CA1128883A - Flavin adenine dinucleotide - labeled conjugates for use in specific binding assays - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE Labeled conjugates of the formula: Wherein Riboflavin-(Phos)2-Ribose represents the riboflavin -pyrophosphate-ribose residue in flavin adenine dinueleotide (FAD), ? = 2 through 6, and ?CO)L is flavin adenine dinueleotide ligand, or a binding analog thereof, and is preferably an iodothyronine such as thyroxine, bound through an amide bond; and intermediates produced in the synthesis of such FAD-labeled conjugates. The FAD-labeled conjugates are useful as labeled conjugates in specific binding assays for determining the ligand or a specific binding partner thereto in liquid media such as serum.

Description

BACKGROUND OF THE INVENTION
.
1 . FIELD OF T~E INVENTI ON

This invention relates to novel labeled corijugates for use in specific binding assays for ligands or their binding partners in a liquid medium. In partiçular, the invention relates to flavin adenine dinucleotide (FAD) - labeled conju-gates for use in such assays, particularly for determining an iodothyronine such as thyroxine in serum. The invention further relates to intermediate compounds produced in the synthesis of the novel labeled conjugates.
The iodothyronines have the following general formula:

HOOC-CH CH2- ~ o ~ OH
Bl ~2 wherein ~1 and ~2 are, independently, hydrogen or iodine. The principal iodothyronines of clinical interest are listed in Table 1 below.

TABl,E 1 Iodothyronine ~1 ~2 3,5,3'5'-tetraiodothyronine iodine iodine (thyroxine; T-4) 3,5,3'-triiodothyronine iodine hydrogen (liothyronine; T-3) 3,3',5'-triiodothyronine hydrogen iodine ("reverse" T-3) 3,3'-diiodothyronine hydrogen hydrogen The quantitative determination of the concentration of the various iodothyronines, particularly the hormones T-3 and T-4, in serum and of the degree of saturation of the iodothy-ronine binding sites on the carrier protein thyroid binding globulin (TBG) are valuable aids in the diagnosis of thyroid disorders. Li~ewise, the determination of other components of body fluids including serum is useful in assessing the well -being of an individual. Examples of other substances of clinical interest are evident from the description below.

2. BRIEF DESCRIPTIO~ OF THE PRIOR ART
:
Specific binding assay methods have undergone a techno-logical evolution from the original competitive binding radioimmunoassay (RIA) in which a radioisotope-labeled antigen is made to compete with antigen from a test sample for binding to specific antibody. In the RIA technique, sample antigen is quantitated by measuring the proportion of radioactivity which becomes associated with the antibody by

- 3 ~ 28~83 binding of the radiolabeled antigen (the bound-species of the labeled antigen) to the radioactivity that remains unassociated rom antibody (the free-species) and then comparing that proportion to a standard curve. A comprehensive review of the RIA technique is provided by Skelly et aZ, C~n. Chem. 19:
146(1973). While by definition RIA is based on the binding of specific antibody with an antigen or hapten, radiolabeled - binding assays have been developed based on other specific binding interactions 9 such as between hormones and their binding proteins.
From the radiolabeled binding assays have evolved non-radioisotopic binding assays employing labeling substances such as enzymes as described in U.S. Patents Nos. 3,654,090 and 3,817~837. Recently further improved nonradioisotopic binding assays have been developed as described in German O-ffenlegungschriften Nos. 2,618,419 and 2,618,511, based on U.S. Serial Nos. 667,982 and 667,996, filed on March 18, 1976 and assigned to the present assignee, employing particularly unique labeling substances, including coenzymes, cyclic reac-tants, cleavable fluorescent enzyme substrates, and chemilum-inescent molecules. Flavin adenine dinucleotide is mentioned as being useful as a coenzyme label since FAD functions as ; a coenzyme in useful monitoring reactions. In U.S. Patent Application Serial No. 917,961, filed June 22, 1978 and assigned to the present assignee, FAD is further described as useful in improved specific binding assays employing a prosthetic group as the label because FAD also functions as a prosthetic group in select biochemical systems.

Various methodologies exist for the determination of iodothyronine concentrations in serum. A significant advance in iodothyronine assays was the development of the competitive protein binding assay by Murphy and Pattee, J. C~in. Endocri~o~.
Metab. 2~ :187 (1964) in which radiolabeled iodothyronine com-petes with serum iodothyronine for binding to TB~. The development of specific antiserum for the various iodothyro-nines permitted radioimmunoassays to be devised in which radiolabeled and serum iodothyronine compete for binding to antibodies rather than to lBG. In both the competitive protein binding assay and the radioimmunoassay for an iodothy-ronine, the radiolabeled material consists of the native iodothyronine in which one or more of the iodine atoms are replaced by a radioactive iodine isotope, usually 125I. The above-mentioned nonradioisotopic binding assays have offered even more advantageous methods for determining iodothyronines, particularly those methods described in U.S. Patents Nos.

4,043,872 and 4,040,907 and most especially in OLS's 2,618,419 and 2,618,511 and U.S. Serial No. 917,961 mentioned above.

SUMMARY OF THE INVENTION

Novel flavin adenine dinucleotide (FAD) - labeled conjugates have been devised for use in binding assays ~or determining ligands, or binding partners thereof, of analyti-cal interest, such as the iodothyronines, and particularly for use in the assay referred to hereinbefore employing a pros~hetic group label. The FAD-labeled conjugates have the general formula:

NH-~c~l2~-rN~ co)L

~IN~
Riboflavin-(Phos)2-Ribose wherein Riboflavin-~Phos)2-Ribose represents the riboflavin -pyrophosphate-ribose residue in FAD; n = 2 through 6, and preferably is 2 or 6; and tCO)L is a specifically bindable ligand, or a specific binding analog thereof, and preferably is an iodothyronine such as thyroxine, bound through an amide bond.
The specifically bindable ligand or analog thereof in the present labeled conjugates, in terms of its chemical nature, usually is a protein, polypeptide, peptide, carbo-hydrate, glycoprotein, steroid, or other organic molecule for which a specific binding partner is obtainable. In func-tional terms, the ligand will usually be an antigen or an anti-: body thereto; a hapten or an antibody thereto; or a hormone, vitamin, or drug, or a receptor or binding substance therefor.
Most commonly, the ligand is an immunologically-active poly-peptide or protein of molecular weight between 1,000 and 4,000,000 such as an antigenic polypeptide or protein or an antibody; or is a hapten of molecular weight between 100 and ~ 1,500.
- 20 FAD-labeled conjugates wherein the ligand therein is an iodothyronine are particularly useful in specific binding assays to determine the iodothyronine in liquid media such as serum and preferably have the general formula:

~1 ~CH2~nN}I~C-cHcHZ~

Riboflavin-~Phos)2-Ribose wherein Riboflavin-(Phos)2-Ribose represents the riboflavin -pyrophosphate-ribose residue in flavin adenine dinucleotide, = 2 through 6, and ~1 and ~2 are, independently, hydrogen or iodine.
The FAD-labeled conjugates are used in binding assays for the ligand or a specific binding partner therefor and are deter-mined, i.e., monitored, for the purposes of the assay by measur-ing FAD activity, e.g., the coen~yme or prosthetic group activity generated upon combination of such conjugate with an apoenzyme that requires FAD to perform its catalytic function as described in detail in the above-mentioned U.S. Serial No. 917,961.
The present FAD-labeled conjugates can be prepared by a variety of synthetic routes. Exemplary of such available synthetic routes is the following general reaction procedure:
Cl ~O N
,~ ~0~ (1) 1~
co~o Reaction of 6-chloro-9-(2',3'-O-isopropylidine-~-D-ribofuranosyl) purine (1) [Hampton et a~, J. Am. Chem. Soc. 83:150(1961)] with an ~,~-diaminoal~ane selected from those listed in Table 2 D~

n ~ diaminoalkane 2 1,2-diaminoethane 3 1,3-diaminopropane 4 1,4-diaminobutane l,S-diaminopentane 6 1,6-diaminohexane yields the intermediate 6-(~-aminoalkyl)-9-(2',3'-0-isopropylidine-~-D-ribofuranosyl) purine (2).

NH~CH2~ NH2 <~N ~NJN
\V~ (2) ~ n = 2-6 X

The amino-purine intermediate ~2) is then linked by formation of a peptide or amide couple with either the ligand, where such contalns a carboxylic acid function, or a binding analog of the ligand (e.g., a derivative of the ligand) which analog contains the desired carboxylic acid function, to form the ligand or analog substituted adenosine intermediate (3) NH-~CH2 ~ NH-~CO)L

HO <N ~N'J
O ~ (3) n = 2-6 O O
H C X CH

8 ~ ~3 wherein -~CO)L is the ligand or analog thereof bound by an amide bond. Such condensation reactions can be accomplished by reacting the amino-purine intermediate (2) directly with the carboxylic acid-containing ligand or ligand analog using conventional peptide condensation reactions such as the carbodiimide reaction [Science 144:1344(1964], the mixed anhydride reaction [Erlanger et a~, ~ethod~ In Immuno-~ogy and Immunochemistry, ed. Williams and Chase, Academic Press (New York 1967) p. 149], and the acid azide and active ester reactions [Kopple, Peptides and Amino Acids, W.A.
Benjamin, Inc. (New York 1966)]. See also for a general review C~in. Chem. 22:726(1976).
It will be recognized, of course, that other well known methods are available for coupling the ligand or a derivative thereof to ~he amino-purine intermedia~e (2). In particular, conventional bifunctional coupling agents can be employed for coupling a ligand, or its derivative, containing a car-boxylic acid or amino group to the amino-purine intermediate (2). For example, amine-amine coupling agents such as bi~- isocyanates, bi~- imidoesters, and glutaraldehyde [Imm~nochem. 6: 53(1969)~ can be used to couple a ligand or derivative containing an amino group to the amino-purine intermediate (2). Also, appropriate coupling reactions are well known for inserting a bridge group in coupling an amine (e.g., the amino-purine intermediate) to a carboxylic acid (e.g., the ligand or a derivative thereof). Coupling reactions of this type are thoroughly discussed in the literature, for instance in the above-mentioned Kopple mono-graph and in Lowe ~ Dean, Affinity Chromatography~ John Wiley ~ Sons (New York 1974).

Such coupling techniques will be considered equivalents to the previously discussed peptide condensation reactions in preparing useful labeled conjugates. The choice of cou~-ling technique will depend on the functionalities available in the ligand or analog thereof for coupling to the amino -purine intermediate (2) and on the length of bridging group desired. In all cases, for purposes of this disclosure, the resulting condensation product will comprise the amino-purine intermediate, which ultimately is converted to FAD, bound to the remaining portion of the product, or ultimately to the remaining portion of the FAD-labeled conjugate, through an amide bond. Such remaining portion of the condensation product, or conjugate, will be considered as a residue of a binding analog of the ligand, unless the ligand itself is directly coupled to the amino-purine intermediate (2). Thus, in this description and in the claims to follow, the abbrevia-tion -~CO)L represents the ligand or a binding analog thereof coupled through an amide bond, wherein such analog can be a derivative of the ligand coupled by peptide condensation or can be the ligand or derivative thereof coupled through a bridging group inserted by coupling of the ligand or derivative with a bifunctional coupling agent.
~` It is evident that in coupling the ligand or derivative thereof to the amino-purine intermediate (2) it may be desir-able to protect certain reactive groups in such ligand or derivative from participating in side reactions during coup-ling. Protection of reactive groups may also be desirable to - 10 - ~ .`, ~ 8 ~ ~ ~

prevent interfering reactions during the synthetic steps described below for completing the preparation of the FAD
-labeled conjugate. Depending upon the specific ligand or derivative involved and the coupling technique chosen, the S addition of protecting groups at the reactive sites on the ligand or derivative can be accomplished before or after the coupling to the amino-purine intermediate (2). One skilled in the art will have a wide variety of conventional blocking reactions from which to accomplish the desired protection of reactive groups such that the blocking group added can be readily removed in a subsequent synthetic step to yield the original ligand or derivative coupled to FAD.
; For instance, where the ligand is an iodothyronine, it is preferably treated to protect the amine group prior to conden-sation or linkage with the amino-purine intermediate. The amine-protected iodothyronine intermediate has the formula:

HOOC-CHCH

, H or I

wherein Y is an amine-protecting group. It will be recognized tha* protection of the amine group is a conventional procedure and the amine-protecting group can be selected from a wide variety of groups, including trifluoroacetyl, which is pre-ferred, and the like, such as others of the acyl type (e.g., ~ 3 formyl, benzoyl, phthalyl, p-tosyl, aryl- and alkylphosphoryl, phenyl- and benzylsulfonyl, tritylsulfenyl, o-nitrophenyl-sulfenyl and o-nitrophenoxyacetyl), those of the alkyl type (e.g., trityl, benzyl and alkylidene) and those of the urethane type (e.g., carbobenzoxy, p-bromo-, p-chloro- and p-methoxycarbobenzoxy, tosyloxyalkyloxy-, cyclopentyloxy-, cyclohexyloxy-, t-butyloxy, l,l-dimethylpropyloxy, 2-(p-biphenyl)-2-propyloxy- and benzylthiocarbonyl.
The substituted adenosine in~ermediates formed by con-densation or linkage between the amino-purine intermediate (2) and the amine-protected iodothyronine intermediate (4) are of the formula (3) wherein -~CO)L is:

- Il CHCH2 ~ 0 ~ 0H (5) = H or I

wherein Y is an amine-protecting group as above.

Treatment of intermediate (3) with phosphorous oxy-chloride produces the phosphorylated ligand or analog sub-stituted adenosine intermediate (6) NH-~CH2 ~ NH-~CO)L

1l <N ~ NJ (6) HO-P-O

O o n = 2-6 which upon hydrolysis yields the ligand or analog sub-5stituted 5'-adenylic acid intermediate (?).

NH~CH2~NH~CO) L

1l < ~N (7) OH ~ ~
n = 2-6 OH OH

Condensation of riboflavin-5'-monophosphate with inter-mediate (7) ac~ivated to a phosphorimidazolidate by treatment with N,N'-carbonyldiimidazole yields FAD-labeled conjugates (8).

N --f CH2~n NH~CO) L
<N~¢ N (8) n = 2-6 Riboflavin-(Phos)2-Ribose In the preferred embodiment wherein the ligand is an iodothyronine, and thus -~CO)L is represented by formula (S) above, the resulting FAD-iodothyronine conjugates are of the formula:

NH-~CH2~-nNH-~Ci-CHCH2 ~ O ~ H
~N I N ~1 ~2 N N~ n = 2-6 ; Riboflavin-(Phos)2-Ribose ~1 ~2 = H or I
f wherein Y is an amine-protecting group or, upon conventional treatment for removal of such protecting group, Y is hydrogen.
As illustrated above, the novel intermediate compounds (2,3,6 and 7) produced in the course of synthesizing the FAD-labeled conjugates have the following general formulae [the amino-purine intermediates ~2) correspond to formula A
below and the intermediates (3,6 and 7) correspond to formula B below~:
, ~

formula A

NH~CH2 3~NH2 HO ~N--l~N
'~~,1 ~1 .

X

wherein n = 2 through 6; and formula B
NH-~CH2 ~ NH-~CO)L

'J
~0\ l wherein tCO)L is a specifically bindable ligand, or a bind-ing analog thereto, and preferably is of formula (s), bound through an amide bond; n = 2 through 6; ~l and ~2 are, independently, hydrogen or iodine; Rl is -OH or -O-P-OH

when R2 and R3 together form the group l X l , or R is -O-~-OH when R .and R3 are -OH.
-~H
: As stated hereinabove, the ligand which is comprised in the labeled conjugate or whose binding analog is comprised in the labeled conjugate is in most circumstances an immunologically-active polypeptide or protein of molecular weight between l,000 and 4,000,000 9 such as an antigenic poly-: peptide or protein or an antibody, or is a hapten of molecu-lar weight between 100 and 1,500. Various methods for coupling such ligands or analogs thereof to the aminQ-purine inter-mediate (2) through an amide bond in the synthesis of the present FAD-labeled conjugate will now be presented.

PoZypeptides c~nd Proteins Representative of specifically bindable protein ligands are antibodies in general, particularly those of the IgG, IgE, IgM and IgA classes, for example hepatitis antibodies;
and antigenic proteins such as insulin, chorionic gonadotropin (e.g., HCG), carcinoembryonic antigen (CEA), myoglobin, hemo-; globin, follicle stimulating hormone, human growth hormone, thyroid stimulating hormone (TSH), human placental lactogen, ; thyroxine binding globulin ~TBG), instrinsic factor, trans cobalamin, enzymes such as alkaline phosphatase and lactic dehydrogenase, and hepatitis-associated antigens such as hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBCAg). Representative of polypeptide ligands are angiotensin I and II, C-peptide, oxytocin, vasopressin, neurophysin, gastrin, secretin, and glucagon.
Since, as peptides 7 ligands of this general category possess numerous available carboxylic acid and amino groups, coupling to the amino-purine intermediate (2) can proceed according to conventional peptide condensation reactions such the carbodiimide reaction, the mixed anhydride reac~ion, and so forth as described hereinabove, or by the use of conven-tional bifunctional reagents capable of coupling carboxylic acid or amino functions to the amino group in the amino-purine intermediates (2) as likewise described above. General references concerning the coupling of proteins to primary amines or carboxylic acids are mentioned in detail above.

Haptens Haptens, as a class, offer a wide variety of organic substances which evoke an immunochemical response in a host animal only when injected in the form of an immunogen conju-gate comprising the hapten coupled to a carrier molecule, almost always a protein such as albumin. The coupling reac-tions for forming the immunogen conjugates are well developed in the art and in general comprise the coupling of a carboxy-lic acid ligand or a carboxylic acid derivative of the ligand to available amino groups on the protein carrier by formation of an amide bond. Such well known coupling reactions are directly analogous to the present formation of labeled conju-gates by coupling carboxylic acid ligands or binding analogs to the amino-purine intermediate (2).
Hapten ligands which themselves contain carboxylic acid functions, and which thereby can be coupled directly to the amino-purine intermediate (2), include the iodothyronine hormones such as thyroxine and liothyronine, as well as other materials such as biotin, valproic acid, folic acid and certain prostaglandins. Following are representa~ive synthetic routes for preparing carboxylic acid binding analogs of hapten ligands which themselves do not contain an available carboxylic acid function whereby such analogs can be coupled to the amino-purine intermediate (2) by the aforementioned peptide condensation reactions or bifunctional coupling agent reac-tions (in the structural ormulae below, n represents an integer, usually 1 through 6, and Me represents methyl).

Carbamazepine Dibenz[b,f]azepine is treated sequentially with phosgene, an ~-aminoalkanol, and Jones reagent (chromium trioxide in sulfuric acid) according to the method of Singh, U.S. Pat. No. 4,058,511 to yield the following series of carboxylic acids:

CONH~C~ ~ COOH

Quinidine Pollowing thP method of Cook et aI~ Pharma¢oZog~st 1~:
219(1975~, quinidine is demethyla~ed and treated wi~h

5-bromovalerate followed by acid hydrolysis to yield a suitable carboxylic acid derivative.

Digoxin and Di~-itoxin The aglycone of ~he cardiac glycoside is treated with succinic anhydride and pyridine according to the method of Oliver et aZ, J. CZin. Invss~. 47:1035~1968) to yield the following:

Me ~J
R ~ l I H Z ~ H or OH
HOOCtCH2~CO `

38~

Theophylline Following the method of Cook et aZ, Res. Comm. Chem.
Path. Phar~. 13:497~1976), 4,5-diamino-1,3-dimethylpyrimidine -2,6-dione is heated with glutaric anhydride to yield the ~ollowing:

3 N ~ N
1 ~ ~ ~ C~ ~ COOH
I

Phenobarbital and Primidone SGdium phenobarbital is heated with methyl 5-bromovalerate and the product hydrolyzed to the corresponding acid derivative of phenobarbital [Cook et aZ, Quantitative AnaZytic Stud*es in EpiZepsy~ ed. Kelleway and Peterson, Raven Press (New York 1976) pp. 39-5~]:

~ 2 0~1~0 CC~ ~ CGOH

~ 19 -To obtain the acid derivative of primidone following the same Cook et aZ reference method, 2-thiophenobarbital is alkylated, hydrolyzed, and the product treated with ~aney nickel to yield:
., O
T~C2H5 O
(CH2 ~ COOH

Diphenylhydantoin Following the method of Cook et aZ, Re~. Comm. Chem.
Path. Pharm. 5:767(1973), sodium diphenylhydantoin is reacted with methyl 5-bromovalerate followed by acid hydrolysis to yield the following:

~
CH2 ~ COOH

Morphine Morphine free base is treated with sodium ~-chloroacetate : according to the method of Spec~or et aZ, Science 16B:1347 (1970) to yield a suitable carboxylic acid derivative.

~ 8 Nicotine According ~o the method of Langone et aZ, Biochem. 12~2~J:
5025(1973)~ trans-hydroxymethylnicotine and succinic anhydride are reacted to yield the following:

N

Hooc~cH23~ c-ocH

Androgens Suitable ca~boxylic acid derivatives of testos~erone and androstenedione linked through either the 1- or 7-posi~ion on the steroid nucleus are prepared according to the method of Bauminger et aZ~ J. Steroid Bioahem. 5: 739(1974). Follow-ing are representative tes~osterone derivatives:

l - pos*tion HOOC~LH

?-posi tion S~CH2 ~ COOH

Estrogens Suita~le carboxylic acid derivatives of estrogens, e.g., estrone, estradiol and estriol, are prepared according to the method of Bauminger et a~, $upra, as represented by the following estrone derivative:

HO
N ~H2 COO~

Progesterones Suitable carboxylic acid deriva~ives of progesterone and its metabolites linked ~hrough any of the 3-, 6- or 7-posltions on the steroid nucleus are prepared according to the method of Bauminger et aZ, supra, as represented by the following progesterone deri~atives:

.
3-posi tion Me

6- pos~ tion C=O
Me I
,~
~ '.
S~C~ ~COOH

7-position ~H3 C= O

O ~ ~ S-SC~ ~ COON

The methods described above are but ~xamples of the : many known techniques for forming suitable carboxylic acid deri~atives of haptens of analytical interest. The ; principal derivation techniques are discussed in CZin. Chem.
22:726(1976) and include esterification of a primary alcohol with succinic anhydride lAbraham and Grover, Princip~e6 of Competitive Prote*n-Bind*ng Assays, ed. Odell and Daughaday, J.B. Lippincott Co. (Philadelphia 1971~ pp. 140-157], forma-tion of an oxime from reac~ion of a keton~ group wi~h carboxyl- -methyl hydroxylamine [J. Bio~. Chem. 234:1090(1959)], intro-duction of a carboxyl group into a phenolic residue using chloroacetate lSc*enae 168:1347(1970~], and coupling to di;~-zotized p-aminobenzoic acid in the manner described in J. Rio2.
Chem. 235:1051(1960).

.

The general reaction scheme described above is exempli-fied by the following descriptions of the synthesis of the ethyl (n=2) and hexyl (n=6) analogs o the FAD-labeled conju-gates wherein the ligand is the iodothyronine thyroxine [i.e., -~CO)L is o the formula (5) wherein ~1 and ~2 are both iodine]. Also provided are descriptions of assay methods, and results therefrom, employing the exemplified analogs as labeled conjugates in a specific binding assay for thyroxine.

8~

1. Ethy~ AnaZog l-I. PREPARATION OF THE LABELED CONJUGATE

6-(2-Aminoethyl)amino-9-(2',3'-O-isopropylidine-~-D
-ribofuranosyl) purine (2).

13.56 grams (g) [41.5 millimoles (mmol)] of 6-chloro-9-(2',3'-O-isopropylidene-~D-ribofuranosyl) purine (1) [Hampton et a~, J. Am. Chem. So~. ~3:150(1961)] was added with stirring over a 15 minute period ~o a cold excess of 1,2-diaminoethane [75 milliliters (ml)]. The resulting solution was allowed to stand at room temperature for 24 hours. The solution was evaporated in vacuo and the resulting yellow oil was stirred - with 50 ml of cold, sa~urated sodium bicarbonate. The mixture was evaporated in vacuo and the resulting residue was further repeatedly evaporated in vacuo first from water (3 times from 50 ml) and then from 2-propanol ~4 times from 50 ml) to ob-tain a yellow glass (15 g). A portion (3 g) of the glass was dissolved in a small volume of water which was then applied to the top of a 25x55 centimeter (cm) Dowçx 50W-X2 cation exchange column in the ammonium form (Bio-Rad Laboratories, Richmond, California USA).
The column was eluted with a linear gradient generated with 2 liters (L) each of water and 0.5 molar (M) ammonium bicarbonate. The elution was completed using a linear gradient generated with 2 L each of 0.5 M and 1 M ammonium bicarbonate.
The effluent from the column was collected in 19 ml fractions and monitored by elution on silica gel thin layer chromatography (TLC) plates ~E. Merck, Darms~adt, West Germany) with a 9:1 (v:v) mixture of ethanol and ammonium hydroxide. The developed TLC plates were examined under ultraviolet light, then sprayed with ninhydrin reagent [Randerath, ~hin Layer Chromatography, Academic Press (1966)]. Fractions numbered 250 through 350 ~ ~ ~8 8 ~ ~

from thg column chromatography were combined and evaporated in va~uo leaving the desired purine (2) as a pale yellow amorphous glass (1.5 g).

Analysis: Calculated for C15H22N6O4: C, 51.42;
H, 6.33; N, 23.99 Found: C, 50.92; H, 6.54; N, 23.01 NMR (60 MHz, CDC13): ~ 1.37 (s,3H, isopropylidene), 1.63 (s,3H, - isopropylidene), 5.92 (d, lH, l'-ribosc), 7.90 (s, lH, purine), 8.26 (s, 1ll, purine) Optical Rotation [a]20 = -74.85 (c 1.0, (H3O~I) The remaining crude product (lZ g) was purified by chroma-tography on Dowex 50W-X2 as described above. The overall yield was 8 g (55%).

a-(N-Trifluoroacetyl)amino-~-[3,5-diiodo-4-(3',5'-diiodo-4' -hydroxy~ noxy)phenyl] propanoic acid (4).

This compound was prepared by the method of Blank, J . ~'harm .
Sc3, 53:1333(1964)~ To a cooled (0C), stirrcd suspen~ion of 5 g (6.4 mmol) of L-thyroxine ~Sigma Chemical Co., St. Louis, Missouri USA) in 60 ml of dry ethyl acetate was added 11.5 ml of trifluoroacetic acid and 1.9 ml of ~rifluoroacetic anhydride.
After 30 minutes the resulting clear solution was washed threc times with 30 ml of water, once with 30 ml of 5% sodium bicar-bonate, and twice with 50 ml of saturated sodium chloride. The combined aqueous washings were extracted twice with 20 ml of ethyl acetate. The ethyl acetatc layers were combine~ and washed with 30 ml of water, then dried over magnesium sulfate.
The dried ethyl acetate solution was evaporated in va~o leavin~
a white solid. Recrystallization from a mixture of ethyl ethcr and petroleum ether gave a pinkish-white solid (3.95 g, 70.5O
yield) having a melting point (m.p.) of 228-230C with decomposition.

Analysis: Calculated for C17HloF3I4NO5: C, 23.~9;
H, 1.15; N, 1.60 Found: C, 23.00; H, 1.05; N, 1.65 MMR [60 MHz, DCON(CD3)2] ~ 7.28 (s, 21~, aromatic), 8.03 (s, 2H, aromatic),

9.7 (m, lH, amido) IR (KCl): 1700 (>C=O) Optical Rotation [~25 = -14.97 (c 1.0 dimethylsulfoxide) A second recrystallization produced a second precipitate (0.95 g) m.p. 224-228~C with decomposition. The overall yield 15was 87.5~.

N-{2-[N-~Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl3 aminoethyl}-2'~3~-O-isopropylidene adenosine (3).

A solution of 8.72 g (10.0 mmol~ of ~-(N-trifluoroacetyl) amino-~-[3,5-diiodo-4-(3',5'-diiodo-4'-hydroxyphenoxy)phenyl]
propanoic acid (4~ and 3.86 g (11.0 mmol) of 6-(2-aminoethyl) amino-9-(2',3'-O-isopropylidene-~-D-ribofuranosyl) purine (2) in 50 ml of dry dimethylacetamide was prepared under a dry argon atmosphere at -20C. To this cold stirred solution was added a solution of 3.04 g (11.0 mmol) of diphenylphosphoryl azide (Aldrich Chemical Co., Milwaukee, Wisconsin USA) in 10 ml of dry dimethylacetamide followed by the addition of l.fi ml (11.0 mmol) of dry triethylamine. The solution W(IS lert ;It room temperature for 2Z hours. The solution was then a~de~
dropwise to 300 ml of cold (0C) water with stirrin~. ~he 27 ~

resulting white precipitate was collected by filtration an~
dried in vaouo (56C) to give 13.0 g of a light cream colored solid. The solid was dissolved in 500 ml of acetonc alld tl-c solution was concentrated by boiling. The white soli(l whi~ll precipitated from the boiling acetone solution was collectcd by filtration while hot. Continued boiling of the filtrate produced two additional precipitates. The three precipitatcs were combined to gi~e 8 g (66.6% yield) of a white solid, nl.p.
198-200C (decomposed).
; 10 Analysis: Calculated for C32H30F3I4N7O8: C, 31.89;
H, 2.51; N, 8.14 Found: C, 31.95; H, 2.60; N, 7.86 NMR 1220 MHz, ~CD3)2SO] ~ 1.32 (s, 31~, isopropylidene), 1.55 (s, 3H, iso-propylidene), 6.14 ~d, lH, l'-ribose), 7.02 (s, 2H, thyroxine), 7.82 (s, 2ll, thyroxine), 8.25 (s, lH, purine), 8.36 (s, lH, purine), 8.41 (t, 1~l, J-6, amido), 9.64 (d, lH, J-8, trifluoroacetamido) Optical Rotation [a]25 = -11.82 (c 1.0, pyridine) N-{2-[N-~Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl) aminoethyl}-2',3'-O-isopro~ylidene-5'-adenylic acid monotriethylamine salt. monohydrate (6).

A solution of 1.2 g (1.0 mmol) of N-{2-[N-(trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-isopropylidclle adenosine (3) in 10 ml of dry triethylphosphate was l-repared under a dry argon atmosphere at 0C. To the cold, stirred solution was added 0.45 ml (5 mmol) of phosphorous oxychlorid~.
The resulting solution was kept for 24 hours at 0C, then ~dc~
dropwise with stirring to 1 L of ice water. The resulting precipitate was collected by filtration and dried in vacu~
to give 1.23 g of a white solid. The solid was dissolved in acetone and 0.32 ml (2 2 mmol) of triethylamine was added. A
precipitate formed. The mixture was e~aporated in vacuo an~l the resulting residue lixiviated with dry acetone, then rc-crystalized from a mixture of dry methyl alcohol an~ dry ethyl ether to gi~e 390 mg ~27.8~ yield) of a whi~e solid, m.p. 173-183C (decomposed).

Analysis: Calculated for C38H48F3I4N8O12P C, 32-50;
H, 3.45; N, 7.93 Found: C, 32.24; H, 3.08; N, 7.58 NMR [60 MHz, (CD3)2S0] ~ 1.53 ~s, 3H, isopropylidene), 6.2 (d, lH, l~l-ribose), 7.1 (s, 2H, thyroxine arom~tic), 7.87 (s, 2H, thyroxine aromatic), 8.27 (s, ll~, purine), 8.52 (s, lH, pl1rine) Optical Rotation [~]25 = -17.50 (c 1.0, ~H3~11) N-{2-[N (Trifluoroa etyl)-3,3',5,5'-tetraiodothyronyl~
aminoethyl}-5'-adenylic acid ~?).

200 milligrams (mg) (0.14 mmol) of N-{2-[N-(~ri~luoroacetyl -3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-isopro~ylidenc -S'-adenylic acid monotriethylamine salt monohydrate (6) was suspended in 1 ml of water (0C) and trifluoroacetic acid (9 ml) was added dropwise with stirring. After 30 minutcs a clc.lr solution was obtained. The solution was kept cold (0~) ror an additional 15 hours, then evaporated in vaCuo (30C). I`hc re-sulting residue was evaporated four times in vaCuo (25C) fr~m 20 ml volumes of anhydrous ethyl alcohol and then dricd in vacuo (25C) leaving a white solid.
The solid was stirred for 30 minutes with 10 ml o~ cold methyl alcohol, then collected by filtration and dried in vacuo (25C) to give a white solid (135 mg, 76% yie]d) wh-ich slowly melted with decomposition above 188C.
Analysis Calculated for C29H27F3I4N7OllP C, 27-97;
H, 2.19, N, 7.87 ; Found: C, 28,11; H, 2.31; N, 7.65 NMR [220 MHz, (CD3)2S0] ~ 5.95 (d, lH, l'-ribose), 7.04 (s, 2H, thyroxine aromatic), 7.84 (s, 2H, thyroxine aromatic), 8.25 (s, lH, purine), 8.36 (s, lH, purine), 8.43 (m, Il~, ; amido), 9.66 (d, lH, trifluoroacetamido) Optical Rotation [a325 = -2.72 (c 1.0, pyridine) Flavin adenine dinucleotide - thyroxine conjugate (8).

498 mg (0.4 mmol) of N-{Z-[N-(trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-5'-adenylic acid (7) was dis-solved in 10 ml of dry dimethylformamide and tri-n-butylamine [96 microliters (~1), 0.4 mmol] was added followed by the addition of l,l'-carbonyldiimidazole (320 mg, 2.0 mmol). Art~r stirring for 18 hours at room temperature in the ab.sence of moisture, water (280 ~1) was added and then the solvent evaporated in vacuo.
The resulting oil was dried by repeated in va~uo evapol-ation from dry dimethylformamide (4 times from 10 ml). Thc resulting phosphorimidazolidate was redissolved in 10 ml o~
dry dimethylformamide and added dropwise to a 0.4 mmol so]ution of the tri-n-octylamine salt of riboflavin-5'-monophosphate in

10 ml of dry dimethylformamide. The salt was preparcd hy ing a solution of the ammonium salt of riboflavin-5' -monophosphate (192 m~, 0.4 mmol) in 10 ml of water to a stirred solution of tri-n-octylamine (176 ~1, 0.4 mmol) ill 100 ml of acetone. After 30 minutes, the resulting mixturc was evaporated in vacuo. The residue was dried by repeated evaporation in vacuo from dry dimethylformamide leavin~ the salt as an orange solid.
The above solution containing the phosphorimidazolidate of (7) and the riboflavin-5'-monophosphate salt was divide~
into two equal aliquots after 24 hours and one aliquot was evaporated in va¢uo. The resulting residue was chromatographe~
on a column (2.5x78 cm) prepared from 100 g of Sephadcx 1.~1-20 ~Pharmacia Fine Chemicals, Uppsala, Sweden~ which had becn preswollen (18 hours) in a 19:1 (v:v) mixture of dimethyl-formamide and triethylammonium bicarbonate (1 M, pH 7.5). Thc column was eluted with the above 19:1 (v:v) mixture and 10 m]
fractions were collected. The effluent from the column was monitored by elution on silica gel 60 silanised RP-2 I`LC
places (E. Merck, Darmstadt, West Germany).
The TLC plates were developed using a 40:40:25:1:1 (v:v) mixture of acetone, chloroform, methyl alcohol, watcr, ~n~
triethylamine. Fractions numbered 11 throu~h 17 from the above-mentioned column chromatography were combined and evaporated in vacuo. The residue was chromatographed on ~ co~lmn (2.5x75 cm) prepared from 125 g of Sephadex Ll1^20 which h~
been preswollen (18 hours) in 0.3 M ammonium bicarbonate. Ille column was eluted with 0.3 M ammonium bicarbonate collectinl~
10 ml fractions. The effluent was monitored by absorption ~f ultraviolet light at 254 nanometers (nm). The volume ol the fractions was increased to 20 ml beginning with fraction number 150. The salt concentration of the eluent was decrease~
in a stepwise fashion as follows: 0.15 M ammonium bicarbonate --at fraction number 295, 0.075 M ammonium bicarbonate at frac-tion number 376, and ~ater at fraction number 430. A total of 480 fractions was collected. Fractions numbered 200 througl 235 were combined and evaporated ~n vacuo leaving the labele~
conjugate (8) as a yellow-orange residue. An alkaline, a~ueolls solution of this residue exhibited ultraviolet absorption maxiln;
at the following wavelengths: 266 nm, 350 nm, 373 nm, and 450 nm. The yield, estimated from the absorption at 450 was about 5~.
A phosphodiesterase preparation (Worthington Biochemical Corp., Freehold~ New Jersey USA) isolated from snake venom (Crota~us Adamanteus) hydrolyzed the above product to riboflavin-5'-monophospha~e and the thyroxine substituted 5'-adenylic acid (7) wherein the trifluoacetyl blockinp grou~
had been removed.

l-II. BINDING ASSAY FOR THYROXINE

The above-prepared labeled conjugate was used in a prosthetic group-labeled specific binding assay as follows (further details regarding such an assay method may he found in the U.S. Patent Application - Serial No. 917,961-referred to hereinbefore):
- ~2 -A. Preparation of apoglucose oxidase Purified glucose oxidase with low catalase activity obtained from the Research Products Division of Milc~ bor~-tories, Inc., ElXhart, Indiana USA was twice dialyze~ for 12 hours each against 0.5% (w:v) mannitol (30 volumes eacll).
Aliquots of the dialyzate containing 100 mg of glucosc n~i~ase each were lyophilized and stored at -20C.
~ Bovine serum albumin (200 mg) was dissol~ed in 12 ml of ; water adjusted to pH 1.6 with concentrated sulfuric acid, mixe-l with 150 mg charcoal (RIA grade from Schwarz-Mann, Orangcburg, New York USA), and cooled to 0C. Lyophilized gluco~e oxida~-(100 mg) was redissolved in 3.1 ml of water and 3 ml was adclc~
to the stirred albumin-charcoal suspension with continu~d stirring for three minutes. The suspension was then ~iltcrc-l through a 0.8 micron, 25 millimeters (mm) diameter Millil)orc filter (Millipore Corp., Bedford, Macsachusetts USA) moulltc~
in a Sweenex filter apparatus (Millipore Corp.) on a sn ml disposable plastic syringe. The filtrate was quickly neutr~-lized to pH 7.0 by addition of 2 ml of 0.4 M phosphatc buffcr ~pH 7.6)-and thereafter 5 N sodium hydroxide. Dry cJIarcoal ~150 mg) was then added and stirred for one hour at 0C. The resulting suspension was filtered first through a 0.8 micro Millipore filter and then through a 0.22 micron Millipore filter. To the filtrate was added glycerol to 25% (v:v) an~
; 25 the stabilized apoglucose oxidase preparation was store~l at 4C.

B. Assay Reagents 1. Labeled conjugate - The ethyl analo~ l~beled eonjugate prepared as in section l~ above was diluted in 0.1 M phosphate buffer (pll 7) to ; 5 concentration of 1 micromolar (~M).
2. Apoenzyme - Apoglucose oxidase was diluted with 0.1 M phosphate buffer (pH 7) to a concentration of 0.6 ~M FAD binding sites. The FAD binding site concentration of the apoenzyme preparation was determined experimentally by measuring the minimum amount of FAD required to give maximum glucose oxi--dase activity when incubated with the apoen~yme.
3. Insolubilized antibody - A washed, moist cake of Sepharose 4B gel (Pharmacia Fine Chemicals, IJppsala, Sweden) activated by cyanogen bromide accordinx to the method of March et aZ, Ana~ . Biochem. ~0 :1 ] 9 (1974) was added to a soll-tion of 85 mg o~ antibody, (isolated from antiserum agains~ a thyroxine-bovinc serum albumin conjugate~ in 20 ml of 0.1 M phosphate buffer (pH 7.0) and agitated slowly for 36 hours at 4C. Upon completion of the coupling reaction, 1 m~
of 1 M alanine was added and shaking continued for four more hours to block unreacted sites. The result-ing Sepharose-bound antibody was washed on a scintere~
funnel with 400 ml each of 50 mM sodium acetate -~ 500 millimolar (mM) sodium chloride (pH 5) and 50 mM
- phosphate buffsr - 500 mM sodium chloride ~pH 7), .~n~
~ 800 ml of 100 mM phosphate buffer (pH 7). The moist ;~ ~ilter cake was then suspended in 100 mM phosphatc 3(~ buffer (pH 7) containing O.n1~ so~iium nzidc to giv~ 2~ ml ot an about 50~ suspension.
' ' 4. Standard - A 1.15 mM stock solution of thyroxine in 5 mM sodium hydroxide was diluted to 2 ~M in ().1 M
phosphate buffer (pH 7).
5. Monitoring reagent - A glucose oxidase assay reauent was prepared to contain ~he following mixture ~er 130 ~1: 25 ~1 of 1.2 mg/ml peroxidase (Si~ma Chemical Co., St. Louis, Missouri USA) in 0.1 M phosphate buffer (pH 7), 5 ~1 of 10 mM
4-aminoantipyrine in water, 20 ~1 of 25 mM
3,5-dichloro-2-hydroxybenzene sulfonate in O.l M
phosphate buffer ~pH 7), 30 ~1 of 16.5% bovine serum albumin in 0.1 M phosphate buffer (pH 7), and 50 ~l of l M glucose in aqueous saturated benzoic acid solutionO

C. Assay Procedure Binding reaction mixtures were prepared by mixing 150 ~]
of the insolubilized antibody suspension, 80 ~1 of the labeled conjugate solution, various amounts of ~he standard thyroxine solution to ~ive varying concentrations of thyroxine in the reaction mixtures, and a sufficient volume of 0.1 M phosphate buffer (pH 7~ to ma~e a total volume of 500 ~1. lhe reaction mixtures were incubated with shaking for two hours at 25C.
Each reaction mixture was ~hen vacuum filtered through a ~lass wool plugged, dry pasteur pipette pre~iously trea~ed sequcn-tially with periodate and ethylene glycol solutions to eliminate possible FAD contamination To a 300 ~1 aliquot of each filtrate was adde~ 130 ~1 of the monitoring reagent and 50 ~1 of the apoenzyme solution. After one hour, the absorbance of each reaction mixturc was measurcd at 520 nm.

D. Results Following is Table 3 showing the results of the assay procedure in measuring thyroxine. The absorbance results .Ire expressed as the average of duplicate runs corrected for residual enzyme activity in the apoenzyme solution (absorbance of 0.522) and for endogenous FAD in the antibody suspension (absorbance of 0.142).

TABlE 3 Volume of Thyroxine Absorbance (520 nm) Standard Added (~1) .. _ _ . . . ..

0.223 0.281 250 0.286 The results demonstrate that the present labeled conjugates are useful in a specific binding assay method for determining a ligand in a liquid medium.

~8~

2. He~yZ AnaZog 2- I. PREPARATION OF THE LABELED CONJUGATE

6-(6-Aminohexyl)amino-9-(2',3'-O-isopropylidene-~-D-ribofuranosyl) purine t2).

16 0 g ~50 mmol) of 6-chloro-9-(2',3'-O-isopropylidene-~-D-ribofuranosyl) purine (1) [Hampton et a~, J. Am. Chem. Soc.
~3:1501tl961)] was added with stirring to a molten (70C) sample of freshly distilled 1,6-diaminohexane (58 g, 500 mmol).
The resulting mixture was stirred under argon at 40C for 18 hours. The excess diamine was removed by distillation under reduced pressure ~60C, 0.01 mm Hg). The resulting pale yellow residue was adsorbed onto 150 g o silica gel 60 (E.
Merck, Darmstadt, West Germany) and used to top a chromato-graphic 9:1 (v:v) mixture of absolute ethyl alcohol and tri-ethylammonium bicarbonate (pH 7.5, 1 M). The column was eluted wikh the above 9:1 (v:v) solvent mixture and 900 20 ml fractions were collected. The fractions were examined by thin ; layer chromatography (TLC) on silica gel 60 eluting with a 7:3 (v:v) mixture of absolute e~hyl alcohol and triethylammonium bi-carbonate ~pH 7.5, 1 M). Fractions numbered 391 through 900 ` from the column chromatography were combined and evaporated in ; vacuo leaving 15.0 g of a gl~ssy residue (74% yield). A 1 g sample of the glass was dissolved in a small volume of methyl alcohol and applied to the top of a column prepared from 80 g of Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) preswollen in methyl alcohol. The column was eluted with methyl alcohol. A total of ninety 8 ml fractions were col-lected. The fractions were examined by TLC on silica gel 60 eluting with a 7:3 (v:v) mixture of absolute ethyl alcohol and triethylammonium bicarbonate (pH 7.5, 1 M). Fraction~
numbered 19 through 27 from the column chromatography wer~
combined and evaporated in vaauo leaving 910 mg (91~, re~overy) of a white ~lass.

Analysis: Calculated for C19H30N604: C, 56.14;
H, 7.44; N, 20.68.
Found: C, 53.91; H, 7.33; N, 19.18 NMR (60 MHz, CDC13): ~ 1.40 (s, 3H, isopropylidene), 1.63 (s, 3H, i~opro-pylidene) 5.98 (d, lH, l'-ribose), 7.92 (s, lH, purine), 8.36 (s, 1~l, pur ine ) Optical Rotation [c~] 25 = 50 .11 (c 1. O, methyl alcohol) N-{6-[N-Trifluoroacetyl)-3,3',5,5'-tetrai_dothyronyl]
aminohexyl}-2',3'-0-isopropylidene adenosine ~3).

A solution of 4.36 ~ (5.0 mmol) of ~-(N-trifluoroacetyl) amino-~-[3,5-diiodo-4-(3',5' diiodo-4'-hydroxyphenoxy)-phenyl]
~; propanoic acid (4), prepared as descri~ed in section l -I ~hovc, and 2.24 g (5.5 mmol) of 6-(6-aminohexyl)amino-9-(2',3'-()-isopropylidene-~-D-ribofuranosyl) purine (2) in 100 ml of dry dimethylformamide was prepared under a dry argon atmosphere at -20C. To ~his cold stirred solution was added a solution of 1.52 g (5.5 mmol) of diphenylphosphoryl azide (Aldrich Chemical Co., Milwaukee, Wisconsin USA) in 50 ml of dry dimethylforma-mide followed by the addition of 0.8 ml ~5.5 mmol) of dry triethylamine. lhe solution was left a~ room temperature for 22 hours. The solution was then added dropwise to 60() ml of cold (0C) water with stirring. The resulting white precipi-tate was collected by filtration and dried in vacuo (60C) to ~ 3 give 4,90 g (78% yield) of white solid. A sample of thi~
solid was recrystallized from a mixture of acetone alld water giving a white solid~ m.p. 205-207C (decomposed).

Analysis: Calculated for C36}l38F3I4N7O8 C, 34-Z~;
H, 3.04; N, 7.77 Found: C, 34.22; H, 2.99; N, 7.41 Mass Spectrum (20 ma) m/e: 1262 [M~l ], 1164 [M minus COCF3]
Optical Rotation ~]25 = -21.89 (c 1.(), yyridine) N-{6-[N-~Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]
; aminohexyl}-2' 9 3'-O-isopro~ylidene-5'-adenylio acid monotriethylamine salt monohydrate (6).

A solution of 1.89 g (1.5 mmol) of N-{6-N-(trifluoroacetyl)-3,3',5,5'-tetraiod~hyronyl]aminollexyl}-2',3'-O-isopropylidene adenosine (3) in 15 ml of dry tri-ethylphosphate was prepared under a dry aTgon atmosphere at -10C. To the cold stirred solution was added 0.68 ml -~ (7.5 mmol) of phosphorous oxychloride. The resulting solution was kept for 18 hours at -15C then added dropwise with stirring to 1.5 L of ice wa~er. The resulting precipitate was collected by filtration and dried in vacuo to give 1.~1 R
(87% yield) of a white solid. The solid was dissolved in 10 ml methyl alcohol and 0.38 ml (2.6 mmol) of triethylaminc was added. This solution was evaporated in vacuo and the resulting residue was recrystallized from a mixture of methyl alcohol and ethyl ether to give 720 mg (33~ yield) of a white solid, m.p. 151-154C (decomposed).

:

Analysis: Calculated for C42H56F3I4N80l2 H, 3.86; N, 7.67 Found: C, 35.24; H, 3.88; N, 7.75 Mass Spectrum (20 ma) m/e: 1342 L~H l, , 1244 [M minus COCF3]
Optical Rotation [~]25 = -17.20 (c 1.0, CH30H) N-{6-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]
aminohexyl}-5'-adenylic acid (7).

600 mg (0.41 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,5' -tetraiodothyronyl]aminohexyl}-2',3'-0-isopropylidene-S'-adenylic acid monotriethylamine salt monohydra~e (6) was suspended in 0.6 ml of water (0C) and trifluoroacetic aci~
(6 ml) was added dropwise with stirring. After 50 minutes , clear solution was obtained. The solution was kept col~
~ (0C) for an additional 15 hours the~ evaporated in v~cu~
; (30C). The resulting residue was evaporated in vacuo f ive times from 20 ml ~olumes of anhydrous ethyl alcohol then triturated with 30 ml water and washed with a small volume of methyl alcohol. The resulting whlte solid (430 mg) was re-crystallized from methyl alcohol to give 290 mg (54.6~ yiel~) of white solid, m.p. 180-183C (decomposed).

Analysis: Calculated for C33H35F3I4N
H, 2.71; N, 7.54 Found: C, 30.77; H, 2.55; N, 7.29 Mass Spectrum (20 ma) m/e: 1302 [MH ], 1204 [M minus COCF

~ ~ 2 ~

Flavin adenine dinucleotide - thyroxine conjugate (8).

130.13 mg (0.1 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,5' -tetraiodothyronyl]aminohexyl}-5'-adenylic acid (7) was ~lace~l in an argon atmosphere. To this sample was added a solu~io of 14 ~1 (0.1 mmol) of triethylamine in 1 ml of dry dimethylformamide followed by the addition of a solution of 16.2 mg (0.1 mmol) of l,l'-carbonyldiimidazole in 1 ml of dry dimethylformamide. After 24 hours, a second equivalent of l,l'-carbonyldiimidazole (16.2 mg) in 1 ml of dry (limethyl-formamide was added. The above reaction was allowed to proceed a total of 48 hours at room temperature excluding moisture.
A sample of 47.3 mg (0.1 mmol) of the ammonium salt of riho-1avin-5'-monophosphate was converted to the correspondin~
tri-n-octylamine salt as described in section l-I above. lhis salt was dissolved in 3 ml of dry dimethylformamide and ad~cd to the above solution containing the phosphorimidazoli~ate of the adenylic acid intermediate (~).
The resulting solution was allowed ~o stand in the dark at room temperature excluding moisture for 24 hours. lhe solvent was cvaporated in vacuo and the resulting residue w~s chromatographed on a column l2.5x78 cm) prepared from 100 ~
of Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) which had been preswollen (18 hours) in a 19:1 (v:v) mixturc of dimethylformamide and triethylammonium bicarbonate (1 M, pH 7.5). The column was eluted with the above 19:1 (v:v) mixture and 5 ml fractions were collected. The effluent from the column was monitored by elution on silica ~el 6() silanised RP-2 TLC plates (E. Merck, Darmstadt, West Germany).
The TLC plates were developed using a 40:40:25:1:1 (v:v) mix-.~(i ture of acetonc, ~hloroform, methyl alcohol, watcr, an~l tri-ethylamine.

Fractions numbered 24 through 38 from the colulnn chromatography were combined and evaporated i~l v~c~o. 'I'he residue was chromatographed on a column (2.5x85 cm) I-reparc~
from 125 g of Sephadex LH-20 which had been preswollen (18 hours) in 0.1 M ammonium bicarbonate. The column was elute~
with a linear gradient generated from 2 L of 0.7 M ammonium bicarbonate and 2 L of water and 23 ml fractions collected.
The effluent was monitored by ultraviolet absorption (254 ~m).
Fractions numbered 170 through 182 were combined and eva~orat~d in vacuo. The residue was chromatographed on a column (2.5x55 cm) prepared from 80 g of Sephadex LH-20 which had been pre~wollen in 0.05 M ammonium bicarbonate. ~`he ~olum was eluted with a linear gradient generated from 2 L of 0.05 M ammonium bicarbonate and 2 L of 0.02 M ammonium hical-bonate. The effluent was monitored by ultraviolet absorptio (254 nm). F.lutlon was continued with 2 L of O.Z M ammonium bicarbonate, collecting 23 ml fractions. A total o~ 257 fractions was collected. Fractions numbered 70 through ll( were combined and evaporated in vac~o leaving -the labeled conjugate (8) as a yellow-orange residue. An alkaline, aqueous solution of this residue exhibited ul~raviolet ilbsorl--tion maxima at the following wavelengths: 270 nm, 345 nm, ;Ind 450 nm. The yield, estimated from ~he absorption at 45() nm, was about 5%.
A phosphodiesterase preparation (Worthington Biochemic.~l COTP., Freehold~ New Jersey VSA) isolated from snake venolll (Crota~us Adama~teus) hydrolyzed the above pro~uct to ribo-flavin-5'-monophosphate and the thyroxine substituted 5'-adenylic acid (~) wherein the trifluoroacetyl blockillg ~() group had been removed.

2-II. BINDING ASSAY FOR THYROXINE

The above-prepared labeled conjugate was used il~ a prosthetic-group labeled specific binding assay as fo~lows (further details regarding such an assay method m~y be ~olJn-l in the U.S. Patent Application - Serial No.917,961- referrcd to hereinbefore):

A. Preparation of apoglucose oxidase The apoenzyme used was prepared by the method descril-ed in section 1 II, part A above. -~

.
B. Assay Reagents i 1. Labeled conjugate - The hexyl analog labeled conjugate prepared as in section 2-I above was .
diluted in 0.1 M phosphate buffer (p~ 7) to a concentration of 100 nM.
2. Apoenzyme - This reagent was the same as tllat described in section l-II, part B-2 above.
3. Insolubilized antibody - This reagent was the same as that described in section l -II, part B-3 abovc.
4. Standard - A 1.15 mM stock solution of thyroxine in 5 mM sodium hydroxide was diluted to 1 ~M in 0.1 M phosphate buffer (pH 7).

- ~3 -~81~

-S. Monitoring reagent - A glucose oxidase reagent was prepared to contain the following mixture per 117 ~1: 25 ~1 of 1.2 mg/ml peroxidase (Sigma Chemical Co., St. Louis, Missouri USA) in ().] M
phosphate buffer (p~l 7), 5 ~1 of 10 mM
4-aminoantipyrine in water, 20 ~1 of 25 m~l 3,5-dichloro-2-hydroxybenzene sulfonate in ~.l M
phosphate buffer (pll 7), 17 ~1 of 30% bovine serum albumin in 0.1 M phosphate buffer (~ 7), and 50 ~1 of 1 M glucose in aqueous saturated h~nzoi~ -acid solution.

C. Assay Procedure Binding reaction mixtures were prepared by mixin~ 30 ~1 of the insolubilized antibody suspension, 100 ~1 of the l~hclc~l conjugate solution, either 100 ~1 or none of the standard thyroxine solution, and a sufficient volume of 0.1 M phosphato buffer (pH 7) to make a total volume of 500 ~1. The reactio mixtures were incubated with shaking for two hours at 25~.
Each reaction mixture was then vacuum filtered througil a glass wool plugged, dry pasteur pipette previously treated sc<luen-tially with periodate and ethylene glycol solutions to eliminate possible FAD contamination. To a 350 ~1 aliquot ; of each filtrate was added 117 ~1 of the monitoring rca~ent and 50 ~1 of the apoenzyme solution. Af~er one hour, the absorbance of each reaction mixture was measured at 520 nm.

D. Results ~ ollowing is Table 4 showing the results of tho ~lss~y procedurc in measuring thyroxine. lhe absorbancc reslllts ;Irc expressed as the avera~e of duplicate runs corrected for ~28~3~3 residual enzyme activity in the apoenzyme solution (al).sorban~
of 0.467) and for endogenous FAD in the antibody susl)ension (absorbance of 0.041).

TA~LE 4 ~. , `l 5 Volume of ThyroxineAbsorbance (520 nm) Standard Added (~1) , o 0.231 : 100 0.295 `, :
. The results demonstrate that the present labeled conjugates : 10 are useful in a specific binding assay method for determillin~ a ligand in a liquid medium.

:`
'

Claims (35)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A flavin adenine dinucleotide - labeled conjugate of the formula:

wherein Riboflavin-(Phos)2-Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide, n = 2 through 6, and ?CO)L is a specifically bindable ligand, or a binding analog thereof, bound through an amide bond.

2. The labeled conjugate of claim 1 wherein said specifically bindable ligand is an antigen or an antibody thereto; a hapten or an antibody thereto; or a hormone, vita-min, or drug, or a receptor or binding substance therefor.

3. The labeled conjugate of claim 1 wherein said specifically bindable ligand is an antigenic polypeptide or protein, a hapten, or an antibody.

4. The labeled conjugate of claim 1 wherein said specifically bindable ligand is an antigenic polypeptide or protein of molecular weight between 1,000 and 4,000,000.

5. The labeled conjugate of claim 1 wherein said specifically bindable ligand is an antibody.

6. The labeled conjugate of claim 1 wherein said specifically bindable ligand is a hapten of molecular weight between 100 and 1,500.

7. The labeled conjugate of claim 1 wherein said specifically bindable ligand is an iodothyronine hormone.

8. The labeled conjugate of claim 7 wherein said hor-mone is thyroxine.

9. The labeled conjugate of claim 1, 2 or 3 wherein n = 2.

10. The labeled conjugate of claim 1, 2 or 3 wherein n = 6.

11. A flavin adenine dinucleotide - iodothyronine conjugate of the formula:

wherein Riboflavin-(Phos)2-Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide, n = 2 through 6, and .beta.1 and .beta.2 are, independently, hydrogen or iodine.

12. The conjugate of claim 11 wherein n = 2.

13. The conjugate of claim 11 wherein n = 6.

14. The conjugate of claim 11, 12 or 13 wherein and .beta.2 are both iodine.

15. The conjugate of claim 11, 12 or 13 wherein .beta.1 is iodine and .beta.2 is hydrogen.

16. The conjugate of claim 11, 12 or 13 wherein .beta.1 is hydrogen and .beta.2 is iodine.

17. A flavin adenine dinucleotide - iodothyronine conjugate of the formula:

wherein Riboflavin-(Phos)2-Ribose represents the riboflavin pyrophosphate-ribose residue in flavin adenine dinucleotide, n = 2 through 6, Y is an amine-protecting group, and .beta.1 and are, independently, hydrogen or iodine.

18. The conjugate of claim 17 wherein n = 2.

19. The conjugate of claim 17 wherein n = 6.

20. The conjugate of claim 17 wherein Y is trifluoro-acetyl.

21. The conjugate of claim 17, 18 or 19 wherein .beta.1 and .beta.2 are both iodine.

22. The conjugate of claim 17, 18 or 19 wherein is iodine and .beta.2 is hydrogen.

23. The conjugate of claim 17, 18 or 19 wherein is hydrogen and .beta.2 is iodine.

24. A method for preparing a conjugate of flavin adenine dinucleotide coupled to a specifically bindable li-gand or a binding analog thereof, which method comprises the steps of:
(a) reacting 6-chloro-9-(2',3'-O-isopropylidine-.beta.-D-ribofuranosyl)purine with an .alpha.,.omega.-diaminoalkane selected from 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, and 1,6-diaminohexane to yield a 6-(.omega.-aminoalkyl)-9-(2',3'-O-isopropylidine-.beta.-D-ribofuranosyl)pur-ine;
(b) coupling said ligand or analog thereof to the primary amino group in said 6-(.omega.-aminoalkyl)-9-(2',3'-0-iso-propylidine-.beta.-D-ribofuranosyl)purine by formation of an amide bond to yield a ligand or analog substituted adenosine inter-mediate;
(c) treating said ligand or analog substituted ade-nosine intermediate with phosphorus oxychloride to yield a phosphorylated intermediate;
(d) hydrolyzing said phosphorylated intermediate to remove the isopropylidine group therefrom yielding a 5'-ade-nylic acid intermediate; and (e) condensing riboflavin-5'-monophosphate with the phosphorimidazolidate formed by treatment of said 5'-adenylic acid intermediate with N,N'-carbonyldimidazole to yield the desired flavin adenine dinucleotide-labeled conjugate.

25. The method of claim 24 wherein said specifically bindable ligand is an antigen or an antibody thereto; a hap-ten or an antibody thereto; or a hormone, vitamin, or drug, or a receptor or binding substance therefor.

26. The method of claim 24 wherein said specifically bindable ligand is an antigenic polypeptide or protein, a hap-ten, or an antibody.

27. The method of claim 24 wherein said specifically bindable ligand is an antigenic polypeptide or protein of molecular weight between 1,000 and 4,000,000.

28. The method of claim 24 wherein said specifically bindable ligand is an antibody.

29. The method of claim 24 wherein said specifically bindable ligand is a hapten of molecular weight between 100 and 1,500.

30. The method of claim 24 wherein said specifically bindable ligand is an iodothyronine hormone.

31. The method of claim 30 wherein said hormone is thyroxine.

32. The method of claim 31 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).

33. The method of claim 32 wherein said protecting group is trifluoroacetyl.

34. The method of claim 32 or 33 wherein said diaminoalkane is 1,2-diaminoethane.

35. The method of claim 32 or 33 wherein said diaminoalkane is 1,6-diaminohexane.