Spectrofluorometric method and compounds that are of value for the method

• US 5,216,134 A
• Filed: 03/06/1991
• Issued: 06/01/1993
• Est. Priority Date: 10/23/1989
• Status: Expired due to Term

• Current Assignee: Wallac Oy (Revvity, Inc.)
• Original Assignee: Wallac Oy (Revvity, Inc.)
• Inventors: Sund, Christian, Mukkala, Veli-Matti, Kwiatkowski, Marek
• Primary Examiner(s): Rizzo, Nicholas S.
• Assistant Examiner(s): Grumbling, Matthew V.
• Application Number:
  US07/665,279
• Time in Patent Office: 818 Days
• Field of Search: 546/257, 546/258, 546/2, 546/5, 546/6, 544/4, 530/391.3, 436/546, 534/15, 534/16
• US Class Current: 534/15
• CPC Class Codes: C07D 213/22 containing two or more pyridine rings directly linked together, e.g. bipyridyl
• CPC Class Codes: C07D 213/38 having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
• CPC Class Codes: C07D 213/53 Nitrogen atoms
• CPC Class Codes: G01N 2458/40 Rare earth chelates
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

A chelate formed between Eu³⁺, Tb³⁺, Dy³⁺ and Sm³⁺ and a compound having the formula ##STR1## where A_1-6 are single carbon or nitrogen atoms; n=1 or 2; R_1-6 is nothing when A is a nitrogen, and hydrogen or an organic group when A is a carbon; Z and Z'"'"' are selected among --N(CH₂ CH₂ COO^-)₂, --N(CH₂ COO^-)₂, --N(CH₂ OPO₃^2-)₂ and --N(CH₂ PO₃^2-)₂ ;--species that --X--Y is a substituent replacing a hydrogen anywhere in the parent compound; and --X--Y represents an organic group containing no chelating heteroatom closer than four atoms from a chelating heteroatom in the parent compound and X is a stable bridge containing certain groups and Y is selected from (a) specified relative groups allowing coupling to other compounds and (b) residues of compounds participating in biospecific affinity reactions.

Claims

1. A chelate formed between Eu³⁺, Tb³⁺, Dy³⁺ and Sm³⁺ and a compound having the formula

where

i A₁ -A₆ are single atoms selected from the group consisting of a carbon atom and a nitrogen atom,

ii n is 1 or 2,

iii each of R₁ -R₆ is nothing when the A to which it is attached is nitrogen;

and for each A that is a carbon atom R₁ -R₆ are selected from the group consisting of

(a) hydrogen, hydrocarbon group, cyano, halo, nitro and

(b) carboxy (--COOH), amido (--CONH₂), amino (--NH₂), hydroxy (--OH) and substituted forms of these four groups in which hydrogen is replaced with a hydrocarbon group according to (a) and for amino and hydroxy the hydrogens also have the possibility of being replaced with an acyl group (R--CO--) where R is a hydrocarbon group according to (a),

iv Z and Z'"'"' represent identical or different chelating structures selected from the group consisting of N-biscarboxyethyl amino, N-biscarboxymethyl amino and the analogous phosphate (--N(--CH₂ --O--PO₃^2-)₂ and phosphonate --N(--CH₂ --PO₃^2-)₂, respectively),

v B is selected from the group consisting of a direct link, --NH--, --CO-- and --O--,

vi--specifies that the group --X--Y is substituent replacing a hydrogen anywhere in the parent compound,

vii X--Y represents an organic group in which X is stable in the sense that does not deteriorate when the compound is used and X--Y has no chelating heteroatom closer than four atoms from a chelating heteroatom in the parent compound of formula (IV), and Y is

(a) a functional group selected among isothiocyanato, bromoacetamido, iodoaceteamido, succinimido, pyridyldithio, mercapto, carboxyl and its active esters, hydroxyl, aldehyde, amino, diazonium, tosyl, mesytylyl, trexyl, phosphodiester or phosphotriester, or

(b) a residue of a biologically active molecule having the ability to participate in biospecific affinity reactions, and

X consists of groups selected among --NR-- (secondary or tertiary amine), --CONR-- and --RNCO-- (substituted amide), --S--S-- (aliphatic disulfide), --S-- (aliphatic thioether), --O-- (ether), --COO-- and --OOC-- (ester), --N═

N-- (diaza) and pure hydrocarbon chain containing from 1 to 12 carbon atoms, in which groups R is an alkyl group having less than 5 carbon atoms, with the proviso that, when XY is attached to the heterocyclic rings of formula IV, X has a methylene group (--CH₂ --).

2. The chelate of claim 1 wherein Z and Z'"'"' are identical.

3. The chelate of claim 2 wherein each of Z and Z'"'"' are N-biscarboxymethyl.

4. The chelate of claim 1 wherein n=1, and X--Y replaces one of R₁ -R₆, while the A to which X--Y is bound is carbon.

5. The chelate of claim 1 wherein X--Y replaces a hydrogen in at least one of Z and Z'"'"'.

6. The chelate of claim 1 wherein Y is a residue of a residue of a biologically active molecule having the ability to participate in biospecific affinity reactions and selected from the group consisting of antigens, haptens and antibody active components and nucleic acids.

7. The chelate of claim 1 wherein Y is a functional group selected among isothiocyanato, bromoacetamido, iodoacetamido, succinimido, pyridyldithio, mercapto, carboxyl and its active esters, hydroxyl aldehyde, amino, diazonium, tosyl, mesytylyl, trxyl, phosphodiester or phosophotriester.

8. The chelate according to claim 1 wherein only one of A₁ -A₆ is nitrogen.

9. The chelate according to claim 1 wherein only one of A₁ -A₃ is nitrogen and only one of A₄ -A₆ is nitrogen.

10. A chelate of 4-ethoxy-6-(N-(carboxymethyl)-N-(1-(5-p-isothiocyanatobenzamido))-1-carboxypentyl)aminomethyl)-6'"'"'-(N,N-bis(carboxymethyl)aminomethyl)-2-2'"'"'-bipyridine that is bound to an antibody via its p-isothiocyanato group wherein the chelated metal ion is selected from Eu³⁺ and Tb³⁺.

11. A chelate of 6-(N-(carboxymethyl)-N-(1-(5-p-isothiocyanatobenzamido))-1-carboxypentyl)aminomethyl)-6'"'"'-(N,N-bis(carboxymethyl)aminomethyl)-2-2'"'"'-dipyridyl ketone that is bound to an antibody via its p-isothiocyanato group wherein the chelated metal ion selected from Eu³⁺ and Tb³⁺.