Solid phase fluorescence labeling reagents and uses thereof

Patent metadata

Field	Value
Publication number	US-9896522-B2
Application number	US-201113819668-A
Country	US
Kind code	B2
Filing date	Aug 26, 2011
Priority date	Aug 27, 2010
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

A solid phase reagent and method for simultaneously capturing and fluorescently labeling an analyte with multiple reactive sites to provide a mono-labeled analyte are disclosed. The reagent can be used in the method and comprises a plurality of analyte-reactive groups tethered to a porous solid phase, wherein each analyte-reactive group is covalently attached to a fluorescent group either directly or indirectly, wherein the distance between adjacent analyte-reactive groups is greater than the gyration radius of a captured analyte; the fluorescent group is covalently attached to a cleavable anchor group either directly or through a first spacer; and the cleavable anchor group is covalently attached to the solid phase either directly or through a second spacer, wherein the solid phase is a porous solid or a porous gel.

First claim

The invention claimed is:

1. A solid phase reagent for simultaneously capturing and fluorescently monolabeling an analyte having multiple reactive sites, comprising a solid phase and a plurality of moieties tethered to the solid phase, wherein each moiety of the plurality of moieties comprises:
(a) a single analyte-reactive group for capturing an analyte to provide a captured analyte having a gyration radius,
(b) a single fluorescent group, and
(c) a single cleavable anchor group,
wherein
the analyte-reactive group is covalently attached to the fluorescent group either directly or indirectly, and wherein any two analyte-reactive groups of the plurality of moieties are separated by a distance greater than the gyration radius of the captured analyte;
the fluorescent group is covalently attached to the cleavable anchor group either directly or through a first spacer; and
the cleavable anchor group is covalently attached to the solid phase either directly or through a second spacer, and
wherein the fluorescent group is selected from the group consisting of:

wherein:
R 1 , independently at each occurrence, is hydrogen, alkyl C1-12 , or hetero(backbone)alkyl C1-12 , wherein when substituted, at least one hydrogen atom of the alkyl C1-12 , or hetero(backbone)alkyl C1-12 moiety is replaced with a functional group carrying a net zero, net negative, or net positive charge;
R 2 , independently at each occurrence, is hydrogen, alkyl C1-12 , or hetero(backbone)alkyl C1-12 , wherein when substituted, at least one hydrogen atom of the alkyl C1-12 , or hetero(backbone)alkyl C1-12 moiety is replaced with a functional group carrying a net zero, net negative, or net positive charge; and
R 3 is divalent alkyl C1-12 or divalent hetero(backbone)alkyl C1-12 .

2. The solid phase reagent of claim 1 , wherein the functional group is a carboxylate, a sulfonate, a sulfate, a primary amino group, a secondary amino group, or a tertiary amino group.

3. The solid phase reagent of claim 1 , wherein the analyte-reactive group is an amine-reactive group.

4. The solid phase reagent of claim 1 , wherein the solid phase is selected from the group consisting of a particle-based porous solid phase, a monolith porous solid phase, and a gel.

5. The solid phase reagent of claim 1 , wherein the first spacer, the second spacer, or both comprise an oligo(oxyethylene) group with a mer-number between 1 and 100.

6. The solid phase reagent of claim 1 , wherein the cleavable anchor group is a 1,3-dioxolane selected from the group consisting of

wherein R a is selected from the group consisting of
(a) H and
(b) CH 3 .

7. The solid phase reagent of claim 1 , wherein the solid phase is a monolith solid phase comprising a functional group selected from the group consisting of epoxy, carboxylic acid, hydroxyl, and combinations thereof.

8. The solid phase reagent of claim 1 , wherein the solid phase is a 2-hydroxyethylmethacrylate-based monolith solid phase.

9. A method of simultaneously immobilizing and fluorescently monolabeling an analyte having multiple reactive sites in an analyte-containing solution, comprising:
(a) contacting an analyte having multiple reactive sites with a solid phase reagent of claim 1 ;
(b) reacting the analyte with an analyte-reactive group of the solid phase reagent to provide an immobilized monolabeled analyte;
(c) reacting any analyte-reactive group of the solid phase reagent not reacted with the analyte with a quencher to provide quenched analyte-reactive groups;
(d) releasing the immobilized monolabeled analyte and quenched analyte-reactive groups from the porous solid phase by cleaving the cleavable anchor group to provide a monolabeled analyte; and
(e) isolating or analyzing the monolabeled analyte.

10. The method of claim 9 further comprising analyzing the monolabeled analyte by a method selected from the group consisting of capillary electrophoresis, liquid chromatography, absorption spectroscopy, fluorescence spectroscopy, mass spectrometry, and combinations thereof.

11. The method of claim 9 , wherein the analyte comprises an amino group that reacts with the analyte-reactive group.

12. The method of claim 9 , wherein the analyte is selected from the group consisting of a peptide, a protein, and a carbohydrate.

13. The method of claim 9 , wherein the solid phase is a porous solid phase.

14. The method of claim 9 , wherein the solid phase is a gel.

15. A solid phase reagent for simultaneously capturing and fluorescently monolabeling an analyte having multiple reactive sites, comprising a solid phase and a plurality of moieties tethered to the solid phase, wherein each moiety of the plurality of moieties comprises:
(a) a single analyte-reactive group for capturing an analyte to provide a captured analyte having a gyration radius,
(b) a single fluorescent group, and
(c) a single cleavable anchor group,
wherein
the analyte-reactive group is covalently attached to the fluorescent group either directly or indirectly, and wherein any two analyte-reactive groups of the plurality of moieties are separated by a distance greater than the gyration radius of the captured analyte;
the fluorescent group is covalently attached to the cleavable anchor group either directly or through a first spacer; and
the cleavable anchor group is covalently attached to the solid phase either directly or through a second spacer, and
wherein the fluorescent group is:

16. The solid phase reagent of claim 15 , wherein the analyte-reactive group is an amine-reactive group.

17. The solid phase reagent of claim 15 , wherein the solid phase is selected from the group consisting of a particle-based porous solid phase, a monolith porous solid phase, and a gel.

18. The solid phase reagent of claim 15 , wherein the first spacer, the second spacer, or both comprise an oligo(oxyethylene) group with a mer-number between 1 and 100.

19. The solid phase reagent of claim 15 , wherein the cleavable anchor group is a 1,3-dioxolane selected from the group consisting of

wherein R a is selected from the group consisting of
(a) H and
(b) CH 3 .

20. The solid phase reagent of claim 15 , wherein the solid phase is a monolith solid phase comprising a functional group selected from the group consisting of epoxy, carboxylic acid, hydroxyl, and combinations thereof.

21. The solid phase reagent of claim 15 , wherein the solid phase is a 2-hydroxyethylmethacrylate-based monolith solid phase.

22. A method of simultaneously immobilizing and fluorescently monolabeling an analyte having multiple reactive sites in an analyte-containing solution, comprising:
(a) contacting an analyte having multiple reactive sites with a solid phase reagent of claim 15 ;
(b) reacting the analyte with an analyte-reactive group of the solid phase reagent to provide an immobilized monolabeled analyte;
(c) reacting any analyte-reactive group of the solid phase reagent not reacted with the analyte with a quencher to provide quenched analyte-reactive g

Assignees

• Vigh Gyula
• Estrada Iii Roy T
• Texas A & M Univ Sys

Inventors

• Vigh Gyula
• Estrada Iii Roy T

Classifications

• G01N33/54306

  Solid-phase reaction mechanisms · CPC title

• Y10T436/173845

  Amine and quaternary ammonium · CPC title

• C07C2603/50

  Pyrenes; Hydrogenated pyrenes · CPC title

• C07D317/22

  etherified · CPC title

• G01N33/54393

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title