Colors for chromogenic IHC and ISH staining with multi-dye quinone methide and tyramide conjugates

Patent metadata

Field	Value
Publication number	US-12130284-B2
Application number	US-202117318541-A
Country	US
Kind code	B2
Filing date	May 12, 2021
Priority date	Jun 28, 2016
Publication date	Oct 29, 2024
Grant date	Oct 29, 2024

Abstract

Disclosed herein are novel chromogenic conjugates, the conjugates comprising at least two detectable moieties.

First claim

The invention claimed is:

1. A multi-dye conjugate comprising a tissue reactive moiety and at least two chromophores, wherein the tissue reactive moiety is a quinone methide precursor moiety and wherein the at least two chromophores are different; wherein the at least two chromophores of the multi-dye conjugate are covalently conjugated to a heterobifunctional linker or a polyamine linker having between 2 and 10 amine groups, and wherein the heterobifunctional linker or the polyamine linker is covalently conjugated to the quinone methide precursor moiety, and wherein the quinone methide precursor moiety has the structure of Formula (II):

wherein
R 1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar;
R 2 is a halide;
R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms; and
R 7 is —(CH 2 ) w NH—, —O(CH 2 ) w NH—, —N(H)C(O)(CH 2 ) w NH—, —C(O)N(H)(CH 2 ) w NH—, —(CH 2 ) w O—, —O(CH 2 ) w O—, —O(CH 2 CH 2 O) w —, —N(H)C(O)(CH 2 ) w O—, —C(O)N(H)(CH 2 ) w O—, —C(o)N(H)(CH 2 CH 2 O) w —, —(CH 2 ) w S—, —O(CH 2 ) w S—, —N(H)C(O)(CH 2 ) w S—, —C(O)N(H)(CH 2 ) w S—, —(CH 2 ) w NH—, —C(O)N(H)(CH 2 CH 2 O) w CH 2 CH 2 NH, —C(O)(CH 2 CH 2 O) w CH 2 CH 2 NH—, —C(O)N(H)(CH 2 )NHC(O)CH(CH 3 )(CH 2 ) w NH—, or —N(H)(CH 2 ) w NH—, where w is an integer ranging from 1 to 12; and wherein the wavy line indicates covalent linkage point of the heterobifunctional linker or the polyamine linker.

2. The multi-dye conjugate of claim 1 , wherein the at least two chromophores are selected from the group consisting of TAMRA, Dabsyl, Cy5, Dabcyl, Cy3, Cy7, Cy3.5, Cy3B, Cy5.5, rhodamine 800, fluorescein, and derivatives or analogs thereof.

3. The multi-dye conjugate of claim 1 , wherein a first of the at least two chromophores comprises a yellow or a yellow-like color.

4. The multi-dye conjugate of claim 1 , wherein a second of the at least two chromophores comprises a cyan or a cyan-like color.

5. The multi-dye conjugate of claim 1 , wherein a first of the at least two chromophores comprises a rhodamine or a derivative or an analog thereof.

6. The multi-dye conjugate of claim 5 , wherein the rhodamine or the derivative or analog thereof comprises rhodamine 800.

7. The multi-dye conjugate of claim 6 , wherein a second of the at least two chromophores is selected from the group consisting of TAMRA or a derivative or an analog thereof, Dabsyl or a derivative or an analog thereof, Cy5 or a derivative or an analog thereof, Dabcyl or a derivative or an analog thereof, Cy3 or a derivative or an analog thereof, Cy7 or a derivative or an analog thereof, Cy3.5 or a derivative or an analog thereof, Cy3B or a derivative or an analog thereof, Cy5.5 or a derivative or an analog thereof, and fluorescein or a derivative or an analog thereof.

8. The multi-dye conjugate of claim 1 , wherein a first of the at least two chromophores comprises Cy5 or a derivative or an analog thereof.

9. The multi-dye conjugate of claim 8 , wherein a second of the at least two chromophores is selected from the group consisting of a rhodamine or a derivative or an analog thereof, Dabsyl or a derivative or an analog thereof, TAMRA or a derivative or an analog thereof, Dabcyl or a derivative or an analog thereof, Cy3 or a derivative or an analog thereof, Cy7 or a derivative or an analog thereof, Cy3.5 or a derivative or an analog thereof, Cy3B or a derivative or an analog thereof, Cy5.5 or a derivative or an analog thereof, and fluorescein or a derivative or an analog thereof.

10. The multi-dye conjugate of claim 1 , wherein the heterobifunctional linker or the polyamine linker comprises a molecular weight ranging from between about 50 g/mol to about 300 g/mol.

11. The multi-dye conjugate of claim 1 , wherein the heterobifunctional linker or the polyamine linker is derived from lysine or a derivative or analog thereof.

12. A conjugate comprising (i) quinone methide precursor moiety, (ii) a rhodamine or a derivative or an analog thereof, and (iii) Cy5 or a derivative or an analog thereof; wherein the rhodamine or the derivative or the analog thereof and the Cy5 or the derivative or the analog thereof are conjugated to a heterobifunctional or a polyamine linker having between 2 and 10 amine groups, and wherein the heterobifunctional linker or the polyamine linker is covalently conjugated to the quinone methide precursor moiety; and
wherein the quinone methide precursor moiety has the structure of Formula (II):

wherein
R 1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar;
R 2 is a halide;
R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms; and
R 7 is —(CH 2 ) w NH—, —O(CH 2 ) w NH—, —N(H)C(O)(CH 2 ) w NH—, —C(O)N(H)(CH 2 ) w NH—, —(CH 2 ) w O—, —O(CH 2 ) w O—, —O(CH 2 CH 2 O) w —, —N(H)C(O)(CH 2 ) w O—, —C(O)N(H)(CH 2 ) w O—, —C(o)N(H)(CH 2 CH 2 O) w —, —(CH 2 ) w S—, —O(CH 2 ) w S—, —N(H)C(O)(CH 2 ) w S—, —C(O)N(H)(CH 2 ) w S—, —(CH 2 ) w NH—, —C(O)N(H)(CH 2 CH 2 O) w CH 2 CH 2 NH, —C(O)(CH 2 CH 2 O) w CH 2 CH 2 NH—, —C(O)N(H)(CH 2 )NHC(O)CH(CH 3 )(CH 2 ) w NH—, or —N(H)(CH 2 ) w NH—, where w is an integer ranging from 1 to 12; and wherein the wavy line indicates covalent linkage point of the heterobifunctional linker or the polyamine linker .

13. The conjugate of claim 12 , wherein the rhodamine or the derivative or the analog thereof and the Cy5 or the derivative or the analog thereof are conjugated to the tissue reactive moiety through a multi-functional linker, wherein the multi-functional linker comprises a molecular weight ranging from between about 50 g/mol to about 300 g/mol.

14. The conjugate of claim 13 , wherein the multi-functional linker is derived from lysine or a derivative or analog thereof.

15. The conjugate of claim 12 , wherein the rhodamine or the derivative or the analog thereof comprises rhodamine 800.

16. A multi-dye conjugate comprising a tissue reactive moiety and at least two different chromophores, wherein the tissue reactive moiety is a quinone methide precursor moiety and the multi-dye conjugate produces a green or green-like color; wherein the at least two different chromophores of the multi-dye conjugate are covalently conjugated to a heterobifunctional linker or a polyamine linker having between 2 and 10 amine groups, and wherein the heterobifunctional linker or the polyamine linker is covalently conjugated to the quinone methide precursor moiety; and
wherein the quinone methide precursor moiety has the structure of Formula (II):

wherein
R 1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar;
R 2 is a halide;
R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms; and
R 7 is -(CH 2 ) w NH—, —O(CH 2 ) w NH—, —N(H)C(O)(CH 2 ) w NH—, —C(O)N(H)(CH 2 ) w NH—, —(CH 2 ) w O—, —O(CH 2 ) w O—, —O(CH 2 CH 2 O) w —, —N(H)C(O)(CH 2 ) w O—, —C(O)N(H)(CH 2 ) w O—, —C(o)N(H)(CH 2 CH 2 O) w —, -(CH 2 ) w S—, —O(CH 2 ) w S—, —N(H)C(O)(CH 2 ) w S—,

Assignees

• Ventana Med Syst Inc

Inventors

• Ashworth-Sharpe Julia
• Kelly Brian D
• Lefever Mark
• Polaske Nathan W

Classifications

• C09B23/083

  five >CH- groups · CPC title

• C09B11/24 Primary

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• G01N33/532

  Production of labelled immunochemicals · CPC title

• G01N33/583

  with non-fluorescent dye label · CPC title

• G01N33/582

  with fluorescent label · CPC title