

21. The composition according to claim 1 wherein the deposition enhancer is selected from 2-hydroxypyridine, pyrimidine or 2-hydroxypyrimidine.

22. A method for detecting a target in a sample by proximally depositing a detectable moiety, comprising:
contacting the sample with a recognition solution, the recognition solution including a specific binding moiety specific to the target;
labeling the specific binding moiety with an enzyme;
contacting the sample with a detection solution, the detection solution comprising an enzymatic substrate so that the detectable moiety deposits proximally to the target in the presence of a first deposition enhancer and a second deposition enhancer, the first deposition enhancer having a formula

wherein R 1 , R 2 , R 3 , and R 4 are independently selected from aliphatic, aryl, halogen, a heteroatom-containing moiety, and hydrogen; R 1 and/or R 3 can be bound to R 2 to form a fused, aromatic ring system; R 5 is a heteroatom-containing moiety; A is carbon, a heteroatom other than sulfur, and any combination thereof; and n is 1-5; and
detecting the detectable moiety.

23. The method according to claim 22 where the second deposition enhancer has a formula

where:
A is carbon, a heteroatom other than sulfur, or any combination thereof;
B is carbon, nitrogen or oxygen; and
R 8 , R 9 , R 10 and R 11 independently are aliphatic, aryl, halogen, a heteroatom-containing moiety, hydrogen, or any combination thereof.

24. The method according to claim 23 where the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR 6 R 7 where R 6 and R 7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, or any combination thereof.

25. The method according to claim 23 where A is carbon and B is nitrogen.

26. The method ac

Assignees

• Ventana Med Syst Inc

Inventors

• May Eric J
• Murillo Adrian E
• Kosmeder Ii Jerome W

Classifications

• G01N33/5306 Primary

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N33/581 Primary

  with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates) · CPC title

• G01N33/539

  involving precipitating reagent {, e.g. ammonium sulfate} · CPC title

• G01N33/531

  Production of immunochemical test materials · CPC title

• C12Q1/6813

  Hybridisation assays · CPC title