Flavin adenine dinucleotide-labeled conjugates for use in specific binding assays

Claim 2

In a labeled conjugate for use in specific binding assays of the general formula: labeling substance(NH--CO)L wherein --CO)L is a hapten of molecular weight greater than 100 or an antigenic polypeptide or protein bound to said labeling substance through an amide bond, characterized in that said labeling substance (NH-- has the formula: ##STR29## wherein Riboflavin--Phos) 2 Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide and n=2 through 6.

Claim 3

The labeled conjugate of claim 1 wherein --CO)L is an antigenic polypeptide or protein of molecular between 1,000 and 4,000,000.

Claim 4

The labeled conjugate of claim 2 wherein said antigenic polypeptide or protein is an antibody.

Claim 5

The labeled conjugate of claim 1 wherein --CO)L is a hapten of molecular weight between 100 and 1,500.

Claim 6

The labeled conjugate of claim 1 wherein --CO)L is an iodothyronine hormone.

Claim 7

The labeled conjugate of claim 5 wherein said iodothyronine hormone is thyroxine.

Claim 8

The labeled conjugate of any of claims 1-4 wherein n=2.

Claim 9

The labeled conjugate of any of claims 1-4 wherein n=6.

Claim 10

A flavin adenine dinucleotide-iodothyronine conjugate of the formula: ##STR30## wherein Riboflavin--(Phos) 2 --Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide, n=2 through 6, and β 1 and β 2 are, independently, hydrogen or iodine.

Claim 11

A flavin adenine dinucleotide-iodothyronine conjugate of the formula: ##STR30## wherein Riboflavin--(Phos) 2 --Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide, n=2 through 6, and β 1 and β 2 are, independently, hydrogen or iodine.

Claim 12

The conjugate of claim 9 wherein n=2.

Claim 13

The conjugate of claim 9 wherein n=6.

Claim 14

The conjugate of any of claims 9-11 wherein β 1 and β 2 are both iodine.

Claim 15

The conjugate of any of claims 9-11 wherein β 1 is iodine and β 2 is hydrogen.

Claim 16

The conjugate of any of claims 9-11 wherein β 1 is hydrogen and β 2 is iodine.

Claim 17

A flavin adenine dinucleotide-iodothyronine conjugate of the formula: ##STR31## wherein Riboflavin--Phos) 2 --Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide, n=2 through 6, Y is trifluoroacetyl, and β 1 and β 2 are, independently, hydrogen or iodine.

Claim 18

A flavin adenine dinucleotide-iodothyronine conjugate of the formula: ##STR31## wherein Riboflavin--Phos) 2 --Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide, n=2 through 6, Y is trifluoroacetyl, and β 1 and β 2 are, independently, hydrogen or iodine.

Claim 19

The conjugate of claim 15 wherein n=2.

Claim 20

The conjugate of claim 15 wherein n=6.

Claim 21

The conjugate of any of claims 15-17 wherein β 1 and β 2 are both iodine.

Claim 22

The conjugate of any of claims 15-17 wherein β 1 is iodine and β 2 is hydrogen.

Claim 23

The conjugate of any of claims 15-17 wherein β 1 is hydrogen and β 2 is iodine.

Claim 24

A compound of the formula ##STR32## wherein --CO)L is a hapten of molecular weight greater than 100 or an antigenic polypeptide or protein bound through an amide bond; n=2 through 6; R 1 is --OH or ##STR33## when R 2 and R 3 together form the group ##STR34## or R 1 is ##STR35## when R 2 and R 3 are --OH.

Claim 25

A compound of the formula ##STR32## wherein --CO)L is a hapten of molecular weight greater than 100 or an antigenic polypeptide or protein bound through an amide bond; n=2 through 6; R 1 is --OH or ##STR33## when R 2 and R 3 together form the group ##STR34## or R 1 is ##STR35## when R 2 and R 3 are --OH.

Claim 26

The compound of claim 21 wherein --CO)L is an antigenic polypeptide or protein of molecular weight between 1,000 and 4,000,000.

Claim 27

The compound of claim 22 wherein said antigenic polypeptide or protein is an antibody.

Claim 28

The compound of claim 21 wherein --CO)L is a hapten of molecular weight between 100 and 1,500.

Claim 29

The compound of claim 21 wherein --CO)L is an iodothyronine hormone.

Claim 30

The compound of claim 25 wherein said hormone is thyroxine.

Claim 31

The compound of any of claims 21 or 22-25 wherein n=2.

Claim 32

The compound of any of claims 21 or 22-25 wherein n=6.

Claim 33

The compound of claim 21 wherein --CO)L is ##STR36## wherein Y is trifluoroacetyl and β 1 and β 2 are, independently, hydrogen or iodine.

Claim 34

The compound of claim 29 wherein n=2.

Claim 35

The compound of claim 29 wherein n=6.

Claim 36

The compound of any of claims 29-31 wherein both β 1 and β 2 are iodine.

Claim 37

A method for preparing a conjugate of claim 1, which method comprises the steps of: (a) reacting 6-chloro-9-(2',3'-O-isopropylidine-β-D-ribofuranosyl)purine with an α,ω-diaminopropane, selected from 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, and 1,6-diaminohexane to yield a 6-(ω-aminoalkyl)-9-(2',3'-O-isopropylidine-β-D-ribofuranosyl)purine; (b) coupling a ligand to be labeled selected from a hapten of molecular weight greater than 100 and an antigenic polypeptide or protein to the primary amino group in said 6-(ω-aminoalkyl)-9-(2',3'-O-isopropylidine-β-D-ribofuranosyl)purine by formation of an amide bond to yield a substituted adenosine intermediate; (c) treating said substituted adenosine intermediate with phosphorous oxychloride to yield a phosphorylated intermediate; (d) hydrolyzing said phosphorylated intermediate to remove the isopropylidine group therefrom yielding a 5'-adenylic acid intermediate; and (e) condensing riboflavin-5'-monophosphate with the phosphorimidazolidate formed by treatment of said 5'-adenylic acid intermediate with N,N'-carbonyldiimidazole to yield the desired flavin adenine dinucleotide-labeled conjugate.

Claim 38

The method of claim 33 wherein said ligand is an antigenic polypeptide or protein of molecular weight between 1,000 and 4,000,000.

Claim 39

The method of claim 33 wherein said antigenic polypeptide or protein is an antibody.

Claim 40

The method of claim 33 wherein said ligand is a hapten of molecular weight between 100 and 1,500.

Claim 41

The method of claim 33 wherein said ligand is an iodothyronine hormone.

Claim 42

The method of claim 37 wherein said hormone is thyroxine.

Claim 43

The method of claim 38 wherein the thyroxine is treated to protect the amino group therein prior to coupling step (b) and wherein the protecting group added thereby is removed subsequent to condensing step (e).

Claim 44

The method of claim 39 wherein said protecting group is trifluoroacetyl.

Claim 45

The method of claim 39 or 40 wherein said α,ω-diaminoalkane is 1,2-diaminoethane.

Claim 46

The method of claim 39 or 40 wherein said α,ω-diaminoalkane is 1,6-diaminohexane.