Method of chemical analysis employing molecular release tag compounds

• US 4,650,750 A
• Filed: 03/19/1984
• Issued: 03/17/1987
• Est. Priority Date: 02/01/1982
• Status: Expired due to Term

• Current Assignee: Roger W Giese
• Original Assignee: Roger W Giese
• Inventors: Giese, Roger W.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Teskin, Robin Lyn
• Application Number:
  US06/591,262
• Time in Patent Office: 1,093 Days
• Field of Search: 435/7, 435/188, 435/175, 435/181, 435/183, 424/177, 260/112 B, 260/112 R, 436/2, 436/56, 436/501
• US Class Current: 435/7.72
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

A method of chemical analysis is disclosed which employs a release tag compound of general formula

Rx--Re--S

in which Rx is a reactivity group capable of forming a covalent bond with another molecule, Re is a release group capable of being cleaved, and S is a signal group. The release tag is covalently bonded to a substance of interest which is to be determined in the course of a chemical analysis for an analyte, release group Re is cleaved at an appropriate point in the analytical procedure, and signal group S is determined, thereby determining the substance of interest. Where the substance of interest is the analyte, determination of S determines the analyte. Where the substance of interest is not the analyte but is related to the analyte concentration, determination of S allows indirect determination of analyte.

Claims

1. In an assay which includes contacting an analyte-containing sample with a labeled reagent, or with a conjugate made by covalently reacting a labeled reagent with an analog of, ligand for, or specific binding partner of said analyte, incubating, and measuring the amount of label, the improvement which comprises:

using as the labeled regent a release tag compound, S-Re-Rx, wherein S is a signal group covalently bound to group Re, and Re is a cleavable release group which is covalently bound to group Rx which is a reactivity group;

and S is further defined as comprising a halogenated electron-absorbing group which can be measured by electron capture detection;

and Re is further defined as comprising a functionality selected from the group consisting of methionylamides, vicinal glycols, olefins, β

-phenylethylamine oxides and benzyl ethers, and is capable of releasing the signal group in a volatile form suitable for measuring by electron capture detection;

and Rx is further defined as comprising a functional group which is capable of forming a covalent bond with the analyte to be detected, or with an analog of, ligand for, or specific binding partner of said analyte.

2. The method of claim 1 wherein the signal group S is selected from the group consisting of pentafluorobenzoyl, heptafluorobutyryl, p-(pentafluorophenoxy)-2,3,5,6-tetrafluorobenzoyl, and pentachlorophenyl.

3. The method of claim 1 wherein the release group Re is selected from the group consisting of methionyl-glycinyl, α

-methyl methionyl-glycinyl, methionylsulfoxide-glycinyl, and 6-amino-4-methyl-3,4-dihydroxyhexanoyl functionalities.

4. The method of claim 1 wherein the reactivity group Rx is selected from the group consisting of N-hydroxysuccinimide ester, p-nitrophenyl ester, phenylisothiocyanate, and phenylsulfonyl chloride.

5. The method of claim 1 wherein the reactivity group Rx is selected from the group consisting of silyl halides, sulfonyl halides, acid halides, acid anhydrides, α

-halo ketones, diones, maleimides, diazonium salts, imidoesters, aldehydes, halogenated nitrophenyls, arylazides, isothiocyanates, epoxides, carbenes, nitrenes, sulfenyl halides, amines, and hydrazides.

6. The method of claim 1 wherein the release tag compound is selected from the group consisting of:

(a) N-pentafluorobenzoyl-α

-methyl-methionyl-glycine-N-hydroxysuccinimide ester;

(b) N-pentafluorobenzoyl-methionyl sulfoxide-glycine-N-hydroxysuccinimide ester;

p1 (c) N-pentafluorobenzoyl-6-amino-4-methyl-3,4 dihydroxy hexanoic acid-N-hydroxysuccinimide ester;

(d) p-(4-pentachlorophenoxy-benzyloxy)-phenylsulfonyl chloride.

7. The method of claim 1 wherein the release tag compound is N-pentafluorobenzoyl-methionyl-glycine-N-hydroxysuccinimide ester, having the formula ##STR4##

8. The method of claim 1 wherein the released signal group S is determined by means of gas chromatography with electron capture detection.

9. In an assay which includes contacting an analyte-containing sample with a labeled reagent, or with a conjugate made by covalently reacting a labeled reagent with an analog of, ligand for, or specific binding partner of said analyte, incubating, and measuring the amount of label, the improvement which comprises:

using as the labeled reagent a release tag compound, S-Re-Rx, wherein S is a signal group covalently bound to group Re, and Re is a cleavable release group which is covalently bound to group Rx which is a reactivity group;

and S is further defined as comprising a halogenated electron-absorbing group which can be measured by electron capture detection;

and Re is further defined as comprising a functionality selected from the group consisting of disulfides, tryptophan, tyrosine, thioesters, azo compounds, p-toluenesulfonic esters of β

,γ

-acetylenic alcohols, alkyl phenyl ethers, hydrazones, thioethers, benzoyloxycarbonylamines, alkylsulfonylethoxycarbonylamines, arylsulfonylethoxycarbonylamines, alkylthioethyloxycarbonylamines, arlythioethyloxycarbonylamines, tosylamines, S-benzylethers, o-nitrobenzylamides, 2-nitro-4,5-dimethoxy-benzyloxycarbonylamines, amine oxides, and xanthates, and is capable of releasing the signal group in a volatile form suitable for measuring by electron capture detection;

and Rx is further defined as comprising a functional group which is capable of forming a covalent bond with the analyte to be detected, or with an analog of, ligand for, or specific binding partner of said analyte.

10. The method of claim 9 wherein the signal group S is selected from the group consisting of pentafluorobenzoyl, heptafluorobutyryl, p-(pentafluorophenoxy)-2,3,5,6-tetrafluorobenzoyl, and pentachlorophenyl.

11. The method of claim 9 wherein the reactivity group Rx is selected from the group consisting of N-hydroxysuccinimide ester, p-nitrophenyl ester, phenylisothiocyanate, and phenylsulfonyl chloride.

12. The method of claim 9 wherein the reactivity group Rx is selected from the group consisting of silyl halides, sulfonyl halides, acid halides, acid anhydrides, α

-halo ketones, diones, maleimides, diazonium slats, imidoesters, aldehydes, halogenated nitrophenyls, arylazides, isothiocyanates, epoxides, carbenes, nitrenes, sulfenyl halides, amines, and hydrazides.

13. The method of claim 9 wherein the released signal group S is determined by means of gas chromatography with electron capture detection.

14. In an assay which includes contacting an analyte-containing sample with a labeled reagent, or with a conjugate made by covalently reacting a labeled reagent with an analog of, ligand for, or specific binding partner of said analyte, incubating, and measuring the amount of label, the improvement which comprises:

using as the labeled reagent a release tag compound, S-Re-Rx, wherein S is a signal group covalently bound to group Re, and Re is a cleavable release group which is covalently bound to group Rx which is a reactivity group;

and S is further defined as comprising a halogenated electron-absorbing group which can be measured by electron capture detection;

and Re is further defined as comprising a functionality selected from the group consisting of methionylamides, vicinal glycols, olefins, γ

-phenylethylamine oxides and benzyl ethers, and is capable of releasing the signal group in a volatile form suitable for measuring by electron capture detection;

and Rx is further defined as comprising a functional group which is capable of forming a covalent bond with the analyte to be detected, or with an analog of, ligand for, or specific binding partner of said analyte;

cleaving said release group to free said signal group; and

measuring said free signal group.