Electrophoretic tag reagents comprising fluorescent compounds

• US 6,673,550 B2
• Filed: 11/09/2001
• Issued: 01/06/2004
• Est. Priority Date: 04/30/1999
• Status: Expired due to Term

• Current Assignee: Virologic, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings)
• Inventors: Hernandez, Vincent, Matray, Tracy, Singh, Sharat
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US10/008,495
• Publication Number:
  US 20020146726A1
• Time in Patent Office: 788 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/25.32
• US Class Current: 435/6.13
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/16 involving encoding steps
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Electrophoretic probes comprising fluorescent compounds as detection groups and mobility modifiers are disclosed for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. In one embodiment, detection involves the release of identifying tags as a consequence of target recognition. Target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters. Typically, uncleaved or partially cleaved e-tag probes are removed and the mixture of e-tag reporters is separated by any technique that provides for separation by mass or mass to charge ratio and the like and detected to provide for target identification.

Claims

1. A probe set for detecting the presence or absence of one or more target compounds, the probe set comprising a plurality of electrophoretic probes selected from the group defined by the formula:

2. The probe set of claim 1 wherein said cleavable linkage is selected from the group consisting of olefins, thioethers, sulfoxides, and selenium analogs of thioethers or sulfoxides.

3. The probe set of claim 2 wherein said active species is selected from the group consisting of singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals.

4. The probe set of claim 3 wherein said target-binding moiety is a monoclonal antibody or a polyclonal antibody.

5. The probe set of claim 4 wherein k is in the range of from 1 to 20.

6. The probe set according to claim 1, 2, 3, 4 or 5 wherein said plurality is in the range of from 5 to 100.

7. The probe set according to claim 6 wherein each said detection group, D, of each electrophoretic probe has substantially the same spectral properties.

8. The probe set of claim 6 wherein said fluorescent molecule is selected from a group defined by the formula:

wherein;

Z′

is COOH,

A′

is O,

D′

is OH, OR^3′

wherein R^3′

is lower alkyl, substituted lower alkyl, aryl, or substituted aryl,

W^1′

, W^2′

, W^3′

, W^4′

and W^6′

are independently H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromide, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, or imino,

W^5′

is H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, or imino,

X^1′

-X^{4 ′}

are independently H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, or imino,

wherein W^2′

and W^3′

may be taken together to form one or more rings comprising 4 to 14 atoms and comprising 1 to 7 unsaturations, and

wherein W^4′

and W^5′

may be taken together to form a ring comprising 4 to 14 atoms and comprising 1 to 7 unsaturations.

9. The probe set of claim 6 wherein said fluorescent molecule is selected from the group defined by the formula:

wherein

Z″

is COOH,

A″

is N(R^1″

)(R^2″

) wherein R^1″

and R^2″

are independently lower alkyl, or substituted lower alkyl,

D″

is OH, OR^3′

wherein R^3′

is lower alkyl, substituted lower alkyl, aryl, or substituted aryl,

W^1″

and W^6″

are independently H, lower alkyl, substituted lower alkyl, COOH, chloro, or fluoro,

W^2″

and W^5″

are independently H, lower alkyl, substituted lower alkyl, COOH, chloro, or fluoro,

W^3″

and W^4″

are independently H, lower alkyl, substituted lower alkyl, COOH, chloro, or fluoro,

wherein W^2″

and W^3″

may be taken together to form one or more rings comprising 4 to 14 atoms and comprising 1 to 7 unsaturations, and

wherein W^4″

and W^5″

may be taken together to form a ring comprising 4 to 14 atoms and comprising 1 to 7 unsaturations,

X^1″

-X^4″

are independently H, chloro, fluoro, COOH, bromo, or iodo.

10. The probe set of claim 9 wherein Z″

is carboxyl, W^6″

and W^1″

are lower alkyl, W^5″

and W^2″

are halogen, X^2″

and X^3″

are hydrogen or carboxyl and X^1″

and X^4″

are hydrogen or halogen.

11. The probe set of claim 9 wherein Z″

is carboxyl, W^6″

and W^1′

are methyl, W^5″

and W^2″

are chloro, one of X^2″

and X^3″

are hydrogen and the other is carboxyl and X^1″

and X^4″

are hydrogen.

12. The probe set of claim 9 wherein Z″

is carboxyl, W^6″

and W^1″

are methyl, W^5″

and W^2″

are chloro, one of X^2″

and X^3″

are hydrogen and the other is carboxyl and X^1″

and X^4″

are chloro.

13. The probe set of claim 1 wherein k is in the range of from 1 to 20.

14. The probe set according to claim 13 wherein said plurality is in the range of from 5 to 100.

15. The probe set according to claim 14 wherein each said detection group, D, of each electrophoretic probe has substantially the same spectral properties.

16. The probe set of claim 15 wherein said fluorescent molecule is selected from a group defined by the formula:

wherein;

Z′

is COOH,

A′

is O,

D′

is OH, OR^3′

wherein R^3′

is lower alkyl, substituted lower alkyl, aryl, or substituted aryl,

W^1″

, W^2′

, W^3′

, W^4′

and W^6′

are independently H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, or imino,

W^5′

is H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, or imino,

X^1′

-X^4′

are independently H, lower alkyl, substituted lower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, cycloalkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aromatic, substituted aromatic, phenyl, substituted phenyl, polycyclic aromatic, substituted polycyclic aromatic, heterocyclic, substituted heterocyclic, chlorine, fluorine, bromine, iodine, COOH, carboxylate, amide, nitrile, nitro, sulfonyl, sulfate, sulfone, amino, tethered amino, quaternary amino, or imino,

wherein W^2′

and W^3′

may be taken together to form one or more rings comprising 4 to 14 atoms and comprising 1 to 7 unsaturations, and

wherein W^4′

and W^5′

may be taken together to form a ring comprising 4 to 14 atoms and comprising 1 to 7 unsaturations.

17. The probe set of claim 15 wherein said fluorescent molecule is selected from the group defined by the formula:

wherein

Z″

is COOH,

A″

is O, N(R^1″

)(R^2″

) wherein R^1″

and R^2″

are independently lower alkyl, or substituted lower alkyl,

D″

is OH, OR^3″

wherein R^3″

is lower alkyl, substituted lower alkyl, aryl, or substituted aryl,

W^1″

and W^6″

are independently H, lower alkyl, substituted lower alkyl, COOH, chloro, or fluoro,

W^2″

and W^5″

are independently H, lower alkyl, substituted lower alkyl, COOH, chloro, or fluoro,

W^3″

and W^4″

are independently H, lower alkyl, substituted lower alkyl, COOH, chloro, or fluoro,

wherein W^2″

and W^3″

may be taken together to form one or more rings comprising 4 to 14 atoms and comprising 1 to 7 unsaturations, and

wherein W^4″

and W^5″

may be taken together to form a ring comprising 4 to 14 atoms and comprising 1 to 7 unsaturations,

X^1″

-X^4″

are independently H, chloro, fluoro, COOH, bromo, or iodo.

18. The probe set of claim 17 wherein Z″

is carboxyl, W^6″

and W^1″

are lower alkyl, W^5″

and W^2″

are halogen, X^2″

and X^3″

are hydrogen or carboxyl and X^1″

and X^4″

are hydrogen or halogen.

19. The probe set of claim 17 wherein Z″

is carboxyl, W^6″

and W^1″

are methyl, W^5″

and W^2″

are chloro, one of X^2″

and are hydrogen and the other is carboxyl and X^1″

and X^4″

are hydrogen.

20. The probe set of claim 17 wherein Z″

is carboxyl, W^6″

and W^1″

are methyl, W^5″

and W^2″

are chloro, one of X^2″

and X^3″

are hydrogen and the other is carboxyl and X^1″

and X^4″

are chloro.