Molecular analytical release tags and their use in chemical analysis

• US 4,709,016 A
• Filed: 02/01/1982
• Issued: 11/24/1987
• Est. Priority Date: 02/01/1982
• Status: Expired due to Term

• Current Assignee: Northeastern University
• Original Assignee: Northeastern University
• Inventors: Giese, Roger W.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/344,394
• Time in Patent Office: 2,122 Days
• Field of Search: 260/112.5, 260/543 R, 424/177, 424/94, 424/1, 424/12, 436/2, 436/536, 436/501, 436/547, 435/7, 435/188, 435/12, 435/424, 435/260, 435/175, 435/181, 435/183, 548/542, 530/389, 530/390, 530/391, 536/27
• US Class Current: 530/391.5
• 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 new class of chemical reagents called release tags which comprise signal, release and reactivity groups is disclosed and a release tag involving a pentafluorobenzoyl signal group, a methionylamide release group, and an active ester reactivity group is used to analyze the hormone, thyroxine, in serum, involving quantitation of the released signal group by gas chromatography with electron capture detection.

Claims

1. A molecular conjugate, comprising:

a release tag compound covalently bound to an analyte to be analytically determined or to a specific binding partner thereof;

said specific binding partner being selected from the group consisting of antibodies for the analyte, and DNA probes;

said release tag compound comprising;

a signal group S, a release group Re, and a reactivity group Rx, group S being covalently linked to group Re, and group being further covalently linked to group Rx to define structure S--Re--Rx for said compound;

Rx further comprising a functional group capable of forming a covalent bond with another molecule, said functional group being 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;

Re further comprising a functionality selected from the group consisting of methionylamides, vicinyl glycols, olefins, β

-phenylethylamine oxides, and benzyl ethers; and

S further comprising a halogenated electron-absorbing organic group;

groups S and Re being mutually selected such that upon cleavage of said release tag at release group Re, signal group S is released in a volatile form suitable for electron capture determination in the gas phase.

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

4. The molecular conjugate 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.

3. The molecular conjugate of claim 34 wherein the release group Re is selected from the group consisting of methionyl-glycyl, α

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

5. A release tag compound, 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;

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

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

6. A release tag compound, comprising:

N-pentafluorobenzoyl-methionyl-glycine-N-hydroxysuccinimide ester, having the formula ##STR4##

7. A molecular conjugate, comprising:

a release tag compound covalently bound to an analyte to be analytically determined or to a specific binding partner thereof;

said specific binding partner being selected from the group consisting of antibodies for the analyte, and DNA probes;

said release tag compound comprising;

a signal group S, a release group Re, and a reactivity group Rx, group S being covalently linked to group Re, and group Re being further covalently linked to group Rx to define structure S--Re--Rx for said compound;

Rx further comprising a functional group capable of forming a covalent bond with another molecule;

said functional groups being 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;

Re further comprising a functionality selected from the group consisting of disulfides, tryptophan, tyrosine, thioesters, azo compounds, p-toluensulfonic esters of β

, γ

-acetylenic alcohols, alkyl phenyl ethers, hydrazones, thioethers, benzoyloxycarbonylamines, alkylsulfonylethoxycarbonylamines, arylsufonylethoxycarbonylamines, alkylthioethyloxycarbonylamines, arylthioethyloxycarbonylamines, tosylamines, S-benzylethers, o-nitrobenzylamides, 2-nitro- 4,5-dimethoxy-benzyloxycarbonylamnes, amine oxides, and xanthates; and

S further comprising a halogenated electron-absorbing organic group;

group S and Re being mutually selected such that upon cleavage of said release tag at release group Re, signal group S is releases in a volatile form suitable for electron capture determination in the gas phase.

8. The molecular conjugate of claim 7 wherein the signal group S is selected from the group consisting of pentafluorobenzoyl, heptafluorobutyryl, p-(pentafluorophenoxy)-2,3,5,6-tetrafluorobenzoyl, and pentachlorophenyl.

9. The molecular conjugate of claim 7 wherein the release group Re is selected from the group consisting of methionyl-glycyl, α

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

10. The molecular conjugate of claim 7 wherein the reactivity group Rx is selected from the group consisting of N-hydroxysuccinimide ester, p-nitrophenyl ester, phenylisothiocyanate, and phenylsulfonyl chloride.