Polyfluoreno[4,5-cde]oxepine conjugates and their use in methods of analyte detection

Patent metadata

Field	Value
Publication number	US-10316136-B2
Application number	US-201815860965-A
Country	US
Kind code	B2
Filing date	Jan 3, 2018
Priority date	Mar 28, 2016
Publication date	Jun 11, 2019
Grant date	Jun 11, 2019

Abstract

The invention provides for polyfluoreno[4,5-cde]oxepine conjugates and their use in methods of analyte detection.

First claim

What is claimed is:

1. A method of detecting an analyte in a sample, comprising
a) combining said sample with a detection reagent comprising a polymer under conditions under which said detection reagent will bind said analyte; and
b) detecting the detection reagent bound analyte by fluorescence
wherein the polymer comprises
monomer of formula A

wherein X is the number of monomer A units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein X is from 6 to 100; and one or more monomers selected from the group consisting of
monomer of formula B

wherein Y is the number of monomer B units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein Y is from 0 to 99, and
monomer of formula C

wherein Z is the number of monomer C units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein Z is from 0 to 99;
wherein R1 to R6 independently is hydrogen, an alkyl, a polyethyleneglycol (PEG), an aryl, a heteroaryl group, or a biological substrate conjugated via a linker (L-BS);
wherein SG1 to SG6 independently is an alkyl, a water soluble group or a L-BS; and
wherein the polymer includes end groups HG1 and HG2 wherein HG1 and HG2 independently is a hydrogen, an alkyl, a halogen, a boronyl, an aryl, a heteroaryl group or a L-BS;
wherein the ratio of X to Y+Z is >1,
wherein the sum of X+Y+Z is >10; wherein the polymer comprises at least one L-BS wherein L is a linker and BS is a biological substrate wherein the biological substrate is a complementary binding member capable of specifically binding to the analyte to be detected.

2. The method of claim 1 , wherein BS is an antibody.

3. The method of claim 1 , wherein BS is an anti-digoxigenin antibody, a goat anti-mouse IgG antibody, goat anti-rabbit IgG antibody, goat anti-human IgG antibody, donkey anti-mouse IgG antibody, donkey anti-rabbit IgG antibody, donkey anti-human IgG antibody, chicken anti-mouse IgG antibody, chicken anti-rabbit IgG antibody, or chicken anti-human IgG antibody.

4. The method of claim 1 , wherein BS is an avidin, streptavidin, neutravidin, avidinDN, or avidinD moiety.

5. The method according to claim 1 , wherein the analyte is a target protein expressed on a cell surface.

6. The method of claim 1 wherein monomer unit B and monomer unit C are different.

7. The method of claim 1 , wherein the monomer units are directly connected to one another.

8. The method of claim 1 , wherein the linker comprises an alkyl, a PEG, a carboxamide, a thioether, an ester, an imine, a hydrazine, an oxime, an alkyl amine, an ether, an aryl amine, a boronate ester, an N-acylurea or anhydride, a platinum complex, an aminotriazine, a triazinyl ether, an amidine, a urea, a urethane, a thiourea, a phosphite ester, a silyl ether, a sulfonamides, a sulfonate ester, a 1,2,3-triazole, a pyradazine, a thiazolidine, a 2-diphenylphosphonyl-benzoamide, an isoxazole or a succinimide group.

9. The method of claim 1 wherein:
(i) R1 to R6 independently represent hydrogen, methyl, or ethyl; and/or
(ii) SG1 to SG6 independently represent a PEG, an alkyl, a carboxyalkyl, a sulfonylalkyl, a phosphonylalkyl, an aminoalkyl or L-BS; and/or
(iii) L is an alkyl chain or a PEG chain; and/or
(iv) BS is an antibody, a peptide, a protein, an oligonucleotide, a nucleic acid or a carbohydrate; and/or
(v) HG1 and HG2 independently represent a hydrogen, an aryl, a halogen or a boronyl; and/or
(vi) x is an integer from 11-80 and y and z are each an integer independently selected from 0-80, provided that (1) the ratio of BS/polymer is 1-2, (2) the ratio of x/(y+z) is >1, and (3) the sum of x+y+z is >20.

10. The method of claim 1 wherein:
(i) R1 to R6 are hydrogen; and/or
(ii) SG1 and SG2 are PEG; and/or
(iii) SG3 to SG6 independently represent a PEG, an alkyl, a carboxyalkyl, a sulfonylalkyl, a phosphonylalkyl, an aminoalkyl or a L-BS.

11. The method of claim 1 wherein:
(i) SG3 to SG6 independently represent a PEG, an alkyl, a carboxyalkyl, or a L-BS; or
(ii) SG3 to SG6 independently represent a PEG, an alkyl, an aminoalkyl or a L-BS.

12. The method of claim 11 wherein alkyl is a methyl group.

13. The method of claim 1 wherein R1 to R6 are hydrogen; wherein SG1 to SG6 independently represent a PEG, an alkyl, a carboxyalkyl, a sulfonylalkyl, a phosphonylalkyl, or an aminoalkyl; wherein HG1 and HG2 independently represent a hydrogen, an aryl, a halogen, a boronyl or a L-BS; wherein L comprises an alkyl chain or a PEG chain; wherein BS is an antibody, a peptide, a protein, an oligonucleotide, a nucleic acid or a carbohydrate; and wherein x is an integer from 16-80 and y and z are each an integer independently selected from 0-80, provided that (1) the ratio of BS/polymer is 1-2, (2) the ratio of x/(y+z) is >1, and (3) the sum of x+y+z is >20.

14. The method of claim 1 wherein SG1 and SG2 are independently PEG6 to PEG18.

15. The method of claim 1 wherein the ratio of BS/polymer is 1; x is an integer from 16-80, y and z are each an integer independently selected from 0-64 and wherein the sum of x+y+z is 30-80.

16. The method of claim 1 wherein:
(i) HG1 and HG2 independently represent a hydrogen, a carboxyaryl, or a L-BS; or
(ii) HG1 and HG2 independently represent a halogen, a boronyl, a carboxyaryl, or a L-BS.

17. A method of detecting an analyte in a sample, comprising
a) combining said sample with a detection reagent comprising a polymer under conditions under which said detection reagent will bind said analyte; and
b) detecting the detection reagent bound analyte by fluorescence,
wherein the polymer comprises
monomer of formula D

wherein W is the number of monomer D units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein W is from 6 to 100; and one or more monomers selected from the group consisting of
monomer of formula E

wherein X is the number of monomer E units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein X is from 0 to 98,
monomer of formula F

wherein Y is the number of monomer F units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein Y is from 1 to 20, and
monomer of formula G

wherein Z is the number of monomer G units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein Z is from 0 to 98; and
wherein R1 to R6 independently is hydrogen, an alkyl, a polyethyleneglycol (PEG), an aryl, a heteroaryl group, or a biological substrate conjugated via a linker (L-BS);

Assignees

• Aat Bioquest Inc
• Affymetrix Inc

Inventors

• Diwu Zhenjun
• Guo Haitao
• Peng Ruogu
• Zhao Qin
• Jennings Travis
• Funatake Castle

Classifications

• C09K2211/145

  containing oxygen as the only heteroatom · CPC title

• C08G2261/94

  Applications in sensors, e.g. biosensors · CPC title

• C08G61/12 Primary

  Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule · CPC title

• C08G2261/344

  containing heteroatoms · CPC title

• C08G2261/3142

  fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene · CPC title