CA1137981A - 6-(.omega.-AMINOALKYL)-9-(2',3'-O-ISOPROPYLIDINE- .beta.-D-RIBOFURANOSYL)PURINES - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Compounds of the formula:

Description

BACKGROUND OF THE INVENTION

1 . FI ~3LD OF THE I N VI~NTI ON

This invention relates to novel labeled conjugates for use in specific binding assays for ligands OT their binding partners in a liquid medium. In particular, 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 ~o intermediate compounds produced in the lS synthesis of the novel labeled conjugates.
The iodothyronines ha~e the following general formula:

HOOC-CH-CH2 ~ ~ OH

wherein ~l`and ~2 are, independently, hydrogen or iodine. The principal iodothyronines of clinical interest are listed in Table 1 below.
.

- 2 -~.3~

Iodothyronine ~1 B2

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 ~he 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. Likewise, 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 DESCRIPT~OD 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 ~rom a test sample for binding to specific antibody. In the RIA technique9 sample antigen is quantitated by measuring the proportion of radioactivity which becomes associated with the antibody by -~3~

binding of the radiolabeled antigen (the bound-species of the labeled antigen) to the radioactivi-ty that remains un-associated from antibody (the free-species) and then com-paring that proportion to a standard curve. A comprehensive review of the RIA technique is provided by Skelly, et aZ., CZin. Chem. Z9:146(1973). While by definition RIA is based on the binding of specific antibody with an antigen or hap-ten, radiolabeled binding assays have been developed based on other specific binding interactions, such as between hor-mones and their binding proteins, From the radiolabeled binding assays have evolvednonradioisotopic 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 Offenlegungsehriften Nos. 2,618,419 and 2,618,511, assigned to the present assignee, employing particularly uni~ue la-beling substanees, including coenzymes, eyclie reaetants, eleavable fluoreseent enzyme substrates, and ehemilumines-~0 eent moleeules. Flavin adenine dinucleotide is mentionedas being useful as a coenzyme label since FAD functions as a eoenzyme in useful monitoring reactions. In Canadian Patent Application No. 330,233, filed June 21, 1979 and assigned to the present assignee, FAD is further deseribed ~5 as useful in improved speeifie binding assays employing a prosthetie group as the label beeause FAD also funetions as a prosthetie group in seleet bioehemieal systems.

~, .

.:: .; , .

Various methodologies exist for the determination of iodothyronine concentrations in serum. A significant ad-vance in iodothyronine assays ~as the development of the competitive protein binding assay by Murphy and Pattee, J.
CZin. ~ndoe~inoZ . Metab . 24:187(1964) in which radiolabeled iodothyronine competes with serum iodothyronine for binding to TBG. The development of specific antiserum for the various iodothyronines permitted radioimmunoassays to be devised in which radiolabeled and serum iodothyronine com-pete for binding to antibodies rather than to TBG. In both the competitive protein binding assay and the radioimmuno-assay for an iodothyronine, the radiolabeled material con-sists of the native iodothyronine in which one or more o~
the iodine atoms are replaced by a radioactive iodine iso-tope, 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 Canadian patent application No. 330,233 mentioned above. `
;~
SUMMARY OF THE INVENTION

Novel flavin adenine dinucleotide (FAD) - labeled conjugates have been devised for use in binding assays for determining ligands, or binding partners thereof, of anal-ytical interest, such as the iodothyronines, and particu-larly for use in the assay referred to hereinbefore employ-ing a prosthetic group label. The FAD-labeled conjugates have thQ general formula:

~ ~ 5 ~

. .. .. , . ,. . i . ~ ...

NH-~CH2~-nNH-~cO)L
~INXN~

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 -~CO)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 OT 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 lOO and 1,500.
FAD-labeled conjugates wherein the ligand therein is an iodothyronine are particularly use~ul in specific binding assays to determine the iodothyronine in liquid media such as serum and preferably have the general formula:

NI~ ~CH2~nNH - C - CHCH~

~IN ~N~ Bl 2 Ribofla~in-(Phos)2-Ribose wherein Ribofla~in-(Phos)2-Ribose represents the riboflavin -pyrophosphate-ribose residue in flavin adenine dinucleotide, n = 2 through 6~ and ~1 and ~2 are, independently, hydrogcn or iodine.
S ` 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 coenzyme or prosthetic group activity ~cncrated upon combination of such conjugate with an apoenzyme that requires FAD to perform its catalytic function as described in de ail in the abov~-n~ntioned Canadian pat~nt a~plication No. 330,233.
The present FAD-labeled conjugates~ can be prepared by a ~ariety of synthetic routes. Exemplary of such available synthetic routes is the following general reaction procedure:
Cl ~N~ ' ' ' ' ; ''`
H0 N ~ j ~i I - ' (1,) 0\/0 H3 ~ CH3 :
Reaction of 6-chloro-9-(2',3'-0-isopropylidine~ -ribofuranosyl) purine (1) ~Hampton et aZ, J. Am. Chem. Soc. 83:150(1961)3 with an ~,w-diaminoalkan~ selected from those listed in Table 2 ~ 7 n ~ diaminoalkane . . . _ 2 1,2-diaminoethane 3 1,3-diaminopropane 4 1,4-diaminobutane 1,5-diaminopentane 6 1,6-diaminohexane yields the intermediate 6-(~-aminoalkyl)-9-(2',3'-0-isopropylidine-~-D-ribofuranosyl) purine (2).

H-~CH2 ~ NH2 ~ ~J
O ~ ( 2 ) n = 2- 6 0~ 0 H3C~CH3 , The amino-purine intermediate (2) is then linked by formation of a peptide or amide couple with either the ligand, where such contains 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 ~ J
O ~ (3) n = 2-6 0\,0 H C ~ CH

.

~L~ 3~

wherein tCO)L is the ligand or analog thereof bound by an amide bond. Such condensation reactions can be accomplishe~
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 1~4.1344(1964], the mixed anhydride reaction [Erlanger et a~, Methods In lmmuno-~ogy and Immunochemistr~, 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: 72~(1976).
It will be recognized, of course, that other well known methods are available for coupling the ligand or a derivativc thereof to the amino-purine intermediate (2). In particular, conventional bifunctional coupling agents can be employed for coupling a ligand, or its derivative, containing a car-bo~ylic acid or amino group to the amino-purine intermediate (2). For example, amine-amine coupling agents such as bis-isocyanates, bis-imidoesters, and glutaraldehyde [Immunoohem. 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 ChromatographyJ John Wiley ~ Sons ~New York 1974).

: . .

Such coupling ~echniques 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 a~ailable 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 pep~ide condensation or can be the ligand or derivative thereof coupled through a bridging group inserted by coupling of the ligand or derivative ~ith 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 reactlons during coup-ling. Protection of reactive groups may also be desirable to .
, .~ .....

.

prevent interfering reactions during the synthetic ste~s described below for completing ~he preparation of the FAD
-labeled conjugate. Depending upon the specific ligand or derivative involved and the coupling technique chosen, the addition of protecting groups at the reac~ive sites on the ligand or derivative can be accomplished before or after the coupling to the amino-purine intermediate (2). One skille~
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 pro~ect the amine group prior to conden-sation or linkage with the amino-purine intermedia~e. The amine-protected iodothyronine intermediate has the formula:

HOOC-CHCH2~ ~O~OH (~) 2 = H or I
. `,:
wherein Y is an amine-protecting group. It will be recogni~ed ~ .
that 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 7 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 S 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 intermediates 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:

- C-CHCHz ~ O ~ OH (5) ~ 2 = H or I

wherein Y is an amine-protecting group as above.

.

~ ~ 3t~

Treatment of intermediate (3) with phosphorous oxy-chloride produces the phospho~ylated ligand or analog sub-stituted adenosine intermediate (6) NH-~GH2 ~ H-~CO)L

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

~ .
n = 2-6 -O O :.
H3CXcH3 which upon hydrolysis yields the ligand or analog sub-stituted 5'-adenylic acid intermediate (7).

NH-~CH2 ~ NH-~CO)L

Il <N ~ N~J (7) HO-IP-O ~ ~

n = 2-6 H H

Condensation of riboflavin-5'-monophosphate with inter-mediate (?) activated to a phosphorimidazolidate by treatment i`
with N,N'-carbonyldiimidazole yields FAD-labeled conjugates ~8).

.
NH-~cH2~-n-NH~co)L

<~N ~ N (~) 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 (5) above, the resulting FAD-iodothyronine conjugates are of the formula:

NH-~CH2~-nNH-ICI-CHCH2 ~ O ~ H

~N ~ N ~1 ~2 \N ~ N ~ n = 2-6 Riboflavin-(Phos)2-Ribose ~1 ~2 = H or I

S 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 PAD-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 ~NH2 ~N
HO N
\1/0 I I
0\~0 ~ :"

wherein n = 2 through 6; and .. . .... .

formula B
NH-~CH2 ~ NH-~CO)L
~ ~ N

R ~ O

~herein -~CO)L is a specifically bindable ligand~ or a bind-ing analog thereto, and preferably is of formula (5), bound through an amide bond; n = 2 through 6; ~1 and ~2 are, independently, hydrogen or iodine; Rl is -OH or -O-P-OH
l l OH
when R2 and R3 together form the group X ~ or Rl is Il 2 3 H3C CH3 -O-~-OH when R and R 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 1,000 and 4,000,000, 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 amino-purine inter mediate (2) through an amide bond in the synthesis of the present FAD-labeled conjugate will now be presented.

. .

- 1~3~?~

~o7ypeptides and Proteir~

Representative of specifically bindable protein ligands are antibodies in general, particularly those of the IgG, IgE, IgM and IgA classes, for example hepa~itis antibodies;
and antigenic proteins such as insulin, chorionic gonadotropin (e.g., HCG), carcinoembryonic antigen (CEA), myoglob;n, 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 peptidesg ligands of ~his general category possess numerous available carboxylic acid and amino groups, coupling to the amino-purine intermediate (2) can proceed ~0 according to conventional peptide condensation reactions such the carbodiimide reaction, the mixed anhydride reaction, and so forth as described hereinabove, or by the use of conven-tional bifunctional reagents capable of coupling carboxylic `
acid or amino functions to ~he amino group in the amino-purine ~5 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 ~or 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 prostag~andins. Following are representative 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 formulae below, n represents an integer, usually 1 through 6, and Me represents methyl).

.3 Carbamazepine Dibenz[b,f]azepine is treated sequentially with phosgene, an ~-aminoalkanol, and Jones re~gent (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 Following the method of Cook et aZ, PharmacoZogist 17:
219tl975), quinidine is demethylated and treated with ~ .

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

Digoxin and Di~itoxin The aglycone of the cardiac glycoside is treated with succinic anhydride and pyridine according to the method of .
Oliver et aZ, J. C~in. ~nvest. 47 :1035~1968) to yield the following: ::~
z ~0~0 ~ .
I Me 7 _ .. :
~ ~J ~ ' ' ~ H Z c H or OH , HOOC~CH2~CO J

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

3 ~N ~ ~ ~ ~ C~ ~ COOH

Phenobarbital and Primidone Sodium phenobarbital is heated with methyl 5-bromovalerate .
and the product hydrolyzed to the corresponding acid derivative of phenobarbital lCook et aZ, Quantitative AnaZytic Studies in EpiZep~y, ed. Kelleway and Peterson, Raven Press tNew York 1976~ pp. 39-58]:

HN ~ C2 0~1~0 CC~ ~ COOH

~ 19 r To obtain the acid derivative of primidone following the same Cook et aZ reference me~hod, 2-thiophenobarbital is alkylated, hydrolyzed, and the product treated with Raney nickel to yield:
., o ~ ".
HN~C2~

~CH2 ~ COOH

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

~,0 HN~ N ~ CH2 ~ COOH
O .~
h~or~hine -.
: ' Morphine free base is treated with sodium g-chloroacetate according to the method of Spector et aZ, Soience 168 :1347 ~1970) to yield a suitable carboxylic acid derivative.
: ', . . .

Nicotine According to the me~hod of Langone et aZ~ Biochem. ~2~24):
5025~1973), tran8-hydroxymethylnicotine and succinic anhydride are reacted to yield the following:

. ~N ~ fH3 ~ N~

HOOC~CH2~C-OCH2J--i Andro~ens Suitable carboxylic acid derivatives of testosterone and androstenedione linked through either the 1- or 7-position on the steroid nucleus are prepared according to the method of Bauminger et aZ, J. Steroid Biochem. 5: 739(1974). Follow-ing are representative testosterone derivatives:

l-position HC~CH2)n ~1 O~V

?-position o S~CH2 ~ COOH

-; ; ,.

~3~

Estrogens Suitable carboxylic acid derivatives of estrogens, e.g., estrone, estradiol and es~riol, are prepared according to the method of Bauminger et aZ~ gUpra ~ as represented by the following estrone derivative:

Progesterones Suitable carboxylic acid derivatives of progesteron~
and its metabolites linked through any of the 3- J 6- or `
7-positions on the steroid nucleus are prepared according to the method of Bauminger et a~ ~upra, as represented by the following progesterone derivatives:

3-posi ~ion :
MeOH

.
.
"

- 22 - : :

- .. - , , . ~, , ~, : ~3'7~

6- posi tion ~3 C=O
Me ¦

0~
S~C~ ~COOH
-

7-position ~H3 ¦
C=O
Me f~-O ~ SW~C~ ~ COOH

The methods described abo~e are but examples of the many known techniques for forming suitable carboxyl.ic acid derivatives of haptens of analytical interest. The principal derivation techniques are discussed in CZ~n. Chem.
22:726~1976) and include esterifîcation of a primary alcohol with succinic anhydride lAbraham and Grover, pr~oip~eB of Compe~itive Protei~-Binding Assays, ed. Odell and Daughaday, J.B. Lippincott Co. (Philadelphia 1971) pp. 140-157~, forma-. tion of an oxime from reaction of a ketone group with carboxyl~
methyl hydroxylamine [J. ~ot. C~em. 234:1090(1959)], intro-.. .. ..
duction of a carboxyl group into a phenolic residue using chloroacetate lSaience 168;1347(1970)], and coupling to di~-zotized p-aminobenzoic acid in the manner described in J. ~io~.
Chem. 235 :1051(1960).

, . . :, . . .. .

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

1. Ethyl Analog 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-(2~3l-o-isopropylidene-~-D-ribofuranosyl)purine (1) [Hampton et al, J. Am. Chem. Soc. 83:150(1961)] was added with stirring over a 15 minute period to 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, saturated 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 obtain a yellow glass (15 g). A portion (3 g) of the glass was dis-solved in a small volume of water which was then applied to the top of a 25x55 centimeter (cm) *Dowex 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. Merch, Darmstadt, 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, Thin Layer Chromatography, Academic Press (1966)]. Fractions numbered 250 through 350 *Trade Mark ..
- . ,~ .

~-.3~

from the column chromatography were combined and evaporated in vacuo 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'-ribose), 7.90 (s, lH, purine), 8.26 ~s, lH, purine) Optical Rotation [~]D = -74.85 (c 1.0, C~30H) The remaining crude product (12 g) was purified by chroma-tography on Dowex 50W-x2 as described above. The overall yield was 8 g (55~).

~-(N-Trifluoroacet~l)amino-~-[3,5-diiodo-4-(3',5'-diiodo-4' -hydroxyphenoxy)phenyl]propanoic acid (4).

~ :
This compound was prepared by the method of Blank, J.
Pharm. Sci~ 53:1333(1964). To a cooled (0~C), stirred suspen~
sion of 5 ~ (6.4 mmol) of L-thyroxine (Sigma Chemical Co., St.
~0 Louis, Missouri USA) in 60 ml of dry ethyl acetate was added -~
11.5 ml of trifluoroacetic acid and 1.9 ml of trifluoroacetic anhydride. After 30 minutes the resulting clear solution was washed three times with 30 ml of water, once with 30 ml of 5 sodium bicarbonate, and twice with 50 ml of saturated sodium chloride. The combined aqueous washings were extracted twice with 20 ml of ethyl acetate. The ethyl acetate layers were com-bined and washed with 30 ml of water, then dried over magnesium sulfate. The dried ethyl acetate solution was evaporated in vacuo leaving a white solid. Recrystallization from a mixture of ethyl ether ~-~

3~

and petroleum ether gave a pinkish-white solid (3.~5 ~, 7n.5 yield) having a melting point (m.p.) of 228-2303C with decomposition.

Analysis: Calculated for C17HloF3I4N05: C, 23.39;
H, 1.15; N, 1.60 Found: C, 23.00; H, 1.05; N, 1.65 NMR [60 MHz, DCON(CD3)2] ~ 7.28 ~s, 2~, aromatic), 8.03 (S9 2H, aromatic), 9.7 (m, lH, amido) IR (KCl): 1700 (>C-O) Optical Rotation E~25 = -14.97 (c 1.0 dimethylsulfoxide) A second recrystallization produced a second precipitate (0.95 g) m.p. 224~228C with decomposi~ion. The overall yield 15was 87.5%.

N-{2-[N-~rrifluoroacetyl)-3,3'~5,5'-tetraiodothyronyl]
aminoethyl}-2'~3'-0-isopro~ylidene adenosine (3).

A solution of 8.72 g (10.0 mmol) of a-(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'-0-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 tAldrich 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 wus ~ert ;It room temperature for 22 hours. The solution was then a~de~
dropwise to 300 ml of cold (0C) water with stirring. ~he .

~3 ~

resulting white precipitate was collected by filtration and dried in vacuo (56C) to give 13.0 g of a light crea~ colored solid. The solid was dissolved in 500 ml of acetone and the solution was concentrated by boiling. The white solid which precipitated from the boiling acetone solution was collected by filtration while hot. Continued boiling of the filtrate produced two additional precipitates. The three precipitates were combined to give 8 g (66.6% yield) of a white solid, m.p.
198-200C (decomposed).
Analysis: Calculated for C32H30F3I4N7O8: C, 31.89;
H, 2.51; N, 8.14 Found: C, 31.95; H, 2.60; N, 7.86 NMR [220 MHz, (CD3)2SO] ~ 1.32 (s, 3H, isopropylidene), 1.55 (s, 3H, iso-propylidene), 6.14 (d, lH, l'-ribose), 7.02 (s, 2H, thyroxine), 7.82 (s, 2H, ~ -thyroxine), 8.25 (s, lH, purine),

8.36 (s, lH, purine), 8.41 (t. lH.
J=6. amido). 9.64 (d. lH. J=8.
~0 trifluoroacetamido~
~ptical Rotation [~]D = -11.82 (c 1.0, pyridine) N-{2-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]
aminoethyl}-2',3'-O-isopropylidene-5'-adenylic acid monotriethvlamine salt monohydrate (6).

A solution of 1.2 g (1.0 mmol) of N-{2-[N-(trifluoro- ;;
acetyl)3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-isopropyl-idene adenosine (3) in 10 ml of dry triethy]phosphate was pre-pared under a dry argon atmosphere at 0C. To the cold, stirred `

'.; . ` . `

~3~

solution was added 0.45 ml (5 mmol) of phosphorous oxychloride.
The resulting solution was kept for 24 hours at 0C, then added dropwise with stirring to 1 L of ice water. The resulting precipitate was collected by filtration and dried in vacuo 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 evaporated in vacuo and the resulting residue lixiviated with dry acetone, then re-crystalized from a mixture of dry methyl alcohol and dry ethyl ether to give 390 mg (27.8% yield) of a white solid, m.p. 173-183C (decomposed).
Analysis: Calculated for C38H48F3I4N8 12 H, 3~45; N, 7.98 Found: C, 32.24; H, 3.08; N, 7.58 NMR [60 MHz, (CD3)2SO] ~ 1.53 (s, 3H, isopropylidene~, 6.2 (d, lH, l'H-ribose), ~ ;
7.1 (s, 2H, thyroxine aromatic), 7.87 (s, 2H, thyroxine aromatic),8.27 (s, lH, purine). 8.52 (s, lH, Purine) Optical Rotation [~] D = -17.50 (c 1.0, CH30H) N-{2-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]
aminoethyl}-5'-adenylic_acid (7) 200 milligrams (mg) (0.14 mmol) of N-{2-[N-(trifluoro-acetyl-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-o-isopropyl-idene-5'-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 minutes a clear solution was obtained. The solution was kept cold ~0C) for an additional 15 hours, then evaporated in vacuo (30C). The resulting residue was evaporated four times in vacuo (25C) from 20 ml volumes of anhydrous ethyl alcohol and then dried in vacuo (25C) leavina a white solid.
The solid was stirred for 30 minutes with 10 ml of cold methyl alcohol, then collected by filtration and dried in vacuo (25C) to give a white solid (135 mg, 76% Yield) which slowly melted with decom~osition above 188C.
Analvsis: 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, (CD3~2SO] ~ 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, lH, amido), 9.66 (d, lH, trifluoroacetamido) `~
Optical Rotation ~]25 = -2.72 (c 1.0, pyridine) Flav~n aden n dinucl otide _ thyroxine conjugate (8) 498 mg (0.4 mmol) of N-{2-[N-(trifluoroacetyl)-3,3',-5,5'-tetraiodothyronyl]aminoethYl}-5'-adenylic acid (7) was dissolved in 10 ml of dry dimethylformamide and tri-n-butyl-amine [96 microliters (~1?, 0.4 mmol] was added followed by the addition of l,l'-carbonyldiimidazole (320 mg, 2.0 mmol).
After stirring for 18 hours at room temperature in the absence of moisture, water (280 ~1) was added and then the solvent evaporated in vacuo.

-;

~L~l3~

The resultinq oil was dried by repeated in vacuo evapor-ation from dry dimethylformamide (4 times from 10 ml). The resultin~ ~hos~horimidazolidate was redissolved in 10 ml of dry dimethylformamide and added dropwise to a 0.4 mmol solution of the tri-n-octylamine salt of riboflavin-5'-monophosphate in 10 ml of dry dimethylformamide. The salt was prepared by add-ing a solution of the ammonium salt of riboflavin-5'-monophos-phate (192 mg, 0.4 mmol~ in 10 ml of water to a stirred solution of tri-n-octylamine (176 ~1, 0.4 mmol3 in 100 ml of acetone.
After 30 minutes, the resulting mixture was evaporated in vacuo-The residue was dried by repeated evaporation in vacuo from dry dimethylformamide leaving the salt as an oran~e solid.
The above solution containinq the phosphorimidazolidate of (7) and the riboflavin-5'-monoPhosphate salt was divided into two equal aliquots after 24 hours and one aliquot was evaporated in vacuo. The resulting residue was chromatographed on a column (2.5x78 cm) prepared from 100 g of *Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) which had been preswollen (18 hours) in a 19:1 (v:v) mixture of dimethyl-formamide and triethylammonium bicarbonate (1 M, P~I 7.5). Thecolumn was eluted with the above 19:1 (v:v) mixture and 10 ml fractions were collected. The effluent from the column was monitored by elution on silica gel 60 silanised RP-2 TLC
places (E. Merch, Darmstadt, West Germany).
The TLC plates were developed usinq a 40:40:25:1:1 (v:v) mixture of acetone, chloroform methvl alcohol, water, and triethylamine. Fractions numbered 11 through 17 from the *Trade Mark , . ..

akove-mentioned column chromatography were combined and eva-porated in va~uo. The resldue was chromatographed on a co-lumn (2.5 x 75 cm) prepared from 125 g of Sephadex LH-20 which had been preswollen (18 hours) in 0.3 M ammonium bi-carbonate. The column was eluted with 0.3 M a~monium bicar-bonate collecting 10 ml fractions. The effluent was moni-tored by absorption of ultraviolet light at 254 nanometers (nm). The volume of the fractions was increased to 20 ml be~inning with fraction number 150. The salt concentration o~ the eluent was decreased in a stepwise fashion as follows:
0~15 M ammonium bicarbonate at fraction number 295, 0.075 ammonium bicarbonate at fraction number 376, and water at fraction number 430. A total of 480 fractions was collected.
Fractions numbered 200 through 235 were combined and evapo-rated in va~uo leaving the labeled conjugate f8) as a yellow-orange residue. An alkaline, aqueous solution of this res-idue exhibited ultraviolet absorption maxima at the follow-in~ wavelengths: 266 nm, 350 nm, 373 nm, and 450 nm. The yield, estimated from the absorption at 450 was about 5%.
~0 A phosphodiesterase preparation (Worthington Biochem-ical Corp., Freehold, New Jersey USA) isolated from snake venom (~rotaZus A~amanteusJ hydrolyzed the above product to riboflavin-5'-monophosphate and the thyroxine substituted 5'-adenylic acid (7) wherein the trifluoacetyl blocking ~5 ~roup 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 be found in the Canadian Patent Application No. 330,233 referred to hereinbefore):

~ .
~ ~ ,.

3~

A. Preparation of apoglucose oxidase Purified glucose oxidase with low catalase activi ty obtained from the Research Products Division of Milcs l.lbor~-tories, Inc., Elkhart, Indiana USA was twice dialyze~ for 12 hours each against 0.5~ (w:v) mannitol (30 volumes e~c~
Aliquots of the dialyzate containing l00 mg o~ glucosc o~ se each were lyophilized and stored at -20C.
Bovine serum albumin (200 mg) was dissol~ed in 12 ml of water adjusted to pH l.6 with concentrated sulfuric aci~, nlix~
with 150 mg charcoal (RIA grade from Schwarz-Mann, Or~n~eburg, New York USA), and cooled to 0C. Lyophilized glucose oxi-la~-(l00 mg) was redissolved in 3.l ml of water and 3 ml was addc~
to the stirred albumin-charcoal suspension with continued stirring for three minutes. The suspension was then rilterc-l through a 0.8 micron, 25 millimeters (mm) diameter Millil)orc filter ~Millipore Corp., Bedford, Massachusetts USA) moulltc~
in a Sweenex filter apparatus (Millipore CorpO) on a 5n ml disposable plastic syrings. The filtrate was quickly ne~ltr~-lized to pH 7.0 by addition of 2 ml of 0.4 M phosphate huffcr ~pH 7.6) and thereafter 5 N sodium hydroxide. Dry charcoal ~l50 mg) was then added and stirred for one hour at 0C. The resulting suspension was filtered first through a 0.8 miero Millipore filter and then through a 0.22 micron Millipore filter. To the filtrate was added glycerol to 25~ (v:v~ ~n~ ;
the stabilized apoglucose oxidase preparation was stored ~t 4C.

~ 33 -.

~ 3~

B. Assay Reagents 1. Labeled conjugate - The ethyl analog labeled conjugate prepared as in section l-I above was diluted in 0.1 M phosphate buffer (pH 7) to a 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 apoenzyme.
3. Insolubilized antibody - A washed, moist cake of Sepharose 4B gel (Pharmacia Fine Chemicals, Uppsala, Sweden) activated bv cvanoaen bromide accordin~ to the method of March et al, Anal. Biochem. 60:119 (1974) was added to a solution of 85 mg of antibody, (isolated from antiserum against a thyroxine-bovine serum albumin conjugate) in 20 ml of 0.1 M phosphate ~0 buffer (pH 7.0) and a~itated slowlv for 36 hours at 4C. Upon completion of the coupling reaction, 1 ml of 1 M alanine was added and shaking continued for four more hours to block unreacted sites. The re-sulting Sepharose-bound antibody was washed on a scintered funnel with 400 ml each of 50 mM sodium acetate - 500 millimolar (mM) sodium chloride IpH 5) and 50 mM phosphate buffer - 500 mM sodium chloride (pH 7), and 800 ml of 100 mM phosphate buffer (pH 7).

The moist filter cake was then suspended in 100 mM
phosphate buffer (pH 7) containinq 0.01~ sodium azide to give 22 ml of an about 50% suspension.

:.

r~

4. Standard - A 1.15 mM stock solution of thyroxine in 5 mM sodium hydroxide was diluted to 2 ~M in U. 1 M
phosphate buffer (pH 7).
5. Monitoring rea~ent - A glucose oxidase assay rea~ent was prepared to contain the following mixture ~er 130 ~1: 25 ~l of 1.2 mg/ml peroxidase (Si~ma Chemical Co., St. Louis, Missouri USA) in 0.1 M phosphate buffer (pH 7), 5 ~l of lO mM
4-aminoantipyrine in water, 20 ~1 of 25 mM
3J5-dichloro-2-hydroxybenzene sulfonate in 0.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 solution.

C. Assay Procedùre Binding reaction mixtures were prepared by mixin~ 150 ll3 of the insolubilized antibody suspension, 80 ~1 o~ the labeled ~ `
conjugate solution, various amounts of the standard thyroxine solution to give varying concentrations of thyroxine In the reaction mixtures, and a sufficient volume of 0.1 M phosphate `buffer ~pH 7) to make a total volume of 500 ~1. lhe reaction mixtures were incubated with shaking for two hours a~ 25~C.
Each reaction mixture was then vacuum filtered through a ~lass wool plugged, dry pasteur pipette previously treated sequcn-tially with periodate and ethylene glycol solutions to eliminate possible FAD contamination. To a 300 ~l ali~uot of each filtrate was adde~ 130 ~l of the monitoring rea~ent and 5Q ~1 of the apoenzyme solution. After one hour, the absorbance of each reaction mixturc was measurcd at 520 nm.

f~

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

Volùme of Thyroxine Absorbance (520 nm) 10Standard Added ~

0 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 mediùm.

~ 36 2. HexyZ AnaZo~

2- I. PREPARATION OF THE LABELED CONJUGATE

6-(6-Aminohexyl)amino-9-(2' 93' -O-isopropylidene-~-D-ribofuranosyl) purine (2).

16.0 g (50 mmol~ of 6-chloro-9-(2',3'-O-isopropylidene-~-D-ribofuranosyl) purine (1) [Hampton et a~, J. Am. Chem. Soc.
83:1501(1961)] 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 lS hours. The excess diamine was remo~ed by distillation under reduced pressure (60C, 0.01 mm Hg). The resulting pale yellow residue was adsorbed onto 150 g of silica gel 60 (E.
~5erck, 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 with 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 ethyl alcohol and triethylammonium bi-carbonate (pH 7.5, 1 M). Fractions numbered 391 through 900 ~`
~rom the column chromatography were combined and e~aporated in vacuo leaving 15.0 g of a glassy 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 . , 3r~

and triethylammonium bicarbonate (pH 7.5, 1 M). ~:raction~
numbered 19 through Z7 from the colu~n chromatography were combined and evaporated in vaCuo leaving 910 mg (91~, rc~ovcry) of a white glass.

Analysis: Calculated for ClgH30N~O4: 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, isopro-pylidene) 5.98 (d, lH, l'-ribose), ;
7.92 (s, lH, purine), 8.36 (s, 1~l, purine) Optical Rotation [a]25 = -50.11 (c 1.0, methyl alcohol) N-{6-[N-Trifluoroacetyl)-3,3',595'-tetraiodothyronyl]
aminohexyl}-2_',3'-O-isopro~ylidene adenosine (3).

A solution of 4.36 g (5.0 mmol) of ~-tN-trifluoroacetyl) amino-~-[3,5-diiodo-4-(3',5'-diiodo-4~-hydroxyphenoxy)-phenyl]
propanoic acid t4), prepared as described in section 1 r abovc, 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 this cold s~irred 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. l`he solution was left at room temperature for 22 hours. The solution was then added dropwise to 600 ml of cold (0C) water with stirring. The resulting white precipi-tate was collected by filtration and dried in v~cuo (60C) to give 4.90 g (78~ yield) of white solid. A samplc of thi~
solid was recrystallized from a mixture of acetone ~ w.lter giving a white solid, m.p. 205-207C (decomposed).

Analysis: Calculated for C36H38F3I4N7O~: C~ 34-28;
S H, 3.04; N, 7.77 Found: C, 3q.22; H, 2.99; N, 7.41 Mass Spectrum (20 ma) m/e: 1262 [Mll ], 1164 ~M minus COCF3]
Optical Rotation [~]25 = -21.89 (c 1.(), pyridine) N-{6-[N-~Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl~
aminohexyl}-2',3'-O-isopropylidene-S'-adenylic acid monotriethylamine salt monohydrate (6~.

A solution of 1.89 g (1,5 mmol) of N-{6-N-~trifluoroacetyl)-3,3',5,5'-tetraiod~tllyronyl~aminohexyl}-2',3'-O-isopropylidene adenosine (3) in 15 ml of dry tri-ethylphosphate was prepared under a dry argon atmospherc at -10C. To the cold stirred solution was added 0.68 ml ~7.5 mmol) of phosphorous oxychloride. The resultin~ solut iOIl was kept for 18 hours at -15C then added dropwise with stirring to 1.5 L o ice water. The resulting precipitate was colle~ted by filtration and dried in vacuo to give 1. 91 R
t87% yield) of a white solid. The solid was dissolved in 10 ml methyl alcohol and 0.38 ml (2.6 mmol) of tr~ethylaminc 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 C42H56F3I4N8Ol2 H, 3.86; N, 7.67 Found: C, 35.24; H, 3.88; N, 7.75 Mass Spectrum (20 ma) m/e: 1342 LMH 1, 1244 [M minus COCF3]
Optical Rotation [~]25 = -17.20 (c 1.0, CH30H) N-{6-[N-(Trifluoroacetyl)-3~3l95~5l-te~raiod4thyr aminohexyl}-5'-adenylic acid (7).

600 mg (0.41 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,8' -tetraiodothyronyl]aminohexyl~-2' 3 3'-O-isopropylidene-5'-adenylic acid monotriethylamine salt monohydrate (6) was suspended in 0.6 ml of water (0C) and trifluoroacetic aci~
(6 ml) was addéd dropwise with stirring. After 50 minutes clear solu~ion was obtained. The solution was kept cold (0C) for an additional 15 hours the~ evaporated ~n v~cu~
(30C). The resulting residue was evaporated in vacuo f ive times from 20 ml volumes of anhydrous ethyl alcohol then triturated with 30 ml water and washed with a small volum~ o~
methyl alcohol. The resul~ing white solid (430 mg) was re-crystallized from methyl alcohol ~o give 290 mg (54.6% yiel~) of white solid, m.p. 180-183C (decomposed).

Analysis: Calculated for C33H35F3I4N7O
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~]

. - , . . .
.. . .

J~

Flavin adenine dinucleotide - thyrox ne conju~ate (83.

130.13 mg (0.1 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,5' -tetraiodothyronyl]aminohexyl}-5'-adenylic aci~ (7) was ~lace~l in an argon atmosphere. To this sample was added a ~oluti~n 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 n~l of dry dimethylformamide. After 24 hours, a second equivalent of l,l'-carbonyldiimidazole (16.2 mg) in 1 ml of dry (~imethyl-formamide was added. The above reaction was allowed to proccc~
a total of 48 hours at room temperature excluding moisture.
A sample of 47.3 mg (0.1 mmol) of the ammonium salt of ribo-flavin-5'-monophosphate was converted to the corresponding tri-n-octylamine salt as described in section l-I above. l`hi~
salt was dissolved in 3 ml of dry dimethylformamide and addcd to the above solution containing the phosphorimidazolidate of the adenylic acid intermediate (7).
The resulting solution was allowed to stand in the dark at room temperature excluding moisture for 24 hours. The solvent was evaporated in vacuo and the resulting residue w~s chromatographed on a column (2.5x7B cm) prepared from lO0 ~
of Sephadex LH-20 (Pharmacia Fine Chemicals 9 Uppsala, Swe~en) which had been preswollen (18 hours) in a 19:1 (v:v) mixture of dimethylformamide and triethylammonium bicarbonate (1 M, 2S 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 (~. Merck, Darmstadt, West ~.ermany). ~ -The TLC plates were developed using a 40:40:25:1:1 ~v:v) mix-~t) ture of acetone, chloroform, methyl alcohol, watcr, ~nd tri-ethylamine.

.. . . . . .. ... . .

Fractions numbered 24 ~hrough 38 from ~he c~lunln chromatography were combined and evaporated in v~cuo. 'I'l~e residue was chromatographed on a column (2.5x85 cm) ~-re~arc~l from 125 R of Sephadex LH-20 which had been preswollen (18 hours) in 0.1 M ammonium bicarbonate. The column wa~ eluted with a linear gradient generated from 2 L of 0.1 M ammoniuln bicarbonate and 2 L o~ water and 23 ml fractions col]~ctc~.
The effluent was monitored by ultraviolet absorption (254 ~ln).
Fractions numbered 170 through 182 were combined and eva~rat~?~l n ~n vacuo. The residue was chromatographed on a column (2.5xS5 cm) prepared from 80 g of Sephadex LH-20 which ha~
been preswollen in 0.05 M ammonium bicarbonate. l`he colunln was eluted with a linear gradient generated from ~ L of 0.05 M ammonium bicarbonate and 2 L of 0.02 M ammonium ~)icar-bonate. The effluent was monitored by ul~raviole~ abxorptio~
~254 nm). Elutlon was continued with 2 L of 0.2 M ammoniun bicarbonate, collecting 23 ml fractions. A total of 257 fractions was collected. Fractions numbered 70 throu~h 11l) were combined and evaporated in vacuo leaving the labeled ~0 conjugate (8) as a yellow-orange residue. An alkalinc, aqueous solution of this residue exhibited ultraviolet absor~
tion maxima at the following wavelengths: 270 nm, 345 nm, ;Ind 450 nm. The yield, estimated from the absorption at 45() nm, was about 5%.
A phosphodiesterase preparation (Worthington Biochen~ic~l `
Corp., Freehold, New Jersey USA) isolated from snake venom (CrotaZus Adamanteus) hydrolyzed the abo~e product to ribo-flavin-5'-monophosphate and the thyroxine substituted 5'-adenylic acid (?) wherein the trifluoroacetyl hlochi ~() grou~ had been remove~.

--II. BINDING ASSAY FOR THYROXINE

The above-prepared labeled conjuga-te was used in a prosthetic-group labeled specific binding assay as follows tfurther details regarding such an assay method may be found in Canadian Patent Application No. 330,233 referred to hereinbefore):

A. Preparation of apoglucose oxidase The apoenzyme used was prepared by the method descri-bed in section l-II, part A above.

B. Assay Reagents 1. Labeled conjugate - The hexyl analog labeled conjugate prepared as in section 2-I above was diluted in 0.1 M phosphate buffer (pH 7) to a concentration of 100 nM.

2. Apoenzyme - This reagent was the same as that 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 above.

~0 4. Standard - A 1.15 nM stock solution of thyroxine in 5 nM sodium hydroxide was diluted to 1 ~M in 0.1 M phosphate buffer (pH 7).

~ _ 43 _ , . .. . ~ ....... . : . ~
.. . ,. , ~ :

3~

5. Monitoring reagent - A glucose oxidase rea~ent W;IS
prepared to contain the following mixture ~cr 117 ~1: 25 ~1 of 1.2 mg/ml peroxidase (Si~ma Chemical Co ~ St. Louis, Missouri USA) in ~).] M
phosphate buffer (pH 7), 5 ~1 of 10 mM
4-aminoantipyrine in water, 20 ~1 of 25 m~
3,5-dichloro-2-hydroxybenzene sulfona~e in 0.l M
phosphate buffer (pH 7), 17 ~1 of 30% bovine serum albumin in 0.1 M phosphate buffer (~i 7), and 50 ~1 of 1 M glucose in aqueous saturated henzoi~
acid solution.

C. Assay Procedure Binding reaction mixtures were prepared by mixin~ 30 ~1 of the insolubilized antibody suspension, 100 ~1 of the lahclc(l conjugate solution, either 100 ~1 or none of ~he stand~rd thyroxine solution, and a sufficient volume of 0.1 M phosl-hate buffer ~pH 7) to make a to~al volume of 500 ~1. The reuctio mixtures were incubated with shaking for two hours a~ 25~.
Each reaction mixture was then vacuum filtered through a glass wool plugged, dry pasteur pipette previously treated sequen-tially with periodate and ethylene glycol solutions to eliminate possible FAD contamination. To a 350 ~1 aliquot o~ each filtrate was added 117 ~1 of the monitoring rcagent and 50 ~1 of the apoenzyme solution. After one hour, the absorbance of each reaction mixture was measured at 520 nm.

D. Results ~ `ollowing is Table 4 showing the results of the ilS~y procedurc in measuring thyroxine. lhe absorbancc reslilts ;lre expressed as the average of duplicate runs corrected for - . . . . " ~.-residual enzyme activity in the apoenzyme solution (al)sorl~an~e of 0.467) and for endogenous FAD in the antibody susl~ension (absorbance of 0.041).

~A Bl,E 4 S Volume of Thyroxine Absorbance (520 nm) Standard Added (~

0.231 100 0.295 The results demonstrate that the present labeled conj Ugiil~CS
are useful in a specific binding assay method for d~tcrmioin~
a ligand in a liquid medium.

- 45 - :
.
. ~

Claims (3)

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

1. A compound of the formula:

wherein ? = 2 through 6.

2. The compound of Claim 1 wherein ? = 2.

3. The compound of Claim 1 wherein ? = 6.